Phytosfere'99 - Highlights in European Plant Biotechnology Research and Technology Transfer (Developments in Plant Genetics and Breeding)

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Developments in Plant Genetics and Breeding, 6

Phytosfere ‘99 Highlights in European Plant Biotechnology Research and Technology Transfer

Developments in Plant Genetics and Breeding

1A ISOZYMES IN PLANT GENETICS AND BREEDING, PART A edited by S.D. Tanksley and T.J. Orton 1983 x +516 pp. 1 6 ISOZYMES IN PLANT GENETICS AND BREEDING, PART B edited by S.D. Tanksley and T.J. Orton 1983 viii +472 pp. 2A CHROMOSOME ENGINEERING IN PLANTS: GENETICS, BREEDING, EVOLUTION, PART A edited by P.K. Gupta and T. Tsuchiya 1991 xv + 639 pp. 2B CHROMOSOME ENGINEERING IN PLANTS: GENETICS, BREEDING, EVOLUTION, PART B edited by T. Tsuchiya and P.K. Gupta 1991 vi + 630 pp. GENETICS IN SCOTS PINE edited b y M. Giertych and Cs. Matyas 1991 280 pp. BIOLOGY OF BRASSICA COENOSPECIES edited by C. Gomez-Campo 1999 x + 490 pp. PLANT GENETIC ENGINEERING: TOWARDS THE THIRD MILLENNIUM edited by A.D. Arencibia 2000 x + 272 pp. PHYTOSFERE '99 - Highlights in European Plant Biotechnology Research and Technology Transfer edited by G.E. de Vries and K. Metzlaff 2000 Vlll + 286 pp.

Developments in Plant Genetics and Breeding, 6

Phytosfere ‘99 Highlights in European Plant Biotechnology Research and Technology Transfer Proceedings of the Second European Conference on Plant Biotechnology, held in Rome, Italy, 7-9 June 1999 Chief Editors

Gert E. de Vries Karin Metzlaff Editorial Board: C. Bachem, I. Benediktsson, C. Bowler, C. Castresana, M. Delseny, S. de Vries, 0. Doyle, R . Nehls and B. Reiss

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Library of Congress Cataloging-in-Publication Data European Conference on Plant Biotechnology (2nd : 1999 : Rome, Italy) Phytosfere '99 - highlights in European plant biotechnology research and technology transfer : proceedings of the second European Conference on Plant Biotechnology, held in Rome, Italy, 7-9 June 1999 / chief editors, Gert E. de Vries, Karin Metz1aff.-- 1 st ed. p. cm. -- (Developments in plant genetics and breeding ; 6) Includes bibliographical references (p.). ISBN 0-444-50326-9 (alk. paper) 1. Plant biotechnology--Congresses. I. Vnes, Gert E. de. 11. Metzlaff, Karin. 111. Title. IV. Series. SB106.B56 E87 1999 631,5'233--dc21 00-057644

ISBN: 0 444 50326 9

All contributions in this publication have undergone peer review by the Advisory Board members of the European Plant Biotechnology Network (EPBN) @The paper used in this publication meets the requirements of ANSINIS0 239.48-1992 (Permanence of Paper). Printed in The Netherlands.

Preface Coinciding with the first proposals for research projects being submitted to the Fifth Framework Programme (FP 5) the Conference "Phytosfere '99" was held in Rome, highlighting the results of the Fourth Framework Programme (FP 4). This was as much an occasion to look back and assess the achievements made as to look forward and estimate future challenges. In the Biotechnology Programme of FP4 forty five plant biotechnology projects have been funded, some of which will continue work until the end of the year 2000. Particularly welcomed is the high industrial participation in these projects. Only three projects have no industrial partner and most have more than one. The 45 projects were grouped in six clusters based on their thematic focus and the EPBN was set up to further network the participating laboratories and to interact with a wider audience. The conference "Phytosfere'99" has been such an interaction, where a wide spectrum of stakeholders met to discuss the advances made through these projects. While it remains a primary necessity for scientists to communicate with each other to follow rapid technological advances and to develop new ideas, the communication with the "outside world" becomes ever more important. The Commission wishes to encourage this process of bringing people of different background together to improve the understanding between them and to prevent scientific advances from being locked up in "academic monasteries". When designing FP5, European science policy-makers wished to focus more on what plant sciences might be used for. The question of utitity is crucial at present. Awareness is rising for the possible environmental costs of intensive agricultural technology and consumers are increasingly concerned for the safety of their food, so that commodity production and trading is more than ever in demand of science-based standards. In addition, future markets and employment demand are becoming highly technical throughout and the ethical debate raises questions that go beyond human values in the strict sense, to include consideration of the limits of world resources. Perhaps not surprisingly, this new perspective for plant biotechnology has now become reality but it also encompasses an opportunity. An opportunity for scientific pioneers in the field to sow their skills and knowledge into fertile ground, while cultivating their own awareness of the expectations of the society. Responding to this challenge FP5 offers possibilities through its key actions for fundamental scientists to team up with engineers, entrepreneurs, investors, regulators or consumers to optimise the conditions for beneficial use of knowledge. In thi's new ~ogramme, the elucidation of plant systems will be part of many distinct activities, including breeding, primary production, disease control, raw materials upgrading (key action 5), food processing (key action 1) or investigation and exploitation of genetic and metabolic diversity, as well as the innovative and safe use of transgenes (key action 3). The trend is towards widening the scope of plant sciences and will be helped by the researchers themselves widening their thinking to address the arguments of the economic and society players most concerned. Everyone acknowledges the importance of interdisciplinary research for science. The coming together of biology, chemistry and mathematics gave birth to modem molecular genetics. Countless other disciplines played various parts. It is in the interest of the European Colvanunity that this interaction is taken even further to include areas outside experimental research, e.g. consumer issues, socio-economic considerations and private investment. Only the active and constant dialogue between all parties can guarantee the realisation of the huge potential of scientific and technological development. V

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CONTENTS

Preface

...........................................................................................................................

Section I

Introduction

v

Prof. Paolo Fasella (1 930 - 1999). .......................................................................................... 1 Owen P. E. Doyle, The European Plant Biotechnology Network and Phytosfere ‘99. ......... 3 Karin Metzlaff, AMICA, supporting the development of plant biotechnology in Europe. ................................................................... 7 Gert E. de Vries, Technology transfer support by the Plant Industrial Platform. ................ 1 1

Section I1

OpportunitiesAnd Challenges Of European Plant Biotechnology

Sylvia Burssens en Marc Van Montagu, Why do we need genetically modified plants? . 13 Jeff Schell, Science and agriculture in the 21st century. ...................................................... 17 Etienne Magnien, Opportunities and challenges of EU plant biotechnology. ................... 27 Steve Bowra et al., The forgotten area of plant biotechnology. .......................................... 29 Beatrix Tappeser, Biotechnology, food, agriculture, public policy and consumer concerns. ................................................................................. 37 Terry L. Medley, U S . biotechnology regulatory system. An industry view. ....................... 43

Section I11

Control Of Genes

Bernd Reiss, The cluster: “Control Of Genes” ..................................................................... 45 Tesfaye Mengiste and Jerzy Paszkowski, The molecular genetics of homologous recombination in plants. ................................................................................. 47 Phil J. Dix, Developments in plastid transformation 59 Michael Metzlaff, Post-transcriptional gene silencing in plants: Pain or delight in transgene research? ........................................................... 67 Karin Hollricher, Comments from the session rapporteur. ................. ........ .... 75

Section IV

Mapping Gene Location

Michel Delseny, The cluster: “Gene Location Mapping Cluster”. ...................................... Christiane Gebhardt et a]., Function maps of potato. .................................... Dona1 M. 0’Sullivan and Keith J. Edwards, Novel traits for cereal biotechnology - Positional cloning revisited. ........................................................................ Elly Speulman and Andy Pereira, Insertional mutagenesis of the Arabidopsis genome. .......................................................................... Helen Gavaghan, Comments from the session rapporteur. ...............................................

VII

77 81 91 10 1 105

SectionV

Controlling Development And Architecture

Chris Bowler, The cluster: “Controlling Developmental Processes And Architecture”. .. 109 Sacco de Vries, The European Plant Embryogenesis Network (EPEN). ............................ 1 13 Caroline Dean et al., Molecular analysis of flowering time and vernalization response in Arabidopsis, a minireview. .......................... 115 Domenico De Martinis, Modification of plant development by genetic manipulation of the ethylene biosynthesis and action pathway. ...................................... 123 Livio Trainotti et al., Molecular aspects of the strawberry fruit softening. ...................... 133 Sacco de Vries, Signals and their transduction in early plant embryogenesis. ................. 141 Malcolm J. Bennett, Mutational studies of root architecture in Arabidopsis thaliana. .. 149

Section VI

Response To Challenges Of The Environment

Carmen Castresana, The cluster: “Responses To Challenges Of The Environment”. ..... 157 Godelieve Gheysen et al., Concerted efforts to develop handles for plant parasitic nematode control. ........................................................... 159 P.S. Puzio and F.M.W. Grundler, Isolation and application of nematode induced promoters, genes and proteins from Arabidopsis thaliana. ........................ 169 S. Sanz-Alfdrez et al., Cis-elements in nematode-responsive promoters. .......... ..... 177 Andrew Maule, Virus resistance models in a EU crop plant, Pisum sativum. .................... 183 Kriton Kalantidis et al., Generation of 13k-gene sugar beet transformants and evaluation of their resistance to BNYVV infection. ........................... 189 G . Perrone et al., Phytotoxic activity of Mycosphaerella graminicola culture filtrates. .................................. ...................... 195 L.C. van Loon, Helping plants to defend themselves: biocontrol by disease-suppressing Rhizobacteria. .............. ................................ 203 Olav Keerberg et al., C 0 2 exchange of potato transformants with reduced activities of glycine decarboxylase .......................... Marianne Heselmans, Comments from the session rapporteur ......................................... 22 1

Section VII Uncovering Metabolic Pathways Christian Bachem, The cluster: “Uncovering Metabolic Pathways”. ............................... 223 Nicolaus von WirCn et al., Improving fertiliser use efficiency in agro-ecosystems and nutrient efficiency in plants. ................................................................. 225 Babette Regierer et al., A European approach towards phosphate efficient plants. ........ 235 Jean-Paul Vincken et al., Remodelling pectin structure in potato. .................................... 245 F. Damiani et al., Toward the identification of the genes for the synthesis of condensed tannins in forage legumes. ..................................................... 257

Section VIII Entrepreneurship In Plant Science Gwilym Williams, An overview of important market and technology transfer issues for commercialising academic plant biotechnology. ..................................

265

Author Index ....................................................................................................................... 283 VIII

Prof. Paolo Fasella (1930- 1999)

Professor Paolo Fasella, former Director-General of DG XII died unexpectedly on Friday, 11 June 1999 in Rome. Paolo Fasella, who retired from the Commission's services at the end of 1995, headed DG XII for 14 years and worked successively with five Commissioners - Davignon, Narjes, Pandolfi, Ruberti and Cresson. He was one of the main architects and promoters of European policies and programmes in the field of research and technological development (RTD) and it was particularly during the years when he was in charge of DG XII that the idea of increased European co-operation in science and technology gained substantial ground and led to the EU RTD Framework Programme becoming one of the main platforms for European co-operative efforts. Before joining the Commission in 198 l, Paolo Fasella had a brilliant international career in science. Trained and specialising in medicine, he soon discovered biology as an essential neighbouring discipline, a domain in which he held major positions in laboratories in his native Italy as well as in Germany, the former Soviet Union, Japan and the United States. After his appointment as Director-General of DG XII, he never ceased to be a man of science, who continued to follow scientific developments and trends to the point that many of his former colleagues asked for his opinion and advice on scientific matters many years after he had left the laboratory. And yet Paolo Fasella was never the representative of any single discipline. His scientific interests and knowledge stretched far beyond medicine and biology and there were few areas in which he was not a respected partner for serious discussions with specialists from other disciplines. Moreover, outside science and research Paolo Fasella was equally impressive. His outstanding educational background and wide cultural understanding enabled him to draw on many fields of learning to enliven and enrich meetings and discussions. As Director-General he combined his scientific knowledge and interests with a deep understanding of Community politics. His enthusiasm and never-ending energy impressed all, as did his extraordinary qualities as a very accessible, compassionate and warm human being, who listened and gave advice to the most junior as well as to the most senior of his staff and who was genuinely concerned about the welfare of all. Paolo Fasella enjoyed not only the respect and admiration but also the affection of all his staff. Those who had the pleasure and privilege of working with him will never forget him.

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

The European Plant Biotechnology Network And Phytosfere '99 P r e f a c e

Welcome to the proceedings of Phytosfere 99, the second pan European meeting for Plant Biotechnologists. Phytosfere 99, the premier event of European Plant Biotechnology Week provides a platform to highlight the major advances in plant biotechnology research, from the European Commission funded fourth framework programme. A total of 45 individual projects involving 400 laboratories in 20 countries represents a combined investment of 150 million Euros in plant biotechnology research. Phytosfere 99 and European Plant Biotechnology Week are organised by the European Plant Biotechnology Network (EPBN; figure 1), which is a joint initiative between AMICA, the Plant Industrial Platform and the Framework Programme IV plant biotechnology projects. EPBN is fully supported by the European Commission until November 2000. It is run by a management group with representatives from AMICA and PIP, and advised by an advisory board represented by co-ordinators from each of the six clusters within the Framework projects. Each cluster concentrates on specific topics: - Cluster I investigates gene control within plant cells - Cluster II focuses on mapping genes in different crops - Cluster III researches development processes in plants - Cluster IV concentrates on embryo development - Cluster V works on pathogen and environmental stress - Cluster VI analyses metabolic pathways and studies sink/source relationships.

The objectives of EPBN are: to add value to the scientific output of European plant biotechnology projects by increasing communications, interactions, and providing opportunities for networking between all participants; to promote the achievements of European plant biotechnology projects to the wider public; to stimulate technology transfer by bringing scientists into contact with industrial partners and technology developers: and to provide a 'one stop shop' contact point for information on plant biotechnology in Europe. The key to the success for EPBN is communications, between scientists, with industry and the public. The European Plant Biotechnology Database, has been constructed with an extended list of over 6,500 records representing scientists within and outside of the European Frame-

O w e n

E E. Doyle, Chairman of EPBN, BioResearch Ireland, University College, Dublin, Ireland

Introduction F i g u r e 1. Organisation of the European Plant Biotechnology Network

(Accompanying measure BIO4 CT98 4818) Advisory Board & activities: AMICA Science EEIG,

Karin Metzlaff Owen Doyle

Heads of the 6 FP4 clusters:

Christian Bachem Chris Bowler Carmen Castresana Sacco de Vries Michel Delseny Bernd Reiss

Website: http://www.epbn.org database Phytosfere'99 Industry contact meetings '98 & 2000

Plant Industrial Platform:

Gert de Vries Reinhard Nehls

NewsBriefs, NewsFlashes Brochures PB'99 & 2000 Startup Company Workshop '99 Daily support: [email protected]

work Programmes, EU plant biotechnology companies, policy makers at national and EU level, journalists and public relations contacts. A web-site at http://www.epbn.org was established and regular newsbriefs issued which resulted in several articles about plant biotechnology research being published in the trade and popular press, the newsbriefs were also issued to existing fourth framework researchers via email. A total of 4,000 copies of the EPBN news brochure were distributed to industry, scientists, policy makers and journalists and at various conferences and meetings. This publication which is in a very attractive newspaper format has proved very successful in communicating plant biotechnology to a wider audience. A second news brochure is presently being prepared. The first industry contact meeting was organised in June 1998 at DANISCO in Copenhagen, Denmark. A total of more than 70 participants were registered at this successfully industryscientist meeting. A future industry contact meeting will be arranged in October 2000 at Wageningen International Conference Centre, in addition to the planned Startup Company Workshop (Majorca 4-6 th November 1999) which is designed for scientists / entrepreneurs to learn more about starting their own company or transferring technology derived from plant biotechnology research to industry. Humans face the challenge of producing enough food to meet the demands imposed by economic, biological and agricultural factors" rising population; rising income; and an expectation of higher quality food and a more diverse diet; decreasing amount of land available for food production; lowering environmental impact of agricultural practices and preserving biodiversity. B iotechnology is one of the most exciting and dynamic industries of our day. It offers us the possibility of reducing our dependence on intensive farming. Plant biotechnology is central to the search for effective, environmentally safe and economically sound alter-

The European Plant Biotechnology Network natives to the use of chemical pesticides and the exhaustion of natural resources. To-day, applied plant science has four overall goals: increased crop yield, improved crop quality, reducing production costs and reducing negative environmental impact. B iotechnology is proving its value in meeting these goals. It offers farmers higher yielding crops with lower costs of production and new outlets such as nutraceuticals and crop-based bio-factories. It offers the European economy the potential of high quality, knowledge based job creation and the European consumer better quality, tastier and more nutritious food. Though there is public concern of genetic engineering, those who are close to the science understand that this is the next big frontier to be crossed. The potential and opportunities offered by plant biotechnology must not be missed. We must go forward on that basis rather than turning our backs on the science. The programme for Phytosfere 99 consisted of: a panel discussion "Opportunities and Challenges of European Biotechnology"to inform the public about the aims and methods of European Plant Biotechnology both at present and in the future; highlights from the Framework four projects that reflect the interaction between academia and industry; discussions on entrepreneurship in science; an introduction to Framework five; and a poster and trade show. These contributions made this a very successful and unique conference. This kind of communication between academia, industry, regulatory bodies, consumer organisations, policy makers and the general public is reflected as well in these proceedings. You will find contributions representing the science itself and contributions showing various views on it. We thank the folowing sponsors of the conference: Advanta Seeds, Rilland-Bath, the Netherlands Elsevier Science B.V., Amsterdam, the Netherlands PlantTec Biotechnologie GmbH, Potsdam, Germany PLANTA Pflanzengenetik Biotech.GmbH, Einbeck, Germany Rh6ne Poulenc S.A., Lyon, France Stazione Zoologica di Napoli, Italy Zeneca Agrochemicals, Bracknell, United Kingdom The European Plant Biotechnology Network is funded by the European Commission Research Directorate (formerly DGXII)

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

AMICA, Supporting The Development Of Plant Biotechnology In Europe AMICA Science EEIG (A Molecular Initiative in Community Agriculture) was founded in 1993, during Framework Programme 3, as a pan European non-profit making company. The need for such an organisation developed with the start of complex plant molecular genetics research projects, funded by the European Commission, in 1991. The John Innes Centre in Norwich and the Max-Planck-Institut far Ztichtungsforschung (Max Planck Institute for Plant Breeding Research) in Cologne translated this into reality. Since last year Bioresearch Ireland in Dublin has joined them in this effort. In addition to its 3 members AMICA has a Science Board (Fig.l) with well-recognised representatives from academia and industry to assist in its policy. The aim is to support the development of Plant Biotechnology in Europe. Currently AMICA is inviting more organisations to join in order to widen its base and activities. "Good fundamental research on Molecular Plant Biology is difficult to finance fight now in Europe. It is therefore most important that the best plant scientists in Europe should join forces to revert this trend" summarised the AMICA chairman, Professor Jeff Schell, the current situation in Europe at the last AMICA Science Board meeting. AMICA's mission is 1) to form a multinational platform that represents and increases visibility and impact of the EU plant science community; 2) to communicate between academia, industry & the general public for independent dissemination of plant science information; 3) to represent the vision of the European Plant Science community for the future and support decision making of funding agencies at European and national level on long term strategies to support independent basic and applied plant science; 4) to contribute to sustainable agriculture, horticulture and forestry. AMICA's activities are aimed at the support for scientific work and added value to the scientific work. In Framework Programme 3 these activities were combined in the PTP, Project of Technological Priority, linking 15 scientific themes together, including research, training, meetings, publications and administration. In FP4 these tasks are divided up into 1) project management as a basis and 2) EPBN, the European Plant Biotechnology Network, as an umbrella activity for all Plant Biotech projects. EPBN connects scientists, academia and industry, and finally these with the general public. Due to the development in Europe this is getting even more important now, the time of FP5, where AMICA will again apply for activities like EPBN to help solving existing problems in these three levels of collaboration and communication.

Karin Metzlaff, AMICA Project & EPBN Manager, University of Gent, VIB, Gent, Belgium

Introduction Figure 1. AMICA Science EEIG, Supporting the development of Plant Biotechnology in Europe Science Board & activities:

Northern Europe: Carol Barrett, IE Caroline Dean, UK Claus Christiansen, DK

Central Europe: Ton Bisseling, NL Christian Dumas, F Georges Freyssinet, F Marc Van Montagu, B

Southern Europe: Fotis Kafatos, G Maria Pais, P Javier Paz-Ares, E Francesco Salamini, I

Jeff Schell, chairman, D Stage 1: 1993 - 1996

FP3: Project of Technological Priority

Stage 2: 1995- 2000

FP4: Project management & EPBN

Stage 3:1999 -

FP5: Project management & EPBN-2

Communicate achievements, needs & future visions of Plant Biotechnology in EU

AMICA organised Framework Programme 3 projects from 130 of Europe's most outstanding plant biotechnology laboratories into 15 themes, under the guise of "Project of Technical Priority" or PTE Throughout its involvement, AMICA aided communications between the projects through three major publications, various workshops, and biotechnology transfer events such as the Phytosfere'97 conference. As part of the PTP, AMICA devised, implemented, and managed a highly successful training scheme that was open throughout the European Union to European scientists. Through this very popular scheme, 129 young European scientists were able to take short-term placements in different laboratories where they learnt the latest techniques and skills. In Framework Programme IV, five AMICA co-ordinated projects have finished: the "Arabidopsis genome project, ESSA2", the "Arabidopsis insertional mutagenesis, AIM" and the "Search for the function of Myb transcription factors in plants, MYB" projects developed new underpinning technologies and produced valuable scientific data. From all three networks new EU projects developed already, building on their experience and information. The other two projects, the "Control of source - sink relations by carbohydrate regulation of gene expression, Source-Sink" and the "Structure / function analysis of LRR proteins and their ligands in plant pathogen interactions and engineered resistance, SLIPPER", investigated successfully phenomena from a specific area. Five more research projects are still ongoing in the year 2000. RECOMBIO, Nitrogen Signalling and GMOF focus on specific biological questions, whereas ESSA3 and EGRAM further develop horizontal technologies. RECOMBIO, a consortium of 8 laboratories from 5 European countries has started two years ago to identify and analyse molecular mechanisms of genetic recombination and the genes involved in its control in plants. Important results obtained thus far are the identification of a role of chromatin in homologous recombination and resistance to DNA damage in plants, the isolation of several key recombination genes from plants, and an assessment of strategies to

AMICA improve the efficiency of gene targeting in plants. Upon completion of the project this year the development of new strategies to improve gene targeting and to alter the stability and fluidity of the chromosomes in plants are expected.

Nitrogen Signalling, a programme that aims to increase the effectiveness of nitrate uptake and use by (a) producing plants with altered expression of proteins that are important for nitrate uptake in the roots, and altered expression of nitrate reductase in the leaves, (b) analysing the signalling mechanisms that normally down-regulate the expression or activity of these proteins in vivo, and manipulate them, and (c) generating mutant plants which help to identify and isolate genes that play an important role in the signal transduction chain of nitrate and other nitrogen metabolites has completed the second year of research. Transformants are currently being isolated, the first mutants in nitrogen-signalling processes analysed and the impact on parameters like nitrate and protein levels, root architecture and flowering time investigated. GMOF, a consortium of eleven scientific partners, including four industrial, has made considerable progress in dissecting the complex network of processes and pathways that control flowering time. New genes involved in controlling flowering have been identified and how some of the genes work is being elucidated. Genes from the model plant Arabidopsis are being used to alter flowering time of a range of crops. ESSA3, the third Plant Genome Sequencing Project working on the model plant Arabidopsis, collaborates with the CWU consortium in the US and the Kazusa Institute in Japan to complete the sequencing of the chromosomes 4 and 5 of the model plant. Chromosome 4 is sequenced by now, work on chromosome 5 is well in progress. The sequence generated is available for public use. In addition to this a unified database for chromosome 4 containing DNA and protein sequence and structure, chromosomal features and data from functional analysis is under construction. At the end of the project advanced methods and strategies are available for future sequencing approaches. The sequences determined from flowering plants especially help to uncover genes elaborating a vast variety of chemicals from sunlight, air and water and to investigate the very sophisticated defense mechanisms of plants. EGRAM, a consortium of 13 partners in seven European countries, started two years ago with the objective of developing comparative genomics in cereals and demonstrating the potential of this method by finding and isolating genes for disease resistance in the cereals and related grass species. The project is divided into the Resources and the Diseases section. Using rice as a model species the Resources section has developed and released to partners two sets of comparative RFLP probes providing over 200 anchored markers across the cereal and grass genomes. In the final year a further set of probes will be developed from rice, maize and wheat ESTs providing comparative information on possible known function genes. Partners in the Disease section are fine mapping genes for resistance to powdery mildew, leaf scald and net blotch of barley and rusts of wheat and ryegrass. As project manager, AMICA helps with the preparation and submission of the proposals, admisters the finances for the project, advices on Intellectual Property and writes a consortium agreement acceptable to all participants. It assists in the production of project reports,

Introduction material for public relations and the organisation of scientific meetings. The project manager links the participants with other EU funded projects and partners from industry. All plant biotechnology projects in FP4 are complemented by EPBN, an AMICA project, in which AMICA and PIP, the Plant Industrial Platform, share the management and the day to day work. The EPBN advisory board supports them - two representatives from AMICA and PIP and six scientists, the cluster heads of the six FP4 Plant Biotech clusters representing altogether 45 projects. They work in close collaboration with the European Commission. The EPBN activities are described in detail in the "Introduction to Phytosfere and EPBN" in this book.

Future development AMICA co-ordinates two new projects developing genetic technologies, REGIA and EXOTIC, in the fifth Framework Programme. The REGIA project links 30 participants among Europe to undertake functional characterization of transcription factors from Arabidopsis, opening the way to their maximal biotechnological exploitation. In addition the consortium aims to obtain insights into regulatory hierarchies, gene redundancies, evolution and functional interdependencies among transcription factors. The EXOTIC project initiates a largescale programme aimed at determining the expression patterns of approximately 5000 genes from Arabidopsis. These expression patterns reveal one of the important facets of gene function that can be linked to others, such as phenotypes from loss-of-function and mis-expression mutants, and predictions based on protein sequence and structure, to reveal an holistic and predictive view of the cellular roles of gene products. This approach may reveal aspects of the functions of essential genes that are not amenable to genetic analysis. In addition to this, AMICA will again apply for activities similar to the current EPBN work to provide common actions for European Plant Science. In response to the latest developments of plant science in Europe, AMICA will focus more to build a multinational platform that represents and increases visibility and impact of the EU plant science community (Fig.2). It aims to communicate the achievements, needs and future vision of Plant Biotechnology in Europe. This will facilitate basic and applied research for sustainable agriculture, horticulture and forestry.

Secretariat The AMICA Project & EPBN manager, Karin Metzlaff, is located in Belgium at the University of Gent, VIB. (Tel. / Fax: +32-9-264-8724/5349, e-mail: , , ). Vanessa Kent and Derek Fripp at John Innes Centre (Norwich, UK) handle the administration. The AMICA chairman is Jeff Schell at the Max Planck Institute for Plant Breeding Research in Cologne, Germany.

10

Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

Technology Transfer-Support By The Plant Industrial Platform Introduction The Plant Industrial Platform (PIP) was established in 1992 and is since 1997 a society according to Dutch law. Membership to the platform is open to all (EU) plant biotechnology companies, plant breeders and seed producers. The platform provides means to interact with academic researchers and to gain easy access to the results of funded research programmes. A Steering Committee of approximately 5 industrial members supervises the activities of the PIP and interacts with EU officials on a regular basis. The composition of the Steering Committee is changed regularly to stimulate close involvement of PIP members. PIP members are invited to attend research meetings of EU funded research programmes, participate in PIPdriven workshops and are consulted on developments in the design and management of EU research programmes. Many research programmes have already been financially supported by the European Union since 1985 (BAP, BRIDGE, BIOTECH, ECLAIR, FAIR, AIR). The first modem biotechnology products are on the market and it is clear that plant biotechnology will have significant economical and social impacts. In support of this process the Plant Industrial Platform has established mechanisms for the distribution of novel research results or initiatives and stimulated improved contacts between the EU scientific research community and end users from industry. Several modalities have been initiated to reach these goals. Membership of the Plant Industrial Platform is therefore essential to gain rapid access to information concerning EU plant biotechnology. Activities In the Plant Industrial Platform Newsletter new research activities are highlighted as well as information on EU funding schemes, programme developments, company profiles, full address details of new contacts, proceedings of past meetings, progress reports of specific research fields and upcoming activities. If necessary the Newsletter is distributed in electronic form, if the volume of information excludes printing. Company requests for sample copies submissions for contributions can be directed to the PIP permanent secretary. The PIP NewsAlert is an email service for urgent matters, general news items and information on a range of publications, pertaining to agro-biotechnology business information, collected

Gert E. de Vries, PBP / PIP / EPBN management, Overschild, the Netherlands

11

Introduction

from the Internet and a range of other sources. This service is available only for PIP members. The PIP Internet site functions as an information source and introduction to the activities of the Platform. Its web-site address is: PIP members may be invited to otherwise restricted research meetings, as organised by the project co-ordinators from the different EU funded programmes. Yearly workshops organised in 1996 - 1998 have proven to be instrumental in bringing together specialists from different fields of interest: science, companies, patent lawyers. These and the annual members meeting offer a great opportunity to exchange ideas, contribute to education and build lasting relationships. The PIP is also directly involved in management activities of the European Plant Biotechnology Network (EPBN). Objectives of this network are: promotion of achievements from FP4 plant biotech research projects, enhancement of interactions between EU industry and academia and stimulation of the awareness of the benefits of plant biotechnology to society. PIP members profit from PIP's involvement through direct access to up to date information.

New developments In order to develop and expand the required academic - industrial partnership during FP5, the Plant Industrial Platform has developed a scheme which commits both the PIP members as well as partners of a research proposal to make use of opportunities for technology transfer and networking. Many FP-5 research proposals will, as they have in the past, include one or more European plant biotech-oriented companies as a research partner. Technically, the requirement for exploitation has been fulfilled. However for all partners a great potential for dissemination and exploitation may be missed if no further action is taken. Other European companies, small and large, could be interested in results or technology which may be irrelevant to the (industrial) partner(s) in the project. It is also possible that exploitation should be carried out by an external party as soon as property fights have been secured by the project partners. Finally, just dissemination of results is an essential step to investigate above described opportunities. It is therefore that the PIP offers direct, contractually binding, involvement in research projects as subcontractor to the co-ordinator of the project. This commitment will be documented in the submitted proposal. Contractual (principal or assistant) partners in shared cost actions are expected to carry out independent research and all share access fights to results, generated during the project. Since the PIP will not contribute to research activities, and thus will not share prior or current knowledge, the status as full contractor is not possible. It is therefore, that the PIP would act as a sub-contractor to the project management. The PIP has a legal status (association according to Dutch law), its activities therefore can be documented in the proposal. Involvement of the PIP would clearly demonstrate optimal possibilities for future application of research results, if a clear agreement is made on a non-obstructive method of technology transfer, while protecting original IPR.

Secretariat A permanent secretary is located in the Netherlands: dr. Gert E. de Vries, Meerweg 6, 9625 PJ Overschild, the Netherlands, phone: ++31-596.566.321, fax: ++31-596.566.508, email: 12

Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

Why Do We Need Genetically Modified Plants?

Summary

1

Biotechnology is a rapidly evolving field with a great potential to bring solutions for sustainable approaches for agriculture. The effort of the EU to stimulate plant molecular biology, and the international co-operations around plant genomes have generated the tools for developing good plant-biotechnology programs. The decisions of major agrochemical companies in Europe and in the US to profile themselves as Life Science companies, brings a major stimulus to EU universities for co-operating on possible applications of the findings of fundamental research. However, a survey of ongoing plant biotech research indicates that the environmental benefits of this work are not enough highlighted.

Introduction The first genetically modified plant was only obtained in 1983. Antibiotic resistance derived from a bacterial gene, was genetically engineered in a tobacco variety making use of the natural transforming capacity of a soil microorganism, Agrobacterium tumefasciens. Since then, the progress in plant biotechnology has been enormous. For most major and minor crops, transformation procedures have been established. Moreover, many plant genes have been isolated and the complete sequencing of several plant genomes, spurred by the Human Genome Project, is well advanced. The genomic sequences of the model plant in genetics, Arabidopsis thaliana, but also important crops such as rice and maize, are almost completely known. Recent development of novel methodologies, in particular functional genomics, is accelerating the unravelling of the genetic and molecular basis of plants, widening the potential for plant biotech applications. During the nineties large-scale field trials with transgenic crops, engineered to contain new traits of importance to large-scale agriculture, were undertaken. These trials have generated the necessary confidence for commercialisation to start the production of transgenic crops and in 1998, nearly 30 million hectares of land were planted with transgenic crops for commercial use. Almost three-quarters of this took place in the United States, 15% in Argentina and 10% in Canada. Despite the fact that field tests are also being performed in Europe and the effort of EU to stimulate research in plant biotechnology, Europe is lagging far behind, mainly due to a general concern about the potential risks of genetically modified plants and a general negative public perception. Nevertheless, major agrochemical companies in Europe

Marc Van Montagu, Department of Molecular Genetics, Ghent University, Belgium

13

Opportunities and Challenges like Novartis, Aventis, Bayer, BASE ICI/Zeneca have realised that modem agriculture has become highly polluting and that innovative but sustainable approaches are needed.

Genetically modified plants: what are the benefits? B iotechnology gives us the possibility to introduce directly or to modify input, output and physical traits into crops. To cope with the demands, in terms of food and feed production, of a spectacularly increasing world population, agriculture will always need the improvement of yields. Plants with a genetically modified architecture or that process solar energy in an ameliorated way, may produce a higher critical mass resulting in for example more or bigger fruits. Biotechnology can also contribute in many ways to a sustainable economy, reducing significantly harm to the environment as much as possible. One of the first successes of plant genetic engineering was the development of an insect resistant plant by introducing a Bacillus thuringiensis (Bt) endotoxin gene. Since then many different types of toxins, targeting specific insect groups, have been isolated. Currently, strategies for the introduction of virus, bacterial, fungal and nematode resistance have been developed. Engineered pest-resistant plants do not only have higher yields, but are also less dependent on environmental harmful pesticides that are, especially in the developing countries, still used at a large scale. Current research on plant endophytes may lead to the development of transgenic, non-leguminous plants, such as sugarcane or rice, capable to associate nitrogen fixing bacteria. This will enable them to fix their own nitrogen, avoiding the use of chemical fertilisers. Plants can also be engineered to produce chemicals for the generation of new, biodegradable polymers. Production of chemicals in transgenic plants is not only protecting the environment because a petroleum based production is avoided but also because of the good use of solar energy. Another example of a biotechnological application to a healthier environment is the use of plants for phytoremediation in order to clean away the pollution, e.g. heavy metals. Also resistance against abiotic stress can be engineered in plants. Most developing countries are located in semi-arid or arid regions where drought, salinity, flooding and heat are major problems that have to be tackled in agriculture. The production of transgenic crops that grow on marginal lands is a major asset to increase the food capacity in these poor countries. Particularly since, due to water shortage, salinisation is continually reducing the acreage of valuable land. People living in developing countries can also not afford expensive medication. Transgenic plants may function as mini-factories to generate low-cost drugs from secondary plant metabolites. At the moment it is already possible to express vaccines in plants; a striking example is the production of transgenic bananas that contain a vaccine against cholera or diarrhoea. The generation of antibodies in plants, opens the way to the development of lowcost diagnostic tests and clinical analyses. The creation of value from all parts of a plant is an aspect to be estimated and learned. For example, sugar cane is not only cultivated for sugar production. Biotechnology will lead to the improvement of sugar cane fibres for their use in either the production of paper or animal feed. Molecular techniques are also of great value to capture and create value of the natural biodiversity of the tropical rain forests. Unfortunately, forests and woodlands are diminishing 14

Why do we need GM plants ? at a fast rate. By use of the AFLP fingerprint technique the genetic structure within natural stands of tropical tree species can be rapidly defined in order to elucidate the dynamics of the tropical forest ecosystem. This information is of high significance for the valorisation of the tropical rain forest and for the reuse of devastated areas by replantation. By genetic improvement, combined with intensive management based on biodiversity studies, it will be feasible to produce more wood on less soil. So it will be economically interesting to surround the remaining tropical forest with extensive plantations. This might bring the insight to protect the source of the newly captured value, the natural forest, rather than pursuing its destruction.

Conclusions and perspectives Due to novel methodologies and the combination of several disciplines in plant sciences, the progress of biotechnology has been enormous during the last years and the scope of applications has become extremely interesting for modern society. It is of no doubt that, mainly due to the rapid increase of the world population and the global pollution, an industry based on plant biotechnology will be of one of the major solutions in the coming century in order to solve food shortage and environmental problems. Some seemingly well-intentioned NGO's try to stop this evolution by taking aggressive and irresponsible actions against the introduction of genetically modified plants. This can be very detrimental for the funding under the fifth framework of the EU. It is the task of the scientists to explain the political authorities and industrial leaders the value and the rationale behind transgenic plants. They should in the first place enter into dialogue with ecologists to inform them about the environmental benefits and advantages for the consumer. A major task is also restore the confidence of the public at large in science and scientists.

Authors of this publication Sylvia Burssens en Marc Van Montagu* Department of Molecular Genetics, Ghent University, Belgium. (*) Corresponding author.

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

Science And Agriculture In The 21 st Century

Potential There is little doubt that agriculture in the 21st century will be more and more "knowledge-driven" and that genetic engineering will play a major role in this process. Politicians, industrialists, farmers and scientists agree on this point. Countries like Austria and others that, for whatever reason, ignore this at this critical time when relevant knowledge is accumulating and when this knowledge is covered by intellectual property rights (i.e. with economic consequences because patents are taken) will unavoidably not be able to compete in a global economy based on agriculture. One might even fear that food processing will suffer if, as is to be expected, such processes will be made more economical by breeding of "tailor-made" crops especially with the aim to make processing easier. As evidence for this development we can take the increasing number of field trials from 1986 till 1992 (unfortunately for us mostly in the U.S.). Let us take an example. There is presently more and more interest in starch production (indeed, more than 600 commercial products involving starch are marketed). The uses of starch are not only plentiful but are bound to increase especially in non-food applications e.g. biodegradable plastics. Since starch is a biological and renewable commodity produced by agriculture, the following considerations are relevant with regard to the possible role that could be played by plant biotechnology and - genetic engineering. Many of the present procedures used to produce commercial products, are based on starch chemistry and would be considerably easier and cheaper if one could biologically separate the two forms of starch i.e. i) the branched form with ~(1-->6) linkages responsible for the densely packed glucan chains in the crystalline structure of plant starch (called amylopectine), and ii) the unbranched form resulting from cz(1--->4) links (called amylose). Two enzymes are critical in starch synthesis: one is the starch synthase (SS) and is responsible for the ~(1-->4) links and the other is the appropriately called branching enzyme (BE) responsible for the c~(1-+6) links and, therefore, for the synthesis of branch points. Since in plants the storage of starch takes place mostly in the form of granules, in the so-called amyloplasts, it is of importance to know the precise starch synthase and branching enzyme that is responsible for the synthesis of starch in amyloplasts. The storage form of starch in plants is a mixture of amylose and amylopectin. Amylopectin is by far the major compound, it is very long and has more than5 % of ~(1-->6) branches. Amylose is unbranched (less than

Jeff Schell, Max-Planck-Institut fiir Ztichtungsforschung, K61n, Germany

17

Opportunities and Challenges 1%), smaller and is found associated with amylopectin in the starch granules of amyloplasts. The goal of having plants only making amylose or amylopectin has been realized in Germany by the group of L. Willmitzer and world-wide by Unilever. First, before Willmitzer or Unilever, one has obtained amylose free potatoes by mutation and later in transgenic plants by antisensing the starch granule bound starch synthase. One has also obtained amylopectine free potatoes in transgenics with antisense constructs of branching enzymes. Thus, although it has not been possible to obtain amylopectin free mutants, one has obtained transgenic potato plants producing only amylose (and not amylopectin) by inhibiting the formation of amylopectin by expressing in transgenic plants a gene coding for BE in the reverse orientation (antisensing). AVEBE (The Netherlands) is doing field trials with potatoes in which the starch composition is modified at their breeding station Karna. Large field trials such as those for the trials in 1997 and 1998 were stopped in view of the EU situation. Thus it should be possible to provide industry with plants, e.g. potato or maize, that only contain amylose or amylopectin or other starch derivatives that have industrial value. As a result the future of starch based non-food products is very promising to those who have people trained in biotechnology but not for those who have ignored this field. A global economy will benefit mostly those countries who have invested financially and politically in new technologies (e.g. biotechnology) that provides them with a competitive edge. This potential has been recognized by some countries such as the U.S., Japan and China, whereas most European countries are lagging behind and are inhibited by problems of acceptancy, notwithstanding the fact that a considerable and largely successful effort was made mostly in terms of research but was not sufficiently translated into commercial products.

Positive developments In this regards it is important to note that unfortunately exactly those organisations or political parties the support of which is based on the protection of the environment and on providing optimal social conditions, have been most active in rejecting this new technology because of its real and largely imagined risks. A word of caution for politicians is, however, appropriate, then attitudes could rapidly change: At a meeting in February 1997 in London organised by the Green Alliance on the theme "How can Biotechnology Benefit the Environment" the representative Jens Katzek of the Department for International Environmental Affairs of BUND in Germany said that there is a discussion going on within some environmental groups and political parties about whether the advantages of genetic engineering for the environment are, in some limited cases, so overwhelming that the concerns could be put aside. Because up to now, however, the arguments are not convincing, BUND maintains its position of rejection. For the optimist in me this means, environmental organisations, such as the BUND, are preparing themselves to reverse their opposition/f and when the arguments are convincing. The main consequence of this attitude is that this technology has been developed mostly abroad for the benefit of some regions and for industries of the rich world and that its potential for protection of the environment has been largely ignored (not exploited) in Europe. Indeed, in some countries of Europe, more than anywhere else, the public view new science and 18

Science and agriculture in the 21 st century

technology with caution. There is a lack of scientific facts and uncertainty of the potential risks involved. The experience gained from the use of nuclear power as well as the pollution resulting from the use of chemicals has made it obvious that new technologies can bring new and unexpected or unpredicted dangers. Therefore, government and industry are expected by the public to make sure that technical innovation is either not dangerous and that adequate means have been adopted to protect them from danger. Regulations, therefore, are crucial but should be based on solid and rational facts and considerations. Let us quickly review what biotechnology - and in particular genetic engineering - could do for protection of the environment and to improve the social conditions of the population involved in agriculture. Let me quote from a text called "The Bioengineering of Crops" and prepared by R. Beachy, F. Gould, T. Eisner, R. Herdt, H.W. Kendall, P. Raven, M.S. Swaminathan and J. Schell as a report of the World Bank Panel on Transgenic Crops: "What can be accomplished now. Plant scientists can now transfer genes into many crop plants and get stable intergenerational expression of encoded traits. Promoters can be associated with transferred genes to ensure expression in particular plant tissues or at particular growth stages. Transformation can be achieved with greater efficiency and more routinely in some dicots than in some monocots, but with determined efforts nearly all plants can be transformed.

G e n e transformation Transformation and marker assisted breeding have been used toward four broad goals: to change product characteristics, improve plant resistance to pests and pathogens, increase output, and produce unique metabolites. Changed product characteristics are illustrated by one of the first genetically engineered plants to pass regulations and to be made available for general consumption by the public, the FLAVR SAVR TM tomato, whose fruit ripening characteristics have been modified so as to provide a longer shelf life. Enhanced quantitative resistance against fungal disease by combinatorial expression of different antifungal proteins in transgenic tobacco has also been achieved. Biotechnology has also been used to change the proportion of fatty acids in soybeans, modify the composition of canola. "Horizontal" gene transfer from a transgenic potato line to a bacterial pathogen (Erwinia chrysanthemi) occurs - if at all - at an extremely low frequency (Bio/Technology 13, 10941098, 1995), and change in the starch content of potatoes as well as enhanced protection against fungal attack by constitutive co-expression of chitinase and glucanase genes in transgenic tobacco has been achieved. Natural variability in the capacity to resist damage from insects and plant diseases has long been exploited by plant breeders. Biotechnology provides new tools to add to this capacity. Bacillus thuringiensis (bt), which produces an insect toxin particularly effective against lepidoptera, has been applied to crops by gardeners for decades. Transformation of several different plants with the gene that produces bt toxin was one of the first demonstrations of how biotechnology could be used to enhance plants' ability to resist damage from insects (Ref.: EJ. Perlak and D.A. Fishoff in Advanced Engineered Pesticides (L. Kim, ed.), pp 199-211, Marcel Dekker. 1993). Transgenic cotton that expresses bt toxin at a level providing protection against cotton bollworm has been developed, and a large number of bt transformed crops 19

Opportunities and Challenges are currently being field tested. Other strategies to prevent insect damage include transfomation of crops with genes of plant origin for proteins that retard insect growth, such as lectins, amylase inhibitors, protease inhibitors and cholesterol oxidase. It might be of interest to combine several different strategies, including the use of environmentally friendly insecticides in order to obtain not only better protection through synergistic effects but also in order to dramatically reduce the probability of the emergence and selection of insects resistant to these insecticidal strategies. Genes providing resistance to viral plant pathogens have been derived from the viruses themselves, most notably with coat protein mediated resistance (CP-MR). Following extensive field evaluation, a yellow squash with CP-MR resistance to two plant viruses was approved for commercial production in the United States. Practical resistance to fungal and bacterial pathogens has been more elusive, although genes encoding several enzymes that degrade fungal cell walls or inhibit fungal growth have been explored. More recently classical genes for resistance to pathogens have been cloned, manipulated, and shown to function when transferred to susceptible plants. While protecting plants against insects and pathogens promises to increase harvested output by saving a higher percentage of the present yield, several strategies seek to increase the potential crop yield, including the exploitation of hybrid vigor, delayed senescence, early flowering and increased starch. Several strategies to produce hybrid seed in new ways will likely contribute to increasing yield potential. Cytoplasmic male sterility has been widely used even before modern biotechnology, but strategies to exploit nuclear-encoded male sterility required biological manipulations that can only be carried out using molecular biology tools, and several of these are quite far advanced. A number entail suppression of pollen formation by exploiting sensitivity to temperature or day length. Delayed senescence or "stay-green" traits enable a plant to continue producing photosynthate beyond the period when a non-transformed plant would, thereby potentially producing higher yield. Potatoes that produce higher starch content than nontransformed controls have been developed. Plants have been designed to produce a range of lipids, carbohydrates, pharmaceutical polypeptides and industrial enzymes, leading to the hope that plants can be used in place of microbial fermentation. One of the more ambitious of such applications is the production of vaccines in plants against animal or human diseases. The hepatitis B surface antigen has been expressed in tobacco, and the feasibility of using the purified product to elicit an immune response in mice has been demonstrated.

Gene markers Far-reaching possibilities for closely identifying genome composition has been made possible through various molecular marker techniques with exotic names like RFLP, RAPD, micro-satellites and others. These techniques allow scientists to follow genes from one generation to subsequent generations, adding powerfully to the tools at the disposal of plant breeders. In particular, it enables plant breeders to combine several resistance genes, each of which may have different modes of action, leading to longer-acting or more durable resistance against pathogens. It also facilitates the combining of several genes, each of which individually may provide only a weakly expressed desirable trait. 20

Science and agriculture in the 21 st century

On-going research Research continues to improve the efficiency and reduce the costs of the various means of transformation and the several types of genetic markers. As this research succeeds, it will be applied to more different plants and genes. By far the greatest proportion of current crop biotechnology research is being conducted in industrialised countries on the crops of economic interest in those countries. Plant biotechnology research in the 15 countries of the European Union probably is a fair reflection of global current plant biotechnology research. As of 1996 almost 2000 projects were underway, 1300 actually using plants (as opposed to plant pathogens, theoretical work and so forth). Of those about 210 were for work on wheat, barley and other cereals, 150 on potato, 125 on oil seed rape and about 90 on maize. Field trials reflect the composition of earlier research activities, and those data show the work on cereals was started somewhat later than on other plants. Some 1024 field trials had been conducted world wide through 1993; 88 % were in OECD countries, with 38 % in the United States, 13 % in France, 12 % in Canada, with Belgium, The Netherlands and the United Kingdom each hosting about 5 % of the total number of field trials. Argentina, Chile, China and Mexico led in numbers of field trials in developing countries, but none had more than 2 % of the total. The largest of field trials was conducted in potato (19 %), with oilseed rape second (18 %); tobacco, tomato and maize each accounted for about 12 %, with more than 10 trials each on alfalfa, cantaloupe, cotton, flax, sugar beet, soybean and poplar. Nine tests were done on rice, and fewer than that on wheat, sorghum, millet, cassava and sugar cane, the food crops that, aside from maize, provide most of the food to most of the world's people who live in the developing countries. Herbicide tolerance has been the most widely tested trait, accounting for 40 % of the field trials for agronomically useful genes. Twenty-two percent of tests were conducted on 10 different types of modified product quality - including delayed ripening, modified processing characters, starch metabolism, modified oil content. About 40 % of field trials in developing countries were for virus resistance, one-quarter of the field trials were for crops modified for herbicide resistance, another one-quarter for insect resistance, with the balance for product quality, fungal resistance or agromic traits." Let us see whether we can agree on a definition of plant genetic engineering (PGE). We propose that the broad definition of PGE is the changing of the genetic properties of plants to provide plant growers (farmers) with cultivars capable of providing us (the customers) with products and processes that will satisfy our needs. (This definition is a quote from Dr. Boulter in Critical Reviews in Plant Sciences 16, 231-251, 1997). PGE is, therefore, not really new but is as old as plant breeding or fermentation. It should be said quite clearly that today PGE is to be preferred over some traditional and well accepted methods used by plant breeders. Indeed, by introducing new well defined genes into plants via recombinant DNA technology, the probability of introducing unknowingly potentially deleterious genes in cultivars is much lower than by introducing them via sexual crosses. By saying this we do not want to imply that the present products of the seed industries are potentially dangerous: not only do plant breeders eliminate all products from their crosses that would be dangerous or do not represent a clear improvement over previous cultivars, but they have a very long and satisfying experience with most of the products of agriculture.

21

Opportunities and Challenges What we do say is that, provided plant breeders incorporate recombinant DNA procedures or PGE in their methods for the production of better cultivars, this should not increase the potential for unwanted properties of their products but, in fact, result in potentially even safer products. Of course, if one wanted to make more detrimental products (e.g. for warfare) this would also be possible. In order to discuss the concerns for potential dangers of gene technology I shall quote from the article by P. Boulter in Critical Reviews in Plant Sciences 16, 231-251, 1997. It is clear that society will have to rely on the advice of knowledgeable and scientifically qualified experts for the formulation of a proper attitude. The politicians and administrators responsible for the formulation and implementation of proper regulations will also have to rely on scientific proven facts and rationale considerations. On top of all this one has to realise that in a global economy competition will be fierce and that, if Europe wants to keep its high standards of living and civilisation, it will have to be competitive. In view of the fact that most economically relevant developments will be more and more knowledge rather than exclusively tradition driven, Europe can in the future only maintain its tradition if they are complemented by a more liberal attitude towards new mostly knowledge driven, technologies. That is the main reason why, in the field of plant biotechnology, a number of well known and respected scientists and industrialists have formed AMICA (Advanced Molecular Initiative in Community Agriculture). AMICA is an initiative by independent plant scientists.

National versus EU responsibility The uncontested mandate of competence for the EU-Commission is: "The strengthening of the scientific and technological basis of industry with a view to strengthen the capacity of European industry to be competitive in the international marketplace". Support and organisation of basic research in contrast should remain the main responsibility and in the competence of national organisations. This division does not withstand the test either of pragmatic application or of critical examination. Although there can be no doubt that national authorities have to play a prime role in supporting and implementing basic research of the highest quality, it is nevertheless a fact that the selection of action lines (projects) is very often very difficult if not impossible at the national level. An international evaluation system is therefore essential if European basic science is to be internationally competitive. This may not always be possible if this evaluation process is the prime responsibility of national authorities. The Commission should, therefore, continue to play a major role in selecting basic research action lines. The EU Commission should, however, make sure that the selected action lines (projects) are successfully implemented and transferred to industry if appropriate. One of the dominant principles governing Community research is subsidiarity. Community research ought to be complementary to national research, and should therefore not duplicate, substitute or counteract investments made in the Member States. The involvement of national organisations and their experts in the process of defining specific community initiatives to complete national efforts would be very effective. The value of science can only be measured in international terms and high quality scientific progress can only be achieved and measured 22

Science and agriculture in the 21" century

in international terms. By providing national research institutions with international standards for scientific decision-making and evaluation, one could help to mobilise the best of the research that is carried out in national institutions for a common European policy thereby contributing to resolving the tension between national and EU competences. Role of scientists versus science administrators in decision making, problems of the autonomy of science: Process in science and technology cannot be achieved without a high

level of motivation of the scientists involved, and also progress is essentially of a very complex rapidly evolving and largely unpredictable nature. Active scientists should, therefore, be involved as much as possible in i) decisions relative to the content of a research programme and ii) decisions relative to quality standards (because they can tell the difference between good and less good research), but also be responsible for the implementation of these programmes. Cohesion versus competitiveness:_By integrating European and national efforts and establishing a pan-European research network wasteful funding and waste in scientific manpower will be avoided. The exchange of ideas, materials, techniques within the network can be promoted.

However, this is only the beginning. Indeed, the most far reaching importance of molecular biology and genetic engineering (GE) will be its use as a powerful research tool since it will have a revolutionary impact on all classical biological disciplines not only genetics, physiology, biochemistry, development but also taxonomy and ecology. A considerable part of all this fast increasing knowledge will undoubtedly have great applied significance. Indeed, one shall in the near future know how plants resist biotic and abiotic (e.g. climatic) stresses; how they develop; why and how they adapt themselves to their biological or geographical environment, as well as how they synthesise products that are important for our health (nutrition, pharmaceuticals) or can be made to produce important commodities (such as e.g. biodiesel and biodegradable plastics etc., etc.). It will then be relatively straightforward to use this knowledge for the realisation of a more competitive, more productive and more sustainable agriculture, not by using only new knowledge and techniques but by integrating this new knowledge and new techniques with those that were developed on the basis of tradition and that define for many of us "quality of life". Internationally, as opposed to what happened and happens in most of Europe and especially in German speaking regions, politicians, industrialists, farmers and scientists agree and identify PGE as rapidly developing and promising field. However, some people have concerns. Here I would like to quote from a paper published by Mrs. Julie Hill of the "Green Alliance" which appeared in The Implications of Plant Science - a study document, published by the British BBSRC and the Gatsby Charitable Foundation: "Environmental groups are concerned that ... - genes inserted into microorganisms, plants and animals, that could never have got there by conventional breeding, will over time be spread to other organisms. - We do not know enough about ecological interactions to be able to predict accurately what the long term consequences will be of these introduced genes in the environment. - Changes to the environment may not be noticed early. 23

Opportunities and Challenges the regulatory system controlling releases to the environment has not taken on board the concept of"genetic pollution" - in other words, the spread of genes in the environment,when they could not have got there by natural means, is not seen as environmental damage in itself. - Work with viruses poses particular risks. - Genetic modification may not further the development of more "sustainable" agriculture. - The development of herbicide resistant plants could cause changes in the patterns of herbicide use in agriculture in ways that will be more environmentally damaging than at present. - Efforts to engineer top predators such as fish could lead to serious ecological disruption. - Liability for damage caused by GMOs need special provision. - The regulatory system does not give enough scope for consultation with the public." -

In effect the public is concerned about whether PGE is i) risky and ii) whether it is morally wrong. In my opinion D. Boulter in an article in Critical Reviews in Plant Sciences 16, 231251 (1997) answered this question very appropriately, I quote freely from this article: A.

GE

i s r i s k y . Risk is often expressed as the chance of causing a number of deaths or illnesses or as economic loss or as loss of quality of life. - Perceived risk is risk modified by a so-called "outrage reaction". The components of the "outage reactions" are familiar versus unfamiliar (car versus airplane), knowable vs unknowable (one does not like difficult science and fears scientists), diffuse or concentrated, non dreaded or dreaded (snakes or mice), voluntary versus involuntary (voluntary risks are more acceptable than involuntary ones - debate on labelling of GMOs), natural versus artificial (artificial or technological risks are exaggerated). For example: one has a tendency to underestimate risks associated with e.g. car travel and overestimate the risks associated with air travel (whereas the most dangerous part of a voyage is the trip to the airport by road). Similarly the risk of radiation when sunbathing is underestimated and the risk from radiation of food is overestimated.

B.

PGE

is morally wrong~ Because it is blasphemous or unnatural or disrespectful or unfair on ethical grounds, the extent of risk is not at issue. - "PGE is blasphemous because by crossing species boundaries PGE and those promoting it would be playing God." However, many religious people accept that evolution has forced the properties of living organisms to change (natural selection) and these "believers" recognise that interference can be reasonable and acceptable. - "PGE is unnatural". Implied is that all that is natural is good whereas man-made or technological realities are bad. This is obvious, but unfortunately wrong. Indeed, nothing is more 'natural' than 'EBOLA' and plants are not programmed to serve man's needs but as all other living organisms to survive by competing with pests and illnesses. "Expressions such as "nature knows best" and J.J. Roussau's contention that without human tempering, balance and harmony reigns and all will be well, show that nostalgia is confused with reality. It is by no means certain that nature if left alone, will arrive at a perfect, balanced, harmonious ecosystem." Furthermore, the vector most used in PGE is a bacterium: Agrobacterium which lives by PGE. - "PGE will deplete biodiversity". Here one confuses high tech or intensive agriculture and GE. In fact, since GE is dependent on natural biodiversity as a source of new genes, this

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Science and agriculture in the 21 st century

-

technology should promote measures to secure biodiversity resulting from natural evolution rather than endangering it as modern agriculture does. Everyone agrees plant resources should be sustained. "GE is unfair. It is generally accepted that the benefits from GE will not go to support those that sustain its greatest risks or in general will not be shared in a fair way." This is true and is further stimulated by the fact that "Greens" and "Reds" oppose this technology thereby ensuring that only "rich" people and countries will benefit and that this technology will not be used to protect, reasonably, our environment.

Conclusion In conclusion, whereas care has to be taken in order to make sure that Plant Biotechnology does not engender unacceptable dangers, I do not think that this is exceptionally true for B iotechnology. I am convinced that those will loose out dramatically in a world of global economy, who ignore the potential of this new technology to the point of wanting to have nothing to do with the support of a development and use of this technology.

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

Opportunities And Challenges Of EU Plant Biotechnology

The US Secretary of Agriculture Dan Glickman, at the world agricultural congress this year, reported his visit of the Wheat Research Centre in Mexico, where some of the research was carried out on the wheat gene Norin 10 which helped India and Pakistan increase their wheat harvest by 60%. At this centre, he read an inscription on a wall saying "A single gene has saved 100 million lives". Let us think how many other genes would potentially withhold, unless they can be accessed and elucidated, the solutions to other ill-defined agricultural practices, to uncontrolled environmental damage (chemicals, wastes, water depletion), to food-chain contaminations, to post-harvest losses, etc. The genes, like the whole fabric of secondary pathways in plant tissues, altogether are the new knowledge which will bring about further progress for our world of demographic explosion and shrinking resources. This new knowledge is characteristic of the rise of a "knowledge-based economy" announced and described by the OECD, and retains the capacity to drive innovation. DNA sequence banks double their size every 18 months. And with known released GMOs, we are just scratching the surface (most used genes have been lent by generous micro-organisms: think when plant genes become understood and tailored to specific goals!). Historically, agricultural progress has been pushed forward by mobilising land, stocks, capital, techniques (the "hard" foundations) and now, this would have to continue by mobilising genetic information (the "soft"). Here, we have a difficulty: Elected policy-makers, and their voters alike, have been educated normally as land-owners, as capitalists, as technologists and engineers, and they all are the products of this industrial culture inherited from the 19th century. Faced with the knowledge revolution in genetics and in molecular ecology, they come to all sorts of misconceptions and mistrusts. The mistrust is even increased by an accumulation of failures, perceived as knowledge abuses whereas demonstration could be made these had been repeatedly failures of the technologies, and from the most traditional ones through e.g. dangereous upscaling. Hence, the lack of familiarity with the new knowledge has rendered the "precautionary principle" an attractive solution. Policy-makers, more and more, being accountable to their constituencies, will hesitate whether to abstain from making any use of a new technology, or to rather make a responsible use of it within limits defined by the state of knowledge.

Etienne Magnien, Head of Unit of Co-ordination of Horizontal Aspects, Life Sciences Directorate, European Commission, Brussels, Belgium

27

Opportunities and Challenges The policy-demand, today, particularly as the world experiences an accelerated global metabolism, will be towards the reinforcement of demand-driven predictive capabilities, as a knowledge basis for decision-making: - crop yields environmental cost disease outbreaks -

-

Predictive scenarios would have to encompass parameters such as: - genetic erosion contaminations consumer attitudes - commodity pricing water availability -

-

-

Moreover, instead of making the land more productive in bulk commodities, turning capital into boosted yields (high input agriculture), one should rather use the unprecedented genetic and ecological knowledge to broaden the range of alternative opportunities (food, feed, specialities) from an infinite diversity of newly deciphered biological traits. The second will not be straight forward as long as the advanced knowledge is not made usable by non-specialists and formatted to become a policy instrument. This explains what is expected from plant biotechnology in the new EU programme. The thrust must be: -on problem-solving objectives -on the incorporation of ethical principles -centred on the citizen, and with the citizen's understanding It is hoped that the programme may be perceived as a facilitator for front-line scientists to add value to knowledge, for socio-economic experts to envision new models of sharing the benefits from bio-alternatives, for industry to ensure ethically inspired innovation, and for end consumers to participate in responsible choices. The programme is about knowledge indeed, knowledge of genetic and metabolic variability, knowledge of species, communities and sustainable self-reproducible systems. It is an underlying principle that this knowledge should be an instrument of human dignity and prosperity, as made available through the selected key actions with an obligation for the researchers, while offered a much broader range of opportunities, to use these opportunities towards the formation of partnerships, the organisation of collaboration around anticipated results and an extensive amount of work-sharing.

28

Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

The Forgotten Area Of Plant Biotechnology

Summary This review article attempts to address the wider issues relating to Plant B iotechnology and leads on to develop a case for a completely integrated agricultural policy which recognises the huge potential benefits that would be derived from developing under utilised plant species for industrial feedstock. More specifically we attempt to discuss how this could be achieved and, indeed, the role and responsibility of the scientific community, and emphasise the need for facilitating networks to foster policy as well as commercial development of agricultural biotechnology.

Background From when Agrobacterium was first shown to transfer and integrate DNA into the plant genome [1], the whole field of plant biotechnology has been gathering momentum. With more and more crop plants becoming amenable to regeneration and transformation, the potential for genetically modified crop plants to change the appearance of world agriculture has increased. We are now, perhaps, at the dawning of a new era of agriculture that could be described as an agricultural biotechnology (Ag-biotech) revolution. Increasingly Ag-biotech is promoted as the way to feed the ever-increasing world population, estimated to be 10 billion people in 2050. The first steps to substantiate the claim have already been made and can be recognised in the first wave of genetically modified plants. This first wave relates to input traits and includes herbicide and insect resistance [2]. The second wave will, it is hoped, address output traits such as yield and quality etc [3]. The third anticipated wave is the plant-based production of pharmaceutically important proteins such as vaccines etc. [4]. However, with such potential comes opportunities and responsibilities. Therefore, to realise the potential it is important to see plant biotech, and in particular genetic modification, in a larger context. That is, whilst very important, genetic modification is only one of many useful tools available to develop sustainable, socio-economically, and environmentally responsible agriculture.

Need to feed the world? The following quotes illustrate the need to find ways of securing food supply: "More than 800 million people in the world go to bed hungry every night, they are chronically

Steve Bowra, John Innes Centre, Norwich Research Park, Norwich, United Kingdom

29

Opportunities and Challenges undernourished. Millions more suffer from hunger during times of seasonal or periodic food shortages. Every year nearly 13 million children do not reach the age of five, they die as a direct or indirect result of hunger and malnutrition." "The three biggest killers of children in developing countries are malaria, diarrhoea, and respiratory infections work hand in hand with malnutrition. Health programs cannot be successful without food security." "In the next hour, 9,000 babies will be born. By this time tomorrow, the world will have an additional 200,000 mouths to feed. Each baby will be a unique individual with the potential to live a full life and to be a contributing world citizen. Unfortunately, most of these babies will be born into poverty and hunger. Many will die in infancy." (Future Harvest) "We already have 840 million people going hungry and 3 billionwho are malnourished and are going to have 2 billion additional people on the planet in developing countries before the population stabilises." "The ratio of the world's population in the developing countries and developed countries in 1950 was 67/33. This had changed to 78/22 by 1995 and it is expected to reach 83/17 in 2050." ( Ismail Serageldin Chairman of CGIAR) However, the much-vaunted population explosion is unlikely to materialise. The global annual birth rate reached the all time high of 2.1% in 1968, when Paul Ehrlich and George Harrar both independently warned of the dangers of rapid population growth. It has since steadily decreased to the current level, 1.4%, and is predicted to be 0.8% in 2050 [5]. According to economists this would not indicate a population explosion. Whilst in percentage terms the decline is self-apparent, the population is still expected to reach 9-10 billion by 2050 [6]. Clearly this is a significant increase in the global population, especially when in 1960 it was approximately 3 billion. However, perhaps more importantly, the growth will occur in developing countries which current food surpluses produced by developed countries cannot reach. Historically, cereal production per person peaked during the mid 1980s and declined towards the mid 1990s after which it began to increase again [7]. This has been shown to be the direct consequence of European and North American efforts to control overproduction. Therefore, although an over simplification, the growth in the global demand for food could be met by the world's agricultural system as a whole. However, developing regions of the world, in particular sub-Saharan Africa, may not have enough income to pay for food imports. Additionally, many of the world's poor and hungry live where there are no roads or other infrastructure to deliver the food to the needy. These people are best helped if they are enabled to grow their own nourishment. Therefore food security is becoming a global issue to be solved by the collaboration between the developed and developing countries. Plant biotechnology is considered to be one of the most promising, environmentally friendly technologies for global food security, particularly if incorporated into a fully integrated crop management approach [8]. Thus it appears well accepted that in the medium term the world food production will expand at a rate sufficient to match growth of effective demand [9]. This is explained by a deceleration in effective demand for food, which comprises both positive and negative factors. The positive aspects are the overall decrease in birth-rate and the ever-growing proportion of the population achieving sufficient levels of nutrition. The negative aspect is the role of poverty in reducing demand for food. Demand is decelerating because areas with inadequate consumption also lack the relevant money to buy food and therefore create a demand in the market place. 30

Forgotten area of plant biotechnology However, the fact that economic predictions indicate a global food crisis will not materialise would appear comforting. Despite this, the need to continue to achieve a growth rate in food production, however small, will remain a challenge. Moreover, the process of increased yields and agricultural advance is extremely complex and is influenced by a multitude of factors such as education, health conditions, technology, transport and communication etc. Perhaps central is socio-political stability and the economics of developing world trade to try to offset the "local food crisis". With all the above factors aligned, agricultural research is still required to continue to develop not only the potential of plant biotech, using such tools as genetic modification, but also develop more integrated agricultural practises. However, it is alarming to realise that financial support for agricultural research has been in decline for a number of years. It is expected to continue to decline because of the steady drop in world food prices and because developed nations are focusing on internal issues rather than deal with the problems facing developing countries [10].

Sustainable agricultural development Plants are the basis of the human food supply. Through a long process of conscious and unconscious selection, an estimated 400 000 plant species have been whittled down to thirty crops that today account for 95% of all human calories and proteins production [11]. Currently about half of our food is derived from only four plant species (rice, maize, wheat and potato); our survival as a species is now based on these plant species. It is humbling to realise that prehistoric farmers selected these central crops on which we are now so dependent. Given that until relatively recently in terms of crop domestication a greater number of plant species were used for feed, fibre, energy, construction, manufacturing and environmental protection, some argue that agriculture based on these four major crops is too narrow and fragile [12]. The increasing population pressure compounds this concern. It would seem probable, therefore, that there should be a push to expand the agricultural base but all the evidence seems to indicate that the opposite is in fact true. Three basic options are: - genetic improvement and more efficient production of existing crops, investment in under utilised crops and - screening plant diversity to discover completely new crops. -

The first option has received the most political and financial support from those with vested interest, growers, processors and, in particular, agrochemical companies that have mutated into life science companies. Thus the core four crops have received the bulk of research, while agricultural production has been reinforced by expensive subsidies and tax incentives in both the EU and in North America. The situation is further compounded by the focus of ag-biotech on altering major crops rather than minor ones. Monocultures now dominate vast areas of the agricultural regions of the world. The continued drive towards this preferred system of agriculture has replaced natural ecosystems that once contained many hundreds of plant, insect and vertebrate species. Thus, the simplification of agriculture has reduced diversity and created an inherent instability in the food supply chain. This fact was illustrated in 1970 by the devastating epidemic in the USA of southern corn leaf blight (fungal pathogen Bipolaris maydis Nisikado Shoemaker, race T). The impact was attributed to the fact that the male sterile genetically uniform varieties of corn all contained T-cytoplasm [ 13]. 31

Opportunities and Challenges However, the facts remain that world food production has almost doubled from 1961 to 1996, a clear indication of success. But at what cost? To achieve the dramatic increases in yield, obviously better yielding varieties were developed. In addition the achievement was assisted by increased use of herbicides for weed control, and insecticides and fungicides for pest and disease control. Furthermore, nitrogen and phosphorous application increased by 6.87 and 3.48 fold respectively. Land utilisation increased by 10%, and the amount of irrigation doubled [14]. It would be naive to assume that continued increase in global demand for food could be met by a linear increase in the use of nitrogen, phosphorous, pesticides, irrigation and land utilisation. The long-term impact of increased application of agricultural nitrogen and phosphorous is well-documented [ 15], although the extent will depend on the levels that accumulate in non-agricultural systems which, in turn, are influenced by the complex biogeochemistry of nitrogen and phosphorous cycling. It is recognised that runoff and ground water contamination by nitrogen and phosphorous is of great concern. However, impact on the ecosystem should be the principal concern as the elevated levels of nutrition allow the dominance of few species and therefore the loss of species diversity [16]. Pest control has become an increasingly serious constraint on agricultural production as a direct result of reduced plant species diversity, in spite of dramatic advances in pest control. A survey of the development of chemical control begins with the introduction of copper-based insecticides in the late 1800s followed by the dichlorodiphenyl-trichlorethane (DDT) in the 1930s. This was originally shown to be harmless to humans, animals and plants, a point to remember given the current issues surrounding GMs. However, the analogy does not stop there because, as a consequence of the success of the DDT research, funds were directed away from basic entomology, therefore delaying the development of integrated pest management (IPM) and at the same time allowing the chemical companies to expand the market for chemical led pest control. As late as the 1970s, there appeared legitimate public concern about a "pesticide conspiracy" between chemical manufacturers and agricultural interests in an attempt to further delay the development of IPM. This was despite the fact that during the 1950s it was recognised that the benefits of increased yield were achieved at significant cost. Not only was there clear evidence of resistance developing in the target population, but also beneficial insects and other wildlife were suffering. Indirectly, so was human health, e.g. as a result of exposure to dioxins [17]. As of the mid 1990s, the first wave of genetically engineered herbicide resistant and insect resistant crop varieties, once again led by agrochemical companies, have resulted in new concerns. It could be argued that the failure to promote and direct research funds to develop an integrated crop management approach over the last fifty years has been because it does not make money for shareholders. Thus chemical led pest control has been allowed to further reduce diversity. Although estimates vary it is generally thought that as part of keeping pace with the demand for food, land utilisation will have to increase by 18 %, an area equivalent to 268 million hectares. The direct consequence will be further destruction of ecosystems and therefore further loss of diversity. It is also important to appreciate that nonagroecosystems also provide benefit to agricultural ecosystems. For example, although insects, birds and mammals are pollinators, they are also agents of biological control, i.e. as predators and parasites. In addition the water flow through the ecosystem can be controlled by the flora, thereby reducing the chances of drought or flood. Attempts have been made to estimate the value of these "ser32

Forgotten area of plant biotechnology vices" [18] and whilst it has proved difficult to derive definitive figures, the work has illustrated the need for policy to allow the non agroecosystem to provide these vital services for agriculture. Thus it is important to find ways of decreasing the environmental impact of agriculture while improving its productivity, sustainability and stability. Aside from technical improvements associated with point application of nutrients and pesticides and genetic modification, it is possible that an examination of the principles governing how ecosystems function may help establish ways of creating a stable sustainable agricultural system. As summarised by Tilman [21] there are three main factors that influence how ecosystems function: 1) the traits of the species within the ecosystem, 2) the number of species within the ecosystems and 3) the physical conditions; the first two illustrate the influence and importance of diversity. The plant species diversity and composition within an ecosystem influences the primary productivity [18][19]. It is claimed that increasing species diversity from one to twenty increases primary productivity by 35-70% [20]. Protection against disease could be enhanced by diversifying crops grown, diversity in substitute crops and introducing diversity of genetic resistance within crops [21]. Higher plant diversity reduces the loss of limiting nutrients via leaching etc [22]. The direct result of lower concentrations of unconsumed soil nutrients decreases the number of other species that can invade the ecosystem. Therefore, the balance of nonagroecosystems with agriculture may be an important part of reducing the invasion of weeds [23]. The summary of the ecological principles would lead to the conclusion that monocultures will be relatively unstable, be subject to high levels of nutrient leaching, be susceptible to invasion of weedy species and attract high levels of disease; clearly this is the case. Sustainable development and global food security will only be possible when it is recognised that the land, water and biological diversity that create the world's food supply exist in a very delicate balance. Only diversity can promote sustainability and support economic development [24].

Agricultural diversification Having established a case for diversification and an integrated approach to agriculture, the next questions are how, via what strategies and what are the possible scenarios. Clearly, any approach has to take into account current agricultural practice and the economic environment. The highly complex nature of the issue allows the discussion to be started from many levels. For the purpose of this discussion we will focus on the issues facing agriculture in the developed world, ostensibly because the pseudo free markets maintained by the western governments are shareholder led. Therefore, unless financial return can be generated by agriculture in the developed world, there is going to be a continuation of the reduction in investment in agricultural research. The knock-on effects are best illustrated by the considerable reduction in funding received by CGIPR, a collaborative agricultural research body focused on the development of sustainable agriculture in the developing world. Why is agriculture in both North America and the EU subsidised to the tune of $335 billion annually? [25] In simple terms, over capacity contrives to reduce prices, hence reduced returns, leading to less profit. The issue of funding agricultural research to facilitate cheap food has to stop. The price of a ton of wheat in real terms is $110 cheaper today than it was 50 years ago [26] and the amount of money spent on food as a ratio of per capita income in the developed world is now 11 [27]. We cannot continue to promote agricultural research on the basis of cheap food. Sustainable 33

Opportunities and Challenges and economically viable agriculture will only be achieved when it can be shown to pay, i.e. give the shareholder a return. Over production of cheap food could be avoided by agricultural diversification. The introduction of alternative food crops, and more importantly non-food crops, could provide the necessary tools to develop an integrated strategy for sustainable, environmentally responsible agriculture. This appears to be the forgotten area of plant biotechnology. The development of under utilised or completely new crops for their potential to provide valuable industrial feedstock would provide a complementary source of income for the agricultural community. This would have the effect of reducing the farmers' dependence on the production of food to generate their income, therefore reducing the pressure to increase yields to make an adequate living. The net product may even be a slight increase in food prices, which may then promote the development of integrated methodologies. The creation of new industries on renewable agricultural based feedstock will substitute for the current non-renewable petroleum based products. In turn, rural economies will be stimulated as illustrated in North Dakota where growing crambe has stimulated local and rural based industries, such as processing, and provided general economic stability [28]. Besides crambe there have been numerous reports and surveys that have demonstrated the potential of alternative non-food crops that could provide a range of novel oils, starch, fibres and proteins [29]. However, despite the huge potential, which has frequently been cited for at least the past ten years, the progress to realising the benefits is almost imperceptible. Why? clearly, whilst it is easy to state potential, the proposition has to be financially viable. The difficulty in carrying out life cycle analysis (LCA) and cost benefit analysis (CBA) has led to: 1) there being very few examples, and 2) the assessment of viability is usually based on comparisons with existing industrial feedstock. These feedstocks are less expensive petroleum based products; however, while almost always less expensive than agricultural products, their price may not completely reflect the true cost to society. For example, they are unsustainable and non-renewable and carry the hidden cost of pollution and increased cost of disposal etc. Often therefore, financial viability is difficult to ascribe to alternative crops when attempting to meet industrial financial requirements. This is particularly the case when the socio-economic and environmental values are not introduced into the LCA/CBA or adequately weighted. From the preceding discussion it should be clear that diversity and sustainability not only impact on the ecosystem but also on the economy. The result is that price (a purely economic quantity) is not a sustainable platform for agricultural development. Therefore the driving force behind alternative non-food crops has to change and become socio-economic and environmental rather than purely financial. If socio-economic and environmental issues become the driving forces, the information derived from LCA/CBA will provide an insight to where the current bottlenecks exist and provide a focus for further R&D rather than the basis for creating a case for preventing development. In addition, developing LCA and CBA models should also capture some of the forward linkage of agriculture such as processing and marketing. This could be enhanced by utilising "biocascading", a process that identifies and develops many secondary products from one agricultural product. Full utilisation of biocascading will further enhance the financial viability of non-food crops [30]. Therefore, it can be seen that increasing diversity of the agricultural base by developing under utilised crops or, indeed, 34

Forgotten area of plant biotechnology totally new crops, allows the development of a completely integrated approach to agricultural. Creating sustainability, stability and economic advantage, both at rural and national levels enables the principles of ecosystems to be better utilised in reducing the impact on the environment with a concomitant reduction in inputs and pesticide use.

Conclusions We started this review by illustrating the direction of agriculture as driven by genetic modification. This was followed by a discussion of the increasing pressure being placed on agriculture to feed an increasing world population yet, at the same time, having to cope with the decreasing investment in agricultural research. In the face of increasing environmental concern and questions about sustainability, we then went on to take an historical perspective as to how agriculture developed into its current form, identifying the forces that drive it. From here, we led into a debate about how introducing species diversity into agriculture would assist the transformation of a presently unsustainable and inherently unstable agricultural system into an environmentally responsible industry. To round off the debate, we have attempted to put forward scenarios whereby the introduction of alternative crops and in particular non-food crops, would have numerous benefits. Throughout the discussion, the need for appropriate funding for agricultural research has been emphasised and, as all that are involved in agricultural research know, this is essential. We also illustrate the fact that agricultural research has been and is currently led by shareholders, although the realisation that these shareholders are directly responsible for who is fed and, more importantly, who is not is not very reassuring. We have tried to work with the current economics and create ways of meeting the shareholders' requirements and, at the same time, provide a route to sustainable agriculture. Ultimately, with developed agriculture, both sustainable and profitable, we believe that the chances of appropriate technology being transferred to the developing countries will be enhanced, enabling them to develop their own economies and providing adequate nutrition to the expanding population. Plant biotechnology has a central role to play in achieving these goals although, however, it is perhaps not the area currently being pursued. In addition, at the global level, the work of CGIAR, the FAO and the World Bank are vital to provide the collaborative forums that create the push to change and direct policy relating to agriculture and therefore directly plant science research. Therefore, only when scientific potential and global agricultural policy are integrated will we begin to appreciate the real benefits of plant biotechnology.

Authors of this publication Steve Bowra*, Douglas Hobbs and Eddie Arthur. John Innes Centre, Norwich Research Park, Colney Lane, Norwich, United Kingdom. *Corresponding author

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2.

Schell, J., Van Montagu, M., De Beuckeleer, M., De Block, M., Depicker, A., De Wilde, M., Engler, G., Genetello, C., Hernalsteens, J.P., Holsters, M., Seurinck, J., Silva, B., Van Vliet, E, Villarroel, R. Interactions and DNA transfer between Agrobacterium tumefaciens, the Ti-plasmid and the plant host. Proc. R. Soc. Lond. B Biol. Sci. 204 (1979) 251-266 Dempsey, D.A., Silva. H., Klessing, D.F. Engineering disease and pest resistance in plants. Trends in

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

Biotechnology, Food, Agriculture, Public policy And Consumer Concerns Introduction

Since 20 years genetic engineering is being discussed. Since the very beginning there have been serious concerns, also by the scientists involved in the first GMO experiments. Since then, billions of dollars have been invested in the development in the different application fields and millions of dollars in public education and acceptance. An international analysis of associated risk research or research on possible ecological and health impact came to the conclusion that less than 1% of the world wide development budget has been used for research regarding safety aspects [1]. The consumer and the public as a whole feels more and more ill at ease given these facts. There is more and more distrust in industry and in the regulatory framework, because what they say and how they try to assure the public does not fit the facts. For me, dealing with risk assessment in genetic engineering since 15 years, there is an extreme contradiction between the emerging data and the handling of these data in the context of evaluation and decision making. It is obvious that having different value systems and different interests, scientists, the public, industry representatives and others may come to different conclusions based on the same facts. The problem seems to be how to integrate these different evaluations in a fair and impartial manner in decision processes. I like to discuss - as examples - two areas of concerns in more detail. But before doing that I like to give a short overview on the main questions concerning ecological or health impacts. Main

areas of concern

- cross-hybridisation with related species/weeds (example canola: [2-5]) - horizontal gene transfer [6] resulting in uncontrolled spread of new gene combinations - rapid resistance development of important plant pathogens to biological pest-management tools with an accompanying destruction/exhaustion of the naturals means to defend against pests and pathogens (resulting in an even greater dependence on chemical pest control in the long term) [7] - problematic impacts on non-host organisms (examples: reduced fertility in lady bugs when fed with aphids who ingested bt-containing plant sap [8], toxic impacts of corn-borer larvae on lacewing-flies [9])

Beatrix Tappeser, Institute for Applied Ecology, Freiburg, Germany

37

Opportunities and Challenges -

-

development of new virus combinations using virus resistant plants [ 10, 11] pleiotropic and position-effects influencing ecological traits and/or digestibility of the plants (see Pusztai case) allergenicity [ 12, 13] horizontal transfer of antibiotic resistance genes [ 14]

Some of these concerns are interconnected. For example it is known that proteinase-inhibitors very often have allergenic potential and belong to the set of proteins which cause certain plant to be allergenic to sensitive people [ 15]. Different proteinase-inhibitors are being cloned in a wide range of crop plants to confer insect-resistance. It has been shown that proteinase-inhibitors have the potential to harm benefical insects such as bees, inpairing their ability to recognise flower smells and shortening their lives [ 16]. Being a biologist especially such effects make me very concerned. The long term impact of less or even lack of pollination capacity in nature may be enormous. Many of our fruit trees and vegetables, but also a lot of other plants are dependent on insect pollination as the main possibility of pollen transfer for fruit and seed production. Until now we could take such service by insects for granted. What would it mean - for agricultural production, for the functioning of ecosystems, for biological diversity as a whole, if there will be a wide range of different crop plants producing such proteins. There will be no quick answer but lots of questions and concerns. Given our absolute dependence on agriculture for food production precaution should precedent any other evaluation scheme. We have too few knowledge to take a responsible decision now. The example given indicates possible problems that we may face a long way down the road we have already taken. Although allergenicity problems are known and there are hints for serious non-target impacts the perceived economic benefits seem to be big enough to continue along this path of development.

Horizontal

gene

transfer

Another question of great concern is the possibility of spread of the new recombinant sequences via horizontal gene transfer. It was one of the first questions raised after the creation of the first transgenic microorganism by Paul Berg and collaborators. It is exactly 25 years ago that Paul Berg and others published their famous letter ,,Potential Biohazards of Recombinant DNA Molecules" in Science [17]. Amongst others, they were especially worried about horizontal gene transfer of antibiotic resistance genes. Since then the likelihood of gene transfer events has been heavily debated. The main assumptions had been that the probability would be extremely low because of highly restricted transfer under natural conditions. As additional support it was stated that DNA released into the environment or in the digestive tract would be quickly and easily degraded. Since the end of the seventies the message for any risk assessment has been: Don't worry about horizontal gene transfer. There is almost no chance for this to happen. Both assumptions on gene transfer possibilities and on DNA stability have been proven wrong. DNA is quite stable in different environments and transfer events can be demonstrated under different conditions [18]. Even the long doubted transfer of recombinant plant genes to bacteria could be shown to occur under laboratory conditions [6]. 38

Public policy and consumer concerns Another area of concern is horizontal gene transfer in the gastrointestinal tract of insects and vertebrates. In contrast to earlier, long standing views, DNA is not quickly fragmented in the intestine but instead remains stable for a surprisingly long period. Moreover, DNA ingested with food can be excreted after only partial digestion. DNA can also pass into the bloodstream to be taken up by leukocytes and cells of the liver and spleen [ 19, 20]. The quoted experiments were performed on naked DNA, while DNA ingested with food is normally complexed with proteins and thus better protected. Beside this, the environment of the human or animal gastrointestinal tract changes in the course of digestion depending on what types of food are eaten together. This means that the resistance of proteins or DNA to digestion is not always constant but may vary. Laboratory experiments on the degradability of DNA in synthetic gastrointestinal liquids, as they are usually carried out in studies on transgenes, do not take such effects into account because they use a constant pH and purified DNA [ 10, 21 ]. Recently it has even been possible to demonstrate "natural" transformation in an artificial mammalian gastrointestinal model. [22] Such evidence has been available some 15 years when Orpin et al. [23] were able to show that Selenomonas ruminantium, a bacterial species inhabiting the bovine gastrointestinal tract, is naturally transformable. Furthermore, Tebbe et al. [24] reported that gene transfer can occur from orally administered GMMs to various recipients (Arthrobacteria) via transformation in the intestine of springtails (Folsomia candida). Perreten et al. [14] documented the development of a plasmid carrying multiple antibiotic resistances which they had isolated from raw milk cheese. These resistances originated from four different microorganisms and probably developed through the action of antibiotics in the microflora of the lactating cows. The authors take a clear stance on the issue: "To preserve the life-saving potential of antibiotics, the spread of resistance genes at all levels must be stopped. Distribution routes like those between animals, food and consumers have to be interrupted." ([14], p.802). Only recently transformation events in an artificial mammalian gastro-intestinal tract could be detected with a probability of one in 10 million. Given the billions of bacteria inhabiting our intestine that is not really a rare event. [22].

Vertical gene transfer Another central issue that has featured in discussions on the cultivation of transgenic plants since its very beginning is that of outcrossing from such plants and introgression of the recombinant genes to related weed and wild plants. It was more or less accepted, at least in the beginning of the debate, that spread of transgenes should be avoided as best possible, as this may have problematic effects on the composition of wild flora and biocoenoses in general. A further point now attracting increasing attention are the implications of resistance development through outcrossing (e.g. herbicide resistance or insect resistance), as this may not only have consequences for non-cultivated ecosystems but in particular also for agricultural land use systems. The results of hybridisation experiments clearly demonstrate the possibility of a gene flow from rape to wild herb populations. Potential hybridisation partners of Brassica napus are not only to be found in the genus of Brassica but also in other groups of the mustard family [25]. Potential hybridisation partners of rape include in particular wild herbs, which are probably 39

Opportunities and Challenges all subject to a high degree of cross-fertilisation. According to Darmency [26] this crossfertilisation facilitates the transmission of transgenes from rape to associate herbs. Under field conditions rape has proven capable of hybridisation with wild turnip (Brassica rapa), brown mustard (Brassicajuncea), black mustard (Brassica nigra) hoary mustard (Hirschfeldia incana, synonymous with Brassica adpressa), wild radish (Raphanus raphanistrum) and wild mustard (Sinapis arvensis) [27]. All experience and data gained in the course of the past years point to a high probability of rape populations prevailing outside cultivated areas and the possibility of gene flow to nontransgenic populations and related wild herbs. Many of Europe's major crop species have been equipped with identical herbicide resistance genes. Their large-scale use will therefore produce an enormous selective pressure towards corresponding resistant weeds. While rape will be the plant to initiate rapid resistance development, other plants equipped with the same resistance but lacking crossable wild relatives in the region will sustainably promote the onesided selection of weeds rendered resistant by the former. This development will also be accompanied by a further impoverishment in farmland-associated floral species and insects, because of the constantly increasing usage of broad-spectrum herbicides instead of selective herbicides. Furthermore, the cloning of multiple resistances into one and the same crop species also gives related wild herbs the opportunity to acquire multiple resistance. The largescale resistance management schemes now being discussed in anticipation of herbicide resistance problems may have to accommodate whole regions and extend over several rotation periods in order to be effective [28]. That will need a high planning and control effort. No less in conflict with the requirements of sustainability, and with the principles of sustainable utilisation and conservation laid down in the Convention on Biological Diversity, is the endangerment of species diversity entailed in the present herbicide resistance strategies. In the light of the knowledge on horizontal and vertical gene transfer that has accumulated during the past years, the rapid commercialisation of a multitude of herbicide-resistant transgenic plants also constitutes a violation of the precautionary principle. Speakers at international debates are often heard to invoke another principle, namely that decisions should only be made on the basis of scientific knowledge. There is no objection to this, just as long as such knowledge-based decisions really take account of all the relevant scientific evidence available. In concluding I would like to quote Heinemann : "The risk of genetically engineered organisms for commercial preparation is the potential for the engineered product to demonstrate unexpected "monster" qualities or for the genes to escape into the wild fauna and flora and thereby create genetic "monsters". These are risks which cannot be excluded with present data. The potential for escape of a resistance gene introduced into the genome of a commercially desirable plant cannot be gauged by small-scale gene escape experiments. By known examples of gene transfer frequencies in nature, the potential for exchange is too great to be excluded by argument. (...) What we know the least about is the nature of the evolutionary forces that determine the success or failure of monsters. We have limited or no predictive power for the fate of recombinant genes. In the case of an antibiotic resistance, we may be able to say that its known functions pose no additional threats. But we cannot be sure that its known functions are all of its potential functions either on its own or in conjunction with the 40

Public p o l i c y a n d c o n s u m e r concerns m a n y n o v e l g e n o m i c c o n t e x t s in w h i c h it m i g h t be f o u n d s h o u l d it be t r a n s f e r r e d horizontally. Therefore, as a qualitatively different t e c h n o l o g y , genetic e n g i n e e r i n g s h o u l d be c o m m e r c i a l i s e d w i t h e x t r e m e c a u t i o n until the a p p r o p r i a t e scientific e x p e r i m e n t s can be c o n d u c t e d " ([29], p.23).

References 1.

2.

3. 4. 5. 6. 7. 8. 9.

10. 11.

12. 13. 14. 15.

16. 17.

18. 19. 20.

Sukopp, H.; Sukopp, U. (1997): 0kologische Begleitforschung und Dauerbeobachtung im Zusammenhang mit Freisetzung und Inverkehrbringen gentechnisch ver~inderter Kulturpflanzen; edited by: Thtiringer Ministerium ftir Landwirtschaft, Naturschutz und Umwelt (TMLNU) in: Chancen und Risiken der Gentechnik im Umweltschutz; 43-51 Jorgensen, R.B., Andersen, B. (1994): Spontaneous hybridization between oilseed rape (Brassica napus) and weedy B. campestris (Brassicaceae). A risk of growing genetically modified oilseed rape. American Journal of Botany 12, 1620-1626 Mikkelsen, T.R., Andersen, B., Jorgensen, R.B. (1996) The risk of crop transgene spread. Nature 380, 31 Lefol, E.; Fleury, A.; Darmency, H. (1996): Gene dispersal from transgenic crops. II. hybridization between oilseed rape and the wild hoary mustard. Sexual Plant Reproduction 9 (4)189-196 Chevre, A.-M.; Eber, F.; Baranger, A.; Renard M. (1997): Gene flow from transgenic crops. Nature, vol.389, p.924 Gebhard, E; Smalla, K. (1998): Transformation of Acinetobacter sp. strain BD413 by transgenic sugar beet DNA. Applied and Environmental Microbiology 64 (4) 1550-1554 Mellon, M.; Rissler, J. (1998): Now or never: Serious New Plans to Save a Natural Pest Control, Union of Concerned Scientists (UCS) Birch, A. N. E.; Geoghegan, I. E.; Majerus, M. E. N.; Hackett, C; Allen, J (1997): Interaction between plant resistance genes, pest aphid populations and beneficial aphid predators. Soft fruit & perennial crops p.68-72 Hilbeck, A., Baumgartner; Fried, E M.; Bigler, E (1998): Effects of transgenic bacillus thuringensis corn-fed prey on mortality and development time of immature Chrysoperla carnea (Neuroptera: Chrysopidae); Environmental Entomology 27 (2) 480-487 Eckelkamp, C.; J~iger, M.; Weber, B. (1997): Risikotiberlegungen zu transgenen virusresistenten Pflanzen. Gutachten i. A. des Umweltbundesamts (UBA) Texte 59/97 Rubio, T.; Borja, M.; Scholthof, H. B.; Jackson, A. O. (1999): Restoration of wild-type virus by double recombination of tombusvirus mutants with a host transgene; Molecular Plant-Microbe Interactions 12 (2) 153-162 Nordlee, J. A.; Taylor, S. L.; Townsend, J. A.; Thomas, L. A.; Bush, R. K. (1996): Identification of a brazilnut allergen in transgenic soybeans. The New England Journal of Medicine 334 (11) 688-692 Nestle, M. (1996): Allergies to transgenic foods - questions of policy. The New England Journal of Medicine 334 (11)726-727 Perreten, V.; Schwarz, E; Cresta, L.; Boeglin, M.; Dasen, G.; Teuber, M. (1997): Antibiotic resistance spread in the food. Nature 389, 801-802 Franck-Oberaspach, S. L.; Keller, B. (1996): Produktsicherheit von krankheits- und sch~idlingsresistenten Nutzpflanzen: Toxikologie, allergenes Potential, Sekund~ireffekte und Markergene; in: Gentechnisch ver~inderte krankheits- und sch~idlingsresistente Nutzpflanzen. Eine Option ftir die Landwirtschaft? Band I, Materialien, Publikation des Schwerpunktprogramms Biotechnologie des Schweizerischen Nationalfonds, Bern Pham-Delegue, M.-H. (1997): Risk assessment of transgenic oilseed rape on the honeybee. Personal communication; see also: Sting in the tale for bees. New Scientist 16.8.1997 Berg, E; Baltimore, D.; Boyer, H. W.; Cohen, S. N.; Davis, R. W.; Hogness, D. S.; Nathans, D.; Roblin, R.; Watson, J. D.; Weissmann S.; Zinder, N. D. (1974): Potential biohazards of recombinant DNA molecules. Science, July 1974 vo1.185, p.303 Eckelkamp, C.; J~iger, M.; Tappeser, B. (1998): Verbreitung und Etablierung rekombinanter Desoxyribonukleins~iure (DNS) in der Umwelt. Gutachten i. A. des Umweltbundesamtes (UBA) Texte 51/98 Schubbert, R.; Lettmann, C.M.; Doerfler, W. (1994): Ingested foreign DNA persists in the gastrointestinal tract and enters the bloodstream of mice. Molecular and General Genetics 242, 495-504 Schubbert, R.; Renz, D.; Schmitz, B.; Doerfler, W. (1997): Foreign (M13) DNA ingested by mice reaches peripheral leucocytes, spleen, and liver via the intestinal wall mucosa and can be covalently linked to mouse

41

Opportunities and Challenges DNA. PNAS 94, 961-966 21. Eckelkamp, C.; J~iger, M.; Weber, B. (1997a): Antibiotikaresistenzgene in transgenen Pflanzen, insbesondere Ampicillin-Resistenz in Bt-Mais. Oko-Institut e.V., Freiburg. 22. New Scientist (1999): Gut reaction. 30 January (Debora MacKenzie) 23. Orpin, C.G.; Jordan, D.J.; Hazlewood, G.E; Mann, S.E (1986): Genetic transformation of the ruminal bacterium Selenomonas Ruminantium. J. Appl. Bacteriol. 61:xvi 24. Tebbe, C.C.; Vahjen, W.; Munch, J.C.; Feldmann, S.D.; Ney, U.; Sahm, H.G.; Amore, R.; Hollenberg, C.E (1994): f0berleben der Untersuchungsst~mme und Persistenz ihrer rekombinanten DNA. BioEngineering 6/ 94, 14-21 25. Scheffier, J.A., Dale, EJ. (1994) Opportunities for gene transfer from transgenic oilseed rape (Brassica napus) to related species. Transgenic Research 3, 263-278 26. Darmency, H. (1994): The impact of hybrids between genetically modified crop plants and their related species: introgression and weediness. Molecular Ecology 3, 37-40 27. Eckelkamp, C.; Mayer, M.; Weber B. (1997c): Basta-resistenter Raps. Vertikaler und horizontaler Gentransfer unter besonderer Beriicksichtigung des Standortes W61fersheim-Melbach. Werkstattreihe Nr. 100, Oko-Institut e.V., Freiburg. 28. Korell, M.; Schittenhelm, S.; Weigel, H.J. (1997): Aufstellen yon Kriterien ftir die nachhaltig umweltgerechte Nutzung gentechnisch ver~inderter Kulturpflanzensorten. Umweltbundesamt, Texte 88/97 29. Heinemann J.A. (1997): Assessing the risk of interkingdom DNA transfer. In: Nordic seminar on antibiotic resistance marker genes and transgenic plants. Norwegian Biotechnology Advisory Board (ed.), Oslo, 17-28

42

Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). O Elsevier Science B.V. All rights reserved.

U.S. Biotechnology Regulatory System. An Industry View

Biotechnology is an enabling technology with broad application to many different areas of industry and commerce Broadly defined, biotechnology includes any technique that uses living organisms or parts of organisms to make or modify products; to improve plants or animals; or to develop micro-organisms for specific use. Public acceptance, gene transfer, mandatory labelling, and transparency of information are some of the issues surrounding the first generation of Agricultural biotechnology products While these issues constitute appropriate questions for national biosafety reviews, the global dialogue must also focus on the future applications of these new technologies The real challenge is to determine how to best utilise these new technological tools to create sustainable solutions for the existing and emerging global needs in the areas of quality of health care, environmental enhancement, and food production The significant contribution of biotechnology in the development of new pharmaceuticals and diagnostic tools for treating and diagnosing human health and disease conditions is well documented For agriculture, biotechnology is demonstrating it's potential to increase productivity, enhance the environment and improve food safety and quality. For the chemical industry it can lower costs and provide raw materials for current products; allow rapid development of more cost effective new products; and the technology can also be used for the development of products otherwise unavailable In the United States products of biotechnology have entered mainstream American commerce in human health care and now in agriculture. In agriculture ten different crops and over 35 different varieties of those crops have been approved or acknowledged by the responsible US regulatory agencies for commercialisation The products of biotechnology incorporated into the American health care arena and agriculture and their ability to have a significant beneficial impact in the United States have been aided by oversight structures that assure regulatory safety standards are met while facilitating technology transfer The US regulatory oversight is through a risk-based system, which focuses on the end product and its uses. The three Federal agencies with responsibility for protecting agriculture, the human environment and the food and drug supply developed, where necessary, regulations and oversight processes consistent with existing statutory authorities Broadly speaking, the Terry L. Medley, Regulatory and External Affairs DuPont Nutrition and Health, Wilmington, Delaware, USA

43

Opportunities and Challenges roles adopted by these agencies are; the Animal and Plant Health Inspection Service (APHIS) at United States Department of Agriculture (USDA) assures that new trangenic plant varieties are as safe to use in agriculture as conventional varieties; the Environmental Protection Agency assures the safe use in the environment of novel plant-pesticidal substances introduced into plants, or new uses of herbicides in conjunction with trangenic plants; and the Food and Drug Administration (FDA) consults with developers of trangenic plants to assure that the new varieties, and foods produced from them are as safe to consume as conventional varieties. Another fundamental aspect of the US regulatory oversight process is the commitment to public involvement in the decision making process. The Administrative Procedure Act (APA) requires that all Federal agencies provide the public with an opportunity for "notice and comment" before adopting final regulations. Additionally, APHIS, EPA and FDA have sought to achieve transparency in their oversight system through use of public meetings and web pages. Lastly, the agencies have been open to adjusting their regulations based upon experience and advancements in the technology in this regard, it should be noted that extensive scientific evaluation by USDA, EPA, and FDA have identified no safety problems unique to the biotechnological process.

44

Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

The Cluster: "Control Of Genes"

Introduction

A cell or an entire organism has to co-ordinate various functions in its daily live or in development. It is generally believed that the integration of various functions is accomplished by controlling the expression of genes. This control can be exerted at different levels. The best studied case is control of transcription. Genes are connected to control units immediately upstream of the regions encoding the structural genes, the promoters. The activity of the gene is regulated by the presence or absence of proteins which recognise specific regions in these promoters and have either activating or repressing activities. The understanding of this regulatory network is one of the top issues in modern molecular biology and the key to understand gene regulation. On top of these basic regulatory mechanisms there are chromatin and genome structure. Although both received considerable attention lately, these mechanisms are by far less well understood. All networks in the control of genes cluster have one common theme, the understanding of basic mechanisms that regulate the activity of genes in plants at one or the other level. The cluster consists of five thematic networks: - BIO4-CT96-0242, Activation tagging as a means of plant gene isolation is co-ordinated by Bernd Reiss - B IO4-CT96-0253, Control of gene expression and gene silencing in transgenic plants is co-ordinated by Peter Meyer - BIO4-CT97-2028, The mechanisms and control of genetic recombination in plants is coordinated by Karin Metzlaff, Bernd Reiss, and Charles White. - BIO4-CT97-2160, Analysing the function of existing and novel genetic promoters for tissue specific expression of transgenes in Zea mays is co-ordinated by Howard Thomas. - BIO4-CT97-2245, Plastid transformation in crop plants is co-ordinated by Philip John Dix. The network "activation tagging as a means of plant gene isolation" is devoted to the development of a novel method to identify new plant genes. This method is based on Agrobacteriummediated transformation and the ectopic activation of plant genomic sequences. This newly

Bernd

Reiss, Max-Planck-Institut fuer Zuechtungsforschung, Carl-von-Linne-Weg 10, KOln, Germany

45

Control of genes developed method is used to isolate genes involved in homologous recombination and regulatory genes involved in plant development and signal transduction pathways. Major elements in the control of genes are promoters. The network "analysing the function of existing and novel genetic promoters for tissue specific expression of transgenes in Zea mays" develops tools for controlled expression of transgenes in maize. Regulatory sequences from maize genes are isolated and the motifs conferring the specificity of expression defined. This analysis will help understanding how genes are specifically expressed during senescence, during kernel, pollen, and seedling development, or in flowers and roots. The plant cell contains more than one genome. In addition to the nucleus, the chloroplasts are of major importance to plant life since the apparatus for energy delivery and major pathways are localised in this compartment. To make this compartment accessible to molecular methods, transformation procedures need to be developed. The network "plastid transformation in crop plants" works on improvements of methods for plastid transformation in crop plants like oilseed rape and tomato. In addition, the transformation technology is used to explore fundamental aspects of plastid gene expression and interactions between nuclear and plastid genomes. Other, less defined mechanisms operate in plants to control the expression of genes. In contrast to promoters, these mechanisms are complex, work at another level of gene regulation and often lead to instability of gene expression. The network "control of gene expression and gene silencing in transgenic plants" deals with one of these mechanisms. In addition to leading to instabilities of transgene expression, gene silencing became an important tool to reduce the activity of genes. The network focuses on the understanding and exploitation of gene silencing mechanisms in plants and the environmental and physiological conditions which determine stability of transgene expression. The position of the transgene in the genome has an important impact on expression. However, the point of integration of a transgene cannot be pre-determined with present day technology. The network "the mechanisms and control of genetic recombination in plants" is focused on this point and works on the development of tools for targeted DNA integration. Since these tools depend on homologous recombination, the effort is s o m mutants can release silencing in t r a n s made to understand this basic from a silent hygromycin resistance gene biological process in plants and use this knowledge to devise new tools for gene targeting. In addition, recombination plays also a major role in genome stability and the ability of a plant to protect its genome from environmental damage. Therefore knowledge to imtester line A tester line A x som2 prove plants in this respect is F2 seedlings also to be expected from this network. 46

Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

The Molecular Genetics Of Homologous Recombination In Plants

Abstract The genetic components of the plant homologous recombination pathway are poorly understood. The identification of factors influencing the levels of plant homologous recombination may lead to improved precision in plant genetic engineering. Growing sequence databases reveal plant genes encoding proteins similar to those involved in homologous recombination in other organisms. This evolutionary conservation should accelerate the deciphering of elements of the plant homologous recombination pathway and help define limiting factors. The first such factor was recently determined and characterized at the molecular level through the isolation of an Arabidopsis mutant displaying DNA-damage hypersensitivity. The mutant is affected in a gene related to the structural maintenance of chromosomes (SMC) protein family and shows a defect in intrachromosomal recombination in somatic cells.

Introduction Transgenic plants promise new added value in food, feed and fibre production and should enhance crop productivity. Molecular biology techniques at present have a significant influence on plant breeding. The development of new molecular marker systems has stimulated marker-assisted breeding of many crops and developing linkage maps for many species have been combined with phenotypic data to identify genes coding for agronomic traits. Several of these traits have been characterized and modified in breeding programs [ 1]. These efforts will be further supported by emerging molecular techniques such as DNA arrays and by automation. Introducing a gene into a crop species is no longer a major obstacle and transgenes have been introduced with relative ease into most major cultivated species. However, the precision of plant genetic engineering is very low. Specific modification or disruption of endogenous genes in vivo in a directed way is not yet possible and the generation of transgenic crops relies on random insertion of foreign DNA. Consequently, the expression level of an introduced gene can vary greatly or is even prevented by the gene silencing process [for review see 2]. The transmission of introduced genes to progeny does not always follow the Mendelian pattern of inheritance due to the instability of transgenic inserts. Individuals with the desired stable phenotype and with reliable transgene expression must be selected among transformed lines, and even then there is no guarantee of the stability of the introduced trait. This situation

Tesfaye Mengiste, Novartis Agribusiness Biotechnology Research, Inc. (NABRI), Research Triangle Park, USA.

47

Control of genes

would greatly be improved by placing foreign DNA at predetermined chromosomal positions by exploiting homologous recombination. Transgenes introduced into an organism may be incorporated into the genome either randomly by non-homologous end joining (NHEJ) or by homologous recombination (HR). In plants, gene targeting frequencies, expressed as the ratio of the number of transgenic inserts resulting from HR events and the number obtained by random non-homologous insertions, is in the range of 10-5-10.3 [3-6]. Thus non-homologous integration is the predominant pathway in plants. Our limited knowledge of plant factors directing the choice of transgene integration pathway further exacerbates the problem. Transgene integration exploits pathway repairing DNA double-strand breaks (DSBs). Thus genetic factors mediating DSBs repair also determine features of transgene integration. In yeast, HR is the predominant process and mutations in components of HR belonging to Rad52 group of genes lead to a defect in DSB repair and hence hypersensitivity to DNA-damaging treatments. Integration of gapped or linearized plasmids is also dependent on Rad52 genes in yeast [7]. In contrast, a defect in elements of NHEJ in yeast is not accompanied by increased DNA-damage sensitivity unless HR is prevented [8, 9]. In animal cells, where transgenes integrate mainly randomly, mutations in the NHEJ pathway result in DNA-damage hypersensitivity. Recent in vitro studies indicate that the HR pathway suffers competition in higher organisms such as plants from protein factors favouring NHEJ. The choice between HR and NHEJ may be determined by competition between different DNA-end-binding proteins which direct the repair of DSBs into alternative pathways [10]. HR repairs DNA lesions precisely using the homologous template in the genome (e.g. the homologous chromosome, a sister chromatid or other identical or nearly identical sequences at ectopic positions). NHEJ repairs DSBs by joining the two ends of a broken DNA through a process that is independent of terminal DNA sequence homology. NHEJ is non-conservative, unprecise and often results in chromosomal inversions, deletions, translocations and duplications [ 1 i-17]. It also produces junctions varying in sequence composition [for review see 15]. The selection of the repair pathway has important consequences for genomic integrity. In yeast, the vast majority of genomic DNA comprises coding sequences and hence accurate repair by HR is preferable to prevent rapid accumulation of mutations [18]. In higher organisms, however, coding DNA accounts for only a small proportion of a larger genome. Therefore it is possible that the advantages of rapid repair of DSBs by NHEJ outweighs the need for accuracy. The two pathways share responsibility for the maintenance of genome integrity and some division of labour during the cell cycle. In mammalian cells, repair of DSBs in G1 and early S phase occurs almost exclusively by NHEJ. In late S and early G2 phase, however, when the cells have replicated DNA, HR is also active [ 19]. The determination and characterization of plant components of the HR and NHEJ pathways is required to reveal the key regulators. This should lead to the ability to alter the equilibrium between HR and NHEJ during the introduction of genomic modifications.

Transgene integration / non-homologous end joining In plants, NHEJ has been studied by the molecular analysis of transgene insertion sites and the sites of excision of transposable elements [20-25]. The structure of transgenic loci created by direct gene transfer have revealed characteristic features of NHEJ, such as short patches of 48

Homologous recombination sequence homology between vector and target DNA and regions of duplication in the target DNA around the integration site. Rearrangements of genomic DNA, including deletions and translocations, have also been observed [26]. T-DNA integration is also accompanied by duplications of short DNA around the target site and in the incoming T-DNA [22, 24]. Sequences at p l a n t / T - D N A junctions show no conservation but only a few base-pair microhomologies with one or both of the T-DNA ends. Agrobacterium-mediated transformation of plant cells leads to integration of transgenes at random positions but promoter-less reporter genes present either at the left or right border repeat of the T-DNA often become activated after integration [27]. This supports the hypothesis of a possible preference for insertion into transcribed genomic regions. A recent study in tobacco cells revealed that, in contrast to mammalian cells, repair is often accompanied by large filler insertions and extensive DNA degradation [28]. Also, the repair of DSBs introduced at a predetermined locus by transient expression of the restriction enzyme I-SceI was followed by filler genomic sequences [29]. Such a repair mechanism may lead to new genetic variation in plants. T-DNA harbouring long stretches of DNA homology to the plant genome almost exclusively integrates by NHEJ in plants. However, when the T-DNA shares homology with the yeast or Aspergillus awamori genomes, it uses HR for integration in both organisms [30, 31]. This suggests that host factors are the key players in foreign DNA integration [31, 32]. The plant proteins involved in NHEJ are unknown, although plant homologues of NHEJ components can be found in current plant sequence databases.

Homologous recombination In somatic cells, the main role of recombination is the precise repair of DSBs and hence the maintenance of genetic identity. In meiosis, HR contributes to genetic variation by the creation of novel genetic linkages. In Saccharomyces cerevisiae, the RAD52 epistasis group of genes encodes proteins for the recombinational repair of DSBs [7, 33-35]. Several of these genes are also required for meiotic recombination. In somatic cells, a range of processes (gene conversion, mitotic crossover, and the integration of gapped or linearized plasmids) rely on RAD52 genes [7]. Numerous studies have revealed that HR proteins are conserved from yeast to mammals [33, 36-39]. The mammalian and plant genomes contain large segments of repetitive sequences and multigene families that are prone to recombine. Yet such complex genomes appear to escape frequent genome rearrangement that could destabilize the genome. Presumably, this is due to tight regulation of the equilibrium between genome stability and plasticity. Multiple levels of regulation have been implicated in guarding mammalian genomes against genetic instability [40]. Recombination is already suppressed by sequence divergence of only 1.2%, which reduces DSB-promoted gene conversion in embryonic stem cells eightfold [41]. Furthermore, significantly reduced recombination has been observed between repeats at ectopic positions. This is further supported by the fact that, in mammalian cells, interchromosomal recombination between allelic as well as between ectopic substrates is reduced compared with intrachromosomal HR [42]. These observations support the hypothesis of active suppression 49

Control of genes of ectopic recombination [40]. In plants germ cells arise only late in development [43, 44] and, as a result, somatic mutations in plant cells may be inherited by the progeny. This was illustrated by the creation of genetic diversity at the Zein locus in maize [45]. Thus somatic recombination may also result in reorganisation of genetic information which, together with meiotic recombination, increases variation in the progeny. The frequency of recombination between genomic repeats in mammalian cells is low but can be enhanced by treatments causing damage to chromosomal DNA [46, 47]. The plant genome appears to respond by a significant increase in genomic rearrangement in response to environmental stress. In plants, ionizing radiation, UV, high temperature and salinity increase the frequency of intrachromosomal recombination by up to tenfold, measured using assays involving plants transgenic for ICR substrates [48, 49]. In addition, tissue culture and/or nitrogen starvation cause changes both in copy number and structure of chromosomal repeats [50]. Also, somatic intrachromosomal recombination can be induced by a transposable element, as shown for the P locus of maize (the P gene of maize is required for red phlobaphene pigmentation). Recombination in somatic cells between two direct repeats flanking the P gene excises a 4.5-kb Ac element and 12.5 kb of P sequences flanking both sides of Ac [51 ]. In addition, plants adapt to emerging races of pathogens by rapid evolution of resistance genes. Recent recombination events between members of a large disease-resistance gene familiy in maize [52-54], rice [55] and tomato [56] have been documented. This resulted in new loci conferring novel resistance.

Genetic approaches to homologous recombination The identification, characterization and functional analysis of plant proteins involved in recombination or its regulation has been slow. Plant orthologues of yeast recombination genes such as RAD51 and the meiosis-specific RAD51 homolog DMC1 have been isolated using evolutionary sequence conservation to other organisms [57-59]. However, the biological functions of these genes in plants have not been elaborated.

AT(I

Plant mutants impaired in recombination help identify factors involved in recombination and elaborate their role. Radiation-hypersensitive plant mutants have been described [60-64]. A subset of these mutants exhibits altered extrachromosomal and meiotic recombination frequencies [65]. The molecular cloning of the affected loci should shed light on elements of the HR and NHEJ pathways. Recently, we isolated a collection

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I l'i

50

Genomic organization of the mim locus and structural features of the putative MIM protein. (A) Structure of the MIM gene and position of the T-DNA insert. Exons are shown as filled boxes. The T-DNA is not drawn to scale. (B) Structural features of the MIM protein showing the five conserved regions.

Homologous recombination of T-DNA tagged Arabidopsis mutants hypersensitive to mitomycin C, MMS and X-rays [66, 67]. The T-DNA tagging allowed us to isolate the affected genes. One of these mutants had drastically reduced levels of intrachromosomal homologous recombination. Genetic and molecular analysis of the tagged locus confirmed that DNA-damage hypersensitivity is a single recessive Mendelian trait linked to the T-DNA insertion. The mutation was named mim (hypersensitive to MMS, Irradiation and MMC, table 1). Additionally, mim plantlets are senA

MIM

radl8 rhcl8

XCAPC Cut3 XCAPE scII curl4 smcl

DPY27 SUDA SMC2 SMC3

B

MIM

radl8 rhcl8 xcape scII

cutl4

xcapc

cut3 smcl

DPY27 SUDA SMC2 SMC3 SMC4

c

20 95 80 76 124 1 1 1 4 91 1 1 1

952 1025

i010 1066

1064 1067 1166

1211 Iii0

1226 1091 1068 III0 1047

GSILRIKVENFMCHSYLQI EFGEWVNFITGQNGSGKSAILTALC GVIECIHLVNFMCHDSLKI NFGPRINFVIGHNGSG~Y~AILTGLT GYIKKVILRNFMCHEHFEL ELGSRLNFIVGNMGSGKSAILTAIT LMITHIVNQNFKSYAGERILGPFHKRFSCIIGPNGSGKSNVIDSML LVVYILRLTNFKSYAGTQIYGPFHPSFSSIVGPNGSGKSNVIDALL MHVKSIIIDGFKSYAQRTEINGFDPLFNAITGLNGSGKSNILDSIC MYIKSIVLEGFKSYAQRTEIRTFDPLFNAITGLNGSGKSNILDSIC MKIEELIIDGFKSYAVRTVISNWDDQFNAITGLNGSGKSNILDAIC LVGLEL SNFKSYRGVTKVGFGESNFTSIIGPNGSG~NMMDAIS MIILNLYVENFKSYAGKHILGPFHKNLTMILGPMGSGKSNVIDALL MYVKQIIIQGFKSYKDQTVIEPFSPKHNVIVGRNGSGKSNFFAAIR MKVEELIIDGFKSYATRTVITDWDPQFNAITGLNGSGKSNILDAIC MYIKRVIIKGFKTYRNETIIDNFSPHQNVIIGSNGSGKSNFFAAIR

IEVKMPQDATSNVVR DTKGLSGGER RNLATAHNRHEKSKV SVQGLSOGEK .......... DEKAR N V D T L S G G E K DGLEFKVALGNTWKE NLTELSGGQR DGLEFRVGLGDIWKE NLTELSGGQR DGLEIHVKIGSIWKD SLAELSGGQR EGIMFSVRPPKKSWK KIFNLSGGEK EGVLFSVMPPKKSWK NISNLSGGEK AGIKYHATPPLKRFK DMEYLSGGEK GGIKFSVRPAKKSWK LIENLSGGEK VGISVSFNSKHDDQQ RIQQLSGGQK QGLEVKVKLGNIWKE SLIELSGGQR VSISVSFNSKQNEQL HVEQLSG~K SEGVTFSVMPPKKSWRNITNLSGGEK

SFSTLCFALALHEMTE SFATICMLLSIWEAMS SFSQMALLLATWKPMR SLVALSLILAMLLFKP SLAALSLILAILLFKP SLVALALIMSLLKYKP TLSSLALVFALHHYKP TLSSLALVFALHNYKP TVAALALLFAINSYAP TLASLCFVFAMHHYRP SLCALALVFAIQACDP SLIALSLIMALLQFRP TVCAIALILAIQMVDP TLSSLALVFALHKYKP

V A F G C R A R G T Q AATLKDFI ICLGAKASNT N APNMKSLV I G L G A K A S E T N GSSLKDLI F V F G Y R . A Q K I SKKLSVLI F V F G F R . A S K I QSKASALI FLLGISNLTQ ASNLQDLV FLLGISNLSQ ASSLQDLV FVLGITNMST AQNLQDLI F V L G V R S . N H LI SNILKDLI F V F G F K A G K I i TKKLSALI F V L S D A Y T H L G i iEERQALLH F V L G I A S M S T V !~ S S L Q D L I F V L S D D Y S N L K i EERQGLII

APFP~F~/FM~VSKKISLDALVDFAIGEG S~WMFITPHD CPLRCLDEFDVFMDAVNRLVSIKMMVDSAKDSSDKQFIFITPQD SRIIALDEFDVFMDQVNRKIGTTLIVKKLKDIARTQTIIITPQD APIYILDEVDAALDLSHTQNIGQML..RTH.FRHSQFIVVSLKD APIYILDEVDAALDLSHTQNIGQML..HAH.FKQSQFLVVSLKD APMYILDEIDAALDLSHTHPLEDLL..KQS.LEGSQFIIVSHKE TPLYFMDEIDAALDFKNVSIVAFYI...YEQTKNAQFIIISLRN TPLYVMDEIDAALDFKNVSIVANYI...KERTKNAQFIVISLRS SPFFVLDEVDAALDITNVQRIAAYI..RRHRNPDLQFIVISLKN TPLYVMDEIDAALDLNNVSLIANYI KHSERTRNAQFIIISLRN APFYLFDEFDVFMDAQYRTAVAQML KTISDSTNGQFICTTFRP APMYILDEVDAALDLSHTQNIGHLI KTRFKGSQFIVVSLKE ASFYLFDEIDAALDKQYRTAVATL LKELSKNAQFICTTFRT TPLYVMDEIDAALDFRNVSIVANYI KERTKNAQFIVISLRN

MIM

19 R • K • E N F M C H S Y L Q I E F G E W V N F I T G Q N G S G K S A I L T A L C I A F G C R A • G T Q R A A T L K D F I K T G C S Y A - - V V Q V E M K N S G E D A F K .... SEIY 4 Q L T I S N F A I V R E L E I D F H S G M T V I T G E T G A G K S I A I D A L G L C L G G R A E A ........ D M V R T G A A R A D L C A R F S L K D T - P A A L R W L E E N Q L E

MIM

GGVIIIERRITESATATVLKDYL-GKKVSNKRDELRELVEHF-NIDVENPPCVVMSQDKAGSSYI•ECKGNSSSFLRNLLQQVNDLLQ•I -y DGHECLLRRVISSDGRS--RGFINGTAVP--LSQLR~LGQLLIQIHGQHAAHQLLTKPEH-QKFLLDGYANETSLLQ~MTARYQLWHQSCRDLAHH

MIM

RecN

EHLTKATAIVDELENTIKPIEKEISELRGKIKNM~QVEEIAQ~QQLKKKLAWSWVYDVGRQLQEQTEKIVKLK~RIPTCQAKIDWELGKVESL~ QQLSQERAARAEL---LQYQLKELNEFNPQPGEFEQIDEEYKRLA NSGQLLTTSQNALALMAD GEDANLQ

MIM RecN

D T ~ T K K_~.A~Q_~ A_C~~_D E STAMKRE I E S F H Q S ~ K T A V.__ R~K IALQE_ ~_ F. ~~ K_ ~~ /YVQK I K ~~ R .~ V .G D ~ I.N E Q T ~ ....... T~AE~ SQ~YT~QL~SE~I GMDSKLSGVLDMLEE-~TI Q I~--S~L~DRLDLDP~~QRI SKQ I S LAR~HHVS P E A L P ~

MIM RecN

E I~KLK~EREVE~RSRLKEE

RecN

RecN

LL~Q- Q~DI~ADSQ'~'~LNVTKH

D MIM RecN MIM

RecN

L~A~FD~K~LEIEASKEQN~INQ~MRRKRE~EN~EEL~LKv~QLKK~R~QAEKvLTTKE~EM~D-~KNT~I~S~PS~--S~LQREIM E L G Q L L I ~ H G Q H A H Q L L TK P ~ H Q K F L L D G Y ~ T ~ L Q ~MTA~YQ L ~ ....... C R ~ Q Q ~ S Q E ~ L ~ Q Y Q L K E ~ F N P Q P

-

KDL~E~DEKEAFLEK~%CLKEAELKANKLTALFENM~ESAKGEIDAFEEAENELKKIEKD~QsAEAEKIHYEN~KNKVLPDIKNAEANYEED~ -GEFEQX~E---YKR~SGQLLTTSQNALALMADGEDANLQSQLYTAKQLVSE~IGMDSKL-SGVLDMLEEAT~QIAEASDELRHYCDRLDLRL

MIM

KA~EICPESEIESLG~WDGST~EQLSAQITRMN~RLH~ENQ~FSESIDDLRMMYES~--~KIAKK~KS~QDHRE~CKNAL~RW~KFQRNA RISKQISLARKHHVSp--EALPQYYQSLLEEQQQLDDQADS~ETLALAVTKHHQQALEIARALHQQRQQ~AEELAQ~IT ..... ~

MIM

SLLRRQLTW--QFNAHLGKKGISGHIKVSYENKTLSIE•KMPQDATSNVVRDTKGL•GGERSFSTLCFALALHEMTEAPFRAMDEFDVFMDAVSR S M P H G Q F T I D V K F D E H - - H L G A D G A D R I E F R - - - V T T N P G Q P M Q P I A K V A ...... S G G E L S R I A L A I Q V I T A R K M E T P A L I F D E V D V G I S G P T A

MIM

KISLDALVDFAIGEGSQWMFIT--PHD AVVGKLLRQ--LGESTQVMCVTHLPQV

RecN RecN SecN

-L

Figure 2 A-D. The SMC family. Alignments of MIM with the N-terminal (A) and the C-terminal (B) conserved sequences of the SMC protein family and the N-terminal (C) and C-terminal (D) domains of E. coli RecN. Dark shading indicates amino acids conserved in all entries and light shading shows amino acids identical to the MIM sequence. In C and D dark shading indicates amino acids identical to MIM and light shading indicates amino acids similar to MIM.

51

Control of genes Table 1. Summary of stress factors tested and responses of mim and wild-type seedlings sitive to elevated temperature (30~ Agent Concentration/condition tested Sensitivity but are not different to the wild-type +++ Methyl methanesulphonate (MMS) 0,25, 50 ", 75, 100, 150 ppm plants in all other stress responses. +++ Mitomycin C (MMC) 0, 2.5, 5 ~, 10, 15, 20 mg/l This excludes the possibility of mim +++ UV-C (254nmY 0, 2, 3, 4, 5 b, 6 kJ/m-'/s being a mutant affected in general +++ X-rays 0, 80, 150Gy b stress response (table 1). The mim +++ Temperature (30~ exposed to 30~ for 6 h, 7 h, 16 h, 3 days, 7days ' mutant phenotype is typical of a Salinity (NaCI) 0, 0.04, 0.08, 0.12 M DSB-repair defect [7, 33, 34]. The Osmoticum (mannitol) 0, 0.1,0.2, 0.4, 0.8, 1 M mira plants are hypersensitive to Oxidant (Rose bengal, H,00 0, 0.1, 0.5, 1, 2, 4 laM MMC, MMS, UV-C and X-rays. Antioxidant (N-acetyl-L- cysteine) 0, 0.3, 1, 3, 6, 12 mM Ethylene Applied at 10 ppm for 5 days MMC causes DNA cross-links, Abscisic acid 0, 0,5, 1, 3, 5, 10, 15 laM which represent a unique class of '50cA survival mutant seedlings at 3 kJ/m"/s (LD50). "doses causing 100% seedling lethality in the mutant. +++. hypersensitive. - v,lld-type level Procedures and the assay conditions are described in detail Ill Albmlsky 1999. Revenko'~a 1999. DNA lesions repaired in yeast by Menglste 1999 components of the excision and recombination repair pathway [68]. Hamster cell lines (irsl and irslSF) which display a wide range of DNA damage hypersensitivity (ionizing radiation, UV, EMS and MMC) can be complemented by the human Rad51-family genes XRCC2 and XRCC3, which are implicated in HR [69]. Thus, the sensitivity of mim to agents causing DSBs supports a defect in a recombinational repair. of

et a l .

et a l .

et a l .

Genomic and cDNA clones of the wild-type gene have been isolated and sequenced. Alignment of MIM cDNA to the genomic sequence revealed that the MIM gene is interrupted by 28 introns (Fig. l a). The T-DNA in the mim mutant is inserted in the 22 "d intron. The large TDNA of about 6kb caused loss of MIM gene A B ~ d~ ~ transcript (Fig. 3a). We mapped the MIM lo~ cus, using an ordered yeast artificial chromor ~0~ / J some genomic library of Arabidopsis [70], to the bottom of chromosome 5 between mark~,~.,. ers LFY3 and SEP5A [71]. The DNA-damage hypersensitivity phenotypes of the mu25S NA t m I l l N i tant were rescued to the wild-type level in complementation experiments using the MIM C MMStreatment(h) wild-type genomic clone, suggesting that the 0 2 24 48 mutation in the MIM gene is solely responsible for the mim-specific phenotypes. The expression of the MIM transcript correlates well with cell division activity. The MIM transcript is of low abundance in RNA extracted from 2-week-old seedlings and stems,

wt mm i

Wlm'

MM / ~1

25SR rN~ A ] 9

" ,

Figure 3 A-C. Expression patterns of the

MIM gene. (A) Northern blot of total RNA (10 lag) extracted from wild-type and mim mutant callus hybridized with a fragment of MIM eDNA spanning the first 22 exons left of the T-DNA insert (B) Northern blot of total RNA (10 lag) extracted from different plant tissues, callus and suspension culture cells, hybridized to a full-length MIM cDNA. (C) Northern blot of total RNA (5 ~g each) extracted from wild-type suspension culture cells treated before harvest with MMS (100 ppm) for the periods indicated. Cells in lane 0 were not treated with MMS. Blots were hybridized to a full-length MIM eDNA probe. All blots were rehybridized to the constitutively expressed RAN gene (A) or to 25S rDNA (B, C) as loading controls. Blots hybridized to the MIM probe in C were exposed for shorter times than in B.

52

Homologous recombination but is high in young inflorescences, rapidly growing roots and particularly in callus and suspension culture cells (Fig. 3b). Furthermore, the MIM transcript is induced by exposure to the DNA-damaging agent MMS (Fig. 3d).

Table 2. Comparison of MIM, Radl8, RHC18 and RAD50 proteins Character

MIM

Rad 18/RHC 18

RAD50

Size (amino acids)

1055

1140/1114

1312

Hydrophilic

Y

Y

Y

NTP-binding site

Y (49-56)

Y(122-131

Y (32-41)

Heptad repeats

Y

Y

Y

Matches to myosin

Y

Y

Y

The MIM cDNA contains an open Essential N Y N reading frame able to encode a proThe Rad50 sequence is from Alani et al. (1989), and RHC18 and Radl8 sequences are from tein of 1055 amino acids with ex- Lehmann et al. (1995).Y, yes; N, No. tensive homology to proteins of the SMC family [reviewed in 72] (Fig. 2a,b). Typically, proteins of the SMC family contain five, conserved structural features: an amino-terminal globular domain with an NTP-binding motif (Walker A type), -helical regions with a potential to form a coiled-coil structure, a hinge region, the second coiled-coil region, and a second globular domain in the carboxyl-terminal region harbouring the DA-box. The DA-box is a signature motif for the SMC family [73] and includes an NTP-binding motif (Walker B) [74] (Fig. l b). Mutational analysis showed that the DA-box or NTP-binding domains are required for SMC function [75, 76]. The MIM gene product is the first plant protein identified with all these structural attributes. SMCs are important modulators of chromosome architecture and are involved in chromosome condensation and segregation [76-78], sister chromatid cohesion [79, 80], transcriptional repression [75] and possibly HR [81, 82]. Sequence alignment and phylogenetic analysis showed the radl8 gene of Schizosaccharomyces pombe to be Character mim Radl8/rhcl8 rad50 the closest relative of MIM with an Sensitivityto MMS Y n.d n.d overall amino acid identity of 26.8 Sensitivityto MMC Y n.d n.d %. Sequence conservation is higher Sensitivityto X 0rT-rays Y Y Y in a 121-aa region covering the NSensitivityto UV Y Y Veryslight terminal NTP binding site (47% idenRecombinationdeficient Y 9" Y tity), and in 53 aa around the DA"Suggestedto function in post replicationrepair pathwayby Lehmannet al. (1995). box motif at the C-terminus (54% Y, yes; n.d, no data. identity) (Fig. 2a,b). Importantly, the radl8 gene is epistatic to RHP51 (S. pombe homologue of RAD51), which is implicated in HR [81]. However, the effect of the radl8 mutation on recombination has not been shown directly. The DSB-repair protein RecN of E.coli [83] also shows sequence similarity to the MIM protein (Fig 2 c,d).

Table 3. Comparisonof mim,radl8, rhcl8 and rad50 mutant phenotypes

The RAD50 protein from various organisms shows some structural and functional similarity to SMCs, and thus to MIM and RHC18/Radl8 (Table 2). Also, the mim mutant phenotypes correspond to the radl 8/rhc 18 and rad50 mutants of yeast (Table 3). The rad50 mutants of yeast and mim show deficiency in recombination and are sensitive to ionizing and UV radiation [84]. We used an intrachromosomal recombination assay to determine the role of the MIM gene product of HR in Arabidopsis. The assay makes use of a recombination substrate 53

Control of genes derived from the 6-glucuronidase (GUS) gene (Fig. 4a) [85]. After crossing we obtained plants harbouring the recombination substrate in the mutant or wild-type genetic background. The frequency of intrachromosomal recombination could be evaluated from the number of somatic sectors expressing the GUS gene restored by HR. The frequency in the mim/mim mutant background was reduced approximately fourfold relative to the wild-type control plants (Fig. 4b). This, together with the DNA-damage hypersensitivity of mim, implies that the MIM protein is involved in recombinational DNA repair in Arabidopsis. SMC proteins are necessary for structural changes to chromatin affecting activities of the DNA template (transcription, repair) [reviewed in 86" 72, 87, 88]. Our finding of an SMC-like gene as a component of the plant recombination apparatus reinforces the importance of chromatin structure in HR. SMC proteins have been implicated in HR. In vitro, SMC proteins can perform recombination related activities such as DNA renaturation [82, 89]. The bovine recombination complex (RC1), which recombines DNA substrates and repairs gaps and deletions in vitro, contains two SMC sub-units essential for its function [82]. However, all eukaryotic SMCs tested to date are essential for viability; depletion of functional protein, including S. pombe radl8, is lethal [81; for review see 72]. Since proficiency in recombination is not essential for yeast survival, multiple A functions of Rad 18 can be envisaged. Thus direct GUSin vivo evidence for the involvement of SMC proteins in HR was lacking. In this context, it is rather surprising that homozygous mim plants develop normally in the absence of genotoxic stress. However, this enabled us to provide the first in vivo evidence for the involvement of proteins with SMC features in recombination. It showed that the MIM protein has a more-specialized function than other 9 G U S known SMCs. + The exact contribution of SMCs and SMC-like

~

R

+

Figure 4. Intrachromosomal recombination assay. (A) A recombination substrate derived from the E. coli b-glucuronidase (GUS) gene integrated into the Arabidopsis line N1DC1 no.ll as a single copy (Swoboda et al., 1994) including a promoter (Pr) and terminator (ter) linked to the hygromycin phosphotransferase resistance gene (HYGR). (B) Homologous recombination frequencies in Arabidopsis lines homozygous for the recombination substrate and the wild-type allele of MIM (GU-US/GU-US;MIM/MIM) and lines harbouring the mim mutation (GU-US/GU-US;mim/ mim). Recombination frequency was determined by scoring the number of somatic recombination events in 30-50 single plant progeny (F3) seedlings (thin black bar) of both genotypes. Wide bars represent the mean recombination frequencies for 20 and 22 F3 populations of GU-US/GUUS;MIM/MIM and GU-US/GU-US;mim/mim genotypes, respectively. Standard errors are indicated.

54

12 lO

~ ~g

8

~,"

6

on. o (9,-

~-~ 4

L'I111,i11 ,1,I111,

IqLIIIIILIIII

I

Idlllll][llll.i ,i, i_.,_.,_

IIIIIIllrlll:l ilrl,rllrl, fl i

GU-US/GU-US;MIM/MIM

GU-US/GU-US;mim/mim

Homologous recombination

proteins to DNA repair is still unknown. However, the structural features of chromatin conferred by SMCs might be required for recognition of DNA damage and the recruitment of repair complexes. In plants, chromosomal recombination is suppressed in contrast to the efficient extrachromosomal recombination of naked DNA [for review see 90]. Thus, chromatin structure could be a critical step controlling in vivo accessibility of chromosomal DNA for repair in plants and thus also for homologous recombination.

Authors of this publication Tesfaye Mengiste and Jerzy Paszkowski*, Friedrich Miescher Institute, P. O. Box 2543, CH4002 Basel, Switzerland, *corresponding author.

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Homologous recombination tion between duplicated chromosomal sequences in mouse L cells. Mol Cell Biol 8, 196-202. 47. Bhattacharyya NP, Maher VM, McCormick JJ. (1990) Intrachromosomal homologous recombination in human cells which differ in nucleotide excision-repair capacity. Mutat Res 234, 31-41. 48. Lebel E, Masson J, Bogucki A and Paszkowski, J. (1993) Stress-induced intrachromosomal recombination in plant somatic cells. Proc. Natl. Acad. Sci. USA. 90, 422-426. 49. Puchta H, Swoboda P, Hohn B. (1995). Induction of intrachromosomal homologous recombination in whole plants. Plant J 7, 203-210. 50. L6rz H, Scowscroft WR. (1983) Variability among plants and their progeny regenerated from protoplasts of Su/su heterozygotes of Nicotiana tabacum. Theor Appl Genet 66, 67-75. 51. Athma P, Peterson T. (1991) Ac induces homologous recombination at the maize P locus. Genetics 128, 16373. 52. Das OE Poliak E, Ward K, Messing J. (1991 a) A new allele of the duplicated 27kD zein locus of maize generated by homologous recombination. Nucleic Acids Res 19, 3325-30. 53. Das OP, Ward K, Ray S, Messing J. (1991b) Sequence variation between alleles reveals two types of copy correction at the 27-kDa zein locus of maize. Genomics 11,849-56. 54. Richter TE, Pryor TJ, Bennetzen JL, Hulbert SH. (1995) New rust resistance specificities associated with recombination in the Rpl complex in maize. Genetics 141, 373-81. 55. Song WY, Pi LY, Wang GL, Gardner J, Holsten T, Ronald PC. (1997) Evolution of the rice Xa21 disease resistance gene family. Plant Cell 9, 1279-87. 56. Parniske M, Hammond-Kosack KE, Golstein C, Thomas CM, Jones DA, Harrison K, Wulff BB, Jones JD. (1997) Novel disease resistance specificities result from sequence exchange between tandemly repeated genes at the Cf-4/9 locus of tomato. Cell 91,821-32. 57. Doutriaux ME Couteau F, Bergounioux C, White C. (1998) Isolation and characterisation of the RAD51 and DMC1 homologs from Arabidopsis thaliana. Mol. Gen. Genet. 257, 283-291. 58. Klimyuk V, Jones J. (1996) AtDMC1, the Arabidopsis homologue of the yeast DMC1 gene: characterization, transposon induced allelic variation and meiosis specific expression of a pAtDMCI: GUS fusion. Plant J. 11, 1-14. 59. Kobayashi T, Hotta Y, Tabata S. (1993) Isolation and characteriztion of a yeast gene that is homologous with a meiosis specific cDNA from a plant. Mol. Gen. Genet. 237, 225-232. 60. Masson JE and King PJ, Paszkowski J. (1997). Mutants of Arabidopsis thaliana hypersensitive to DNAdamaging treatments. Genetics 146, 401-407. 61. Davies C, Howard D, Tam G, Wong N. (1994) Isolation of Arabidopsis thaliana mutants hypersensitive to gamma radiation. Mol Gen Genet 243, 660-5. 62. Jenkins ME, Harlow GR, Liu Z, Shotwell MA, Ma J, Mount DW. (1995) Radiation-sensitive mutants of Arabidopsis thaliana. Genetics 140, 725-32. 63. Jiang CZ, Yen CN, Cronin K, Mitchell D, Britt AB. (1997) UV- and gamma-radiation sensitive mutants of Arabidopsis thaliana. Genetics 147, 1401-9. 64. Albinisky D, Masson J, Bogucki A, Afsar K, Vass I, Nagy F, Paszkowski J. (1999) Plant responses to genotoxic stress are linked to ABA/salinity signaling pathway. Plant J 17, 73-82. 65. Masson JE, Paszkowski J. (1997) Arabidopsis thaliana mutants altered in homologous recombination. Proc. Natl. Acad. Sci. U S A 94, 11731-11735. 66. Revenkova E, Masson J, Koncz C, Afsar K, Jakovleva L, Paszkowski J. (1999) Involvement of Arabidopsis thaliana ribosomal protein $27 in mRNA degradation triggered by genotoxic stress. EMBO J. 18, 490-499. 67. Mengiste T, Revenkova E, Bechtold N, Paszkowski J. (1999). An SMC-like gene is required for efficient homologous recombination in Arabidopsis. EMBO J 18, 4505-4512. 68. Jachymczyk WJ, von Borstel RC, Mowat MR, Hastings PJ. (1981) Repair of interstrand cross-links in DNA of Saccharomyces cerevisiae requires two systems for DNA repair: the RAD3 system and the RAD51 system. Mol Gen Genet 182, 196-205. 69. Liu N, Lamerdin JE, Tebbs RS, Schild D, Tucker JD, Shen MR, Brookman KW, Siciliano M J, Walter CA, Fan W, Narayana LS, Zhou ZQ, Adamson AW, Sorensen KJ, Chen DJ, Jones NJ, Thompson LH. (1998) XRCC2 and XRCC3, new human Rad51-family members, promote chromosome stability and protect against DNA cross-links and other damages. Mol Cell 1,783-93. 70. Creusot F, Fouilloux E, Dron M, Lafleuriel J, Picard G, Billault A, Le Paslier D, Cohen D, Chaboue ME, Durr A. (1995) The CIC library: a large insert YAC library for genome mapping in Arabidopsis thaliana. Plant J 8, 763-70. 71. Schmidt R, Love K, West J, Lenehan Z, Dean C. (1997) Detailed description of 31 YAC contigs spanning the majority of Arabidopsis thaliana chromosome 5. Plant J. 11,563-573.

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Control of genes 72. Strunnikov AV. (1998) SMC proteins and chromosome structure. Trends Cell Biol. 8, 454-459. 73. Strunnikov AV, Larionov VL, Koshland D. (1993) SMCI: an essential yeast gene encoding a putative headrod-tail protein is required for nuclear division and defines a new ubiquitous protein family. J Cell Biol 123, 1635-48. 74. Walker, J.E., Saraste, M., Runswick, J, Gay, N.J. (1982) Distantly related sequences in the a- and 13-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1,945-951. 75. Chuang PT, Albertson DG, Meyer BJ. (1994) DPY-27:a chromosome condensation protein homolog that regulates C. elegans dosage compensation through association with the X chromosome. Cell 79, 459-74. 76. Strunnikov AV, Hogan E, Koshland D. (1995) SMC2, a Saccharomyces cerevisiae gene essential for chromosome segregation and condensation, defines a subgroup within the SMC family. Genes Dev. 9, 58799. 77. Saitoh N, Goldberg IG, Wood ER, Earnshaw WC. (1994) SclI: an abundant chromosome scaffold protein is a member of a family of putative ATPases with an unusual predicted tertiary structure. J Cell Biol 127, 30318. 78. Saka Y, Sutani T, Yamashita Y, Saitoh S, Takeuchi M, Nakaseko Y, Yanagida M. (1994) Fission yeast cut3 and cutl4, members of a ubiquitous protein family, are required for chromosome condensation and segregation in mitosis. EMBO J 13, 4938-52. 79. Guacci V, Koshland D, Strunnikov A. (1997) A direct link between sister chromatid cohesion and chromosome condensation revealed through the analysis of MCD1 in S. cerevisiae. Cell 91, 47-57. 80. Michaelis C, Ciosk R, Nasmyth K. (1997) Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell 91, 35-45. 81. Lehmann AR, walicka M, Griffiths DJ, Murray JM, Watts FZ, McCready S, Carr AM. (1995) The radl 8 gene of Schizosaccharomyces pombe defines a new subgroup of the SMC superfamily involved in DNA repair. Mol Cell Biol 15, 7067-7080. 82. Jessberger R, Riwar B, Baechtold H, Akhmedov AT. (1996). SMC proteins constitute two subunits of the mammalian recombination complex RC-1. EMBO J. 15, 4061-4068. 83. Rostas K, Morton SJ, Picksley SM, Lloyd RG. (1987) Nucleotide sequence and LexA regulation of the Escherichia coli recN gene. Nucleic Acids Res 15, 5041-9 84. Alani E, Subbiah S, Kleckner N. (1989) The yeast RAD50 gene encodes a predicted 153-kD protein containing a purine nucleotid-binding domain and two large heptad-repeat regions. Genetics 122, 47-58. 85. Swoboda R Gal S, Hohn B, Puchta H. (1994) Intrachromosomal homologous recombination in whole plants. EMBO J 13, 484-9. 86. Koshland D, Strunnikov A. (1996) Mitotic chromosome condensation. Annu Rev Cell Dev Biol 12, 305-33. 87. Jessberger R, Frei C, Gasser SM. (1998). Chromosome dynamics: the SMC protein family. Curr Opin Genet Dev 8, 254-9. 88. Hirano T. (1999) SMC-mediated chromosome mechanics: a conserved scheme from bacteria to vertebrates?. Genes Dev 13, 11-9. 89. Sutani T, Yanagida M. (1997) DNA renaturation activity of the SMC complex implicated in chromosome condensation. Nature 388, 798-801. 90. Puchta H, Hohn B. (1996). From centiMorgans to base pairs: homologous recombination in plants. Trends Plant Sci. 1,340-348.

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

Developments In Plastid Transformation

Introduction In recent years there has been a growing appreciation that transformation of the plastid genome (plastome) has numerous attractions as an alternative to nuclear transformation for the genetic modification of crops. It has already been demonstrated that the large copy number of the plastome results in transgene amplification, which can lead to accumulation of very high levels of recombinant protein [1 ]. Furthermore consistency of expression can be ensured through precisely targeted integration, exclusion of vector DNA, and absence of any gene silencing mechanism. Particularly attractive are those features of this technology which address some of the main consumer concerns over the use of genetically modified crops. The use of antibiotic insensitivity mutations for selection eliminates the controversial use of bacterial antibiotic resistance genes, and indeed provides the prospect of restricting the heterologous DNA solely to the gene of interest and its regulatory elements. Predominantly maternal inheritance of the plastome in most crop plants reduces the risk of pollen spread and provides natural containment of the transgene. Furthermore, transgene expression can be driven by efficient plastid promoters, avoiding the use of promoters derived from plant pathogens (eg cauliflower mosaic virus, Agrobacterium tumefaciens). The main limitation to the potential applications of plastid transformation lies in the fact that no system for export of recombinant protein from the plastids has been identified. Therefore the plastid itself has to be an appropriate final location for the recombinant protein. For many agronomic traits including insect and herbicide resistance, for which its efficacy has already been demonstrated [2, 3], as well as "cell factory" applications for the production of valuable protein, this appears to be the case. The year 2000 will mark the end of the first decade of higher plant plastid transformation. The first report [4], in common with most subsequent reports, used Nicotiana tabacum (tobacco) as the target species and a biolistic method of DNA delivery. In order to select transformants a pUC-based vector was used into which a fragment of tobacco plastid DNA containing a 16S rRNA gene including mutations conferring resistance to streptomycin and spectinomycin, had been cloned. This strategy, utilising so-called "binding-type" antibiotic resistance (or antibiotic insensitivity) markers was subsequently abandoned in favour of heterologous antibiotic resistance genes, such as a chimeric aadA gene [5]. The efficiency which can be achieved using the original antibiotic insensitivity approach was however confirmed by a number of

Phil J. Dix, Biology Dept., NUI Maynooth, Co. Kildare, Ireland.

59

Control of genes

investigations in the laboratory of E Medgyesy at the Biological Research Center, Szeged, Hungary, culminating in a protocol combining it with a delivery method using PEG-mediated uptake of DNA into protoplasts [6]. The relative merits of different selectable markers and DNA delivery methods were reviewed by Dix and Kavanagh [7] and remain a critical issue in the refinement and extension of plastid transformation technology. Aware of the enormous biotechnological potential of plastid transformation, the EU Commission approved a project (entitled: "Plastid Transformation in Crop Plants") under the 4 thFramework Biotech Programme, bringing together eight laboratories to further develop the technology. Several of the partners were already experienced in plastid transformation procedures, while the others provided complementary skills in molecular biology, plant physiology, photosynthesis and control of plastid gene expression. The rationale behind the project was that the improvement and extension of the technology needs to be underpinned by an improved understanding of fundamental aspects of the structure, function and regulation of the plastome, as well as its interaction with the nuclear genome. Therefore in addition to improvements in plastid transformation procedures, there is a very strong focus on these fundamental questions. The results of the project will provide the foundations for further, mission-orientated, applied research projects, aimed at fulfilling the undoubted promise of plastid transformation technology. The following sections summarise some of the main results obtained by the mid-point of this three year project. They all either relate to the transformation procedures themselves, or provide information relevant to the longer term application of the technology.

Vectors based on plastome mutations Development of an efficient system for the induction and selection of plastome mutations conferring structural insensitivity to antibiotics [8] provided the opportunity to develop plastid transformation vectors based on these mutations. Aside from the aesthetic attractions of such vectors (avoidance of heterologous resistance genes and excess heterologous DNA), these vectors also have practical advantages, leading to rapid establishment of homoplasmy, without the repeated cycles of selection required with heterologous markers [6, 7]. A double plastome mutant of Solanum nigrum carries a mutation in the 16S rRNA gene conferring resistance to spectinomycin, and a second in the ribosomal protein gene, rps 12 (3'), conferring resistance to streptomycin [9]. These two genes are located conveniently close together and are included in the same plasmid pSSH1, which has been used for numerous successful tobacco plastid transformation experiments [ 10, 11 ]. The 7.8 kbp S. nigrum plastome insert in pSSH1 has now been modified as follows: approximately 1 kbp sequence has been removed from either end and a multiple cloning site has been introduced between the two genes containing the selectable mutations. This provides a versatile vehicle into which a range of expression cassettes can be introduced, and is believed to have wider utility than just for Nicotiana species. To date vectors containing uidA (encoding GUS) and bar (conferring BASTA resistance) genes have been produced and are being used for plastid transformation. One limitation of binding type resistance markers is that integration is restricted to the location of the native genes where the resistance mutations reside. For some applications it may be important to introduce foreign genes at other sites on the genome. It has already been 60

Plastid transformation demonstrated that when the heterologous marker gene aadA is used, a second unlinked sequence can also be integrated at a high frequency [ 12]. In order to demonstrate that this is also the case with the binding type markers, tobacco protoplasts were co-transformed with both pSSH 1 and with a second plasmid containing a mutant form of the unlinked psbA gene. This mutation confers resistance to triazine herbicides [ 13] and also imparts a disinctive change in chlorophyll fluorescence. Of thirty spectinomycin resistant plants obtained (not all of which have been confirmed as transformants), seven exhibited the characteristic triazine resistance and fluorescence parameters associated with the psbA mutation. This suggests a sufficiently high frequency of integration of a non-linked sequence to suggest that it should be possible to introduce transgenes at any point on the plastome by constructing a vector with the appropriate flanking sequences. Transformants will be detectable by screening the spectinomycin resistant lines for the presence of the transgene.

Homologous and homeologous recombination Stable plastid transformation occurs exclusively through homologous recombination, and excludes the foreign plasmid DNA. The plasmid pSSH1 contains a 7.8 kbp sequence from the Solanum plastome. This exhibits incomplete homology (ie. is homeologous) with the Nicotiana plastome, and provides the opportunity to explore the tolerance limits on partial homology which still allow efficient integration. This information may be critical for the design of versatile vectors amenable to plastid transformation in a broader range of crop species. The presence of a number of diagnostic molecular markers for the two species also permits a detailed dissection of the recombination events associated with integration across the homeologous region, which was not possible in earlier studies [eg. 6] in which completely homologous Nicotiana plastid DNA was used. Plastid transformation of tobacco with S. nigrum plastid DNA, involved selection for either streptomycin+spectinomycin resistance, or for spectinomycin resistance alone, and proved to be jus'~ as efficient as with comparable vectors based on Nicotiana sequences [11]. This indicates that the 2.4% sequence divergence between the two species across this 7.8 kbp region can be tolerated without effecting transformation efficiency, a finding in marked contrast to the situation in Chlamydomonas where a 1% divergence results in a 10-100 fold reduction in recombination frequency [14]. Detailed molecular characterisation of the Nicotiana transplastomics revealed that most had resulted from multiple recombination events. For example, in one experiment only one out of eleven transformants had all the Solanum diagnostic sites indicating uninterrupted integration of Solanum sequence through a double recombination event (subject to the limitations of the analysis). Two others could have arisen from a double crossover but were missing a peripheral Solanum marker so a shorter sequence was integrated. All the other transformants displayed a mosaic of Nicotiana and Solanum sequence indicative of multiple (4, 6 or 8) crossover events. This too differs from the situation reported for Chlamydomonas where transformation proceeds exclusively through one double exchange [14]. Both the high tolerance of incomplete homology, and the high recorabination frequency in the higher plant plastid genome are indic~tive of a diminished mismatch repair system and augur well for development of plastid transformation vectors based on these sequences. Another positive observation [ 11 ] is that recombination is nonrandom, with a bias in favour of peripheral regions adjacent to the completely heterologous vector DNA. 61

Control of genes

Reporter genes The GUS reporter system has previously been shown to function in tobacco plastids [ 15] and provides a valuable tool for rapid screening of plastid transformants. Our pSSH1 based vector (pSSH-GUS) containing uidA (GUS) with prrn promoter and the psbA 3' untranslated region, has allowed both transient and longer term expression of GUS in Brassica napus (oilseed rape) leaf tissue transformed by microprojectile bombardment. Studies on plastid promoter activity, and gene expression in different plastid types, which are of central importance to the application of plastid transformation technology, would however benefit from a rapid, convenient, non-destructive, transient expression assay. Such a system has now been developed, utilising the g/~ (green fluorescent protein) reporter gene from Aequoria victoria [ 16]. A plasmid was constructed in which the coding sequence for the GFP chromophore was flanked by the rrn promoter and the psbA untranslated region. Biolistic delivery of this construct allowed the demonstration of gfp expression in chloroplasts of tobacco and the arc6 mutant of Arabidopsis thaliana (which has a small number of very large chloroplasts). Furthermore, modified constructs in which g ~ is driven by the bacterial trc promoter, were used to demonstrate that efficient transgene expression can be achieved in chloroplasts using bacterial promoters. Transient g ~ expression was also achieved in non-photosynthetic plastid types including potato tuber amyloplasts and marigold petal chromoplasts, which augurs well for the modification of traits dependent on these plastid types. The GFP reporter constructs also provide a valuable method for monitoring homoplasmy/heteroplasmy in stable plastid transformants. For example, in a tobacco plastid transformant obtained using the leaky selection system based on the heterologous aadA gene, the presence of both gfp expressing and non-expressing plastids could be clearly visualised. The gfp reporter constructs have also assisted in the development of a micro-injection system for the delivery of DNA directly into individual chloroplasts and other organelles [17].

Transcriptional control of plastid genes The plant plastid genome is transcribed by at least two different types of RNA polymerase [ 18]. One (PEP) is procaryotic-like and multimeric, with a plastid-encoded core enzyme. The other (NEP) is nuclear-encoded, probably monomeric, and has homology to phage and mitochondrial enzymes. Plastid transformation is being used to analyse expression from plastid promoters recognised by NEP or PEP, and more complex promoter structures that are recognised by both enzymes, perhaps with a temporal or spatial bias. To achieve this, various promoter-reporter constructs are being made. Those produced so far focus on two chloroplast promoters (for psbA and rbcL). Both intact and truncated promoters have been isolated by PCR. Both uidA and g ~ have been inserted between the cloned promoters and rrn terminator and their expression is being examined in tobacco chloroplasts. In parallel with these investigations, in vitro transcriptional assays are being used to determine the upstream DNA sequence of a given gene or operon needed to give full promoter activity. Several transcription vectors have been constructed harbouring NEE PEP or mixed promoter regions. NEP and PEP promoter recognising transcriptional activities have been separated from crude plastid extracts and used to characterise the different promoters by in vitro transcription. The following promoter regions have been tested so far: rbcL (PEP), rrn P1/PC/P2 (mixed), rrn P3 (NEP) and clpP (mixed). All tested promoter regions are from 62

Plastid transformation

spinach because the in vitro transcription extracts are made from spinach. Observations have been made on the promoter regions as follows: - rbcL: recognised by PEP but not NEE - rrn: PC promoter is recognised by NEE but only in the presence of the transcription factor

CDF2. Neither P 1 nor P2 is recognised by the NEP enzyme, nor are the corresponding promoter elements important for transcription initiation at PC. The P3 promoter is recognised by NEP but its promoter strength is very weak and recognition is dependent on the deletion of the preceding P1/PC/P2 promoter region. The probable reason for this is that the P3 promoter is always occupied by running-down transcription complexes that were initiated at PC. - clpP: The promoter region contains three overlapping promoters. One is recognised by T7 RNA polymerase and by NEE The second is recognised by E. coli RNA polymerase and NEE The third region is not recognised in vitro by either of the two transcriptionally active fraction enzymes, but is the main transcription start site in vivo. It is not yet clear which enzyme (NEP or PEP) transcribes from the clpP promoter in vivo, or whether the negative in vitro results are due to the loss of a specific transcription factor. Similar studies also confirm differences in transcription between different species. For example, the tobacco rrn operon is mainly transcribed by PEP, while the equivalent spinach operon is transcribed exclusively by NEE Observations such as this have important implications for the design of versatile expression cassettes, based on plastid promoters, for use in a range of crop plants.

The plastid NAD(P)H dehydrogenase complex The plastid genome of higher plants contains 11 genes homologous to mitochondrial genes encoding subunits of the NADH dehydrogenase complex (complex I). Recently, a large complex showing NADH dehydrogenase activity, and similar to bacterial NADH dehydrogenase complex, has been purified from pea chloroplasts [ 19]. In order to elucidate the physiological role of this respiratory complex, inactivation of ndh genes has been undertaken. Targeted inactivation of a plastidial ndh gene ( n d h B ) was carried out by plastid transformation [20]. The transformation plasmid pSSH1 (see section 1) includes the n d h B gene. A frame shift was introduced into the coding sequence leading to translational termination. The modified plasmid was introduced into tobacco chloroplasts by PEG mediated uptake into protoplasts. The resulting plants grew normally and could not be distinguished from wild type plants under normal growing conditions. Southern analysis confirmed the homoplasmy of these lines and Western analysis showed the n d h H gene product was absent from the transformants, indicating that the NDH complex disappeared in response to n d h B inactivation. Chlorophyll fluorescence measurements showed no obvious differences between wild type and ndhB- plants during a dark to light transition. However in the dark period following illumination, the normal transient increase in fluorescence was almost completely suppressed in ndhB-plants, thereby indicating an inability of the NDH complex to reduce the plastoquinone pool in the dark. Further characterisation of these plants has revealed impairment of photosynthetic capacity under certain stress conditions including anaerobiosis [21]. Further experiments are aimed at precise determination of the phenotype of the transformants, which should reveal the role of the plastid NAD(P)H dehydrogenase complex during photosynthesis. 63

Control of genes

Nucleo-cytoplasmic interactions The production of a functional photosynthetic apparatus is dependent on a balanced co-operation between products of nuclear and plastid genomes. Frequently, the production of alloplasmic lines (containing the nucleus of one species with cytoplasmic genomes of a second) results in impairment of chloroplast function due the disruption of this balance [22]. In the present study cybrids (alloplasmic lines produced by protoplast fusion) between Solanaceous species, are being characterised to establish the nature of these defects. Ultimately, the contribution of the plastome will be explored through complementation tests in which restoration of normal chloroplast function will be achieved by plastid transformation with defined plastome sequences. The investigations have centred on cybrids with the nucleus of Nicotiana tabacum, and chloroplasts of either Atropa belladonna (cybrid Nt(Ab)) [23] or Salpiglossis sinuata (cybrid Nt(Ss)) [24]. In Nt(Ab) plants the protein profiles of the light harvesting complex II (LHC II) are found to be altered compared to either parent [25], with two novel protein bands. A possible cause of this could be that protein processing is affected in the cybrid, but so far no pronounced physiological or biochemical consequences of this modification have been detected (except for a slight reduction in trimeric LCH II). In the case of Nt(Ss), there were more pronounced effects on chloroplast structure and function [25]. The cybrid exhibits an increased chlorophylla/b ratio, suggesting that chlorophyll a+b-binding proteins of the antenna system of photosystem II (PS II) are missing. This leads to a lower photosynthetic efficiency (Fv/Fm) in the leaves of the cybrid, which becomes more pronounced as the leaves age. The older leaves are severely effected by photoinhibition. These cybrid specific features suggest an involvement of nucleus-chloroplast genome interaction in the processing of LCH II, and in chloroplast development. The cybrid plants also showed a far greater sensitivity to high light stress, an additional finding which may have implications for the use of alloplasmic lines in crop breeding.

Expanding the species range The biotechnological application of plastid transformation technology is dependent on extending the crop range for which transformation procedures are available. Part of the current Work Programme is to extend the procedures to two important European crops, Lycopersicon esculentum (tomato), and Brassica napus (oilseed rape). Tomato is closely related to Solanum nigrum, on which the current set of vectors (pSSH1 and its derivatives), utilising binding-type resistance, is based. Also, protoplast culture of tomato is a routine procedure within the Consortium, so PEG-mediated uptake of pSSH1 into protoplasts has been the delivery method of choice. To date, antibiotic resistant protoplast-derived plants have been obtained, but stable plastid transformation has yet to be confirmed. A highly responsive leaf and cotyledon regeneration system has been developed for oilseed rape variety Drakkar, and transformation is proceeding using pSSH1 derivative (pSSH-GUS) containing uidA with plastid expression signals (rrn promoter and psbA downstream sequences). A biolistic delivery method has resulted in transient GUS expression (confirming the effectiveness of the vector), and stable GUS expressing structures from which it has not yet been possible to regenerate shoots. Additional transformation vectors are also being produced, specifically tailored to the Brassica plastome. 64

Plastid transformation

Future prospects We believe that this project is laying important groundwork for the extension of plastid transformation to deliver important biotechnological products in key European crops. By the end of the project, in the year 2000, we will have an enhanced understanding of the regulation of native plastid genes, which will help us to optimise transgene expression both in chloroplasts and in other plastid types. We will have unravelled some of the remaining mysteries regarding the function of plastid genes, and their sensitive interaction with the nuclear genome, both of which have to be taken into account when modifying the plastome. Versatile plastid vectors, avoiding heterologous antibiotic resistance genes, will be available, and the precise criteria for tissue culture-selection to make optimal use of these vectors, will have been defined. This will pave the way to a major extension of the crop base for which plastid transformation procedures are available. The fundamental thrust of this 4 th Framework project will give way to mission-orientated applied research projects in which the potential of the plastome will be harnessed to deliver specific medical and agronomic products in key crops.

Authors of this contribution R J. Dix ~, J. C. Gray2, T.A. Kavanagh 3, S. Lerbs-Mache 4, R Medgyesy 5, A. Mordhorst 6, G. Peltier 7, C. Sch~ifer8 and B. Uijtewaal 6 ~Biology Dept., NUI Maynooth, Co. Kildare, Ireland, 2Dept. of Plant Sciences, University of Cambridge, Downing St., Cambridge, U.K., 3Dept. of Genetics, University of Dublin, Trinity College, Dublin, Ireland, 4Laboratoire de Genetique Moleculaire des Plantes, Universit6 Joseph Fourier, Grenoble, France, 5Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary, 6Nunhems Zaden B.V., Voort 6, AA Haelen, The Netherlands, 7CEA/Cadarache, DSV, DEVM, Laboratoire d'Ecophysiologie de la Photosynth6se, 13108 Saint-Paul-lez-Durance, France, 8Lehrstuhl ftir Pflanzenphysiologie, Universit~it Bayreuth, Bayreuth, Germany.

References 1. 2.

3. 4. 5. 6.

7. 8. 9.

Staub JM, Maliga P (1993) Accumulation of D1 polypeptide in tobacco plastids is regulated via the untranslated region of the psbA mRNA. EMBO J 12:601-606 McBride KE, Svfib Z, Schaaf DJ, Hogan PS, Stalker DM, Maliga P (1995) Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco. Bio/ Technology 13:362-365 Daniell H, Datta R, Varma S, Gray S, Lee S-B (1998) Containment of herbicide resistance through genetic engineering of the chloroplast genome. Nature Biotechnol 16:345-348 Svab Z, Hajdukievitz P, Maliga P (1990) Stable chloroplast transformation in higher plants. Proc Natl Acad Sci USA 87:8526-8530 Svab Z, Maliga P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci USA 90:913-917 O'Neill C, Horv~ith GV, Horvfith E, Dix PJ, Medgyesy P (1993) Chloroplast transformation in plants: polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems. Plant J 3:729-738 Dix PJ, Kavanagh TA (1995) Transforming the plastome: genetic markers and DNA delivery systems. Euphytica 85:29-34 McCabe PF, Timmons AM, Dix PJ (1989) A simple procedure for the isolation of streptomycin resistant plants in Solanaceae. Mol Gen Genet 216:132-137 Kavanagh TA, O'Driscoll KM, McCabe PF, Dix PJ (1994) Mutations conferring lincomycin, spectinomycin,

65

Control of genes

10.

11.

12. 13. 14.

15.

16. 17.

18. 19.

20.

21.

22. 23. 24. 25.

and streptomycin resistance in Solanum nigrum are located in three different chloroplast genes. Mol Gen Genet 242:675-680 Dix PJ, Thanh ND, Kavanagh TA, Medgyesy P (1995) Integration of Solanum DNA into the Nicotiana plastome through PEG-mediated transformation of protoplasts. In M Terzi, R Cella, A Falavigna eds) Current issues in Plant Molecular and Cellular Biology. Kluwer Acad Publ, pp 297-302 Kavanagh TA, Thanh ND, Lao NT, McGrath N, Peter SO, Horv~ith E, Dix PJ, Medgyesy P (1999) Homeologous plastid DNA transformation in tobacco is mediated by multiple recombination events. Genetics 152:1111-1122 Carrer H, Maliga P (1995) Targeted insertion of foreign genes into the toobacco plastid genome without physical linkage to the selectable marker gene. Bio/Technology 13:791-794 Pfiy A, Smith MA, Nagy F, Mfirton L (1988) Sequence of the psbA gene from wild-type and triazineresistant Nicotiana plumbaginifolia. Nucl Acids Res 16:8176 Newman SM, Boynton JE, Gillham NW, Randolph-Anderson BL, Johnson AM (1990) Transformation of chloroplast ribosomal RNA genes in Chlamydomonas: molecular and genetic characterisation of integration events. Genetics 126:875-888 Seki M, Shigemoto N, Sugita M, Sugiura M, Koop HU, Irifune K, Morikawa H (1995) Transient expression of glucuronidase in plastids of various plant cells and tissues delivered by a pneumatic particle gun. J Plant Res 108:235-240 Hibberd JM, Linley PJ, Khan MS, Gray JC (1998) Transient expression of green fluorescent protein in various plastid types following microprojectile bombardment. Plant J 16:627-632 Knoblauch M, Hibberd JM, Gray JC, van Bel, AJE (1999) The galinstan expansion femtosyringe allows micro-injection of fluorochromes and DNA into eukaryotic organelles and prokaryotes. Nature Biotech. (submitted) Iratni R, Diederich L, Harrak H, Bligny M, Lerbs-Mache S (1997) Organ-specific transcription of the rrn operon in spinach plastids. J Biol Chem 272:13676-13682 Sazanov LA, Burrows PA, Nixon PJ (1998) The plastid ndh genes code for an NADH-specific dehydrogenase: isolation of a complex I analogue from pea thylakoid membranes. Proc Natl Acad Sci USA 95:13191324 Horv~ith EM, Peter SO, Cuin6 S, Guedeney G, Rumeau D, Horv~ith GV, Sch~ifer C, Peltier G, Medgyesy P (1998) Targeted inactivation of the plastidic ndhB gene in tobacco reveals its role in higher-plant photosynthesis, abstr V-2. International Congress of Plant Mitochondria, Aronsborg, Sweden JoEt T, Cerovic Z, Rumeau D, Cournac L, Guedeney G, Horvfith EM, Medgyesy P, Peltier G (1998) Increased sensitivity of photosynthesis to anaerobic conditions induced by targeted inactivation of the chloroplast ndhB gene. In: (Garab G ed) Photosynthesis: mechanism and effects. Kluwer Acad Publ, pp 1967-1970 Medgyesy P (1990) Selection and analysis of cytoplasmic hybrids. In: (Dix PJ ed.) Plant cell line selection, VCH Weinheim, pp.287-316 Kushnir SG, Shlumukov LR, Pogrebnyak NJ, Berger S, Gleba Y (1987) Functional cybrid plants possessing a Nicotiana genome and an Atropa plastome. Mol Gen Genet 209:159-163 Thanh ND, P~ly A, Smith MA, Medgyesy P, M~irton L (1988) Intertribal chloroplast transfer by protoplast fusion between Nicotiana tabacum and Salpiglossis sinuata. Mol Gen Genet 213:186-190 Peter S, Spang O, Medgyesy P, Sch~ifer C (1999) Consequences of intergeneric chloroplast transfers on photosynthesis and sensitivity to high light. Aust. J. Plant Physiol. (in press)

66

Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

Post-Transcriptional Gene Silencing In Plants: Pain Or Delight In Transgene Research? Introduction The introduction of transgenes into plants can either result in transcriptional gene silencing (TGS) [1] or in post-transcriptional silencing (PTGS) [2](Figure 1). Mechanistically, TGS has been linked with extensive DNA methylation in promoter regions resulting in a drastic reduction in the rate of transcription [3]. However, in many cases the rate of transcription did not change in transgenic plants but the steady state level of transcripts was drastically reduced [4,5,6]. It is an accepted view that this reduction is due to enhanced post-transcriptional RNA degradation [7,8,9]. However, the mechanisms acting in the recognition, targeting and decay of a specific type of transcript in a pool of transcripts of a given plant or tissue are still obscure although progress has been made in unrevealing the prerequisites for the induction of PTGS and in defining specific RNA species detectable during the three phases of post-transcriptional gene silencinginitiation, development and maintenance.

Post-transctiptional silencing

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Gene Silencing]

Why is it important to invest into re- IT~lnscripti~162 ? e n e I search with the aim to understand the mechanisms of PTGS? In addition to - transcription rate not reduced the thrilling academic question how - drastic reduction of the - primary transcript made but the post-transcriptional control of transcription rate fast R N A decay and occurrence gene expression is performed in - no primary transcript of aberrant RNAs - coding region methylated plants, PTGS can be used as a pow- - promoter hypermethylated erful tool in functional genomics. Its transient action offers the opportuni1 ties to perform rapid function assess...... ,~ -----~/ ment, to target whole gene families 9 9 9 9 9 9 9 9 9 or single members of families, to choose between complete or incomplete knock-out of genes and to be able to perform conditional knock-outs for studying the developmental and tissue-specific aspects of gene expression and their temporal and spatial distribution patterns. On the other hand the understanding "}

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Michael Metzlaff, Aventis CropScience N.V., Gent, Belgium

67

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Control of genes of the post-transcriptional control of gene expression in plants will provide us with new insights for finding ways to stabilize transgene expression. Which are the prerequisites for the induction of PTGS? In contrast to TGS, PTGS does not need promoter homology to be initiated. Short regions of homology in coding regions of multiple gene copies are sufficient to trigger PTGS. The smallest regions reported up to now which have been sufficient to induce silencing are about 60 nucleotides in size [10]. It has been shown that even promoter-less transgene constructs can induce PTGS but in these cases the transgene locus had an inverted repeat structure and the expression via nearby located promoters has not been excluded. Inverted repeats of transgenes appear to be in general very effective silencers. This can be explained by their ability to form distinctive secondary structures at the DNA and/or RNA level [11]. However, in many reported cases expression of transgenes is needed for PTGS induction. Some authors have shown that elevated levels of a transgene transcript also elevate the frequency of PTGS occurring [ 12]. These findings count towards the existence of a threshold for a given mRNA. Once such a threshold is exceeded the RNA is directed into a decay pathway. How such a threshold may be measured is not understood. In other cases it has been shown that also low levels of specific transcripts can induce PTGS [ 11 ]. Thus, not only quantities but also qualities of RNA species seem to be important for the decision whether the RNA is targeted for degradation or not. For several cases it has also been demonstrated that developmental and environmental regulation of gene expression can influence the frequency of PTGS [13]. Also viruses can induce PTGS which then is called 'virus induced gene silencing' (VIGS) [14]. In a so-called recovery phenomenon, virus infected plants can develop 'immunity' against the virus infection. It has been shown that this virus 'immunity' is maintained post-transcriptionally by degradation of the virus RNA, a striking parallelism to what has been observed for PTGS of endogenous genes and transgenes. By integrating short coding regions into the virus genome which have homology to either nuclear endogenous genes or transgenes, it has been demonstrated that such chimeric viruses are powerful tools for the knock-out of genes. Vice versa, by integrating parts of virus genomes into plant genomes stable virus resistance has been achieved [15]. In summary, the induction of PTGS seems to be coupled on the existence of multiple copies of coding regions with sequence homology, the degree of sequence homology between these copies and the transcription of at least one of these copies. Both forms of gene silencing, PTGS and TGS, can also be defined as 'homology-dependent gene silencing' (HDGS) because in any case the structural basis for the induction of gene silencing seems to be the basepairing between either homologous DNA-DNA or DNA-RNA or RNA-RNA molecules [ 1].

Range of transcripts Which specific RNA species can be found in plant tissue displaying PTGS? As pointed out earlier PTGS is accompanied by a drastic reduction in the steady state level of the mRNA of the silenced genes [6,16,17,18]. Several authors have published the occurrence of aberrant RNAs instead of mature mRNAs in silenced plants (Figure 2). Aberrant RNA can be defined as any RNA carrying structural irregularities either due to gene structure rearrangements or premature termination of transcription or inefficient processing of primary transcripts. Aberrant RNA can also be defined as an unproductive RNA in the sense that it is not directed into 68

Gene silencing translation but rather into an RNA decay pathway [2]. In some of the plants showing PTGS longer-thannormal transcripts have been observed most likely caused by read- ILo,ge~-!han-,o~,aRNAs through transcription or inefficient transcript processing [19]. How- [Vu~lengthRNAs]

Northern RNA Filter]

l--] --]~ _ _ ~ t RNAs

ever, in an increasing number of shorter-than-normalRNAst. cases of PTGS shorter-than-normal (truncated RNAs) I transcripts have been found. Some t a ..... iatingLMWRNAs, of these truncated RNAs can accu- ~sense,antisense,aouble-stra,ae~ m mulate to surprisingly high levels and seem to be protected against [-~ transgenic fast degradation [6,18]. This is most obvious for small RNA fragFigure 2. Specific RNAs found in plant tissue displaying PTGS. In plant tissues displaying PTGS the level of mature full-length ments carrying sequences of the 5' mRNA is drastically reduced. Instead, shorter and longer RNA and 3' ends of the transcripts. Some species can be observed, which are called aberrant RNAs. In of the observed truncated RNAs some cases the accumulation of RNAs with low-molecularbelong to the polyA- RNA fraction weight (LMW RNAs) has been observed. but there is increasing experimental evidence that the RNA fragments are polyadenylated first before they are targeted for further degradation [20]. Also antisense RNAs have been found in silenced tissues [21]. Finally, there is increasing evidence ' "l"tAberrantRNA I for double-stranded RNAs (dsRNA) occurring in silenced tissues. In this case the most convincTranslation-independentRNA degradation: ] ing experimental evidence for the Step 1: endonucleolyticclevages involvement of dsRNA in gene silencing resulted from experiments which have not been performed on Step2: exonucleolyticcleavages plants but on the nematode worm Ceanorhabditis elegans [22]. In this worm the injection of dsRNA Step3"polyadenylationof RNA3' ends was triggering very efficient gene AAA silencing events, dsRNA can either . . . . l, F u r t h e r d e g r a d a t i o n be formed by intermolecular pair- Degradati0n-resistant LMWRNAs: . . .sen.se,. . . .antisense, ... dsRNAI -~.~-- --/ ing between two RNA molecules Figure 3. A model for a specific RNA decay pathway in PTGS. The most recent data point towards the involvement of 'multiprotein machines' in the decay of plant RNA. A model for the decay of RNA in plant tissues displaying PTGS would involve initial cleavages of aberrant RNAs by endonucleases followed by exonucleolytic digestion of the RNA cleavage products towards stabilizing stem-loop structures. The addition of secondary poly(A)-tails at the remaining RNA 3' ends would act as 'toeholds' for further RNA degradation. The PTGS RNA decay pathway is translation-independent.

69

carrying regions of sequence complementarity or by intramolecular pairing within a single selfcomplementary RNA molecule. Taking the experimental data of all the different silencing systems into account it becomes obvious that the occurrence of aberrant RNA struc-

Control of genes

tures coincides not only with the early phase of initiation of PTGS but also with the later maintenance phase.

Aberant RNA's: result or cause? Are the aberrant RNAs result or cause of PTGS and how are the produced? The experimental data, which are available today, seem to show that the aberrant RNAs can be both, result and cause. This conclusion follows a model for an autoregulatory and cyclic modus of gene silencing [23]. In this model an effector molecule produces in multiple autocatalytic cycles more effector molecules in a rate which is higher than its own turnover. As pointed out earlier some of the observed small aberrant RNAs can accumulate to high levels and resist fast degradation. If these small RNAs are in antisense orientation it can be easily envisaged that they can base-pair with sense-oriented mRNA and thereby form double-stranded RNA regions. In consequence of this either the processing of primary transcripts in the nucleus is inefficient or dsRNA-specific endoribonucleases recognize these aberrant RNA structures and cleave them within the nucleus and/or in the cytoplasm. Also small sense RNAs carrying regions of complementarity with sense mRNAs could act in a similar way like small antisense RNAs [24,6]. There are several (theoretical!) possibilities for the origin of aberrant RNAs. One possibility is that they result directly from aberrant transgene structures. Transgenes integrated into plant genomes are artificial structures because they carry non-gene-specific 5' and 3' regions and are normally intronless. These artificial structures may cause premature termination of transcription or inefficient processing of primary transcripts. Because transgenes integrate randomly into the plant genome they may be affected by position effects that interfere with the transcription process. A second possibility could be that aberrant RNAs result from 'read-through-transcription'. Larger- than-normal transcripts have been observed in several PTGS systems. Especially in the case of transgene loci with an inverted repeat structure a read-through in transcription would produce primary transcripts capable of forming duplex structures. Thus, dsRNA structures, which are known to trigger PTGS, would be formed intramolecularly in a single transcript. It is known that the 3' ends of mRNAs can form extensive secondary structures. Inefficient 3' end processing of read-through transcripts may result in unusual stem-loop structures, which are a target for enzymatic activity, like endonucleolytic cleavages by endoribonuclease activity or synthesis of antisense RNA by RNA-dependent RNA polymerase (RdRP) activity [25]. The involvement of a protein with homology to RdRP in PTGS has been shown recently for Neurospora crassa [26]. However, most of the experimental data point towards the release of aberrant RNA from specific RNA degradation pathways (Figure 3). These degradation pathways seem to be translation-independent and to differ from the known translation-dependent RNA decay pathways [8,9]. Experimental evidence has been accumulated for several plant systems that the degradation of transcripts in silenced tissue starts with endonucleolytic cleavage by specific endoribonucleases, e.g. RNaseElike enzymes, followed by exonuclease activities [6,16]. RNA helicases and RNA-binding proteins interfering with RNA secondary structures are assumed to be involved, too. For some of the studied transcripts it has been shown that the splicing process is delayed and that unspliced pre-mRNA can accumulate [ 19,27]. This points towards the involvement of very early mechanisms of transcript modifications taking place in the nucleus of silenced cells. It has also been found that many 3' truncated RNAs carry a polyA-tail [6,8]. The sites of polyadenylation seem to be scattered all over the transcript. This is in parallel with findings for degradosomes 70

Gene silencing identified in E.coli, in plant chloroplasts and mitochondria and in yeast that polyadenylation can accelerate RNA degradation by giving a 'toehold' to exonucleases [20]. If such a complex degradosome is involved in PTGS remains to be investigated. However, some of the smallest intermediate degradation products may survive further degradation and may act as effector molecules as discussed earlier. They may act as primer for antisense synthesis by RdRP activity (Figure 4). In summary, dsRNA structures are very likely to be the stable intermediate effector molecules in an autocatalytic RNA degradation cycle. In this scenario, in a phase of iniIntramolecular } tiation of PTGS aberrant RNAs would be the result either of aberrant gene structures or inefficient processing of primary transcripts or high transcript levels. These processes are not exclusive and can proceed in parallel. In the case of FL--q stable integrated transgenes these processes may start very early in y gene expression already in the -- -LMW RNAs with sequence complementarity nucleus. However, also the cytoplasm can be the site of initiation if transcripts are excluded from translation and accumulate to high Figure 4. Possible origin of antisense and dsRNAs. There is levels or in the case of incoming experimental evidence from several PTGS systems for the inviruses. In this case small RNA volvement of antisense and double-stranded RNA. Doubledegradation products produced in stranded RNA can either be formed intramolecularly (A) by hairpin formation at a single RNA molecule or intermolecuthe cytoplasm may feed-back into larly (B) by interaction of two (partially) complementary molthe nucleus and interfere there with ecules. In both cases the regions of double-stranded RNA may nuclear processes of gene expresbe extended by the activity of RNA-dependent RNA polymerase sion. They may also feed-back to (+RdRP). the DNA templates and trigger denovo methylation in coding regions. The involvement of an RNA-mediated de-novo methylation in gene silencing has been suggested for viroid silencing and for some cases of PTGS in plants [28,29,30]. In the phase of development of PTGS the level of aberrant RNAs increases constantly and the aberrant RNAs become the cause for the production of more aberrant RNAs via an autoregulatory process discussed earlier. In the phase of maintenance of PTGS the autoregulatory process has switched into a self-maintaining cycle and does not need any longer the input of aberrant RNAs, e.g. even if the integrated transgene would not be expressed any longer the PTGS status would be maintained. Gene expression in plants is highly dependent on developmental and environmental factors. Therefore all internal and external factors inducing or reducing the level of gene expression have to be regarded as factors influencing the development of PTGS in a spatial and temporal mode.

rIntormoleco'ar r

Is there a systemic silencing signal involved in the induction of PTGS? Experiments in which scions of non-silenced transgenic tobacco plants have been grafted onto silenced transgenic tobacco stocks have revealed that the non-silenced scion can be silenced within days [31]. 71

Control of genes This suggests that from silenced cells signal molecules are released which trigger the induction of gene silencing in cells receiving the signal. The receiving cells may then be a secondary source for the signal. This phenomenon is called systemic acquired silencing (SAS). Because of its sequence-specificity the signal is likely to be RNA or DNA which can travel either short distances from cell to cell through plasmodesmata or long distances through the phloem. Also the local delivery of DNA into plants by leaf infiltration or biolistical bombardment results in systemic PTGS supporting the view that a systemic signal exists [32,33,34]. However, the signal has not been isolated yet.

Concluding remarks Only very recently it has become clear that the post-transcriptional events of pre-mRNA processing and RNA decay are important and sensitive control levels in plant gene expression. Plants have to react fast and efficiently to a large number of developmental and environmental factors. Post-transcriptional RNA processing and decay may be the level of tuning gene expression accordingly. The RNA level may also be the level of intra- and intercellular signaling needed for the spatial and temporal synchronization of plant gene expression patterns [35]. Gene silencing research was able to identify some of the involved candidate molecules because the introduction of foreign genes into plant genomes has disturbed the balance in gene expression patterns, which resulted in detectable levels of specific RNA molecules. The infection of plants with RNA viruses may induce the same control mechanisms at the RNA level. The first mutants defective in PTGS, isolated from Neurospora (qde) [36] and Arabidopsis (sgs) [37], will provide us with more information about structural proteins and enzymes involved in PTGS. Finally, the stabilization of transgene expression and targeted knock-out of individual allels of genes, members of gene families or whole gene families are extremely potent applications of PTGS and VIGS in transgene technology and functional genomics.

Acknowledgement I thank Dick Flavell, now at Ceres Inc. Malibu USA, for making me thinking about and working on gene silencing at the John Innes Centre Norwich UK during the last six years. It was an exciting and demanding time I spent with him, Mike O'Dell and Roger Hellens in our small Petunia group. I also thank all members of the EU program 'Control of gene expression and silencing in transgenic plants' for all the stimulating discussions we had during our 'nonsilent' silencing meetings.

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4. 5.

E Meyer, H. Saedler. Homology-dependent gene silencing in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 23-48, 1996. A. Depicker, M. Van Montagu. Post-transcriptional gene silencing in plants. Curr. Opin. Cell Biol. 9, 373382, 1997. Y.-D. Park, I. Papp, E.A. Moscone, V.A. Iglesias, H. Vaucheret, A.J. Matzke, M.A. Matzke. Gene silencing mediated by promoter homology occurs at the level of transcription and results in meiotically heritable alterations in methylation and gene activity. Plant J. 9, 183-194, 1996. C. Napoli, C. Lemieux, R. Jorgensen. Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2, 279-289, 1990. A.R. van der Krol, L.A. Mur, M. Beld, J.N.M. Mol, A.R. Stuitje. Flavonoid genes in petunia: Addition of a

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limited number of gene copies may lead to suppression of gene expression. Plant Cell 2, 291-299, 1990. M. Metzlaff, M. O'Dell, ED. Cluster, R.B. Flavell. RNA-mediated RNA degradation and chalcone synthase A silencing in petunia. Cell 88, 845-854, 1997. R. van Blokland, N. van der Geest, J.N.M. Mol, J.M. Kooter. Transgene-mediated suppression of chalcone synthase expression in Petunia hybrida results from an increase in RNA turnover. Plant J. 6, 861-877, 1994. J.J.M.R. Jacobs, K. Liti6re, V. van Dijk, G.J. van Eldik, M. Van Montagu, M. Cornelissen. Post-transcriptional beta-l,3-glucanase gene silencing involves increased transcript turnover that is translation-independent. Plant J. 12, 885-893, 1997. H. Holtorf, H. Schoeb, C. Kunz, R. Waldvogel, E Meins Jr. Stochastic and nonstochastic post-transcriptional silencing of chitinase and beta-l,3-glucanase genes involved increased RNA turnover - Possible role for ribosome-independent RNA degradation. Plant Cell 11,471-483, 1999. T. Sijen, J. Welling, J.-B. Hiriart, A. van Kammen. RNA-mediated virus resistance: role of repeated transgenes and delineation of targeted regions. Plant Cell 8, 2227-2294, 1996. M. Stam, R. De Bruin, S. Kenter, R.A.L. van der Hoorn, R. van Blokland, J.N.M. Mol, J.M. Kooter. Posttranscriptional silencing of chalcone synthase in Petunia by inverted transgene repeats. Plant J. 12, 63-82, 1997. Q. Que, H.-Y. Wang, J.J. English, R.A. Jorgensen. The frequency and degree of cosuppression by sense chalcone synthase transgenes are dependent on transgene promoter strength and are reduced by premature nonsense codons in the transgene coding sequence. Plant Cell 9, 1357-1368, 1997. R.A. Jorgensen. Cosuppression, flower color patterns, and metastable gene expression states. Science 268, 686-691, 1995. D.C. Baulcombe. Mechanisms of pathogen-derived resistance to viruses in transgenic plants. Plant Cell 8, 1833-1844, 1996. D.C. Baulcombe. Fast forward genetics based on virus-induced gene silencing. Curr. Opin. Plant Biol. 2, 109-113, 1999. M.M. Tanzer, W.E Thompson, M.D. Law, E.A. Wernsman, S. Uknes. Characterization of post-transcriptional suppressed transgene expression that confers resistance to tobacco etch virus infection in tobacco. Plant Cell 9, 1411-1423. K.Y. Lee, C. Baden, W.J. Howie, J. Bedbrook, E Dunsmuir. Post-transcriptional gene silencing of ACC synthase in tomato results from cytoplasmic RNA degradation. Plant J. 12, 1127-1137, 1997. G.J. van Eldik, K. Liti6re, J.J.M.R. Jacobs, M. Van Montagu, M. Cornelissen. Silencing of beta-l,3glucanase genes in tobacco correlates with an increased abundance of RNA degradation intermediates. Nucl. Acids Res. 26, 5176-5181, 1998. R. van Blokland, N. van der Geest, E De Lange, M. Stam, J.N.M. Mol, J.M. Kooter. Post-transcriptional suppression of chalcone synthase genes in Petunia hybrida and the accumulation of unspliced pre-mRNAs, in: D. Grierson, G.W. Lycett, G.A. Tucker, (Eds.), Nottingham University Easter School, Nottingham University Press, 1996, pp. 57-69. A.J. Carpousis, N.E Vanzo, L.C. Raynal. mRNA degradation - a tale of poly(A) and multiprotein machines. Trends in Genet. 15, 24-28, 1999. D. Grierson, R.G. Fray, A.J. Hamilton, C.J.S. Smith, C.E Watson. Does co-suppression of sense genes in transgenic plants involve antisense RNA? Trends Biotechnol. 9, 122-123, 1991. A. Fire, S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, C.C. Mello. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811, 1998. E Meins Jr., C. Kunz. Gene silencing in transgenic plants: A heuristic autoregulation model. Curr. Top. Microbiol. Immunol. 197, 105-120, 1995. EH. Cameron, EA. Jennings. Inhibition of gene expression by a short sense fragment. Nucl. Acids Res. 19, 469-474, 1991. W. Schiebel, T. Pelissier, L. Riedel, S. Thalmeir, R. Schiebel, D. Kempe, E Lottspeich, H.L. Sanger, M. Wassenegger. Isolation of an RNA-directed RNA polymerase-specific cDNA clone from tomato. Plant Cell 10, 1-16, 1998. C. Cogoni, G. Macino. Gene silencing in Neurospora crassa requires a protein homologous to RNAdependent RNA polymerase. Nature 399, 166-169, 1999. K.K. Mishra, A.K. Handa. Post-transcriptional silencing of pectin methylesterase gene in transgenic tomato fruits results from impaired pre-mRNA processing. Plant J. 14, 583-592, 1998. M. Wassenegger, S. Heimes, L. Riedel, H.L. Sanger. RNA-directed de novo methylation of genomic sequences in plants. Cell 76, 567-576, 1994. M. Wassenegger, T. Pelissier. A model for RNA-mediated gene silencing in higher plants. Plant Mol. Biol.

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Control of genes 37, 349-362, 1998. 30. M. O'Dell, M. Metzlaff, R.B. Flavell. Post-transcriptional gene silencing of chalcone synthase in transgenic petunias, cytosine methylation and epigenetic variation. Plant J. 18, 33-42, 1999. 31. J.-C. Palauqui, T. Elmayan, J.-M. Pollien, H. Vaucheret. Systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions. EMBO J. 16, 4738-4745, 1997. 32. O. Voinnet, D.C. Baulcombe. Systemic signaling in gene silencing. Nature 389, 553, 1997. 33. O. Voinnet, E Vain, S. Angell, D.C. Baulcombe. Systemic spread of sequence-specific transgene RNA degradation is initiated by localized introduction of ectopic promoterless DNA. Cell 95, 177-187, 1998. 34. J.-C. Palauqui, S. Balzergue. Activation of systemic acquired silencing by localized introduction of DNA. Curr. Biol. 9, 59-66, 1999. 35. R.A. Jorgensen, R.G. Atkinson, R.L.S. Forster, W.J. Lucas. An RNA-based information superhighway in plants. Science 279, 1486-1487, 1998. 36. C. Cogoni and G. Macino. Isolation of quelling-defective (qde) mutants impaired in posttranscriptional transgene-induced gene silencing in Neurospora crassa. Proc. Natl. Acad. Sci. USA 94, 10233-10238, 1997. 37. T. Elmayan, S. Balzergue, E Beon, V. Boudon, J. Daubremet, Y. Guenet, E Mourrain, J.C. Palauqui, S. Vernhettes, T. Vialle, K. Wostrikoff, H. Vaucheret. Arabidopsis mutants impaired in cosuppression. Plant Cell 10, 1747-1757, 1998.

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

Comments

f r o m the S e s s i o n R a p p o r t e u r

Public understanding of science - an experiment The European Plant B iotechnology Network (EPBN), organiser of the Phytosphere'99 conference 1999 in Rome, started an experiment. The EPBN staff mixed European plant biotechnology scientists with a handful of science journalists in the presence of some EU-officials and representatives of biotech-industry. In excess of audience this mixture incubated for three days. By inviting science writers from several European countries the EPBN officials tried to improve the media coverage on plant biotechnology in general and during this conference in particular. The main reason for this is that European plant biotechnologists are under hard pressure. Many Europeans compare their work with the development of "Frankenstein food". Not only scientists wonder why Europeans consumers seem to think that food made from transgenic plants is of no benefit to them, while American consumers seem to have readily accepted the development. During the whole meeting I did not hear satisfying explanations for that phenomenon, although it predominated many discussions - not only during panel debates but also during coffee breaks. The European attitude may be the result of a combination of being more conservative in lifestyle (food is a very traditional topic) and being more sceptical about "experts " (such as scientists). Occasionally scientists complained that journalists do not write "positive green science stories". I really want to make the point that this is not t r u e - and this has been shown by a study of the University Hohenheim. However it must be noted that, mostly well written, stories about vitamin A rice or salt-resistant plants are placed on the science pages in the middle of the newspapers whereas spectacular actions of Greenpeace or "shocking" cloning stories appear on the front page and consequently gain more attention of the reader. Often for newspapers bad news is good news - politicians have to live with that fact of life and so do scientists. During the conference the journalists were asked to attend and summarise specific sessions. However, journalists are not scientists and therefore it was not easy to critically follow the often complicated presentations. In order to bring this task to a good end we had intensive discussions with the speakers. Fortunately the organisers had planned enough breaks and the

Karin Hollricher, Science journalist, Neu-Ulm, Germany

75

Control of genes speakers were open for even the most naive questions, which I greatly appreciated and therefore would encourage on other occasions. Many of the presentations were essentially overviews on objectives and results of complete research networks. It was therefore very difficult to find "breaking news" that could make a story on the science pages of German newspapers. Certainly there have been a lot of "milestones" but all had been published in scientific journals and all had made their way through the German press before Phytosfere'99 took place. It would therefore be of great help if journalists could get detailed press information about the highlights of such a conference beforehand. On the other hand, for me personally, the talks were an investment into future. I left the conference with the satisfactory feeling that I roughly know what is going on in European plant biotechnology labs and how European science funding works. Together with a large collection of names, faces and phone numbers this is a good basis for future writing. The personal and easy contacts to the EPBN staff have already been advantageous, i.e. for my investigations about the objectives and development of Framework V. Therefore I think the experiment has succeeded and it will pay off in the future for the public understanding of science.

76

Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

The Cluster: "Gene Location Mapping" Why mapping genes? Mapping gene location on the chromosome is a key action in modern plant breeding and in plant molecular biology. Genes are identified either by a mutation which gives a specific phenotype to the mutant or by a molecular marker. The plant breeder is usually interested in selecting a few specific genes which confer favourable properties to his crop. These favourable properties can be selected among the progenies of various crosses on the basis of the phenotype but this is space and time consuming and often quite costly. The expression of the phenotype might be dependent on the developmental stage, on climatic conditions, on the presence of a given pathogen or other environmental conditions. In addition it is difficult to cumulate and combine different advantageous characters within the same line. With the availability of molecular maps, it has become possible to associate phenotypic characters with molecular markers and to select plants using the appropriate molecular markers at an early stage. This process is known as marker assisted selection. From the point of view of the molecular biologist, gene mapping is also an essential tool to isolate genes for which the final protein product is not available: the gene again is identified by a mutation which, in a first step, is flanked by molecular markers. The closest ones are then used to initiate a chromosome walk and search new markers until no recombination event is observed between the mutation and the markers. Therefore, mapping is essential both for breeding and for gene discovery.

An overview of the projects in the cluster The five projects which have been grouped in this cluster are all aiming at developing gene mapping and map-based gene discovery in cultivated crops. The five programmes are the followings: - BIO4-CT96-0443: Tomato Genome Project, molecular and physical dissection of the tomato genome, TAG-A-MAP, co-ordinated by Mark Van Haaren (Wageningen). - BIO4-CT97-2125: Comparative genome mapping in conifers, ANACONGEN, coordinated by David Marshall (Dundee).

Michel Delseny, Universit6 de Perpignan, Laboratoire de Physiologie et Biologie Mol6culaire des Plantes, UMR 5545 CNRS, 66860 Perpignan, France.

77

Mapping Gene Location - BIO4-CT97-2170: Comparative genome analysis in dicotyledonous crop species and Arabidopsis, EuDicotMap, co-ordinated by Michel Delseny (Perpignan). -BIO4-CT97-2220: The European comparative gramineae mapping programme, EGRAM, co-ordinated by Steve Quarrie (Norwich). - BIO4-CT97-2312: Map-based cloning of agronomically important genes directly from Zea mays, co-ordinated by Keith Edwards (Bristol). Two reports by Christiane Gebhardt (EuDicotMap) and Donal O'Sullivan (Maize map) are presented in these proceedings and illustrate some of the major results in these projects. In this short paper, the activities in the cluster and in the EuDicotMap project are briefly reviewed. The various target crops in these five projects are at different stages of development. Very little is known at the moment on the conifer genetics and the goals of the ANACONGEN project were to set up a few tools for accelerating mapping of these complex genomes, which are in average 8 times bigger than the human and 250 times larger than the Arabidopsis genomes. These tools are cytogenetic localisation of major repetitive sequences versus coding sequences, development of a set of microsatellite markers, development of ESTs, and construction of a second generation genetic map based on PCR markers. The proposed application in this project was the location of QTL for wood quality. On the opposite, a lot more information and tools are already available in maize and tomato and the two projects could address more specific targets. In tomato the idea was to create a collection of 500 tomato transgenic lines, each containing a construction with a transposon and a few additional elements (tool box) which would allow one to extensively mutagenize and rearrange regions of interest for gene function identification. This project has been highly successful because the lines have been constructed, most of them are mapped on the tomato genome and a few regions of interest are already under intensive study, namely those containing resistance genes In maize, the objective was to construct a BAC library with large insertions in order to facilitate gene cloning by direct chromosome walking. Meanwhile the partners were interested in isolating candidate genes for digestibility and sileage quality and they have targeted an early maturity gene, a gametophytic male fertility gene and a fungal resistance gene for chromosomal walking. A major characteristic of this project is the high number of private partners and the involvement of a biotech company for high throughput AFLP mapping. Most of the known genes involved in lignin biosynthesis in other species have now been cloned and characterised in maize and a major gene resistance cluster, the Rpl locus, with complex organisation, is being characterised. In the EGRAM project, the idea was to exploit the already well established synteny conservation between cereal genomes, to create a common resource of anchor molecular markers and a reference rice mapping population. This would allow one to use rice as a resource to map and clone genes in the other cereals with much larger genomes (wheat is about 40 times larger than rice genome). The second part of the project aimed at developing this type of application with attempts to clone and characterise genes for disease resistance (rusts) in wheat, barley and ryegrass. 78

The gene location mapping cluster

The EuDicotMap project Finally in the EuDicotMap project the goal was to investigate possible synteny conservation between the model genome Arabidopsis and major dicotyledonous crops. In this programme we initially identified 300 genes, the nucleotide sequence of which is conserved, based on extensive comparison between Arabidopsis and rice ESTs. All these sequences have been mapped on the Arabidopsis genome and this activity is now being considerably accelerated by the availability of more than 60% of the genomic sequence. These conserved sequences were distributed to the different partners for mapping in the different crops of interest: Brassica napus and oleracea, pea, alfalfa, potato, sugar beet, sunflower, almond and peach trees. Out of the 200 initially distributed probes more than a 100 homologous genes could already be mapped in the other crops. Two difficulties were met in this approach: the first one is that each Arabidopsis probe usually detects several loci in the cultivated crops and the second is that a number of probes give poor signals with crop DNA. Alternatives have been developed, namely for potato, sugar beet and sunflower. In these crops, already mapped, but anonymous, cDNA are being partially sequenced as EST and compared to other sequences in databases. Using this strategy considerably facilitates comparison between crops and identification of homologues. We also systematically mapped in Arabidopsis a number of members of multigene families. This approach is now revealing several large duplications in this genome, which presumably arose by reciprocal translocations. Such duplications are important to identify before using comparative mapping as a tool to search homologous genes in other species. Preliminary comparisons between maps of different genomes with common markers identified a few chromosomal regions in which synteny seems to be conserved. In the future, the overall analysis will be completed by mapping additional common markers and we shall attempt to establish more precise limits for syntenic regions. Meanwhile several partners are comparing, on a fine scale, regions which have been sequenced in Arabidopsis with their homologues in Brassica oleracea and Capsella bursa-pastoris .Two types of situations were observed: regions which are very well conserved, with same gene order, with sometimes amplification of a given gene in one of the compared species, and with comparable size of intergenic regions. Several cases in which the intergenic regions are expanded and where genes are rearranged have also been observed. Altogether, these results suggest an extensive conservation of synteny within botanical families, but a very limited one when moving from one family to another .At the extreme we did not detect any synteny between rice and Arabidopsis except possibly in a very short region. Nevertheless, this programme turns out to be very useful in identifying in crops a number of genes already known in model genomes and in revealing some key features of the evolution of the plant genomes.

Cluster activities The advantage of having the various programmes organised into a cluster is that partners from the different projects can meet each other, communicate and generated added value to their research. Examples of interactions are exchange of probes information between

79

Mapping Gene Location EuDicotMap and EGRAM and creation of a common database. A joint meeting of the two projects has been organised by the end of February 1999 in Montpellier, to which the coordinators of the three other programmes in the cluster were invited to present their project and their non confidential results. A meeting of the co-ordinators also took place in September 1998 and we try to keep in touch and be informed of the progress of our programmes. We plan to have a general cluster meeting at the end of the year 2000 to highlight our main results.

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

Function Maps Of Potato

Introduction In 1831, it was the mission of the HMS Beagle, joined by Mr. Charles Darwin as the captains accompanying person, to chart South Americas coast lines in order to explore new trade routes and ports for the British empire (1). The utilitarian purpose of this voyage is today largely forgotten. Now it is remembered as having provided Darwin with the experience for developing his revolutionary concept on the origin of species. In our days, 170 years later, the era of charting unknown continents has passed. Instead, we chart the genomes of humans, animals and plants in order to identify locations of genes which control, for example, disease or, in the case of crop plants, agronomic performance. Positional information on genes of known phenotypic effect but unknown molecular identity can be used to identify and manipulate them at the molecular level. Besides the utilitarian purpose of finding better treatments for inherited diseases or producing better crop plants, we may make discoveries which will change our understanding of nature in a similar way as Darwins theory of evolution. Whereas positions on geographical maps are determined by longitude and latitude, positions on genome maps are determined by a framework of DNA markers which are positioned relative to each other based on genetic linkage (molecular linkage maps). More precise than linkage maps are physical maps consisting of cloned genomic fragments which are ordered in the same linear order as they occur on the chromosomes of the organism considered. The ultimate precision of a genome map is reached when the DNA sequence of the whole organism has been determined. This stage has been achieved for the genomes of yeast, Caenorabditis elegans and a growing number of bacterial genomes and is well underway for the human genome and model species like Arabidopsis thaliana (see, for example http://www.tigr.org) Due to the financial resources required for constructing a complete physical map and for sequencing a whole genome, most genome maps of today and particularly those of most crop plants are molecular linkage maps. A variety of DNA markers has been used for constructing molecular linkage maps for crop plants. The most common ones are RFLP (Restriction Fragment Length Polymorphism), RAPD (Random Amplified Polymorphic DNA), AFLP TM (Amplified Fragment Length Polymorphism) and SSR (Simple Sequence Repeat) markers. The common molecular basis of all

Christiane Gebhardt, Max-Planck Institut fiir Ztichtungsforschung, K61n, Germany

81

Mapping Gene Location marker assays are point mutations and/or insertion/deletion mutations which have accumulated during evolution in the DNA of the individuals of a species without having a detrimental effect on their ability to survive and to propagate. For providing a molecular framework, it is of no relevance whether the DNA markers originate from functional genes or from intergenic regions. Most molecular maps, particularly those based on RAPD, AFLP or SSR markers, are anonymous with respect to function. DNA polymorphisms are, on the other hand, also the molecular basis of the genetic component of phenotypic variability. Ideal for diagnostic purposes are, therefore, DNA markers based on molecular variants of those gene(s) which control the phenotype we are interested in, as in this case absolute linkage is observed between a specific marker allele and a phenotype. For example, sickle cell anemia is diagnosed most precisely by a marker specific for the mutant amino acid causing the defective haemoglobin. In the case of agronomic characters of crop plants the identity of the genes responsible for the observed phenotypic variability is not known. More is known on the metabolic pathways which are relevant at least for some of those characters. For example, we do not know the identity of the genes which control the variability of the tuber starch content in the field. We do know, however, many of the enzymatic steps and the genes required for starch biosynthesis and degradation. Moreover, the basic gene repertoire of plants is being catalogued and will be available within few years in databases. The observation of tight linkage between unknown genetic factors controlling an agronomic character and known genes functionally relevant for the same character provide candidate genes that can be used for diagnostic purposes. Moreover, candidate gene alleles can be isolated and tested for effect on agronomic performance. Function mapping consists of two activities, therefore: - Genetic factors controlling agronomic characters are positioned in the genome relative to a framework of Mendelian DNA markers and - DNA fragments encoding functional proteins or showing significant similarity to such proteins are used for constructing molecular function maps. Besides being used for the identification of candidate genes for agronomic characters, molecular function maps can be also used for comparative genome analysis based on DNA sequence similarity between homologous genes in different species.

Function m a p of potato for pathogen resistance As a vegetatively propagated crop, potato cultivation is particularly vulnerable to pathogen attack because infections can be transferred to the next growing season via tubers. Selecting cultivars with field resistance to major pests is an important goal of potato breeding. Since the beginning of this century, genes for resistance have been introgressed into cultivated germplasm from several wild and cultivated South American potato species by sexual hybridisation (2). Only since the last ten years, DNA markers made possible the localisation of a number of these resistance factors on the molecular map of potato. The cyst-forming nematode species Globodera rostochiensis and Globodera pallida parasitize the roots of potato causing considerable yield reduction. Single dominant genes and major QTLs (quantitative trait loci) for resistance to G. rostochiensis or G. pallida have been 82

Function maps of potato mapped to potato chromosomes V, VII and XII (3, 4, 5, 6, 7, 8, 9). A gene for resistance to another nematode species, Meloidogyne chitwoodi, a root knot nematode, was localised on potato chromosome XI (10). Potato viruses are transmitted by aphids to the leaves, infect the tubers and are, therefore, a threat for seed tuber production. Single dominant genes conferring either an extreme or a hypersensitive type of resistance to potato virus X I II III IV (PVX), potato virus Y (PVY) rbcS- l-stC[9(c) rG3~ Stl.2.1(b) GPt8 1 R G L ( c ) or potato virus A (PVA) have StCfg(b) StCJ')(a) been mapped to potato chrorbcS-c~ Sti,2.3 TG24-- GIuA St3.3.13(d) GP22 ,&l.Z4(e) CP6~ mosomes V, IX, XI and XII (11, CPt 08-- Stl.2.4(a) Ght.~ GP26I | Pi~ l 12, 13, 14, 15, 16). ce.| TG20(b)'-

Late blight caused by the o o m y c e t e Phytophthora infestans is the most important fungal disease in potato cultivation world wide. When not controlled, late blight can lead to complete loss of the crop yield as it was the case in Ireland of the 19th century causing the Irish famine. Resistance is controlled by single dominant factors (R genes) expressing hypersensitive resistance and by QTLs. Factors for both types of resistance have been located on the potato molecular map, R genes on chromosomes IV, V and XI (17, 18, 19, 20) and QTLs on ten of the twelve chromosomes (21, 22, 23).

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Figure 1: Function map of potato for pathogen resistance. Twelve linkage groups corresponding to the chromosomes of potato are shown schematically with few anchor RFLP markers on the left. GP and CP markers correspond to potato genomic and cDNA clones, respectively. TG markers correspond to tomato genomic clones and have been provided by S.D. Tanksley, Cornell University, USA. Map segments having genes controlling quantitative resistance to late blight are shown as green rectangles. R1, R2, R3, R6 and R7 are genes conferring race specific resistance to late blight (Phytophthora infestans). Rxl, Rx2, Nb and Nx are genes for resistance to potato virus X. Ry and Ra are genes for resistance to potato virus Y and A, respectively. Grol, GroV1, Grpl, H1, Gpa, Gpa2 and Mci are nematode resistance genes. Sen1 is a gene for resistance to potato wart (Synchytrium endobioticum). Marker loci functionally related to pathogen resistance are shown on the right of the linkage groups. St loci contain genes with sequence similarity to known plant resistance genes. Nt loci were detected with pathogenesis related genes of tobacco. The remaining loci were detected with defense related genes of potato. Small letters in parenthesis indicate that more than one locus is detected with the same marker probe.

83

Mapping Gene Location Potato wart, caused by the fungus Synchytrium endobioticum, destroys the tubers and is a quarantine disease as it cannot be effectively controlled by chemicals. A single dominant gene for resistance to wart has been identified on potato chromosome XI (24). All mapping studies have been carried out in different genetic backgrounds using different sets of DNA markers, in most cases RFLP markers of potato and/or tomato. The information on the positions of resistance loci in the potato genome can be integrated, however, into a function map (Figure 1) based on linkage of genes for resistance to anchor RFLP markers of known position on the available molecular maps of potato (25, 26, 27, 28, 29) that were used in different mapping experiments. It is apparent from the function map that some regions of the potato genome are ,,hotspots" for resistance, containing closely linked genes for resistance to different types of pathogens. Most notably in this respect is one region on chromosome V, containing genes for resistance to late blight, the nematode G. pallida and the PVX virus, both distal map segments of chromosome XI and one distal map segment of chromosome XII. Genetic factors controlling quantitative resistance (QTLs) have not been mapped with the same precision as major genes. Nevertheless, QTL for resistance to late blight have also been found to be linked to hotspots for major gene resistance (Figure 1).

Molecular function map of potato for pathogen resistance Host-pathogen interactions have been studied in plants for decades by geneticists, physiologists, biochemists and molecular biologists. Based on such studies, many genes have been identified, characterised and, more recently, molecularly cloned which have been shown to function in or to be correlated with plant defense reactions. These genes are (i) resistance genes which recognise the pathogen and trigger the resistance response (recent review in 30), (ii) genes which are involved in the signal transduction pathway(s) downstream from the trigger and which are just started to be unravelled at the molecular level and (iii) the large group of PR (Pathogenesis Related) genes which are expressed in response to pathogen attack (reviewed by 31). It is now well established that many plant genes for resistance to different types of pathogens are members of a superfamily of genes sharing structural domains like a nucleotide binding site (NBS) domain and a leucine rich repeat (LRR) domain (30). DNA sequence motifs conserved among functional resistance genes of tomato, tobacco and Arabidopsis were used to obtain by a PCR-based approach potato gene fragments with high similarity to known plant resistance genes. These DNA fragments were mapped like conventional RFLP markers to potato RFLP maps. Most RGL (Resistance Gene Like) markers detected gene families located on ten of the twelve potato chromosomes, among others families corresponding to the tomato resistance genes Pto and Cf9 (Figure 1, 32, Zimnoch-Guzowska et al., submitted). Different RGL loci were tightly linked to the nematode resistance gene Grol on potato chromosome VII and to resistance hot spots on chromosomes XI and XII suggesting that at least some potato genes for resistance are members of the NBS-LRR superfamily. When anchor RFLP markers are used in related species such as potato, tomato and tobacco, a comparison of the genomes across species borders is possible. Such a comparison with mark84

Function maps of potato ers tagging both distal segments of potato/tomato linkage group XI reveals that, in the tobacco genome, the position of the N gene for resistance to tobacco mosaic virus (TMV) is syntenic with the resistance gene cluster ,,on top" of potato linkage group XI including Ry, Ra, Senl and Mci (Fig. 2). The cluster of potato genes for resistance to late blight ,,at the bottom" of linkage group XI is syntenic with the tomato 12 gene for resistance to the fungus Fusarium oxysporum (Fig. 2). Both N and 12 genes have been cloned and shown to be members of the NBS-LRR superfamily (33, 34, 35). The first potato gene for resistance which has been characterized at the molecular level is located in the resistance hotspot on linkage group XII, encodes resistance to PVX and is also a member of the NBS-LRR superfamily (36). The structure of cloned resistance genes of tobacco, tomato and potato corroborate the significance of the observation that RGL markers tightly linked with resistance loci are indeed candidates for the resistance genes (24, 32).

XI CP58

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stl.Z l
R3,R6 Figure 2: Hotspots for resistance on potato c h r o m o s o m e XI. The syntenic tobacco N gene for resistance to tobacco mosaic virus and the tomato 12 gene for resistance to Fusarium oxysporum are shown on the left. Further explanations as in figure 1.

Candidates for participating in the control of quantitative resistance to pathogens are PR genes. Cloned PR genes of potato and tobacco have been used as conventional RFLP marker probes for mapping (28). The majority of PR loci identified so far does not correlate well with positions of QTLs for late blight. Exceptions are the 4CL locus (linkage group III) detected by a cDNA encoding 4coumarate:CoA ligase and prpl and PAL loci (linkage group IX) detected by marker probes for a pathogenesis related glutathione-S-transferase and phenylalanine ammonia-lyase, respectively (Figure 1). No PR locus identified so far is linked to one of the hot spots for resistance in the potato genome. It must be kept in mind, however, that the information on number and position of resistance loci as well as of candidate gene loci is incomplete.

Conclusion Mapping of single genes and QTL for pathogen resistance on one hand and of cloned genes functionally correlated with plant defense on the other reveals that the potato genome contains clusters of genes for resistance to various pathogens (hotspots for resistance) which are closely linked to RGL loci. This suggests that members of the NBS-LRR gene superfamily are candidates for controlling qualitative and quantitative resistance in potato.

85

Mapping Gene Location

Function map of potato for tuber traits Besides disease resistance, characters affecting the tuber are considered most important in potato genetics and breeding. These characters are tuber yield, dry matter, content and quality of starch and protein, cooking and chipping quality, tuber shape, eye depth, flesh and skin colour, taste, glycoalkaloide content, tuberization and tuber dormancy. Most of those traits show quantitative variation determined by environmental and genetic factors. Using DNA markers, genetic factors controlling tuber traits have been identified and positioned on the potato molecular map. Major loci for tuber flesh colour and tuber skin colour have been mapped to potato chromosomes III and X, respectively (25, 26, 27). Also on chromosome X, a major QTL for tuber shape has been located (37). QTL for tuberization, tuber dormancy, chip colour and specific gravity have been analysed in interspecific crosses between dihaploid Solanum tuberosum lines and diploid wild potato species (38, 39, 40, 41, 42). QTL analysis of tuber starch content and tuber yield in two genetically different diploid potato mapping populations identified factors controlling these traits on all twelve potato chromosomes (43). Most QTLs for tuber yield were linked with QTLs for tuber starch content suggesting that the effects on both traits are controlled by the same genetic factors. The quantitative character ,,earliness" affects tuber traits indirectly and is an indicator for the time required to complete the vegetative annual life cycle of the potato plant. Time from planting seed tubers to flowering, to tuber initiation and maturity are different phenotypic aspects of the character ,,earliness" which affects the whole plant. The character ,,earliness" is controlled by genetic factors and day-length. A major QTL for ,,earliness" has been localised on potato chromosome V (22,23). Interestingly, this QTL is closely linked with the hotspot for resistance on the same chromosome.

Molecular function map of potato for tuber starch content Due to its agricultural importance, physiology, biochemistry and molecular biology of starch accumulation in plant storage organs has been extensively studied (review in 44). During potato tuber growth and maturation, starch is synthesised in the amyloplasts from sucrose. Sucrose is synthesised in photosynthetically active source leaves and transported to the sink tubers. Several metabolic pathways participate in this process: photo-assimilation (Calvin cycle), sugar metabolism and transport, starch biosynthesis and degradation. Many of the genes controlling these pathways have been cloned from potato or other plant species and DNA sequence information is available in databases. We have started to construct a function map for genes involved in carbohydrate metabolism and transport by using cloned potato genes as RFLP markers (28). Alternatively, PCR products are generated from potato genomic DNA using as primers oligonucleotides derived from DNA sequences in the public database. In few cases, polymorphic PCR products are obtained that can be mapped directly. In most cases, PCR products are mapped only after digestion with four-cutter restriction enzymes (CAPS markers = cleaved amplified polymorphic sequence markers). Around seventy loci have been identified to date (unpublished results) which are distributed on all twelve potato chromosomes. A subset of them is positioned in the same map segments where QTL for tuber starch content and yield were identified.

86

Function maps of potato

Authors of this publication Christiane Gebhardt o), Ralf Sch~ifer-Pregl (1) Pea Oberhagemann o), Xinwei Chen (~),Catherine Chatot-Balandras (2),Enrique Ritter (3), Luigi Concilio (4), Eric Bonnel % Josef Hesselbach (~), Francesco Salamini (~) (1)Max-Planck Institut ftir Ztichtungsforschung, Carl von Linne Weg 10, D-50829 K61n, Germany (2)GERMICOPA, 29334 Quimper Cedex, France (3) Centro de Investigacion y Mejora Agraria (CIMA), Granja Modelo de Arkaute (Alava), E01080 Vitoria-Gasteiz, Spain (4) Consorzio ,,Mario Neri", Via Emilia Levante 18, 40026 Imola (BO), Italy

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Mapping Gene Location 246-252. 16. Tommiska TJ, H~im~il~iinen JH, Watanabe KN, Valkonen JPT (1998) Mapping of the gene NXph,, that controls hypersensitive resistance to potato virus X in Solanum phureja ivP35. Theor Appl Genet 96: 840-843. 17. Leonards-Schippers C, Gieffers W, Salamini F, Gebhardt C (1992) The R1 gene conferring racespecific resistance to Phytophthora infestans in potato is located on potato chromosome V. Mol Gen Genet 233: 278-283. 18. E1-Kharbotly A, Leonards-Schippers C, Huigen DJ, Jacobsen E, Pereira A, Stiekema WJ, Salamini F, Gebhardt C (1994) Segregation analysis and RFLP mapping of the R1 and R3 alleles conferring race specific resistance to Phytophthora infestans in progenies of dihaploid potato parents. Mol. Gen. Genet. 242" 749-754. 19. E1-Kharbotly, Palomino-Sanchez C, Salamini F, Jacobsen E, Gebhardt C (1996) R6 and R7 alleles of potato conferring race-specific resistance to Phytophthora infestans (Mont.) de Bary identified genetic loci clustering with the R3 locus on chromosome XI. Theor Appl Genet 92: 880-884. 20. Li X, van Eck HJ, Rouppe van der Voort J, Huigen D-J, Stam P, Jacobsen E (1998) Autotetraploids and genetic mapping using common AFLP markers: The R2 allele conferring resistance to Phytophthora infestans mapped on potato chromosome 4. Theor Appl Genet 96: 1121-1128. 21. Leonards-Schippers C, Gieffers W, Sch~ifer-Pregl R, Ritter E, Knapp SJ, Salamini F, Gebhardt C (1994) Quantitative resistance to Phytophthora infestans in potato: a case study for QTL mapping in an allogamous plant species. Genetics 137" 67-77. 22. Oberhagemann P, Chatot-Balandras C, Bonnel E, Sch~ifer-Pregl R, Wegener D, Palomino C, Salamini F, Gebhardt C (1999) A genetic analysis of quantitative resistance to late blight in potato: Towards marker assisted selection. Mol Breeding, in press. 23. Collins A, Milbourne D, Ramsay L, Meyer R, Chatot-Balandras C, Oberhagemann P, de Jong W, Gebhardt C, Bonnel E, Waugh R (1999) QTL for field resistance to late blight in potato are strongly correlated with earliness and vigour. Mol Breed, in press. 24. Hehl R, Faurie E, Hesselbach J, Salamini F, Whitham S, Baker B, Gebhardt C (1999) TMV resistance gene N homologues are linked to Synchytrium endobioticum resistance in potato. Theor Appl Genet 98: 379-386. 25. Bonierbale M, Plaisted RL, Tanksley SD (1988) RFLP maps based on a common set of clones reveal modes of chromosomal evolution in potato and tomato. Genetics 120" 1095-1103. 26. Gebhardt C, Ritter E, Debener T, Schachtschabel U, Walkemeier B, Uhrig H, Salamini F, (1989) RFLP analysis and linkage mapping in Solanum tuberosum. Theor. Appl. Genet. 78" 65-75. 27. Gebhardt, C., Ritter E, Barone A, Debener T, Walkemeier B, Schachtschabel U, Kaufmann H, Thompson RD, Bonierbale MW, Ganal MW, Tanksley SD, Salamini F (1991) RFLP maps of potato and their alignment with the homoeologous tomato genome. Theor. Appl. Genet. 83: 49-57. 28. Gebhardt C, Ritter E, Salamini F (1999) RFLP map of the potato. In: DNA-based markers in plants, edited by Phillips RL, Vasil IK, Advances in Cellular and Molecular Biology of Plants, Kluwer Academic Publishers, 2nd edition, in press. 29. Tanksley SD, Ganal MW, Prince JP, de Vicente MC, Bonierbale MW, Broun P, Fulton TM, Giovannoni JJ, Grandillo S, Martin GB, Messeguer R, Miller JC, Miller L, Paterson AH, Pineda O, R6der MS, Wing RA, Wu W, Young ND (1992) High density molecular linkage maps of the tomato and potato genomes. Genetics 132" 1141-1160. 30. Hammond-Kosack KE, Jones JDG (1997) Plant disease resistance genes. Annu Rev Plant Physiol Plant Mol Biol 48: 575-607. 31. Kombrink E and Somssich IE (1997) Pathogenesis-related proteins and plant defense. In: The Mycota V Part A Plant Relationships, Carroll/Tudzynski (Eds.), Springer-Verlag Berlin Heidelberg: 107-128. 32. Leister D, Ballvora A, Salamini F, Gebhardt C (1996)A PCR based approach for isolating pathogen resistance genes from potato with potential for wide application in plants. Nature Genetics 14: 421429. 33. Whitham S, Dinesh-Kumar SP, Choi D, Hehl R, Corr C, Baker B (1994) The product of the tobacco mosaic virus resistance gene N: similarity to Toll and the Interleukin-1 receptor. Cell 78:1101-1115. 34. Ori N, Eshed Y, Paran I, Presting G, Aviv D, Tanksley S, Zamir D, Fluhr R (1997) The I2C1 family from the wilt disease resistance locus 12 belong to the nucleotide binding, leucine-rich repeat superfamily of plant resistance genes. Plant Cell 9" 521-532. 35. Simons G, Groenendiijk J, Wijbrand J, Reijans M, Groenen J, Diergaarde P, Van der Lee T, Bleeker M, Onstenk J, de Both M, Haring M, Mes J, Cornelissen B, Zabeau M, Vos P (1998) Dissection of the

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Fusarium 12 gene cluster in tomato reveals six homologs and one active gene copy. The Plant Cell 10: 1055-1068. Bendahmane A, Kanyuka K, Baulcombe DC (1999) The Rx gene from potato controls separate virus resistance and cell death response. The Plant Cell 11: 781-791. Van Eck HJ, Jacobs JME, Stam P, Ton J, Stiekema WJ, Jacobsen E (1994) Multiple alleles for tuber shape in diploid potato detected by qualitative and quantitative genetic analysis using RFLPs. Genetics 137: 303-309. Freyre R, Warnke S, Sosinski B, Douches DS (1994) Quantitative trait locus analysis of tuber dormancy in diploid potato (Solanum spp.). Theor Appl Genet 89: 474-480. Van den Berg JH, Ewing EE, Plaisted RL, McMurry S, Bonierbale MW (1996a) QTL analysis of potato tuberization. Theor Appl Genet 93: 307-316. Van den Berg JH, Ewing EE, Plaisted RL, McMurry S, Bonierbale MW (1996b) QTL analysis of potato tuber dormancy. Theor Appl Genet 93: 317-324. Douches DS, Freyre R (1994) Identification of genetic factors influencing chip color in diploid potato (Solanum spp.). Am Potato J 71: 581-590. Freyre R, Douches DS (1994) Development of a model for marker-assisted selection of specific gravity in diploid potato across environments. Crop Sci 34: 1361-1368. Frommer WB, Sonnewald U (1995) Molecular analysis of carbon partitioning in solanaceous species. J Exp Bot 46: 587-607. Sch~ifer-Pregl R, Ritter E, Concilio L, Hesselbach J, Lovatti L, Walkemeier B, Thelen H, Salamini F, Gebhardt C (1998) Analysis of quantitative trait loci (QTLs) and quantitative trait alleles (QTAs) for tuber yield and starch content. Theor Appl Genet 97: 834-846.

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

Novel Traits For Cereal Biotechnology Positional Cloning Revisited

Summary ....,,.

.

. .

Recent technological developments have made a huge impact on the feasibility of map based cloning for the 'largegenome' cereals. For example, the numbers of markers required to saturate a very large genome would have been impossible to obtain before the AFLP technique was described in 1995. Progress has also been made in the construction of large-insert libraries, such that representative YAC and BAC libraries now exist for all the main cereals except bread wheat and even here it is certain that such libraries will become available within the next few years. Moreover, new resources and techniques for contig assembly and retrieval of expressed sequences from large genomic clones make the final step of gene identification much simpler than in the past.

Exploitation of all of these developments has led to the recent cloning by a positional approach of the broad-spectrum pathogen resistance gene mlo from barley. As discussed during the 'Gene Mapping cluster session of Phytosfere 99', maize is now the next genome being targeted for positional cloning in a concerted way. Under the Map Maize Framework IV Programme, the initial mapping steps for cloning a male sterility gene, an early maturity gene and genes involved in a silage quality QTL have already been carried out. The Map Maize Consortium aims to use chromosome 'landing' to identify the gene(s) controlling these important traits. If successful this programme will lead to important advances in understanding of the molecular processes which in turn will be highly relevant to tomorrow's agriculture.

Introduction - novel genes for the next generation of i m p r o v e d crops Crop breeding as a science-based discipline has existed for more than 100 years. It is therefore quite remarkable that the basic breeding techniques used to select and combine favourable agronomic traits have hardly changed at all. However, it is clear that we are on the threshold of a new era where the fruits of three decades of molecular genetics are finally making their way out of the research labs, into the breeding programs and thence into our fields and onto our tables [ 1,2]. After studying the heredity of countless genetically determined characters for over a century, we are now able to explain an ever expanding number of them in terms of cloned genes, and characterised protein products. Most of the current traits are simply inherited dominant genetic factors. Race-specific resistance genes (reviewed in [3]) are good ex-

Donal M. O' Sullivan, IACR-Long Ashton Research Station, University of Bristol, Long Ashton, Bristol, UK.

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Mapping Gene Location amples of this class. Single dominant alleles for useful traits such as disease resistance can be exploited simply by transforming them into genotypes with recessive alleles. Characterisation of simple Mendelian characters will only get us so far. Even a simple understanding of biology demonstrates that much of the plants biochemical activity is carried out through complex, multi-step signalling pathways which hold the key to cellular metabolism, and through an intricate web of developmental pathways which give rise to the spatial and temporal variations that govern plant architecture and life cycle. Full molecular elucidation of these processes would go to the heart of plant cell biology, and open the door to a second generation of modified plants based on knowledge of complex, non-linear pathways and polygenic traits. In terms of agronomy, the traits which belong to this latter category are yield, nutrient uptake, durable resistance to pathogens and pests, tolerance to abiotic stresses (e.g. drought, heat), taste, aesthetics, processing qualities, and nutritional value to name but a few. Only minor aspects of these traits can be modified through manipulation of single genes. However, increasingly sophisticated methods are now available to break these complex characters down into their component parts. Quantitative Trait Loci (QTL) analyses, using the composite interval method popularised by Lander and Botstein [4], have led to the identification and mapping of individual genetic components of quantitative disease resistance, fruit weight, drought, water stress, early maturity and yield [5-8] to cite but a few examples. Cloning of the multiple genes that underlie QTLs is now at the centre of scientific attention, and will surely underpin the second wave of molecular biotechnology applied to crops.

Use of model genomes for gene isolation There are many possible approaches to gene cloning in plants. Since many agriculturally important plants have relatively large genomes (as discussed later), positional cloning has been the least used of the possible methods. In part this is because of the technical difficulties involved in precisely mapping, locating and proving the functionality of genes in a background of overwhelmingly repetitive or redundant sequence. To avoid these technical problems, much effort has been invested in systematic elucidation of the molecular biology of the model plant Arabidopsis [9,10]. The underpinning assumption has been that discovery of Arabidopsis genes involved in such basic plant processes such as photosynthesis, cell cycle, hormonal responses, flowering, etc. will facilitate elucidation of the same processes in any other plant. As was clearly seen in "Phytosfere 99", the heavy investment of European researchers in Arabidopsis research has led to some remarkable spin-offs in crop improvement. Rice has been proposed as an alternative cereal model to Arabidopsis for a number of reasons [ 11,12]. Firstly, it is a major food crop in its own right, one of three cereals produced annually at world wide levels of approximately half a billion tons. Secondly, comparative mapping studies have shown major blocks of synteny between rice chromosomes and homeologous chromosome segments in other grass species, leading to the development of the paradigm of the grasses as a single genetic system [ 13,14]. Therefore, it was expected that gene discovery in rice would greatly expedite gene cloning in related cereal species. Indeed, cereal synteny is being actively pursued as a means to map-based clone wheat and barley genes. The barley rust resistance genes Rpgl and rpg4 have been fine mapped using markers derived from the syntenous segments of the rice genome [15,16]. Synteny with rice has also been used in an 92

Positional cloning revisited attempt to physically span the rice equivalent of the wheat Phl locus [17]. However, there will be limitations to the power of the rice model for dissecting agronomic traits. There are many documented cases of the breakdown of cereal synteny. A particular note of caution for map-based cloning strategies using cereal synteny is struck by the discovery of breakdown of micro-synteny within a region of overall macro-synteny [ 15].

Direct cloning methods - applications and limitations There are many different possible approaches to direct gene cloning in plants. However, our discussion here will be limited to two of the most powerful and popular methods : transposon tagging and differential display. Tagging has been most successful in maize. This is not surprising since transposons were first discovered in maize [18]. The Ac-Ds, Spm, and Mutator elements have all been used to tag genes, and have contributed enormously to the isolation of maize genes. In fact, the fate of maize genes and transposable elements has been linked from the beginning with the simultaneous cloning of waxy with Ac-Ds [19]. Opaque-2, a transcriptional activator involved in seed development was cloned by transposon tagging [20] many years before other possible approaches had been developed. The isolation of Viviparous 1, again by transposon tagging, is a further example of the seminal breakthroughs in plant developmental genetics facilitated by the transposon tagging system [21]. More recently, the maize rust resistance gene Rpl was cloned through isolation of a rust susceptible mutant tagged by the Mutator element (S. Hulbert, personal communication]. In each of these cases, the tagged individual (an insertional mutant) could be detected from a large population of untagged siblings by a simple phenotypic screen. Therefore, a discrete qualitative mutant phenotype is a crucial requirement for tagging to be successful, and so precludes the dissection of quantitative variation by this method. Another route to pinpointing a gene is to focus on cDNAs, which by definition, correspond to transcribed sequences, a large proportion of which correspond to functional genes. Differential display (which is technically similar to AFLP; [22]) allows transcripts that are differentially expressed in different tissues/isogenic lines to be detected. For example Johnson et al. has recently used the technique to identify cDNAs expressed in embryos of dormant and nondormant Avena fatua [23]. However, it has to be said that this type of approach is generally considered more as a 'black box' strategy, whereby discrete functions related to a particular developmental stage or state of induction are randomly cloned and functionally characterised. The approach therefore has the virtue of sometimes leading in unexpected new directions. On the other hand, it can be prone to false positives and has not yet been used to isolate a specific gene underlying a known phenotype. Furthermore, si~ace a difference in expression is required, small variations or constitutive expression may not be detected.

Positional cloning- a 'purist' view As noted in the preceding sections, each cloning method makes one or more starting assumptions. These are summarised in table 1. It is a logical truism that the more an investigation hinges on the truth of its intial assumptions, the less new knowledge can be gained during the investigation. So for example, if we limited ourselves to the study of cereal relatives of Arabidopsis genes, while making sensible use of a wonderful resource, we will certainly 93

Mapping Gene Location overlook an incredible array of monocot-specific genes of crucial importance to agriculture. Similarly, the limitation of the transposon tagging technique to single gene traits, means that if it were our only tool, we would be locked into the 'first generation' biotechnology described in the introduction.

TABLE 1 : Summary of inherent limitations of different cloning methods Cloning Method Use equivalent Arabidopsis

Underlying assumptions

Associated limitations

1. Arabidopsis has a similar function

This assumption holds well only for genes involved in

gene to find cereal

fundamental pathways

homologue

2. Identical functions are governed by homologous genes

Duplication in cereal genomes means that homologues

Rice as intergenomic

1. Synteny exists around trait of interest

Breakdown of synteny at both macro and micro-level is

2. Gene is conserved between rice and other cereals

This may not apply to fast-evolving genes such as race-

may have acquired new functions

cloning vehicle

well documented

specific resistance genes (24) Differential display

1. Candidate differences can be related to function

Prone to false positives (spurious products and artefactual differences in expression)

2. Presence/absence of a particular transcript results from Can only be applied to isogenic/clonal material. Not

a narrowly-defined difference in genotype/growth

useful for complex traits from exotic accessions

conditions Transposon tagging

1. Interruption of the gene will lead to a visible

Good assumption for genes already known to be single

phenotype

Mendelian traits; useless for polygenic traits

Positional cloning, which will be the focus of the remaining sections of this paper, is dependent on one precondition: it assumes that there exists a pair of interfertile accessions with a measurable difference in phenotype. Serendipitously, manipulation of measurable differences in phenotype between diverse accessions has formed practically the sole basis for the whole previous century of crop breeding. Therefore, it is important to realise that this assumption, rather than limiting our horizons, focuses decades of agronomic and breeding expertise into the initial steps of delimiting the position of the gene(s) of interest. Recent refinements in QTL analysis mean that even complex polygenic traits can be accurately and efficiently located with a view to cloning the genes involved. The approach does not depend on similarity with any other plant species, and so a trait of interest can in principle be cloned from the background in which it has been identified. The overall level of expression of the target gene(s) does not affect the outcome of a positional cloning exercise, nor are between tissue variation in or induction of expression required. Neither is biochemical or physiological knowledge of the biological processes being investigated required to direct a search for candidate genes. In summary, it is the surest way of proceeding from a trait for which the molecular basis is not known (nor even hypothesised) to a gene. From a purely academic perspective, the prospect of dissecting hitherto intractable polygenic traits is inherently appealing. From the applied perspective, it is particularly noteworthy that genes discovered by positional cloning (by very definition) have a functional significance which merits attention and support. Positional cloning in large-genome cereals has been seen for some time as being time-consuming, technically troublesome, and not very elegant. However, it is the authors' opinion 94

Positional cloning revisited that very recently the technological balance has tipped in favour of positional cloning, regardless of the size of genome being analysed. The following sections are devoted to a review of the history of positional cloning in plant systems, its modern variants, and current efforts to pursue the objective stated in the title of this paper.

Positional cloning- a brief history The general strategy of positional cloning was developed in mammalian systems, one of the notable early successes being the isolation of the cystic fibrosis gene [24]. The first plant genes to be cloned through a map-based strategy were the Arabidopsis genes ABI3 and fad3 genes, fad3, controlling omega-3-fatty acid desaturation, was located on chromosome 2 at a genetic distance of 0.4 cM from an RFLP marker. This marker was used to screen YAC libraries, and one of the positive YACs hybridised to a cDNA which was subsequently shown to restore the wild-type phenotype [25]. The cloning of ABI3, mediating responsiveness to abcissic acid, by Giraudat et al. [26] made use of the just-completed physical map of the Arabidopsis genome [27] to isolate a cosmid contig containing both flanking RFLP markers and Agrobacterium-mediated transformation was used to prove the functionality of the candidate gene identified. These successes were no doubt aided by the relatively small size of the Arabidopsis genome (100 Mb), and by the favourable relationship of physical to genetic distance (100 kb/cM). Therefore the segregating populations used and the number of markers surveyed were indeed small. However, the examples serve to illustrate the impact of appropriate genomic resources - a saturated RFLP map, YAC and cosmid libraries, a 95% coverage physical map, and a rapid and efficient transformation system - on the process of positional cloning. Even before Arabidopsis had gained momentum as a model, attention had turned to a real crop species; tomato. Tomato was attractive due to its reasonable genome size (950 Mb), existing high-density RFLP linkage map, and newly created YAC library [28]. Before long, the map-based cloning of the Pto bacterial resistance gene had been reported [29]. In this study, a tightly-linked RFLP marker was used to isolate a 400 kb YAC clone whose left and fight arms mapped to either side of Pto, thereby encompassing the gene on a single clone. Other groups who failed to isolate a single clone spanning the target locus faced two problems. The chief problem was the huge proportion of repetitive DNA in the large plant genomes. This fact meant that it was difficult to find low copy probes from the extremities of large insert clones which would permit further chromosome walking steps, and furthermore hampered the restriction mapping, contig building with clones initially isolated. Our understanding of the nature and organisation of repetitive DNA is progressing rapidly [30,31 ], and in the case of maize, the ubiquitous nature of certain repeated elements has been turned to an advantage through their use as a YAC clone fingerprinting tool which permits rapid contig building and detection of chimaeric clones [32]. Another problem encountered was the high proportion of chimaeric clones frequently encountered in YAC libraries [33-35]. With these problems in mind, the experience of Martin et al. [29], whereby the target locus was somewhat fortuitously spanned on a single clone, was subsequently developed into a general scheme for map-based cloning in crop plants. The new approach was dubbed 'chromosome landing' [36], and was touted as a way to avoid much of the technical trouble associated with chromosome walking in genomes containing high proportions of repetitive DNA. 95

Mapping Gene Location

Chromosome landing- a triumph of technology As noted above, chromosome landing was first performed in tomato, through the cloning of Pto. The next report of chromosome landing in plants was the isolation of another disease resistance gene, Xa21 from rice [37]. However, before it was applied to larger genomes, a further technical development was required. In 1995, a new type of DNA marker called AFLP was described [38]. The AFLP technique proved to be extremely robust and amenable to high-throughput analysis and was quickly adopted as the method of choice where large numbers of markers had to be rapidly screened for polymorphisms. The coupling of Bulked Segregant Analysis [39] and AFLP made it possible to completely saturate target genomic regions relatively quickly. However, while the number of markers used can easily be scaled up according to the size of genome, the number of F2 individuals required to order these markers increases in tandem. So, for the large cereal genomes, to obtain a genetic resolution of 0.1 cM, several thousand gametes must be obtained and their DNA extracted. These are then screened for at least two flanking markers to obtain the subset of individuals harbouring recombination events within the target interval, Fine mapping is performed using only the informative individuals. Therefore, while new technology has made the exercise feasible, it is clear that chromosome landing remains a serious commitment in terms of resources. Recently, the mlo broad-spectrum resistance gene from barley was successfully fine mapped [34] and cloned [40] in a chromosome landing strategy. Key elements in the successful cloning of mlo were a barley YAC library, examination of > 1,900 AFLP primer combinations for polymorphisms in the mlo and Mlo bulks, fine mapping in a population of >2,000 individuals, BAC sub-cloning of YACs, and use of AFLP for contig assembly. In the past year, the first steps in the positional cloning (again by chromosome landing) of the barley Rarl gene, encoding a disease response signalling factor have been reported [35,41]. Two general points are to be noted from the above historical perspective. Firstly, once certain key resources are in place for a given crop species, history tells us that they translate very quickly into cloned genes. The key resources in question are high density linkage maps, and large-insert libraries. Secondly, the chromosome landing approach shifts the emphasis from cumbersome and time-consuming chromosome walking to regional marker saturation. The process of saturating a genetic interval with markers whether by using the RAPD or AFLP technique is only a question of how big the target genome is. A simple calculation taking genome size, rate of polymorphism, and primer redundancy is needed to determine the number of primer combinations that may be needed in order to find a given number of polymorphisms in the target region. Chromosome landing, therefore, is technically feasible, even in the large-genome cereals. Moreover, at the present time it seems that efforts are well underway to clone the genes which underlie QTLs in number of different species. Alpert & Tanksley [42] have reported high resolution mapping of a fruit weight QTL. A YAC contig spanning the QTL has been constructed, and the QTL has been genetically mapped to within a 150 kb physical interval. The large number of QTL studies which have been carried out in rice [6,43] will very likely soon lead to the isolation of quantitative effect genes for a variety of agronomic traits, since a rice physical map [ 12,44] and a large EST database [45] are now available.

96

Positional cloning revisited

Towards chromosome landing in maize In 1999, we have reached the point where considerable investment, both public and private, has been made in cereal genomics technology [46,47]. The structural genomics phase has so far involved investment in the production of high-density maps, large insert libraries, and large EST databases. The functional genomics phase is now developing methods to rapidly assign phenotypes to the growing number of putative genes coming from genomic and EST sequencing projects. The net result of this investment is that there are high density genetic maps based on RFLP, RAPD and SSR marker technology, major EST databases and large insert libraries for almost all major cereals. Table 2 summarises the resource status of all the major cereal crops. TABLE 2 : Genomic resources for major cereal crops

Genome Size

Cereal Crop

YAC library

BAC library

Public EST dB (no. of sequences)

(Mb)

R&e

440

Bread Wheat

16,000

no

no

no

Maize

2,5'00

yes

yes

yes (>10,000)

800

no

yes

no

4800

yes

yes

no

Sorghum Barley

......

yes

yes

yes (35,000)

As we have seen above, chromosome landing has so far been carried out in rice and in barley. A large insert library is still lacking for wheat, and so positional cloning in wheat is not feasible at present. Indeed there are other potential problems for those who would positionally clone from wheat, not least its hexaploid genome and a distinct lack of polymorphism between cultivated varieties. However, isolation of genes by positional cloning from two of the top five cereals grown world wide, maize and its smaller-genome relative sorghum, is more than feasible, but has yet to be performed. In the case of maize, the incentives to positionally clone are overwhelming. The crops economic importance is indisputable, and from a European perspective, the exponential growth in hectarage within the EU means that strategic research cannot simply be left to the US, as consumer requirements and growing conditions in Europe are significantly different, requiring a different emphasis for breeding and hence for 'Euro-friendly' biotechnological research. Maize has long been pre-eminent as a model for genetic studies. Therefore, the detailed knowledge of its genetics, and the considerable public resources that have been acquired over the years (a very dense RFLP/SSR genetic map, a veritable mass of mutant stocks and genetic markers etc.) means that it is better placed than most other cereals to join the ranks of the positionally cloned. There are publicly available maize YAC and BAC libraries [48]; these are being complemented by the construction (in progress) of a new BAC library from the European inbred line 'F2' as part of the EU Framework IV Map Maize program [49]. The main objective of the Map Maize program is the isolation by chromosome landing of three targets of agronomic importance. A gametophytic sterility gene (GaMS-1), an early

97

Mapping Gene Location maturity gene and genes involved in silage quality QTLs are being targeted. The Map Maize Consortium has put together the resources needed to grow, phenotypically score and screen for recombinants, F2 segregating populations of up to 10,000 individuals; to carry out 1,000s of AFLP reactions for BSA to saturate local genomic regions around each of the targets; to screen for, and isolate large insert clones corresponding to the target loci. Each of these operations is carried out by a partner that has maximum experience in the particular field of expertise. The first steps of fine mapping and marker saturation have already been carried out, and were presented orally to Phytosfere '99. It is therefore our belief that in the near future, the 'second wave' of crop biotechnology will be a reality in maize and in other large-genome cereals.

Acknowledgements IACR - Long Ashton is funded by the Biotechnology and Biological Sciences Research Council of the United Kingdom. A u t h o r s o f this p u b l i c a t i o n Donal M. O' Sullivan and Keith J. Edwards. IACR-Long Ashton Research Station, Department of Agricultural Science, University of Bristol, Long Ashton, BRISTOL BS41 9AE UK.

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9. 10. 1 1. 12. 13. 14. 15.

D. Duvick (1996) Plant breeding, an evolutionary concept. Crop Sci. 36:539-548 M.E. Sorrells, W.A. Wilson (1997) Direct classification and selection of superior alleles for crop improvement. Crop Sci. 37:691-697 B.J. Staskawicz, EM. Ausubel, B.J, Baker, J.G. Ellis, J.D.G. Jones (1995) Molecular genetics of plant disease resistance. Science: 268:61-667 E.S. Lander, D. Botstein (1989) Mapping mendelian factors underlaying quantitative traits using RFLP lingage maps. Genetics 121 : 185-199 K.B. Alpert, S. Grandillo, S.D. Tanksley (1995) FW-2.2 - A major QTL controlling fruit weight is common to both red-fruited and grenn-fruited tomato species.Theor. Appl. Genet. 91:994-1000 S.R. McCouch, R.W. Doerge (1995) QTL mapping in rice. Trends Genet. 11:482-487 N.D. Young (1996) QTL mapping and quantitative disease resistance in plants. Annu. Rev. Phytopathol. 34:479-501 S. Pelleschi, S. Guy, J-Y Kim, C. Pointe, A. Mah6, L. Barthes, A. Leonardi, J-L. Prioul (1999) Ivr2, a candidate gene for vacuolar invertase activity in maize leaves. Gene-specific expression under water stress. Plant Mol. Biol. 39:373-380 D.W. Meinke, J.M. Cherry, C. Dean, S.D. Rounsley, M. Koornneef 1998) Arabidopsis thaliana: A model plant for genome analysis. Science 282:662-682 E.M. Meyerowitz (1989) Arabidopsis: a useful weed? Cell 56:263-269 I.J. Havukkala (1996) Cereal genome analysis using rice as a model. Current Opinion in Genetic Development 6(6):711-714 S.A. Goff (1999) Rice as a model for cereal genomics. Curr. Opinion Plant Biol 2:86-89 G. Moore, K.M. Devos, Z Wang, M. Gale (1995) Grasses line up and form a circle. Curt. Biol. 5:737739 G. Moore, M. Roberts, L. Alcaide, T. Foote (1997) Centromere sites and cereal genome evolution. Chromosoma 105:321-323 A. Kilian, J. Chen, F. Han, B. Steffenson, A. Kleinhofs (1997) Towards map-based cloning of the barley stem rust resistance genese Rpgl and rpg4 using rice as an intergenomic cloning vehicle. Plant Molec. Biol. 35:187-195

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Positional cloning revisited 16. E Han, A. Kleinhofs, S.E. Ullrich, A. Kilian, M. Yano, T. Sasaki (1998) Synteny with rice: analysis of barley malting QTLs and rpg4 chromosome regions. Genome 41:373-380 17. T. Foote, M. Roberts, N. Kurata, T. Sasaki, G. Moore (1997) Detailed comparative mapping of cereal chromosome regions corresponding to the Phl locus in wheat. Genetics 147:801-807 18. B. McClintock (1984). The significance of responses of the genome to challenge. Science. 226:792801 19. C.F Weil, S. Marillonnet, B.Burr, S.R. Wessler (1992). Changes in state of Wx-m5 allele of maize are due to intragenic transposition of the Ds element. Genetics 130:175-185 20. D. Michel, H. Hartings, S. Lanzini, M. Mhel, M. Motto, G.R. Riboldi, F. Salamini, H.E Doring. (1995) Insertion mutations at the maize opaque2 locus induced by transposable element families AC, EN/SPM and B G . Mol. Gen. Genet., 248:287-292 21. D.R. McCarty, T. Hattori, C.B. Carson, V. Vasil, M. Lazar, I.K. Vasil (1991) The viviparous-1 developmental gene of maize encodes a novel transcriptional activator. Cell 66: 895-905. 22. G.R. Heck and D.E. Fernandez (1997). Differential Display of mRNA. In Differentially Expressed Genes in Plants. (ed. E. Hansen and G. Harper). pp45-61. 23. R.R. Johnson, H.J. Cranston, M.E. Chaverra and W.E. Dyer (1995). Characterisation of cDNA clones for differentially expressed genes in embryo of dormant and nondormant Avena fatua L. caryopses. Plant Molecular Biology, 28:113-122 24. J.M. Rommens, M.C. Iannuzzi, B. Kerem, M.L. Drumm, G. Melmer, M. Dean, R. Rozmahel, J.L. Cole, D. Kennedy, N. Hidaka, M Zsiga, M. Buchwald, J.R. Riordan, L.C. Tsui, F.S. Collins (1989) Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245:1059-1065 25. V. Arondel, B. Lemieux, I. Hwang, S. Gibson, H.M. Goodman, C.R. Somerville (1992) Map-based cloning of a gene controlling omega-3 fatty acid desaturation in Arabidopsis. Science 258:1353-1354 26. J. Giraudat, B.M. Hauge, C. Valon, J. Smalle, F. Parcy, H.M. Goodman (1992) Isolation of the Arabidopsis ABI3 gene by positional cloning. Plant Cell 4:1251-1261 27. B.M. Hauge, J. Giraudat, S. Hanley, I. Hwang, T Kohchi, H.M. Goodman (1991) Physical mapping of the Arabidopsis genome and its application In Plant Molecular Biology 2, R.G. Hermann and B. Larkins 28. R.A. Wing, H-B. Zhang, S.D. Tanksley (1994) Map-based cloning in crop plants. Tomato as a model system: I. Genetic and physical mapping of jointless. Mol. Gen. Genet. 242:681-688 29. G.B. Martin, S.H. Brommonshenkel, J. Chunwongse, A. Frary, M.W. Ganal, R. Spivey, T. Wu, E.D. Earle, S.D. Tanksley (1993) Map-based cloning of a protein kinase gene conferring disease resistance in tomato. Science 262:1432-1434 30. L. Ramsay, M. Macaulay, L. Cardle, M. Morgante, S. degli Ivanissevich, E Maestri, W. Powell, R. Waugh (1999) Intimate association of microsatellite repeats with retrotransposons and other repetitive elements in barley. Plant Journal 17:415-425 31. E SanMiguel, A. Tikhinov, Y. Jin, N. Motchoulskaia, D. Zakharov, A. Melake-Berhan, P.S. Springer, K.J. Edwards, M. Lee, Z. Avramova, J.L. Bennetzen (1996) Nested retrotransposons in the intergenic regions of the maize genome. Science 274:765-768 32. Edwards, K.J., Veuskens, J., Rawles, H., Daly, A., and Bennetzen J.L. (1996). Characterisation of Four Dispersed Repetitive DNA Sequences from Zea mays and their use in Constructing Contiguous DNA Fragments using YAC clones. Genome 39: 811-817. 33. E.D. Green, M.V. Olsen (1990) Chromosomal region of the cystic-fibrosis gene in yeast artificial chromosomes - A model for human genome mapping. Science 250:94-98 34. G. Simons, T. van der Lee, P. Diergaarde, R. van Daelen, J. Groenendijk, A. Frijters, R. Bfischges, K. Hollricher, S. T6psch, P. Schulze-Lefert, F. Salamini, M. Zabeau, EVos (1997) AFLP-based fine mapping of the Mlo gene to a 30-kb DNA segment of the barley genome. Genomics 44:61-70 35. T. Lahaye, K. Shirasu, E Schulze-Lefert (1998) Chromosome landing at the barley Rarl locus. Mol Gen Genet 260:92-101 36. S.D. Tanksley, M.W. Ganal, G.B. Martin (1995) Chromosome landing: a paradigm for map-based gene cloning in plants with large genomes. Trends Genet. 11:63-68 37. W-Y. Song, G-L. Wang, L-L Chen, H-S Kim, L-Y Pi, J. Gardner, B. Wang, W-X Zhai, L-H. Zhu, C. Fauquet, P.Ronald (1995) A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21. Science 270:1804-1806 38. E Vos, R. Hogers, M. Bleekers, M Reijans, T. van der Lee, M. Hornes, A Frijters, J. Pot, J. Peleman, M. Kuiper, M. Zabeau (1995) AFLP: A new technique for DNA fingerprinting. Nucl. Acids Res. 23:4407-4414

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Mapping Gene Location 39. J.J. Giovannoni, R.A. Wing, M.W. Ganal, S.D. Tanksley (1991) Isolation of molecular markers from specific chromosomal intervals using DNA pools from existing mapping populations. Nucleic Acids Res. 19:6553-6558 40. R. Btischges, K. Hollricher, R. Panstruga, G. Simons, M. Wolter, A. Frijters, R. vanDaelen, T. van der Lee, P. Diergaarde, J. Groenendijk, S. Topsch, P. Vos, F. Salamini, P. Schulze-Lefert (1997) The barley mlo gene : a novel control element of plant pathogen resistance. Cell 88:695-705 41. T. Lahaye, S. Hartmann, S. T6psch, A. Freialdenhoven, M. Yano, P. Schulze-Lefert (1998) Highresolution genetic and physical mapping of the Rarl locus in barley Theor. Appl. Genet. 97:526-534 42. K.B. Alpert, S.D. Tanksley (1996) High-resolution mapping and isolation of a yeast artificial chromosome contig containing fw2.2: A major fruit weight quantitative trait locus in tomato Proc. Natl. Acad. Sci. USA 93:15503-15507 43. M. Yano, T. Sasaki (1997) Genetic and molecular dissection of quantitative traits in rice Plant Mol. Biol. 35:145-153 44. K. Yamamoto, T. Sasaki (1997) Large-scale EST sequencing in rice. Plant Mol. Biol. 35:115-127 45. G. Hong, Y. Qian, S. Yu, X. Hu, J. Zhu, W. Tao, W. Li, C. Su, H Zhao, L. Qiu, et a1.(1997) A 120 kilobase resolution contig map of the rice genome. DNA Sequence 7:319-335 46. J.L. Bennetzen (1999) Plant genomics takes root, branches out. Trends Genet. 15:85-87 47. S. Rounsley, X. Lin, Ketchum K.A. (1998) Large-scale sequencing of plant genomes. Curr. Opinion Plant Biol 1:136-141 48. K.J. Edwards, H. Thompson, D. Edwards, A. de Saizieu, C. Sparks, J.A. Thompson, A.J. Greenland, M. Eyers, W. Schuch (1992) Construction and characterisation of a yeast artifical chromosome library containing three haploid maize genome equivalents. Plant Mol. Biol. 19:299-308 49. D.M. O'Sullivan, P.J. Ripoll and K.J. Edwards, unpublished results

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All riglats reserved.

Insertional Mutagenesis Of The Arabidopsis Genome

The Arabidopsis Insertional Mutagenesis (AIM) project was initiated to establish a link between the International Sequencing projects and research aimed at the structural and functional analysis of genes. Its objective was to achieve a saturation of the Arabidopsis genome using T-DNA-, heterologous transposon-, retrotransposon-, gene-trap and enhancer trap insertions, in order to be able to identify insertional mutations in any gene of interest. Over the past decade, these mutagens have been shown to represent one of the most efficient ways of studying gene function. (1-7). The development of several populations carrying different mutagens by members of the consortium, would enable insertional mutagenesis to evolve from being a technique used for individual gene isolation to a method used for systematic functional analysis of the genome using reverse genetics. Genome analysis of the model plant Arabidopsis will contribute in assigning a function to a large variety of novel Arabidopsis genes of potential and agricultural and industrial importance and will also contribute to providing genes of functional importance to other crop plants. In order to develop an effective reverse genetics strategy in Arabidopsis based on transposons, our laboratory used the maize transposable element En-I (Spin) system, based on the autonomous Enhancer (En) and nonautonomous Inhibitor (I/dSpm) elements, to saturate the Arabidopsis genome with I-elements. The En-I (Spm) system was modified into a two-component system comprising a mobile transposon component and an immobilized transactive transposase source. A stable En/Spm transposase source under control of the CaMV 35S promoter was shown to mediate frequent transposition of I/dSpm elements which occurred continuously throughout plant development (8). This continuous, frequent transposition observed for this transposon system was used to our advantage for the generation of a small population of 2592 lines containing multiple I/dSpm elements per line. After 6 generations of selfing and propagation by single seed descent and based on an independent transposition frequency of 10-30%, we estimate that this population contains approximately 20-30 Ielements per line and 50,000 to 75,000 I-element insertions in total. This number of insertions corresponds to 1-1,5 insertions every 2 kb in the Arabidopsis genome. To identify insertions in specific genes of interest using PCR-based reverse genetics screens, the population was divided into pools of plants in order to decrease the amount of PCRs

Elly Speulman, CPRO-DLO, Department of Molecular Biology, Wageningen, The Netherlands.

101

Mapping Gene Location necessary to identify an insertion. In the three-dimensional pooling strategy used, the population was divided into 3 sets, containing 9 blocks of 96 plants each and plant material was pooled in such a way that each plant was represented by a unique combination of one block, one row and one colPlant Population pools Set I-IX + Set X-XVIII+ Set XIX-XXVII umn (Figure 1). 87 DNA Pools To minimize the effort needed to iden[ F .... tify an insertion in a I primers ~ / gene of interest, we 8 Rows designed the three ditarget gene m e n s i o n a l screen with in total 87 DNA 12 Columns PCR and Hybridisation pools and used two IBlocks Rows Columns element specific I V I G I 11 I primers in combination with a gene-specific primer in one reaction. After gel-electrophoresis and Southern blotting of the PCR products, the blots are hybridised with a genespecific probe, and pools with an insertion in the gene of interest can be identified. Figure 1 shows an example of such a three-dimensional screen. In this example, positives were found in three dimensions: block V, row G and column 11. This screen therefore identified plant VI 1-G in the population. i

3-D Pooling

[

. . . . .

I

i_'.'.:

itir 2

---'--:....

itir 3

E

This population was further used for the identification of insertions in a total of 150 genes (Table 1). These genes belonged to a variety of gene families and were screened for by members of our laboratory and by members of several other international laboratories who visited the laboratory or who sent us their gene-specific primers. In general, three-dimensional insertions were found in 75% of the genes tested, which were shown to be heritable in 50% of the initial positives. These insertions are currently being used in functional and Tablel: Number of genes, belonging to different families, tested for insertions in the multiple I population. Random Insertions Transcription factors

# Genes

35

(Myb-, MADS-box-, Homeodomain-genes) 24

Signal transduction

(Kinases, G-protein, Hormone interacting proteins) 21

Transporters

(Proteins, Ammonium, Sucrose, Antiporter) Defense related (homologs of DAD1, Mlo, R-genes, R-gene interacting proteins, signal transduction) Wax and Lipid biosynthesis ( Cer homologs) Translocators

13 12

(Phosphate, Malate, Secretory proteins) 15 10

Plant Development Photosynthesis related Drought resistance related Ageing

102

Insertional mutagenesis genetic analysis in laboratories world-wide. Our results show that this population can be used for reverse genetics purposes. The multiple I Arabidopsis lines can be used for the identification of insertions in any gene or DNA sequence and are a valuable addition to the other populations generated as part of the Arabidopsis Insertional Mutagenesis programme. Members of the consortium can be contacted for further reverse genetics screens.

Members of the consortium A. Pereira (AMICA Science EEIG co-ordinator), CPRO-DLO, Department of Molecular Biology (NL); J.D.G. Jones, Sainsbury Laboratory (GB); G. Coupland, John Innes Centre, Genetics Department (GB); C. Dean, John Innes Centre, Genetics Department (GB); M. Caboche, INRA (F); C. Koncz, MPIZ, Genetic Principles of Plant Breeding Department (D); H. Lucas, INRA (F); H. Saedler, MPIZ, Molecular Plant Genetics Department (D).

Authors of this publication Elly Speulman and Andy Pereira, Department of Molecular Biology, CPRO-DLO, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.

References 1. 2. 3. 4. 5. 6. 7. 8.

K . A . Feldmann. Plant J. 1, 71-82 (1991). C. Koncz, K. Nemeth, G. P. Redei, J. Schell. Plant Mol.Biol. 20, 963-976 (1992). M . G . M . Aarts, W. G. Dirkse, W. J. Stiekema, A. Pereira. Nature 363, 715-717 (1993). I. Bancroft, J. D. G. Jones, C. Dean. Plant Cell 5, 631-638 (1993). D.A. Jones, C. M. Thomas, K. E. Hammond-Kosack, P. J. Balint-Kurti, J . D . G . Jones. Science 266, 789-793 (1994). J. Okuley, et al.. Plant Cell 6, 147-158 (1994). R. Azpiroz-Leehan, K. A. Feldmann. Trends in Genetics 13, 152-156 (1997). M . G . M . Aarts, P. Corzaan, W. J. Stiekema, A. Pereira. Mol.Gen.Genet. 247, 555-564 (1995).

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). O Elsevier Science B.V. All rights reserved.

Comments from the Session Rapporteur Attending Phytosphere as a rapporteur - usually I would attend such a conference as a journalist- proved to be thought provoking and prompted a few conclusions about perceptions that may irritate and can certainly be challenged. For the sake of argument, then, and in case the resulting polemic is of any use, I thought I'd write an opinion piece as my contribution to the Phytosphere conference report. First, the role of rapporteur and journalist are and should remain mutually exclusive. This is not as insignificant a point as it might seem at first glance. To be rapporteur at a scientific k conference prevents the journalist doing a journalist's job, unless, of course, one just does a journalists job and ignores the task of being a rapporteur. It may of course be a question of language or perception, but it seems to me important to realise that the job of rapporteur is not the same as that of a reporter, and that even if they were the same thing, reporting is only one small aspect of a journalist's skill. More importantly, though, is the fact that unbiased reporting itself can be used to serve a strongly held view. This thought has recurred strongly to mind several times since the Phytsophere conference. On one occasion, for example, I was reading a story in a British paper- the Daily Mail. This paper has consistently been distrustful of gm food and agriculture, so any journalist covering the area needs to know what it is saying. What caught my eye this time, however, was a story about cloning. I was interested, and a little suprised, to read unbiased reporting of a story about human cloning that seemed to go out of its way not to whip up negative emotion - it could have been the work of a rapporteur. Two days later, and I didn't make any connection at the time, the UK government banned research on human embryos even in the first 14 days of life prior to the formation of the primitive streak. The ban was announced despite a scientific advisory panel's opinion that research on embryos up to 14 days of age should go ahead for therapeutic purposes. Now there may or may not have been a connection between the story in the Mail and the government ban, but I wonder. When listening to people chat on buses and in the pub, I realised that what had seemed to me to be unbiased reporting of science was seen by some only as a profoundly shocking interference in human life.

Helen Gavaghan, Freelance journalist, Hebden Bridge, UK.

105

Mapping Gene Location My perception that cloning profoundly shocks many people is, of course, based purely on anecdote. Yet despite my distant training in biophysics, which showed me the deceptive nature of anecdote and averages, I also remember that anecodote can provoke the formation of a good questions. Perhaps one of those questions should be to ask whether rationality is the best way to debate or explain the role of biotechnology in our lives. This thought brings me to my second conclusion, namely that the main mistake made by scientists and, until recently by politicians in the UK, is to attempt to debate the issue of biotechnology and geneticallymodified food on a rational basis, when the debate is, in fact, fuelled by emotion and often unspoken and unrecognised attititudes about the nature of life. Let me be even more irritating. It seems to me, too, that the rationalism of the scientific camp is often a cloak for an emotional attachment to the position that gentically-modified food is safe or an emotional reaction against a perception which may or may not be true. To illustrate the point about perception I'd like to refer to the story of Arpad Pusztai and the potatos. During Phytosphere someone, who shall be nameless, told me all the things Dr Pusztai had said and claimed on the TV programme that provoked the storm. I had, however, just watched a recording of this programme about three times for something I was wroking on, and I was fascinated by how little resemblance the supposedly rational disputation of Dr Pusztai's statements had to do with what he had actually said. Now for the question about whether genetically-modified food is safe. My personal opinion - as a journalist writing an opinion piece? I can't see any reason intrinsically why it should not be safe. But what do I know? Studying biophysics 19 year ago or researching the history of satellites scarcely qualifies me to take a strong stance either way. Hence I am left only with the journalist's - and not the rapporteur's role - of thinking of questions to ask, no matter how stupid they may be, and reporting the answers. A few of those questions - but remember my dilletantish mind is already thinking of my next deadline - are: - can you explain to me what significance I should attach to the fact that there is no way of knowing where the gene will insert itself? could you please justify that opinion if, as I assume, the answer to the question is that I should attach no significance to it from a safety point of view? -

OK, so you say that its a bit silly to be preoccupied by the insertion of one gene in a genome when natural reproduction shakes up the whole pack, but is that a fair dismissal? Surely natural reproduction leads often to unviable genomes, would the same happen if the gene were inserted in such a way as to make the gm genome unviable? Why is that a silly question? Also, how do scientists know there are not good biophysical or biochemical reasons precluding certain genetic arrangements? Why should I not view the addition of a gene from some other organism as an entirely different biological proposition to shuffling the entire pack? I could go on asking questions for a long time, and I daresay they'd all seem very ignorant to the biotechnology community. The questions are doubtless the wrong questions, but I'd love to report a good ding dong of a debate reflecting honest scientific disagreement, if there is any. That's the journalistic instinct, of course, and it is the same instinct that is distrustful of 106

Session rapporteur people when they ask me why I can't just write the good news. I realise that such a debate if it happened would be held in more ratified magazines and journals, but some of the serious papers would print the arguments providing journalists and editors made a supreme effort to translate the arguments to lay language. And yes, I do realise the arguments could be misquoted, but, hey, it happens to everyone. I wonder, too, if an emotionally charged but scientifically solid and open argument between scientists might not - in the long run - be more reassuring in a strange way than the repeated assertion that everything in the garden is rosy and that gm foods will feed the world and cure many ills. I suspect they may well do so. but when people ask me to explain just why gm foods are so safe, I don't know how to answer, and that reminds me of my biophysics training, and suggests I am about to flunk an exam. Helen Gavaghan is a freelance journalist based in Hebden Bridge, West Yorkshire who contributes to US, UK and French science and technology magazines.

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

The Cluster: "Controlling Developmental Processes And Architecture" Introduction The eight projects within this cluster focus on the elucidation of the basic cellular processes that regulate plant development and architecture. Several projects focus on the role of key plant hormones such as abscisic acid, auxins and gibberellins, another addresses the influence of the plant photoreceptors phytochrome and cryptochrome, and two others dissect the role of key signal transduction intermediates, e.g., the second messenger calcium and the MADS-box family of transcription factors. The remaining two projects address specific issues in plant development, the control of flowering time and grain filling in cereal crops. Although much of the research is of a basic scientific nature, in many cases it is possible to utilise the information to control important agronomic characters such as plant stature, seed dormancy and root architecture. The industrial partners involved within each of these projects are attempting to achieve these goals. Two major highlights within this cluster have been the molecular identification of the genes utilised to improve yields during the green revolution, and the ability to optimise root architecture for nutrient uptake from the soil.

Overview of the projects within the cluster BIO4-CT96-0101 - Calcium and activated oxygens as signals for stress tolerance, co-ordinated by Chris Bowler, Napoli, IT, <www.szn.it/,--cast/welcome.html> The manipulation of signal transduction pathways controlling responses to adverse environmental conditions is likely to be the most effective way to generate stress tolerant crop plants. Before this new generation of resistance strategies can be realised, it is necessary to identify the signalling molecules involved, to understand how they interact with each other and with other pathways, and to determine how specificity in response is achieved. The project is addressing these questions by systematically dissecting the signal transduction pathways involved in activating pathogen defence responses and cold tolerance. In particular, much research is focusing on the functions of calcium and activated oxygen species (AOS), as both play essential signalling roles in each response. The research groups are uniquely able to combine well characterised simplified experimental systems with highly advanced technologies that allow the dynamic visualisation of individual

Chris Bowler, Laboratory of Molecular Plant Biology, Stazione Zoologica, Naples, Italy.

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Embryogenesis, Development and Architecture signal transduction events at the cellular level. The combined studies will allow an understanding of how common signalling intermediates such as calcium and AOS can control different responses and will lead to new resistance phenotypes for agriculture. BIO4-CT96-0062 - Characterizing and engineering abscisic acid action, co-ordinated by Jerome Giraudat, Gif sur Yvette, France, <www.cnrs-gif.fr/isv/JG/biotech.html> The plant hormone abscisic acid (ABA) controls a diversity of essential processes. During seed development and germination, ABA controls seed dormancy, the acquisition of dessication tolerance, and the mobilisation of food reserves. During vegetative growth, ABA regulates the aperture of stomatal pores and hence the water-loss via transpiration, and triggers adaptive responses to environmental stresses such as drought and cold. The first objective of this project is to identify and characterise novel key elements regulating the plant responses to developmental and environmental signals transduced by ABA. The second objective is to assess the potential of ABA-biosynthesis, ABA-signalling and ABA-target genes for improving the seed characteristics and the growth performance of crop species. BIO4-CT96-0487 - Molecular control of lateral initiation, co-ordinated by Malcolm Bennett, Nottingham, UK, <www.transgene.ndirect.co.uk/latin/> The objective of this project is to improve understanding of the regulation of branching in plants using Arabidopsis as a model. Despite its importance, little is known about the molecular mechanisms controlling root and shoot lateral initiation and maturation. Physiological studies indicate that the plant hormones auxin and cytokinin are of central importance. Arabidopsis molecular genetics provides a powerful approach to dissect phytohormone-regulated branching, and has already resulted in the identification of a selection of root and shoot branch mutants and their genes by members of the project. The information and molecular tools which arise from these studies on Arabidopsis will underpin efforts to manipulate branching in transgenic crops. Eucalyptus, poplar and oil-seed rape are important European crops, providing raw materials for the paper, construction and chemical industries, respectively. The ability to engineer novel branching phenotypes within these target crops will have major economic consequences for agronomically-important qualities such as plant propagation, processing and yield. BIO4-CT96-0621 - Regulation of plant development through gibberellin signal transduction, co-ordinated by Richard Hooley, Bristol, UK. Gibberellins (GAs) are a class of plant hormones that are essential endogenous regulators of growth and development. They influence a range of events during the plant life cycle, such as cell growth, flower and fruit development, and cereal seed germination. GAs are perceived at the cell surface. Mutations that confer insensitivity to GA, such as the Arabidopsis GAI gene, may identify components of the GA signalling pathway. GA-regulated expression of genes is mediated by sequence elements in their promoters and proteins that interact with them. The specific aims of the project are to identify GA receptors, to isolate GA signal transduction intermediates, to unravel GA-regulation of gene expression and to examine the potential of the Arabidopsis GAI gene for modifying crop growth. Because agriculture, horticulture and citriculture exploit the growth-regulating properties of GAs, an advanced understanding of GA signal transduction should lead to new opportunities to enhance crop quality and crop efficiency. 110

Controlling developmental processes and architecture BIO4-CT97-2124 - The photoregulation of plant architecture and performance, co-ordinated by Harry Smith, Leicester, UK. Plants are extremely sensitive to light signals, detected by two principal photoreceptor families, the phytochromes and the cryptochromes. Recent developments have demonstrated that the architecture of crop plants growing in the field can be drastically altered by genetic engineering techniques that modify photoreceptor action. This project is designed to provide fundamental understanding of phytochrome and cryptochrome action and to use that information to improve the performance of crop plants. The overall objectives are to elucidate mechanisms of light signal perception and signal transduction, to select new photomorphogenic mutants, to engineer gene constructs for transformation of crops, to create new crop plants with altered architecture, and to propose approaches for the introduction of new crop varieties into European agriculture. BIO4-CT97-2158 - Manipulation of transfer cells to improve grain filling, co-ordinated by Richard Thompson, K61n, Germany, <www.mpiz-koeln.mpg.de/~riehl/grainfilling/index.html> Agronomic selection in seed crops during domestication has usually resulted in many-fold increases in seed weight. This has been achieved by corresponding increases in solute transfer from the plant body during seed development, which is mediated by specialised transfer cells. This project investigates the possibility of modifying, through the specific expression of transgenes, the transport function of these cells with regard to both the selectivity and efficiency of solute partitioning. BIO4-CT96-2217 - The role of the MADS-box family in plant architecture, co-ordinated by Brendan Davies, Leeds, UK. In a typical dicot plant, MADS-box genes are a family of between 40-50 transcription factors that play a major role in plant development, such as in transition to flowering, organ identity determination, fruit development and lateral root initiation. MADS-box proteins act as dimers and are involved in a complex network of molecular interactions. The aims of this project are to complete the isolation of MADS-box genes in snapdragon, petunia, and rice, to determine the full extent of MADS-box gene function in plant development by reverse genetics, to compare sequence, expression pattern and functions between gene orthologs, and to generate improved crop species by manipulation of these functions. Potential applications include induced male sterility, delayed or abolished flowering, modified flower structure, and modified shoot and root architecture. BIO4-CT97-2340 - Genetic and molecular control of the transition to flowering, co-ordinated by Caroline Dean and George Coupland, Norwich, UK. Flowering time is an important agronomic trait. The vegetative parts such as leaves, stems or roots that the plant forms before flowering, or the fruits and seeds it produces following this process, are the agricultural commodities of numerous crop plants. The aim of this project is to obtain a better understanding of the mechanisms controlling flowering time. Experiments are being conducted in three inter-related areas: the identification and isolation of new flowering time genes, the functional analysis of already isolated flowering time genes, and testing of whether these genes can be applied for purposeful modification of flowering behaviour in crop species such as oil-seed rape and lettuce.

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Embryogenesis, Development and Architecture

Cluster activities Meetings of all project co-ordinators have taken place once a year, beginning in 1998. These meetings have been useful to compare project progress and to generate common formats for project management. Furthermore, they have been a useful platform to discuss the importance of Europe-wide collaborative research projects, to reflect on the role of the EU in promoting European research, and to propose the means by which the European plant research community can best be supported in the future. A general cluster meeting is planned in early 2000 to highlight the major results from the individual projects.

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

The European Plant Embryogenesis Network (EPEN)

Introduction At the time of the call for projects for the Fourth Framework programme, the idea was launched to combine projects dealing with plant embryogenesis into a larger network. This led to a combination of first 5, and later 6 projects that together formed the European Plant Embryogenesis Network or EPEN. This idea was then well-received by the EU and rewarded with an additional modest amount of funding. Before describing in more detail the six individual projects, I would like to briefly introduce several of the activities of EPEN. In the Technical Annex of the six original projects the aims of the network were listed as follows: The first aim of EPEN is to understand and control plant embryogenesis and the formation of the embryonic root meristem. It is essential to integrate knowledge from different approaches. This is now possible through the contributing projects that range from genetic analysis of embryo pattern formation in Arabidopsis, Zea and Oryza to in vitro systems such as microspore and somatic embryogenesis and finally to root development. These areas have much more in common then is perhaps evident at first glance, and it will be a challenge to exchange results and to integrate the research in order to increase the scientific scope of the entire network. This confluence is important for the control of embryogenesis with the widest range of applications possible. The second aim is to provide access to research results for industrial partners that have an active interest in control of embryogenesis in many different forms. This means that the industrial partners and perhaps other interested small and medium sized companies must be given the opportunity to attend meetings, receive information and be able to contact directly those laboratories that are working on subjects of interest to them. The third aim is to clarify the importance and impact of this type of research for European agriculture. This must be done on a regular basis and in a way that addresses all potential interest groups.

Summary The most important means to achieve the aims of the project are summarised below. EPEN website at http://epen.tran.wau.nl. The website of EPEN has been established since October 1996. At present, close to 60,000 files have been sent to more then 2,500 individual

Sacco de Vries, Wageningen Agricultural University, Department of Molecular Biology, Wageningen, the Netherlands

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Embryogenesis, Development and Architecture email addresses all over the world. The site has been kept very simple in layout and design and offers: brief introductions into the six participating projects, links to participants' homepages, links to other sites of direct interest, facilities for electronic reporting, news and meetings section, all addresses of participants with direct email connections and passwordprotected Annual Reports of all projects. The name "EPEN" has gathered quite a high level of exposure, judged by the substantial amount of emails received with requests for information. An interesting discovery is that a substantial number of PhD students have taken up positions with other EPEN partners after graduation. This suggests a positive effect on the number of people employed and for employees offers the advantage that the students are highly trained in this specific area of research.

EPEN meetings Two general meetings have been held. The first was in 1997 in Wageningen from 30 Nov-2 December and had slightly over 100 participants. It was highly valued as an important way to get many collaborations initiated, not only between members of one project, but also between the different projects. The second was in Barcelona from 19-21 December 1998 and because of a joint venture with the fifth Plant Embryogenesis Workshop was considerably larger with 200 participants. The structure of both meetings was the same, with a general part including guest speakers from abroad and extensive poster sessions followed by closed project meetings for each of the six individual projects in order to protect IE All projects had biannual meetings.

Posters In early 1998 seven different posters, one for the EPEN and one each of the different projects were designed, printed and distributed as full-size A0 versions and smaller A4 sized versions. From the A4 version 1,000 copies were printed of each, 700 delivered to the EU and the remainder distributed amongst the EPEN participants and also distributed at meetings. A small black and white version of each posters serves as an introduction in this issue of the PIPNewsletter. It turns out that especially the smaller A4 sized posters are highly effective and are in very high demand at all meetings where they have been presented. Contacts. Important links have been established between the European Plant Biotechnology Network (EPBN) and EPEN. The EPEN co-ordinator is a member of the EPBN board. Also contacts with the Plant Industrial Platform (PIP) is maintained at co-ordinators level. The common aims are to have mutually fruitful contacts between industry and universities as well as to inform the public of the importance of plant reproduction research. As far as I have been informed, most if not all participants perceive EPEN as a worthwhile service. Two aspects are mentioned most frequently, the successful meetings and the very high number of collaborations made possible. The initial difficulties concerning confidentiality have been largely solved to satisfaction. Both awareness of the need to retain confidentiality amongst researchers as well as recognition of the value of basic work amongst company representatives has increased. Names of all participants as well as more detailed descriptions of the research activities can be found at the EPEN website at http://epen.tran.wau.nl.

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V.

Molecular Analysis Of Flowering Time And Vernalization Response In Arabidopsis, A Minireview Summary The complex network of pathways that regulate flowering time in Arabidopsis is being dissected using genetic and molecular analyses. The Dean group has focused on those pathways regulating whether plants require vernalization for early flowering. We also characterising genes that mediate the vernalization response in order to understand the molecular basis of the acceleration of flowering by cold temperature.

Introduction The acceleration of flowering by a long period of cold temperature is known as vernalization. We have been studying this process using a molecular genetic approach in Arabidopsis. Unlike the rapid-cycling types used for most Arabidopsis research, the majority of Arabidopsis ecotypes are classified as winter annual types, that is they flower late unless they have been vernalized. This ensures the plants overwinter vegetatively and flower in the favourable conditions of spring. We are investigating the genes that determine whether Arabidopsis plants require vernalization for early flowering. We have cloned and analysed FRIGIDA (FRI), a gene that represses flowering whose action is antagonized by vernalization. We have also cloned and analysed FCA, recessive mutant alleles of which confer late flowering. This late flowering can be reversed to wild-type early flowering by vernalization. In addition to analysing genes conferring a vernalization requirement, we are also investigating the molecular basis for how plants sense and respond to the cold temperature signal. The genetic basis for the perception and response to vernalization has so far not been actively explored in any plant species. We have identified a set of mutants (vrn) that define the genes which mediate vernalization.

FRI

confers a dominant vernalization requirement

Active FRI alleles delay flowering flowering (fig.l) unless the plants have been vernalized and as such FRI confers a dominant requirement for vernalization rather like that conferred by QTLs in Brassica species [1] and Shl alleles in barley [2]. Genetic analysis of a range of Arabidopsis ecotypes with different flowering times has shown that allelic variation at FRI, is the major determinant for flowering time variation in Arabidopsis [3, 4, 5]. The late flowering conferred by FRI requires the action of additional genes, the best characterised being FLC.

Caroline Dean, Department of Molecular Genetics, John Innes Centre, Norwich, UK.

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Embryogenesis, Development and Architecture

Vernalization requirement and response Genes conferring a vernalization requirement for early flowering

Genes mediating the vernalization response

vernalization

vernalization

FRIGIDA

Figure 1. Vernalizationrequirement and response. Dominant alleles of FRIGIDA confer late flowering that is reversed to early flowering by vernalization. Mutants carrying recessive alleles of FCA are late flowering and, again, this is reversed to early flowering by vernalization, vrnl, 2, 3, 4 mutants identify genes that mediate the vernalization response.

vernalization 4

fca

vrn 1 vrn2 vrn3 vrn4...

FLC encodes a MADS-box containing protein and is likely to function as a transcriptional regulator [6, 7]. FLC steady state m R N A levels are substantially increased in genotypes carrying active FRI alleles. We have recently cloned FRI using a map-based approach and found it encodes a protein with no strong homology to known proteins or domains currently in the databases and no clear cellular localisation signals. FRI RNA is also a low-abundance message. Our current work is aimed at determining whether vernalization acts directly on FRI function which then affects FLC levels, or if FRI activity functions antagonistically with vernalization to affect the levels of expression of genes such as FLC.

We are also addressing the molecular basis for how allelic variation at FRI correlates with flowering time. Sequence analysis of FRI alleles has shown that Landsberg erecta and Columbia, two rapid cycling ecotypes, carry independent deletions that cause loss of function FRI alleles. Polymorphism analysis of a larger set of ecotypes has shown that the majority of early flowering ecotypes carry one of the two deletions. Thus, rapid cycling ecotypes have evolved independently at least twice through loss of FRI function. There is a correlation between an ecotype carrying a loss of function FRI allele and its collection from southern European locations. This is consistent with loss of FRI function conferring a selective advantage to ecotypes growing in locations with less extreme winters.

fca Mutations

confer a recessive vernalization requirement

Recessive mutations in genes of the autonomous floral promotion pathway (FCA, FVE, FPA, FY, LD) confer late flowering which can be corrected to early flowering if the mutant seedlings are vernalized (fig.l) [8]. The autonomous pathway and vernalization promotion path-

116

Vernalization response in Arabidopsis way are therefore considered functionally redundant in the early flowering ecotypes in which these mutations were identified. The genes of the autonomous promotion pathway thus confer a recessive requirement for vernalization rather like the B locus in sugar beet [9] and the Vrnl locus in wheat [ 10]. We have been studying the action of the FCA gene in this pathway and have shown that FCA is part of a post-transcriptional regulatory cascade which regulates flowering time [ 11 ]. FCA encodes a protein with two RNA-binding motifs and a WW protein interaction domain. The FCA transcript is alternatively spliced at introns 3 and 13 (figure 2). Transcript f3 results from processing and polyadenylation within intron 3. A protein isoform encoded by the 13transcript would contain only a portion of the first RNA recognition motif. Overexpression of the FCA genomic clone using a 35S promoter resulted in a ~100 fold increase in transcript 13levels and little change in transcript 31suggesting that the processing of intron 3 is a regulated step in vivo. Transcript y (which has all the introns spliced out) encodes the predicted full length FCA protein and is the only transcript that complements the late flowering phenotype offca-1. Analysis of the protein isoforms encoded by transcript y shows the expected 90kD isoform and another ~ 10kD larger (Dijkwel, R and Dean, C. unpublished results). The functional significance of these isoforms is currently being analysed. A second alternative splicing of the FCA transcript occurs at intron 13 resulting in transcript ~5. A larger intron is spliced out as compared to transcript ], using non-canonical splice sites that are novel and have not been found to function as splice sites in any other eukaryotic RNA. The functional significance of the alternative splicing in FCA function is under investigation. So far we have demonstrated that splicing of the FCA transcript does limit FCA protein levels which in turn limits flowering time (Macknight, R. and Dean, C. unpublished results). In addition, comparison of 13-glucuronidase (GUS) activity from fusions where GUS was fused either in frame to FCA exon 5 or at the ATG indicate that intron 3 splicing is both temporally and spatially regulated (Macknight, R., Duroux, M. and Dean, C. unpublished results). We are currently combining a transgene overexpressing the FCA protein (a 35S-y transgene) with mutations affecting flowering time in the autonomous, photoperiod and gibberellin floral promotive pathways in order to fully establish the role of FCA in the hierarchy of genes controlling flowering time (Simpson, G. and Dean, C unpublished results). The late flowering conferred by ft, fd andfwa mutations is epistatic to the early flowering conferred by the 35S-y 117

F C A g en e

RNA-binding domains

[--I

II I III IIl i I I I 1 ~

~ [I : .....J It

FCAtranscripts ct t ~ ~

WW domain

Ir--]

!!!~ 5!i;5i!! ~i..ii!!!:i.li ~F

n ....

potential ORFs

~'

....

~

....

Figure 2. Alternative processing of the FCA transcript. The FCA gene is composed of 21 exons and covers 8.1kb. Exons encoding the RNA-binding motifs and the WW protein interaction domain are shown in black. The FCA transcript is alternatively spliced at introns 3 and 13. Transcript 13is a very low abundance transcript which still contains intron 3. Transcript ~ results from processing and polyadenylation within intron 3. Transcript 7 is a fully spliced transcript and it is the only transcript that fully complements the late flowering phenotype offca-1. A larger intron 13, extended both 5' and 3' is spliced out in transcript ~5. This results in a frameshift and premature translation stop just upstream from the WW domain. The potential and observed open reading frames are shown as black lines.

Embryogenesis, Development and Architecture transgene. Thus, FT and FD function and suppression of FWA function are required for earliness mediated through FCA expression (Simpson, G. and Dean, C unpublished results). Conversely, the early flowering conferred by the 35S- T transgene is epistatic to late flowering conferred by active FRI alleles (Simpson, G. and Dean, C unpublished results). Either FCA and FRI interact quantitatively through an antagonistic effect on a common target (eg. FLC) or FCA acts downstream of FRI function. We are currently undertaking experiments to distinguish between these two possibilities. In order to define the targets of FCA we used SELEX experiments to select RNA sequences that have a high affinity for FCA RNA-binding domains (Sivadon, E and Dean, C. unpublished results). The selection was performed against two synthetic RNA libraries, one with a 13-mer random sequence located within the loop of a hairpin structure, and one with a linear 25-mer random sequence that can have any secondary structure according to its sequence. We selected about 50 sequences from each library after seven rounds of selection. Analysis and comparison of these sequences enabled us to define two different consensus sequences (some sequences containing both) that may correspond to the RNA sequences specifically bound by FCA in vivo. A good match to one of the consensus sequences is located within the 5' untranslated region (5'UTR) of all FCA mRNAs. The FCA 5'UTR is predicted to fold in a hairpin secondary structure with the putative consensus sequence lying within the loop, at the tip of the hairpin. Gel shift experiments demonstrated that FCA RRM domains bind to the FCA 5'UTR in vitro with high affinity, in the micromolar range. Our current hypothesis is that the binding of the 5'UTR by an FCA protein isoform may regulate use of alternate translation start sites. The functional significance of this remains to be established. The FCA protein contains a putative WW protein-interaction domain. These have not been found in many plant proteins but in other systems they act like SH3 domains, interacting with proline-rich ligands or phosphoserine / phosphothreonine residues in interacting proteins. In order to identify putative FCA protein partners, we have screened both yeast two-hybrid libraries and E.coli expression libraries. No FCA-WW interacting proteins have been identified using these approaches (Dijkwel, E Simpson, G. and Dean, C. unpublished results). However, a specific FCA-WW-binding activity has been identified using affinity precipitation (Dijkwel, E and Dean, C. unpublished results). Proteins from Arabidopsis extracts were passed over a column carrying an immobilised C-terminal peptide of FCA (carrying the WW domain). The bound proteins were then eluted, run on a gel, blotted to nylon membrane and probed with a labelled WW-domain fragment. An 86kD protein was detected in wild-type plants that specifically bound to the FCA WW domain. When the affinity precipitation was done using a mutated WW-domain, binding was completely abolished. We are now analysing the presence of this 86kD binding activity in different plant extracts and genotypes. In order to identify some of the downstream targets of FCA function, we have undertaken suppressor mutagenesis experiments with the strong allele, fca-1. Ten independent suppressor mutations (termed acf, for reversal of fca) have been identified and characterised with respect to their flowering time in long and short day photoperiods, their dominance, and whether they suppress other late flowering mutations. Seven of the ACF loci have been mapped. One mutant, acf20, maps to chromosome 5 and shows a semi-dominant suppression of the fca-1 late flowering phenotype. It causes a reduction in FLC RNA levels suggesting it acts as 118

Vernalization response in Arabidopsis a dose-dependent repressor in the flowering pathway between FCA and FLC (Smart, B. and Dean, C unpublished results). We have also investigated the interaction of FCA with the meristem-identiy genes TFL1, AP1 and LFY and the floral repression gene EMF1 [ 12]. The results support a model where FCA function promotes flowering in multiple pathways, one leading to activation of LFY and AP1, and another acting in parallel with LFY and AP1. The results are also consistent with AP1 and TFL1 negatively regulating FCA function. FCA contributes to the early flowering of emfl mutants and EMF1 and FCA are likely to operate in different floral pathways. Double mutant analysis has been used to investigate the role of gibberellins, abscisic acid and phytochrome B in the promotion of flowering by FCA and vernalization (Chandler, J. MartinezZapater, J. and Dean, C submitted for publication). Vernalization was unaffected in genotypes where fca-1 was in combination with gal-3, gai, abil-1, abi2-1, abi3-1 and phyB. However, the mutations did interact with fca-1 to change flowering time in the absence of vernalization. The results suggest that gibberellin action mediated via GA1 and GAI, abscisic acid action mediated through ABI1 and ABI2 and phytochrome B, function to control floral transition in pathways that act independently of vernalization.

Identification of genes mediating the vernalization response In addition to analysing genes that confer a vernalization requirement, the Dean lab is also undertaking a comprehensive analysis of all the genes needed for a vernalization response (figure 1). Mutation of fca-1 has yielded nine mutants (termed vrn mutants) conferring a reduced vernalization response [13]. These currently define at least four complementation groups. The vrnl and vrn2 mutations did not affect the acclimation response as judged by expression of cold-induced transcripts and freezing tolerance assays, vrnl affected the shortday vernalization response of Landsberg erecta and reduced the vernalization response of other late-flowering Arabidopsis mutants. The mutations are therefore considered to define the Translocation C o l d Activation genes (VRN1 & VRN2) acting in the vernaliza~" , -stabilization , "~ - phosphorylation tion promotion pathway. VRN1 (Levy, Y. and ~ ,a ~~---.~-conformational Dean, recentlyUnpublishedmap_basedresults)and VRN2 ////~@ ~ \\ (Gendall,C. T. and Dean, C. unpublished results) have also been cloned and // \\ they encode predicted proteins with hallmarks of cy,op,. . . .II, . ,e,, transcriptional regulators. Initial studies on the I "VRN transcripts show that vernalization does not "~"~ I Fl~ I significantly alter levels of their steady state RNA. This suggests that the activation of VRN gene function by vernalization is at a post-transcrip- Figure 3. Models for activation of VRN gene functional or post-translational level. Possible mod- tion by vernalization. If VRN transcript levels are els for VRN activation are shown in figure 3. Our unaffected by vernalization, then the cold temperawork has focused on the molecular interaction of ture may activate VRN proteins or affect their subcellular localization. Alternatively, vernalization FRI, FCA and VRN gene function which dictates may activate a VRN protein partner and therefore whether Arabidopsis plants need and can respond activate the VRN genes indirectly. 119

Embryogenesis, Development and Architecture

to vernalization. It will be interesting to examine the interactions of these genes in Arabidopsis ecotypes that show a range of flowering behaviour. Knowledge of the changing interactions and predominance of the different flowering time pathways in Arabidopsis ecotypes may provide the paradigm to analyse flowering time control in plant species that show different flowering strategies.

Characterisation of the floral repressor, WLC In addition to our work focused on vernalization, we have also analysed an early-flowering Arabidopsis mutant caused by insertion of the maize transposon Ds [14]. The mutant was called wlc-1 for wavy leaves and cotyledons, but a more striking phenotype is that it is extremely early flowering in short-day conditions. The WLC gene encodes a protein with no homology to proteins or protein domains currently in the database (Hutchison, C and Dean, C. unpublished results). Analysis of the mutant shows that the floral organ identity genes, AG and AP3 are ectopically expressed in the leaves and this is likely to be caused by the hypomethylation of certain regions of the genome in the wlc mutant, wlc co mutants are almost as early flowering as wlc whereas fca wlc mutants flowered with an intermediate leaf number, fi wlc and fwa wlc mutants flowered late (Hutchison, C and Dean, C. unpublished results) placing WLC function in the FT, FWA sub-pathway [ 15] downstream of the photoperiod pathway. The wlc and fwa mutants have distinct but non-overlapping hypomethylated genomic regions [ 16] and define a sub-pathway that can act downstream of at least two of the major promotion pathways (photoperiod and autonomous). Whether the effects of accelerated flowering in antisense methyltransferase plants [17] can be accounted for by effects through this sub-pathway rather than the vernalization promotion pathway [ 18] remains to be established.

Authors of this contribution Caroline Dean ~, Paul Dijkwel, Meg Duroux, Tony Gendall, ~Claire Hutchison, Urban Johanson, Yaron Levy ~, Clare Lister', Richard Macknight, Bonita Smart, Gordon Simpson ~, Pierre Sivadon, Keri Torney ~ and Joanne West. ~Current lab members, Department of Molecular Genetics, John Innes Centre, Norwich, UK.

Acknowledgements We thank Mervyn Smith for looking after the Arabidopsis plants so well and Leah Clissold for plant transformation and plant genetic analysis. The Dean lab has been funded by the grants BBSRC CSG, BBSRC208/PG0606, BBSRC208/MOL04649, BBSRC208/CAD05634, EC BIO4-CT97-2340 and HFSPO RG0303/1997-M. Reprinted with permission from Flowering Newsletter, Nov. 1999, issue 28, pp. 6-11.

References 1. Osbom,T. C., Kole, C., Parkin, I. A. E, Sharpe,A. G., Lydiate,D. J. And Trick, M. Comparisonof flowering time in Brassica rapa, B. napus and Arabidopsis thaliana. Genetics 146:1123-1129, 1997. 2. Laurie,D., Pratchett, N., Bezant, J. H. And Shape, J. W. RFLPmappingof five majorgenes and eight QTL

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controlling flowering time in a winter x spring (Hordeum vulgare L.) cross. Genome 38: 575-585, 1995. Burn, J. E., Smyth, D. R., Peacock, W. J. And Dennis, E. S. Genes conferring late flowering in Arabidopsis thaliana. Genetica 90: 145-157, 1993. 4. Clarke, J. H. And Dean, C. Mapping FRI, a locus controlling flowering time and vernalization response in Arabidopsis thaliana. Mol.Gen.Genet. 242: 81-89, 1994. 5. Lee, I., Bleecker, A. And Amasino, R. Analysis of naturally occurring late flowering in Arabidopsis thaliana. Mol. Gen. Genet. 237: 171-176, 1993. 6. Michaels, S. D. And Amasino, R. M. Flowering locus C encodes a novel MADS domain protein that acts as a repressor of flowering. Plant Cell 11: 949-956, 1999. 7. Sheldon, C. C., Burn, J. E., Perez, E E, Metzger, J., Edwards, J. A., Peacock, W. J. And Dennis, E. S. The FLF MADS box gene: A repressor of flowering in Arabidopsis regulated by vernalization and methylation. Plant Cell 11: 445-458, 1999. 8. Koornneef, M., Hanhart, C. J. And Van Der Veen, J. H. A genetic and physiological, analysis of late flowering mutants in Arabidopsis thaliana. Molecular General Genetics 229: 57-66, 1991. 9. Boudry, E, Wieber, R., Saumitou-Laprade, E, Pillen, K., Van Dijk, H. And Jung, C. Identification of RFLP markers closely linked to the bolting gene B and their significance for the study of the annual habit in beets (Beta vulgaris L.). Theor. Appl. Genet. 88: 852-858, 1994. 10. Snape, J. W., Quarrie, S. A. And Laurie, D. Comparative mapping and its use for the genetic analysis of agronomic characters in wheat. Euphytica 89: 27-31, 1996. 11. Macknight, R., Bancroft, I., Page, T., Lister, C., Schmidt, R., Love, K., Westphal, L., Murphy, G., Sherson, S., Cobbett, C. And Dean, C. FCA, a gene controlling flowering time in Arabidopsis, encodes a protein containing RNA-binding domains. Cell 89: 737-745, 1997. 12. Page, T., Macknight, R., Yang, C.-H. And Dean, C. Genetic interactions of the Arabidopsis flowering time gene FCA, with genes regulating floral initiation. Plant J. 17: 231-239, 1999. 13. Chandler, J., Wilson, A. And Dean, C. Arabidopsis mutants showing an altered response to vernalization. Plant J. 10: 637-644, 1996. 14. Bancroft, I., Jones, J. D. G. And Dean, C. Heterologous transposon tagging of the DRL1 locus in Arabidopsis. The Plant Cell 5:631-638, 1993. 15. Simpson, G., Gendall, A. And Dean, C. When to switch to flowering. Ann. Rev. Cell Dev. Biol. 15: 519550, 1999. 16. Soppe, W.J.J., Jacobsen, S.E., Alonso-Blanco, C., Kakutani. T., Peeters, A.J.M. And Koornneef, M. The gain of function epi-mutant fwa causes late flowering. Abstract Int. Arab. meeting Melbourne 1999. 17. Finnegan, E. J., Genger, R. K., Kovac, K., Peacock, W. J. And Dennis, E. S. DNA methylation and the promotion of flowering by vernalization. Proc. Natl.Acad.Sci.USA 95: 5824-5829, 1998. 18. Burn, J. E., Bagnall, D. J., Metzger, J. D., Dennis, E. S. And Peacock, J. E. DNA methylation, vernalization, and the initiation of flowering. Proc. Natl. Acad. Sci. U.S.A. 90: 287-291, 1993. 3.

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V.All rights reserved.

Modification Of Plant Development By Genetic Manipulation Of The Ethylene Biosynthesis And Action Pathway Introduction Ethylene is one of the five "classical" naturally occurring plant hormones [1 ]. It is a key regulator of plant growth and development, as it is involved in most of the developmental processes of the plant, from seed germination, stem and root elongation, flowering, to leaves and flower senescence, fruit development and ripening. Moreover ethylene is inducible also as a response to biotic and abiotic stress, such as pathogen attack, wounding, water logging and drought [2]. In the last decade, the biosynthetic pathway of the chemically simple gas molecule ethylene (C2H4)has been established and several steps of the mechanism of perception and transduction pathway of the ethylene signal have been elucidated (Fig. 1). Ethylene derives from the "Yang" cycle [3]; methionine is converted to ethylene with S-adenosylmethionine (SAM) and 1aminocyclopropane-l-carboxylic acid (ACC) as intermediates. These two reactions are catalysed by ACC synthase (ACS) and ACC oxidase (ACO). Ethylene signal is perceived by receptor proteins and transduced trough a phosphorylation cascade that lead to transcription factor activation and consequently ethylene-related gene expression [4]. Due to its role in regulation flower senescence and fruit ripening, the control of ethylene production and perception has always been a major issue to prevent fruit/flower spoilage and prolong marketability. Plant physiologists have been challenged in the search of the optimal treatment of harvested fruit, to prevent the metabolic processes that are induced by ethylene. Plant biotechnology enabled to control ethylene biosynthesis and perception in plants. Mutants and transgenic plants altered in ethylene perception or production have been generated either to elucidate ethylene function and the mode of action but also to improve fruit and flower shelf-life. The aim of this review is to illustrate how ethylene affects plant development by illustrating the plant phenotype of the transgenic plants and the mutants described so far.

Seed germination, the "triple response" of Arabidopsis thaliana During seed germination and emergence from the soil, the terminal part of the shoot axis of certain dicotylodenous plants exhibits an apical arch-shaped structure, called "apical-hook". The apical hook may protect the delicate apical tissues of the growing meristem from injury

Domenico De Martinis, Ente Nazionale per Le Nuove Tecnologie, Energia e Ambiente (ENEA), Roma, Italy.

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while the stem is emerging from the soil into open air. During germination in the dark (etiolated seedlings), ethylene is produced locally in the apical hook region and is involved in determining the shape of the plantlet. As soon as the etiolated plantlet tissues become photosynthetically competent, they lose sensitivity to ethylene. The application of ethylene to the etiolated seedlings results in the "triple response"; this effect consists of three distinct morphological changes in the shape of the seedling: inhibition of stem elongation, radial swelling of the stem and absence of normal geotropic response (diageotropism). This effect, that was first described in the beginning of the century [5] enabled to identify in the model plant Arabidopsis thaliana (Arabidopsis) several mutations mutation related to ethylene production and perception [6]. In Arabidopsis the seedling phenotype is easily scored by visual inspection or with the aid of a dissecting microscope. After germination in the dark seedlings germinated in 10~l/ethylene show inhibition of root Methionine MTRand hypocotyl elongation, exaggerated tightenPPi+P~ ADP YANG ~ ATP ing of the apical hook and swelling of the hypoATP J I cyc i e ~a~[2~2;~[ ......... MTR SAM cotyl, whiIe air germinated seedlings are highly elongated and exhibit the apical hook at the ter~ I ~-----AVG MTA ACC synt ha s e minal part of the shoot axis. More than a dozen ACC ethylene response mutants have been identified 1/2 < AIB by screening for alteration in the triple response. HCN+C02 ACCoxidase Two major classes of mutants affected in the ethethylene C2H4 ylene production or perception pathway are Per cep t9~q-present: a) mutants that show a constitutive triple response in absence of ethylene, namely, etol ( ethylene overproducer) eto2, eto3 and ctrl (for (ERS1, ETR2, EIN4) constitutive t_fiple response) b) mutants that do not exhibit a triple response if germinated in the presence of ethylene, namely etrl (for ethylene response) etr 2, ein2 (for ethylene insensitive) ein3, ein4, ein5, ein6, ein7 and ainl (for ACC Phosphorilation cascade insensitive). Molecular cloning of the ETR gene revealed that it encodes a receptor for ethylene v K// [7], and CTR1 encodes for a Raf-like serine / ,~~m (~.IT.Z, ~.Ir.2) Primary Targets threonine kinase [8], while genetic analysis have 3 ~ gene r e s p o n s e I -I placed these genes in a genetic pathway [9]. More recently, molecular approaches enabled the clon~,~ (EREBPS) Secondary Targets gene response ing of at least other four ETR l-like genes [ 10,11 ] I and genetic studies indicated the mode of action Figure 1. Ethylene biosynthesis and action. of the ethylene signalling pathway [ 12]. The conAdapted from ref. 2,3,7-12,39,40. ACC, 1stitutive triple response of the eto mutation is a aminocyclopropane-l-carboxylic acid; ATE result of a dramatic difference in ethylene proadenosine triphosphate; KMB, 2-keto-4methylthiobutyrate; MACC, malonyl-ACC; duction, at least 40-fold higher than the correMTA, 5'-methylthioadenosine; MTR, 5'sponding wild-type etiolated seedling and two to methylthioribose; MTR-I-P, MTR 1-phosfive fold higher in light-grown eto seedling and phate, SAM, S-adenosylmethionine. adult tissue if compared to the ethylene producAVG aminoethoxyvinilglycine; AIB t~-aminoisobutyric acid. tion relative to wild type. These observation were 1 -P

0

~

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124

Ethylene biosynthesis and action pathway confirmed by reversion of the eto seedlings to wild-type like phenotype if chemical inhibitors of ethylene biosynthesis (AVG, AIB) were added to the germination media. Ethylene insensitive mutants (etr and ein ) showed at least a threefold difference in the length of the hypocotyl in the presence of ethylene if compared with ethylene treated wild-type hypocotyl. In these mutants, the apical hook was either absent or showed some curvature at shoot apex. The easily scored triple response phenotype of an ethylene-treated Arabidopsis seedlings provides a powerful screen to identify ethylene-related mutants, observation of mutant plant lines growth to maturity indicated alteration in the growth habits, indicating that the effect of the mutation is not seedling specific. Ethylene overproducer mutants grew more slowly and showed reduced size of the rosette, however, they bolted at the same time than in wild-type plants. Moreover, the rosette leaves were darker green and the main inflorescence and lateral branches showed purple pigmentation. In contrast the rosette of the ethylene insensitive mutants was larger than the wild-type rosette and delay in bolting was observed. Finally, no dramatic effects were observed in mutant fertility, despite it was reported that the gynoecium of mutants that show an ethylene constitutive response (ctrl) mature earlier in development relative to the rest of the flower. In ethylene-insensitive Arabidopsis mutants (Ein) maturation of the gynoecium is delayed relative to the rest of flower development [8]. These observations related to the plant growth habits and flower development will be better evaluated further in this review.

Plant development Ethylene does act to slow growth of many plant tissues. This can be easily seen in transgenic tobacco in which auxin overproduction and its concomitant ethylene overproduction causes a strong apical dominance and a large reduction in internode elongation. The introduction in the transgenic lines of an ACC deaminase gene, (that reduces to wild-type levels ethylene production) eliminates the reduction of internode length, without to affect the auxin related apical dominance effect [13]. This example shows that there are complex interactions between the plant hormones and sometimes is difficult to distinguish the hormone specific effect on plant growth and effects consequent to the overall hormonal rate. In any case, due to its involvement in response to stress and in the control of ripening and senescence, several plant species have been engineered for reduced production or sensitivity. The first breakthrough in the biotechnology of ethylene was the generation of transgenic tomato that was inhibited in ethylene synthesis by antisense gene silencing approach [14]. By introducing a chimaeric gene consisting of the antisense TOM13 gene under the control of the 35S CaMV promoter resulted in reduced ethylene synthesis. Notably, that was the first demonstration that the antisense approach could lead to the identification of gene of unknown function, indicating that TOM13 encodes for an ACC-oxidase involved in the conversion of ACC to ethylene. Other examples of constitutive reduction of ethylene production and perception are also in other plants species, among which tobacco [15, 16] and petunia [17]. Despite these plants were mainly characterised for fruit ripening (tomato) and for flower senescence (petunia) or regarding biotic stress (tobacco) they provided an opportunity to study how reduced ethylene action affects the vegetative development of the plant. Transgenic tomato transformed either antisense ACS [18] and ACO [14] transgenes evolve greatly reduced levels of ethylene. Silencing rest~lted in a reduction of ethylene production by 68% in wounded leaf and by 87% up to 99.5% in ripening fruit. In transgenic "low ACO" tomatoes, leaf senescence that is 125

Embryogenesis, Development and Architecture characterised by visible chlorophyll loss, was delayed by 10-14 days. Despite the decreased ethylene production levels, leaf senescence was not arrested although it was significantly delayed. Constitutive expression of ACS in transgenic tomato plants [ 19] resulted in high rates of ethylene production by many tissues of the plant and induced petiole epinasty and premature senescence and abscission of flowers, usually before anthesis. Still, there were no obvious effects on senescence in leaves of ACS overexpressers. Taken together, these comparison between low and high ethylene producers in tomato suggest that, although ethylene may be important, it is not sufficient alone to determine tomato leaf senescence. Thus, together with ethylene other signals must be involved. A second feature of low-ethylene producing transgenic tomatoes was a characteristic dark-green pigmentation, comparable to the pigmentation of the ethylene insensitive Arabidopsis mutants (see previous paragraph), that pigmentation is correlated with a clear retention of photosynthetic activity. Molecular analysis [20] demonstrated that the expression pattern of two photosynthetic-associated genes the chlorophyll a/ b-binding protein (cab) and the ribulose biphosphate carboxylase / oxygenase small subunit (rbcs) was higher in transgenic "low-ACO" tomatoes than in wild-type leaves of the same age; the mRNA for cab and rbcs decline during normal leaf senescence, thus indicating that in transgenic tomatoes the delay in leaf senescence was not only a visual characteristic but also reflected a the molecular composition of the transgenic plant tissues. Moreover, the analysis of the photosynthetic activity by mean of chlorophyll content, oxygen evolution, and quantum yield of photosystem II in vivo (Fv/Fm, ref. 21), indicated a clear retention of photosynthetic capacity in leaves of transgenic tomatoes, thus supporting the hypothesis that photosynthetic decline is coupled with the senescence syndrome [22]. Also in tobacco, transgenic plants were generated to express sense and antisense transcripts corresponding to ACS and/or ACO, but ethylene production resulted inhibited with less efficiency than in tomato (by 74%), while by overexpressing the gene encoding for ACS the highest level of ethylene was by 320% (overproduction). High-ethylene producer tobacco plants had a shorter internodes and resulted in a reduced size and/or internodal length if compared to wild type or low-ethylene producer plants. This result is similar to that described for auxin/ethylene overproducer already mentioned [13], and for potato expressing antisense SAM-decarboxylase [23], which resulted in high ethylene levels. Also in transgenic tobacco, low-ethylene production corresponded with an higher content of chlorophyll in leaves than is wild type leaves of the same age. Another successful approach to study ethylene action has been the inhibition of ethylene perception by expressing the gene encoding for a mutant ethylene-receptor from Arabidopsis in heterologous plants [ 16, 17]. The introduction of the etrl-1 sequence encoding in tomato, petunia and tobacco conferred ethylene insensitivity to the transformed plants. In tobacco, the absence of ethylene sensitivity seemed to reduce their perception of neighbouring plants. In general, when wild type seedling a were grown together, plant growth slowed before leaves of neighbouring plants overlapped. These plants remained relatively small and exhibited accelerated leaf senescence. In contrast ethylene-insensitive plants did not appear to perceive their neighbouring plants. They did not show a reduction in growth, resulting in a "crowding effect" of interdigitating leaves, which, moreover, remained fully green. Roots too, respond markedly to ethylene. Numerous studies provided evidences that ethylene is a positive regulator root epidermal development. Early studies it was shown that ethylene induced a mass of root hairs on Elodea in conditions where this plant normally formed no root hairs [24]. Analysis of ethylene-related Arabidopsis mutants provided a versatile tool to 126

Ethylene biosynthesis and action pathway observe root hairs development. In Arabidopsis, cells in the root epidermis are arranged in "hair-forming" and "non-hair" forming files [25], hair files are located over the anticlinal (radial) walls of underlying cortical cells and non-hair files are located over the outer periclinal (tangential) walls of cortical cells. The phenotypic characterisation of ctrl-1 mutant roots has implicated ethylene in the process of cell patterning and differentiation in the epidermis [8, 26]; root-hair spacing in ctrl-1 is abnormal and root hair cells differentiate in the position normally occupied by non-hair cells. Despite this observations, as the CTR 1 gene encode presumably for a protein kinase of the Raf family, it is possible that the mutation affects other pathways independent of ethylene in the specification of epidermal pattern. Surprisingly, ethylene insensitive mutants, ein2 and etrl do produce normal root hairs [27], anyhow, it is possible that those mutants retain a residual ethylene response sufficient for some root hair development. To elucidate the role of ethylene in root development the study of mutants was elegantly complemented by the use of chemical inhibitors and inducers of ethylene production and perception. The combination of mutant analysis and pharmacological studies indicated that ethylene promote root air elongation. As proposed in a model for the development of root hairs in relation of ethylene has been proposed [26], during normal development nonhair forming cells are not exposed to exogenous ethylene or are exposed to levels below the threshold necessary to induce the differentiation of root hairs. Ethylene inhibition results in complete inhibition of root hairs (either in non-hair and hair-cells), whether ethylene exogenous delivery of ethylene precursor ACC or ethylene constitutive-response in mutants results in root-hair development where non-hair cells are located. It is possible that in normal conditions, root anatomy determines the delivery of ethylene or its precursor ACC only to the "hair-cell" files thus, specifically inducing root hair formation.

Flower development Pollination-induced ethylene production and flower senescence have been the most widely studied ethylene-related physiological changes in the flower [28]. Still, ethylene may act also in flower induction and sex determination. The induction of flowering by ethylene is of considerable commercial importance in pineapple and in other tropical fruits [29]. Despite its economical importance, no information is yet available on the mechanism by which ethylene induces flowering in these species. Induction of flowering could be linked to the events that determines sex determination in the flower; it has been demonstrated that application of ethylene (or ethylene-releasing compounds) to seedlings would dramatically change the ratio of male to female flowers in members of Cucurbitaceae [3]. Recently it was reported the isolation of an auxin-inducibile gene encoding for an ACC synthase, tightly associated with the F locus that determines female sex expression in cucumber, supporting the hypothesis that ethylene plays a pivotal role in the determination of sex in cucumber flowers [30]. In monocots, namely orchid, [31 ] it was shown that pollination and auxin regulate ethylene production and ovary development. When inhibitors of ethylene were used, pollination- or auxin-induced ovary development were inhibited. More recently it was hypothesised that an unknown pollination factor has a synergistic effect with auxin in stimulating ethylene biosynthesis and consequently ovary development in orchid [32]. Also in Petunia flowers, the expression of the ACO gene family, is temporally regulated during pistil development [33], and it was suggested that ethylene plays a role in reproductive physiology by regulating the maturation of the secretory tissues of the pistil. A direct evidence that ethylene is necessary to induce female 127

Embryogenesis, Development and Architecture gametophyte development was provided in tobacco [34]. The isolation and characterisation of a tobacco pistil-specific ACO gene revealed expression in the tobacco ovary when the first events of megasporogenesis occur. The pattern of expression of the ACO gene isolated was specifically linked to the reproductive tissues of the pistil suggesting a specific role of this gene in the reproductive physiology of the tobacco flower. Transgenic tobacco plants in which that pistil-specific ACO gene was silenced showed a flower phenotype with a reduced size and female sterility. Cytological analysis revealed that in the transgenic plants, ovules did not complete megasporogenesis and did not produce an embryo sac. Moreover, the supply of an ethylene source was sufficient in itself to recover fully developed and functional ovules, clearly demonstrating that ethylene alone induces ovule maturation in tobacco. Outside of orchids, in which ethylene-related ovary development was triggered by pollination, this is the first evidence of a direct role of ethylene in ovule development. Despite these evidences, it is surprising that no clear data on the reproductive biology of ethylene-related Arabidopsis mutants or ethylene insensitive transgenic plants were never provided. Recent advances in the studies on mechanism of ethylene perception and transduction in Arabidopsis [10, 11 ], have shown that the ethylene receptors are encoded by a gene family that in Arabidopsis is comprised of at least five members that may possibly posses different ethylene binding affinities and signaling activities. It is possible that redundancy of these genes masks ethylene effect on flower development and fertility. One of these gene family members, the gene encoding for the ethylene receptor ETR2, shows an expression pattern enhanced in the developing carpels especially in the funiculi and in the ovules since the early stages of megasporogenesis. Though the flower phenotype of these ethylene insensitive mutants has never been described so far, these observations together with the data provided on cucumber, orchids, and tobacco suggest that ethylene plays a role in female gametophyte development in several plant species. Once the flower has been pollinated, major developmental changes occur, involving an interorgan signalling within the flower. Upon pollination a transient increase in ethylene production is induced in several flowers such as, i.e., orchids, petunia and tobacco [31, 33, 35]. This transient ethylene production is responsible for petal senescence, but it is also necessary to induce deterioration of pistil transmitting tissue that is though to facilitate pollen tube growth toward the ovary. The senescence-related ethylene responses of the flower are very important for floriculture, as many important floricultural products are extremely sensitive to ethylene. Genetic engineering to reduce the rate of ethylene biosynthesis maybe not sufficient as the presence of external ethylene may anyhow induce flower senescence and reduce drastically marketability. To overcome this problem, genetic manipulation to reduce ethylene sensitivity rather than production seems to be a more successful strategy. Transgenic petunia plants containing the etrl-1 cDNA have been characterised for the lack of ethylene sensitivity [ 17]. In particular, the senescence and abscission of flowers following pollination were monitored as indication of ethylene sensitivity. Corollas of pollinated flowers from transgenic petunia remained turgid and structurally intact for at least 5 days longer than the corresponding wild type control flowers. The molecular basis of the extended flower-life and delayed abscission in the transgenic petunia was shown to be related to ethylene perception rather than ethylene synthesis, as flower from transgenic petunia was found to exceed that of control flowers, indicating that the capability to respond to pollination by triggering a burst of ethylene was still intact in the transgenic plants. Despite a more through characterisation of the transgenic plants produced with this technology will be necessary to reveal any alteration in growth, 128

Ethylene biosynthesis and action pathway fertility, disease susceptibility and responsiveness to environmental stresses, this work demonstrated that the production of ethylene-insensitive transgenic plants may represent a valid alternative to the use of biochemicals to prolong flower shelf-life.

Fruit ripening The stimulation of fruit ripening is one of the earliest reported effects of ethylene. Fruits are classified as climacteric or non-climacteric according to their respiratory output at the onset of ripening process and the ability of ethylene to stimulate autocatalytic production of ethylene. Classic climacteric fruits such as bananas, apples, pears and tomatoes show a clear increase in respiration at the onset of ripening concomitant with a dramatic increase in the rate of ethylene production. The melon, in which a substantial rise in ethylene production occurs before the onset of ripening, is not typical of climacteric fruits. Non-climacteric fruits are i.e. the citrus family and strawberries, that do not show increase in respiratory activity neither ethylene evolution at the onset of ripening. Ripening correspond generally with the alteration of colour, flavour, aroma and texture, and the role of ethylene in ripening of climacteric fruits, especially in tomato has been studied not only for his scientific value, but also for the economic importance of the tomato fruit as a major food crop. The first evidence that the ethylene biosynthetic pathway could be manipulated in transgenic plants was provided in tomato[ 14, 18]. As already mentioned in this review the stable integration and expression of the ACO gene in antisense orientation resulted in a clear reduction of the rate of ethylene biosynthesis in fruits by 97%. Using the same approach, the expression of an antisense ACS gene resulted in an inhibition by 99.5% of ethylene production in fruits. These two example of manipulation of the ethylene biosynthetic pathway opened the way to other successful attempts of manipulation of ethylene production to achieve extended fruit storage life, such as melon fruits [36]. In these three example of "low ethylene" transgenic plants (tomato ACO antisense, tomato ACS antisense and melon ACO antisense) reduction of ethylene biosynthesis resulted in increased resistance to over-ripening either on plants or detached and stored in air. Transgenic ACO antisense developed normally after fertilisation, and colour changes at the onset of ripening resulted normal if compared to wild-type tomatoes. However, despite the timing of the onset of ripening was unaffected, the subsequent ripening process was moderated; reddening of the fruit was reduced and once fruits were stored at room temperature, fruits from ACO antisense transgenic tomatoes were noticeably more resistant to over-ripening and shrivelling than control fruits. The reduction of reddening was consistent with a clear reduction of carotenoid pigments, specifically lycopene, and reduction of the expression of the mRNA encoding for the phytoene synthase, an enzyme involved in carotenoid biosynthesis. A similar phenotype was observed in transgenic ACS antisense tomato fruits that shown reduction of red coloration derived from lycopene accumulation and a progressive loss of chlorophyll that resulted in a yellow colour of the fruit. Fruits kept on air or on the plants for 90 to 120 days will eventually develop an orange colour but never turn fully red and soft or develop an aroma. Different behaviour was observed in fruits from transgenic ACO antisense melon; in those fruits, ethylene production was reduced by 99%. The ripening processes of chlorophyll degradation, fruit softening and activation of the peduncular abscission zone resulted to be ethylene-dependent and were therefore reduced, whilst flesh pigmentation resulted to be ethylene-independent. This is consistent with the observation that in melon the phytoene synthase gene is expressed before the onset of ripening. One remarkable feature of the transgenic 129

Embryogenesis, Development and Architecture melon was the absence of activation of the peduncular abscission zone; as a consequence fruit did not drop from the plant even at very late stage of development this results in a higher sugar contents in the fruits from transgenic melon. In general all the three transgenic "low ethylene" plants produced fruits with longer shelf-life, but resulted to be affected in normal development of colour (tomato) and aroma (tomato and melon, ref. 37). Fruit phenotype could be reverted upon delivery of external ethylene, or its analogue propylene, either in tomato than in melon, but while ripening seems to be completely reverted in transgenic ACS antisense tomato and transgenic ACO antisense melon, it did not seem to be completely reverted in transgenic ACO antisense tomatoes. In contrast with ACS antisense tomatoes, ACO antisense tomato fruits detached from the plant failed to accumulate lycopene to wild-type levels. The authors [38] suggest that ethylene is not the only trigger of fruit ripening and suggested the involvement of another "ripening-factor-X" associated with attachment to the plant which can modulate ripening in conjunction with ethylene.

Conclusions The plant hormone ethylene is involved in a wide range of developmental processes, as well as in biotic and abiotic stress responses in plants. Despite its relative chemical simplicity, its biosynthesis involves two key enzymes (ACS and ACO) each encoded by a multigene family and differentially expressed in response to different environmental or developmental stimuli. Ethylene perception and signal transduction too is mediated by a family of receptors that activate a phosphorylation cascade that lead to transcription factor activation and ethylenerelated gene-expression [7, 8, 9, 10, 11, 12, 39, 40]. The diverse role that ethylene plays and the specificity of its action suggests complexity in the regulation of its synthesis and in its signal transduction pathway. In this review were described the alterations in the plant developmental processes upon manipulation of ethylene biosynthesis and action by mutagenesis or genetic engineering (figure 2). Several approaches demonstrated to be successful in the modification of the ethylene signal, and delayed ripening/senescing fruits and flowers have been generated. The production of ethylene-insensitive transgenic plants is a complementary strategy to the alteration of ethylene biosynthesis. The latter is necessary in climacteric fruits in which an ethylene treatment is necessary to induce proper maturation when desired (before release on the market or before processing). Reduction of ethylene sensitivity could be successful in floriculture but also in those cases in which ethylene causes post-harvest spoilage as Fruit ripening lycopene biosynthesis fruit abscission

Figure 2. schematicrepresentationof the effects of ethylene during plant growth.

Ethylene ~ ~ ,

Seedling Etiolated 'triple seedling response'

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development

130

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Ethylene biosynthesis a n d action p a t h w a y

for lettuce and c u c u m b e r s [ 17]. Still, in this review it was described how constitutive modification of ethylene production/action in the plant could produce u n e x p e c t e d side-effect, as the wide n u m b e r of genes involved in ethylene m e t a b o l i s m determine the rate of biosynthesis and the sensitivity b e t w e e n different plant species, b e t w e e n tissues and b e t w e e n different develo p m e n t a l stage of the plants. The analysis of the plants genetically altered in ethylene metabolism will enable to understand the different responses to ethylene and distinguish between ethylene-related and e t h y l e n e - i n d e p e n d e n t plant responses [41 ]. In the future, it is conceivable that the dissection of the ethylene p a t h w a y of biosynthesis and action will enable us to generate plants with targeted modification of specific responses by the use of tissue-specific, developmental-regulated chimaeric genes.

Acknowledgment D.M. acknowledges the EC, FP4 for the support grant FAIR CT98-4211 "Fruta Fresca: imp r o v e m e n t of natural resistance in fruit".

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Embryogenesis, Development and Architecture Nature Biotechnology, 15, 444-447. 18. EW., Oeller, M.W., Lu, L.E, Taylor, D.A., Pike, A. Theologis, (1991) Reversible inhibition of tomato fruit senescence by antisense RNA. Science, 254, 437-439 19. H.C.Lanahan, J.J., Yen, Giovannoni and H.J. Klee (1994) The never ripe mutation blocks ethylene perception in tomato The Plant Cell Vol 6, 521-530 20. J., Isaac, R., Drake, A., Farrell, W., Cooper, L., Lee, E, Horton, and D., Grierson. (1995). Delayed leaf senescence indeficient ACC-oxidase antisense tomato plants: molecular and physiological analysis. Plant J. 7(3),483-490 21. O. Bjorkman and B., Demming. (1989) Photon yeld of O 2 evolution and chlorophyll fluorescence characteristics at 77K among vascular plants of diverse origin. Planta, 170, 489-504. 22. L.L., Hensel, V. Grbic, D.A., Baumgarten, and A.B. Bleecker (1993) Developmental and age-related processes that influence the longevity and senescence of photosynthetic tissues in Arabidopsis. The Plant Cell, 5, 553-564. 23. Kumar, M.A. Taylor, S.A. Mad Arif, H.V. Davies (1996) Potato plants expressing antisense and sense Sadenosylmethionine decarboxylase (SAMDC) transgenes show altered levels of polyamines and ethylene: antisense plants display abnormal phenotypes. Plant J. 9:147-158 24. H., Cormack, (1935) The development of root hair by Elodea canadensis. New Phytol. 34, 19-25 25. Do!an Duckett, C.M., Grierson, C., E, Linstead, Schneider, K., Lawso, E., Dean, C., and Roberts, K., (194) Clonal relationships and cell patterning in the root epidermis of Arabidopsis. Development, 120, 2465-2474. 26. Tanimoto, K., Roberts, and L. Dolan. (1995) Ethylene is a positive regulator of root hair development in Arabidopsis thaliana. Plant J. 8(6) 943-948 27. Pitts, A. Cernac, and M. Estelle. (1998) Auxin and ethylene promote hair elongation in Arabidopsis Plant J. 16(5), 553-560. 28. S. D. ,O'Neill and J.A., Nadeau. (1997) Postpollination flower development. Hortic. Rev., 19, 1-58. 29. M.S. Reid. Ethylene in plant growth, developmen and senescense, in: P.J. Davies (Eds.) Plant Hormones, 2nd edition, Kluwer Acad. Publishers pp.486-508. 30. T., Tova, J.K.E., Staub and S.D., O'Neill (1997) Identification of a 1-Aminocyclopropane-l-Carboxylic Acid Synthase gene linked to the Female (F) locus that enhances female sex expression in Cucumber. Plant Physiol. , 113:987-995. 31. X.S., Zhang and S.D., O' Nei11,(1993) Ovary and gametophyte development are coordinately regulated by auxin and ethylene following pollination. The Plant Cell, 5, 403-418. 32. A.Q., Bui and S.D., O'Neill, (1998) Three 1-Aminocyclopropane-l-Carboxylate Synthase genes regulated by primary and secondary pollination signals in orchid flowers. Plant Physiol., 116, 419-428. 33. X.,Tang, A.M.T.R., Gomes, A., Bhatia and WoR., Woodson (1994) Pistil-specific and ethylene-regulated expression of 1-aminociclopropane-l-carboxytate oxidase genes in Petunia flowers. The Plant Cell, 6, 12271239. 34. D., De martinis and C., Mariani (1999) Silencing gene expression of the ethylene-forming enzyme results in a reversible inhibition of ovule development in transgenic tobacco plants. The Plant Cell, 11, 1061-1071. 35. Wang H, Wu HM and Cheung AY, Pollination induces mRNA poly(A) tail-shortening and cell deterioration in flower transmitting tissue. Plant J. 9 (5), 715-727 (1996). 36. R., Ayub, M., Guis, M.B., Amor, L., Gillot, J.E, Rousten, A., Latche, M., Bouzayen, and J.C., Pech, (1996). Expression of ACC oxidase antisense gene inhibits ripening of cantaloupe melon fruits. Nature Biotech. 14, 882-886. 37. A.D. Bauchot, E, John, D.S., Mottram. Role of ethylene on aroma formation n cantaloupe charentais melons, in: A.K., Kanellis (Eds.) Biology and biotechnology of the plant hormone ethylene II,NATO-ASI series, Kluwer Acad. Publishers, in press. 38. S., Picton, S.L., Barton, M., Bouzayen, A.J. Hamilton, and D., Grierson. (1993) Altered fruit ripening and leaf senescence in tomatoes expressing an antisense ethylene-forming enzyme transgene. Plant J. 3(3), 469481. 39. M., Ohme-Takagi, and H., Shinshi, (1995) Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element. The Plant Cell, 7, 173-182. 40. R., Solano, A., Stepanova, C.,Qimin and J.R., Ecker. (1998) Nuclear events in ethylene signaling: a trascriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1. Genes & Dev.3703-3713 41. A. Theologis (1993) Use of a tomato mutant constructed with reverse genetics to study fruit ripening, a complex developmental process. Dev. Genet. , 14, 282-295.

132

Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V.All rights reserved.

Molecular Aspects Of The Strawberry Fruit Softening

Summary The involvement of endo-B-1,4-glucanases (EGases, EC 3.2.1.4) in the ripening of strawberry fruits has been investigated by means of structural, biochemical, molecular and cytological techniques. Endo-g-l,4-glucanase activity starts to be detectable at the end of fruit cells expansion (i.e. large green stage), has a steep increase during the ripening process and reaches a maximum in red fruits. It is caused by two different isoforms both increasing along the ripening process. Two full-length cDNA clones (faEG1 and faEG3) coding for divergent EGases have been obtained by screening a library representing transcripts from red strawberry fruits. The transcripts related to the two cDNAs accumulate during ripening, however faEG3 related mRNA can also be detected, although at very low levels, in green berries and in other green developing tissues. In fruits, both transcripts are down-regulated by exogenous application of an auxin analogue (1-naphthalene acetic acid), thus confirming that auxin has a fundamental role in the ripening of the nonclimacteric strawberry. The protein encoded by faEG3 has an extra peptide of about 130 amino acids at its C-terminus. In particular, this peptide shows striking homologies to the cellulose binding domains of some microbial cellulases. The structural and cytological findings are well in agreement with the expression pattern of the two different endo-B-1,4-glucanases during the ripening of strawberry fruits.

Introduction The softening of fleshy fruits is a physiological process that has a very strong economic impact. For instance, fruit texture is of paramount importance for the processing industry, being a key parameter for the preparation of frozen and canned fruit, juices, jams, purees and other derivatives. Furthermore, it has been shown that fruit firmness is a key factor for the post-harvest life of many fleshy fruits. Hard fleshy fruits can better tolerate the accidental damages which might occur during their harvesting and shipping and are more resistant to microbial infections than soft ones [1], therefor they can have a better commercial quality when they reach the end of the distribution chain, i.e. the customer. For the same reasons

Livio Trainotti, Department of Biology, University of Padua, Padua, Italy

133

Embryogenesis, Development and Architecture harder fruits can better tolerate prolonged storage, thus extending their availability and reducing losses due to rotting. In many cases fleshy fruits are harvested before optimal ripening, e.g. when tissues are still hard enough to tolerate the stress that the fruit will meet before reaching the customer's table. Although the ripening process can continue after the fruit harvesting, there are two major disadvantages in harvesting fruits before their complete ripening: organoleptic characteristics are lower than in fruits ripened naturally and storage life is not yet under full control. In the physiology of fruit ripening a lot of efforts has been put to unravel the skein of fruit softening and ultrastructural [2, 3 and others] and biochemical [review in 4] researches have been focused to the understanding of the cell wall metabolism. The dismantling of middle lamella and primary cell wall, which leads to loss of adhesion between adjoining cells, was observed in many different fruit species [2, 3, 5 and others]. These observations revealed the expression of many different classes of wall degrading enzymes. However the most commonly expressed enzymes degrade either pectins (polygalacturonases) or glucans (endo-131,4-glucanases). These are the two classes of enzymes that are more deeply involved in fruit softening and therefore characterised with more details. Many different fruits have been studied, although tomato and avocado have become the model fruits for studying polygalacturonase (PG) and endo-13-1,4-glucanase (EGase), respectively. Initially, the work done on the tomato PG gave evidence that this hydrolase was responsible for the fruit softening occurring during ripening [review in 1]. However, subsequent experiments carried out with the gene silencing technology led to the conclusion that this single enzyme is not the only responsible of the tomato fruit softening, thus giving the genetic evidence that cell wall degradation is the result of the co-ordinated action of different cell wall degrading enzymes [1 ]. Biochemical data demonstrating the presence of endo-f~-1,4-glucanase (EGase, formerly called cellulase, EC 3.2.1.4) activity during fleshy fruits ripening date back to the 60's [6]. However, the observation that cellulose was neither dissolved nor reduced in molecular weight during ripening relegated this enzyme to a marginal role. Only in recent years new efforts have been put in order to find a role for EGases in fruit softening. Pivotal experiments carried out with avocado fruits allowed the isolation of the ripening EGase. Interestingly, the availability of the pure enzyme made it possible to understand that the physiological substrate for this enzyme was not the crystalline cellulose [7]. However after many years of work and with many EGase sequences known, conclusive proofs about the natural substrate(s) for these hydrolases are not yet available, though it is clear that they play a key role in the disassembly of the cellulose-hemicellulose network [8]. In strawberry, early data about the presence of EGase activity in ripening fruits [9] were not given further attention until 1990, when Abeles and Takeda [10] observed an ethylene-independent rise in EGase activity during the ripening process proper. In this paper we show that two isoenzymes with different isoelectric points contribute to the EGase activity observed in ripening strawberry fruits. The characterization of two different EGase cDNAs (faEG1 and faEG3) well correlates with the presence of two different isoenzymes. Expression of the mRNAs related to both cDNAs has been examined in a number of different tissues. In particular, during the fruit ripening the two EGase genes show differences in temporal but not in spatial expression. The effect of auxin treatment has been analysed in fruits both at the onset and at 134

Molecular aspects of the strawberry fruit softening the end of the ripening process proper. Analysis of the two cDNA sequences has revealed that, while the EGase encoded by faEG 1 is a typical E-type plant EGase, faEG3 codes for a protein which is novel for higher plants. In fact, besides having the usual E-type EGase polypeptide [11 ], at the C-terminus this protein has an extra peptide of about 130 amino acids sharing some similarity with a number of microbial EGases and containing a putative type-1 [12] cellulose binding domain.

Results and discussion When strawberry (Fragaria x ananassa) fruits of the cultivar Chandler start the ripening process proper, a steep increase in endo-13-1,4-glucanase (EGase) activity is observed. In the few days needed for the fruits to ripen (from 2 to 5, depending on the growing condition), the rise in EGase activity continues to be so pronounced that, at the end of the process, the activity is almost doubled (about 90% more in red T a b l e 1: EGase activity in strawberry fruits at five stages of development fruits when compared to white ones, see Developmental stage EGase activity Standard Deviation table 1). Isoeloctrofocusing experiments ....units/100~t~ prot SG 0.10 n.a. allowed the detection of two basic EGase LG 1.00 0.70 isoforms (pI 7.9 and 9.0, respectively) in W 14.88 6.99 both white and red berries, and the acP 17.43 4.79 R 28.39 1.63 tivities of both isoenzymes increased dur(SG: small green; LG: large green; W: white; P: pink; R: ripe red). ing ripening. The finding that the EGase activity observed during ripening was due to the presence of two different isoforms was confirmed by the cloning of two divergent EGase cDNAs. The screening of a library, representing transcripts from red strawberry fruits, with two previously isolated RT-PCR cDNA fragments allowed the isolation of the corresponding full-length cDNA clones which were named faEG1 and faEG3, respectively. Table 2:faEG1 a n d f a E G 3 g e n e e x p r e s s i o n in s t r a w b e r r y fruits The expression profiles of the transcripts reat five stages o f d e v e l o p m e n t lated to faEG1 and faEG3 (table 2) clearly show that they are both encoded by ripenD e v e l o p m e n t a l stage faEG1 faEG3 SG ing-related genes although some differences LG + could be found. The faEG 1 mRNA starts to W * ++ be detected in white fruits, that is at the bep ** +++ R ****** ++++ ginning of the ripening process proper, and increases up to an extremely high amount (SG: small green; LG: large green; W: white; P: pink; R: ripe red). Increasing numbers of either asterisks or crosses indicate higher amounts of transcripts. in red berries (0.13 % of the mRNA populaThe expression level between faEG1 and faEG3 are not comparable. tion as deduced from the number of positive clones obtained in the first screening of the cDNA library). On the contrary, faEG3 related transcripts can be detected starting from large green fruits, thereafter their amount increases steadily during ripening to reach a maximum in red berries, though this amount is approximately half that of faEG 1 (0.06% of the mRNA population as deduced from the number of positive clones obtained in the first screening of the cDNA library). Interestingly, the finding that transcripts related to faEG3 can also be detected (although at very low levels) in young developing tissues other than fruit (data not shown) allows us to define as both ripening- and fruit-specific the only faEG1 gene.

135

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The different time course of expression observed in fruits Figure 1: Tissue printing experiments, suggested that differences in spatial expression patterns demonstrating the spatial expression of the two EGase genes. In both cases the might also exist between the two EGase mRNAs. Tissue expression mainly occurs at the level of printing analyses have therefore been performed with fruits the fruit cortical parenchyma; however, both at the beginning (i.e. white) and at the end (i.e. red) contrary to faEG3, the faEGl-related of the ripening process. These analyses, besides confirm- transcripts are not clearly detectable in white fruits. ing the differences in temporal expression with the faEG3 transcripts detectable earlier than those related to faEG 1, have shown an overlapping spatial expression for faEG1 and faEG3 (figure 1). In particular, the expression of both mRNAs is mostly confined to the outer part of the fruits. This observation is in accordance with a report by Neal [13] who found that the main morphological changes taking place during maturation of strawberries were associated with the cortex parenchyma cells. T a b l e 3: E f f e c t o f a u x i n treatment and p o s t - h a r v e s t o n the e x p r e s s i o n o f the It has previously been shown that in the nont w o E G a s e g e n e s in either w h i t e ( W ) or red (R) fruits. climacteric strawberry a crucial role in the Developmental stage Treatment faEG 1 faEG3 ripening process is played by auxin [14]. W time 0 control ** ++ W 48 h H 2 0 **** ++ Accordingly, fruits treated with the auxin W 48 h N A A * + analogue 1-naphthalene acetic acid (NAA) R time 0 control **** ++++ R 48 h H 2 0 ***** ++++ were used to test whether or not this horR 48 h N A A *** +++ mone could influence the expression of the Time 0 control: fresh fruits; 48 h H20:48 hours of treatment with a solution without NAA; 48 h two EGases (table 3). In the NAA treated NAA: 48 hours of treatment with a solution containing 2 mmol/L NAA. Increasing numbers of either asterisks or crosses indicate higher amounts of transcripts. The expression level between faEG1 and faEG3 are not comparable. white and red fruits both transcripts turned out to be down regulated when compared with the non-treated controls, although the hormone effect was more pronounced in the white berries. These results confirm previous data on the auxin inhibitory effect on a number of genes in strawberry [ 14-16].

The same experiment (table 3) gives also information about the post-harvest effect on the two EGase transcripts. In fact, faEG1 mRNA accumulates quickly and at high levels in berries 136

Molecular aspects of the strawberry fruit softening detached from the plant, so its related transcripts, although very high in fresh fruit, can accumulate to even higher amounts in stored ones. The faEG3 mRNA shows a similar expression pattern, but its accumulation is less pronounced than that of faEG1. These data confirm that faEG1 is the "softening" EGase although the similarity of response to auxin treatments and post-harvest strengthens the idea that both EGases are ripening enzymes. The finding of two temporally and spatially overlapping EGases during the ripening of strawberry fruits might appear strange should their sequences be similar. However, this is not the case since the two proteins are very different. The deduced faEG1 protein has a molecular mass of about 54 kDa, thus falling in the same range of most higher plant EGases [17-23]. On the contrary, faEG3 has a deduced molecular weight of 68 kDa, which is uncommon, but not unique, for higher plant EGases. EGases with a similar molecular mass have been described in bean [24], pea [25] and in Coleus blumei [26]. The differences in molecular mass suggest that the two proteins might also have differences in biochemistry such as, for instance, a different substrate specificity. This hypothesis is further strengthened by the presence of a peptide of about 130 amino acids at the C-terminus of the faEG3 sequence. This peptide is of particular interest because it contains a region with homology to bacterial cellulose binding domains (CBDs, see figure 2). The homology does not prove that the faEG3 CBD can actually bind to cellulose, however further support to this possibility is also given by the high proline, serine and threonine content of the faEG3 C-terminal peptide, feature that is commonly found in linker sequences of microbial EGases with real CBDs. Figure 2" Alignment of the deduced amino acid sequences of six cellulose-binding domains (CBD): five putative CBDs from higher plants and one CBD from a microbial protein. Protein sequences used to construct the alignment were deduced from nucleic acid ones and were the following (with EMBL/GeneBank accession numbers): strawberry faEG3 (AJ006349); tomato tomCel8 (AF098292); cotton cottonEG (D88417); Arabidopsis EST clone AtEST (T04443) and genomic AtF2P3 (AF080120, clone F2P3, from the genome sequencing project); Dictyostelium discoideum cellulose binding protein CelB (M33862). Conserved tryptophans are shown in white, while shaded boxes and white boxes indicate identical and conserved amino acid residues, respectively. 494 491 192 36 493 392

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Embryogenesis, Development and Architecture The finding that faEG3 contains a putative cellulose-binding domain suggests that this unusual EGase might be specially active against the xyloglucans which coat the cellulose microfibrils and act as "molecular tethers" holding the microfibrils in place [27]. The activity of this peculiar EGase would therefore cause a severe weakening of the cell wall structural properties by letting loose the cellulose microfibrils. As a matter of fact, a cytochemical assay carried out during the ripening of strawberry fruits s h o w e d the p r e s e n c e of loose and disorganised cellulose fibrils in the cell walls of red fruits [28] and those fibrils started to be visible at the stage of white fruits, that is a stage when the amount of the faEG3 m R N A is already high. The anticipated expression of faEG3, compared to that of faEG1, would therefore cause a gross dismantling of the cellulose-xyloglucan network and make more readily hydrolyzable the substrate(s) specific for the other glucanase. This synergistic and co-ordinate mode of action might explain why the strawberry fruits of the cultivar Chandler soften so much in a few days. Moreover, these findings assign a biotechnological role to both enzymes and experiments carried out with transgenic plants expressing an antisense faEG3 gene in order to verify our hypothesis are in progress.

Authors of this contribution Livio Trainotti, Silvia Spolaore, Anna Pavanello, Giorgio Casadoro* * Corresponding author, Department of Biology, University of Padua, Viale G. Colombo 3, 35121, Padua, Italy

Acknowledgement This work was supported by the European Community FAIR Program (Contract FAIR-CT973005).

References 1. J. Gray, S. Picton, J. Shabbeer, W. Schuch, D. Grierson, Molecular biology of fruit ripening and its manipulation with antisense genes, Plant Mol. Biol. 19 (1992) 69-87. 2. M. Knee, J. Sargent, D. Osborne, Cell wall metabolism in developing strawberry fruits, J. Exp. Bot. 28 (1977) 377-396. 3. R. Ben-Arie, N. Kislev, C. Frenkel, Ultrastructural changes in the cell walls of ripening apple and pear fruits, Plant Physiol. 64 (1979) 197-202. 4. R. Fischer, A.B. Bennett, Role of cell wall hydrolases in fruit ripening, Annu. Rev. Plant Physiol. Plant Mol. Biol., 42 (1991) 675-703. 5. K.A. Platt-Aloia, W.W. Thomson, R.E. Young, Ultrastructural changes in the walls of ripening avocados: transmission, scanning and freeze fracture microscopy, Bot. Gaz. 141 (1980) 366-373. 6. C.B. Hall, Cellulase in tomato fruits, Nature 200 (1963) 1010-1011. 7. R. Hatfield, D.J. Nevins, Characterization of the hydrolytic activity of avocado cellulase, Plant Cell Physiol. 27 (1986) 541-552. 8. J.C.K. Rose, A.B. Bennett, Cooperative disassembly of the cellulose-xyloglucan network of plant cell walls: parallels between cell expansion and fruit ripening, Trends Plant Sci. 4 (1999) 176-183. 9. M.E Barnes, B.J. Patchett, Cell wall degrading enzymes and the softening of senescent strawberry fruit, J. Food Sci., 41 (1976) 1392-1395. 10. EB. Abeles, E Takeda, Cellulase activity and ethylene in ripening strawberry and apple fruits, Sci. Hort. 42 (1990) 269-275. 138

Molecular aspects of the strawberry fruit softening 11. D.A. Brummell, C.C. Lashbrook, A.B. Bennett, Plant endo-l,4-b-D-glucanases: structure, properties and physiological function, Am. Chem. Soc. Symp. Ser. 566 (1994) 100-129. 12. E BEguin, J.P. Aubert, The biological degradation of cellulose, FEMS Microbiol. Rev. 13 (1994) 25-58. 13. G.E. Neal, Changes occurring in the cell walls of strawberries during ripening, J. Sci. Food. Agr. 16 (1965) 604-611. 14. K. Manning, Changes in gene expression during strawberry fruit ripening and their regulation by auxin, Planta 194 (1994) 62-68. 15. N. Medina-Escobar, J. Cardenas, E. Moyano, J.L. Caballero, J. Munoz-Blanco, Cloning, molecular characterization and expression pattern of a strawberry ripening-specific cDNA with sequence homology to pectate lyase from higher plants, Plant Mol. Biol. 34 (1997) 867-877. 16. A.S.N. Reddy, B.V. Poovaiah, Molecular cloning and sequencing of a cDNA for an auxin-repressed mRNA: correlation between fruit growth and repression of the auxin-regulated gene, Plant Mol. Biol. 14 (1990) 127136. 17. C. Bonghi, N. Rascio, A. Ramina, G. Casadoro, Cellulase and polygalacturonase involvement in the abscission of leaf and fruit explants of peach, Plant Mol. Biol., 20 (1992) 839-848. 18. J.K. Burns, D.J. Lewandowski, C.J. Nairn, G.E. Brown, Endo-l,4-b-glucanase gene expression and cell wall hydrolase activities during abscission in Valencia orange, Physiol. Plant. 102 (1998) 217-225. 19. E. del Campillo, L.N. Lewis, Occurrence of 9.5 cellulase and other hydrolases in flower reproductive organs undergoing major cell wall disruption, Plant Physiol. 99 (1992) 1015-1020. 20. L. Ferrarese, L. Trainotti, P. Moretto, E Polverino de Laureto, N. Rascio, G. Casadoro, Differential ethyleneinducible expression of cellulase in pepper plants, Plant Mol. Biol. 29 (1995) 735-747. 21. R. Sexton, E. del Campillo, D. Duncan, L.N. Lewis, The purification of an anther cellulase (b(1:4)4-glucan hydrolase) from Lathyrus odoratus L. and its relationship to the similar enzyme found in abscission zones, Plant Sci. 67 (1990) 169-176. 22. E Tonutti, L.G. Cass, R.E. Christoffersen, The expression of cellulase gene family members during induced avocado fruit abscission and ripening, Plant Cell Envir. 18 (1995) 709-713. 23. S.T.J. Webb, J.E. Taylor, S.A. Coupe, L. Ferrarese, J.A. Roberts, Purification of b-l,4-glucanase from ethylene-treated leaflet abscission zones of Sambucus nigra, Plant Cell Envir. 16 (1993) 329-333. 24. ET. Lew, L.N. Lewis, Purification and properties of cellulase from Phaseolus vulgaris. Phytochemistry 13 (1974) 1359-1366. 25. T. Matsumoto, E Sakai, T. Hayashi,: A xyloglucan-specific endo-l,4-b-glucanase isolated from auxintreated pea stems, Plant Physiol., 114 (1997) 661-667. 26. Y. Wang, L.E. Craker, Z. Mao, Purification and characterization of cellulase from leaf abscission zones of Coleus. Plant Physiol. Biochem. 32 (1994) 467-472. 27. C.T. Brett, K.W. Waldron, Physiology and biochemistry of plant cell walls, 2n~ edn., Chapman & Hall, London, 1996. 28. L. Trainotti, L. Ferrarese, E Dalla Vecchia, N. Rascio, G. Casadoro, Two different endo-b-l,4-glucanases contribute to the softening of the strawberry fruits. J. Plant Physiol. 154 (1999) 355-362.

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). O Elsevier Science B.V.All rights reserved.

Signals And Their Transduction In Early Plant Embryogenesis Abstract Several observations suggest that the development of somatic embryos from suspension cells in vitro depends on signals that derive from other suspension cells. If there is a correspondence between somatic and zygotic embryos, then the signals found in vitro must also be acting on zygotic embryos. Two examples of such signalling systems will be described. The first stems from the observation that carrot EP3 class IV endochitinases can rescue somatic embryos of the temperature-sensitive cell line ts 11. Employing whole mount in situ hybridisation it was found that a subset of the cells in embryogenic and non-embryogenic suspension cultures, including ts 11, express EP3 genes. No expression was found in somatic embryos. In carrot plants EP3 genes are expressed in the inner integument cells of young fruits and in a specific subset of cells, located in the middle of the endosperm of mature seeds. No expression was found in zygotic embryos. These results suggest that the EP3 endochitinase has a "nursing" function during zygotic embryogenesis, and that this function can be mimicked by suspension cells during somatic embryogenesis. Signals aimed at the embryo must also be perceived, and as the second example of a signalling pathway involved in embryogenesis, the leucine-rich repeat containing Somatic Embryogenesis Receptor Kinase (SERK) will be discussed. During somatic embryogenesis, SERK expression is detected first in single cells and disappears at the early globular stage. During zygotic embryogenesis, SERK expression was detectable transiently in young zygotic embryos of up to 100 cells. These results demonstrate that competent cell formation and early somatic embryogenesis require a highly specific signal transduction chain also found during zygotic embryogenesis. Whether both examples of signal transduction chains are related is not known.

Introduction Carrot cell cultures secrete many different proteins into the medium, a process that contributes to the conditioning of the medium. Different cell types contribute to the total pattern of proteins secreted into the culture medium [1 ]. Conditioned media are reported to have a promoting effect on the initiation of somatic embryogenesis [2, 3]. A causal relationship between secreted proteins and embryogenic potential exists that has led to the identification of the extracellular protein 3 (EP3), identified as a chitinase. EP3 was originally purified as a protein

Sacco de Vries, Agricultural University Wageningen, Department of Molecular Biology, Wageningen, the Netherlands

141

Embryogenesis, Development and Architecture capable of rescuing somatic embryos in the mutant carrot cell line ts 11 at the non permissive temperature [4]. The acidic endochitinase EP3 was found to be a member of a small family of class IV chitinase genes [5]. Those highly homologous isoenzymes are encoded by at least 4 EP3 genes. Two of these proteins, EP3-1 and EP3-3, were purified and shown to have subtly different effects on the formation of somatic embryos in newly initiated ts 11 embryo cultures. Since the effect of the chitinases was mimicked by Rhizobium-produced Nod factors [6], it was proposed that the chitinases are involved in the generation of signal molecules essential for embryogenesis in ts 11 [6]. The roots of leguminous plants are known to produce chitinases and during the interaction with Rhizobium these plant produced chitinases have been suggested to control the biological activity of Nod factors by cleaving and inactivating them. In this way, chitinases are proposed to have the potential to control plant morphogenesis and cell division [7]. In carrot, the formation of embryogenic cell cultures usually commences with the incubation of seed-derived seedling hypocotyl explants in auxin-containing medium [8]. The origin of embryogenic cells, that are usually present as clusters of small cytoplasm-rich cells [9] is not clear and is thought to involve an auxin dependent transition stage occurring in single cells. It is generally assumed that the formation of plant embryos requires the activation of specific sets of genes [reviewed by 10, 11] and many studies have employed differential screening techniques to identify such genes. Often, genes found to be expressed in early somatic embryos appeared to encode genes normally expressed late in zygotic embryogenesis or throughout plant development [reviewed by 12]. Others, such as LTP [13] and EMB-1 [14] are expressed at the corresponding, globular, stage in zygotic embryogenesis. Several genes have been reported [reviewed by 12] that are putative markers for embryogenic cell clusters, but none have been described to date that are reliable markers for the preceding stage of competent cells. Several screens were carried out employing a series of carrot cell cultures with widely differing numbers of single competent cells as the starting material. One of the genes found encoded a receptor-like kinase that appears to mark competent and embryogenic cells and is also expressed in early somatic and zygotic embryos.

The EP3 genes Cell specific expression of the EP3 genes and localisation of the encoded proteins in suspension cultures. To identify the suspension cells that express the EP3 genes, whole mount in situ mRNA localisation was employed on entire, immobilised suspension cultures. EP3 mRNAs could only be detected in between 4-6% of the total number of cells in an embryogenic culture. The number of embryo-forming cells in a comparable culture does however not exceed 1%, [15], suggesting there is no quantitative relation with EP3 expressing cells. The highest concentration of EP3 mRNAs, in both embryogenic as well as in non-embryogenic cultures, was found in single cells that were elongated and often strongly curved, coiled, elongated or rounded. Whole mount in situ on globular, heart or torpedo shaped somatic embryos never revealed EP3 mRNAs in embryos, but only in a few cells still adhering to embryogenic cell clusters (figure 1). In order to determine when the first EP3 expressing cells appeared during embryogenic cell formation, hypocotyl explants were treated with 2,4-D for a period of ten days, during which embryogenic cell formation occurs [16]. In this system, cell division was 142

Signals in early plant embryogenesis reinitiated in cells of the vascular tissue, and generated a mass of rapidly proliferating cells. Only after ten days a very small number of cells at the periphery of the proliferating mass was found to contain EP3 mRNA. This is three days later as the appearance of the first cells that are competent to form somatic embryos in this system [17]. We conclude therefore that EP3 gene expression is not directly correlated with embryogenic cell formation, nor with somatic embryo development. In plants, EP3 mRNAs could be found in developing and mature seeds. In early stages of seed development, approximately 5-6 DAP, EP3 mRNA was detected in the inner integument cells, lining the surface of the embryo sac in which the zygote or the early embryo is located. Up to at least 20 DAP, no expression of the EP3 genes was detected in the developing endosperm. In the endosperm of mature seeds, EP3 mRNA was restricted to a narrow zone of endosperm cells starting at the cavity in which the embryo is located up to almost the opposite end of the endosperm. Because EP3 chitinases are secreted proteins, we localised these enzymes during the development of seeds and in germinating seeds by tissue printing followed by EP3 antibody staining. The EP3 protein was uniformly spread in the integuments at 10 DAP but also in the developing endosperm at 20 DAP. This A points to transport developing seeds (3 - 20 DAP) of the EP3 proteins from the maternal integument tissues t o w a r d s the entntegumentl) dosperm. How~~deveioptngendosperm ever, we c a n n o t ~p3,,~

zygote / globutar embryo

B mature seeds

EP3 mRNA ....

"

e~dosperm

seed coat

mature embryo

Figure 1. EP3 gene expression in suspensioncultures. Plant material was analysed by whole mount in situ hybridization with an antisense EP3 RNA probes. Light microscopy, coupled to Nomarski optics was used for visualization of the precipitate of the enzymatic detection using alkaline phosphatase. Cells present at the periphery of the proliferated cell mass on a section of a hypocotyl explant that was cultured in the presence of 2,4-D for 10 days are shown. The arrow points to a single EP3 expressing cell.

completely rule out that a very low level of EP3 gene expression that was below detection limits, occurred in the peripheral endosperm cells. In mature seeds the protein was restricted to the inner cell layer of the cavity that surrounds the embryo and to a zone of cells that starts at this cavity and ends almost at the opposite side of the endosperm, which corresponds precisely with the expression pattern of the EP3 genes as determined by in situ hybridisation. Figure 2. Summary of EP3 gene expression in seeds. (I) integuments surrounding the developingembryo and endosperm; (SC), seed coat; (E), endosperm; (EM), embryo. Bar: 50 lam. (A) Longitudinal section of a fruit, 7 DAE (B) Longitudinal section of a mature seed.

143

Embryogenesis, Development and Architecture To summarise, the EP3 genes are expressed in the maternal integument cells. The EP3 chitinases however, were detected in the developing endosperm, at a time that EP3 chitinase gene products cannot be detected in the endosperm and as a consequence they must be largely of maternal origin. In mature seeds the integuments are completely degraded and then the EP3 proteins are produced in a subset of the endosperm cells [18].

The SERK gene Expression of the SERK gene during somatic and zygotic embryogenesis in carrot. Labelled probes for differential screening were obtained from RNA out of a <30 om sieved subpopulation of cells from either embryogenic or non-embryogenic cell cultures. Employing these probes in a library screen of approximately 2000 plaques yielded 26 plaques that failed to show any hybridisation to either probe. These so-called cold plaques were purified and used in spot-dot Northern analysis. One clone showed low expression in all embryogenic cultures and in one non-embryogenic culture, but not in the others. The predicted amino-acid sequence shows homology with the structural features of plant and animal receptor protein kinases, and therefore the gene was named Somatic Embryogenesis Receptor Kinase (SERK). The SERK protein contains an N-terminal domain with five leucine-rich repeats (LRRs). Between the extracellular LRR domain of SERK and the membrane-spanning region there is a 32 amino acid region with 13 prolines, partly arranged in the sequence SPPPP, that is conserved in extensins, a class of universal plant cell wall proteins [ 19]. The significance of this proline-rich box is not clear, it might act as a hinge region by providing flexibility to the extracellular part of the receptor, or act as a region for interaction with the cell wall. In extensins, usually all prolines in the SPPPP repeat are hydroxylated and are considered to be used as target for O-linked glycosylation. The proposed intracellular domain of the protein contains the 11 subdomains characteristic of the catalytic core of protein kinases, while sequence homology suggests a function as a serine / threonine kinase [20]. A bacterially expressed SERK fusion protein is indeed able to autophosphorylate on serines and threonines, confirming the predictions [ 17]. To determine directly whether SERK expressing cells indeed develop into somatic embryos, transformed carrot suspension cultures containing a SERK promoter-luciferase construct were analysed for luciferase expression in cell cultures sieved through a 50 ~tm mesh to enrich for single cells and small cell clusters. Development of the immobilised cells after recording the luciferase images was determined using automated cell tracking [15]. The origin of nine torpedo stage somatic embryos was determined this way. Of these, three developed from a single cell that showed luciferase activity luciferase at day 1, four developed from cell clusters consisting of 2-6 luciferase expressing cells while two embryos day1 day1 day2 day3 day6 day9 day13 developed from single cells that failed to show a detectable level of Figure 3. Luciferase expression under control of the SERK promoter. Luciferace activity of immobilised cells was recorded at day luciferase activity at day 1. The 1 with a CCD camera (left image). The single pixel in the left imsomatic embryo shown in Figure 3 age measures 30 by 30 ~tm. Video cell-tracking of the cells was originated from a luciferase-experformed for a period of 13 days (light microscopic images). The pressing two-celled cluster. These light microscope images were sized to match the CCD image.

/

144

Signals in early plant embryogenesis results demonstrate that somatic embryos develop from single cells and small cell clusters expressing SERK. Somatic embryos up to the globular stage show luciferase expression, confirming the transient SERK gene expression pattern obtained with in situ mRNA hybridisation (results not shown).

Discussion We have shown that only a subset of cells present in an embryogenic carrot culture produce EP3 carrot chitinases. On the basis of the number, cell type and presence of EP3 mRNA producing cells in embryogenic and non-embryogenic cultures, there was no correlation with the ability to produce somatic embryos and the presence of EP3 mRNA. No EP3 gene expression in somatic embryos was found. Because cells that produce EP3 do not develop into embryos themselves, it appears that EP3 chitinases, or products of their enzymatic activity, diffuse via the conditioned medium to cells that are able to help somatic embryos develop and in this way play a "nursing" role in the process of somatic embryogenesis. In plants, EP3 mRNA was found in the inner integuments, while the EP3 proteins are found in the endosperm. Later, cells in the centre of the endosperm do express the EP3 genes and this is likely to be responsible for the presence of EP3 chitinases during imbition and germination. No EP3 gene expression was found in zygotic embryos. It could therefore be expected that the chitinases play a similar "nursing" role in zygotic embryogenesis. The EP3 chitinase proteins found in the endosperm of 20 DAP seeds are most likely produced by integument cells. This indicates a maternal contribution to the proteins that are present in the endosperm. Recent evidence for a role of maternal tissues in endosperm formation comes from the analysis of gametophytic mutations in Arabidopsis. In this species, fie (fertilisationindependent endosperm) mutations are known that are female gametophytic and specifically affect endosperm formation [21]. Regarding the biological function of the EP3 chitinases in plant embryogenesis, we propose that they are involved in reinitiating cell division in embryogenic cells and embryos as part of a "nursing cell" system, that is required for embryogenesis. This hypothesis is based on the following observations: - The EP3 chitinases promote embryogenic cell formation and the number of somatic embryos as well as their progression in development when added to tsl 1 cultures [4]. - The expression of EP3 genes in cells that do not develop into embryos in culture and an absence of expression in somatic embryos. The expression of EP3 genes in maternal tissue and subsequent secretion of the encoded chitinase proteins, resulting in the presence of EP3 in the extracellular matrix of the endosperm surrounding globular-stage zygotic embryos. - The absence of expression in zygotic embryos and expression in endosperm cells prior to and during germination. -

The role of EP3 could be direct and involve a structural property such as the chitin-binding domain of the protein. It appears more likely to envisage a function involving the catalytic properties of the enzyme through the release or modification of an N-acetylglucosamine con145

Embryogenesis, Development and Architecture

taining signal molecule. Recently we obtained evidence that a particular class of soluble arabinogalactan proteins (AGPs) contains N-acetylglucosamine, and can be cleaved by endochitinases. We believe that this result will help to further unravel the role of chitin-based signalling molecules in plant embryogenesis. The process of cellular reactivation and the subsequent formation of embryogenic cells in carrot explants has been described by Guzzo et al. [16]. That work showed that a particular elongated cell type appeared in culture, derived from small rapidly proliferating cytoplasmic cells that themselves derived from reactivated provascular cells. It was further shown cytologically, that some of the elongated cells underwent an asymmetrical division. After continued culture, small clusters of dividing cytoplasmic cells appeared that resemble the proembryogenic masses seen in established embryogenic suspension cultures (Guzzo et al. 1994, De Vries et al. 1988). To determine which single cells were competent to form embryos, markers are needed that distinguish precisely between competent and non-competent cells. The expression of the SERK gene described here was obtained by following the development of living SERK-expressing cells as visualised by luciferase expression under control of the SERK promoter. The predicted SERK protein sequence resembles a leucine-rich repeat (LRR) receptor kinase protein, a class of plant proteins that was originally described by Chang et al. [22]. While the avarage primary cell wall has a thickness of approximately 50 nm [23] and the maximum size of the entire extracellular domain can only be about 15 nm when present as an ? helix, the extracellular ligand binding domain is likely to be completely embedded within the cell wall. The most likely type of ligand for SERK will therefore consist of a cell walldiffusable small molecule such as a (glyco)peptide. Peptides effective in inducing plant responses, such as systemin [24] and ENOD40 [25] have been described. Thus, it appears that while LRR containing receptor-like protein kinases play several roles in plant development, intercellular peptides are now being uncovered that are likely signal molecules that can activate developmental processes mediated through such receptors. In the plant embryo sac and in the activated explant a situation may exist whereby an unknown inducer is present uniformly, while embryo formation awaits the presence of the SERK protein. Such a model may fit with the restricted expression pattern found for the SERK gene both in vivo and in vitro. It is also in line with the hypothesis that in plants inductive interactions mediated by diffusable signal molecules are an important regulatory mechanism [reviewed in 17]. The SERK gene described here may represent a significant part of a mechanism that is essential for the formation of plant cells destined to become embryo. Whether molecules resulting from the activity of the EP3 chitinase are possible ligands for SERK remains to be investigated.

Acknowledgements Research in our group is supported by The Netherlands Organization for Scientific Research (A.v.H. and E.S.), and by the European Commission Biotechnology program PTP-Biotech (V.H.). I thank Arjon van Hengel, Ed Schmidt and Val6rie Hecht for their help in preparing this abstract. 146

Signals in early plant embryogenesis

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Van Engelen FA, De Vries SC (1993) Secreted proteins in plant cell cultures. In KA Roubelakis-Angelakis, K Tran Thanh Van, eds, Markers of Plant Morphogenesis. Plenum Press, New York, pp 181-200. Hari V (1980) Effect of cell density changes and conditioned media on carrot cell embryogenesis. Z. Pflanzenphysiol. 96:227-231 Smith JA, Sung ZR (1985) Increase in regeneration of plant cells by cross feeding with regenerating Daucus carota cells. In M Terzi, L Pitto, ZR Sung eds, Somatic Embryogenesis. IPRA, Rome, pp 133-137 De Jong AJ, Cordewener J, Lo Schiavo E Terzi M, Vandekerckhove J, Van Kammen A, De Vries SC (1992) A carrot somatic embryo mutant is rescued by chitinase. Plant Cell 4:425-433 Kragh KM, De Jong AJ, Hendriks T, Bucherna N, HCjrup P, Mikkelsen JD, De Vries SC (1996) Characterization of chitinases able to rescue somatic embryos of the temperature-sensitive carrot variant ts 11. Plant Molecular Biology 31:631-645 De Jong AJ, Heidstra R, Spaink HP, Hartog MV, Meijer EA, Hendriks T, Lo Schiavo E Terzi M, Bisseling T, Van Kammen A, De Vries SC (1993) Rhizobium lipooligosaccharides rescue a carrot somatic embryo mutant. Plant Cell 5:615-620 Staehelin C, Schultze M, Kondorosi E, Mellor RB, Boller T, Kondorosi A (1994) Structural modifications in Rhizobium meliloti Nod factors influence their stability against hydrolysis by root chitinases. Plant Journal 5:319-330 De Vries, S. C., Booij, H., Meyerink, E, Huisman, G., Wilde, H. D., Thomas, T. L. and Van Kammen, A. (1988a) Acquisition of embryogenic potential in carrot cell-suspension cultures. Planta 176, 196-204. Komamine, A., Matsumoto, M., Tsukahara, M., Fujiwara, A., Kawahara, R., Ito, M., Nomura, K. and Fujimura, T. (1990) Mechanisms of somatic embryogenesis in cell cultures-physiology, biochemistry and molecular biology. In Progress in plant cellular and molecular biology (ed. H. J. J. Nijkamp, L. H. W. Van Der Plas, A. Van Aartrijk), pp. 307- 313. Kluwer Academic Publishers, Dordrecht. Goldberg, R. B., de Paiva, G. and Yadegari, R. (1994) Plant embryogenesis: zygote to seed. Science 266, 605-614. Thomas, T. L. (1993) Gene expression during plant embryogenesis and germination: an overview. Plant Cell 5, 1401-1410. Zimmerman, J. L. (1993) Somatic embryogenesis: a model for early development in higher plants. Plant Cell 5, 1411-1423. Sterk, E, Booij, H., Schellekens, G. A., Van Kammen, A. and De Vries, S. C. (1991) Cell-specific expression of the carrot EP2 lipid transfer protein gene. Plant Cell 3, 907-921. Wurtele, E.S., Wang, H., Durgerian, S., Nikolau, B.J. and Ulrich, T.H. (1993) Characterization of a gene that is expressed early in somatic embryogenesis of Daucus carota. Plant Physiol. 102, 303-312. Toonen, M. A. J., Hendriks, T., Schmidt, E. D. L., Verhoeven, H. A., Van Kammen, A and De Vries, S. C. (1994) Description of somatic-embryo-forming single cells in carrot suspension cultures employing video cell tracking. Planta 194, 565-572. Guzzo F, Baldan B, Mariani P, LoSchiavo F, Terzi M (1994) Studies on the origin of totipotent cells in explants of Daucus carota L. J. Exper. Botany 45:1427-1432 Schmidt EDL, Guzzo F, Toonen MAJ, De Vries SC (1997) A leucine-rich repeat containing receptor-like kinase marks somatic plant cells competent to form embryos. Development 124:2049-2062 Van Hengel, A.J., Guzzo, E, van Kammen, A., de Vries, S.C. (1998) Expression pattern of the carrot EP3 endochitinase genes in suspension cultures and in developing seeds. Plant Physiol. 117, 43-53. Varner, J. E. and Lin, L.-S. (1989) Plant cell wall architecture. Cell 56, 231-239. Hanks, S. K., Quinn, A. M. and Hunter, T. (1988) The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241, 42-52. Ohad N, Margossian L, Hsu Y, Williams C, Repetti P, Fischer RL (1996) A mutation that allows endosperm development without fertilization. Proc. Natl. Acad. Sci. 93:5319-5324 Chang, C., Schaller, G.E., Patterson, S.E., Kwok, S.E, Meyerowitz, E.M. and Bleecker, A.B. (1992) The TMK1 gene from Arabidopsis codes for a protein with structural and biochemical characteristics of a receptor protein kinase. Cell 4, 1263-1271. Pmitt, R. E., Hulskamp, M., Kopczak, S. D., Ploense, S. E. and Schneitz, K. (1993) Molecular genetics of cell interactions in Arabidopsis.. Develop. 1993 suppl., pp. 77-84. McGurl, B., Pearce, G., Orozco-Cardenas, M. and Ryan, C.A. (1992) Structure, expression, and antisense inhibition of the systemin precursor gene. Science 257, 1570-1573. Van de Sande, K., Pawlowski, K., Czaja, I., Wieneke, K., Schell, J., Schmidt, J., Walden, R., Matvienko, M.,

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Embryogenesis, Development and Architecture Wellink, J., Van Kammen, A., Franssen, H. and Bisseling, T. (1996) Modification of phytohormone responseby a peptide encoded by ENOD40 of legumes and a nonlegume. Science 273,370-373.

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

Mutational Studies Of Root Architecture In Arabidopsis thaliana Summary A major determinant of plant architecture is the degree of lateral branching in the root and shoot systems. The obvious importance of branching on plant form is reflected in its profound influence on plant function. The ability to modify branching patterns would therefore be a powerful tool for plant breeders. The EC Framework IV Biotechnology funded LATIN consortium aims to improve basic understanding of the molecular regulation of lateral initiation in order to develop sophisticated strategies to manipulate branching in transgenic crops. In order to realise this goal, one of the primary objectives of LATIN involved the identification by mutation of genes controlling branching in the model plant, Arabidopsis thaliana. LATIN researchers have collectively screened a transposonmutagenised population of Arabidopsis [ 1]. We describe a series of novel screens to identify root architectural mutants within an En-1 transposon mutagenised population. Several classes of root architectural mutants have been identified including the anchor root mutant tripod and the adventitious root mutants, medusal and medusa2. Attempts to identify candidate En-1 transposon elements that co-segregate with the root mutant phenotypes have been frustrated by the high frequency of transposon excision and footprinting. We describe a novel bulk segregant analysis based approach that negates many of the problems associated with plants mutagenised with autonomous transposable elements such as En-1.

Introduction Plants and animals employ contrasting developmental strategies for organogenesis. Animal organogenesis takes place during embryogenesis, whereas plants form only a rudimentary set of root, hypocotyl and cotyledon organs prior to seed maturation. Deferring organogenesis until after germination allows plants to retain a greater degree of freedom to manipulate their development in response to the prevailing environmental conditions as illustrated by the day length control of floral induction in many species. Plants achieve their adult form by using a variety of post-embryonic organogenic processes to elaborate their architecture. A major determinant of plant architecture is the degree of lateral branching in the root and shoot systems. The obvious importance of branching on plant form is reflected in its profound influence on plant function. For example, the pattern of lateral root initiation is central to the efficient

Malcolm J. Bennett, School of Biological Sciences, University of Nottingham, UK

149

Embryogenesis, Development and Architecture exploitation of water and nutrient supplies and the degree of shoot branching affects important factors such as light harvesting and fecundity. The ability to modify branching patterns would therefore be a powerful tool for plant breeders. The EC Framework IV Biotechnology funded LATIN consortium aims to improve basic understanding of the molecular regulation of lateral initiation in order to develop sophisticated strategies to manipulate branching in transgenic crops. In order to realise this goal, one of the primary objectives of LATIN involved the identification by mutation of genes controlling branching in the model plant, Arabidopsis thaliana. LATIN researchers collectively screened a transposon-mutagenised population of Arabidopsis [1]. The autonomous maize element En-1 was selected since it has been demonstrated to be highly active in Arabidopsis [2]. The population, termed the En-1 gene machine, is composed of 3,000 lines, containing approximately 15,000 insertions of the transposable element En-1. The En-1 elements appear to provide good coverage throughout the genome since a selection of genes on every chromosome have been described to contain En-1 insertions [3]. In this paper, we describe the screens employed to isolate a selection of root architecture mutants within the En-1 gene machine population and outline an innovative approach to overcome many of the inherent problems associated with tagging genes using an autonomous, highly mobile, transposon like En-1.

Isolation of

Arabidopsis root

architectural m u t a n t s

Lateral root architecture directly influences the ability of a plant to colonise the soil, maximises nutrient acquisition and provides mechanical support. Adventitious roots derived from stem tissues and anchor roots emerging at the root-shoot junction likewise perform important mechanical and physiological roles in crops like maize. We describe below a series of screens to identify Arabidopsis mutants that modify adventitious, anchor and/or lateral root development.

Adventitious root mutants. Adventitious roots rarely emerge from non-root, light grown tissues such as the hypocotyl in wild-type Arabidopsis. However, we have observed that wildtype hypocotyl tissues are capable of forming significant numbers of adventitious roots following a short period of etiolation. An initial three-day period of seedling etiolation was determined to be optimal to stimulate several adventitious roots per wild-type hypocotyl. These conditions provided the basis to screen the En-1 gene machine for mutants that either failed to develop or formed elevated numbers of adventitious roots. Several mutants that formed elevated numbers of adventitious roots under our screening conditions were identified within the En-1 gene machine. The mutants were termed medusal (Fig. 1A) and medusa2 (figure 1B) because they preferentially formed a ring of adventitious roots close to the apex of the seedling following etiolation. Neither mutant formed elevated numbers of adventitious roots under either constant light or dark grown conditions (figure 1A & B). Crosses between medusal and medusa2 confirmed that they were not allelic with one another. However, crosses with two existing adventitious root mutants, surl [5] and sur2 [6], confirmed that medusa2 was allelic to sur2 [7]. The medusal mutant was not allelic to either surl or sur2 and so potentially represents a novel locus controlling adventitious root formation. 150

Root architecture mutants in Arabidopsis thaliana

A

B

C

~~,

i!j , t J

L

D/L

D

L

D/L

D

Figure 1. Phenotypes of the medusal, medusa2 and tripod mutants. The medusal (A) and medusa2 (B) seedlings were grown on MS agar under the following regimes; L - grown for 15 days in constant white light; D/L - grown for 1 day in the light followed by 3 days in the dark and 11 days in the light; D - grown for 15 days in the dark. Arrowheads indicate the position of the junction between the hypocotyl and the root. Seedlings were moved to a single plate for photography. (C) Three examples of tripod mutant seedlings which have been grown for 15 days on MS agar under constant light conditions. Anchor root m u t a n t s . Arabidopsis is capable of initiating anchor roots at the hypocotyl-root junction (figure 1C). However, anchor roots rarely emerge from wild-type Arabidopsis seedlings (less than 0.5%; Harmston & Bennett, unpublished results). Anchor root mutants were therefore identified on the basis of their ability to form one or more anchor roots. One anchor root mutant, termed tripod (tpd), forms a significantly increased number of anchor roots (figure 1C). Preliminary studies have observed that tpd seedlings initially produce a short primary root which stops elongating after ten days, followed by a pair of anchors roots that elongate to a similar length as the primary root, to create the characteristic three pronged tripod root architecture (figure 1C). Lateral root m u t a n t s . To date, only a handful of mutants have been identified on the basis of their lateral root phenotype in Arabidopsis. Mutations like alf4 and surl either abolish or exhibit an over-proliferation of lateral roots, respectively [8, 5, 9]. Visual screens for mutants exhibiting more subtle alterations in their lateral root development, spacing and/or number within the En-1 gene machine have proved unsuccessful. Mutants that exhibit subtle differences in their lateral root architecture, such as tir3 and pasl, were originally identified as a result of a phytohormone-related defect[10,11]. Such observations have prompted us to perform a selection of phytohormone-based screens in order to identify root elongation and lateral root mutants. The antagonistic effects of auxin and cytokinin have been well documented for lateral root initiation [12]. IAA acts by stimulating cell division within pericycle tissues [13], whereas cytokinin treatment blocks lateral root development (figure 2). A screen for mutants with reduced auxin sensitive root elongation was 151

Embryogenesis, Development and Architecture 0.6

Figure 2. Cytokinin inhibits the formation of lateral roots in wild-type Arabidopsis thaliana. Wild-type (Columbia) seedlings

0.5

were grown in the presence of various concentrations of benzyladenine (BA) with or without the addition of 20mM Ag § The seedlings were grown for 11 days after which time the number of emerged lateral roots per mm of primary root were counted. Error bars represent the standard deviation (n-20).

0.4 0.3 0.2

performed since several auxin response mutants have been described to form fewer lateral roots [ 14]. In par0.1 allel, a screen designed to identify mutants that contin0 1-ued to form lateral roots in the presence of cytokinin [BA]#M 0 0 0.1 0.1 0.01 0.01 was also performed. The latter screen required the in-t[Ag +] 20~ M " + clusion of silver in order to block the inhibitory effects caused by cytokinin-induced ethylene production on root elongation (figure 2). Several mutants were identified which were subsequently characterised at the molecular level. -

-t-

-

Molecular genetic characterisation of En-1 induced mutations The autonomous maize element En-1 is highly active in Arabidopsis [2]. En-1 transposes via a cut and paste mechanism in which excision from the donor site is regularly followed by reinsertion at other positions in the genome. The continuously changing position of En-1 makes the development of multiple copy lines feasible, reducing the number of lines required to be screened 9 However, the ease with which the En-1 element causing the mutation can be identified is largely dependent on the copy number and stability of the element(s) within the mutant line being studied 9 We initially describe a simple example, where an auxin response mutant line contains relatively few En-1 elements. The mutant was out-crossed with wild-type, then the F1 allowed to self-fertilise to observe segregation of the recessive mutant phenotype in the F2 generation. DNA was isolated from a selection of F2 lines that were either homozygous (-/-), heterozygous (-/+) or wild-type (+/+) for the mutation. Southern hybridisation using a probe made to the 3' end of the En-1 gene identified 4 different sized bands, indicative of the number of En1 elements present within the original mutant line (figure 3). A single En-1 element was clearly observed to faithfully co-segregate with the F2 +/-/+/+ Genotype lines that were homozygous or heterozygous for the mutant phenotype, but never with wild-type F2 lines (figure 3). The exact correlation between the presence of 7kb ~ - - 5kb

Figure 3. Molecular analysis of an En-1 mutagenised line containing 4 inserted elements. DNA was prepared from individual F2 plants which were heterozygous (+/-) or homozygous (-/-) for the mutant phenotype and from F2 plants having a homozygous wild-type genotype (+/+). The DNA was restricted with EcoRV, electrophoresed on an agarose gel and Southern blotted. The blot was probed with a DIG labelled probe made to the 3' end of the En-1 element. A single element indicated by an arrowhead was found to cosegregate with the (+/-) and (-/-) plants which was absent from the (+/+) plants, thus representing a strong candidate for the insertion causing the mutant phenotype.

152

~ - - 4kb

.~.~

4 - - 2kb :

.

41"--Ikb

Root architecture mutants in Arabidopsis thaliana

the En-1 element and the mutant phenotype is indicative that the particular transposon is closely linked with (and possibly causes) the mutation. The DNA was size fractionated to isolate just the En-1 element causing the mutation and TAIL-PCR used to isolate flanking sequence [15]. The previous example is somewhat unusual in that most of the gene machine derived mutant lines we have characterised by Southern hybridisation contain up to 15 En-1 elements. More seriously, frequently no single element appears to faithfully co-segregate with a mutant phenotype. The latter observation appears to be due to the tendency of the En-1 element to leave footprints at the original site of transposon insertion following excision [7]. En-1 footprints are short duplicated stretches of 2-4 nucleotides that act to maintain the mutant phenotype in the absence of a transposon. The frequency with which such footprinting events occur acts to reduce the level of confidence with which you can predict the particular En-1 element causing the mutation. In order to circumvent the disruption of segregation caused by footprinting, we have developed a bulked segregation analysis-based protocol (figure 4). Southern analysis is performed on just three pools of F2 DNA in order to identify a single En-1 element which causes the mutant phenotype. Two sets of F2 plants segregating for the mutant phenotype are generated from individual F1 plants. DNA is prepared from 10 homozygous F2 wild-type (+/+) lines (5 from each F2 set) and from 5 F2 homozygous (-/-) or heterozygous (-/+) plants for the mutant phenotype for each F2 set (MS1 and MS2). The DNA is restricted, run on a gel, Southern blotted and then probed with either the 5' or 3' end of the En-1 element. When analysing the Southern data, the En-1 element that causes the mutation should only be found in MS 1 and MS2 samples and should be absent from the wild-type (+/+) sample. Footprinting within one or two of the mutant lines is unimportant since multiple mutant lines are sampled and so some will retain the element within the bulked pool. The plant sequence flanking the particular En-1 element causing the mutation can be cloned by initially size selecting mutant genomic DNA, then using a TAILMS 1 MS2 PCR [ 15] or inverse PCR based protocol to amplify plant DNA. Once + ] + +]- +]the plant sequence is obtained, primers can be designed to allow a 0 0 ,~ diagnostic PCR demonstrating the presence of the En-1 element within 0 0 the gene of interest to be established. For example, we have observed that analysis of 50 F2 seedlings showed the presence of a diagnostic 0 ~:_~ 0 PCR product that co-segregated with the sur2 mutant phenotype in all o

o

,q 0 o

o o

o

o

o

o

Figure 4. A schematic representation of a bulked segregation analysis allowing rapid identification of an En-1 element causing a mutant phenotype. Two independent F2 populations segregating for the mutant phenotype of interest are made. Tissue samplescollectedfrom 5 individualheterozygous(+/-) or homozygousmutant (-/-) F2 plants from each population were used to prepare genomic DNA (MS 1 and MS2). Genomic DNA was also prepared from 10 wild-type F2 plants (+/+). The DNAs were restricted, run on a gel and Southern blotted. A probe made to either the 5' or 3' end of the En-1 element was used to probe the Southern blot. Only bands which are present in both +/- (or-/-) samples and which are absent from the wildtype (+/+) sample represent candidates for the element causing the mutant phenotype as indicated by the arrow head. 153

Embryogenesis, Development and Architecture but 4 cases. Analysis of the gene sequence in these cases revealed a 4 base insertion footprint in 3 of the alleles and a 2 base insertion in the fourth, providing positive confirmation of the identity of the SUR2 gene [7].

Discussion We have described a series of novel screens that have been developed to identify root architectural mutants within the En-1 gene machine population. Several classes of root architectural mutants have been identified including the anchor root mutant tripod and the adventitious root mutants, medusal and medusa2 (figure 1). Attempts to identify candidate En-1 transposon elements that co-segregate with the root mutant phenotypes have been frustrated by the high frequency of transposon excision and footprinting, necessitating the development of a bulk segregant analysis based approach. This has resulted in the identification of the TRIPOD and MEDUSA2 (SUR2) genes. To date, our knowledge of the genetic regulation of lateral root development has come from studying mutants identified using conventional genetic approaches. However, lateral root mutants such as surl and alf4 have been identified on the basis of their extreme root architecture [5, 8, 9]. Other lateral root mutants have been identified indirectly, as a result of a phytohormone-related defect [14, 10]. The paucity of lateral root mutants is likely to reflect the difficulty in identifying mutants with subtle differences in lateral root numbers and the undoubted genetic complexity of this process. Adopting a quantitative genetic approach would allow researchers to rapidly calculate the number, relative importance and chromosomal location of the quantitative trait loci (QTL) regulating lateral root number, providing the basis for future gene isolation. Such a QTL based approach has been used to isolate the gene that represents the major locus regulating branching in maize [16]. The development of well characterised recombinant inbred (RI) populations [ 17, 18] and the impending completion of the Arabidopsis genome sequence makes QTL-based gene mapping a very effective approach to characterise complex genetic traits [19].

Materials and methods Arabidopsis seedlings. All seed was surface sterilised [4] and plated onto MS agar (4.3g/1 MS salts [Sigma, Dorset, U.K.], 1% sucrose and 1% bacto-agar, pH to 6.0 with 1M KOH). The plates were placed at 4~ for 48 hours and then in constant white light at 22~ unless stated otherwise. Phytohormone mutant screens. Auxin resistance and hypersensitivity screens. Seedlings were grown on plates containing either 0.1mM 2,4-dichlorophenoxyacetic acid [Sigma] for the resistance screens or 0.01mM 1naphthaleneacetic acid [Sigma] for hypersensitivity screens. The seedlings were grown for 5 days after which time the primary root lengths were screened for either long roots (resistance screen) or short roots (hypersensitive screen) compared to the wild-type control. Growth of

Cytokinin screens. Seed was germinated on MS agar containing 0. l mM benzyladenine [Sigma] plus 20mM AgNO3 and grown for 11 days after which time the plates were screened for mutants which formed more lateral roots than the wild-type control. Adventitious root mutant screens. Seed was plated on MS agar and the plates placed at 4~ for 48 hours followed by 24 hours in the light, 72 hours in the dark to induce etiolation and then 8 days in the light. After this time seedlings were screened for adventitious roots originating from the hypocotyl. A n c h o r root mutant screens. Seedlings were grown on MS agar plates under constant light conditions for 12 days after which time they were screened for anchor root formation.

154

Root architecture mutants in Arabidopsis thaliana

Acknowledgements We would like to acknowledge funding from the EC framework IV PL96 0487 for supporting this research. A u t h o r s of this p u b l i c a t i o n Alan Marchant, Ranjan Swamp and Malcolm J. Bennett* School of Biological Sciences, University of Nottingham, UK. * Corresponding author References 1. 2. 3.

4. 5.

6. 7.

8. 9. 10.

11.

12. 13. 14. 15. 16. 17. 18.

E. Wisman, G.H. Cardon, P. Fransz and H. Saedler. (1998) The behaviour of the autonomous transposable element En/Spm in Arabidopsis thaliana allows efficient mutagenesis. Plant Mol. Biol. 37, 989-999. G.H. Cardon, M. Frey, H. Saedler, and A.Gierl. (1993) Mobility of the maize transposable element En/Spm in Arabidopsis thaliana. Plant J. 3, 773-784. E. Baumann, J. Lewald, H. Saedler, B. Schulz and E. Wisman. (1998) Successful PCR-based reverse genetic screens using an En-1 mutagenised Arabiclopsis thaliana population generated via single seed descent. Theor. Appl. Genet. 97, 729-734. N.R. Forsthoefel, Y. Wu, B. Schultz, M.J. Bennett and K.A. Feldmann (1992) T-DNA insertion mutagenesis in Arabiclopsis: prospects and perspectives. Aust. J. Plant Physiol. 19, 353-366. W. Boerjan, M-T. Cervera, M. Delarue, T. Beeckman, W. Dewitte, C. Bellini, M. Caboche, H. Van Onckelen, M. Van Montagu and D. Inz6. (1995) superroot, a recessive mutation in Arabidopsis, confers auxin overproduction. Plant Cell 7, 1405-1419. Delarue, M., Prinsen, E., Van Onckelen, H., Caboche, M. and Bellini, C. (1998) Sur2 mutations of Arabidopsis thaliana define a new locus involved in the control of auxin homeostasis. Plant J. 14, 603-611. I. Barlier, M. Kowalczyk, A. Marchant, R. Bhalearo, M.J. Bennett, G. Sandberg and C. Bellini. (2000) SUR2 gene of Arabidopsis thaliana, involved in the control of auxin homeostasis, encodes the Cytochrome P450 CYP83B 1. Proc. Natl. Acad. Sci. USA (submitted) J.L. Celenza, P.L. Grisafi and G.R. Fink. (1995) A pathway for lateral root formation in Arabidopsis thaliana. Genes & Dev. 9, 2131-2142. J.J. King, D.P. Stimart, R.H. Fisher and A.B. Bleecker. (1995) A mutation altering auxin homeostasis and plant morphology in Arabidopsis. Plant Cell 7, 2023-2037. M. Ruegger, E. Dewey, L. Hobbie, D. Brown, P. Bernasconi, J. Turner, G. Muday and M. Estelle. (1997) Reduced naphthylphthalamic acid binding in the tir3 mutant of Arabidopsis is associated with a reduction in polar auxin transport and diverse morphological defects. Plant Cell 9, 745-757. P. Vittorioso, R. Cowling, J-D. Faure, M. Caboche and C. Bellini. (1998) Mutation in the Arabidopsis PASTICCINO 1 gene, which encodes a new FK506-binding protein-like protein, has a dramatic effect on plant development. Mol. Cell. Biol. 18, 3034-3043. P.J. Davies. (1996) Plant hormones and their role in plant growth and development. Kluwer Academic Publishers. Dordrecht, Netherlands. M.J. Laskowski, M.E. Williams, C. Nusbaum and I.M. Sussex. (1995) Formation of lateral root meristems is a two-stage process. Development 121, 3303-3310. L. Hobbie and M. Estelle. (1995) The axr4 auxin-resistant mutants of Arabidopsis thaliana define a gene important for root gravitropism and lateral root initiation. Plant J. 7, 211-220. Y-G. Liu, N. Mitukawa, T. Oosumi and R.E Whittier. (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J. 8 457-463. J. Doebley, A. Stec and L. Hubbard. (1997) The evolution of apical dominance in maize. Nature 386, 485488 C. Lister and C. Dean. (1993) Recombinant inbred lines for mapping RFLP and phenotypic markers in Arabidopsis thaliana. Plant J. 4, 745-750. C. Alonso-Blanco, A.J.M. Peeters, M. Koornneef, C. Lister, C. Dean, N. van den Bosch, J. Pot and M.T.R. Kuiper. (1998) Development of an AFLP based linkage map of Ler, Col, and Cvi Arabidopsis thaliana

155

Embryogenesis, Development and Architecture ecotypes and construction of a

Ler/Cvi recombinant

inbred line population. Plant J. 14, 259-271.

19. C. Alonso-Blanco and M. Koornneef. (2000) Naturally occurring variation in Arabidopsis: an

underexploited resource for plant genetics Trends in Plant Sci. 5, 22-29.

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

The Cluster: "Responses To Challenges Of The Environment" Plants can be confronted to multiple biotic and abiotic stress situations during development and have evolved specific responses to deal with these environmental challenges. The ability of plants to react against adverse conditions represents an advantageous characteristic contributing to their selection. The study of the molecular mechanisms mediating the plant response to external stimuli is therefore of interest to understand the natural processes mediating the adaptation of plants to stress and to generate new methods to improve agriculture productivity. This cluster groups a number of projects directed to the study of the molecular mechanisms activated in plants in response to different stress conditions. Efforts of many groups are focused in the interaction of plants with different type of pathogens, nematodes, fungi, virus, and bacteria. The remaining projects address the responses of plants to salinity and the control of photorespiration, an important trait for productivity and water use efficiency. The ten projects integrated in this cluster are the following: - BIO4-CT96-0318. Basic and development of molecular approaches to nematode resistance, co-ordinated by G. Gheysen (BE). - BIO4-CT96-0352. The molecular and cellular basis of specific Septoria tritici leaf blotch of wheat, caused by Mycosphaerella graminicola, co-ordinated by J. Brown (UK). - BIO4-CT96-0515. Structure/function analysis of LRR proteins and their ligands inplants pathogens interactions and engineered resistance, co-ordinated by J. Jones (UK). - B IO4-CT97-2244. Delivery of elicitors and pathogenicity factors from bacterial pathogens and their interaction with plant cells: application of basic studies, co-ordinated by J. Mansfield (UK). - BIO4-CT97-2300. Composition of plant virus RNA replicases, co-ordinated by J. Bol (NL). - BIO4-CT97-2356. Characterisation of recessive genes that control natural resistance to potyvirus, co-ordinated by E. Jonhansen (DK). - BIO4-CT97-2120. Induced resistance of plants to pathogen infection: triggering and expression, co-ordinated by C. Castresana (ES). - B IO4-CT97-2275. Central role in adaptation of fourteen three proteins, co-ordinated by D. Collinge (DK).

Carmen

Castresana, Campus Universidad Autonoma, C.S.I.C., Centro Nacional de Biotechnologia, Madrid, Spain

157

Challenges of the Environment - BIO4-CT96-0775. Ion transport and signal transduction pathways contributing to salt tolerance in plants, co-ordinated by R. Leigh (UK). - BIO4-CT97-2002. Control of photorespiration in plant leaves by RD technology, co-ordinated by B. Miflin (UK). Two reports by Andy Maule and Hans Helder are presented in these proceedings, illustrating the progress achieved in these particular projects. The main goal in these projects concerns the understanding of the molecular mechanisms controlling the interaction of agronomical important plants with different type of pathogens such as virus (BIO4-CT97-2356) and nematodes (BIO4-CT96-0318). Plant diseases are of economical significance in Europe where are responsible for significant losses in agronomical important crops. Most diseases are controlled by the use of chemicals which are toxic and frequently non-specific. Moreover, these compounds have a negative effect for both the consumers and the environment. The study of these plant-pathogen interactions will provide new fundamental information that will help in the development of alternatives to generate environmental-friendly crop protection strategies.

158

Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

Concerted Efforts To Develop Handles For Plant Parasitic Nematode Control

Abstract Plant-parasitic nematodes - especially root knot and cyst nematodes - are economically important pests in numerous crops. Chemical soil sterilisation and the use of other unselective pesticides to control plant parasitic nematodes are still a common practice in many European countries and at present no realistic alternatives are available. For the identification of handles to control root knot and cyst nematodes we need to know how they interact with their host. Two main types of endoparasitic nematodes can be distinguished: root-knot and cyst nematodes, both inducing a feeding site in the plant root, but in a different way. To get insight into the molecular mechanisms behind this complex interaction, several strategies to analyse plant gene expression in response to nematode infection have been followed. Random in vivo gus fusions have been particularly successful in identifying plant promoter sequences that are highly activated in nematode feeding sites, with very little expression elsewhere in the plant, but the isolation of the corresponding genes is often not straightforward. Many highly transcribed plant genes have been identified in the feeding sites, but few have been characterised in such detail as to know how important they are for a successful infection. The available data are nevertheless providing interesting tools for novel strategies to engineer nematode resistance into crops. Concomitantly, the signals coming from the nematode that are triggering this plant response or that are important in other steps of the infection process are being characterised. This study has revealed that plant-parasitic nematodes produce many different enzymes to enable them to infect the plant root and to protect themselves against the plant defence response.

Introduction Sedentary plant-parasitic nematodes are important pests of agricultural crops. Traditional management of plant-parasitic nematodes is relying on three basic strategies: crop rotation, agrochemicals, and the use of resistant plants. Crop rotation is sometimes economically disadvantageous and is often not effective, for example in the case of the root-knot nematode Meloidogyne incognita that has a very broad host range. Chemical control is expensive, effective nematicides are difficult to contain and have a broad toxic spectrum, including animals and humans. Host resistance is the most environmentally and economically sound method [ 1]. Unfortunately, natural resistance to plant parasitic nematodes is not available for all cul-

Godelieve Gheysen, Departement Plantengenetica, Universiteit Gent, Belgium

159

Challenges of the Environment tivated crop species. Furthermore, a very important limiting factor to the general usefulness of natural resistance genes is the specific nature of their protection, which is often restricted to one nematode species or even certain pathotypes [2]. An alternative strategy that tries to combine effectiveness, broad applicability and durability with environmental friendliness is the engineering of nematode resistance into plants. In the last few years several excellent reviews have been written on this research development, and the reader is referred to them for more general aspects [3-4]. Here, we will summarise the breakthroughs of the last years, mainly in the light of the co-ordinated efforts done by the 12 laboratories involved in the ARENA project of the European Union.

Morphological and histological changes Sedentary nematodes establish and maintain an intimate relationship with their host. After invading the plant root, they migrate to the vascular cylinder in search for a cell that can serve as an initial feeding cell. In response to repeated stimulation by the parasite, this cell develops into either a syncytium (for cyst nematodes, such as Globodera and Heterodera), or several cells are stimulated to form a system of giant cells (as is the case for root-knot nematodes). The combination of light and electron microscopy studies of fixed samples and time-lapse video microscopy on living material has provided detailed insight in the establishment and structure of the nematode feeding site [5-6, reviewed in 3]. The syncytium is formed by breakdown of plant cell wails and subsequent fusion of neighbouring cells. A giant cell is formed as the result of repeated nuclear divisions without cytokinesis. Although these feeding sites differ from each other in ontogenesis and structure, their function is the same: to supply the nematode with sufficient nutrients for growth and reproduction. This common function is reflected in a final analogous ultrastructure. Syncytial and giant cells are hypertrophied and multinucleated cells with a dense granular cytoplasm, an increase in rough endoplasmic reticulum and in the number of mitochondria. Recently the formation of cell wall openings occurring during syncytium differentiation in A. thaliana roots was studied in more detail [7]. The first openings are formed by gradual widening of existing plasmodesmata followed by the fusion of protoplasts from two adjacent cells. At later stages, wall openings form exclusively in cell walls without the involvement of plasmodesmata. The sequence of events leading to these openings is: accumulation of ER membranes accompanying initial wall lesions, expansion of lesions until the middle lamella, and finally dissolution of the middle lamella [7]. Functional plasmodesmata were never found in the outer wall of mature syncytia, although they are present in the walls of the adjacent sieve elements. These plasmodesmata were always closed from the syncytium side by wall material. It can be concluded that the formation of a syncytium is accompanied with many changes in cell wall biology: callose-like deposition around the stylet, cell wall ingrowths adjacent to the xylem, widening or closing of plasmodesmata depending on their location in the syncytium, and de novo formation of wall openings. It will be interesting to see which enzymes are involved in these processes and how they are temporally and spatially controlled. Another aspect of nematode feeding site formation that is being unravelled is the activation of the cell cycle by the nematode stimulus. Sedentary endoparasitic nematodes induce multinucleate feeding cells in the roots of their host plants. These cells undergo multiple rounds of 160

Plant parasitic nematode control shortened cell cycles leading to genome amplification and hypertrophy of the cytoplasm. This aspect of the infection process has been thoroughly reviewed by [8] and more recently by [9]. It is now generally believed that giant cells develop by repeated mitosis without cytokinesis [5]. Jones and Payne [10] showed that cell plate vesicles initially lined up between the two daughter nuclei but then dispersed, resulting in the abortion of the new cell plate formation. No mitosis has been seen in syncytia [11 ], although Piegat and Wilski [ 12] reported an initial mitotic stimulation during syncytium induction by Globodera rostochiensis in infected potato roots. However, it is not clear from the data shown whether this mitosis is really inside the developing syncytium. Indeed, stimulation of cell division has been observed in parenchyma cells surrounding the syncytium and which will be incorporated [13-14]. The enlargement of nuclei indicates that DNA multiplication is taking place within the syncytial tissue during and after the incorporation of new cells through cell wall dissolution [ 11]. Studies with cell cycle inhibiting drugs indicate that mitosis is essential for giant cell development and for normal syncytium expansion [ 14].

Nematode secretions Stylet secretions that originate in the esophageal gland cells of plant parasitic nematodes are believed to play a major role in pathogenesis. Secretions from the dorsal gland cells have always been considered critical for pathogenesis but till recently a role for the subventral glands in the infection process has been questioned. Subventral gland secretions are released into the pharyngeal lumen behind the pump chamber and were thought only to be able to pass posteriorly into the intestine. However, immunolocalisation of HG-ENG-2 demonstrated the presence of this protein in the nematode's subventral glands (where it is produced), as well as in the root cortical tissue after secretion from the stylet [ 15]. This HG-ENG-2 protein belongs to a family of f~-l,4-endoglucanases first identified in secretions of cyst nematodes by [16], and later also in root knot nematodes [ 17]. These enzymes presumably facilitate the migration of the nematode through plant roots by partial cell wall degradation. The corresponding genes represent the first cellulase genes ever cloned from an animal and show highest homology (35-40%) with 13-1,4-endoglucanases from bacteria [16]. Other lytic enzymes identified in the nematode secretions include pectate lyase and proteases (Herman Popeijus and John Jones, unpublished results). Other proteins that are present in nematode secretions (although not necessarily from the esophageal glands) appear to be related to the active protection of the nematode against the plants defense response. GP-SEC2 functions as a fatty acid binding protein capable of binding e.g. linolenic and linoleic acids, that are important precursors in plant defence signalling pathways. In addition, superoxide dismutase and thioredoxin peroxidase secreted by the nematode are involved with inactivation of superoxide radicals and hydrogen peroxide respectively. How important these proteins or the above mentioned lytic enzymes are for nematode pathogenesis, can only be decided if inactivation or absence of these proteins results in a lower percentage of successful nematode infections. Inhibition of the proteins could be achieved by the expression of plantibodies [ 18]. Inactivation of the genes is another theoretical possibility, but transformation of plant-parasitic nematodes is still virtually impossible [19]. A strategy that could be useful is RNA interference, a method capable of efficiently eliminating specific mRNAs in C. elegans [20]. 161

Challenges of the Environment The most intriguing function of the esophageal secretions, their role in formation of the feeding site, is still a black box. Unfortunately, in contrast to comparative studies of nematode development and the genes involved, analysis of the model nematode C. elegans does not help much in respect to pathogenesis-involved genes. However, with the current expertise and molecular technology, at least some clues about possible signalling molecules in the secretions of plant-parasitic nematodes should be identified in the near future.

Screening Screening for nematode-responsive plant regulatory sequences was done by by T-DNA tagging. Plant genes that are up-regulated after nematode infection have first been identified by differential screening of cDNA libraries [21-23] or from subtraction cDNA libraries [24] and later by differential display [25, Vercauteren et al., accepted by MPMI]. To identify promoters that are early and specifically induced in the nematode feeding sites, the most appropriate method is undoubtedly the analysis of promoter-reporter fusions (for example with 13-glucuronidase [gus]). Plant-gus fusions can be made at random in vivo and then screened for specific expression patterns. This "promoter trapping" or "tagging" approach is based on the random integration of a promoterless gus gene after transformation into a plant species, often A. thaliana [26]. When inserted downstream from a plant promoter that is inducible by nematode infection, a higher GUS activity is expected to be seen at the infection site (Fig. 1). The elegance of this method lies in the ability to directly visualise induced expression in nematode feeding sites at various stages of the interaction, while analysing the specificity of expression using uninfected parts of the same plant and control plants. Furthermore, promising plant lines can easily be tested for gus activation upon infection by different types of nematodes. This "tagging" approach has been performed in several labs of the ARENA project and has yielded many interesting promoters [27-30]. The hope was that the homozygous progeny of these T-DNA tagged lines could be knock-outs for the tagged gene,

Figure 1: GUS activity in a Att0025-R/1 syncytium 6 days post inoculation with H e t e r o d e r a s c h a c h t i i (from: Barthels et al. 1997, Plant Cell 9:2119-2134)

,f f

~:~~F::~~ /'

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Plant parasitic nematode control

and would therefore yield information on the role of this plant gene, in general or possibly even during nematode infection. Initially none of the lines analysed had an obvious phenotype [28], strengthening the belief that tagging often yields cryptic promoters [31 ]. Although these cryptic promoters are not less useful for expressing a transgene in the nematode feeding sites, from a more fundamental point of view it would be informative to find the endogenous genes that are specifically activated after nematode infection. However, more recently one of the tagged lines was found to have a T-DNA insertion in the rpe gene (D-ribulose-5-phosphate-3-epimerase) resulting in a mutant phenotype. Homozygous rpe plants have a germination deficient phenotype that can be rescued in dwarf plants on sucrose-supplemented medium [30]. Also, upon further study, several other T-DNA insertions appeared to be linked to a "tagged" gene, although in unexpected configuration, such as inside the promoter instead of downstream, and even several kb away from the gene (line Att0001 (M.Karimi), line Att0025 (S. Ohl), line Att0884 (P.Puzio); unpublished observations).

Transcriptional control of nematode-inducible genes In the light of applications, the most interesting A. thaliana promoters are also introduced into crop species to check their activity after nematode infection. The Art0001 (ARM 1) promoter, as well as some other A. thaliana promoters, are clearly up-regulated after nematode infection in oilseed rape and sugarbeet [32, G. Gheysen unpublished], but not in potato, whereas the rpe promoter is activated in potato nematode feeding sites [B. Favery, unpublished]. Besides possible differences in cell-specific or temporal gene regulation between plant species, another reason could be that in potato, the syncytium is formed in cortex cells [33, 34], while in A. thaliana procambium cells are normally used [13]. Maybe the apparently contradictory results on CaMV35S-promoter activity inside syncytia could also be explained by the different plant species used. Goddijn et al. [27] and Urwin et al. [35] describe down-regulation of the 35S promoter in syncytia induced by H. schachtii in A. thaliana, and this was also observed in sugarbeet (G. Gheysen, unpublished), but not in potato syncytia induced by G. rostochiensis [observed up to 13 dpi, 36]. Now that several nematode-induced promoters are available, it could be speculated that common regulatory sequences should be obvious by sequence comparison. Such comparison is however not that straightforward because these promoters have different temporal and spatial expression patterns besides their common expression in nematode feeding sites. Furthermore, different promoter elements or in different arrays may control expression in feeding sites in different promoters. Therefore, deletion analysis combined with genomic footprinting is being done to identify discrete regions that are involved in responsiveness to nematode infection and to distinguish them from other regions in the promoters that dictate their expression in other plant tissues. In a detailed analysis of the Lemmi9 promoter from tomato, a putative nematode responsive element has been identified which is being tested for functionality by fusion to a minimal promoter and mutational studies [37].

Engineering novel nematode resistance genes The cloning, modification and transformation of natural resistance genes is a promising new field for engineering nematode resistance in crop plants. However, to increase the choice of 163

Challenges of the Environment available strategies, and especially to have resistance constructs that broaden the spectrum of target nematodes, synthetic resistance genes are undoubtedly useful alternatives [38]. Broad-spectrum resistance should be feasible if the strategy focuses on common characteristics in the infection process of different nematode species such as the activation of the cell cycle

[8].

Table 1. Genes expressed in feeding sites

N e m a t o d e sensitivity of the rescued rpe m u t a n t s

M. incognita

H. schachtii

G a l l s / plant a

Cysts / plant a

Eggs / cyst b

Wild type

12.9 _+ 3,1

8.2 + 1.6

301 _+ 16

+/rpe

13.0+3,2

7.4+2.4

271 + 2 1

rpe / rpe

0.1 (small)

3.8 + 1.3

320 + 25

Heterozygous +/rpe and the homozygous rpe/rpe mutant plants were

As described above, several tested, in vitro, in the same plates, on medium supplemented with sucrose 2% and kanamycin. Arabidopsis wild type WS were grown in "plant" promoters have been identhe same conditions except kanamycin. tified that are up (or down-) regua The number of galls and cysts per plant was determined four weeks lated upon nematode infection. after inoculation. Values represent the means + SE obtained on 25 plants, each inoculated with one hundred surface-sterilized J2 of M. Depending on the strategy, a proincognita or H. schachtii. moter can be chosen (or engib The number of eggs per cyst was counted 2 months after infection. (Favery et al. 1998, EMBO J. 17 : 6799-6811) neered) to ensure the desired spatial and temporal expression pattern for expressing a protein that is inhibiting one of the steps in pathogenesis. For sedentary endoparasites, on which this text concentrates, two main approaches can be envisaged: either the nematode is directly attacked by expressing a nematoxic protein (reviewed in [39]), or it is indirectly affected by destroying the feeding cells or inhibiting their functioning. The basic concept here is that genes necessary in the compatible interaction are engineered and used against the nematodes. Blocking the expression of plant genes that are normally up-regulated and important for the induction or maintenance of the feeding site is feasible by the antisense strategy [40] and a promoter which is highly induced but not necessarily uniquely expressed inside the feeding cells would be good for this task. Even a slight decrease in efficiency of food supply might be sufficient to significantly reduce nematode multiplication. Genes known to be expressed at high levels in feeding sites and thus probably needed to induce or maintain these structures are cdc2a [41], hmgl [4] and rpe [30, Table 1]. Therefore, transgenic plants are being made with antisense constructs of cdc2a [42], hmgl [43] and rpe [30] fused to a nematode feeding site specific promoter.

Conclusions The techniques to identify differentially expressed genes are constantly being improved. New opportunities are currently being provided by micro-array technologies and cDNA-AFLP transcript profiling. This will allow to complete the collection of genes that are up- or downregulated in the nematode feeding site as well as in the infecting nematode. The challenge will be to elucidate the role of these genes in the infection process. Because of the obligate biotrophic nature of the parasitic lifestyle of sedentary endoparasitic nematodes and the difficulty to obtain sufficient material for analysis, knowledge on the mo164

Plant parasitic nematode control lecular interaction between these nematodes and their host plants has long been very limited. Recent cloning of nematode genes encoding secreted proteins and a detailed analysis of the plant response by a joint effort of European laboratories is now quickly changing the state of the art, with promising perspectives for engineering nematode resistance into crop plants.

Authors of this contribution Godelieve Gheysen 1,2, Pierre Abad 3, Teresa Bleve 4, Vivian Blok 5, Carmen Fenoll 6, John A. Gatehouse 7, Florian Grundler 8, Keith Lindsey 9, Stephan Ohl 1~ Kristina Sagen 11, Robert Shields 12, and Johannes Helder 13 (all authors are partner in Basis and Development of Molecular Approaches to Nematode Resistance (ARENA, 1996-1999, EC grant BIO4-CT96-0318) 1Laboratorium voor Genetica, Departement Plantengenetica, Vlaams Interuniversitair Instituut voor B iotechnologie (VIB), and 2Vakgroep Plantaardige Productie, Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen, Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium; 3 Laboratoire de Biologie des Invert6br6s, Institut National de la Recherche Agronomique, B.E 2078, F-06606 Antibes, France; 4 Istituto di Nematologia Agraria Applicata ai Vegetali, 1-70126 Bari, Italy; 5 SCRI Nematology Department,DD2 5DA Invergowrie, Dundee, United Kingdom; 6 Departamento de Biologfa, Universidad Aut6noma de Madrid, Cantoblanco, E-28049 Madrid, Spain and Facultad de Ciencias del Medio Ambiente, Universidad de Castilla-La Mancha, E-45071 Toledo, Spain; 7 Plant Insect Group, Department of Biological Sciences, University of Durham, Durham DH1 3LE, United Kingdom; 8 Institut ftir Phytopathologie, Christian-Albrechts- Universit/~t, D-24118 Kiel, Germany; 9 Plant Molecular Biology Group, Department of Biological Sciences, University of Durham, Durham DH1 3LE, United Kingdom; 10Zeneca MOGEN International N.V., NL-2333 CB Leiden, The Netherlands; 11IACR-Rothamsted, Entomology and Nematology Department, AL52JQ Harpenden, United Kingdom; 11 Plant Breeding International Cambridge Ltd., Marls Lane, Trumpington, Cambridge CB2 2LQ, United Kingdom; 12 Wageningen University, Laboratory of Nematology, Binnenhaven 10, 6709 PD Wageningen, The Netherlands

Acknowledgement This review was supported by the EU grants BIO4-CT960318 and FAIR-CT96-1714.

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). O Elsevier Science B.V. All rights reserved.

Isolation And Application Of Nematode Induced Promoters, Genes And Proteins From Arabidopsis r Abstract This article gives a short introduction into the biology of cyst nematodes and plant responses to nematode infection. Furthermore, two strategies are presented to identify promoters or proteins that respond to nematode with an induced activation or synthesis, respectively. Promoters were isolated in a promoter tagging approach in Arabidopsis thaliana. The isolation and analysis of one promoter (ppyk20) is presented. Examples are given for application of ppyk20 in plant biotechnology to engineer resistance to cyst nematodes. In a second approach nematode induced proteins were identified in roots of infected A. thaliana plants. One of them is PYK10, a myrosinase. The cloning approach is described and a new concept to engineering nematode resistance is presented. Resistance is thought to be achieved by the activation of glucosinolates through myrosinases.

Biology of cyst nematodes Nematodes are a group of organisms in which bacterial feeding, but also predatory and parasitic trophism occur. In two orders, Tylenchida and Dorylaimida, plant parasitism has evolved. Plant-parasitic nematodes are obligate biotrophic parasites and draw nutrients from the cytoplasm of living plant cells through a hollow stylet. This stylet is used to pierce the cell wall in order to inject secretions into the plant cell or to ingest nutrients from the cytoplasm. It also serves to pierce and cut openings in the cell walls and to release secretions that dissolve or weaken the cell wall or middle lamella. Plant parasitic nematodes can be grouped into ecto- or endoparasites, depending on whether or not they invade roots for feeding. Some endoparasites, the so-called sedentary nematodes, induce specific feeding sites in the root tissue. Endoparasites comprise economically important groups of nematodes such as root-knot nematodes and cyst nematodes. Here, we concentrate on cyst nematodes, which cause major problems in potato, sugarbeet, weat and soy bean. After invasion the root the juveniles select a single cell, the so called intitial syncytial cell (ISC). The wall of this cell is carefully pierced and secretions are released. The cell responds readily to the nematode's activity by formation of the specific feeding structure. Cyst nema-

P.S. Puzio, Universit~it Kiel, Institut fiJr Phytopathologie, Kiel, Germany

169

Challenges of the Environment todes induce syncytial fusions of mainly procambial, cambial and pericycle cells of the vascular cylinder. Syncytia are formed by partial solution of the cell walls and subsequent fusion of the single protoplasts. In all sedentary nematodes the infective stages lose mobility after initiation of feeding. Thus the developing animals are absolutely dependent on the feeding site. As juveniles grow they assume a saccate shape and undergo three moults until the adult stage is reached. Whereas the globular or lemon-shaped females remain immobile, males become vermiforme again and emerge from the root to find females for mating. After mating the female produces eggs which are mostly retained in the females" body. When egg production is completed the females die whereas the cuticle undergoes changes forming a physically and chemically resistant cover, the cyst. Juveniles develop and moult within the eggs, where they are able to survive for more than ten years. Root exudates stimulate hatching and attraction of the hatched second stage juveniles. The intimate interaction between cyst nematodes and their hosts on the one hand makes it difficult to control the pathogens with conventional methods, one the other hand it should be possible to interfere with the exchange of signals between host and parasite and to inhibit essential processes in the interaction by means of molecular biotechnology. As syncytium formation is an essential prerequisite for nematode development it deserves highest attention to eludicate the underlying mechanisms. In principle two approaches can be followed. One is to identify compounds released by nematodes which stimulate altered plant growth (for review see e.g. [1 ]). The other approach which we present here is to analyse nematode induced responses in infected plants and to isolate genes that are involved in these processes.

The syncytium Syncytium formation can be divided roughly into three phases with blending transitions. During phase I only one single cell, the ISC, is affected. Secretions are released through the stylet which is inserted into the cell but does not perforate the plasmalemma. As first visible changes in the ISC callose-like material is deposited along the cell wall where the stylet is inserted [2], cytoplasmic streaming is accelerated, and the nucleus is enlarged. In further changes most organelles are involved. The vacuolar system is rearranged from a central vacuole to a high number of small separate vacuoles. Ribosomes, mitochondria, and plastids proliferate [3]. At the same time transition to phase II occurs during which surrounding cells are integrated by partial cell wall dissolution and fusion of their protoplasts [2]. This process continues until a syncytium is developed which consists of several hundred former single cells. The cell system is strongly hypertrophied and often fills almost the whole vascular cylinder. Phase III is reached when the system is fully developed and the syncytium is maintained by the adult females for food withdrawal during egg formation. The mature synctial protoplast is characterized by dense cytoplasm, a high proportion of smooth endoplasmic reticulum and lipid droplets [3]. The metabolically highly active tissue induces a strong metabolic sink which leads to a specifc unloading of phloem in the affected area [4].

Differentially expressed plant genes The differentiating cell complex also involves dramatic changes in gene regulation which have been reviewed recently [5, 6, 7, 8]. However, the basic mechanism of formation and 170

Nematode induced promoters, genes and proteins maintenance of the NFS is still unknown. Several molecular techniques have been employed to identify and isolate plant genes with organ-specific expression. In cases like NFS, where only limited amounts of relevant tissue is available, approaches were applied in a combination of PCR techniques, subtractive hybridisation and differential screening of cDNA populations. Using these techniques nematode-induced plant genes have been successfully isolated; e.g., a Lea-like gene, an extensin gene [9], MYB-like genes, H+ATPase genes [10] and a catalase gene [11]. A comparison between nematode infected and non-infected Arabidopsis roots by 2-D gel electrophoresis [12] and differential display of transcribed sequences [13] led to the identification of several proteins and mRNAs, which are up- and down-regulated after nematode infection. Another way to identify nematode responsive genes is to use transgenic plants harbouring a reporter gene under the control of a known promoter. In this way promoters of viral, bacterial and plant origin were found to respond to nematode infections [14]. The potential of this strategy to identify nematode responsive promoters has been shown first by Opperman et al., [15] with the Tob RB7 promoter. In the same way, Niebel et al., [16] observed that the promoters of the cell-cycle-related genes cdc2aAt and cyclAt were activated in NFS. Here, we describe the identification and isolation of two nematode responsive plant genes using the protein based method and the promoter tagging approach.

Tagging of a nematode induced promoter To identify a nematode responsive promoter and gene we deployed a so called promotertagging approach. This strategy is based on the random integration of a promoterless gus reporter gene in the plant genome and its activation by plant regulatory sequences. In this way, several plant promoters [17, 18] and a small number of a plant genes [19, 20] have been already isolated. Recently, a gene coding for D-ribulose-5-phosphate-3-epimerase (rpe) was found to be up-regulated in NFS [21]. In our study, a collection of almost thousand independent transgenic Arabidopsis lines (provided by MOGEN, Leiden, NL) was screened for reporter gene expression in NFS. In this way, several tagging lines have been identified, in which the expression of the reporter gene was observed inside the NFS [22]. One of these selected lines, #884, showed very specific gus activity in nematode feeding structures, leaf hydathodes, and in stipules [23]. The highest number of GUS expressing NFS in this line was detected 7 days after infection. DNA gel blot analysis indicated a single copy of the T-DNA in line #884. To isolate the plant DNA 5" to the T-DNA insertion site the inverse PCR was performed. In this way a 963 bp fragment could be isolated which was then used to screen a genomic library of A. thaliana. Thus, a 4.2 kb fragment containing the insertion site was isolated. No continuous open reading frames were detected close to the site of T-DNA integration [23]. However, at a distance of 744 bp 3" to the T-DNA insertion site an open reading frame encoding the pyk20 gene was found. To elucidate whether transcription of pyk20 responds to nematode infection in the wildtype plants a Northern blot analysis was performed. High levels ofpyk20 mRNA were found in the infected root segments, whereas only a weak signal could be detected in RNAs isolated from uninfected roots. The in situ expression analysis confirmed that pyk20 was indeed strongly 171

Challenges of the Environment expressed in NFS of infected Arabidopsis roots mainly at early stages of infection (1 to 5 days after inoculation) compared to uninfected roots [24]. Taking into account the similar results of the pyk20 gene expression pattern in Arabidopsis wildtype [24] and the reporter gene expression in the tagging line #884 [23] we can state that the T-DNA in line #884 was integrated into the promoter of the pyk20 gene.

Engineering resistance with the tagged promoter As the isolated promoter ppyk20 is strongly activated in syncytia it can be considered to drive expression of genes with products that may help to control nematode development in plants. A number of potential approaches have been presented [25, 26]. In principle two main strategies can be followed. One is to induce expression of genes with products that have nematicidal activitiy. The gene products such as proteinase inhibitors or toxins have to be ingested by the nematodes and are supposed to inhibit further development. The second strategy is not directed against the nematode but against syncytia. By an induced breakdown of the feeding site the associated nematode loses its nutritional basis and will die. Such an effect can be expected from barnase, a highly cytotoxic RNAse from Bacillus amyloliquefaciens [27]. As ppyk20 is induced but not exclusively active in syncytia a specific inhibitor protein, barstar [27], has to be transformed into such plants under the regulation of a promoter that is active in all plant tissues but not in syncytia. This characteristic has been identified in several promoters such as the 35s CaMV or the RolD promoter [28]. The barnase encoding gene will be fused to the NFS-responsive ppyk20 promoter thus leading to an high local expression of the gene product in snycytia. Accidental expression of the barnase in other parts of the plant will then be neutralized by the constitutive expression of his inhibitor (figure 1).

Characterisation of nematode responsive proteins The complex cellular and physiological changes involved in the formation and differentiation of NFS require a highly co-ordinated expression of plant genes in this high specific tissue. There are several ways to determine the nature of such differential expressed genes at protein level. We followed two different approaches which both are based on the altered protein expression patterns in the NFS induced after H. schachtii infection in Arabidopsis roots.

barstax

0

0 barnase

0

withharstar neutralized

0

o nucleus nematode

o

!0 0

9

0

o

9

00 172

Figure 1. Two-component

system to engineer resistance against plant-parasitic nematodes. The system consisting of a toxic and neutralizing component. The highly cytotoxic gene coding a RNA-se (barnase) has been fused to the nematode responsive ppyk20 promoter. Accidental background expression of barnase in other parts of the plant is neutralized by the constitutive expression of its inhibitor.

Nematode induced promoters, genes and proteins The first approach based on the production of monoclonal antibodies against protein extracts of NFS segments. After screening with ELISA techniques against protein extracts of NFS, uninfected roots and nematode, those hybridoma lines were selected which are clearly differentially expressed in infected roots. The identified monoclonal antibody was used to screen a cDNA expression library of Arabidopsis roots in mammalian COS cells [12]. In this way a cDNA of a protein called PYK10 was identified. The primary structure of this protein contains a motif which is common to members of the B-glycosylhydrolase family. Analysis of both amino acid and pyklO cDNA sequence revealed distinct homology to linamarase and myrosinase. The second way for isolation of nematode responsive proteins based on separation of the proteins from infected and non-infected roots using 2D-gelelectrophoresis. From about 700 distinct protein spots separated after the 2D-gelelectrophoresis 9 differential expressed polypeptide spots were selected for further analysis. After collection and purification of these 9 polypeptides the amino acid sequences were determined. On the basis of these sequences, degenerate oligonucleotides were synthesized in order to use suitable constructs as PCR primers to amplify the DNA sequences [12].

Engineering resistance with the isolated protein Currently we try to fevelop a new approach to engineer resistance against plant parasitic nematodes which is based on the myrosinase-glucosinolate system. This system is characteristic of the order Brassicaceae [29]. It includes sulphur- and nitrogen containing secondary metabolites, termed glucosinolates, and the degradative enzyme myrosinase [30]. Upon tissue disruption like wounding or pathogen attack this system is activated as the stored glucosinolate precursors are exposed to the myrosinases. After hydolysis of the glucosinolates their antimicrobial and phytotoxic properties emerge. According to immunocytochemical analyses the myrosinase pyklO is not synthesized in syncytia but in root covering tissue around syncytia [12]. In transgenic plants pyklO will be fused to a syncytium inducable promoter such as ppyk20. In this way the myrosinase be produced in syncytia. As the formation of syncytia in the procambial and cambial cells of the central cylinder involves a large number of physical and ~ .

0 mvrosinase /

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2. Use of the myrosinase-glucosinolate system to engineer resistance against plant-parasitic nematodes. The system consists of highly toxic secondary metabolites, the so-called glucosinolates, and the activating enzyme myrosinase. The NFS responsive ppyk20 promoter drives the local expression of the cloned pyklO myrosinase.

Challenges of the Environment structural changes of these cells, there is high probability that the myrosinase will come in contact with the glucosinolate precursors. In this way one or more of the the active 24 glucosinolates known in A. thaliana [31 ] may be formed. For some glucosinolates cytotoxic and nematicidal activity has been shown which will then cause the degeneration of the syncytium and/or the nematode (figure 2).

Conclusions The identification of syncytium specific i.e. nematode inducible genes or proteins is of basic scientific interest. However, the processes leading to induction, expansion, and maintenance of nematode induced syncytia are far from being understood. Each single identified gene, contributes to the understanding of the complex molecular, structural and physiological changes. Via cloning and gene transfer the isolated genes and proteins can be defined in their biological function. As our work and that of others touch many different aspects of plant biology a collaboration of specialists in the different fields is needed to solve raised problems. As shown in this short review, some of the cloned genes can directly point to an application in nematode control in transgenic plants, while others may need sophisticated approaches to engineer resistance.

Authors of this contribution RS. Puzio and EM.W. Grundler, Institut for Phytopathologie, Universit~it Kiel, HermannRodewald-Strasse 9, 24118 Kiel, Germany

References 1.

R.S. Hussey and F.M.W. Grundler, Nematode parasitism of plants, in R.N. Perry and D.J. Wright, (Eds), The physiology and biochemistry of free-living and plant-parasitic nematodes. Cab International, 1998, pp. 213243. 2. EM.W. Grundler, M. Sobczak and W. Golinowski, Formation of wall openings in root cells of Arabidopsis thaliana following infection by the plant-parasitic nematode Heterodera schachtii, Europ. J. Plant Path., 104 (1998) 545-551. 3. W. Golinowski, EM.W. Grundler and M. Sobczak, Changes in the structure of Arabidopsis thaliana during development of the plant-parasitic nematode Heterodera schachtii, Protoplasma, 194 (1996) 103-116. 4. A.B6ckenhoff, D.A.M. Prior, EM.W. Grundler and K.J. Oparka, Induction of phloem unloading in Arabidopsis thaliana roots by the parasitic nematode Heterodera schachtii. Plant Physiol., 112 (1996) 14211427. 5. G. Gheysen, W. van der Eycken, N. Barthels, M. Karimi and M. van Montagu, The exploitation of nematode-responsive plant genes in novel nematode control methods, Pestic Sci., 47 (1996) 95-101. 6. V.M. Williamson, R.S. Hussey, Nematode Patholgenesis and resistance in plants, Plant Cell, 8 (1996) 17351745. 7. G. Gheysen, J. de Almeida Engler, and M. van Montagu, Cell cycle regulation in nematode feeding sites, in C. Fenoll, EM.W. Grundler, and S.A. Ohl, (Eds.), Cellular and Molecular aspects of plant-nematode interactions, Kluwer Academic, Dordrecht, The Netherlands, 1997, pp. 120-132. 8. C.Fenoll, EA. Aristizabal, S. Sanz-Alferez and EE del Campo, Regulation of gene expression in feeding sites, in C. Fenoll, EM.W. Grundler, and S.A. Ohl, (Eds.), Cellular and Molecular aspects of plant-nematode interactions, Kluwer Academic, Dordrecht, The Netherlands, 1997, pp. 133-149. 9. W. Van der Eycken, J. de Almeida Engler, D. Inz6, M. van Montagu and G. Gheysen, A molecular study of root-knot nematode-induced feeding sites, Plant J., 9 (1996) 45-54. 10. D. McK. Bird and M.A. Wilson, DNA sequence and expression analysis of root-knot nematode-elicited giant cell transcripts, MPMI, 7 (1994) 419-424.

174

Nematode induced promoters, genes and proteins 11. A. Niebel, K. Heungens, N. Barthels, D. Inz6, M. van Montagu and G. Gheysen, Characterisation of a pathogen-induced potato catalase and its systemic expression upon nematode and bacterial infection, MPMI 8 (1995) 371-378. 12. K.-E Schmidt, Proteinanalytische Charakterisierung pathogenesespezifischer Vorg~inge im Wurzelgewebe von Arabidopsis thaliana nach Infektion mit dem Rtibenzystennematoden Heterodera schachtii, Ph.D. Thesis, Christian-Albrechts-Universit~it zu Kiel, Kiel, Germany, (1995). 13. I. Vercauteren, E. van der Schueren, M. van Montagu and G. Gheysen, Isolation of mRNA species expressed upon nematode infection by means of the differential display technique Fourth Annual Meeting of the European Union AIR-CAP on Resistance Mechanisms against plant-parasitic nematode, Toledo, Spain, 1996, pp. 15. 14. O.J.M. Goddijn, K. Lindsey, E M. van der Lee, J.C. Klap and EC. Sijmons, Differential gene expression in nematode-induced feeding structures of transgenic plants harbouring promoter-gusA fusion constructs, Plant J., 4 (1993) 863-873. 15. C.H. Opperman, C.G. Taylor and M.A. Conkling, Root-knot nematode-directed expression of a plant rootspecific gene, Science 263 (1994) 221-223. 16. A. Niebel, J. de Almeida Engler, A. Hemerly, E Ferreira, D. Inz6, M. van Montagu and G. Gheysen, Induction of cdc2a and cyclAt expression in Arabidopsis thaliana during early phases of nematode-induced feeding cell formation, Plant J., 10 (1996) 1037-1043. 17. S. Kertbundit, H. De Greve, F. Deboeck, M. van Montagu and J-E Hernalsteens, In vivo random 13glucuronidase gene fusions in Arabidopsis thaliana, Proc. Natl. Acad. Sci. USA., 88 (1991) 5212-5216. 18. J.E Topping and K. Lindsey, Insertional mutagenesis and promoter trapping in plants for the isolation of genes and the study of development, Transgenic Res., 4 (1995) 291-305. 19. P.S. Springer, W.R. McCombie, V. Sundaresan and R.A. Martienssen, Gene trap tagging of PROLIFERA, an essential MCM2-3-5-1ike gene in Arabidopsis, Science, 268 (1995) 877-880. 20. W. Wei, D. Twell and K. Lindsey, A novel nucleic acid helicase gene identified by promoter trapping in Arabidopsis, Plant J., 11 (1997) 1307-1314. 21. B. Favery, E Lecomte, N. Gil, N. Bechtold, D. Bouchez, A. Dalmasso and E Abad, RPE, a plant gene involved in early developmental steps of nematode feeding cells, EMBO J., 17 (1998) 6799-6811. 22. N. Barthels, E van der Lee, J. Klap, O.J.M. Goddijn, M. Karimi, E Puzio, EM.W. Grundler, S.A. Ohl, K. Lindsey, L. Robertson, W.M. Robertson, M. van Montagu, G. Gheysen and P.C. Sijmons, Regulatory sequences of Arabidopsis drive reporter gene expression in nematode feeding structures, Plant Cell, 9 (1998) 2119-2134. 23. ES. Puzio, D. Cai, EM. S. Ohl, U. Wyss and F.M.W. Grundler, Isolation of regulatory DNA regions related to differentiation of nematode feeding structures in Arabidopsis thaliana, Physiol. Mol. Plant Pathol., 53 (1998) 177-193. 24. ES. Puzio, J. Lausen, J. Almeida-Engler, D. Cai, G. Gheysen and EM.W. Grundler, Isolation of a gene from Arabidopsis thaliana related to nematode feeding structures, Gene, (submitted). 25. ER. Burrows and D. de Waele, Engineering resistance against plant parasitic nematodes using anti-nematode genes, in C. Fenoll, EM.W. Grundler, and S.A. Ohl, (Eds.), Cellular and Molecular aspects of plantnematode interactions, Kluwer Academic, Dordrecht, The Netherlands, 1997, pp.217-237. 26. S. Ohl, F. M. van der Lee and P.C. Sijmons, Anti-feeding structure approaches to nematode resistance, in C. Fenoll, EM.W. Grundler, and S.A. Ohl, (Eds.), Cellular and Molecular aspects of plant-nematode interactions, Kluwer Academic, Dordrecht, The Netherlands, 1997, pp. 250-261. 27. R.W. Hartley, Barnase and barstar. Expression of its cloned inhibitor permints expression of a cloned ribonuclease, J. Mol. Biology, 202 (1988) 913-915 28. Goddijn, O.J.M., Lindsey, K., Van der Lee, EM., Klap, J.C. and Sijmons, EC. (1993) Differential gene expression in nematode-induced feeding structures of transgenic plants harbouring promoter-gusA fusion constructs. Plant J. 4,863-873. 29. E O. Larsen, Glucosinolates, in E.E. Conn (Ed.), The biochemistry of plants, Academic Press, New York, 1981, pp. 501-525. 30. R. Bj6rkman and J-C. Janson, Studies on myrosinases I. Purification and characterisation of myrosinase from white mustard seed (Sinapis alba L.), Biochim. Biophys. Acta, 276 (1972) 508-518. 31. G.W. Haughn, L. Davin, M. Giblin and E.W. Underhill, Biochemical genetics of plant secondary metabolites in Arabidopsis thaliana, Plant Physiol., 97 (1991) 217-226.

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

Cis-Elements In Nematode-Responsive Promoters

The problem Biotrophic plant pathogens that depend on living cells need to use subtle strategies to tame plant cells in way for these to suit the pathogen's needs while being wholly functional. Plant endoparasitic nematodes do so by obliging root cells to differentiate into "nurse cells" from which they feed, through still unraveled mechanisms [1]. Different nematodes induce different types of nurse cells, collectively termed Nematode Feeding Sites (NFSs) [2-4]. For a long time it has been believed that nematode secretions are responsible for NFS induction, but only recently this has received experimental support [5]. How do these secretions act to make a plant cell shift its developmental fate? (figure 1) Although the signal molecules of nematode secretions are still unknown, and so are their primary plant targets, it has now been well established that many genes change their expression patterns at different times during NFS development. For several of these genes, the change in expression occurs at the transcriptional level, and their promoter regions suffice to confer them nematode-responsiveness [6-9]. If promoters are turned on selectively, then nematode signal(s) must somehow influence the transcription transducers ] factor network that control promoter activity. Extensive changes in gene expression during cell differentiation are often due to modulation of transcriptional Transcription of regulatory genes regulation, brought about by signal transduction casActivation of regulatory proteins cades that are triggered by one or more key events. Such events, in the case of NFSs, may involve interactions of nematode secretions or surface molecules transducers with specific cell components of a signal transduction cascade in which transcriptional control is involved. Therefore, analysis of promoters differenTranscription tially regulated during NFS development should

iiiiiiiiili!iiiiiiiiiiililiiii i [iiiiiiiiiiiiiiiiiiiiiiiiiiiii ?!i;iiiiiiiiiiiiii)iliiiil)iiiiiiii iiiiiiiiii!iiiiiiii;iiiiiiiiiii!iiii !

Figure 1. Possible target points in the plant cell for nematode signals during NFS development.

of downstream genes (NFS-specific expression)

S. Sanz-Alf~rez, Departamento de Biologfa, Universidad Aut6noma de Madrid, Spain

177

Challenges of the Environment eventually lead us one step closer to the key events that initiate this developmental process. Finding the sequence elements responsible for nematode-inducibility and their counterpart transcription factors will guide us upstream within the signal transduction pathway linking nematode and plant genes. That will not just give insight into the subtle plant-nematode interaction, but it may also disclose important components of cell differentiation paths. In addition, precise knowledge on how nematodes trigger gene expression has a direct application in the construction of transgenic plants which either produce nematicidal proteins specifically at NFSs, or are unable to differentiate a competent NFS to sustain nematode development.

The question and the approaches Within the just said conceptual frame, the question can be simply formulated (figure 1): what is in there between the nematode signals and the transcription of plant responsive genes? Which are the genes that execute the nematode order to the root cell "acquire the NSF fate"? Which the proteins that regulate the expression of the adequate battery of downstream genes related to the building of NFSs? Which are, among the many a plant has, the signal transduction cascades involved in the process? And, will these cascades lead us to the primary plant targets for nematode signals? The approaches that our group is taking to start answering these questions involve: l) identification of nematode responsive plant genes and study of their promoters (regulatory and downstream genes); 2) identification in the promoters of putative target sites for transcription factors, both as nuclear protein binding regions and as nematode-responsive sequence elements (NREs); 3) cloning the relevant DNA binding proteins that act as direct transcriptional regulators; and 4) disclosing how such transcription factors are activated at NFSs

Nematode-responsive promoters and regulatory sequence elements The first requirement to characterize nematode-responsive promoters is the identification of such promoters. There are already a number of them known to be induced by root-knot and/ or cyst nematodes in which we are working. Some have been identified by others in Arabidopsis in a promoter-tagging effort, partly within the ARENA project [9]. Of the selected tagged sequences which were induced by nematode infection, at least one corresponds to the promoter of a real gene: RPE, which codes for the D-ribulose-5-phosphate 3- epimerase [10]. In collaboration with the groups that cloned the tagged sequences, we have initiated the search for elements involved in their transcriptional regulation, both in NFSs and elsewhere in the plant (Uribe, Herreros and Fenoll, unpublished data) In our laboratory, we have identified several promoters induced by root-knot nematodes, their induction being clearly observed when fused to GUS in transgenic plants [11,12]. Three are from Arabidopsis: the promoters of the HMG1 and HMG2 genes, which code for several isoforms of the hydroxymethyl-glutaryl-CoA Reductase [ 13] and the promoter of the H4A748 gene that codes for the S-phase specific Histone 4 [ 14]. We have also shown that the promoter of a sunflower gene that codes for one small heat shock protein (sHSP17 group) [15] is induced in tobacco plants by Meloidogyne spp. Several promoters from geminiviral genes have also been shown in our laboratory to be expressed at NFSs (unpublished results). 178

Cis-elements in nematode-responsive promoters Several deletions are available for all these promoters, and we have already been able to narrow down them to minimal regions that still confer nematode inducibility. We are presently analysing them in greater detail in terms of time course induction and sequence elements important for both nematode responsiveness and NFS specificity [ 12; unpublished results].

How are promoters activated at NFSs? They could respond to one or a few transcription factors (TFs) of a unique differentiation pathway, or, alternatively, they could be activated by diverse TFs belonging to separate differentiation paths. We have compared several nematode-inducible promoters (among them the said four plus several Arabidopsis tagged sequences) by using a number of programs available for the identification of putative cis elements (Sequence Interpretation Tools, TFSearch, TRANSFAC and TRADA). Such extensive sequence analysis reveals that there is very little in common among the available nematode-inducible promoters, and has failed to identify a conserved putative nematode-responsive element present in all promoters activated in NFSs. From these data, we conclude that the different genes must be regulated in NFSs by an array of different TFs, and that they respond to nematodes through (at least partially) different mechanisms. The hypothesis that we currently favour is depicted in figure 2. Nematodeborne factors will signal in the initial root cell either one or several primary event(s), from which branched transduction cascades, probably part of distinct cell differentiation pathways, carry on downstream the orders to develop a feeding site. Such cascades will certainly involve the activation and/or the de novo synthesis of batteries of tranN e m a t o d e signals scription activators that act (either individually or in a combined manTFa [] TFb 9 ner) on target sequences in the plant nematode-responsive promoters. The fact that some of these promoters are turned on only by [~iFiiJ cyst or by root-knot nematodes, while others respond to both types ~ of parasites, also suggests that multiple routes must be involved in feeding site formation. None of the nematode-inducible promoters tested so far is 100% specific for ~ l ~ NFSs; all are active somewhere else in the plant. This is not sur- Figure 2. The model explains how distinct cell differentiation pathways could mediate the activation of discrete sets of downstream prising, since NFS differentiation promoters (the nematode-responsive promoters described in the text) has to make use of pre-existent de- during NFS development. Different transcription factors (TFa, TFb) velopmental pathways in the ini- are activated, directly or indirectly, by signal molecules from the tial cell, most probably selecting nematode. Each one will, in turn, mediate the expression of other TFs. TF1, TF2 and TF3 directly activate groups of downstream the bits of each that better suit ac- genes, while R acts through additional cascades involving still other complishing the final goal: acquire TFs. For simplicity, direct activation of downstream genes by TFa NFS fate to nurture the nematode. and TFb is not shown. 179

Challenges of the Environment Because of these more or less broad expression profiles, the potential to engineer nematode resistance using the said promoters as they are is limited. Thus, it appears essential further analyses to characterise the NREs in each promoter and, eventually, to separate these from elements responsible for the expression in other tissues. We are currently undertaking these type of analyses, that can be exemplified in the LEMMI9 gene.

Analysis of the LEMMI9 promoter The LEMMI9 tomato gene was identified by differential screening of cDNA libraries by G. Gheysen's group as a highly abundant transcript in nematode-infected roots. Further, they showed by in situ hybridisation a high level of LEMMI9 transcript accumulation specifically in giant cells [ 16]. LEMMI9 codes for a homologue to LEA-14, a cotton late-embryogenesis abundant protein. The function proposed for this class of proteins is to protect the embryo during the dessication of the seed, but some LEAs are also induced during water stress responses in vegetative tissues. In a collaborative work with G. Gheysen's group within the ARENA project [ 17], a genomic clone was isolated from a tomato genomic library, that contained 3500 bp of the LEMMI9 promoter sequence, where the transcriptional start point and the putative TATA box were located. Most of the promoter region from the genomic clone was fused to GUS and used to produce transgenic potato plants. GUS staining revealed that the promoter fragment suffices to confer nematode inducibility. Through the analysis of DNAprotein interactions in electrophoretic mobility gel shift assays, we identified a region close to the putative TATA box that binds in a specific manner nuclear proteins from infected, but not from uninfected roots. Within this region we could map the protein-binding site to a 12-bp repeat [17]. The involvement of this 12-bp repeat in binding of proteins from nematodeinfected root sections and from other expressing tissues has now been confirmed in tomato plants by a ligation-mediated PCR version of in vivo footprinting of the endogenous LEMMI9 promoter (Uribe and Fenoll, unpublished results). We are presently analysing transgenic Arabidopsis plants carrying several deletion-mutation fusions to GUS, to assess the function of this element in the activation of the LEMMI9 promoter (Sanz-Alf6rez, Aristizfibat and Fenoll, unpublished data). An important goal in our studies is to identify the plant protein(s) that bind to the 12-bp sequence in a yeast one-hybrid system, to partially fill the gap between the modification of LEMMI9 expression and the unknown signal coming from the nematode. The results from this work on the LEMMI9 promoter demonstrate that, in spite of the technical difficulties for non-genetic molecular analysis of the plant-nematode interaction, putative promoter elements can be identified through the study of protein-DNA interactions. Our findings also substantiate our initial hypothesis: the existence of putative transcription factors absent (or inactive) in non-infected roots, that act specifically in feeding sites and/or the surrounding root cells.

Making models for further research We believe that the path leading from nematode to mature feeding site could be dissected taking promoter induction as the downstream effect, and walking upstream from this molecular event. The first step in this walk is the identification of the cis elements and the proteins that, upon binding to them, will turn promoters on. Since these promoters must lye down180

Cis-elements in nematode-responsive promoters stream in the cascade initiated by nematode signals (Figure 2), they and their activators will allow us to proceed upstream towards the regulatory molecule(s) that control the fate change of root cells to become NFSs. Our results in the LEMMI9 promoter could be taken as an example of how to tackle the problem from one end of the signal transduction cascade. Recently, we have started to approach the pathway from the other end. Nematode secretions are known to stimulate division in plant protoplasts and cultured animal cells [5], what strongly suggests that secretions are, at least in part, responsible for some of the molecular events in NFS induction. We are at present fractionating nematode secretions [ 18] and using the resulting fractions to induce plant responses in the absence of nematodes. The type of screening we are doing include following changes in gene expression by both testing promoter-GUS fusions, and by cDNA AFLPs. Once we link a secretion fraction to a molecular event, we could address the search for partner molecules in the plant cell, by combining yeast two-hybrid systems with anti-secretions antibody screening of nematode expression libraries. By proceeding simultaneously in the two senses (from plant promoter towards secretions, and vice versa), we hope to disclose elements that participate in this route. Since some of these elements may as well be part of better known signal transduction pathways involved in other differentiation events, we will be able to borrow pieces from those paths to help building the molecular puzzle of NFS differentiation.

Authors of this contribution S. Sanz-Alf6rez 1, X. Uribe 1,3,E A. Aristizfibal TM,E. Herreros 1, E F. del Campo l, C. Fenoll 1,2,g< 1 Departamento de Biologfa, Universidad Aut6noma de Madrid, E-28049 Madrid, Spain, 2Facultad de Ciencias del Medio Ambiente, Universidad de Castilla-La Mancha, E-45071 Toledo, Spain, 3present address: Centro Nacional de Biotecnologfa, Cantoblanco, E-28049 Madrid, Spain, 4 Present address: Departamento de Farmacia, Facultad de Ciencias, Universidad Nacional de Colombia, Ciudad Universitaria, Santa F6 de Bogotfi, Colombia. *Corresponding author

Acknowledgements We want to thank the European Commission for funding this work through grants B IO4CT96 0318 and FAIR3-CT96 1714 to CF.

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

Virus Resistance Models In A EU Crop Plant, Pisum sativum

Introduction The advantages of virus resistance come from improved yield and quality of the crop and environmental benefits resulting from reduced use of pesticides required to control the insect vectors of viruses. Although currently in the EU losses due to virus infection are less than those resulting from fungal pathogens, global climate change and altered agricultural practices associated with extended ranges for insect vectors suggests that virus epidemics will become an increasing problem. Natural resistances to many of the viral pathogens of EU crops exist and have been bred into improved commercial varieties. In these cases, "immunity" (the absence of any detectable in~ fection), hypersensitive resistance associated with an infection localised by death of infected cells and tolerance, where the infection is asymptomatic and inconsequential for crop yield, are all effective. The long term durability of these resistances is dependent predominantly upon the genetic variation in the pathogen pool and the selection pressure provided by monogenic resistance and planting density. In some ways, tolerance may be considered as more durable since it places few restrictions upon the pathogen population. An alternative form of resistance which depends upon the insertion of a genetic component of the pathogen into the host genome, so-called pathogen-derived resistance [PDR; 1, 2], has arisen out of our increased knowledge of the primary structure and organisation of virus genomes. Globally, this strategy has been used effectively in a number of commercial crops where natural resistance genes were not available. This includes melons, papaya, tomato etc., many of which are primary food sources. To date in the EU there are no commercial plantings of crops carrying PDR although some are under review for their toxicological and environmental impact. The use of these engineered crops, subject to successful risk assessment, will depend largely upon their acceptance by the public. Genetic modification (GM) technology is the subject of active media, political and social debate throughout Europe at present and only time will tell whether the public can be convinced of the need and the substantial benefits of the genetic technology. In some ways, plants containing viral genetic components should be of least concern since natural infections with single and complex collections of viruses are common place and their products will frequently be in the food chain.

Andrew Maule, John Innes Centre, Norwich Research Park, Norwich, United Kingdom

183

Challenges of the Environment

Research studies In our studies, we have examined one EU crop, Pisum sativum (pea). Peas are susceptible to a wide range of viruses including Pea enation mosaic virus, Pea early browning virus, and a range of viruses from the Potato virus Y group (Potyviridae). Within this last group, Bean yellow mosaic virus, Bean common mosaic virus, Pea mosaic virus and Pea seed borne mosaic virus (PSbMV) are all important pathogens. In particular, we have studied PSbMV for which all commercial cultivars of peas are susceptible. This susceptibility is compounded by the fact that this virus is not only transmitted from plant to plant by its aphid vector but is also transmitted vertically from generation to generation in the seed. This property has resulted in serious contamination of pea germplasm collections and provides a very effective means to give early and widespread infection of crops soon after seed germination. Consider that a seed transmission efficiency of only 0.1% would result in 10,000 infections after sowing seeds at 10V/hectare, and the importance of seed transmission becomes apparent. At present, this problem is countered by efficient post-harvest testing of seed samples by immunodetection of the virus coat protein, and rejection of contaminated seed lots. As an alternative, and since seed transmission in a range of pea cultivars varies from 0 to 100%, we have investigated whether resistance to seed transmission could be bred into improved pea lines. In test crosses and backcrosses between lines showing no transmission or 60-80% transmission, resistance behaved as a dominant character, although in the F2 and BC2 generations it did not segregate as a Mendelian trait. The quantitative nature of the phenotype suggested that seed transmission would be difficult to include as a resistance trait in a conventional breeding programme. Natural resistance to PSbMV has been identified in pea accessions from North Africa and Asia [3, 4] although, so far, these recessive genes have not been introgressed into commercial lines. Genetic analysis has shown that these genes are clustered with other recessive genes with differing potyvirus specificities at two locations on the pea genome. Genes sbm-1, sbm3, and sbm-4, conferring resistance to PSbMV pathotypes P1, L-1 and P4, respectively, are located on chromosome 6, while sbm-2 also conferring resistance to pathotype L-1 is located on chromosome 2 [5]. Although this organisation is suggestive of local gene conversion and translocation between chromosomes 2 and 6, other evidence suggests that the two gene clusters may have distinct origin and function. Using recombinant hybrid viruses made between different pathotypes the virus avirulence determinant has been defined as the virus genomelinked protein (VPg) for sbm-1 [6]. A structural and functional analysis of the sbm-1 gene is the topic of an EC-Biotechnology project # BIO4-CT97-2356 (www.dias.kvl.dk/eupsbmv) involving research groups and industry from Denmark, Finland, Spain and the UK. Characterisation of the sbm-1 gene will provide particular intellectual and practical rewards. Since, approximately 20% of all virus resistance genes [7] and approximately 40% of genes conferring resistance to potyviruses are recessive [8], understanding how sbm-1 works and what controls the specificity of the adjacent sbm- and other potyvirus resistance genes will be important for a wide range of diseases. However, the sbm- project also has technical challenges, not least in having to deal with the size and redundancy within the pea genome. The pea genome is approximately 5 x 109 base pairs per haploid genome, some 50 times larger than that from Arabidopsis thaliana. Map-based cloning of genes in pea has not been achieved and large insert libraries are not yet available. However, there is the potential in sbm-1 to

184

Virus resistance models in Pisum sativum identify a new class of resistance genes. Resistance genes cloned so far from other species fall into two classes. The dominant resistance genes which function against specific viruses, fungi and bacteria fall broadly into the "NBS-LRR" class [9] and mediate a hypersensitive resistance to infection. The only recessive gene to be cloned (mlo) mediates a non-race specific resistance to powdery mildew in barley and is also associated with localisation of the pathogen in dead cells. Functionally, Mlo acts as a negative regulator of constitutive resistance [10]. In contrast, sbm-1 is race (or pathotype) specific and is not associated with cell death. From these comparisons, several functional mechanisms for sbm-1 seem possible. First, we can view Sbm-1 as a dominant susceptibility factor, required to assist virus replication. This would fit with the probable involvement of the VPg in viral RNA replication and the observation that protoplasts from resistant plants show no detectable virus replication [11]. Second, like Mlo, Sbm-1 could act as a negative regulator of resistance although the specificity differences from mlo would place sbm-1 in a distinct class of resistance genes. Third, sbm1 could be a dominant but dose-dependent weak resistance allele. We favour the first option as the most direct and simple interpretation. For our component in the EC-Biotechnology project we have opted to use genetic approaches to identify the sbm-1 resistance gene product. After identifying suitable pea lines (a BC4 pair of lines carrying homozygous resistance and susceptibility alleles) a cDNA-AFLP strategy has been used to identify expressed genes coming from the introgressed region. So far, ten polymorphic cDNAs have been identified. These are being mapped using recombinant inbred families to confirm their genomic origin. Our alternative strategy is to 'fish' for the sbm1 gene product by using the yeast two-hybrid system with the PSbMV VPg as the bait protein. Two strong candidate cDNAs and eight other cDNAs encoding interactor proteins have been identified from a pea cDNA library made from a susceptible pea line. These cDNAs are also being sequenced and mapped. As part of a previous EC-AIR project (# CT94-1171) involving academic and industrial partners in Denmark, France and the UK, we have also explored the potential for developing PDR to PSbMV in transgenic peas. Since in other systems the viral replicase gene had commonly been used to give PDR by triggering the process of post-transcriptional gene silencing (PTGS), we used the PSbMV replicase cistron (NIb) for transgenic expression in peas [12]. From 35 pea lines, transformed with Agrobacterium tumefaciens T- DNA carrying a 35S promoter -NIb - 35S terminator construct, and the bar gene as a selectable marker for transformed tissue in the presence of the herbicide Bialophos, three lines were shown to be resistant to PSbMV. Two of these lines carried a direct repeat of the 3' end of the NIb gene (NIbIb) since there was some evidence [13] that complex transgene arrangements had more potential to initiate PTGS. All of these lines exhibited a type of PDR termed "recovery" where challenge inoculation results in an initial infection but the plants rapidly recover and show no more symptoms or virus accumulation. The recovered tissues are then resistant to further challenge with the homologous or closely related virus isolates. To assess the significance of this in the field where the plants may be challenged with a population of related viruses, the ability of different isolates of PSbMV to trigger PTGS and to be targeted by induced PTGS was assessed. This showed that viruses with ca. 89% or more identity in the NIb cistron could induce the resistance although the specificity requirements for a second challenge virus to be seen as a target may be higher. For reference, the two most divergent sequenced PSbMV 185

Challenges of the Environment isolates differ by 89% in the NIb region. This distinction in specificity requirements for triggering and targeting in PTGS will be an important consideration for the application of the technology to commercial crops. The relatively broad resistance to PSbMV isolates in NIb transgenic peas contrasts with the extreme pathotype specificity seen for the natural sbmresistance genes where only one or a few changes in the virus avirulence determinant is enough change a PSbMV isolate from avirulent to virulent [6]. Despite the short period of initial infection, the transgenic pea plants showed good growth and seed set after challenge inoculation to give yields under glasshouse conditions equivalent to those seen for uninfected transgenic or non-transgenic lines. We believe that, subject to licensing agreements covering the use of the bar gene for selecting transformed plants, these plants could be useful additions to the panel of pathogen resistance genes to be used in developing new improved pea lines. The transgenic pea plants represent the first legumes displaying PDR against potyviruses and some of the first experimental examples in the Leguminosae of plants showing PTGS. It was valuable, therefore, to establish that the principles governing PTGS and resistance in this system supported those characterised with more commonly used experimental plants (e.g. Nicotiana spp.). As expected for PTGS, induced virus resistance was associated with the degradation of transgene RNA and PSbMV RNA [12]. We also showed that PTGS was mediated in these plants by a systemic signal generated during the initial phase of virus infection, and that this signal had the potential to mediate the spread of PTGS by inducing methylation in the transcribed region of the NIb transgene [ 14]. In conclusion, we have recognised that the agricultural industry would benefit from having stable and effective resistance to PSbMV in peas. The least contentious route to achieve this would be through the incorporation of naturally occurring resistances (either to seed transmission or to virus replication) using conventional breeding strategies. Our relatively poor understanding of the genetic complexity of PSbMV seed transmission means that this is unlikely to be useful in the short term. The sbm- genes are more promising although the lack of closely linked genetic markers and the recessive nature of the resistance create some difficulties. Alternatively, we have demonstrated the potential for creating resistance through the application of transgenic technology although the issues of biosafety and public acceptability will need to be addressed. In addition to these applied considerations, the research has and is generating materials and knowledge that will influence how related approaches can be used in other crop plants. In particular, understanding the mechanisms of action of a new class of virus resistance genes will be important.

Acknowledgements I thank, Stuart Harrison and Frederic Revers for the substantial contributions they made to this work, and Louise Jones, Carole Thomas and Elisabeth Johansen for comments on the manuscript prior to publication. The John Innes Centre receives a grant-in-aid from the UK Biotechnology and Biological Research Council. The other partners in the EC-Biotech programme # BIO4-CT97-2356 are: Danish Institute of Agricultural Sciences, Denmark (Elisabeth Johansen; Co-ordinator), DLF Trifolium A/S, Denmark (Vibeke Meyer), University of Madrid, Spain (Juan Antonio Garcia) and University of Helsinki, Finland (Jari Valkonen). 186

Virus resistance models in Pisum sativum

References 1. 2. 3. 4. 5. 6.

7. 8. 9. 10. 11. 12.

13. 14.

Beachy, R.N. (1997) Mechanisms and applications of pathogen-derived resistance in transgenic plants. Curr. Op. Biotech. 8, 215-220. van den Boogaart, T., Lomonossoff, G.P. and Davies, J.W. (1998) Can we explain RNA-mediated virus resistance by homology-dependent gene silencing. Mol. Plant-Microb. Interact. 7, 717-723. Hagedorn, D.J. and Gritton, E.T. (1973) Inheritance of resistance to the pea seed-borne mosaic virus. Phytopathology 63, 1130-1133. Provvidenti, R. and Alconero, R. (1988) Inheritance of resistance to a third pathotype of pea seed-borne mosaic virus in Pisum sativum. J. Heredity 79, 76-77. Cousin, R. (1997) Peas (Pisum sativum L.). Field Crop. Res. 53, 111-130. Keller, K.E., Johansen, I.E., Martin, R.R., Hampton, R.O. (1998) Potyvirus genome-linked protein (VPg) determines pea seed-borne mosaic virus pathotype-specific virulence in Pisum sativum. Mol. Plant-Microb. Interact. 11, 124-130. Fraser, R.S.S. (1992) The genetics of plant-virus interactions-implications for plant breeding. Euphytica 63, 175-185. Provvidenti, R. and Hampton, R.O. (1992) Sources of resistance to viruses in the Potyviridae. Arch. Virol. Suppl. 5, 189-211 Dangl. J, and Jones, J.D.G. (1998) Affairs of the plant: colonization, intolerance, exploitation and cooperation in plant-microbe interactions. Curr. Op. Plant Biol. 1,285-287. Shirasu, K., Nielsen, K., Piffanelli, E, Oliver, R., and SchulzeLefert, E (1999) Cell-autonomous complementation of mlo resistance using a biolistic transient expression system. Plant J. 17, 293-299. Bak, S., Poulsen, G.B. and Albrechtsen, M. (1998) Two cases of non-host resistance against potyviruses operating at the single cell level. J. Phytopathol. 146, 623-629. Jones, A.L., Johansen, I.E., Bean, S.J., Bach, I. and Maule, A.J. (1998) Specificity of resistance to pea seedborne mosaic potyvirus in transgenic peas expressing the viral replicase (NIb) gene. J. Gen. Virol. 79, 31293137. Sijen, T., Wellink, J., Hiriart, J-E, and van Kammen, A. (1996) RNA-mediated virus resistance: role of repeated transgenes and delineation of targeted regions. Plant Cell 8, 2277-2294. Jones, A.L., Thomas, C.L. and Maule, A.J. (1998) De novo methylation and co-suppression induced by a cytoplasmically replicating plant RNA virus. EMBO J. 17, 6385-6393.

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Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.

Generation Of 13k-Gene Sugar Beet Transformants And Evaluation Of Their Resistance To BNYVV Infection Introduction Sugar beet (Beta vulgaris L.) is one of the main industrial crops in Greece and in many other European countries. Rhizomania, the most important virus disease of the crop, can lead to severe losses in tap root yield and sugar content. The main symptoms of the disease are abnormal proliferation of fine rootlets from the taproot and lateral roots, partial necrosis of these tissues, necrosis of the vascular tissue and stunting of the tap root (reviewed in [1]. The causative agent of the disease is the fungus-transmitted virus beet necrotic yellow vein virus ( BNYVV). The vector of the virus, the soilborne obligate intracellular fungus Polymyxa betae transmits the virus in a persistent fashion. It is difficult to control the disease using environmentally acceptable methods, since the virus can survive in the spores of the fungus several years and soil decontamination is effective only after treatment with methylbromide. Thus, if the disease invades the field, the only alternative for the farmer is the use of resistant cultivars. Generation of resistant varieties using transgenic plant approaches offer an alternative. Several approaches have been developed for pathogen-derived transgenic resistance [2]. Transgenic lines containing and expressing the BNYVV coat protein mRNA have been produced, and were found to contain reduced levels of the virus, although the coat protein was not in detectable amounts [3]. Expression of full length coat protein may have a higher risk because of possible recombination or transencapsidation events. Over-expression of viral sequences in a plant may finally lead to the specific suppression of the gene, in a phenomenon called RNA-mediated resistance [4]. This reaction, which is believed to be the result of a general plant defence mechanism, can lead to virus resistant or even immune plants. As part of a wider sugar beet breeding program aiming to create new sugar beet cultivars resistant to the major diseases of the crop, we present here the first series of experiments for generation of virus resistant plants. BNYVV is a furovirus whose genome consists of four to five plus-sense RNAs. The genes on RNA 1, and 2 encode functions essential for replication and movement in the plant, whereas the genes on RNAs 3, 4, and 5 are implicated in symptom expression or host range and vector-mediated infection of sugar beet (review in [5, 6]). We introduced the viral gene sequence 13K under the control of the CaMV 35S promoter in sugar beet plants. The viral gene 13K, which is located on RNA 2 of the virus in an area

Kriton Kalantidis, Plant Molecular Biology IMBB-FORTH, Heraklio, Greece

189

Challenges of the Environment known as the triple gene block (TGB) [7] encodes for a membrane protein which has a major role in the transport of the virus. Regenerated transgenic plants were challenged with the virus and examined for resistance.

Cloning of the 13K gene 1 2

16~j

st7

i?

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Figure 1. Gel clccm)phorctic analysis of tile IC-PCR products, l.anc M: Marker I)NA. pBR322x Hinfl. Lane I: IC-PCR products from the ~cond stage t~;R. Lane 2: Control reaction without template I)NA

The cDNA of the selected gene of beet necrotic yellow vein virus was cloned using the immunocapture PCR technique as described in [8] Infected plants were tested for the presence of BNYVV by western blotting using a commercially available anti BNYVV antibody (not shown). Plants showing positive signal (truly infected) were then used for the isolation of the virus. B NYVV was first immunocaptured from extracts of infected roots and then the viral particles were used for the reverse transcription and polymerase chain reaction using specifically designed primers. The reaction product was detected only after a second round of PCR (figure 1) using an aliquot of the first PCR and the same primers for amplification. The cDNA 13K product was then cloned in appropriate vectors and sequenced. Integrity of the gene was examined after in vitro transcription and translation. The length of the translation product indicated that the sequence did not contain any PCR derived frame shift (data not shown).

Phenotype of 13K gene transformed sugar beet The 13K cDNA was subcloned in vector p ART7/pART27, a binary plasmid for stable transformation of Agrobacterium. Transformants were regenerated on kanamycin selection medium. The 13K gene was under the control of CaMV 35S promoter. Transgenic sugarbeet plants were regenerated from leaf petioles on selection growth media. Plantlets were transferred on rooting media 2-3 months after the initial shoot induction. Rooted transgenic plants were then transferred to non-sterile conditions in the greenhouse. Regeneration was observed in 5-8% of the initial explants used, depending on the experiment. Transgenic plants that regenerated from neighbouring regions on the leaf petioles were treated as initiating from the same transformed cells and were thus considered 6~,~bp clones. Plantlets grew well and did not show any abnormal development when examined visually.

Figure 2. PCR analysis of lran.sgcnic plants using tile nptll gone specific palmers. lane 1-3: mmsgcnic plan! I)NA, C6. (" 8,132, I.anc 4: WT: Blank: l.ane 6: "'100bp"laddcr

The presence of the foreign gene was examined by PCR using the 13K and nptII specific primers (figure 2). Copy number of the inserted gene was estimated after Southern hybridisation using thel3K gene as a probe. Out of 190

Resistance to BNYVV infection in sugar beet transformants 1

2

3

4

5 !

~

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9kb 7.5kb

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10 sugarbeet transformants tested on Southern hybridisations until now, seven were found to have 1 gene copy per plant, two hybridised to two bands and one revealed three copies (plant 2000, figure 3a and b). Finally, two plants showed no signal and were considered escapes.

Virus

infectivity studies

13K transformed sugar beet plants were tested for resistance to rhizomania in controlled infections Figure3a. Soulhcmhybridisationofst,garbec! in the greenhouse. Transgenic plants were transgcnomic I)NAwitha 13K probe, ferred on infected soil and rootlets were used after l~anc1"/5I)NA, Lancs2-5:gcnomic beet 1)NA, digested with l~c()Rl, plants: C6. WT. B2, C 7 several intervals for ELISA tests. I n o r d e r to avoid differences in local concentration of the virus, samples from three different positions of the root were taken and homogenised together for ELISA assays. We tested in preliminary experiments the efficiency of the infection of a commercially available 1 2 3 4 cultivar for the batch of soil used. 7 out of 10 plants were infected after transfer in the contaminated soil (data not 10 kb shown). In a first series of experiments with transgenic plants, 7 transgenic plants were tested together with 4 W 4--.- 8kb regenerated non transgenic plants of the variety 028 and 4 commercially available plants. All plants tested, except ~I~S 4 - - 5.5 kb non transgenic plant F3, were infected. In subsequent ~@ ,@--- 5 kb experiments, 6 and 4 transgenic plants were tested together with 028 (infected) and healthy controls. All plants were finally infected, but three plants carrying the viral gene (plants B8, C8, C5) showed a one week delay of infection. The expression of the introduced gene was [9] detected by northern hybridisation. 13K expression could be detected in two of the lines analysed so far, line 2000 Figure 3b. Southern hybridi/aiion of sugar beet 13K gene probe (plant C8) and line 1994 (plant B6) (figure 4a and b). [,ane I-3:genomic 1)NA digested wi~h The 13K RNA level detected was similar to that of enBamHI, plants, B5, C8 and WT. I~ane 4:undigested 13K palsmid I)NA(+ marker) dogenous genes.

Discussion Expression of viral sequences, containing either fully functional or truncated ORFs has been shown that can confer resistance in many virus families and several plant species. This type of transgenic resistance can be divided into two groups, one group in which expression of a viral protein is important for resistance (coat protein or other), and another group where expression of RNA sequences are sufficient to confer resistance. The 13K gene of the viruses containing a triple gene block is a suitable target for both approaches: a mutated 13K (or TGB2) protein, when expressed in the plant, has been shown to confer broad spectrum resistance for viruses containing the TGB block, but not for viruses expressing other types of movement proteins 191

Challenges of the Environment [ 10], [ 11 ]. The same genomic segment (about 400 nucleotides) can be theoretically used for obtaining RNA mediated resistance, since it has the necessary homologous RNA length for triggering the resistance mechanism [ 12].

18S rRNA

800bp

We have produced transgenic sugar beet plants containing the BNYVV viral gene 13K, and tested all the transformants for resistance or delay of infection. The transgenic plants obtained (17 plants, from 8 different transformant lines) show low copy number of the inserted genes. Low copy number has been reported to avoid silencing of the introduced genes. However, there are contradictory reports about the importance of DNA or even RNA dosage on the induction and maintenance of PTGS [13, 14]. In a recent study on the effect of Figure 4a. Northern hybridization transgene copy number on gene expression and silenc- of mRNA from transgenic 13K ing through co-supression no direct link was found [15]. sugar beet plants hybridised with a Since the exact mechanism involved in PTGS is still 13K gene riboprobe. unclear it is difficult to know the requirements at the Lane 1: transgene B5, Lane 2: transgene C8, Lane 3: WT (028) mRNA level, for the initiation of this phenomenon. Our results from the northern hybridisations conducted so far have confirmed 13K expression at the RNA level in two of the plants tested. One of the plants (plant C8 ) showing retardation of BNYVV infection did show the highest 13K expression from the plants analysed so far. However, since northern hybridisation experiments and ELISA tests are still in progress it is not possible to draw conclusions about the relation between transgene expression levels and plant tolerance at the moment. RNA mediated resistance is usually characterised by very low levels of virus titer or even absence of virus particles in late stages of the infection (recovery). Our data so far do not support RNA mediated resistance mechanism in the transgenic plants tested. The actual movement protein of the triple gene block has been shown to be TGB 1 [ 16]. TGB 1 binds the viral RNA [9]and mediates cell to cell transport of the viral genome. The 13k gene (TGB2) is an important factor for cell to cell transport of triple gene 1 2 3 4 5 6 7 block containing viruses, 18S rRNA and mutations in this gene ,~'~ ;~'~, affect the expression of the 800 bp first gene of the TBG1 in BNYVV[5]. Possible roles of TGB2 could be a) that it is part of the nucleoprotein complex which, together with TGB 1 transfers the viFigure 4b. Northern Hybridization of mRNA from transgenic 13K sugar beet ral RNA to plasmodesmata plants hybridized with a 13K gene riboprobe. or b) that it is bound to Lanel" WT; Lanes 2-7: transgenic plant mRNA, alfa, F10, C6, B6, F9, beta. 9 :i'

192

.

Resistance to BNYVV infection in sugar beet transformants d e s m o t u b u l e s or other structures near p l a s m o d e s m a t a and mediates the a t t a c h m e n t of T G B 1/ viral R N A c o m p l e x [16]. E x p r e s s i o n of the 13K m R N A resulted in a delay of virus infection in s o m e transgenic plants, but not in a final b l o c k of virus transport. It is assumed, that m o v e m e n t functions of viruses have b e e n obtained f r o m the plant g e n o m e during evolution of viral g e n o m e s in their hosts, and that these viral proteins m i g h t r e s e m b l e cellular ancestors w h i c h have b e e n o p t i m i s e d to transport viral R N A s rather than cellular R N A s [17]. The n o r m a l p h e n o t y p e of the 13k e x p r e s s i n g plants indicates that there is no interference of the transgenic 13k protein with n o r m a l nucl eoprot ei n and protein transport through p l a s m o d e s m a t a , and that 13k is m o s t p r o b a b l y not involved in s y m p t o m formation. Our future plans involve e x p r e s s i o n of m u t a t e d forms of the 13k gene, w h i c h will better interfere with virus transport, as well as the construction of 13k gene specific ribozymes.

Materials and methods Plasmid constructs. All plasmids were constructed according to published routine procedures [18]. The pART7/

27 vector system used for Agrobacterium transformations has been published [19] and was kindly provided by Dr. A. Gleave. Plasmids were inserted in Agrobacteria by tri-parental mating [20]. Immunocapture PCR and Cloning of 13K eDNA. Material from Infected rootlets was extracted in Tris/HCl pH

8.2, 0.25% PVP-360,1% PEG 6000, 140mM NaC1, 0.05%, Tween 20. A commercially available antibody (Bioreba, Switzerland) was used for capturing the virus particles. RT and immuno-capture PCR was performed as described in [8], using oligodT as an RT primer and the oligonucleotide primers 13K5 and 13KR for the PCR reaction at an annealing temperature of 52oC, elongation at 72oC, for 28 cycles. Products were visible only after a second stage PCR (lul of the first PCR reaction were subjected to a second PCR reaction in a volume of 80ul). primer 131~5"5' GCG GAT CCT TTT TGC GAA GAT AGA TAA TGT C 3' ; primer 13KR: 5' ATT CTA GAC AAC AAC ACA ACC GGC AAC TA 3'. The obtained cDNA was cloned in vector pT20 (IMBB) according to published protocols [18], and in the shuttle vector pART7 [19] using the BamHI/Xbal restriction sites. The resulting clone pART7-13K was digested with NotI and the 13K containing fragment subcloned in the final binary vector pART27, which was used for Agrobacterium transformations. Sugar beet transformations. Cultivar 028 of the Greek Sugar Industry were used for all transformations and was kindly provided by Dr. G. Skarakis. Sugar beet petioles from aseptically grown plants were used as primary explants. Transformations were carried out according to [21 ]. Transgenic plants were grown on selection media for at least 3 months. Nucleic acid Hybridizations. Plant genomic DNA was isolated from leaves according to [22], About 8 ug of the

isolated DNA was loaded on the gels. Southern hybridisations were performed according to [18]. The 13K gene probe was labeled with Dig-CTP(Boehringer Manheim-BM) and colour detected according to manufacturers instructions. Plant mRNA was isolated using the "mRNA isolation kit" (BM). Northern hybridisations were performed according to [18]. 13K riboprobe was generated according to [18] labelled with P32 UTP (Amersham). About 1 ~g of mRNA per plant were loaded on the gel for northern analysis. Plant infections. Sugar beet plants were infected by cultivation in infected soil provided by the "Greek Sugar

Industry". The ELISA tests were used for the detection of the virus and the tests were conducted according to manufacturers (BIOREBA) instructions. Samples were taken from 3 different locations of the same plant, homogenized together and loaded for the ELISA assays.

Acknowledgements We thank M. Providaki for technical assistance in subcloning the 13K gene in p A R T 7 / 2 7 , Dr. (3. Skarakis for donating the initial sugar beet plants and Dr. A. G l e a v e for kindly providing the p A R T 7 / 2 7 vectors. This w o r k was s u p p o r t e d by the G r e e k Mi ni st ry of D e v e l o p m e n t , General Secretariat of R e s e a r c h and Technology, F r a m e w o r k IV, p r o g r a m m e E P E T I I 233.

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Authors of this contribution K a l a n t i d i s K. ~, Tsaftaris A. 1,, M a n o u s o p o u l o s J. 2, T z o r t z a k a k i S. 2' T s a g r i s M. 2,3 ~ D e p a r t m e n t o f G e n e t i c s and P l a n t B r e e d i n g ,

Univ. o f

T h e s s a l o n i k i , G r e e c e , 2Institute o f

M o l . Biol. a n d B i o t e c h n o l o g y , E O . B o x 1527, H e r a k l i o n 7 1 1 1 0 , G r e e c e , 3 D e p a r t m e n t o f B i o l o g y , Univ. o f Crete, H e r a k l i o n , G r e e c e .

References 1. 2. 3. 4. 5.

6. 7. 8.

9. 10. 11. 12. 13. 14. 15.

16. 17. 18. 19. 20. 21. 22.

Jupin, I., T. Tamada, and K. Richards, Pathogenesis of Beet necrotic yellow vein virus. Semin. Virol., 1991. 2: p. 121-129. Lomonossof, G.P., Pathogen derived resistance to plant pathogens. Ann. Rev. Phytopathology, 1995. 33: p. 323-343. Mannerlof, M., B.L. Lennerfor, and E Tenning, Reduced titer of BNYVV in transgenic sugarbeets expressing the BNYVV coat protein. Euphytica, 1996. 90: p. 293-299. Baulcombe, D.C., RNA as a target and an initiator of post-transcriptional gene silencing in transgenic plants. Plant Mol Biol, 1996. 32(1-2): p. 79-88. Lauber, E., et al., Cell-to-cell movement of beet necrotic yellow vein virus: I. Heterologous complementation experiments provide evidence for specific interactions among the triple gene block proteins. Mol Plant Microbe Interact, 1998. 11(7): p. 618-25. Richards, K.E. and T. Tamada, Mapping functions of the multipartite genome of beet necrotic yellow vein virus. Ann. Revie of Phytopathology, 1992. 30: p. 291-313. Gilmer, D., et al., Efficient cell-to-cell movement of beet necrotic yellow vein virus requires 3' proximal genes located on RNA 2. Virology, 1992. 189(1): p. 40-7. Koenig, R., E Luddecke, and A.M. Haeberle, Detection of beet necrotic yellow vein virus strains, variants and mixed infections by examining single-strand conformation polymorphisms of immunocapture RT-PCR products. J Gen Virol, 1995.76(Pt 8): p. 2051-5. Bleykasten, C., et al., Beet necrotic yellow vein virus 42 kDa triple gene block protein binds nucleic acid in vitro. J Gen Virol, 1996. 77(Pt 5): p. 889-97. Beck, D.L., et al., Disruption of virus movement confers broad-spectrum resistance against systemic infection by plant viruses with a triple gene block. Proc Natl Acad Sci U S A, 1994. 91(22): p. 10310-4. Seppanen, E, et al., Movement protein-derived resistance to triple gene block-containing plant viruses. J Gen Virol, 1997.78(Pt 6): p. 1241-6. Pang, S.Z., EJ. Jan, and D. Cosalves, Nontarget DNA sequences reduce the transgene length necessary for RNA mediated tospovirus resistance in transgenic plants. PNAS, 1997. 94(15): p. 8261-8266. English, J.J., E. Mueller, and D.C. Baulcombe, Supression of virus accumulation in transgenic plants exhibiting silencing of nuclear genes. Plant Cell, 1996. 8(2): p. 179-188. Vaucheret, H., et al., A transcriptionally active state is required for post-transcriptional silencing (cosuppression) of nitrate reductase host genes and transgenes. Plant Cell, 1997.9(8): p. 1495-1504. Kohli, A., et al., Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination. Plant J, 1999. 17(6): p. 591-601. Lough, T.J., et al., Molecular dissection of the mechanism by which potexivirus triple gene block mediate cell-to-cell transport of infectious RNA. Molecular Plant-Microbe Interactions, 1998. 11(8): p. 801-814. Lucas, W.J. and S. Wolf, Connections between virus movement, macromolecular signaling and assimilate allocation. Curr Opin Plant Biology, 1999. 3(June): p. 192-197. Sambrook, J., E.E Fritsch, and T. Maniatis, Molecular cloning: A Laboratory Manual. 1 ed. Vol. 1. 1989, Cold Spring Harbour: CSH. Gleave, A.E, A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol Biol, 1992. 20(6): p. 1203-7. Topping, J.F., Tobacco transformation. Methods Mol Biol, 1998. 81: p. 365-72. Tertivanidis, K. and A.S. Tsaftaris. regeneration and genetic transformation of sugarbeet (Beta vulgaris L.) by Agrobacterium tumefaciens, in Plant Embryogenesis Conference. 1997. Dublin, Ireland. Dellaporta, S.L., J. Wood, and J.B. Hicks, Isolation of DNA from higher plants. PMB reporter, 1983.4: p. 19-21.

194

Phytosfere.99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 0Elsevier Science B.V. All rights reserved.

Phytotoxic Activity Of Mycosphaerella graminicola Culture Filtrates Abstract Mycosphuerellu graminicola is a high host-specific pathogen, causing Septoria tritici leaf blotch of wheat. Histopathological studies suggested the involvement of toxic compounds in the disease. When tested on various substrates and culture conditions, M. gruminicola expressed the highest toxicity on M1-D in shaken culture (150 rpm) incubated at 22°C in the dark for two weeks. In M-1-D liquid culture M. gruminicola produced phytotoxic acidic compounds with high water affinity. The culture filtrates were tested in different bioassays to assess their phytotoxicity. No correlation was observed when the phytotoxic activity of 24 different strains of M. graminicola was evaluated in relation to their virulence on susceptible and resistant wheat cultivars. This analysis indicates that phytotoxic metabolites of M. graminicola are no determinants of virulence although they may contribute to the extent of disease development

!

Introduction Mycosphaerella gruminicola (Fuckel) Schroeter it7 Cohn (anamorph Septoria tririci Roberge in Desmaz.), is the causal agent of the worldwide leaf spot or speckled leaf blotch of bread and durum wheat. It is a major problem in regions with a temperate, high rainfall environment during the wheat growing season; relative humidity (optimum 85 %) and temperature (2025°C) are considered to be key determinants for successful development of the disesae [ l , 23. The teleomorph is considered to be largely responsible for the oversuminering of the disease, while the anamorph mainly contributes to disease development during the growing season [3, 41. However it was recently reported that M. grumitzicolu is also able to complete several generations of ascospores during the wheat season and this could be of great epidemiological importance [5, 61. The disease, occuring chiefly on leaves, is characterized by the formation of light green to yellow spots between the veins, later evolving in a speckled appearance, with the formation of pycnidia in brown lesions of various shades. The occurrence of elongated chlorosis areas on infected leaves, spreading more rapidly in susceptible varieties suggested the production of toxins by the fungus that could play a role in the pathogenesis on wheat. Very scantly evidence was so far aquired on the toxic activity of specific metabolites produced by M. gruminicola. The phytotoxic activity of M. grutninicolu culture filtrates was investigated in some previous studies [7, 81 and toxic filtrates were proposed for it7 v i m

G. Perrone, Istituto Tossine e micotossine da parassiti vegetali del CNR, Bari, Italy

195

Challenges of the Environment selection of resistance in wheat to the disease [9, 10]. On the other hand, preliminary investigations on the pathogenesis suggested that the resistance mechanism in wheat against M. graminicola might be based on the inhibition of fungal proliferation [11] and to this regard the occurrence of anti-fungal compounds can be evoked [12]. An involvement of soluble toxic compounds in the wheat- M. graminicola pathosystem was suggested also by recent histopathological studies [11] because i) in compatible interactions, wheat mesophyll cells were severely affected without the presence of mycelium in the vicinity, and ii) cell collapse occurred within a short span of time. In this respect, Stagonospora nodorum Berk. the other causal agent of wheat septoriosis, produces aspecific phytotoxic metabolites (e.g. micofenolic acid, mellein and its derivatives, septorin and derivatives) [13, 14, 15]. In this note we report the results of the investigation on the culture conditions that stimulate the in vitro production of toxic compounds by M. graminicola.

P r o d u c t i o n of phytotoxic activity Although various media and different condition were used to investigate the production of phytotoxic activity only the culture filtrates of M-1-D medium showed good toxic activity on wheat leaves tested in the puncture bioassay. The symptoms appeared after 3-4 days on the leaves and showed typical yellow-greyish flecks with irregular lesions and the tissue at the site totally collapsed. The culture filtrates were not active in the bioassay on root inhibition and root absorption.They showed toxicity in leaf injection tests but the puncture bioassay was chosen for its easy handling and reproducibility of the results. This activity was detectable both in culture filtrates from either s h a k e n or static con-

Growth and toxicity of S. tritici on M - I - D

ditions, but the shaken cul6 ture was used in the time~ course experiment because 5 ~ under that condition the activity was produced earlier "I"4 i and in sufficient amounts, ca.3 The results of the time course experiment on 2 growth-toxicity of the fun9 gus showed that although a 1 / toxic activity was detectable 0 ' 10-12 days after inocula5 tion, the peak of activity was at 14-16 days of Figure 1. Time-course growth and activity deity of M. graminicola

0.14 4

" 0.12 0.1 3 ->'~ 0.08 2 :~

~ ~_~

0.06

1 v0 ' ' 10Day s 15

' 20

~.~

.~ 0.04 0.02 0

=>,

experiment on growth and production of toxic activstrain ITEM 2357 cultivated on M-1-D medium at 22~ and shaken at 150 rpm. Bars indicate the confidential interval for p=0.05 of three replicates.

creased in the 20 days old cultures (figure 1). Thus, the best toxic activity was collected from shaken cultures (150 rpm) incubated at 22 ~ C in the dark for two weeks. The pH of the filtrate was lower in correspondence of the best toxic activity, as well as the mycelial dry weight in comparison with other liquid media. M-1-D medium proved to be a good substrate for phytotoxins production. It is used for production and isolation of some fungal phytotoxic compounds such as 13- nitropropionic acid from 196

Phytotoxic activity of Mycosphaerella graminicola Septoria cirsii Niessl., fusaric acid and its derivatives from Fusarium nygamai Burgess and Trimboli [16, 17]. Among the other substrates used, the culture filtrate of Fries modified medium showed also phytotoxicity on wheat leaves, but the non-inoculated control also resulted in a phytotoxic response and was therefore not utilized in next studies. On the contrary, Harrabi et al. [9] used Fries medium for production of phytotoxicity by M. graminicola and proposed to use crude extract in in vitro screening for resistance. In our studies we observed that the production of toxicity appears to have a decline when the fungus cultures are subcultured repeatedly and maintained on solid medium, e.g. potato dextrose or tomato-juice agar. Toxicity production was restored by inoculation of fresh inoculum coming from cryoconserved strains on M-1-D medium.

Extraction of culture filtrate The first step in the extraction and purification of the active fraction from the filtrate was difficult to separate from the aqueous phase. Various attempts to extract the active fraction from the aqueous phase were made with direct solvent extraction using different protocols. The extraction of the culture filtrate at different pH (from 2 to 8) with ethyl acetate showed up the division of the activity in the organic phase when the filtrate was first acidified with HC1 at pH 2. The pH of the crude extract obtained was generally 2.8-3.3, and this is positively correlated with the observation made in the time course experiment, in which the pH of the filtrate decreased in correspondence of the maximum peak of toxic activity. The high water affinity of the M-1-D toxic fractions is a good property for the diffusion and translocation of the compounds in the intercellular fluids between the mesophyll cells of the wheat leaves. To this respect recent studies showed the strictly intercellular growth of the fungus. Hence, the communication between the plant and the fungus takes place in the apoplast [ 11 ]. Moreover, these hystopatological studies, suggested the involvement of soluble toxic compounds. Still, cell collapse was not recorded before 8 days after inoculation, which may indicate that a specific physiological fungal growth state is required to produce toxins and induce necrosis [11 ]. This is in accordance with the time-course experiment in which the toxicity was not observed before 10 days of growth in culture filtrate.

Phytotoxicity and pathogenicity of M. graminicola isolates Eighteen out of 24 isolates, cultured in liquid medium (M-l-D), showed toxic activity on wheat leaves of both cultivars. This activity differs in intensity among the isolates but no difference in response was observed on the two wheat cultivars; in fact the crude extract showed the same level of injuries on leaves of cv. Shafir and cv. KK4500 after a three days incubation period. Ten out of 18 toxic isolates were virulent on cv. Shafir but avirulent on cv. Kavkaz/K4500 (table 1). On the other hand, four of the non-toxic isolates (ITEM 2689, 2692, 2870, 2873) were virulent on cv. Shafir. The durum wheat adapted isolates (ITEM 2361, 2362, 2363, 2364, 2869, 2888) were not virulent on both bread wheat cultivars, but showed toxicity in the leaf bioassay. These data show that there is no relationship between the toxicity of culture filtrates and virulence in the studied M. graminicola isolates. It is evident that susceptible and resistant responses do not depend on the resistance of the mesophyll cells to toxic soluble compounds but probably to other mechanisms, such as the production of antifungal compounds (such as phytoalexins) by the plant [11, 12]. However, the toxic activity 197

Challenges of the Environment Table 1. Toxicity and virulence of ISOLATES

ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM

2356 2357 2358 2359 2360 2361 2362 2363 2364 2689 2691 2692 2867 2868 2869 2870 2871 2872 2873 2884 2885 2886

ITEM 2887 ITEM 2888

Micosphaerellagraminicola isolates

#ORIGIN

HOST

COUNTRY

IPO-87016 IPO-323 IPO-91010 IPO-94269 IPO-95058 IPO-86022 IPO-91014 IPO-91016 IPO-95050 IPO-235 IPO-94265 IPO-94266 IPO-90004 IPO-90020 IPO-91012 IPO-88018 IPO-90012 IPO-89011 IPO-92001 IPO 69323.73 IPO 69323.40 IPO 69323.100 IPO 69323.1 IPO 93035

Bread wheat Bread wheat Bread wheat Bread wheat Bread wheat Durum wheat Durum wheat Durum wheat Durum wheat Bread wheat Bread wheat Bread wheat Bread wheat Bread wheat Durum wheat Bread wheat Bread wheat Bread wheat Bread wheat Bread wheat Bread wheat Bread wheat

Uruguay Netherlands Tunisia Netherlands Algeria Turkey Tunesia Tunesia Algeria Netherlands Netherlands Netherlands Mexico Algeria Tunesia Ethiopia Mexico Netherlands Portugal Netherlands Netherlands Netherlands

Bread wheat Durum wheat

Netherlands Canada

Bioassay with crude extract on a Shafir KK4500 + + +++ +++ + + +/+/++ ++ +/+/+ + ++ ++ + + ++ ++ ++ ++ + + ++ ++ +/+/+ + +/+/. . .

+ ++

+ ++

Virulence b Shafir + nt + + nt nt +/+ + + + + + + + + + .

nt

KK4500 nt nt +/nt -

nt

a =Toxicity data on the basis of this scale: - no symptoms; +/- necrosis 1-2 mm; + necrosis 2-3 mm; ++ necrosis 3-4 mm; +++ necrosis > 4 mm b_ Pathogenicity: += susceptible, -= resistant, nt= not tested

c o u l d be r e l a t e d to a m a j o r c a p a b i l i t y o f the f u n g u s to c o l o n i z e and d e s t r o y the m e s o p h y l l cells (as a p a t h o g e n i c i t y factor), but d o e s not s e e m to be an i n d i s p e n s a b l e f a c t o r for v i r u l e n c e . T h i s is also s u g g e s t e d b y the t o x i c i t y o f the c r u d e extract f r o m d u r u m w h e a t isolates on these t w o b r e a d w h e a t cultivars (see Table 1). A t this r e s p e c t a n a l y s i s o f g e n e t i c v a r i a t i o n for virul e n c e and r e s i s t a n c e in

M. graminicola m a d e b y K e m a et al. [18] s h o w e d that the d i s c r i m i n a -

tion b e t w e e n isolates that w e r e derived f r o m either b r e a d or d u r u m w h e a t w a s less e v i d e n t w h e n c o n s i d e r i n g the n e c r o s i s as the o n l y p a r a m e t e r to asses the disease severity. A l t h o u g h b r e a d w h e a t isolates and d u r u m w h e a t isolates i n d u c e necrosis in d u r u m w h e a t and b r e a d w h e a t , respectively, t h e y do not or h a r d l y p r o d u c e p y c n i d i a c o m p a r e d to the h o s t to w h i c h t h e y are adapted. So p a t h o g e n i c i t y m i g h t be c o n t r o l l e d b y d e t o x i f i c a t i o n o f p h y t o a l e x i n s during the first p h a s e s o f the i n f e c t i o n p r o c e s s (48 hrs), w h i c h e n a b l e c o l o n i z a t i o n o f the m e s o p h y l l tissue and, e v e n t u a l l y the p r o d u c t i o n o f sufficient toxic c o m p o u n d s c o u l d kill h o s t cells [11, 13]. Finally, our findings s u g g e s t the p r o d u c t i o n o f m e t a b o l i t e s b y

M. graminicola w i t h

toxic activity on w h e a t leaves, that i n d u c e n e c r o s i s b o t h on s u s c e p t i b l e and r e s i s t a n t w h e a t cultivars and w h i c h are p r o d u c e d b y b r e a d and d u r u m w h e a t isolates. A l t h o u g h there is s o m e e v i d e n c e o f u s i n g toxic culture filtrates for

in vitro selection for r e s i s t a n c e to M. graminicola 198

Phytotoxic activity of Mycosphaerella graminicola [9], our results suggest that the efficiency might be low. Further investigations are necessary to elucidate the role of soluble toxic compounds and to characterise their toxic activity. Materials and methods M. graminicola isolates. All M. graminicola isolates originated from the DLO-Research Institute for Plant Protection (IPO-DLO) at Wageningen, Netherlands. All the strains received were frozen in sterile 18% glycerol-water and stored at -75~ in the Istituto Tossine e Micotossine da Parassiti Vegetali (ITEM) culture collection at Bari, Italy. C u l t u r e conditions. The fungus was maintained on Petri plates with a medium composed of 20 % (v/v) tomato juice, 0 . 1 % (w/v) CaCO3 and 1.5% (w/v) agar. The plates were inoculated with a conidial suspension (0.5 ml), incubated for 4 days at 25~ 12 hrs dark/light. At these conditions the fungus sporulated abundantly on these plates. Two ml of a spore suspension (~ 107 conidia/ml) obtained from these plates was used for inoculation of the culture flasks for toxin production, containing 200 ml/g of several liquid/solid culture medium in 1 liter of Erlenmyer flask or Roux bottle. Agar blocks containing fungal mycelium transferred from the plates to culture flasks were not effective in producing adequate fungal growth. Two M. graminicola isolates, ITEM 2357 (# IPO 323 from bread wheat) and ITEM 2364 (# IPO 95050 from durum wheat), were grown on a range of media (liquid/solid) and at different culture conditions (light/dark, temperature18~ ~ and shaken/still). Four liquid defined media and three solid-natural media were used in screening the growth and the toxic activity of the fungal filtrate/extract in different bioassays: Liquid defined media: Minimal medium (M-l-D) [19], Fries modified [20], Strobel-medium [15], Malt - yeast-extract [21], Solid natural-media: Rice kernels [22], Wheat kernels [22], Wheat leaf extract agar [23]. M-1D is a defined medium enriched by microelements, its composition is the following: Sucrose (87.6 raM), MgSO 4 (30 mM), Ammonium tartrate (27.1 raM), Ca(NO3) 2 (1.2 raM), KNO 3 (0.79 raM), KC1 (0.87 raM), NaHzPO 4 (0.14 mM), KI (45 laM), H3BO 3 (22 gM), MnSO 4 (30 laM), ZnSO 4 (8.7 pM), FeC12-6H20 (7.4 ~tM). In order to optimize the production of toxic activity a time-course experiment was set up. M-1-D was inoculated with M. graminicola isolate ITEM 2357 (# IPO 323), a good producer of phytotoxic activity, and incubated at 22 ~ C on shaken growth condition in the dark. The cultures were harvested routinely every 4 days until the 20th day of growth, and a curve of toxicity in relation to the age of the culture, the mycelium dry weight and the pH of the culture filtrate was made. The experimentwas conducted with three replicates and was repeated once. The data were statistically analyzed and the confidential interval for p=0.05 was calculated. C u l t u r e extraction. The solid cultures, were air-dried and ground, treated with different liquid-solid extraction protocol: 10 g of the grounded cultures were extracted directly in 100 ml respectively of methanol/water (55/45 v/ v), methanol, and chloroform. The extraction was performed by shaking 2 hours the obtained suspensions in a rotary shaker and by filtrating on Whatman No. 4 filter. The filtrates were concentrated by rotary evaporation in vacuo at 40~ and recovered in 4% methanol in water for the bioassays. The liquid cultures coming from different liquid media, growing at different condition as described above, were filtered through Whatman No. 1 filter and the culture filtrates obtained were tested in different bioassays to screen for phytotoxicity. Culture filtrate concentrated to 1/4 of its original volume, was acidified to pH 2 with HC1 and exhaustively extracted three times with ethyl acetate to obtain a toxic crude extract. The three extracts were combined and reduced to dryness by rotary evaporation in vacuo at 40~ The pH of this extract was generally 2.8-3.3, but at acid pH all test materials produced necrotic lesions in the leaf bioassay (acid reaction). The neutralization (with NaOH 1 M) of crude extract failed to delevolop symptoms in the leaf bioassay, so the crude exctract was dissolved in a biological phosphate buffer (pH 6.5) before being used in the leaf bioassay. In this way the crude extract showed a positive reaction in the bioassay, while the other test material did not. M-1-D medium extract not inoculated with the fungus served as control. Bioassays. Different bioassays (root absorption, seedling root inhibition, leaf puncture, leaf injection) were used to

follow the biological activity, i) root absorption: young wheat plants (15 days after sowing) obtained in the greenhouse were immersed in tubes containing the culture filtrate/crude extract, for 24 hours, under fluorescent light with a fotoperiod of 12 hours in a grown chamber at 22~ Plants were then transferred to distilled water and kept in the same conditions. Symptoms were observed 2-3 days after, ii) seedling root inhibition: wheat seeds were surface sterilized with NaC10 (4%) for 10 min, then seeds were washed with sterile H20 and kept in Petri dishes on sterile wet filter paper for 3 days to allow germination to take place. Homogenous seedlings were then transferred on filter papers in Petri dishes (10 seeds/dish), wet with 10 ml of culture filtrate solution. Seedlings were kept in a grown chamber at 25~ After four days the root length was measured. Symptoms were expressed as reduction of root elongation as compared to the control, iii) leaf puncture: leaves from wheat plant grown in greenhouse were cut and placed in a sealed plexiglass moist chamber. The test solution (20 ~1) was placed over a puncture wound made on the wheat leaf with a syringe. The test leaves were incubated at 25~ 12 hrs light/dark and the effects of

199

Challenges of the Environment the filtrates/crude extratcs were observed after 3-4 days. iv) leaf injection: the test solution (40-50 ~tl) was injected in wheat leaves using a pair of scissors on which an Hamilton syringe was applied. The injected plants were incubated at 25~ 12 hrs light/dark and the symptoms were observed after 5 days. After preliminary experiments the leaf puncture bioassay was chosen for its best response. Culture filtrates were tested both at their original volume and concentrated to 1/2 (v/v). The crude extracts (fractions) were dissolved in methanol and tested after dilution in a phoshate buffer pH 6.5 (4% methanol). Phytotoxicity and pathogenicity of M. graminicola isolates. The toxicity of 24 culture filtrates and crude extracts from isolates with different origin (Table 1) was tested on leaves of a susceptible (Shafir) and a resistant (Kavkaz/K4500) bread wheat cultivar and the activities were compared to the virulence data. The toxicity was assessed on 0-5 scale on the basis of no symptoms (-) to necrosis > 4mm (+++) (Table 1). The experiment was conducted with three replicates and was repeated once. Virulence data were collected at IPO-DLO as described by Kema et al. [24].

Authors of this contribution G. Perrone ~, A. Logrieco ~, G.J.H. Kema 2, A. Ritieni 3 A. Bottalico 4 ~Istituto Tossine e micotossine da parassiti vegetali del CNR, 70125 Bari, Italy, 2DLO Research Institute for Plant Protection, P.O. Box 9060, 6700 GW Wageningen, The Netherlands, 3Dipartimento di Scienza degli Alimenti dell'Universit?a "Federico II", 80055 Portici, Napoli, Italy, 4Istituto di Patologia vegetale dell'Universit?a, 07100 Sassari, Italy.

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J.E. King, R. J. Cook, and S. C. Melville, A review of Septoria diseases of wheat and barley, Ann. Appl. Biol., 103 (1983) 345-373. D.E. Hess, and G. Shaner, Effect of moisture and temperature on development of Septoria tritici blotch in wheat, Phytopathology, 77 (1987) 215-219. M.W. Shaw and D. J. Royle, Airborne inoculum as a major source of Septoria tritici (Mycosphaerella graminicola) infections in winter wheat crops in the UK, Plant Pathol., 38 (1989) 35-43. M.W. Shaw and D. J. Royle, Factors determining the severity of epidemics of Mycosphaerella graminicola (Septoria tritici) on winter wheat in the UK, Plant Pathol., 42 (1993) 882-899. G . H . J . Kema, E. C. E Verstappen, M. Torodova, and C. Waalwijk, Successful crosses and molecular tetrad and progeny analyses demonstrate heterothallism in Mycosphaerella graminicola, Curr. Genet., 30 (1996) 251-258. Hunter, T., Coker, R.R. and Royle, D.J.. The teleomorph stage, Mycosphaerella graminicola, in epidemics of septoria tritici blotch on winter wheat in the UK. Plant Pathology, 48 (1999) 51-57. C.A. Cordo, L. R. Marechal, Toxic action of filtrates of Septoria tritici, Revista de la Facultad de Agronomia, Universidad Nacional de La Plata, 63 (1987) 25-34. H. Malcom, A host specific toxin extracted from Septoria tritici, Proceedings of the Australian Septoria workshop, Agricultural Research Institute, N. S. W. Dept. of Agriculture, (1978) pp. 30-31. M. Harrabi, M. Cherif, H. Amara, Z. Ennaiffer and A. Daaloul, In vitro selection for resistance to Septoria tritici in wheat, Proceedings of a Septoria tritici Workshop, pp. 109-116, 20-24 September 1993, CIMMYT, Mexico. G.D. Voloshchuk, S.I. Voloshchuk and V.S. Girko, Effect of culture filtrates of some fungal pathogen on wheat suspension culture, Tsitologiya i Genetika, 29 (1994) 70-77 (Review of Plant Pathology, 1995, 75, 4441). G.H.J. Kema, D.Z. Yu, EH.J. Rijkenberg, M.W. Shaw, and R.E Baayen, Histology of the pathogenesis of Mycosphaerella graminicola in wheat, Phytopathology, 86 (7) (1996) 777-786. J. Weibull and H.M. Niemeyer, Changes in dihydroxymethoxybenzoxazinone glycoside content in wheat plants infected by three plant pathogenic fungi, Physiological and Molecular Plant Pathology, 47 (1995) 201-212. M. Barbier, M. Devys, J.-E Bousquet e A. Kollmann, Absolute stereochemistry of N-methoxyseptorinol isolated from the fungus Septoria nodorum, Phytochemistry, 35 (1994) 955-957. M. Devys, M. Barbier, J.-E Bousquet and A. Kollmann, Isolation of the Hydroxymellein from the fungus

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Phytotoxic activity of Mycosphaerella graminicola Septoria nodorum, Phytochemistry, 35 (1994) 825-826. 15. S. S. Kent and G. A. Strobel, Phytotoxin from Septoria nodorum, Trans. Br. mycol. Soc., 67 (1976) 355-358 16. J. Hershenhorn, M. Vurro, M. C. Zonno, A. Stierle, G. Strobel, Septoria cirsii, a potential biocontrol agent of Canada thistle and its phytotoxin - 13-nitropropionic acid, Plant Science, 94 (1993) 227-234. 17. R. Capasso, A. Evidente, A. Cutignano, M. Vurro, M. C. Zonno, and A. Bottalico, Fusaric and 9,10Dehydrofusaric acids and their methyl esters from Fusarium nygamai, Phytochemistry, 41 (1996) 10351039. 18. G. H. J. Kema, R. Sayoud, J. A. Annone, and C. H. Van Silfhout, Genetic variation for virulence and resistance in the wheat-Mycosphaerella graminicola pathosystem I. Interaction between pathogen isolates and host cultivars, Phytopathology, 86 (1986) 200-212. 19. E Pinkerton and G.A. Strobel, Serinol as an activator of toxin production in attenuated cultures of Helminthosporium sacchari, Proc. Natl. Acad. Sci., 73 (1976) 4007-4011. 20. J. E Bousquet, H. Belhomme de Franqueville, A. Kollmann, and R. Fritz, Action de la septorine, phytotoxine synth6tis6e par Septoria nodorum, sur la phosphorylation oxydative dans les mitochondries isol6es de Col~optiles de B16, Can. J. Bot., 58 (1980) 2575-2580. 21. N. Zelikovitch, Z. Eyal, and Y. Kashman, Isolation, purification and biological activity of an inhibitor from Septoria tritici, Phytopathology, 82 (1992) 275-278. 22. M. Kostecki, H. Wisniewska, G. Perrone, A. Ritieni, E Glolinski, J. Chelkowski, A. Logrieco, The effects of cereal substrate and temperature on production of beauvericin, moniliformin and fusaproliferin by Fusarium subglutinans ITEM-1434, Food Additives and Contaminants, 1999 in press. 23. N. Zelikovitch and Z. Eyal, Maintenance of virulence of Septoria tritici cultures. Mycol. Res., 92 (1989) 361-364. 24. G. H. J. Kema., Annone, J. G., Sayoud, R., Van Silfhout, C. H., Van Ginkel, M. and De Bree, J. Genetic variation for virulence and resistance in the wheat-Mycosphaerella graminicola pathosystem. I. Interactions between pathogen isolates and host cultivars. Phytopathology, 86 (1996). 200-212.

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Phytosfere’99 - Highlights in European Plant Biotechnology Gert E. de Vnes and Karin MetzlaR (Editors). 0 Elsevier Science B.V. All rights reserved

Helping Plants To Defend Themselves: Biocontrol By Disease-Suppressing Rhizobacteria Abstract Various rhizobacterial strains suppress soil-borne plant diseases by competition with the pathogen for nutrients, siderophore-mediated competition for iron, antibiosis, or production of lytic enzymes. Some strains can also enhance the defensive capacity of plants by mechanisms that are either dependent on the production of salicylic acid and associated with the accumulation of pathogenesis-related proteins in the plant (systemic acquired resistance), or that are not but require responsiveness of the plant to jasmonate and ethylene (induced systemic resistance). To exploit the mechanisms involved in disease suppression by rhizobacteria, the genes responsible are being cloned. combinations of rhizobacterial strains or genetic modification of Pseudomonas bacteria are used to improve biological control of economically important plant diseases. Risk assessment studies have been initiated with regard to the introduction of such genetically-modified microorganisms in the field.

Biological mechanisms that protect plants against pathogens Plants do not develop disease after contact with most potential pathogenic microorganisms. The plant itself may be unsuitable for establishment or multiplication of such microorganisms, but more often plants react by mounting defense responses so quickly that infection remains limited to only a few cells and plants appear essentially immune. Defense responses can be multiple and comprise synthesis of low-molecular-weight antimicrobial phytoalexins, accumulation of pathogenesis-related proteins, and cell wall rigidification [I]. Active resistance in plants is triggered by pathogens that are recognised either directly or indirectly in a gene-for-gene-specific fashion through the action of one or more resistance ( R ) genes in the plant [ 2 ] . Identification and characterization of such R genes allows their transfer into crop plants, providing engineered protection against cognate races of the pathogen. Further elucidation of the mechanisms involved in gene-for-gene interactions will allow additional strategies to be devised in order to protect genetically-modified plants through constitutive expression of key regulatory genes. Not only the plant itself, but also the environment can inhibit pathogens from causing disease. Several nonpathogenic microorganisms that are naturally present in soil and on plant surfaces

L.C. van Loon, Faculty of Biology, Section Phytopathology, Utrecht University, The Netherlands

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Challenges of the Environment can decrease the survival of plant pathogens, reduce the build-up of pathogen populations, or counteract infection of susceptible plants. Such microorganisms are responsible for the disease-suppressive properties of some natural soils in which resident pathogens are unable to cause disease in susceptible crops [3]. The pathogen is not necessarily eliminated, but the disease is absent or decreased [4]. Both the total microbial biomass and specific populations of antagonistic microorganisms can be responsible for disease suppression [5]. Thus, nonpathogenic Fusarium spp. are considered to be primarily responsible for the suppressiveness of the Chateaurenard soil in France [3], whereas fluorescent Pseudomonas bacteria are apparently the prime organisms of suppressiveness in the Salinas Valley soil in California [6]. Specific strains of Pseudomonas spp. rapidly colonize plant roots of various plant species and can effectively control soilborne plant pathogens [7]. Effective and durable genetic resistance against pathogenic fungi causing damping-off and early wilting of seedlings is mostly lacking and these diseases can only be controlled by preventive chemical disinfection or steam sterilisation of soils. Chemical disinfection involves the use of noxious chemicals and is environmentally burdensome, whereas steam sterilisation is costly and seldom durably effective. Thus, using naturally occurring microorganisms antagonistic to soilborne pathogens offers an attractive alternative for disease control. Indeed, biological control of plant diseases is an emerging strategy for the protection of agricultural and horticultural crops [8]. Experiments in commercial greenhouses over a four-year period demonstrated that radish grown from seed coated with the bacterium PseudomoFigure 1. Reduction of Fusarium wilt in radish by biocontrol nasfluorescens strain WCS374 was sigbacteria under commercial greenhouse conditions; left plot: nificantly less affected by Fusarium seeds were treated with a coating containing Pseudomonas fluorescens strain WCS374, middle plot: coating without bac-

teria, fight plot: non-treated,

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oxysporum f.sp. raphani (For) (figure 1), resulting in up to 30% more marketable

Biocontrol by disease-suppressing rhizobacteria produce [9]. Only in rare cases is disease totally suppressed, however, and results can vary depending on environmental conditions [7,10,11 ]. This situation, in which a single bacterial isolate is applied, contrasts with that in naturally disease-suppressive soils, in which the disease-suppressive character appears to depend on combinations of microorganisms acting synergistically [12]. Experiments with carnation have shown that combination of the bacterium Pseudomonas putida strain WCS358 and the nonpathogenic Fusarium oxysporum strain Fo47 resulted in more effective control of Fusarium wilt than treatments with WCS358 or Fo47 alone [13,14]. Improvement of biological control of Fusarium wilt of radish and take-all of wheat, caused by Gaeumannomyces graminis f.sp. tritici (Ggt), was also demonstrated by combinations of Pseudomonas spp. [15-17]. However, there are also examples of co-inoculations which did not result in an improved biological control compared with the separate inoculants [ 18-22]. An important requirement for successful combination of strains is the compatibility of the coinoculated microorganisms [ 16,23,24]. For instance, application of either Pseudomonas strain RE8 or RS 111 to soils suppressed Fusarium wilt in radish to roughly equal extents. When both were combined, no increased protection occurred. In vitro RS 111 is antagonised by RE8, and it seems reasonable to assume that any additional effect that RS 111 might have in suppressing Fusarium wilt in vivo, was offset by the antagonistic activity of RE8. Indeed, a mutant of RS 111, RS 11 l a, was isolated that was no longer antagonised by RE8 in vitro. Combined application of RE8 and RS 111 a to soil in which radish seeds were sown did result in increased protection against For, indicating that bacterial strains must not antagonise each other for enhanced protection to be achievable [17].

Molecular mechanisms of disease suppression by rhizobacteria Negative interactions between antagonistic microorganisms in the rhizosphere can be circumvented by genetically engineering single organisms to optimally express disease-suppressive traits. Moreover, by equipping suitable bacterial strains with additional mechanisms for antagonising pathogenic microorganisms, the effectiveness and reliability of the application can be increased [25]. For genetically engineering resistance genes into plants, each cultivar or variety of each and every crop species has to be transformed anew. B iocontrol bacteria are not specific in colonising plant roots and, thus, can be used in several different crop plants. However, exploitation of the potential of antagonistic microorganisms can only be achieved if the traits responsible are better understood. With regard to disease-suppressing Pseudomonas spp. and the mechanisms involved, much knowledge has become available in the past decade, particularly through the use of transposon mutagenesis and complementation analysis [26].

Competition for nutrients and niche exclusion Root colonisation by Pseudomonas spp. is dependent on the release of organic compounds as exudates and lysates from the plant cells [27]. Fluorescent pseudomonads can utilise a great variety of carbon sources from root exudates, including sugars, organic acids and amino acids. Through competition for nutrients between the bacteria and the pathogen, the amount of substrate available to the pathogen is reduced and, consequently, the disease is suppressed [7]. The ability of fungal pathogens for spore germination and hyphal proliferation is reduced 205

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most if both the antagonistic bacterium and the pathogen have similar nutrient requirements [28]. However, the extent to which Pseudomonas bacteria colonise the root is limited to only a small percentage of the total root surface [29,30], implying that physical exclusion of the pathogen by rhizobacteria can hardly play a role. Several rhizobacterial traits have been identified that are important for root colonisation and competitiveness with other indigenous microorganisms. Isolation and characterisation of colonisation-impaired mutants of P. fluorescens strain WCS365 has shown that traits such as motility, synthesis of the O-antigen of the outer membrane lipopolysaccharide (LPS), prototrophy for amino acids and thiamine, and a high growth rate are all important traits for the colonisation process [31]. The fact that amino acid and thiamine auxotrophic mutants were not able to colonise the root tips of tomato plantlets in a gnotobiotic system unless the compound was added, indicates that the amounts exuded by the roots were too low to physiologically complement the mutation. Moreover, mutants of WCS365 impaired in the utilisation of organic acids showed a reduced colonization ability in competition with the wild type, supporting the notion that nutrient limitation is common in the rhizosphere [32]. Therefore, a high competitiveness in the rhizosphere is essential for bacterial antagonists to suppress pathogenic fungi. Similar screenings indicated that bacterial genes encoding components of the energy-generating NADH dehydrogenase, a colR/colS two-component regulatory system alledgedly involved in nutrient uptake, and a site-specific recombinase, sss, are required for effective colonisation [31 ].

Competition for iron Due to its low solubility, Fe 3§ is often present in limiting amounts in soils. To sequester the scarcely available iron, microorganisms produce low-molecular-weight siderophores with a high affinity for ferric iron. Highly specific outer membrane proteins, inducible by the siderophores and/or iron limitation, function as receptors for delivering the ferric ironsiderophore complex to the cells. Particular strains of Pseudomonas spp. produce fluorescent siderophores, named pyoverdin or pseudobactin, that can effectively suppress disease through competition for iron with pathogens which produce lesser amounts of siderophores or siderophores with a lower affinity for iron, and are unable to utilise the siderophore(s) of the particular Pseudomonas strains [10,21]. Certain strains of Pseudomonas spp. can also produce non-fluorescent siderophores called pyochelins, that contain a salicylic acid (SA) moiety [33]. SA not only functions as an iron-chelating group in pyochelins, but itself can also serve as a siderophore [34,35]. The pseudobactins and pyochelins are complex molecules, with several gene clusters being required for their synthesis [36,37]. Because of substantial structural diversity among the siderophores produced by different strains, most Pseudomonas spp. can utilise only a limited number of these compounds. However, the ability of some strains to utilize siderophores produced by other strains increases their competitiveness in the rhizosphere [21]. Thus, Raaijmakers et al. [ 16] demonstrated that siderophoremediated competition for iron between strains WCS358 and WCS374 decreased root colonisation of the latter. Conversely, transformation of WCS374 with the gene encoding the 206

Biocontrol by disease-suppressing rhizobacteria siderophore receptor PupA for pseudobactin 358, increased population levels of WCS374 in the rhizosphere and enhanced suppression of Fusarium wilt of radish by combination of the two strains. The better disease suppression can be attributed to the reduced competition for iron among the bacterial strains in the rhizosphere, because WCS374 had acquired the ability to utilise pseudobactin 358 in addition to its own pseudobactin 374.

Production of antibiotics In competition with other microorganisms several fluorescent Pseudomonas spp. can produce secondary metabolites with antibiotic activities. Many of these have been implicated in suppression of soilborne diseases, notably 2,4-diacetylphloroglucinol (DAPG), oomycin A, phenazine-l-carboxylic acid (PCA), pyocyanine, pyoluteorin and pyrrolnitrin [26]. Gene loci involved have been cloned and used to enhance the biocontrol properties of Pseudomonas spp. by either overexpressing the genes and, hence, increasing antibiotic production, or introduction in non-expressors, conferring on them the ability to produce the antibiotics. Gutterson et al. [38] placed part of the afu operon involved in the biosynthesis of oomycin A by P. fluorescens strain Hv 37a under the control of a strong heterologous promoter. Consequently, oomycin A production in the rhizosphere was increased up to 1000-fold and damping-off of cucumber resulting from infection by Pythium ultimum was significantly decreased. The non-expressing strain WCS358 was equipped with the PhzABCDEFG genes from P. fluorescens strain 2-79 [39] under the direction of the constitutive Ptac promoter, conferring the ability to produce PCA. The resulting PCA production by WCS358::phz inhibited growth of Rhizoctonia solani, Pythium spp. and Ggt in vitro and reduced take-all of wheat as effectively as the donor strain 2-79 (L.S. Thomashow, unpublished). When applied as a seed coating, WCS358::phz also transiently reduced non-pathogenic soil fungi in the rhizosphere coinciding with high population densities of the introduced bacteria [40]. For antagonistic bacteria to be effective in disease suppression, population levels need to remain elevated. Raaijmakers et al. [41] determined that a minimum of 105 colony-forming units (cfu) per g root was required for WCS358 to suppress For on radish through competition for iron. Similar levels appear to be required for protection against disease through antibiotic production. Under natural conditions, antibiotics are synthesised in response to environmental signals, such as high cell densities and nutrient depletion. Two-component regulatory systems control the production of diffusible autoinducers which, above a threshold concentration, elicit the expression of various activities, including production of antibiotics and lytic enzymes [42]. By overexpression of the global response regulator gacA, production of pyrrolnitrin by P. fluorescens strain BL915 was achieved at lower cell densities, leading to increased suppression of e.g. damping-off caused by R. solani in cotton [43]. Other strategies have involved mutational inactivation of the rpoS gene encoding the stationary phase and stress sigma factor in P. fluorescens strain Pf-5. The rpoS- mutant had lost the capacity to synthesise pyrrolnitrin, but overproduced DAPG and pyoluteorin, and was superior to wild type Pf-5 in suppressing seedling damping-off caused by P. ultimum on cucumber [44]. Thus, increased antibiotic production in the rhizosphere can enhance disease suppressiveness. However, enhanced levels of antibiotics can also have deleterious effects on plant growth, depending on the plant species [4,45]. 207

Challenges of the Environment HCN is a representative of the class of volatile inhibitors which, besides reducing the activity of harmful microrganisms, can also reduce plant growth. HCN contributes to the suppression by P fluorescens strain CHA0 of black root rot of tobacco, caused by Thielaviopsis basicola [46]. Transfer of the hcn biosynthetic gene cluster to non-HCN-producing Pseudomonas strains increased their biocontrol activity against black root rot in tobacco, as well as against the leaf pathogens Septoria tritici and Puccinia graminis in wheat [47].

Production of lytic enzymes Some bacteria can parasitize on and kill fungi by secreting lytic enzymes, such as chitinases, g-l,3-glucanases, proteases and lipases. Growing hyphal tips are particularly susceptible to bursting as a result of chitinase action. Chitinases produced by Serratia marcescens have been associated with biocontrol of fungal diseases on pea [48] and bean [49]. The chiA gene was cloned and expressed constitutively in P putida. The chiA § recombinant afforded increased protection to radish against E oxysporum f.sp. redolens [50]. Similarly, introduction of chiA in Escherichia coli made this bacterium reduce disease caused by Sclerotium rolfsii in bean and R. solani in cotton [51 ]. However, very high doses of the recombinant strain were applied. Secretion of the chitinase by these bacteria is, at best, poor and their effectiveness appears to be due primarily to dying cells releasing the enzyme in the rhizosphere. Expression of chiA in E. coli led to an altered morphology in vitro, suggesting that heterologous bacteria can be affected by the enzyme (J. Folders, J.RM. Tommassen and L.C. van Loon, unpublished).

Induction of systemic resistance in the plant The disease-suppressing activity of biocontrol bacteria is not confined to an antagonistic action towards pathogens, but can also be plant-mediated [52]. The latter is evident when biocontrol bacteria and the pathogen are applied at spatially separated locations on the plant and no contact between the two occurs. This type of disease-suppressive activity has been termed induced systemic resistance (ISR). ISR is phenotypically similar to pathogen-induced systemic acquired resistance (SAR), in which a plant likewise develops an enhanced resistance against challenging pathogens. SAR is dependent on the synthesis by the plant of SA, that acts as the inducer signal, and is associated with the accumulation of novel, pathogenesisrelated proteins (PRs). Transformation of plants with the nahG gene from P putida, which encodes salicylate hydroxylase, causes conversion of the SA produced to catechol, which is inactive as an inducer. As a result, no SA accumulates, no PRs are produced, and no SAR develops [53]. Thus, nahG-transformed plants can be used to determine whether ISR-inducing bacteria trigger the SAR pathway. Bacteria that produce SA as a siderophore under iron-limiting conditions, could by-pass the requirement for plant-produced SA and might directly induce SAR. Several Pseudomonas spp. are able to produce SA in vitro, among which are P aeruginosa strain 7NSK2 [54] and P. fluorescens strains CHA0 [55], WCS374 and WCS417 [56]. Under low-iron conditions 7NSK2 induced resistance in bean and tobacco against gray mold, caused by Botrytis cinerea, and tobacco mosaic virus, respectively. Mutants that had lost the ability to produce SA, did not induce resistance. Moreover, the wild-type strain did not induce resistance in nahG tobacco, clearly implicating SA as the resistance-inducing determinant [57]. In contrast, the SA-pro208

Biocontrol by disease-suppressing rhizobacteria ducing rhizobacterial strain S. marcescens 90-166 induced resistance to wildfire, caused by Pseudomonas syringae pv. tabaci, in both untransformed and nahG-transformed tobacco, indicating that SA was not involved in the resistance induced by this strain [58]. Likewise, in Arabidopsis WCS417 induced resistance against For or P. syringae pv. tomato to the same extent in wild-type and nahG-transformed plants, whereas WCS374 was inactive as an inducer [59]. Hence, rhizobacterially-mediated ISR can be either similar or dissimilar to SAR, with bacterially-produced SA playing a role in only some combinations. By mutant analysis, it was demonstrated that different bacterial determinants are involved in the induction of resistance by rhizobacterial strains, including siderophores, LPS, and other iron-regulated factors [55,56,59-61]. In radish, no accumulation of PRs was observed [62], making it highly likely that, as in Arabidopsis, SA is not involved in the systemic resistance induced by strains WCS374 and WCS417. Using Arabidopsis as a model plant, it was found that rhizobacterially-mediated ISR requires responsiveness of the plant to both jasmonate and ethylene [63,64]. ISR is induced in a plant-species- and bacterial-strain-specific manner, indicative of specific recognition between plant roots and bacterial components [59]. Further characterisation of the regulatory mechanisms involved in ISR offers great potential to protect plants against disease by taking advantage of this naturally occurring type of defence. Whereas ISR usually affords somewhat lesser protection than SAR, recent findings indicate that resistance induced by WCS417 can be boosted by application of SA, suggesting that best protection may be achieved by bacteria that induce resistance by both SA-independent ISR and SAdependent SAR [65]. SA-biosynthetic gene clusters have been cloned from P aeruginosa [66] and from P fluorescens [67], and expressed in non-SA producing Pseudomonas spp. under the direction of a constitutive promoter. SA production rendered P. fluorescens strain P3 capable of protecting tobacco partially from infection with tobacco necrosis virus [68].

Potential of engineered biocontrol bacteria for commercial applications Systemic induced resistance has been shown to be effective against fungal, bacterial and viral pathogens and, once induced, can be maintained for prolonged periods [69]. Engineering rhizobacteria to optimally express resistance-inducing determinants is attractive, because these will stimulate the plant as long as bacterial levels remain sufficiently elevated [41]. In addition, the effect is maintained in the plant afterwards, when bacterial numbers decrease [70]. In contrast, microbial antagonism requires high populations of bacteria to be maintained, often making colonisation a limiting factor in biocontrol [4,7]. So far, none of the mechanisms of bacterial disease suppression is as effective as chemical crop protectants and can afford full protection against diseases. However, mechanisms can be optimised by biotechnological means to enhance biocontrol activity. Moreover, either combinations of strains with complementary mechanisms, or strains engineered to express a combination of mechanisms, may provide sufficient protection for economic losses to be minimised. There is public concern that the introduction of additional antimicrobial activities into rhizobacteria by biotechnological means may provide a hazard to the natural soil community. This problem is being addressed in a field experiment with WCS358 engineered to express PCA in the rhizosphere of wheat [40]. Population levels of the transformed derivatives decreased at the same rate as wild-type WCS358 during the growing season and were no longer 209

Challenges of the Environment detectable 130 days after sowing. Thus, the ability to produce PCA did not confer a selective advantage on the transformant in the rhizosphere. No effects on soil respiration, nitrification potential or cellulose breakdown were measurable, and neither were numbers of soil bacteria r e d u c e d . F u n g a l p o p u l a t i o n levels w e r e t r a n s i e n t l y r e d u c e d w h e n the p o p u l a t i o n of WCS358::phz was above 105 cfu per g root, i.e. levels expected to be effective in antagonising pathogenic fungi. The introduction of large numbers of bacteria on seeds or as a soil drench by itself may be considered to pose risks. However, soil bacterial numbers and composition fluctuate widely under natural conditions, and introduced strains are reduced to background levels within a few weeks to months. There has also been concern about bacterial antibiotic or SA production in the rhizosphere and changes in plant composition due to the induction of systemic resistance. Antibiotic production is a natural phenomenon on the surface of plant roots and largely responsible for natural soil suppressiveness [12,26,71]. SA produced in the rhizosphere can hardly be detected, yet must be sufficient to induce SAR. Non-bacterized, field-grown plants often contain PRs by the harvest stage, indicating that the ambient conditions have been stressful and SA was produced in the plant. So far, rhizobacterially-mediated ISR has not been found to be associated with any changes in plant composition, although the induced state must differ from the non-induced one. It is not clear in how far plants in natural vegetation are induced, but man has historically been dependent on wild plants for food. Unlike synthetic chemicals, the mechanisms of disease suppression by biocontrol bacteria are natural and can be considered environmentally safe.

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Phytosfere.99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 0Elsevier Science B.V. All rights reserved.

CO, Exchange Of Potato Transformants With Reduced Activities Of Glycine Decarboxylase Summary Components of photosynthetic and respiratory CO, exchange in photosynthesising leaves of potato (Solunum tuberosum L., var. Desiree) control plants and of its transformants (P1 and P15) with limited expression of glycine decarboxylase have been determined under normal environmental conditions. The rate of true photosynthesis was highest in leaves of control plants (16.0pmo1 CO,m-*.s-').In transformant P1 it was 2 times lower while PI5 showed an intermediate value. The same relationship was found for the total rate of intracellular decarboxylation in the light: 2.6, 1.3 and 1.8 pmol CO, m-2.s-'in leaves of control plants, P1 and P15, respectively. No differences were detected in the relative rate of intracellular decarboxylation indicating that transformants did not have any advantage with respect to respiratory losses during photosynthesis in the light. In all varieties the main substrates of decarboxylation were primary photosynthates, the contribution of stored photosynthates varied in the range from 15 to 25% the total rate of decarboxylation. The rate of photorespiration exceeded the rate of respiration 7.5 times in control plants and 1.8-2.5 times in transformants. The rate of respiration in the dark was in leaves of control plants 30-40% higher than in leaves of transformants. Light severely, about 20 times, inhibited this component of respiration in control plants but only 1.5 times in PI and 5.5 times in P15. The specificity of Rubisco, determined in intact leaves in vhw, had significantly higher values in transformants compared to control plants. It has been shown that in transformants a portion of glycine was not decarboxylated by glycine decarboxylase but transported out of the glycolate cycle which results in a change of the stoichiometry between RuBP oxygenation and photorespiration and in an apparent increase of Rubisco specificity. In leaves of transformants the content of non-protein glycine was 3-5 times higher and the content of non-protein serine 6-15 times lower than in leaves of control plants.

Introduction Potato is a typical C, plant where a portion of the newly assimilated CO, is lost in the process of photorespiration. Glycine decarboxylase (GDC, EC 2.1.2.10) catalysing the conversion of two molecules of glycine to serine with concomitant evolution of one molecule of CO, is an enzyme immediately responsible for this loss of carbon. Aiming to reduce photoresp&tion and to increase photosynthetic productivity the transformants of potato (Solurzum tuberosum

Olav Keerberg, Estonian Agricultural University, Institute of Experimental Biology, Harku, Estonia

215

Challenges of the Environment L., var. Desire6) with 50-75% suppression of GDC has been constructed [1]. In this study the components of photosynthetic and respiratory CO 2 exchange in photosynthesising leaves of potato control plants and of its transformants were determined. Research was performed as a part of EC project "Control of photorespiration in plant leaves by rDNA technology: effects on plant physiology, agricultural productivity and water use efficiency" (BIO4-CT97-2002).

Material and methods Tubers of potato control plants and transformants P1 and P15 with different degree of GDC expression were obtained from Dr. Hermann Bauwe (Rothamsted Experimental Station, UK). Plants were grown in soil under combined illumination of high pressure sodium discharge lamp LU400/HO/T/40NG (LUCALOX, Hungary) and of high pressure mercury-vapor fluorescent lamp LRF 250W E40 (POLAMP, Poland) at the following conditions: irradiance 250-300 [amol.m-Z.s-~, 12h/12h light/dark, day/night temperature 22/16~ Eight week old control plants and ten week old plants of transformants were used in experiments. Measurements were performed on fully expanded leaves of upper levels under normal environmental conditions: ([CO2] 370jaL.L-', [O2] 210mL.L -', PPFD 750~mol. m -2. s -1, 25~ Using a radiogasometric method [2] the following characteristics of photosynthetic and respiratory CO 2 exchange were determined: rates of net and true photosynthesis, rate of respiration in the light, rate of respiration in the dark, rates of carboxylation and oxygenation of ribulose 1,5-bisphosphate (RuBP) and specificity factor of Rubisco. Four components of respiration were distinguished according to the substrates (primary or stored photosynthates) and mechanisms (photorespiration or respiration) of decarboxylation reactions. To determine the labelling kinetics of glycine the leaves were exposed to 14C02 for different time intervals ranging from 5 s to 10 min. After the exposure leaves were killed in liquid nitrogen. Labelled photosynthates were extracted with cold perchloric acid and separated by paper chromatography combined with the additional separation of amino acids using an analyser T339 (MIKROTECHNA, Prague). The radioactivity of glycine was determined and plotted against the duration of exposure to ~4CO2. Labeling curves of glycine were analysed according to the method described by Keerberg and P~rnik [3].

Results and discussion The rate of true photosynthesis was highest in leaves of control plants (16.0 pmol CO 2. m -2. s-l). In transformant P1 it was 2 times lower, P15 showed an intermediate value (figure 1). The same relationship was found for the total rate of intracellular decarboxylation in the light: 2.6, 1.3 and 1.8 lamol CO 2. m -2. s-1 in leaves of control plants, P1 and P15, respectively (figure 1). No differences were detected in the relative rate of intracellular decarboxylation (15% the rate of true photosynthesis in all varieties, figure 1). It means that transformants don't have any advantage with respect to respiratory losses during photosynthesis in the light. In all varieties the main substrates of decarboxylation were primary photosynthates, the contribution of stored photosynthates varied in the range from 15 to 25% the total rate of decarboxylation (figure 2). The ratio of photorespiration to true photosynthesis was 14.4%, 9.6% and 10.5 % in control plants, P 1 and P 15 Decarboxylation in the light True photosynthesis respectively (figure 2). Lower pho20 torespiration in transformants was compensated by higher respiration 15 ,- 15 I!~,~ of these plants. The rate of photo- ,~2 respiration exceeded the rate of res- ~''E~ 10 E Figure 1. Rates of true photosynthesis and respiratory decarboxylation in the light in leaves of transformants and control plants of potato.

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0 ~ ' 0 The only component of respiration 0 Primary PhotoRespiration Photorespiration Stored respiration Respiration photosynthates operative in the dark is respiratory decarboxylation of stored photosynthates. The rate of respiration in the dark in leaves of control plants (1.3 ~tmol CO 2. m -2. s -1) exceeded that in leaves of transformants (0.8-0.9 ~mol CO 2 m -2. s -1) 30-40% (table 1). Light seTable 1. Rates of respiratory decarboxylation of stored photosynthates in verely, almost 20 times, inhibited this comtransformants and control plants of potato in the light (PPFD 750 lamol m2.s 4) and in the dark at different oxygen concentrations. ponent of respiration in control plants but only 1.5 times in P1 and 5.5 times in P15. P 15 Control [02] P1 Similar phenomenon has been detected in mL L -1 ~tmol C02 m-2.s -1 leaves of winter rye where respiration in the Light 210 0.61 -+0.05 0.15 -+0.02 0.07 _+0.02 light was not inhibited if the photorespiration was suppressed [4].

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Two methods were applied for the determination of the specificity of Rubisco in intact leaves of potato: (A) the specificity factor was calculated from the ratio of rates of carboxylation and oxygenation of RuBP (Pc/Po) according to the formula: S n = P~ [O2]w/Po[CO2]w

(1)

where [ 0 2 ] w and [ C 0 2 ] w a r e oxygen and C O 2 liquid phase concentrations in the reaction centres [2] and (B) from the dependence of CO 2 compensation concentration (7) on oxygen concentration according to the formula [5] S B = 0.5~5[O2]/~57

(2)

Both methods gave significantly higher val- Table 2. Rubisco specificity in leaves of transformants and control plants of potato calculated: from decarboxylation/carboxylation ratio (SA) and from ues of specificity in transformants compared oxygen dependence of CO2 compensation point (SB). to control plants (table 2). No differences in P1 P 15 Control the Rubisco specificity were found in the measurements in vitro (H.Bauwe, personal SA=Pc[O2]w]Po[CO2]w 143.0 +9.9 120.0 __.6.2 104.0 __.5.3 communication). To explain this disagree113.7 +9.2 118.9 + 11.3 101.2 +0.8 ment it must be taken into account that the SB= 0.55[O2]/~' measurements in vivo are based on the determination of the rate of RuBP oxygenation from the stoichiometry of the glycolate cycle assuming that two oxygenation reactions are needed for the production of one molecule of CO 2 in the reaction of glycine decarboxylation. Apparently this assumption is not valid for the 217

Challenges of the Environment Absolute

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Figure 3. Labeling curves of glycine in leaves of control plants and transformant P1 of potato exposed for different time intervals to ~4CO2.

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Circles represent the experimental data. Solid lines are the theoretical functions derived from the model assuming the existence of multiple pools of glycine in different cellular compartments. Dashed lines show the labeling kinetics of different pools: I, active pool in mitochondria, II, cytosolic pool (in diffusion equilibrium with the active pool), III, accumulation of glycine as an end product of potato photosynthesis.

D u r a t i o n o f e x p o s u r e to 14CO2 (s)

transformants. Indeed, analysis of the kinetics of 14C incorporation into glycine revealed in transformants a component corresponding to the accumulation of glycine as an end product of photosynthesis (figure 3, curve III). This component was absent in control plants. It means that in transformants a portion of glycine is not decarboxylated by GDC but transported out of the glycolate cycle resulting in a change of the stoichiometry between RuBP oxygenation and photorespiration. This is consistent with the 3-5 times higher content of the non-protein glycine in transformants compared to control plants (table 3). On the contrary, the content of non-protein serine, the product of glycine decarboxylation, was in transformants 6-15 times lower than in control plants. The accumulation of glycine was detected also in leaves of barley where GDC activity was reduced to 47% and 63% of its activity in wild-type plants [6]. The effect was more pronounced in the conditions supporting photorespiration (low CO 2, high irradiance). In general it may be concluded that in transformants of potato with suppressed expression of glycine decarboxylase a portion of glycine formed in the glycolate cycle Table 3. Content of nonprotein glycine and serine in leaves of transformants is not decarboxylated but transported and control plants of tobacco out of the cycle resulting in lower P1 P15 Control rates of photorespiration and in a pmol. m -2 change of the stoichiometry between RuBP oxygenation and photorespiGlycine 976 _+82 1344 +170 288 _+24 ration. The suppression of photorespiration is compensated by the higher Serine 23 _+12 59 _+28 375 _+49 rates of respiration in transformants.

Authors of this contribution Olav Keerberg, Hiie Ivanova, Hille Keerberg and Tiit P~irnik Institute of Experimental Biology at the Estonian Agricultural University, 76902 Harku, Estonia 218

CO2 exchange of potato transformants

Acknowledgements This work was supported by the grant of European Commission (Project BIO4-CT97-2002) and by the Estonian Science Foundation (Project No 2197).

References 1. 2. 3.

4.

5.

6.

H. Bauwe, cDNA encoding P-protein of the glycine cleavage system in Solanum tuberosum cv. Desire6 (Accession No Z99770). Plant Physiol. 116(1) 445. T. P~irnik, O. Keerberg, Decarboxylation of primary and end products of photosynthesis at different oxygen concentrations. J. Exp. Bot. 46 (9) 1439-1447. O. Keerberg, T. P~irnik, Modelling and quantification of carbon fluxes in photosynthesizing cells of intact plant leaves in vivo, in: C. Larsson, I.-L. Pfihlman, L. Gustafsson, (Eds.), BioThermoKinetics in the Post Genomic Era, Chalmers Reproservice, G6teborg, 1998, pp. 303-306. T. P~irnik, E Gardestr6m, H. Ivanova, O. Keerberg, Regulation of the photosynthetic and respiratory CO 2 exchange in leaves by external factors in the light, in: G.Garab, (Ed.), Photosynthesis: Mechanisms and Effects, Kluwer Acad. Publ., Dordrecht, 1998, pp. 3731-3734. A. Sumberg, A. Laisk, Measurements of C02/02 specificity of ribulose-1,5-bisphosphate carboxylaseoxygenase in leaves, in: E Mathis, (Ed.), Photosynthesis: from Light to Biosphere, Vol. V, Kluwer Acad. Publ., Dordrecht, Boston, London, 1995, pp. 615-618. A. Wingler, EJ. Lea, R.C. Leegood, Control of photosynthesis in barley plants with reduced activities of glycine decarboxylase. Planta 202 (2) 171-178.

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Phytosfere.99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin MetzlaK (Editors). 0Elsevier Science B.V. All rights reserved

Comments from the Session Rapporteur

Attention for non-industrialised agriculture Why has plant-biotechnology such a bad press? ‘Trust’ was an often-used word in the general discussion about plant biotechnology, Monday 7 June at the EPBN-congress in Rome. ,, The public doesn’t trust the scientists anymore”, complained Francesco Salamini from the Max Planck-Institute for plantbreeding in Koln. And Marc van Montagu from the university of Gent analysed: ,,Problem is that the public doesn’t trust the fact that the biotechnology is in hands of the industry.” Lack of ‘trust’ in biotechnologists is also an important explanation for many social scientists that study the controversies about genetic modified food in Europe. In Science from 16 July, researchers from the London School of Economics analysed the results of a survey under more than 10.000 European citizens. The lack of trust in industry, scientists and even national public bodies is indeed shocking. Not more than 1 % from the respondents believes what the industry is saying, only 12 % believes national public bodies, 21,6 % believes scientific committees and 345 % the World Health Organisation. In the US believes 80%-90%what national bodies as the FDA are saying. But not only a lack of trust is the reason for the resistance to food biotechnology in Europe. ,,Various factors are implicated and interrelated”, the English social scientists write. And they name factors like: ,,deep cultural sensitivities, not only toward food and novel food technologies but also toward agriculture and environment.” You often hear: the reason behind the public controversy about plant-biotechnology is a fear that the power in plant breeding and food-technology comes in too little hands. The power in plant-breeding (and thus the decision-making about the quality of our daily food) is coming more and more into the hands from less than ten industrial companies: the Monsanto conglomerate, the DuPontPioneer conglomerate, the ELMPulsar conglomerate, the Novartis conglomerate, the Rhone PoulencLimagrain conglomerate and the ZenecdCosun conglomerate. Twenty years ago there were hundreds independent seed-companies more. The plantscientists in universities and research-institutes did research for their governments and for the scientific scene. Now, they do more and more research for the big conglomerates. What are the social consequences from this development?

Marianne Heselrnans, Science Journalist, Wageningen, the Netherlands

22 1

Challenges of the Environment

The Dutch political scientists Robin Pistorius and Jeroen van Wijk [ 1] recently gave in their thesis 'The exploitation of Plant Genetic Information', more or less the same explanation for the resistance small farmers against patenting seeds. According to them, the real point from NGO's and in the third world is not patenting life, but the increasing industrialisation and globalisation of agriculture. This globalisation, they show with a lot of figures about import and export, further marginalizes small farmers. National governments in the third world stimulate more and more export-crops, like flowers and vegetables, and they took their hands off from breeding crops for national food-security. Basic food like rice and maize come more and more from abroad, the Dutch scientists show, and also plant-material comes more and more from abroad. Ismael Seregeldin says in Science of 9 July the following: ,,The growing gap between the developed and developing worlds in the rapidly evolving knowledge frontier is exacerbated by privatisation of scientific research. An emerging 'scientific apartheid' would further marginalize poor people". What has that to do with working on genes in a laboratory? B iotechnology is an important stimulator of that global, industrialised agricultural order. The London researchers in Science end their article with the question: ,,How should science, industry and governments respond (to the resistance from the public red.)?" Well, several critics give the same answer. Science, industry and governments can respond by stimulating local, non-industrialised agriculture. The possibility to buy locally adapted wheat, tomato's or beans from a farmer, 5 kilometres from home, give the Western consumer more trust in food, and it gives he small farmer in developing countries - who cannot pay for chemicals and genetic modified crops - possibilities for an income. It't is an old story. Already fifteen years ago NGO's, agronomists and politicians plead for more attention to non-industrialised agriculture. And indeed. Now there fire breeders and biotechnologists, working for the small farmers or - in the Western world - working for organic agriculture; there are stimulating-programs initiated by governments and companies to adapt biotechnology for poor farmers, and even there are already special biotechniques developed, like DNA-merkertechnology for cassava and locally adapted varieties for organic agriculture. But normally it's not more then five percent of a research-institute or a company that's working for non-industrialised agriculture. The agreements are highly bilateral and often they are components of philanthropic programs. That's not enough to save local agriculture. ,,Partnerships with legally binding agreements on sharing results have to be developed" suggests Seregaldin in science. Or in other words: ,,More collaboration between the private sector and the 'public-goods' research' in developing countries, while respecting IPR." More attention to the non-industrialised agriculture may be a way to get back the trust of the public.

Reference 1. R. Pistorius and J. van WijkScience, the Plant Revolution, 'The exploitation of plant genetic information', University of Amsterdam, June 1999

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Phytosfere’99 - Highlights in European Plant Biotechnology 0Elsevier Science B.V. All rights reserved

Gert E. ds Vries and Karin Metzlaff (Editors).

The Cluster: “Uncovering Metabolic Pathways” The cluster concept within the EU framework 4 programme was devised to enhance interdisciplinary cross-fertilisation between projects with broadly similar goals. The cluster that has is loosely termed “Uncovering Metabolic Pathways” (UMP) is the largest cluster with 10 networks and has 80 laboratories working on 88 research activities. The cluster contains projects with a wide variety of scientific aims and disciplines ranging from plant-microbe interactions to potato tuber dormancy. At first glance one might be excused from thinking that the UMP cluster is the ragbag of projects that could not sensibly slotted into one of the other more coherent clusters. Although this may indeed have been its genesis, I hope that the papers included here will help to convince the reader of the links and common interests between the projects in the cluster. The UMP cluster is not only unique in its diversity, but also in the general multidisciplinary approach taken in all the projects. Universally, the networks contain not only plant molecular biologists but also plant physiologists and biochemists amongst a wide range of other disciplines. This broad bandwidth puts the UMP cluster projects in the new mould of research that is set to yield high returns for the scientific community and the wider society in general. In addition to the purely scientific objectives within UMP, the projects also all have direct practical applications in a wide range of European crop plants. Several projects have been directed towards improvement of content and enhancement of nutritional characteristics, for example, by remodelling pectins or by increasing the amount of vitamins and of essential amino acids in plants. A number of projects have contributed to a better understanding of crop productivity by analysing the relationships between the productive plant organs (source) and the consumer or storage organs (sinks). Projects aiming at increasing crop productivity and efficiency are not only focusing at the yield characteristics but also at the environmental frugality of the crop in the field. A more detailed description of the achievements of projects in the UMP cluster can be found on www.epbn.org. It may be hoped that the success of this sort of plant research finds a resonance with the general public and is appropriately rewarded by continued funding in the future.

Christian Bachem, Wageningen University Research Centre, Laboratory of Plant Breeding, Wageningen, the Netherlands

223

Uncovering Metabolic Pathways

The projects" "Mechanisms for the regulation of carotenoid production and accumulation in plants", coordinated by P. Bramley (BIO4-CT97-2077) "Ammonium transport in plants: strategic role in nitrogen efficiency" co-ordinated by W. Frommer (BIO4-CT97-2310) "Engineering high quality crops by optimising lysine, methionine and cysteine content" coordinated by M. Jacobs (BIO4-CT97-2182) <[email protected]> "Phosphate and crop productivity" co-ordinated by J. Kossman (BIO4-CT96-0770) "Sugar transport in relation to source/sink interactions in plants" co-ordinated by R. Lemoine (B IO4-CT96-0583) "Regulation and metabolic networks related to nitrogen fixation in the legume nodule" coordinated by A. Ptihler (BIO4-CT97-2319) "Control of source-sink relations by carbohydrate regulation of gene expression" co-ordinated by S. Smith (BIO4-CT96-0311) "Understanding nitrogen signalling and metabolism to tailor plants with improved sink source relations and nitrogen utilisation characteristics" co-ordinated by M. Stitt (BIO4-CT972231) <mstitt@ botanikl .bot.uni-heidelberg.de> "Sink to source transition: an investigation of processes regulating dormancy and sprouting in potato tubers" co-ordinated by R. Visser & Christian Bachem (BIO4-CT96-0529) "Remodelling pectin structure in plants" co-ordinated by E Ulvskov (BIO4-CT97-2224)

224

Phytosfere.99 - Highlights in European Plant Biotechnology Gert E. de Vnes and Karin Metzlaff (Editors). 0 Elsevier Science B.V. All rights reserved.

Improving Fertiliser Use Efficiency In Agro-Ecosystems And Nutrient Efficiency In Plants An essential step towards modern agriculture The continuing growth of world population will require a doubling of cereal production within the next 50 years. This will result in a 2- to 3-fold increase in the use of synthetic nitrogen fertilisers. This global trend will also affect Europe due to the resulting impact on the use and development of agricultural technologies. More nitrogen-efficient crop varieties would help to minimise nitrogen fertiliser application and thereby reduce the broad spectrum of harmful environmental effects resulting from nitrogen fertiliser use. In this context an EU-funded (BIOTEC 4) research project, termed EURATINE, is studying the transport of ammonium, which represents one of the major nitrogen sources to crop plants. Besides in root uptake, ammonium transport has an additional physiological function in leaves through the continuous retrieval of ammonia which is otherwise lost from plant cells by volatilisation. By using yeast as a model genetic system, several plant ammonium transporter genes have been isolated and characterised in terms of their regulation and the biochemical properties of their products. Some of these transporter genes have been used to produce transgenic plants over-expressing or repressing the transport proteins. The analysis of these plants will allow an evaluation of possible environmental and socio-economic benefits of transgenic plants with altered ammonium transport properties in the context of potential reductions in fertiliser application.

Nitrogen fertilisation in agricultural food production Global food production is based on an anthropogenic disruption of the nitrogen cycle by generating excess fixed nitrogen in primarily inorganic (ammonium and nitrate) and organic (mainly urea) forms. This excess fixed nitrogen is introduced into agricultural plant production systems as nitrogen fertilisers or as combined fertilisers mostly together with phosphorus and potassium. However, on average only 40-50% of the applied fertiliser nitrogen is taken up by plants [ 11 and only 50% of this fraction reaches the harvested fruits or organs which serve for human food production [2], (figure 1). The large remainder of the non-absorbed nitrogen escapes directly from agricultural plant production or is converted to various forms which are similar or even more prone to leaching (nitrate) or volatilisation (ammonia, nitrous oxides). Consequently, escaping nitrogen augments the greenhouse effect, diminishes stratospheric

I

Nicolaus von Wirkn, Universitat Tubingen, ZMBP-Pflanzenphysiologie, Germany I

1

225

Uncovering Metabolic Pathways The Fate of Fertilized Nitrogen Nutrients in Agricultural Production 4

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ozone, promotes smog, contaminates drinking water, acidifies rain, causes eutrophication of bays and estuaries and stresses ecosystems [3]. For these reasons ecosystem stabilisation, including nutrient cycling within agricultural production systems, has been set as a goal in modem and sustainable agriculture (EU FP4 & 5). Despite the above considerations synthetic fertiliser application represents a hallmark of the green revolution, together with irrigation, pest management and plant breeding strategies, and has acted as a key factor in improving food security on a global scale [1]. The doubling of agricultural food production in the past 35 years was associated with a 7-fold increase in nitrogen fertilisation [1]. As the global population is growing by 40-80 million people per year, a population size of 9-12 billion has to be expected in roughly 50 years from now [4]. This growth will require another doubling of global food production. Irrespective of whether this increasing food demand is met by an increase in arable land or further intensification of the current production surface, doubling global food production will require another 2- to 3fold increase in global use of synthetic nitrogen [5]. Except South and East Asia, that have the potential to meet their own food demand, there will be an increasing mismatch between the expansion of regional food demand and the potential for food supply, promoting a further expansion of the world cereal trade. Therefore, due to Europe's favorable climate and high level of agricultural technology, its contribution to global food supply is expected to be stable or even to increase. To avoid the expected serious ecological consequences of more intensified agriculture and increased nitrogen fertiliser use in Europe, the entire food production and consumption system (grain, livestock, food distribution and diet) has to be critically examined for opportunities to improve nitrogen use efficiency [3]. This review focuses on the first step in the food production system, which is the use of nitrogen fertiliser by plants. 226

Improving fertiliser use efficiency

Available forms of nitrogen In the biosphere nitrogen is available for plants in different forms, which include molecular dinitrogen, volatile ammonia or nitrogen oxides (NH 3, NO), mineral nitrogen (NO 3- and NH4§ and organic nitrogen (amino acids, peptides etc.). Considering their high nitrogen demand, it is not surprising that plants can use almost all forms of nitrogen, with the exception of molecular dinitrogen which is restricted to plant species living in symbiosis with nitrogen-fixing bacteria. However, the utilisation of these nitrogen sources is strongly influenced by environmental factors, particularly the soil type and conditions can influence the forms of nitrogen present. In well-aerated agricultural soils, mineral nitrogen and especially nitrate is the most abundant form of available nitrogen, while ammonium dominates in soils in which nitrification is inhibited, for example during waterlogging or in cold climates [6]. Under agricultural conditions, soil NO 3 concentrations can range between 0.5 and 10 raM, while ammonium concentrations are usually 10 to 1000 times lower reaching the millimolar range only in exceptional cases such as after fertilisation [6]. However, this difference in soil concentrations does not reflect the uptake ratio of both nitrogen forms, since most plants preferentially take up ammonium when both forms are available and nitrogen supply is limited [7]. Therefore, the contribution of ammonium to nitrogen nutrition of crops can easily be underestimated, as long as low ammonium levels in soils are considered to result only from rapid nitrification. Nitrogen transfer from symbiotic nitrogen-fixing microorganisms contributes to nitrogen nutrition in several plant families and can easily be employed to enhance agricultural plant production. In this case too, ammonium is the main form of nitrogen imported by the plant. Thus, ammonium and nitrate can be regarded as the most important N forms that are available to crop plants.

The uptake of soil nitrogen by plants Since in soils the form and concentration of nitrogen can vary largely over time and site, plants have evolved a broad set of transport systems that adapt to utilise the changing amounts of available nitrogen in the soil solution. These transport systems consist of hydrophobic proteins that can span the plasma membrane several times forming a pore through which substrate import can be controlled. Such transporters have been identified at the molecular level for ammonium, nitrate, amino acids and small peptides [7-9]. To cope with the varying forms of nitrogen supply, there are whole families of transporters with the relative contribution to uptake of each individual member changing, depending on the environmental and physiological conditions. Each of these transporters seems to differ in regulation and substrate affinity [7]. For ammonium transporters in particular plant nitrogen and carbon status as well as soil nitrogen supply determine which transporter is turned on. This allows the plant to control ammonium intake to match the internal nitrogen demand and assimilation capacity, thereby avoiding deleterious effects resulting from the accumulation of toxic ammonium concentrations. In addition, some of these ammonium transporters are localized in the outmost layer of the root cells, in root hairs which are located just behind the root tip [ 10]. This localised expression of ammonium transporters indicates that in these regions at the root tip soil nitrogen can be mined most efficiently. Therefore, it is most likely that ammonium transporters play a strategic role in nitrogen acquisition by plants and contribute significantly to optimal

227

Uncovering Metabolic Pathways growth rates and crop yields. This is supported by the conclusion that further metabolic steps involved in the use of mineral nitrogen may not limit nitrogen assimilation by the plant [ 11].

Nitrogen efficiency In the past decades, classical breeding strategies have been successful in developing new crop varieties with higher yield potential and higher stress resistance, both of which contribute to higher and more stable crop yields [2]. With average harvests of 70 to 80% of the yield potential, modern agriculture may be close to the physiological limit of the presently available crop varieties. However, the development and growth of these improved cultivars has been associated with a higher demand for nitrogen fertiliser input. Unfortunately, both breeders and plant physiologists have not been able to increase the metabolic and assimilatory efficiency of major cereal crops indicating that it will be difficult to increase yield further without overcoming major physiological and morphological constraints [1]. This is a challenge, which currently can only be tackled by a combined use of classical and modem biotechnological techniques that identify limiting processes in yield formation at the molecular level. As many processes that determine an economic use or high acquisition of nitrogen are based on properties and regulation of individual transporters or enzymes, a molecular and functional characterisation of the proteins and their genes involved in nitrogen uptake and metabolism is required. With these genes in hand the limiting steps in the movement and conversion of nitrogen from the soil to the grain can be uncovered. Moreover, targeted biotechnological approaches might be used to alter single steps in this pathway making it possible to overcome physiological bottlenecks or to restrict leaky processes. Then perhaps, plants could be developed which use nitrogen more economically and maintain yield at lower fertiliser application rates. Furthermore, the genes identified may be used as molecular markers to speed up breeding programs. Plant species or cultivars that are able to produce high biomass or to form high crop yield under limiting supply of nitrogen are called nitrogen-efficient [6]. Thus, nitrogen efficiency is an agronomic term expressing a particular trait of one plant species or cultivar relative to another. Nitrogen efficiency has complex causes and depends on different processes. Some of these enhance the uptake of external nitrogen and some the internal utilisation of nitrogen; for example, a rapid and complete remobilization of leaf nitrogen in the grain or roots before leaf fall sets in. It is against this background that a European project, EURATINE (European initiative studying the molecular basis of ammonium uptake and sensing), has set out to examine whether uptake and transport of ammonium might act as a limiting step or bottleneck in overall nitrogen efficiency of plants. Significant advances in our understanding of how plants acquire nutrients and in particular ammonium have been made by the EURATINE team, a group of European scientists with different methodological specialisations (http:// www.uni-tuebingen.de/abot/EURATINE/index.html). To address the primary objective of identifying ammonium transporter genes in bacteria, fungi and plants, the yeast Saccharomyces cerevisiae was chosen as a model organism. A yeast mutant was constructed, in which the endogenous NH4+ transporters were deleted from the genome, thereby producing a mutant strain that was unable to grow on NH4+ as sole 228

Improving fertiliser use efficiency nitrogen source. Genetic transformation of hundreds of thousands of these yeast mutants with individual plant genes and subsequent growth on NH4§ as sole nitrogen source created conditions under which yeast strains only grow when expressing an introduced plant gene coding for an NH4§ transporter. This approach allowed the EURATINE group to isolate a number of NH4+ transporters from different plant species and from yeast itself. Subsequently, efforts were devoted to also investigate regulation and physiological function of each transporter identified. This was favoured by the diversity and complementarity of the various groups within EURATINE. Yeast synthesises three ammonium transporters of the MEP gene family, two of which show high substrate affinity while the third acts as a low-affinity transporter [ 12]. Moreover, one of the transporters, namely Mep2, additionally functions as a sensor for ammonium, releasing a signal that leads to morphological changes in growth when external ammonium is present [13]. Furthermore, expression of these ammonium transporters is highly regulated at both the mRNA and the protein level in response to certain intracellular nitrogen forms and in relation to other transporters for nitrogenous compounds [ 14]. In the model plant Arabidopsis, five ammonium transporter (AMT1) genes have been isolated, and three of them have been functionally expressed in yeast [7]. While two of them transport ammonium in micromolar concentrations, one of them (AtAMT1;1) exhibits transport capacities down to nanomolar concentrations. At the same time, expression of this transporter is strongest under nitrogen deficiency, when ammonium uptake efficiency is most important. This indicates that transporter regulation is linked with its biochemical properties, and associated with the physiological response of the transport process. To further characterise the physiological function of each individual gene, knock-out mutants with deletions in individual transporter genes have been isolated. These will shed light on the physiological function of the individual proteins in vivo. Ammonium transporters have also been isolated from rice, tomato and Lotus japonicus [15, 16], the latter being capable of utilising molecular dinitrogen through symbiosis with nitrogenfixing rhizobia (figure 2). In this case ammonium is probably the key component for nitrogen transfer from bacteria to host plants. This requires a complicated regulation of bacterial ammonium uptake and metabolism, in particular when the bacterial cells switch from a free-living state to a symbiotic (bacteroid) state [ 16]. Moreover, experimental evidence has been obtained

,

Figure 2. The role of nodules in natural nitrogen fixation and nitrogen delivery to plants. VB: vascular bundles; PT: peripheral tissues; CT: central tissues; IC: invaded cells; UC: uninvaded cell; SG: starch grains; PBM: peribacteroid membrane; SB: symbiosome; HC: host cytoplasm.

229

Uncovering Metabolic Pathways that ammonium transport not only regulates nodu4lation efficiency, but also triggers a signal that NH 4 modifies root hair formation of the host plant. Ectopic expression of the silenced ammonium transporter gene amtB under conditions of symbiosis inhibited nodule formation, providing evidence for the proposal that nitrogen is transported from bacteria to the plants in the form of ammonium. In addition, new methods have been developed, for example a new ammonium-selective electrode suitable for measuring ammonium uptake by bacteria and plant roots has been manufactured. This electrode has a good selectivity for ammonium over potassium, the main interfering ion for measurements. The electrode has also been successfully miniaturised to enable the first diNH 4 rect measurements of intracellular ammonium concentrations in single plant cells [ 17]. The elecFigure 3. Schematic model of the topology of the trode measurements allow the ammonium pools MEP/AMT family of ammonium transport proinside plant cells to quantified and the likely teins. mechanisms of transport determined. This methodology serves the whole scientific community engaged in nitrogen transport and metabolism. Finally and very surprisingly, the human Rhesus blood group factors have been identified as belonging to the same superfamily of ammonium transporters as the Mep genes from yeast or the AMTs from bacteria and plants [18]. Whether these Rhesus factors also act as ammonium transporters in blood cells is an exciting topic for future studies.

f

The ammonium transport proteins are conserved in all major life forms from bacteria to animals [12, 19]. This conservation offers the possibility of using the sophisticated genetic tools developed in bacteria to analyze fundamental aspects of the structure and mode of action of the AMT proteins (figure 3). Analysis of the membrane topology of the bacterial AmtB proteins has therefore been able to offer insights not only into the possible mechanisms of the plant ammonium transporters but also into the topology of the human Rhesus proteins. The high efficiency of the EURATINE group was possible through close interactions between all different groups working in very different areas but collaborating on the same topic, namely ammonium transport. Such efficient collaboration was made possible by the stimulating platform generated by the Framework 4 funding system. With the finding of Rhesus factors as candidates for ammonium transporters in humans, the inclusion of a group with experience in transport processes in mammalian systems might have been useful but was not possible in this funding program.

The need for sustainable and efficient practices in agriculture Presently global agriculture is at a new threshold. It has become a major source of nitrogen loading to terrestrial, freshwater and marine ecosystems. If this loading increases, as required 230

Improving fertiliser use efficiency to cover the increasing food demand, agriculture will adversely transform most of the remaining natural ecosystems of the world. Because the global environmental impact of agriculture on natural ecosystems may be as serious a problem as global climate change, the impacts of agriculture and the development of means to reduce these impacts merit considerably more study [ 1]. Continuation of the same approach that has been used during the last 35 years to global food supply will have significant environmental costs. These costs could be lowered by processes that increase the efficiency of fertiliser use, particularly nitrogen, in crops. Moreover, the use of more nitrogen-efficient plants has to be accompanied by the concept of ,,precision farming", in which fertiliser application is adapted to the local demand of single plant patches. Thus, engineering solutions to recognise and distinguish single plant demands and control fertiliser application rates will also be needed. Targeted biotechnological approaches linked to a more information-based agriculture, which includes the development of nitrogen-efficient plants, could provide many solutions to decrease the environmental impact of agriculture, while maintaining or even increasing food production. The regulation of ammonium transporters also needs to be further investigated. It corresponds to the processes plants have evolved to modulate, and in particular to improve, the efficiency of ammonium transport. Thus, understanding and manipulating the endogenous regulatory mechanisms, which may not be specific for ammonium transporters [20], is also a promising strategy to obtain cultivars with higher nitrogen use efficiency.

Outlook The EURATINE consortium has been able to identify genes and proteins that are of central importance for ammonium transport in yeast, bacteria and plants. Moreover, after a thorough characterisation of the regulation of these genes and the biochemical properties of their encoded proteins, some transporter genes have been selected for overexpression or antisense repression in transgenic plants to investigate their role in ammonium uptake from the soil or from nitrogen-fixing bacteria. The investigation and evaluation of these plants will allow an initial estimation of the potential use of ammonium transporters in improving nitrogen efficiency. The results obtained to date provide the basis for manipulating nitrogen fluxes in many divergent systems such as free-living bacteria, fungi including the highly important plant mycorrhizal symbionts, higher plants (and potentially even developing cures for human diseases). Due to the time required to develop transgenic plants, further work is required to generate directly applicable new technologies on this basis.

Conclusion The EURATINE team has specifically addressed one of the key concerns of the European Union, namely the requirement to improve crop plants by means that are consistent with the environmental and socio-economic requirements for agriculture in the 21 st century. To this end they have investigated all aspects of the biology of ammonium transport and have laid the basis for constructing the first generation of transgenic plants with altered ammonium transport properties. This will allow a detailed scientific analysis of the potential benefit from such plants in future. At the same time, the EURATINE contribution will provide an opportunity 231

Uncovering Metabolic Pathways for European citizens, who have expressed many concerns about the use of transgenic plants in agriculture, to consider the merits of a new generation of plants that have been designed to reduce some of the harmful impact of conventional agriculture. This research on ammonium transport has shown that there is considerable potential for manipulation of transport processes, although we are only just beginning to understand the complex regulation of nitrogen transport and metabolism. Although we may not expect to obtain market-ready products within the next few years, it is must be of the highest priority to explore and test the potential benefit of plants with altered transport properties. The environmental and economic constraints that modern agriculture will face during the next decades make this research essential.

Authors of this contribution 1Nicolaus von Wir6n, 2Bruno Andr6, 3Hinrich Harling, 4Alain Gojon, 5Eduardo Patriarca, 6Mike Merrick, 7Anthony Miller, 8Bernd Reiss and 1Wolf B. Frommer 1ZMBP-Pflanzenphysiologie, Universit~it Ttibingen, Morgenstelle 1, D-72076 Ttibingen; 2Institut de Biologie et de Medecine Moleculaires, Rue Pr. Jeener et Brachet 12, B-6041 Gosselies; 3 K W S Saatzucht AG, Box 1463, D-37555 Einbeck, 4Biochimie et Physiologie Mol6culaire des Plantes, INRA, Place Viala, F-34060 Montpellier; 5Institute of Genetics and Biophysics, P.O. Box 3061, Via G. Marconi 10, 1-80125 Naples; 6Dept. Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK ; 7Biochemistry and Physiology, IACRRothamsted, GB-Harpenden AL5 2JQ; SMPI Ztichtungsforschung, Carl-von-Linn6-Weg 10, D-50829 K61n.

Acknowledgements This work was and is supported by the European commission under Framework 4. We would like to acknowledge especially the support by KWS Saatzucht AG. We would like to highlight the excellent contributions of Sonia Gazzarrini, Laurence Lejay, Anne-Marie Marini, Rosarita Tat6, Gavin Thomas & Darren Wells, outstanding students who carried out the experiments with extraordinary success.

References 1. 2. 3. 4. 5. 6. 7.

8. 9.

Tilman D. (1999) Global environmental impacts of agricultural expansion: The need for sustainable and efficient practices. Proc. Natl. Acad. Sci. USA 96, 5995-6000. Cassman K.G. (1999) Ecological intensification of cereal production systems: Yield potential, soil quality, and precision agriculture. Proc. Natl. Acad. Sci. USA 96, 5952-5959. Socolow R.H. (1999) Nitrogen management and the future of food: Lessons from the management of energy and carbon. Proc. Natl. Acad. Sci. USA 96, 6001-6008. Fedorof N.V. and Cohen J.E. (1999) Plants and population: Is there time? Proc. Natl. Acad. Sci. USA 96, 5903-5907. Dyson T. (1999) World food trends and prospects to 2025. Proc. Natl. Acad. Sci. USA 96, 5929-5936. Marschner H. (1995) Mineral nutrition in higher plants. Academic Press, London. Gazzarini S., Lejay L., Gojon A., Ninnemann O., Frommer W.B. & v o n Wir6n N. (1999) Three functional transporters for constitutive, diurnally regulated, and starvation-induced uptake of ammonium into Arabidopsis roots. Plant Cell 11,937-947. Crawford N.M. & Glass A.D.M. (1999) Molecular and physiological aspects of nitrate uptake in plants. Trends Plant Sci. 10, 389-395. Fischer W.-N., Andr6 B., Rentsch D., Krolkiewicz S., Tegeder M., Breitkreuz K. & Frommer W.B. (1998)

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Improving fertiliser use efficiency Amino acid transport in plants. Trends Plant Sci. 3: 188-195. 10. Lauter ER., Ninnemann O., Bucher M., Riesmeier J.W. & Frommer W.B. (1996) Preferential expression of an ammonium transporter and of two putative nitrate transporters in root hairs of tomato. Proc. Natl. Acad. Sci. USA 93, 8139-8144. 11. Gojon A., Dapopigny L., Lejay L., Tillard E & Rufty TW (1998) Effects of genetic modification of nitrate reductase expression on 15NO3- uptake and reduction in Nicotiana plants. Plant Cell Environ. 21, 43-53. 12. Marini A.M., Soussi-Boudekou S., Vissers S. & Andr6 B. (1997a) A family of ammonium transporters in Saccharomyces cerevisiae. Mol. Cell Biol. 17, 4282-4293. 13. Lorenz M.C. & Heitman J. (1998) The MEP2 ammonium permease regulates pseudohyphal differentiation in Saccharomyces cerevisiae. EMBO J. 17, 1236-1247. 14. Marini A.M., Springael J.Y., Frommer, W.B. & Andr6 B. (2000) Cross-talk between ammonium transporters in yeast and interference by the soybean SAT1 protein. Mol. Microbiol. 35: 378-385. 15. von Wirdn N., Bergfeld A., Ninnemann O. & Frommer W.B. (1997a) An ammonium transporter from Oryza sativa. Plant Mol. Biol. 35, 681. 16. Tat6 R., Riccio A., Merrick M. & Patriarca E.J. (1998) The Rhizobium etli gene coding for an NH4§ transporter is down-regulated early during bacteroid differentiation. Mol. Plant Microbe Interact. 11, 188198. 17. Wells D. & Miller A.J. (1999) Intracellular measurement of ammonium in Chara corallina using ionselective microelectrodes. Plant Soil, in press. 18. Marini A.M., Urrestarazu A., Beauwens R. & Andr6 B. (1997b) The Rh (Rhesus) blood group polypeptides are related to NH4§ transporters. Trends Biochem. Sci. 22, 460-461. 19. Thomas G., Coutts, G. & Merrick, M. (2000) The glnKamtB operon: a conserved gene pair in prokaryotes. Trends Genet. 16:11-14. 20. Lejay L., Tillard E, Lepetit M., Olive ED., Filleur S., Daniel-Vedele F. & Gojon A. (1991) Molecular and functional regulation of two NO 3- uptake systems by N- and C-status of Arabidopsis plants. Plant J. 18, 509519.

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Phytosfere.99 - Highlights in European Plant Biotechnology Gett E. de Viies and Karin Metzlaff (Editors). 0Elsevier Science B.V. All rights reserved. ~~~

A European Approach Towards Phosphate Efficient Plants

Introduction Growth and development of plants are to a large extent determined by the availability of mineral nutrients. Apart from nitrogen, phosphate is one of the key nutrients limiting plant growth in terrestrial ecosystems. In plants phosphate is not only a major component of structural elements and metabolic intermediates, but also most metabolic pathways depend on its availability. Under limiting phosphate nutrition conditions, but also under sufficient conditions, the phosphate present in the plant is not allocated in an optimal way. The level of inorganic phosphate (P,) in the cytosol controls most of the metabolic events within a plant cell. Therefore, a constant level must be maintained in this compartment by highly regulated processes [ I ] . These control the acquisition of phosphorus from the soil and its distribution between different organs and cellular compartments. A high supply of phosphate does not necessarily lead to enhanced growth of crop plants. because excess phosphate is mainly stored in the vacuole [2]. The vacuole is the main storage pool of P, in the living plant cell. although in leaf cells the chloroplast probably constitutes an additional important P, compartment [3]. Exchange of P, between vacuole and cytoplasm is relatively slow [3,4] and probably insufficient to meet short-term demands of cytoplasmic PI. This was clearly demonstrated by rapid utilisation of cytoplasmic P, upon feeding cells with P, sequestering agents like mannose [5],despite the presence of abundant vacuolar PI. Therefore, even under sufficient P, supply cells potentially suffer so-called PI-limitation of photosynthesis because the vacuolar P, pool is not metabolically available during short-term limitations [6,7]. Furthermore the way in which P, is metabolised in storage organs is a key determinant for the accumulation of storage carbohydrates and the quality of the harvested organ. Higher plants redistribute PI to their storage organs during later stages of development. In the case of potato this causes a severe internal competition for P, within the plant because the tuber formation begins at a time when leaves are still growing and root expansion needs to continue. This is in contrast to the situation in cereals where grain formation determines the life cycle of the plant. Phosphate in potato tubers is mainly stored as P,, however, a major proportion (up to 40%) is covalently bound to starch. Another considerable amount (12-15% of total phosphate) is stored as phytic acid [8].

Babette Regierer, Max-Planck-Institut fur Molekulare Pflanzenphysiologie, Golm, Germany

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Uncovering Metabolic Pathways To obtain optimal crop yield farmers have to apply fertiliser. Today the use of mineral fertiliser, particularly of phosphorus, must be reduced because of economical as well as ecological reasons. The resources of mineral phosphorus will be limited to the next 50-100 years at the prize and quality at present. 80% of the phosphorus consumed in middle Europe is used as agricultural fertiliser, which is one of the main sources of polluting phosphate leading to eutrophication of surface waters. But in most other parts of the world, mainly the developing countries, the amount of available phosphate in the soil is very low. In these parts of the world farmers can grow crops only with the help of mineral fertiliser. But phosphate fertilisers are not only causing surface water pollution and eutrophication. They are also contributing to soil contamination because they are the main source of cadmium pollution leading to different environmental problems. To avoid the excessive use of mineral fertilisers in the developed countries will contribute to the reduction of environmental contamination problems. The project ,,Phosphate and Crop Productivity (PCP) - A European Approach Towards Phosphate Efficient Plants" aims at the development of crops with enhanced efficiency for the nutrient phosphorus. These new crops will be optimised to produce the same yield with less input of fertiliser. Many approaches to influence sink-source interactions focus on carbon and nitrogen metabolism in leaves as well as storage sink organs. In contrast to the crucial importance of Pi for plant growth and development, very little is known about the mechanisms that influence Pi-efficiency on the whole plant, as well as on the cellular and molecular level. Approaches within the PCP project focus on both the acquisition as well as the utilisation efficiency of phosphate in higher plants.

Acquisition efficiency Phosphate is present in the soil in different fractions. Under normal conditions plants have only access to the soluble phosphate fraction, and in most cases this fraction contains around 10 gM Pi, representing phosphate-limiting conditions [9]. The amount of soluble Pi that is available for plants is mainly dependent on the soil conditions, e.g. pore size, water capacity and substrate composition. Acidic and also alkaline soils have the ability to bind phosphate tightly to largely insoluble inorganic minerals such as Ca, A1 and Fe, so that it becomes unavailable for the plants. Another fraction is the phosphate bound to organic matter. This fraction makes up to 50% of the total phosphate content in some soils. It consists of phosphate bound to carbohydrates, lipids, proteins, nucleic acids and polyphosphates and to a large extent to phytate. During evolution, plants developed different strategies to enhance the acquisition of phosphate from the soil. Individual plant species only follow a subset of these strategies, and, moreover, some of the mechanisms are only induced under Pi starvation conditions. For acidification of the rhizosphere plants can secrete protons via H+-ATPases that are located in the plasmalemma of roots. A decrease in pH can mobilise Ca-, A1- and Fe-phosphate sources. Another strategy to lower the soil pH is the secretion of organic acids such as citrate or malate. Organic phosphate sources can be mobilised by extrusion of unspecific acid phosphatases that cleave the phosphate residues of nucleic acids or phytate. The molecular and biochemical basis of these strategies was investigated and will be used for a biotechnological approach to increase phosphate efficiency in crops. 236

Phosphate efficient plants

Root specific promoters One of the major prerequisites for the specific manipulation of root metabolism is the ability to express genes exclusively in roots. A promoter from the agropine synthase gene of the Ri plasmid (RiAGS) from Agrobacterium rhizogenes was published recently to be functional as a root specific promoter in tobacco plants [ 10]. Fusion products of this RiAGS promoter with a GUS reporter gene showed mainly root specific expression in tobacco, but also callus cells had a high GUS activity. Transformation of the Promoter-GUS fusion product into potato plants did not show any GUS activity in root tissue from plants grown in soil. Therefore, other attempts will be made to identify different root specific promoters, ideally mediating specific expression in different root cell types.

Increasing phosphate uptake capacity Acquisition and also translocation of the mineral nutrient phosphate are essential processes at the root-soil interface. In many plants, phosphate uptake occurs via a phosphate/proton cotransport. This process must be an active transport, because the uptake of Pi into the root is directed against a concentration gradient. Two cDNAs named STPT1 and StPT2 have been isolated from a potato cDNA library encoding different phosphate transporters [11]. An Arabidopsis thaliana EST encoding a phosphate transporter (134M11T7; EMBL accession number T46507) homologous to the PHO84 phosphate transporter from yeast [12] was used for screening a potato root cDNA library. The root cDNA library was made from plants deprived of all nutrients to influence the expression of proteins that can enhance ion uptake. Molecular analysis of these clones showed that the StPT1 transporter is expressed in the whole plant, the expression for the other clone StPT2 is restricted to root tissue and only induced under phosphate starvation conditions. Both transporters have been transformed into a yeast mutant strain that is deficient in phosphate uptake because of a mutation in the PHO84 gene [12]. The StPT1 and StPT2 expressing yeast strains have been tested for 32p orthophosphate uptake and growth rates and resulted in a functional complementation of the deficient yeast strain. Biochemical analysis of both transporters in yeast revealed a K m for StPT1 of 280gM and for StPT2 of 130gM. But the K m in vivo can be different from the one measured in yeast because the expression of the potato transporters in yeast represents an artificial system. The K m in plants could be submitted to posttranslational modifications or regulatory processes. Further biochemical studies will focus on putative phosphorylation sites of the protein to reveal possible regulation in vivo. In order to elucidate the specific function of both phosphate transporters in vivo potato plants have been transformed with antisense constructs for StPT1 and StPT2 to reduce the endogenous phosphate transport activity. In the same way attempts have been made to increase phosphate uptake capacity by overexpressing the phosphate starvation induced transporter StPT2 in potato. All the known PHO84 homologues mediate a H+/Picotransport. [ 11-17]. But recently another class of phosphate transporters has been identified. These transport proteins are homologous to the PHO89 gene from yeast [18] and mediate a sodium-dependent Pi transport activity. Recently a PHO89 homologue was found in Arabidopsis thaliana named AtPperm or Pht2;1. Recent data [19] indicate that AtPperm represents a P transporter that takes part in Pi transport into the shoot. It has a Km of 280 ~/I, thus having the

1

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Uncovering Metabolic Pathways same Pi affinity as the PHO84 homologues. Furthermore, AtPperm shows a pH dependency for Pi uptake. This leads to the conclusion that, although it has a high homology to the Na+/Pi cotransporter PHO89 in yeast, AtPperm represents a low-affinity H + contransporter with a high specificity for Pi- In plants two Pi transporter families have been identified so far: The PHO84 homologous P!H + symporter family (PHS) mediates acquisition at the root/soil interface, the PHO89 homologous transporter family participates in the partitioning of Pi within the shoot.

Solubilising insoluble soil phosphate sources Some plants are able to acidify the rhizosphere under limiting phosphate conditions to mobilise insoluble mineral phosphates. Acidification can be mediated either by proton extrusion or by secretion of organic acids. Both strategies are induced in a specialised root system of white lupin, the proteoid roots. These are clusters of lateral roots densely covered with root hairs. Under phosphate starvation conditions specific metabolic response reactions are induced in mature proteoid roots leading to proton and organic acid extrusion. The proteoid root system can be viewed as a model system to investigate the soil acidification strategy of plants as a phosphate stress response that can be transferred to other crop plants. Secreted organic acids mediate the mobilisation of phosphorus from Ca-, AI-, and Fe-phosphates, mainly by mechanisms of ligand exchange, occupation and dissolution of phosphate sorption sites in the soil matrix [20-22]. Recently the mechanism of the proteoid root metabolism was investigated [23]. High amounts of citric acid accumulate within the proteoid roots during maturation, which are secreted if the roots are fully developed. It could be shown that within the developing proteoid roots PEP carboxylase activity is increased to form malate. Malate is imported into mitochondria via an exchange with citrate. On the other hand aconitase activity in the mitochondria is decreased leading to a net accumulation of citric acid that is finally exported into the rhizosphere. The alterations in organic acid production are accompanied by a higher proton efflux mediated by H+-ATPases. The proton concentration in the rhizosphere controls two different systems: First, it contributes to the acidification of the soil under phosphate limiting conditions. Second, it mediates phosphate uptake. It was shown [ 11-17] that phosphate transporters mediate a phosphate-proton cotransport. Therefore, a higher proton secretion rate will enhance phosphate uptake in roots via high affinity transporters. Transferred to other systems this model will be used to mobilise sparingly available phosphate sources by increasing PEP carboxylase activity, decreasing aconitase activity or changing the TCA cycle turnover maybe in combination with the ectopic expression of organic acid transporters in the root system.

Release of

Pi from

organic compounds

A main proportion of the phosphate present in soils is bound to organic molecules like nucleic acids, phospholipids, inositol phosphates and other phosphoesters. The amount of organically bound phosphate can be as high as 50% of the total phosphate present in soils. The largest fraction is phytate representing 70% of the organic fraction. To get access to these phosphate sources most of the land plants secrete hydrolases into the soil, mainly nucleases and acid phosphatases, via the root system. A secreted ribonuclease RNS 1 was first found in Arabidopsis 238

Phosphate efficient plants [24]. The expression and subsequent secretion of RNS 1 is induced in response to phosphate limitation. But also DNases might be secreted into the rhizosphere to release the P-residues bound to DNA molecules. On the other hand many plant species are able to secrete unspecific acid phosphatases into the rhizosphere, mainly to cleave phosphoester bonds of biomolecules present in the soil [25,26]. The mature proteoid root clusters of white lupin are able to secrete not only organic acids in high amounts, but also acid phosphatase under Pi starvation [22,23]. Ozawa and coworkers [27] characterised the acid phosphatase as an acid purple phosphatase (PAP family) that is very similar to the type 5 acid phosphatases from mammals [28-30]. The name results from the purple colour they display if present in sufficient concentration, which occurs because of a tyrosinate-FeIII charge transfer. This tyrosinate residue is involved in ligand binding and specific for PAPs [31]. Recently a secreted acid phosphatase AtACP5 was found also in Arabidopsis that has high sequence homology to the type 5 purple acid phosphatases from mammals [32]. AtACP5 transcript levels increased under phosphate stress conditions, but the RNA was also present in senescing leaves and flowers similar to RNS 1 [24]. Other environmental stress situations like K- and N-deficiency did not induce the expression of AtACP5. The Arabidopsis phol mutant [33], that is deficient in xylem loading of Pi, has also a higher transcript level of the acid phosphatase AtACP5. But type 5 purple acid phosphatases display also peroxidation activity because of their di-iron-oxo cluster [34,35]. Also the Arabidopsis homologue AtACP5 is inducible by oxidative stress. Therefore, the acid phosphatase AtACP5 is not only mobilising phosphate under starvation conditions, but might also be involved in antioxidation reactions. Application of ABA, salt stress, and HzO2 also induced activity of AtACP5. That leads to the assumption, that the purple acid phosphatase, formation of peroxides leading also to a mobilisation of nutrients as a stress response [36].

Utilisation efficiency Apart from the acquisition also the utilisation of phosphate within a plant is determining the efficiency of a crop. Utilisation efficiency is constituted of various parameters: The distribution of Pi on the whole plant, as well as on the cellular and subcellular level. The distribution of phosphate within a plant is determined by: a) the individual demands of the plant organs depending on its development and physiological status, b) by the translocation rate of the phosphate that is available within the plant; the phosphate is not only taken up by the roots and then transported to the shoot organs, but it is also remobilised from older tissues and redistributed to growing organs. But also the form in which Pi is present in the cells and different organs determines the utilisation efficiency. In addition also the partitioning and storage of Pi in different organs or subcellular compartments of a plant influences the utilisation efficiency.

Distribution of phosphate between cells, tissues and organs Transport proteins play a key role not only at the root-soil interface for phosphate uptake, but they are also regulating the distribution of Pi within the plant. If transport proteins are altered in their activity, certain plant tissues will accumulate phosphate, whereas other parts will contain less. The amount of phosphate and its retranslocation rate within a plant is discussed 239

Uncovering Metabolic Pathways to be an internal signal for phosphate uptake and utilisation. So far two Arabidospsis mutants,

phol and pho2, have been characterised that are altered in phosphate distribution between organs. These have been isolated because of their reduced or increased P~ levels within leaves [36,37]. The phol mutant is defective in the capacity to load Pi into the xylem, and thus contains severely decreased amounts of phosphorus within the shoot. The PHO1 gene, that was affected by EMS mutagenesis will be identified by chromosome walking. It represents either a transport protein involved directly in xylem loading, or it encodes a protein regulating the activity of such a transport system. Overexpression of the PHO1 gene in crop plants will maybe enhance the uptake rate of P~ from the soil because of the facilitated xylem transport.

Subcellular compartmentation of phosphate Apart from the xylem and phloem loading and unloading systems also the distribution of Pi within cells is limiting the utilisation efficiency. Pi has to pass not only the plasmamembrane of cells, but also different membranes of subcellular compartments for the maintainance of metabolism. A main interest will be the isolation of proteins that mediate the P~ exchange across the tonoplast membrane. So far very little is known about tonoplast phosphate transporters that import Pi into the vacuole or mediate the export. Under sufficient Pi -supply, plant cells tend to sequester a large proportion of phosphate in the vacuole. This phosphate is metabolically unavailable under transient conditions where a Pi -limitation of metabolism occurs [6]. On the other hand the vacuolar pool is necessary to maintain the Pi homoeostasis within the cytosol, which is important for the fine tuning of carbon metabolism and photosynthesis. The isolation of cDNAs from the tonoplast would enable a detailed analysis of the role of the vacuole in maintaining Pi homoeostasis. Transgenic plants have been created that express a phosphate transporter from yeast, PHO84, in the tonoplast membrane. These plants will be used as a model system to study phosphate transport across the vacuolar membrane. In addition, plastid and mitochondria membranes will be a target for the manipulation of the exchange of Pi and other phosphorylated intermediates over biomembranes.

Endogenous phosphate pools The main phosphate storage pool in potato plants is phosphate covalently bound to starch contributing to 35-45% of the total phosphate in tubers. But also inositol phosphates, mainly phytate, sequester up to 15% of the total tuber phosphate. If plants are grown under sufficient phosphate nutrition conditions the majority of Pi that is taken up by the plant is directly imported into the vacuole. Under optimal conditions 80-90% of the total phosphate is sequestered in the vacuole [2,38]. If the exogenous availability of phosphate decreases, the phosphate is released out of the vacuole [39]. The mobilisation process, however, is not a rapid response, but a long-term reaction that takes several hours [40]. The vacuolar Pi, therefore, is not used for short-term phosphate limitations that occur during photosynthesis. An increase in the amount of metabolically available Pi in plant cells could, therefore, lead to a higher utilisation efficiency. This can be achieved either by inhibiting the import of phosphate into the vacuole or by increasing the storage capacity of the metabolically active compartments. Within the PCP project another approach was made to increase the utilisation efficiency for phosphate: 240

Phosphate efficient plants By introducing new phosphate containing compounds in the metabolic compartments of the cell, i.e. cytosol, mitochondria or plastids, phosphate stores are established competing with the vacuole. These compounds store phosphate in excess and can release the phosphate faster if limitation occurs. One of these phosphorylated molecules that has been introduced in potato plants was polyphosphate (polyP), a linear polymer of phosphate groups linked by energy-rich phosphoanhydrid bonds. Polyphosphate is utilised in bacteria and fungi as an energy and phosphate store [41] and it has also been detected in algae and animals [42] but not in higher plants. Polyphosphate is synthesised by the enzyme polyphosphate kinase (PPK) that catalyses the following reversible reaction: ATP + (polyP) n => ADP + (polyP)n+l Plants have been transformed with the PPK gene from E. coli [43] to produce polyphosphate in plants. The fusion product of PPK with the CaMV 35S promoter [44] was transformed into potato plants and resulted in the expression of PPK in the cytosol in the whole plant (cytPPK plants). In greenhouse experiments the cytPPK plants had a higher tuber yield under limiting phosphate nutrition conditions compared to the control plants. In these transgenic potato plants low amounts of polyphosphate were detected via 31p-NMR. Furthermore, potato plants were created with only chloroplastic localisation of the PPK to overcome short-term phosphate limitations during photosynthesis. Therefore, the E. coli PPK gene was fused to the plastid targeting sequence of the ferredoxin oxidoreductase from spinach [45] to mediate the import of the protein into the plastids. To restrict expression only to chloroplasts the promoter of the St-LS 1 gene [46] was used for this construct. The presence of polyphosphate could be shown by staining with toluidine blue and by 31p-NMR measurements [47]. Analysis of the transgenic plants (cpPPK) showed a decrease in leaf ATP content due to the fact that ATP is needed for the synthesis of polyR Lower ATP reduces the capacity for 3PGA conversion into triose phosphates leading to a reduction in the flux of carbon through the Calvin cycle resulting in a depressed starch synthesis and a higher level of soluble sugars. As a result, ectopic expression of PPK in potato plants seems to be a model to study the influence of novel phosphorylated molecules on metabolism at the cellular and whole plant level. In the case of cytosolic localisation of the E. coli PPK the transgenic plants are more efficient under limiting phosphate nutrition conditions and are, therefore, an ideal target to study phosphate efficiency in higher plants. Phosphate utilisation and acquisition efficiency are of central importance in creating new plant varieties which show either an increased yield potential at the same input or no loss in yield with a lower input of soil fertiliser. This programme, therefore, represents an important step towards developing new crop varieties with a higher efficiency for the nutrient phosphate, which is a long-term goal of the activities of many plant breeders not only in the EU, but also world-wide.

Authors of this publication Babette Regierer, E Springer and Jens Kossmann* Max-Planck-Institut ftir Molekulare Pflanzenphysiologie, Am Mtihlenberg 1, Haus 4 D- 14476 Golm, Germany, *Corresponding author 241

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Phytosfere’99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 0Elsevier Science B.V. All rights reserved.

Remodelling Pectin Structure In Potato

Summary Pectin is a collection of polysaccharides, which play an important role in controlling the pore size of the plant cell wall, regulating cell-cell adhesion, and providing a source of signalling molecules that elicit a range of cellular responses. Apart from this, pectins are of interest because they are an attractive hydrocolloid for various food applications. The kind and distribution of decorative groups in the pectic molecules largely determines for which application a particular pectin is most suitable. After the extraction of starch from potato tubers, a by-product is obtained, which is relatively rich in pectin. However, the quality of these pectins is poor compared to that from other sources such as citrus and apple. Rather than trying to change the structural characteristics of potato pectin post-harvest, we have embarked on achieving this in the potato plant itself. This paper summarises the structural features of pectin, the distribution of various pectic epitopes in tuber cell walls, the enzymes involved in its biosynthesis and degradation, and strategies employed to alter its fine structure in plaizta.

Introduction Potato is an important EU crop, not only because it is consumed as such (boiled potatoes) or after processing (French fries, chips, purees), but also because it produces a high-quality starch, which can be used in many industrial applications. In the Netherlands, approximately one third of the potatoes grown are used by the starch industry. After extracting the starch from the potato tubers, substantial amounts of by-products (like fibre and proteins) remain, which have mainly found application in animal feed. However, these by-products contain constituents, which have the potential of generating much higher-value products. In the EC project “Remodelling Pectin Structure in Plants” we have embarked on valorising the fibre fraction. This fraction is a collection of various polysaccharides, which together form the packaging material of the cell contents, i.e. the plant cell wall. Of these, pectin is probably the most interesting polymer because it is a known gelling agent in many food applications [ 1,2, and references cited]. The suitability of pectin for food applications is governed by many parameters, including its molecular weight, the proportion of smooth versus hairy regions, the degree of methyl- and acetyl-esterification, as well as the distribution of these ester groups along the homogalacturonan backbone (for structural characteristics of the polysaccharides,

Jean-Paul Vincken, Wageningen University, Laboratory of Plant Breeding, Wageningen, The Netherlands

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Uncovering Metabolic Pathways see further) [2,3, and references cited]. For instance, cross-linking of homogalacturonans promoted when only small amounts of hairs are present, and consequently gels with increased stability can be formed. The primary structure of potato pectin is such that it is of an inferior quality for food applications when compared to, for instance, apple or citrus pectin. In particular, the proportion of hairy regions of potato pectin is too high for good gelling characteristics, and the degree of methylation is too low for use as an emulsifying agent [4]. Thus, adjustments in the potato pectin structure are required to obtain satisfactory gelling properties, and to compete with high-quality pectins. w i t h C a 2+ is

In our EC project, we have chosen to focus on the hairy regions rather than on, for instance, decoration of the homogalacturonan with ester groups. It is our objective to decrease the proportion of hairy regions in planta by genetic modification. In addition to an improvement of the gelling characteristics of potato pectin, we hope that also the starch extraction process will be facilitated (resulting in a higher starch yield). Before discussing the strategies to achieve this, it is important to describe the setting in which these modifications take place. An important part of the project is devoted to characterising wild-type potato tubers in detail, i.e. "What is the chemical fine structure of the polysaccharides?" and "Where are the polysaccharides located in the tuber cell walls?".

Composition and structure of potato tuber walls Figure 1 summarises the structural features of a number of cell wall polysaccharides, which together form pectin [ 1,5, and references cited]. Pectins are best described as a collection of various, covalently linked polysaccharides. It has been suggested that these pectic molecules form an independent network, which determines the porosity of the cell wall. Besides the pectin network, the cellulose-xyloglucan network is thought to form the main scaffolding framework of the wall, but this will not be discussed further. Pectins consist of two parts, an essentially unbranched polymer consisting of galacturonic acid residues (homogalacturonan or smooth region), and a polymer composed of alternating rhamnosyl and galacturonosyl residues, which can be substituted with long neutral sidechains (rhamnogalacturonan I with "hairs" or hairy regions). The hairs are mainly composed of galactosyl and/or arabinosyl residues, which are attached to the rhamnosyl residues. They can either be single unit (13-DGalp-(1-+4)), or polymeric such as arabinogalactan I, and arabinan. Another type of arabinogalactan, arabinogalactan II, is mainly associated with proteins (arabinogalactan proteins). The branching pattern of the hairs is species-dependent. In certain species, the hairs may be cross-linked via ester bonds between diferulic acid residues [6]. It is generally accepted that homogalacturonan (HG) and rhamnogalacturonan I (RG-I) are covalently linked. However, the exact nature of this attachment remains to be determined. HGs can be decorated with ester groups (methyl, acetyl). Stretches of unesterified carboxyl groups of galacturonosyl residues can complex with Ca 2+, cross-link different HG molecules, and form gel-like structures [3]. Further, HGs can contain few clusters of 4 different sidechains with very peculiar sugar residues. These sub-structures of HG are referred to as rhamnogalacturonan II (RG-II). Two molecules of RG-II can form a complex with boron (forming a borate-diol ester), which in principle can cross-link two HG molecules [7, and references cited]. Only the apiofuranosyl residues of the 2-O-methyl-D-xylose-containing sidechains in each of the subunits of the dimer participate in the cross-linking [7,8]. Certain cations (Ca 2§ Pb 2§ Sr 2§ La 3§ promote 246

Remodelling pectin structure Figure 1. Schematic overview of pectic polysaccharide structures. The substitution patterns of the polysaccharides shown in this figure are arbitrary. In potato cell walls arabinan and arabinogalactan carry only very few branches. So far, there is no experimental evidence for the presence of RG-II and arabinogalactan II in potato. In the plant cell wall these structures can be covalently linked (see text), and together they are often referred to as pectin. Pectic polysaccharides can be cross-linked by Ca2+, boron, and diferulic acid residues. These cross-links are indicated by Ca2+, B, and d-FA in gray circles, respectively. Points of attack of various pectin-degrading enzymes are indicated in circles. Monoclonal antibodies raised against various pectic structures are indicated in boxes. Each box spans a number of sugar residues corresponding approximately to the epitope recognized by the antibody. 9 Homogalacturonan (HG) is composed of (1--->4)-cz-D-GalpA residues, which can be esterified at the 0-6 position (methyl) as well as at the 0-2 and/or 0-3 positions (acetyl). The distribution of es~..... ..... :...... homogalacturonan rhamnogalacturonan II homogalacturonan ter groups can either be blockwise or at I I I I random, which determines the properties of the HG to a large extent. The backbone of rhamnogalacturonan II (RG-II) is composed of approximately 9 (1--->4)-o~-DGalpA residues. To this, four complex (but highly conserved among various plant species) sidechains are attached, the exact positioning of which is still unknown. Hairy region arabinan Rhamnogalacturonan I (RG-I) is composed rhamnogalacturonan I I I I I of a backbone with (1--->2)-(z-L-Rhap(1--->4)-(z-D-GalpA repeating units. The vQ " w ve v v; t , Rhap units can be substituted with 6-DGalp-(1-->4) residues, whereas the GalpA residues can carry acetyl groups on 0-2 and/or 0-3. Arabinan has a backbone of i,,~ ................ I t~-t (1 --->5)-o~-L-Arafresidues with [(z-L-Araf(1-->3)]n (n=l,2) and/or (z-L-Araf-(1-->2) arabinogalaetan I I I sidechains. Arabinogalactan I is composed O~ of a (1-->4)-6-D-Galp backbone, which can be branched with single unit cz-L-Araf(1-->3) sidechains. Arabinogalactan II has a backbone of (1-->3)-6-D-Galp residues, Legend which can carry (z-L-Araf-(1--->6)-[6-DO (z-D-GalpA 9 6-D-Apif [ ] I~-L-Rhap ~ (z-D-Xylp O-methyl 9 c(-L-Rhap ~ D-Kdop O 6-D-GalpA 9 6-D-GlcpA o O-acetyl Galp-(1-->6)] n sidechains (n=1,2,3). The ~ ' 6-D-ealp O a-Dhap 9 (z-L-Fucp ~7 c(-D-Galp u O-feruloyl Galp residues in the sidechains can be O &-L-AcefA ~ (z-L-Arap 9 ~-,-Ara, ~ ........ branched with o~-L-Araf-(l-->3) residues. v

dimer formation in vitro in a concentration- and p H - d e p e n d e n t m a n n e r [7,9]. Both Ca 2+ and borate ester cross-linking play an important role to retain pectins in the cell wall. B o r o n in particular seems to be required to control the pore size of the cell wall in vivo [ 10]. In order to obtain reliable information on the native p o l y s a c c h a r i d e c o m p o s i t i o n of potato tuber walls, it is important to consider the p r e s e n c e of e n d o g e n o u s e n z y m e s and h u g e amount s of starch (up to 20 times m o r e than cell wall material). E n d o g e n o u s e n z y m e s should be inactivated at the v e r y b e g i n n i n g of the cell wall i s o l a t i o n p r o c e d u r e . N o t a b l y , the p e c t i n m e t h y l e s t e r a s e (PME) activity is so large that all m e t h y l groups could be r e m o v e d from H G within a very short time span. Starch should be r e m o v e d without extracting cell wall polysaccharides, because it interferes with sugar analysis and fractionation procedures. A m e t h o d has b e e n d e v e l o p e d w h i c h effectively deals with these prerequisites. Figure 2 illustrates the abundance of various p o l y s a c c h a r i d e structures in the wild-type potato tuber cell wall. Pectin (according to the definition given above) comprises m o r e than 55% of the cell wall polysacchatides, which e m p h a s i s e s that they form a very important part of the wall.

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Uncovering Metabolic Pathways Figure 2. Polysaccharide composimannan homogalacturonan tion of the potato cell wall (% w/w). 13%) (14%) The proportion of the various polysaccharides was estimated using rhamnogalacturonan I monosaccharide-composition data of cellulose (6%) starch-free cell wall preparation, (30%) structural information data from the literature and the following assumptions: (i) all rhamnose is part of RGI, which has a strictly alternating galactan Rha-GalA backbone; (ii) about 50% (28%) of the rhamnosyl units carry a single unit galactosyl sidechain [4,11]; (iii) xyloglucan the remaining (non RG-I) GalA (11%) arabinan (8%) forms the HG; (iv) all xylose is part of xyloglucan (minor amounts of xylan are neglected); (v) potato xyloglucan has a XXGG branching pattern, with c. 50% unsubstituted Xyl and equal amounts of arabinosylated and galactosylated Xyl; [12]; (vi) the remainder of Glc is atributed to the cellulose fraction; (vii) galactan and arabinan are mainly present as unbranched polymers [4]; (viii) methyl groups, which can be present on HG, and acetyl groups, which can be present on HG, RG-I, and xyloglucan [12] have not been taken into account in the estimation; (ix) the presence of RG-II has not been considered.

In comparison with other plant materials, the potato cell wall is extremely rich in hairy regions [4]. It has a lower HG to RG-I ratio than for instance apple or beet pectin, which is clearly a disadvantage in many applications. Also the amount and nature of the hairs differs considerably among these three species. Potato has a large amount of (arabino)galactan I hairs and a much smaller amount of arabinan hairs; potato hairs are hardly branched [4]. Sugar beet is rich in heavily branched arabinan hairs, and poor in galactan-containing hairs [13]. The amount of hairs is more or less comparable to that of potato. Apple RG-I is less "hairy" than that of potato or sugar beet [14]. The apple hairs contain similar amounts of arabinogalactan I and arabinan, which are both heavily branched. Currently, a more detailed analysis of the potato cell wall polysaccharides is being conducted, in order to establish the branching patterns of the wall polysaccharides more precisely, and also to map the distribution of ester groups over these polymers. In addition to this, the cell wall composition of potato tubers is monitored during their development. We will try to relate possible modifications in the wall to the presence of endogenous enzymes. Next to the chemical characterisation discussed above, the tuber walls are also being studied microscopically [ 15, and references cited]. Over the years, a number of monoclonal antibodies (mAbs) have been generated, which recognise different, specific pectic structures in the wall. The epitopes recognised by a selection of mAbs are summarised in figure 1. JIM5 and JIM7 recognise low- and high-methylester epitopes of HG, respectively. The epitopes bound by these mAbs are not defined, i.e. it is unknown how many sugar residues they comprise, whether a certain distribution of methylester groups is required, and whether acetylation interferes with recognition. For certain mAbs, such as PAM1 (not used in our studies) and 2F4, this is better documented. PAM1 can bind HG blocks of more than approximately 30 GalA residues [ 16]. It has been suggested by these authors that JIM5 recognises shorter stretches of contiguous GalA residues. 2F4 can bind unesterified oligogalacturonides of at least 9 GalA residues which have dimerized through C a 2+ [ 17]. When C a 2+ w a s replaced by other divalent cations such as Mg 2§ the recognition of HG by 2F4 was prevented. CCRC-M2 binds RG-I 248

Remodelling pectin structure Figure 3. A summary diagram illustrating 2F4 how pectic epitopes are distributed in potato tuber cell walls. Each quarter segment P represents a tuber section labelled with / mAbs JIM 5, JIM 7, 2F4, LM5 and LM6. The outermost line represents the periderm, the cortex proper is shown as two layers of light gray cuboidal cells and the storage parenchyma and vascular tissue in the perimedullary region as large and small light gray polyhedral cells respectively. The cell wall is the space between the cells and 7~ gold labelling is shown in dark gray. Both mAbs JIM 5 and JIM 7 label the entire wall throughout the tuber. The mAb 2F4 labels the entire wall of the cortical cells, but only the middle lamella at corners in the perimedullary region. Only the primary wall is labelled with mAb LM5 and in the cortex there is a labelling gradient, in cortical walls the middle lamella as well as the primary wall labels with mAb LM6, but not in the perimedullary region. Secondarily thickened walls of xylem do not label with any mAb. From [ 15], with permission from Physiologia Plantarum.

from sycamore, but not much is known about the actual epitope [ 18]. Our studies suggest that it does not detect potato RG-I. Therefore, mAbs are now being raised against defined oligomeric RG-I fragments within the project. LM5 recognises stretches of more than three g-(1--->4)-linked galactosyl residues [15, and references cited]. It is uncertain to what extent arabinosylation can affect binding. LM6 recognises an epitope of six c~-(1-->5)-linked arabinosyl residues. Branched arabinans like that from sugar beet are not bound, but after debranching they are. CCRC-R1 (not used in our studies) detects de-esterified RG-II [19]. This mAb has been used to show that RG-II is distributed over the entire wall of suspension-cultured sycamore cells. Only RG-II close to the plasma membrane appears to be unesterified. The antibodies were used in combination with immunogold-labelling to localise the various epitopes in the potato tuber walls [15]. Fig. 3 summarises the results of these studies schematically. Each polysaccharide occupies specific regions of the wall, in a tissue-specific manner. Similar experiments will be done with selected transgenic potato tubers, as well as with tubers from various developmental stages, in order to investigate whether the pectic polysaccharides are differently distributed in these walls.

Altering the cell wall The main objective of our EC project is to increase the ratio of smooth to hairy regions in potato pectin, in order to obtain a higher-value pectin in potato fibre. Genetic techniques (antisense technology, heterologous expression) are being used to realise these modifications in planta. Little is known about the function of the different pectic polymers in the wall, and therefore it is difficult to predict the consequences of down-regulating the amount of hairs for the well-being of the potato plant beforehand. In this respect, it is important to have a collection of signal sequences available, for instance to direct the expression to a particular location within the cell (transit peptides), or to a particular tissue (tissue-specific promoters). In our 249

Uncovering Metabolic Pathways experiments the sequence of interest is often preceded by a tuber-specific promoter, followed by an appropriate targeting signal. Our mai n strategies to alter the structure of potato pectin are discussed below and include: (i) post-depositional modification by expression of fungal genes, and (ii) modification of the biosynthetic machinery.

Post-depositional modification with fungal genes. At the time the project started, it was already realised that a more elegant and efficient approach would be to modify the polysaccharide biosynthetic machinery of potato plants. However, only very few genes involved in cell wall biosynthesis were described at that time. Instead, it was decided to select a number of appropriate fungal, cell wall-degrading enzymes from the large collection of candidates, and try to alter pectin in a more indirect way. The selected enzymes degrade either the hairs themselves, or the RG-I to which they are connected. In Fig. 1 the site of action of a number of these fungal enzymes is summarised. The specificity of the various enzymes is elaborated below, without dealing with the subtle differences in characteristics that may exist among similar enzymes from different species. Furthermore, it should be realised that many of the activities mentioned are also present in plants; however, the corresponding enzymes are usually characterised in much less detail. RG-I is cleaved by two different enzymes, which generate oligosaccharides with 4 or 6 sugar residues in the backbone [20, and references cited]. Endo-rhamnogalacturonan hydrolase (eRGH) splits the backbone at the non-reducing side of a rhamnosyl residue, whereas endo-rhamnogalacturonan lyase (eRGL) cleaves at its reducing side. Another important difference between the two enzymes is that eRGL produces reaction products with an unsaturated GalA residue (double bond between C4-C5), whereas eRGH does not. eRGL requires longer stretches of RG-I for cleavage than eRGH (respectively 12 and 9 glycosyl residues). Both enzymes can cleave an RG-I backbone, which is decorated with single unit galactosyl sidechains. However, the presence of sidechains can influence the catalytic efficiency of eRGH and eRGL. Accessory enzymes are required for efficient RG-I degradation. Deacetylation of the RG-I backbone by rhamnogalacturonan acetyl esterase (RGAE) enhances the action of both eRGH and eRGL. Similar observations have been made in HG degradation, where endo-polygalacturonase (ePG) or endo-pectate lyase (ePAL) act synergistically with pectin methylesterase (PME) and presumably also pectin acetylesterase (PAE). In contrast with ePG and ePAL, endo-pectin lyase (ePL) requires a high degree of methylation to be active [1]. Depending on their degree of branching, the hairs are best degraded by a combination of enzymes [21]. Most endo-galactanases (eGAL) and endoarabinanases (eARA) are not very tolerant to sidechains. An arabinofuranosidase (AF) is needed to linearise the substrate, and enhance the action of the endo-acting enzymes, g-Galactosidase (BGAL) can degrade the arabinogalactan I hairs from the non-reducing end. It is unknown whether these enzymes can by-pass an arabinosyl branch point, although there is some preliminary evidence that a BGAL from Cicer arietinum is capable of doing this. Further experiments to confirm this are being performed. A number of single (eGAL, eARA, eRGH, eRGL, ePG) and double transformants (eRGH + RGAE, en eRGL + RGAE) have already been generated within our EC project. The introduction of eGAL or eARA in potato aims at a "shave" of the corresponding hairs. Introduction of eRGH or eRGL is expected to have an even larger impact on the wall, because it removes all hairs (arabinan and galactan) including the RG-I to which they are attached. The presence of RGAE in the double transformants will presumably increase RG-I degradation, and a larger 250

Remodelling pectin structure effect is anticipated. The plants transformed with ePG serve as a kind of control. Here, the consequences of a reduced amount of smooth regions for the potato tuber will be studied. The endogenous PME(s) may act synergistically with the heterologous ePG. All transgenic plants looked normal except those in which the eARA was introduced. The eARA plants show early senescense, and do not form any tubers. A new generation of eARA transformants is currently being made in which the more tightly regulated, tuber-specific patatin promoter is used (instead of the granule-bound starch synthase promoter), in combination with various targeting sequences (vacuole, ER, Golgi). In case of vacuole or ER targeting, eARA and its substrate will meet only during processing of the tubers. With Golgi targeting of eARA the arabinans might be degraded at the place where they are synthesised. The other transformants are analysed in a step-wise manner, starting with demonstration of transcription from the transgene, gene product accumulation and authenticity, and finally determination of effects on wall composition and architecture in selected transformants. Within the tubers of one series of transformants, the amount of RNA corresponding to the heterologous gene varied considerably. However, in all cases a number of plants with a high transcription of the introduced gene could be selected. For the eRGL transformants the RNA data corresponded very well to eRGL activity in tuber extracts. Surprisingly no rhamnogalacturonandegrading activity was found in the eRGH transformants. The reason for this is unknown; possibly eRGH is incorrectly processed in the tuber after translation. Quite a few of the remaining genes were successfully expressed in the potato plants. Sugar analysis of isolated cell walls from these transformants indeed indicates an altered phenotype. A number of high expressors of the genes mentioned above have been propagated in vitro, and are now grown in the greenhouse in order to obtain more potato tuber material. The cell walls of these transgenic plants will be analysed in detail for modifications in composition, architecture, and targeting of the enzymes.

Modification of the biosynthetic machinery. In principle, four different levels of polysaccharide biosynthesis can be distinguished in plants: (i) maintenance of the pool of nucleotide sugars or other precursors, (ii) polymerisation of a particular backbone, (iii) decoration of these backbones with various substituents (glycosyl residues, methyl- and acetyl groups), and (iv) incorporation of the polymers into the cell wall. At this moment only very few genes directly involved in pectin biosynthesis are known [22, see further]. Many efforts to find "pectin synthases" follow the long and laborious route of purifying a protein of interest, digestion of this protein combined with N-terminal sequencing, preparing probes based on the obtained sequences, and finally screening a cDNA library with these probes. However, the "synthases" are often membrane-bound or part of a protein complex, and upon detergent solubilisation or purification they can lose their activity. Enzymes, which are currently under investigation, include methyltransferases, HG synthase or galacturonosyl transferase, and galactan synthase. Methyltransferases transfer a methyl group from S-adenosyl methionine (SAM) to the C-6 of a galacturonosyl residue [23-25]. It seems likely that there are several methyltransferases in one plant species, (at least) one for HG and two for RG-II. In flax, two pectin methyltransferases with different molecular weight and properties have already been described [24,25]. A galacturonosyl transferase from tobacco catalyses the transfer of a GalA residue from UDP-GalA to the non-reducing terminus of oligogalacturonides with a degree 251

Uncovering Metabolic Pathways of polymerisation greater than nine [26]. In contrast to the membrane-bound galacturonosyl transferase, the solubilised one displays a distributive mode of action (only one residue at a time is added to an acceptor molecule). It has been suggested that this is related to the dissociation of the protein complex. The lack of processivity may also be related to the nature of the acceptor substrate [27]. As part of our EC project, galactan biosynthesis is studied using flax as a model system. In flax there seem to be at least two different galactosyl transferases. One of these catalyses the processive addition of galactosyl residues to a nascent galactan chain, and may be a true galactan synthase. The other shows a more distributive mode of action, and may be involved in the attachment of galactosyl residues to a RG-I backbone. This hypothesis is currently being tested using defined oligosaccharide acceptor substrates generated with eRGH. In parallel with these experiments, the enzymes are purified to homogeneity in order to obtain sequence information. The obtained sequences will be used to clone the potato homologue, and subsequently inhibit the formation of hairs by antisense technology. Most of the plant biosynthetic genes that have been found to date belong to the group of enzymes that are involved in nucleotide sugar conversions [28,29, and references cited]. In all cases they have been recognised as such because a microbial counterpart had been described. In potato, a GDP-D-mannose pyrophosphorylase has been identified, which converts D-mannose-lP to GDP-D-mannose [24]. The MUR1 gene from Arabidopsis thaliana encodes a GDP-D-mannose-4,6-dehydratase, which catalyses the first step in the 3-step conversion of GDP-D-mannose to GDP-L-fucose. Thus, both enzymes are involved in controlling the level of L-fucose, which is a substituent of RG-II. More important for pectin biosynthesis is UDPD-glucose dehydrogenase (Glycine max, Arabidopsis thaliana) [28,29], because it plays a key role in maintaining the pool of UDP-D-galacturonic acid. The dehydrogenase converts UDP-D-Glc to UDP-D-GlcA, which is subsequently epimerised to UDP-D-GalA. The UDP-D-glucose-4-epimerase catalyses the reversible conversion of UDP-D-Glc to UDPD-Gal. Two (putative) isoforms of this enzyme have been found in Arabidopsis thaliana, and one in Pisum sativum and Cyamopsis tetragonoloba. These epimerases may be very important in the supply of buiding units for galactan hair biosynthesis. According to the current biochemical pathways, UDP-D-Gal can only be formed from UDP-D-Glc, at least when no D-galactose is applied exogenously. In Arabidopsis thaliana the UDP-D-Glc-4-epimerase activity has been down-regulated by introducing the gene of one of the isoforms in an antisense orientation [30]. Surprisingly, no decrease in the amount of cell wall galactose was observed. However, when these transgenic plants were grown in a medium containing galactose, an increase in cell wall galactose was found. In the present EC project, two putative UDP-D-Glc4-epimerases from potato have been cloned. Transgenic potato plants in which the amount of both enzymes, alone and in combination, is down-regulated using antisense technology are currently being prepared. It is expected that "hair growth" can be inhibited to a larger extent than in Arabidopsis thaliana, because both isoforms will be antisensed, and because galactan is a much more abundant cell wall polysaccharide in potato than in Arabidopsis. Another interesting gene with respect to pectin biosynthesis is SAM synthethase, because it allows control over the pool of substrate for pectin methyltransferase. The SAM synthethase 252

Remodelling pectin structure has been over-expressed in suspension-cultured flax cells [24]. As a result of this some of the transgenic cell lines produced pectin with a higher degree of methylation, suggesting that the SAM concentration is limiting in the control cell lines. Another multigene family, which has received a lot of attention with respect to cell wall biosynthesis, is cellulose synthase (CesA; A stands for catalytic subunit) and cellulose synthase-like (Csl) genes [31]. Also in this case, the discovery of the first "plant cell wall synthase" gene was preceded by the characterisation of a number of bacterial cellulose synthases. Alignment of the bacterial enzymes showed a number of highly conserved motifs, which were speculated to be involved in UDP-glucose binding. When it was realised that these motifs were present, the search for a plant cellulose synthase was greatly facilitated. Now, the number of putative cell wall synthases is rapidly increasing. In Arabidopsis thaliana 42 CesA or Csl genes have already been found, which can be assigned to 6 different groups. The CesA genes all belong to one group. The biological function of two members of this group has been established by complementation of certain Arabidopsis cell wall mutants. RSW1 encodes a primary cell wall cellulose synthase [32], whereas IRX3 is involved in cellulose production in the secondary cell wall [33]. The specificity of the 5 Csl groups remains to be investigated. Because the binding of nucleotide sugars is a probably a common feature of all "plant cell wall synthases", it has been hypothesised that the Csl genes encode other polysaccharide synthases such as xylan synthase, mannan synthase, etc.. In the present EC project we have set out to isolate a number of CesA or Csl sequences for two reasons. (i) The availability of a true potato CesA gene would enable down-regulation of the amount of cellulose using antisense technology. In this way a number of transgenic potato lines could be generated in which the cellulose content in the tubers is reduced to between 0% (wild-type) and 100% (possibly lethal). It is speculated that these plants will compensate this loss by depositing more pectins in their walls. In order to strengthen the wall, these pectins might be cross-linked to a larger extent than those in wild-type potato tubers (see Fig. 1). Such observations have been made for the walls of suspension-cultured tomato cells that have been grown in the presence of 2,6-dichlorobenzonitrile, an inhibitor of cellulose biosynthesis [34]. (ii) In addition to isolating a true cellulose synthase gene, other polysaccharide synthases might be obtained as well. Discovery of a galactan, arabinan or RG-I synthase would of course be preferred with respect to the aim of the project. Inhibition of these genes by antisense technology could increase the proportion of smooth regions in pectin, which would be an elegant alternative for the introduction of the fungal genes discussed previously. So far, seven CesA or Csl genes have been isolated from potato. These are currently introduced in an antisense orientation in potato plants.

Future prospects From the above it is clear that pectin is an extremely complex component of the cell wall. Although there is a fairly good understanding about the fine structure of the individual polysaccharides, it is obvious that the attachment of the various elements to each other deserves more attention. Considering the complexity of pectin, it is not surprising that there are numerous enzymes involved in its degradation. No doubt the biosynthetic machinery will be equally impressive. Already, a large "toolbox" for pectin remodelling in planta is available. Most of 253

Uncovering Metabolic Pathways the tools are genes encoding cell wall degrading enzymes. Only a limited number of tools for pectin biosynthesis is known. Of these, the genes involved in the interconversions of nucleotide sugars are most abundant. However, the pathways in which these genes participate are very complex, and in many cases there will be alternative routes by which the effect of downregulating a certain activity can be by-passed. Also, altering the level of one particular nucleotide sugar in the plant may have an effect on the structure of various polysaccharides (for instance, many polysaccharides contain galactose). Potentially, more specific modifications of the wall polysaccharides can be achieved by down- or up-regulating the activity of synthases or decorative enzymes. Although there are relatively few genes known in this category, it is anticipated that the toolbox will be supplemented with such genes in the near future. The growing number of T-DNA-tagged mutant lines in Arabidopsis thaliana in combination with the Arabidopsis genome-sequencing project will be very valuable in this respect [29]. A large number of transformants potentially with an altered pectin structure have been generated. Preliminary results with the introgression of heterologous genes look very promising. Larger amounts of these tubers are currently being grown, and will be used for detailed cell wall analysis. Pectins will be extracted and subjected to functional testing in order to investigate whether the desired industrial properties are enhanced. In principle, the tailored hairy region structures may also offer possibilities for new applications. Finally, the remodelling of pectin will be interesting from a plant-developmental perspective. Recombinant DNA technology provides the possibility of probing the significance of individual polysaccharide structures for the architecture and properties of the cell wall. For this, it is very important that the potato cell wall is characterised in great detail, both at the chemical and the microscopic level, so that the background, in which the cell wall modifications are introduced, is precisely known.

Acknowledgement The research described in this paper is funded by a grant from the EC (CT97 2224).

Authors of this contribution Jean-Paul Vincken l, Bernhard Borkhardt 2, Max Bush 6, Chantal Doeswijk-Voragen 3, Berta Dopico 5, Emilia Labrado?, Lene Lange 7, Maureen McCann 6, Claudine Morvan 4, Francisco Mufioz 5, Ronald Oomen I, Isabelle Peugnet 4, Brian Rudolph 8, Henk Schols 3, Susanne SCrensen 2, Peter Ulvskov 2, Alphons Voragen 3, Richard Visser 1. 1Wageningen University, Laboratory of Plant Breeding, Lawickse Allee 166, 6709 DB Wageningen, The Netherlands, 2Biotechnology Group, DIAS, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Denmark, 3Wageningen University, Department of Food Science, Bomenweg 2, 6703 HD Wageningen, The Netherlands, 4University of Rouen, 2Parois et Polymeres Pari6taux 2, SCUEOR ESA 6037 CNRS 76821, Mont Saint Aignan, Cedex, France, 5Universidad de Salamanca, Dept. de Biologia Vegetal (Fisiologia Vegetal), Campus Miguel de Unamuno, Salamanca 37007, Spain, 6john Innes Centre, Department of Cell Biology, Colney Lane, Norwich NR4 7UH, UK, 7Novo Nordisk A/S, Application Technology, Enzyme Development and Applications, Novo All6, 2880 Bagsvaerd, Denmark, 8Hercules, Copenhagen Pectins, Ved Banen 16, DK-4623 Lille Skensved, Denmark. *Corresponding author 254

Remodelling pectin structure

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Phytosfere.99 - Highlights in European Plant Biotechnology Gert E. de Vnes and Karin Metzlaff (Editors). 0Elsevier Science B.V. All rights reserved.

Toward The Identification Of The Genes For The Synthesis Of Condensed Tannins In Forage Legumes Abstract The paper shorty describes the importance of modulating the presence of condensed tannins (CT) in the forage of legume species for improving their nutritive value. The different strategies adopted to modify CT levels in forage legumes are summarised. The paper is focused on the experiments aimed at the modification of CT levels utilising sense and antisense transformation. This strategy is adopted to produce mutants useful for isolating through cDNA comparative analyses the still unidentified genes of the CT pathway.

Introduction Condensed tannins (CT) are phenolic compounds which play a determinant role in the quality of legume forages. In fact these secondary metabolites of the flavonoid pathway bind to proteins and affect enzymatic activity and protein solubility [ 11. These features can have positive and negative draw back in forage quality, in fact moderate amounts of CT in species rich of proteic substances such as forage legumes prevent protein degradation in the rumen [ 2 ] , acted by the ruminal flora and causing great loss in nutritive value, and the insurgence of bloating, a deadly syndrome affecting ruminants feeding with fresh forage of CT free legumes. On the contrary, some other species both legumes and graminae are refused by the animals when too rich of CT which affect negatively palatability for their inhibition of salivary enzymes [3]. The most cultivated forage legumes: white clover and lucerne are tannin negative and there is great interest in producing plants of these species containing moderate amounts of these compounds in the edible tissues, on the other side some neglected species often very productive and adapted to difficult environments are not useful for animal nutrition purposes but could acquire great values once CT levels were reduced. The first attempt to modify CT levels in lucerne were performed through somatic hybridisation with legumes rich in these compounds such as Lotus corniculatus and Onobrychis vicifolia, however the difficulties in producing dividing symmetric hybrids (Paolocci, personal communication), and the lack of CT in the produced asymmetric somatic hybrids [4] discouraged this approach. For the impossibility of a profitable utilisation of close and wide hybridisation, forage breeders turned to genetic transformation as possible tool for modulating CT levels in plants. The first step at this purpose is the clear description of the biosynthetic pathway leading to CT and the identification and cloning of the structural and regulatory genes of the pathway.

F. Damiani, Istituto di Ricerca sul Miglioramento Genetic0 delle Piante Foraggere, Perugia, Italy

257

Uncovering Metabolic Pathways The transformation of these three genotypes with the construct 35S-Sn performed utilising

Agrobacterium rhizogenes produced three populations of transformants highly contrasting for CT average content and variability [12]. The untransformed plants, that resulted highly variable for CT levels in leaves were quite stable for CT levels in other tissues, in fact stems and flowers highly reacted to CT specific staining [20], roots, on the contrary, showed very low levels of CT. The transformation in general had no effect for CT levels in stems and flowers but increased the CT levels in roots, whatever genotype was utilised for transformation. In leaves, the effect of transformation was linked to the starting plant, for instance $41 derived transformants were not different from the starting plant. The variability among transformants increased in $33 derived population and was maximal in $50. This population was carefully investigated and plants scored for CT levels and classified in three groups: unaffected, suppressed and elicited. In the 2nd year of evaluation some plants shifted from one class to another indicating that the phenomenon of both suppression and elicitation could be unstable. In a 3rd year of evaluation no further shifts were observed and some plants were stable for CT suppression and some were stable for CT transactivation. These transformants were investigated at the molecular level. Analyses were made to determine the number of copies of the transgene, their expression and for the expression of some endogenous genes involved in the pathway that were previously cloned in Lotus corniculatus (unpublished data).

U The results (figure 2) clearly indicate that the presence of the transgene can have a double and opposite effect, in fact suppressed plants show the lack of expression of Sn, an endogenous myc-like gene, while DFR expression is significantly reduced. Observations on LAR activity performed in the previous experiments [10] and not repeated here indicated that LAR was absent and therefore also the LAR coding gene should not be expressed. The elicited plants on the contrary show expression of the transgene, of the endogenous myc and over expression of DFR.

~:~*.

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.

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.

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.

.

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Figure 2. Gene expression analysed through northern on control untrasformed $50 plant (U) and on derived suppressed (S) and elicited (E) Sn transgenic. Gene expression has been analysed for Sn, an endogenous myc-like regulator (myc), DFR; as internal control the elongation factor (~EF) has been analysed.

The reasons for the different behaviour of isogenic plants could be caused by the site of insertion and/or by the number of copies of the transgene. Interestingly, stable plants of the suppressed-type showed multiple insertions while stable plants of the elicited-type were single copy transformants. Gene suppression could be considered also in stable suppressed plants as a form of genomic instability occurring in presence of an overflow of gene expression and this could be buffered with a silencing mechanism which can occur in different phases of plant life. We have evidences that some unstable phenotypes with multiple copies of the transgene belong to the elicited plants in the first year of evaluation and moved to the suppressed group in the second year. 260

Synthesis of condensed tannins chalcone and DFR the last enzyme common to CT and anthocyanin pathway [ 13]. The effects of these transformations, carried out utilising Agrobacterium rhizogenes as vector, have been mainly investigated on hairy root, and in some cases on the whole plant [14]. Some generalisations can be deduced from the global analysis of these experiments. The starting genotype has a preponderant role in the phenotypic effects induced by the transformation. In fact three genotypes $33, $50 and $41 have been transformed and one genotype, $41 the one with the high level of CT in any tissue, was never affected by transformation at the phenotypic level.

-

-

The orientation of the gene is not determinant in increasing or decreasing the CT accumulation in fact when $33 plant was transformed with AS CHS the more evident effect observed on hairy roots was the increase of CT, at difference of what expected. Same happened utilising AS DFR transgene which produced some plants with decreased levels of CT in different plant tissues but some other transformants were up-regulated for CT levels.

The explanation that authors claim for both experiments is that the transgene could suppress only one or few genes of the family and that other members of the same family could be overexpressed for compensation [ 14]. This hypothesis is supported by the modification of the levels of hydroxylation of CT which could derive from the different level of hydroxylation of the substrates which are specific for the suppressed and overexpressed genes. In fact it has been demonstrated in other species that different members of the same family have a superior specificity with a specific substrate [15] thus resulting in a slightly different end products. The experiments resulted in the acquisition of useful information about the genetic control of the pathway and were also supported by several experiments concerning the interaction between CT expression and environmental factors such as: light [12], CO 2 concentration [16], temperature levels and water availability [16], phytormones [17]; but did not produced completely reliable mutants useful for isolating the unknown genes of the pathway. Other experiments were more successful using this approach and involved the transformation of L. corniculatus with a heterologous regulatory gene of the pathway in sense orientation. The maize gene Sn [18], a myc like gene transactivating the anthocyanin pathway, was found to effectively induce anthocyanin pigmentation in heterologous hairy roots [19]. The analyses of 35S-Sn hairy root derived Lotus plants showed the leaf specific suppression of CT synthesis, occurred for a supposed mechanism of sense suppression, paralleled with the increase of CT levels in roots [10]. The experiments were repeated in the same selected genotypes previously mentioned $50, $31, $41, also utilised for transformation with AS and S structural genes and the evidences of sense suppression were confirmed as well as it was confirmed the importance of the starting genotype for obtaining transformants with modified CT levels. The three genotypes utilised belong to the same cultivar (Leo) but are very polymorphic for tannin accumulation in leaves. In fact $50 is scored low, $33 medium-low and $41 high. The analyses of CT in hairy roots invert the position of $33 and $50 indicating that CT accumulation is tissue specific, nevertheless $41 has the high rank for CT whatever tissue is examined. 259

Uncovering Metabolic Pathways The transformation of these three genotypes with the construct 35S-Sn performed utilising

Agrobacterium rhizogenes produced three populations of transformants highly contrasting for CT average content and variability [12]. The untransformed plants, that resulted highly variable for CT levels in leaves were quite stable for CT levels in other tissues, in fact stems and flowers highly reacted to CT specific staining [20], roots, on the contrary, showed very low levels of CT. The transformation in general had no effect for CT levels in stems and flowers but increased the CT levels in roots, whatever genotype was utilised for transformation. In leaves, the effect of transformation was linked to the starting plant, for instance $41 derived transformants were not different from the starting plant. The variability among transformants increased in $33 derived population and was maximal in $50. This population was carefully investigated and plants scored for CT levels and classified in three groups: unaffected, suppressed and elicited. In the 2nd year of evaluation some plants shifted from one class to another indicating that the phenomenon of both suppression and elicitation could be unstable. In a 3rd year of evaluation no further shifts were observed and some plants were stable for CT suppression and some were stable for CT transactivation. These transformants were investigated at the molecular level. Analyses were made to determine the number of copies of the transgene, their expression and for the expression of some endogenous genes involved in the pathway that were previously cloned in Lotus corniculatus (unpublished data).

U The results (figure 2) clearly indicate that the presence of the transgene can have a double and opposite effect, in fact suppressed plants show the lack of expression of Sn, an endogenous myc-like gene, while DFR expression is significantly reduced. Observations on LAR activity performed in the previous experiments [10] and not repeated here indicated that LAR was absent and therefore also the LAR coding gene should not be expressed. The elicited plants on the contrary show expression of the transgene, of the endogenous myc and over expression of DFR.

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Figure 2. Gene expression analysed through northern on control untrasformed $50 plant (U) and on derived suppressed (S) and elicited (E) Sn transgenic. Gene expression has been analysed for Sn, an endogenous myc-like regulator (myc), DFR; as internal control the elongation factor (~EF) has been analysed.

The reasons for the different behaviour of isogenic plants could be caused by the site of insertion and/or by the number of copies of the transgene. Interestingly, stable plants of the suppressed-type showed multiple insertions while stable plants of the elicited-type were single copy transformants. Gene suppression could be considered also in stable suppressed plants as a form of genomic instability occurring in presence of an overflow of gene expression and this could be buffered with a silencing mechanism which can occur in different phases of plant life. We have evidences that some unstable phenotypes with multiple copies of the transgene belong to the elicited plants in the first year of evaluation and moved to the suppressed group in the second year. 260

Synthesis of condensed tannins Figure 3. Stability for CT suppression in three somaclonal populations derived from three suppressed Sn transgenic plants. On the ordinate axis is reported the percentage of suppressed plants per population assessed for 8 months consecutively. Each population consisted of about 50 somaclones.

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. ,c ~ . . . c ~ The mechanism of gene suppression 30 20 l""(3... O - - 9 -..0 is still questionable notwithstanding 10 the more recent advances [21 ] but our 0 II ~ ~ I--I data indicate that it is tissue depen1 2 3 4 5 6 7 8 dent. In our experiments it was found that in plants where silencing happens months in leaves the transgene is effective in roots and does not affect the expression of endogenous counterpart in stems and flowers. It is reversible: three populations of three suppressed plants [10], assessed for leaf CT with monthly frequencies reduced progressively and consistently the number of individuals scored negatively for CT (figure 3). It is genotype dependent, there could be different hypotheses to explain the behaviour of $41 transformants which never show any reduction in CT levels in leaves: the absence or the inactivity of the RdRPs (RNA Dependent RNA Polymerases) [22] or compensation mechanisms supported by other non silenced regulatory genes. These hypothesis find confirmations by the fact that in this ~,. :oo. :,0 :,0. ,,0 ,8o ?oo. . . . . . . . genotype, at difference with $50 and $33, CT syns thesis is regulated differently with respect to light ~_~ induction [12].

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$50 untransformed (U), $50 suppressed (S) and $50 elicited (E) plants, cDNA amplification was carried out according to the protocol of AFLP Plant mapping kit for small genomes of PE Applied Biosystems, PCR product were analysed through capillary electrophoresis carried out utilising ABI Prism 300 apparatus of P'erkin Elmer.

developments

The key question of the research is the acquisition of the gene sequences of all the genes still unknown of the pathway. For this purpose experiments are started in the framework of an EU funded project (FAIR 4068) using cDNA-AFLP [23] obtained with RNA isolated from contrasting transformants derived from the same starting genotype. The feasibility of this method seems confirmed from preliminary results (figure 4). We compared the AFLP profile of an elicited transformant and a suppressed one with the common starting plant, taking into account only the fragments longer of 80 bp we observed 32% excess fragments in the first individual and a 32% reduction fragments of the suppressed transformants. There are some fragments present in non-transformed and elicited plant, some 261

Uncovering Metabolic Pathways common to the transformants and absent in the control, some present only in the suppressed plant. The first group of fragments are probably those related with the genes involved in the pathway, the second group are possibly those specific of the transformation (rol and reporter genes), the third group of fragments could be those involved in the mechanism of silencing but also products of RNA degradation. We sequenced only one cDNA fragment, 248 bp long, present in the elicited and untransformed plant and absent in the suppressed one. The derived putative protein sequence showed no significant homology in the Swissprot data bank. This may indicate that novel genes could be isolated when following this strategy. The isolation and characterisation of genes, the assessment of their involvement in CT biosynthesis and of their influence in improving forage quality of legumes will be the scope of the FAIR project carried out in collaboration with IGER, Aberysthwyth - UK and INRA, Lusignan - France.

A u t h o r s o f this c o n t r i b u t i o n Damiani E Paolocci E, V. Turchetti, S. Arcioni Istituto di Ricerca sul Miglioramento Genetico delle Piante Foraggere, CNR via Madonna Alta 130, 06128 Perugia, Italy References 1. 2. 3. 4. 5. 6. 7.

8.

9.

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11.

12.

13. 14.

C.M. Spencer, C. Ya, R. Martin, S. H. Gaffney, P.N. Goulding, D. Magnolato, T.H. Lilley, E Haslam, Polyphenol complexation- some thoughts and observations, Phytochemistry 27 (1988) 2397-2409. G.J. Tanner, A.E. Moore, P.J. Larkin, Proanthocyanidins inhibit hydrolysis of leaf proteins by rumen microflora in vitro, British Journal of Nutrition 71 (1994).947-958. R. Kumar, M. Singh, Tannins: their adverse role in ruminant nutrition, J. Agric. Food Chemistry 32 (1984) 447-453. Y.G. Li, G.J. Tanner, A.C. Delves, P.J. Larkin, Asymmetric somatic hybrid plants between Medicago sativa L. (alfalfa, lucerne) and Onobrychis viciifolia Scop. (sainfoin), Theor. Appl. Genet. 87 (1993) 455-463. G.J. Tanner, K.N. Kristiansen, Synthesis of 3,4-cis-leucocyanidin and enzymatic reduction to catechin, Analytical Biochemistry 209 (1993) 274-277 M.Y. Gruber, B. Skadhauge, J. Stougaard, Condensed tannin mutation in Lotus japonicus, Polyphenols Actualites. Lettre d'information du Groupe polyph6nols. F6vrier 1998 (1998) pp. 4-8. T.R. Carron, M.P. Robbins, P. Morris,. Genetic modifications of condensed tannin biosynthesis in Lotus corniculatus. 1. Heterologous antisense dihydroflavonol reductase down-regulates tannin accumulation in "hairy roots" cultures, Theor. Appl. Genet. 87 (1994) 1006-1015. S.P. Colliver, P. Morris, M.P. Robbins, Differential modification of flavonoid and isoflavonoid biosynthesis with an antisense chalcone synthase construct in transgenic Lotus corniculatus, Plant Mol. Biol. 35 (1997) 509-522. A.D. Bavage, I.G. Davies, M.P. Robbins, E Morris, Expression of an Antirrhinum dihydroflavanol reductase gene results in changes in condensed tannin structure and accumulation in root cultures of Lotus corniculatus, Plant Mol. Biol. 35 (1997) 443-458. E Damiani, E Paolocci, ED. Cluster, S. Arcioni, G.J. Tanner, R.G. Joseph, Y.G. Li, J. deMajnik, EJ. Larkin, The maize transcription factor Sn alters proanthocyanidin synthesis in transgenic Lotus corniculatus plants, Aust. J. of Plant Physiol. 26 (1999) 159-169. M.E Robbins, T.R. Carron, E Morris, Transgenic Lotus corniculatus a model system for the modification and genetic manipulation of condensed tannin biosynthesis, in: R.W. Hemingway and EE. Laks (eds.), Plant Polyphenols: Synthesis, Properties and Significance, Plenum Press, London, 1992, pp. 111-131. E Paolocci, R. Capucci, S. Arcioni S., E Damiani, Birdsfoot trefoil: a model for studying the synthesis of condensed tannins, in: G.G. Groos, R.W. Heminghway, T. Yoshida (Eds), Plant Polyphenols 2. Chemistry and Biology. Kluwer Academic/Plenum Publishers New York, (1999) (in press) (2000) 343-356. R.A.Dixon, N.L. Paiva, Stress-induced phenylpropanoid metabolism, The Plant Cell 7 (1995) 1085-1097. M.E Robbins, A.D. Bavage, C. Strudwicke, P. Morris, Genetic Manipulation of Condensed Tannins in

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15. 16.

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19. 20. 21. 22. 23.

Higher Plants. II. Analysis of Birdsfoot Trefoil Plants Harboring Antisense Dihydroflavonol Reductase Constructs, Plant Physiol. 116 (1998) 1133-1144. T.A. Holton, E.C. Cornish, Genetics and biochemistry of anthocyanin biosynthesis, The Plant Cell 7 (1997) 1071-1083. P. Morris, E. B. Carter, M.K. Theodorou, Environmental effects on condensed tannin accumulation and nutritive value of Lotus corniculatus, in: G.G. Gross,R.W. Hemingway, T. Yoshida (Eds.), Abstract of 3rd Tannin Conference, Bend, Oregon 1998, pp. 139-140. M.P. Robbins, T.E. Evans, P. Morris, The effect of plant growth regulators on growth, morphology and condensed tannin accumulation in transformed root cultures of Lotus corniculatus, Plant Cell, Tissue and Organ Culture 44 (1996): 219-227. C.Tonelli, G. Consonni, S.E Dolfini, S.L. Dellaporta, A. Viotti, G. Gavazzi, (1991). Genetic and molecular analysis of Sn, a light-inducible tissue-specific regulatory gene in maize, Molecular and General Genetics 225 (1991) 401-410. F.Damiani, F.Paolocci, G.Consonni, ECrea, C.Tonelli, S.Arcioni A maize anthocyanin transactivator induces pigmentation in several transgenic dycotiledonous species, Plant Cell Report 17 (1998) 339-344. Y.G. Li, G.J. Tanner, P.J. Larkin, The DMACA-HC1 protocol and the threshold proanthocyanidin content for bloat safety in forage legumes, Journal of Science of Food and Agriculture 70 (1996) 89-101. M. Metzlaff, M. O'Dell, P.D. Cluster, R.B. Flavell, RNA-mediated RNA degradation and chalcone synthase A silencing in petunia, Cell 88 (1997) 845-854. C. Cogoni, G. Macino, Gene silencing in Neurospora crassa requires a protein homologous to RNA dependent RNA polymerase, Nature 399 (1999) 166-169. C.W.B. Bachem, R.S. vand der Hooven, S.M. de Bruijin, D. Vredeugnhil, M. Zabeau, R.F. Visser, Visualization of differntial gene expression using a novel method of RNA fingerprinting based on AFLP: Analysis of gene expression during potato tuber development, The Plant Journal 9 (1996) 745-753.

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Phytosfere.99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 0Elsevier Science B.V. All rights reserved

An Overview Of Important Market And Technology Transfer Issues For Commercialising Academic Plant Biotechnology Summary Biotechnology is projected to become a major global industry in the new millennium, and will have a critical role to play in future European industrial development. Advances in plant biotechnology have potentially far-reaching social and economic benefits, while the continued dependence of agro-chemical and biotechnology companies on new ideas emerging from academic research laboratories represents a significant opportunity to generate an independent source of revenue for such institutions. It is timely to take stock of specific modalities and mobilising factors necessary for the commercialisation of academic plant biotechnology, so that effective technology transfer models for optimising the process may be identified and refined. In the present review, the challenges and opportunities facing bioentrepreneurs working in an academic environment are examined, and recommendations for encouraging effective technology transfer are suggested.

Introduction Conventional definitions of ‘biotechnology’ refer to the application of scientific and engineering principles to the processing of materials by biological agents [ 11, and more recently, the modijkation of biological materials and their subsequent application [2]. While each definition emphasises either the ‘traditional’ (harnessing natural processes, such as fermentation) or the ‘new’ aspects (for example, recombinant DNA), neither acknowledges the great potential for the generation of wealth, nor places the technology in a market context. However, with important exceptions, the inability of entrepreneurs thus far to translate this complex technology into a consistent and sustainable economic growth model, as for example seen in the computer industry, has made the adoption of the term, ‘biocommerce’, seem premature. The business and sociological contexts for many aspects of the biotechnology industry are only beginning to be understood, and although some economic commentators have been keen to draw analogies with the computer industry of thirty years ago, this model is overly simplistic; many factors discriminate biotechnology from other industries, including far-reaching multi-disciplinary, ethical, environmental and social dimensions. It is against this complex backdrop that academic bioentrepreneurs are attempting to translate their ideas into commercial gain. The challenge is somewhat magnified in the case of plant I

I

Gwilym Williams, BioResearch Ireland, University College Dublin, Ireland 1

I

265

Entrepreneurship in Plant Science biotechnology, as the generally perceived social need, the regulatory climate and consequently, the market prospects, are not as clear-cut; this contrasts sharply with the market for human biopharmaceuticals. European consumer acceptance of existing plant biotechnology products has been low to date, while the stance of conventional agriculture in EU member states is difficult to deduce. Possible consumer backlash in Europe is likely to be against supermarkets, food processors and farmers, with potentially serious knock-on effects for the biotechnology sector.

The challenge for bioentrepreneurs in academia The academia-based bioentrepreneur wishing to commercialise a proprietary skill or technology is faced with a number of challenges which relate chiefly to the fact that such activities are tangential to the role of teacher or basic researcher (table 1). Additionally, w h i l e i t h a s been estimated that as much as 50% of the economic growth of developed countries derives specifically from technology [3], with biotechnology a s a classic

Table 1. Common challenges facing bioentrepreneurs working in an academic environment No access to proprietary biobusiness intelligence, poor knowledge of market dynamics (potential commercial developers, competitors, market size, regulatory issues, etc.) Inability to value the worth of technology (necessary for negotiating a license agreement or deciding to incur the expense of patenting or establishment of a spin-out company to exploit the technology) Lack of appreciation of development milestones, time-lines and costs Lack of clarity in key project management issues (maintaining project confidentiality, project demarcation between different study sponsors, technology ownership fights, etc)

example of a pronounced Academic industrial liaison functions are often 'generic' in nature and usually have 'technology push'-type s e c inadequate resources to lend meaningful support to the specific needs of bioentrepreneurs t o r , t h e majority o f E u r o Vor bioentrepreneurs wishing to start a company: inadequate financial/market knowledge to prepare business projections as part of a business plan pean universities (and governments) have been relatively slow to take a lead in implementing the facilitation structures necessary to translate their biotechnology research activities into an engine for economic growth. Major universities in the U.S. have long recognised the importance of a well-funded technology transfer office (for example, ref. 4). European entrepreneurial bioscience continues to lag behind that of the U.S. [5], although countries such as the U.K. and Germany are now taking strong measures to ensure their future international competitiveness. Undoubtedly, the nature of the local and national 'enterprise climate' is important in encouraging academic researchers to become involved in commercialising their work. However, the bioentrepreneur must also be motivated by personal ambition and a market perspective which places financial gain firmly into the technology transfer equation. It is therefore critical that academic institutions have both transparent and equitable reward schemes in place to encourage their researchers to engage in commercial activities; the position of non-tenured research staff should also be addressed in this regard, including the aspect of inventorship rights. The poor global success rate of entrepreneurial bioscience to date is a powerful testament to the difficulties in progressing such ventures. Consequently, there is a pressing need to identify critical areas of importance in the effective commercialisation of academic biotechnology. In broad terms, the decision to embark on this track may be governed by a number of wide-ranging questions: What are the personal career goals of the individual researcher? What is the commercial value and the market for the technology? What is necessary to bring the 266

Commercialising academic plant biotechnology technology to the marketplace? An integrated answer to such questions must be sought from the outset in order to provide the best chances of success.

The transformation from researcher into bioentrepreneur The practical logistics of translating academic research into commercial reality requires a change in approach to how research, personnel and information are managed in the academic environment. The front-line responsibility for this task will fall to the Group Leader, who must now judiciously balance a time commitment to educational and basic research tasks with a burgeoning commercial responsibility. The required attributes of a successful bioentrepreneur have been outlined in detail elsewhere [6, 7], but strong management skills, combined with a good appreciation of technical and finance issues, are critical. The contrast in emphasis on different operational and strategic issues between industry and academia is also illustrative of the required difference in mind-set between researcher and entrepreneur (table 2). Irrespective of whether a Table 2. A comparison of key development issues for academia and industry researcher is seeking to commercialise a laboratory Factor Focus University Company service or a proprietary technology, one of the most Technology emphasis High Medium-high powerful marketing tools, Development emphasis Low High along with strongly proTechnical risk High Low Commercial risk Low High tected intellectual property, Intellectual property Low-moderate High will ultimately be the Market issues/intelligence Low High person's own credibility Management structures Not clearly defined Usually clear Time taken to reach decisions Long Short and track record, which will Ability to value employee time Low High be taken into account by cliRelevance of peer-reviewed publications High Low/moderate Relevance of marketing/publicity Low High ents, investors and collaborators. However, demands on personal time which are additional to the input required for the management of an academic research group may be expected, and will be centrally defined by the significant informational needs of the business world. Both external and university-based technology brokerage functions will require the support of researchers to prepare descriptions of a technology for patent applications, marketing literature and press releases. Researchers will also be called upon to deliver presentations, attend meetings and sometimes speak to journalists in the challenge to bring technology to the marketplace. A second important point which is often overlooked by the academic researcher is that biotechnology is merely one tool in an industrial arsenal which has been assembled to address a real or perceived market need. Visualising early stage technology in the context of a downstream setting (such as the realities of production, government regulation and market response) is an area which has been notably weak in biotechnology as a whole, with the universitybased entrepreneur at a decided disadvantage relative to industrial counterparts. This information deficit has been partly addressed in recent years by many international publishing houses, who now produce a large array of biobusiness publications, of widely varying quality, 267

Entrepreneurship in Plant Science which aim to identify trends, analyse markets and to generally bridge the 'university-industry divide'. Up-to-date biobusiness information is also available on the Internet, as are a number of both free and 'pay-per-view' databases of relevance to biotechnology. The emergence of dedicated biobusiness conferences (such as the annual 'BIO' and 'EuropaBio' events in the U.S. and Europe respectively), which bring together industry and academic biotechnologists to discuss global technology and business trends, also have an important role to play in catalysing information exchange, while providing excellent networking opportunities [8]. Finally, paucity of national funding for basic research in some EU countries (reviewed in ref. 9), with the shortfall being sought from industry, has caused confusion in certain quarters regarding the key difference between applied (commercial) and basic ('blue skies') research. From the researcher's perspective, while a vigorous basic research programme is critical to generate innovative applied research directions, the strategic and day-to-day laboratory management criteria for an already identified valuable piece of intellectual property requires a different operational approach.

Existing plant biotechnology commercialisation models An essential first step on the road to becoming a bioentrepreneur is to look at how others have achieved the transition. 'Benchmarking' progress against established and successful companies will become a recurring theme as part of normal commercial development and growth. The generally agreed biotechnology consensus business model (at the macro-level) shows a strong reliance on academic-derived intellectual capital, and revolves around the synergistic interplay of the established agro-chemical/pharmaceutical industry ('ag-chem') with entrepreneurial bioscience ('biotechnology') companies and academia (figure 1; reviewed in ref. 9). In recent years, the face of the agri-business sector has underAgri-biotech business model gone pronounced change, with food processors and new developA Seedcompanies ments in farming practice having Patent /r '~ EU regulatory important ramifications for the Spin-outcompanies law ~ _ _ . _ _ ~ ~ agro-chemical sector [ 10]. Food processors / A--- -t.---:--l\N Capitalmarkets The two major technology transfer mechanisms operating at the university level within this structure are technology licensing and direct sale of services or product. Technology licensing would appear to be the predominant trend in many branches of plant biotechnology, as similar to the case for human and animal health, the large financial resources required to develop, produce and globally

US regulatory / ~ ~(rto-t;"t;nuwm } X / ~. ~/' N Retailers Venturecapital/ ~ _.~~ # -"-,xN" , f -'-~ Seedcapital

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Figure 1. Consensus business model for the agri-biotech sector. It should also be acknowledged that a significant level of plant biotechnology research is carried out in dedicated government laboratories, reflecting a historical desire for each country to retain a central role in food production.

268

Commercialising academic plant biotechnology distribute a plant biotechnology product are substantial, and are usually only to be found in the long-established industry giants. A noted exception to this rule is the case for diagnostics. In recent years, the agro-chemical sector has been the subject of much industry consolidation (mergers and divestitures), with such companies also taking advantage of new opportunities by acquiring entrepreneurial bioscience companies, seed companies and plant breeders. Strategic in-licensing of technology by the 'ag-chem' sector from biotechnology companies and the universities is now an integral part of the search for new and better products, while these companies have continued to down-size around their core strengths. It is more economic for such organisations to reduce high risk, capital-intensive internal R&D and to source their technology externally. This represents a powerful opportunity for the university-based, market-aware bioentrepreneur. Within spin-out companies from academia, two distinct business models are common. The first, which emphasises a high potential risk/return, technology-centred approach, is based very much along human health biotechnology company lines, where significant private and public investment capital is used to generate a portfolio of patented technologies, and a revenue stream is eventually created, chiefly through licensing to large 'ag-chem' partners. This model requires the input of experienced industry management from an early stage who can maintain investor confidence over the longer-term in the face of inevitable setbacks, and is highly susceptible to market trends and the performance of the sector in general. The second and more prevalent approach is the service or production-based, low risk/return model, where a researcher undertakes to offer an analytical or production-based skill to the market. A number of companies have emerged in recent years offering services such as plant micropropagation, disease detection, and most recently, the application of genetic fingerprinting technologies (such as RAPDs and AFLPs) to the classification of biodiversity, protection of plant breeders' rights and the detection of fraudulent plant produce. The attraction of such companies for many is the fact that they may be grown 'organically', with little initial investment, and in the early stages are able to utilise the 'seed capital' available at the host university (such as laboratory facilities, equipment, reduced overheads, etc.). Additionally, increased European food safety legislation, combined with the influence of quality-conscious supermarket retailers and food processors on the agricultural sector, has created new service/ analytical opportunities. Countering this, managing balanced growth and short-term cash flow can be difficult in such enterprises. The increasing necessity for laboratory accreditation to conduct many analytical services is often incompatible with the university environment. With regard to genetic screening services, the cost of licences for PCR-based technology used as an integral component, and the uncertain legal admissibility regarding plant breeders' rights, has acted as a disincentive to market entry for many fledgling companies. Finally, in the absence of tangible intellectual property being developed in the course of the business venture, the value of the company rests mainly in the goodwill of the client base and expertise of the personnel and founder: successful business exit strategies are consequently more difficult to achieve. The increased, commodity-like use of new bioinformatic (principally genomic) and chemical technologies (such as combinatorial synthesis and robotic screening) in the human 269

Entrepreneurship in Plant Science

biopharmaceutical field is giving rise to an emerging service-based hybrid company model. Although initially capital intensive, the potential to gain significant short-term returns on the basis of offering a highly specialised service to an 'ag-chem' partner can off-set a high cash burn rate incurred as part of the struggle to develop higher value intellectual property. The application of functional genomics, proteomics and glycomics to plant biotechnology will significantly alter the plant biotechnology marketplace [ 11], and this sector will also benefit greatly from continued pharma industry out-sourcing and their need to develop strategic partnerships with such service providers.

The market for plant biotechnology While plant biotechnology strategies are now being deployed for a variety of markets, including the production of medicines, improved forest products and pollution control systems (recently surveyed in ref. 12), the largest and most high profile market sector has been the production of genetically modified (GM) food crops, an area where the influence of down-stream elements such as. governments, regulatory systems, food processors, distributors and endusers/consumers is very clear. Many scientists in Europe have joined the vigorous public debate, primarily in support of the technology as a whole, and few recent plant biotechnology conferences have been complete without a consideration of consumer acceptance of GM crops. In some quarters, the difference encountered in market and regulatory response between the EU and U.S. has generated an erosion of confidence regarding the future growth potential of certain market segments in plant biotechnology. Small European biotechnology companies and universities wishing to commercialise plant biotechnology are more vulnerable than the global agro-chemical sector in this regard, being geographically and financially constricted, and relying heavily on public confidence. Despite this, agricultural biotechnology is currently displaying strong growth, with optimistic projections indicating that the global biotech plant market will increase to US$20 billion by the year 2010 [13]. The technology is at a critical stage of development, and industry marketers are currently mindful of the historical unfavourable precedent set by food irradiation, where European consumer resistance prevented its immediate commercial application [14]: both technologies involve high volume, low value goods intended for human consumption. The EU agri-food market has also been adversely affected by the BSE crisis and lingering public concerns about antibiotic and growth hormone abuse in agriculture; this may be expected to be exacerbated by the requirement for Europe to honour trade commitments with the U.S. on issues such as the admissibility of growth promoters in beef and the use of bovine somatropin for dairy herds. However, despite such difficulties, there are indications that the agricultural commodity sector will indeed be a key biotechnology market for the next millennium, facilitated through the gradual displacement and/or complementation of traditional, chemical-based disease control strategies with biotechnology agents, and the production of GM foods which deliver tangible value to the consumer (especially regarding nutrition and health-promoting properties). The technical opportunity afforded by monogenic traits, combined with the size of the plant disease control market, and the ability to increase the useful commercial life-time of already 270

Commercialising academic plant biotechnology successful products, dictated that the agro-chemical sector would choose herbicide/pest tolerant crops as pathfinder products. Herbicides are currently the largest global agro-chemical market, accounting for about 50% of sales, while insecticides make up 30%, with the remaining 20% largely fungicides [15]. In the U.S., there has been rapid market penetration of GM crops: about 28 million hectares were grown in 1998, and the area is expected to be about 60 million hectares by the year 2000 [16]. However, the strong public reaction to herbicidetolerant GM foods in Europe was unexpected by the industry and bears analysis. Firstly, it must be noted as a matter of interest that genetically modified tomato pastes have not caused the same degree of public distrust observed for herbicide tolerant crops. While one may conjecture that this is primarily due to the association of products such as Roundup Ready TM crops with a ('chemical') herbicide, it strongly suggests that the market acceptance of future plant biotechnology products will be highly case specific. This will heighten the importance of such factors as accurate and comprehensive market research, product labelling and where appropriate/feasible, crop produce segregation for future product launches. Secondly, much public concern has centred on potentially adverse environmental effects due to possible transgene transfer. These concerns are now actively being addressed by industry through the development of second generation products which feature either the use of male sterile clones, the use of hybrids to suppress gene transfer by pollen, and the transformation of mitochondria and chloroplasts [ 17]. Thirdly, the agro-chemical industry failed to discriminate between differences in market factors pertaining to the U.S. and European arenas. The well-documented different eating habits that exist between geographic regions [18] and the differences in how the European public views technology [5] were seemingly ignored by the companies in question. From a marketing perspective, the vociferous adverse public debate may herald the breakdown of the traditionally strong biotechnology industry cohesion which has been observed to date, as individual companies seek to discriminate their technologies from other products or companies with an 'undesirable' image. There are other important factors which will accelerate the market for transgenic food crops. World population continues to increase, with a projected minimum doubling of global food demand predicted by 2050, at which point the population will be about 10 billion people [13]. For example, specific projections for agricultural markets imply a future requirement for cereal production to increase by 41%, meat by 63% and roots and tubers by 40% by 2020 [ 19]. The challenge in producing enough food for the existing population is currently poorly understood by the public. For example, despite an annual investment of US$32 billion on conventional pesticides, crop pests alone reduce global food production by at least one-third [13]. Increased familiarity with biotechnology and modem agricultural practice through government-funded public education, combined with the public endorsement by agencies such as the World Bank [20], will also act to demonstrate the important social and economic role which biotechnological innovation can play. While undoubtedly the market for organically produced foods is growing quickly, this form of agriculture is unlikely to displace conventional agricultural practice in producing quality 271

Entrepreneurship in Plant Science food for the masses. Conversely, the public realisation of the historical dominance of herbicide usage in conventional agriculture (reviewed in ref. 21) is also likely to breed greater acceptance by the markets for technologies which can produce cheap quality food with lower herbicide inputs. In this regard, the benefit of the economic returns from transgenic crops that result from decreased pesticide usage and increased yields must be passed onto the consumer: the Euro has already focused consumer attention on differences in commodity prices between EU member states. Finally, dietary changes in the population and a move to convenience foods which both accompany increased levels of affluence [ 18] will require the maintenance of the quality and volume advantages offered by biotechnology-aided agriculture.

Developing a market-aware project plan While the 'frontier' aspect of biotechnology is still apparent in certain market sectors, it is clear from the poor historic success rate regarding technology transfer in the European academic arena that traditional, 'passive', management of academic research programmes, and accompanying institutional support structures, are not optimal. To try to redress this situation, a number of national biotechnology commercialisation programmes have been established within Europe since the late nineteen eighties, and these have been complemented by the EU Framework research programmes, with both measures providing much-needed support for the fledgling industry (encompassing the provision of finance, training, brokerage services and patent information). While the overall success of such structures is still being assessed, a common feature of many is the promotion of market ('end-user') awareness at an early stage in project life. Additionally, although historically the compartmentalisation of the technology transfer function to post-project completion was common, newer philosophies advocate a more integrated approach to commercialisation. 'Active' project management involves the use of established project planning routines to drive a project in a time and cost-effective fashion to a point where a commercialisation route can be identified and secured. It requires a designated project manager, a comprehensive project plan and a regular written reporting system which charts the progress of the research against agreed targets, some of which will be important signals for the involvement of industrial liaison support staff. Visual representations of project plans, either defined in terms of task duration by horizontal bars (the Gannt chart) or the linkage of tasks through box networks (PERT- Performance, Evaluation and Review Technique - charts) are now commonly used by researchers as a component of research grant applications. The construction of such diagrams has been made easier since the early nineties by the availability of computer software, such as Microsoft Project TM. Time and task dependencies are translated into a series of deliverables and milestones which provide decision points to approve or revise the project plan. Typical research milestones for a biotechnology project would include the demonstration of biological proof of principle, successful trialling, and proof of efficacy. The application of such project plans to academic research can sometimes meet with a degree of resistance from researchers, who often feel that detailed long-term planning (over 1 - 2 years) is incompatible with the unpredictable nature of the research process itself. However, the project plan can be made a more meaningful component of the work if it is composed around a draft, 'ideal' product or service specification, which is drawn up at the project outset 272

Commercialising academic plant biotechnology to reflect both current and anticipated market needs; examples of such factors are provided in table 3.

Table 3: Components of product specification which form the centre-piece of the project plan

Technology/service identification: a succinct working definition of what the project is trying to achieve, defined in a market context Target market: a systematic break-down of all market opportunities. For example, a single disease may affect a number of different crops, thereby adding value to an experimental treatment or diagnostic

The questions which one is Client base: who will buy the service or product and what is known about the dynamics forced to confront in such a of the existing market (usually deduced from strengths/weaknesses of competitor document will quickly products/services) Protection of intellectual property: how will this be achieved (patents? trade marks? eliminate research stratesecrecy?) and is there a precedent available for comparison? gies which are likely to be Registration: what countries represent the most valuable markets? irreconcilable with market Product profile: what is the desired action of a new disease treatment and how should it requirements. Also, it perform in the field to achieve optimal market acceptance? What will be the major selling points of a new diagnostic? The profile is very often defined in terms of the should be noted that new limitations of existing products on the market. technology or service speciPresentation/application/use pattern/storage: in what form will a new biocontrol agent be sold, how should it be applied and what are the required conditions for fications are very often depreservation? Is a diagnostic required at the 'farm-side' or will it be carried out in a fined in the light of defilaboratory? ciencies or strengths of exTarget price and profitability/cost-benefit analysis: what are competitors charging for existing products or services? Can a premium be added for superior performance isting competitor products, characteristics? Can the technology be manufactured cost-effectively? necessitating that such inProduct development milestones: looking beyond the research phase, what safety tests formation be sourced at the will be required by regulatory agencies for product approval? outset. This also has speTime to market: first major market approval wheN? cial relevance to initiating the process of valuing the technology: estimating the worth of biotechnology assets is often refractory to conventional commercial evaluation, with much of the potential in the technology effectively locked up in a researcher's track record, experience and publication history [22]. However, in many cases the technology under development, if successful, will displace or complement existing competitor products for which market data (volume, value, geographical spread) is usually available, or may be inferred. The development of an iterative strategy for determining market data based on the progress of competitor products therefore becomes very important. This working technology specification is not immutable, but should take into account the best available market intelligence at any one point of time. The project plan is assembled around this summary, and is composed in a 'backwards' fashion from this point (reviewed in ref. 6): the ultimate market aim of the project defines the major steps which must be taken to achieve the technical goal(s). Therefore, unexpected results which modify the project plan in the intervening time can be assessed against the technology specification to gauge their potential impact on the development path. Project planning, with inherent success or failure in achieving deliverables and milestones, requires active monitoring. Outside of companies or research development organisations, the concept of employing a dedicated project manager to perform such a function is not common, and it usually falls to the Group Leader to assume this role. The ensuing additional time burden, combined with the possible blurring of academic-commercial goals, can lead to problems for a project with a commercially-focused aim, or where a company sponsor/collaborator requires detailed and regular feed-back on project progress. 273

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Identifying a commercial track Attracting the attention of a potential technology licensee, initiating a technology-based spinout company or offering laboratory services, all share an underlying theme which is based on fulfilling the needs of the market. A demand must either be satisfied or created. A proprietary technology position must be adequately protected at the earliest possible juncture, a suitable development path identified, and the product or services progressed to the commercialisation phase with the minimum of delay so that unnecessary costs may be avoided and returns may be maximised. The conventional technology transfer model present in many European academic institutions relies on the interaction of the research group leader with an in-house industrial liaison function (which usually happens at project close), who then interface in a variety of ways with potential technology developers or service users. The industrial liaison manager will both pro-actively canvass the attention of industry/investment contacts, while also passively acting as a 'gateway' for industry technology acquisition managers. Understandably, unless such a person has a background in biotechnology, the necessary information assimilation challenge to market the technology is magnified many-fold, while the opportunity to achieve technology transfer targets through 'easier to handle' areas (such as computers, information technology, engineering) may dictate that biotechnology is relegated in the priority stakes. However, it is arguably a failure to implement a technology transfer strategy early on in project life, as significant project deliverables and milestones begin to be achieved, that represents the most serious limitation to current practice. Commercially significant results which can be patented may emerge early on in a project, which without expert intellectual property advice may be ignored or compromised. The academic ethos of openness and information exchange, the high turnover of researchers in laboratories and the lack of clear management structures, make it very difficult to maintain confidentiality for long periods of time without taking specific measures. The clear, unambiguous results (proof of principle and efficacy) and exacting laboratory record keeping necessary to obtain and defend a patent require an awareness of competitor prior art (existing patents in the area) and a rigorous operating procedure engendered through proximity to the commercial process. Finally, the long time-lines required to conclude an agreement with potential technology licencees, and the number of different organisations which must usually be approached before success is achieved, dictate that such a process be initiated as soon as an intellectual property protection strategy has been identified. In the absence of industrial liaison staff with an expert knowledge of biotechnology, it will usually fall to the Group Leader (or where available, project manager) to lend meaningful, market-aware support in the technology transfer process. To complement the discussion on the importance of understanding market and project management issues in identifying commercially valuable lines of research, the following sections outline some of the important considerations with respect to protecting and marketing technologies. It is imperative to realise that such issues require the adoption of a credible, team-based approach between an experienced technology transfer professional and the research Group Leader.

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Protecting intellectual property A detailed discussion of intellectual property protection, technology marketing and licensing is beyond the scope of the present paper. However, it is instructive to briefly examine the inter-dependence of these tasks with a view to identifying critical issues which typically arise in the case of biotechnology. The highly competitive bioscience market demands strong intellectual property protection, and the chosen strategy should be decided at project initiation. Available options include patents, plant breeders' rights, trade secrets, trade marks, copyright and encryption (relevant to computer software). A combination of these may be invoked to maximise the productive economic life-time of a technology. Patenting is the most common mechanism in biotechnology and will be discussed here. Accessible overviews of intellectual property protection [23] and the key differences between U.S. and European patent law pertaining to plants are available elsewhere [24, 25, 26]. In filing a patent application, the bioentrepreneur sets in motion a series of events for which prior preparation is essential. The patent system provides the strongest legal implement to protect complex inventions which are typical of biotechnology, and the most important immediate aspect of this is the achievement of a 'priority date' on filing the application, in order to prove when the invention was first registered. A patent application also represents a potent marketing tool for the technology, the existence of which may be productively used to attract the attention of a potential licensee or investor, who may review it (and, if available, a favourable patent search report) under the terms of a confidentiality agreement. In preparing a patent application, the bioentrepreneur needs to be vigilant in providing the answers to questions which may have commercial relevance over the longer term. For example, has a judicious balance been achieved in providing the data necessary for fulfilment of the patent application requirements, without unnecessarily disclosing valuable (though possibly non-patentable) secret know-how? Have all the possible uses for the technology been listed in the claims, thereby removing opportunities to 're-engineer' around the technology? Will cross-licensing of other patents be necessary to make use of the invention, a common facet of DNA inventions [27] and who owns the rights to such technologies? Can the intellectual property protection afforded by the patent be productively consolidated by registering a trade mark? A trade mark may be a useful adjunct to patenting, since it lasts indefinitely, and can identify a product or process for long after a patent has expired. It will be of special relevance to plant biotechnology products which have tangible consumer health benefits. While it sometimes makes good sense for a biotechnology company to 'add value' onto a patent by developing the technology beyond the research phase, thereby affording the chance to negotiate better terms from a major industrial licensee, the high expense incurred in doing this, plus an ignorance of development issues and market dynamics, rarely makes it a viable strategy for a university to undertake. Bioentrepreneurs need also be aware of the significance of the patent term, which is typically 20 years from the date of filing an application, as unnecessary delays or sub-optimal progression through development programmes which adversely affect the date of market launch, will reduce the commercially useful life-time of the patent. Additionally, a patent can be most realistically viewed in the context of a marketing plan, and therefore a product manufacturer and distributor is in the best position to decide which countries (markets) that patent protection is required.

275

Entrepreneurship in Plant Science The decision to patent will ultimately be based on whether the criteria for patentability can be met (novelty, non-obviousness - 'to those skilled in the art'- and industrial applicability) and whether there is an actual market for the invention which will allow a company to recoup the cost of project development and patenting, while making a profit within the patent life-time. The advice of a qualified patent agent should always be sought for the former, since patent law is notoriously complex, with precedents being continually established as to the validity or otherwise of existing patents via the courts. The requirement to attain patent protection in each of the individual countries which represent major markets, and the need to become familiar with typical time-frames which elapse between the patent filing date, issuance of a preliminary search report and formal patent examination and grant, dictate that bioentrepreneurs familiarise themselves fully with national, European Patent Convention (EPC) and Patent Co-Operation Treaty (PCT) rules. While initial patenting costs are manageable by colleges, to avoid the high longer-term costs, the bioentrepreneur must successfully negotiate a technology licensing agreement (and transfer all subsequent patent costs) with a development partner as soon as possible after the filing date. Considerable expense begins to be incurred when a decision must be reached regarding the countries for which protection is sought, with accompanying language translation fees. The costs associated with obtaining and maintaining patent protection over the first 4 years in typical biotechnology markets (US, Europe and Japan) may be expected to be in excess of Euro 60,000. Finally, an aspect of patenting sometimes overlooked by academic bioentrepreneurs is the fact that such tools are only useful if the commercial organisation which brings it to the market has the necessary resolve and financial resources to actively police and enforce its terms. The true strength of a patent is therefore only determined in a court of law, when infringement or unlawful use has occurred, and such disputes are commonplace in the pharmaceutical and biotechnology sectors. Protecting food/feed plant biotechnology inventions 'in the field' will pose additional challenges to the biotechnology sector, including the need to implement new agreement structures with farmers to control transgenic seed use and saving. A logical development of this is the 1998 US Patent No. 5,723,765 ('Control of plant gene expression'), which was granted jointly to the US Department of Agriculture and the American Delta and Pine Land Company, outlining technology to modify plants in order to prevent seeds from germinating in the next generation [28]. This technology, rapidly dubbed 'terminator' by opposition groups, has been the subject of fierce public criticism, being viewed as a potential threat to the livelihoods of third world farmers. Acceptance of such technologies will probably depend on a clear demonstration that the surplus of increased yield due to transgenic varieties will make up for the extra cost of buying new seed every year.

Laboratory security issues An integral component of managing the development of a valuable piece of intellectual property will be taking adequate measures to maintain confidentiality within the research team and also laboratory security. Additionally, it is imperative that projects from different research sponsors be adequately demarcated, with no mixing of different funding sources, so that possible later ownership disputes are avoided. For these reasons, all laboratory staff should 276

Commercialising academic plant biotechnology sign a binding confidentiality agreement which outlines clearly their obligations, and a culture of confidentiality should be engendered throughout the research group so that this awareness is integrated into the normally open university setting. While it goes without saying that publications should be reviewed for the possible inadvertent disclosure of commercially-sensitive information, less obvious sources of disclosure may include posters, student theses and departmental seminars, amongst others. Changes in U.S. patent law permitting foreign applicants to submit laboratory notebooks as proof of 'first to invent' dictates that all research results are recorded as per 'Good Laboratory Practice' (GLP) guidelines, with such provisions as permanently bound notebooks with each entry signed and dated.

Attracting the attention of developers The ultimate goal of the academic bioentrepreneur is to effect the profitable transfer of technology from the academic environment into the market, generating a financial return in the process. Historically, this has been achieved principally through technology licensing, in which the technology is 'leased' to a company in return for a structured, financial reward. In the case of biotechnology, it is rare that a completed 'prototype' can be handed over at this stage, and there is usually further research remaining to be completed within the academic environment. License arrangements may include an up-front cash payment on signing of the agreement and funding for further research, plus an agreed royalty rate on net sales; the latter must usually be negotiated in good faith at a later stage when development issues are examined and understood by both parties, but a provision for external arbitration should be incorporated into the contract as a contingency, in the event that future agreement cannot be reached. Variations in licence structures may include off-setting the up-front payment against future royalties, or linking lump-sum bonus payments to successful completion of development milestones. The area has been reviewed extensively elsewhere [23, 29], but the need for good market information (such as knowledge of precedent deals in similar technology areas) and project management records (existing and projected research costs, patent costs, etc.) in deciding the terms of the license is obvious. However, the route between identifying a valuable piece of intellectual property in the laboratory, to securing a technology development partner, has not received the same level of critical analysis. The emphasis within the pharmaceutical sector on R&D, and the current dependence on bolstering in-house research efforts by appropriating technology externally, represents an advantage to the prospective technology licensor: the 'technology acquisitions manager' is a well-established role in the industry and is pro-active in the search for new technologies which will complement an organisation's portfolio. Countering this, there are now many technology marketers competing for this attention. Personal industry contacts, technology brokerage firms, research support organisations, press releases and mailshots are only some of the common methods by which contact may be established with potential technology licensees. This is an information-driven process, but the importance of people in the technology transfer equation is also paramount. While a final deal may be secured between two organisations on the basis of performing extensive technology, intellectual property and market assessments, a high level of technical and market risk will 277

Entrepreneurship in Plant Science usually remain, and the generation of trust and good faith between the negotiating individuals is essential to sustain a development partnership through the inevitable setbacks. An integral part of this is comprehending and respecting the operational structures of the commercial environment. The goal of the academic bioentrepreneur must be to convert the company contact into a 'technology advocate', who will 'champion' the project through the industry line management system: this may be through such means as being responsive to information requests within specified time deadlines or demonstrating a good awareness of market considerations and constraints. The basic unit for marketing biotechnology inventions is the 'Non-Confidential Project Summary', which will be used by the recipient to decide whether a technology is worth more detailed investigation. The required contents of such a document are best appreciated if considered in the context of the needs of a potential technology licensee or investor. For example, a technology evaluation/acquisitions manager from a large pharmaceutical company will typically receive numerous unsolicited approaches by academic and entrepreneurial bioscience companies in search of development relationships. The first goal, therefore, must be to compile a document which will stand out from competitors, and interest the reader sufficiently to move onto the next stage of contact. The technology should be explained in non-confidential terms, highlighting advantages over existing products, and demonstrating a good level of market awareness. The stage of development of the technology should be outlined, including details on the achievement of commonly employed research milestones (especially the existence of a patent application). These details should be summarised in an executive summary at the start of the document. The emphasis at the start of a search for a technology licensee should be to contact as many companies as possible. Many factors influence the appeal of technology opportunities to companies, including the existence of novel protected intellectual property, the credibility and track record of the research group, and the strategic fit for the company's technology portfolio. Serendipity and perseverance also aid this process. All meetings and more detailed confidential discussions which result should be conducted under the terms of a 'Confidential One-Way - Non-Disclosure Agreement', or if an exchange of information is envisaged, a 'Two-Way Secrecy Agreement'. Negotiations may also involve an evaluation of a biological material by a potential developer, who will undertake to independently verify results as part of their 'due diligence' procedures, and this should be conducted under the terms of a 'Materials Transfer Agreement'. Agreements define the rights and obligations of negotiating parties and tend to follow a set format, depending on the specific undertaking. An excellent account of agreement structures has been published elsewhere [23]. While the 'small print' on legal agreements should always be studied carefully, there are at least two 'standard' aspects which bear mentioning. Firstly, such agreements require the signature of an 'authorised person'; in the case of academic institutions, this is rarely an academic member of staff, and appropriate advice should be sought. Secondly, the binding law under which the terms of an agreement will be examined in the case of a dispute is relevant: wherever possible, the academic institution should insist that their own national laws should apply. Litigation is an extremely costly process without the added expense to travel to another country to pursue a legal action. 278

Commercialising academic plant biotechnology If a potential technology licensee is sufficiently interested in the technology on offer, an extensive assessment of intellectual property strength, strategic fit, technical sophistication and market potential will be undertaken, with success at this stage heralding the start of a license negotiation. As per the research project plan, time deadlines should be set by the college for this process to be achieved, and where appropriate, a suitable option payment secured in exchange for a halt to discussions with other interested commercial parties.

Conclusions It is a widely held belief that considerable untapped potential for novel industrially-relevant biotechnology exists in the European academic sector. Such beliefs usually do not take into account the practical challenges of the commercial world: it has been reported that the chances of success for a new idea in research science may be of the order of less than 1 in 1000 [22]. However, in comparison to the U.S., there is no doubt that Europe is less efficient at exploiting academia as a source of national economic development. Complex and entrenched cultural and organisational factors permeating European institutions [30], combined with 'macro' factors (such as lack of venture capital, risk-averse ethos, absence of regulatory clarity) [5] have all been identified as being responsible for the failure to effectively capitalise on such opportunities. Both national governments and the European Parliament have begun the process of altering the climate for investment in this sector, through such mechanisms as the establishment of dedicated biotechnology support organisations [9], fostering the formation of biotechnology clusters and science parks [31], provision of EU Framework Programme funding and establishing ancillary structures for technology networking. The success of such 'top-down' structures is beginning to become apparent, but major challenges which remain to be addressed, and which have a profound effect on plant biotechnology, include the absence of clarity in the patenting of certain biotechnology inventions and the need for regulatory harmonisation among EU member states. This review has concentrated on key information and organisational aspects of developing, protecting and marketing biotechnology intellectual property when working from an academic base. In considering this brief, 'bottom-up', analysis of the challenges facing academic bioentrepreneurs, it is apparent that much of the problem lies in a failure to adequately appreciate the commercial perspective- there is a need to adopt the 'language' of biocommerce. Additionally, despite the fact that biotechnology arguably represents the archetypal 'technology push' type product area, with few precedents against which to benchmark development strategies, conventional approaches to technology transfer are routinely employed in the European arena (with notable exceptions) which adhere to structures established for older technology sectors. Academic institutions have a crucial role to play in changing the landscape for biotechnology development. Within undergraduate science degree courses, there are real opportunities to complement traditional technical learning with market perspectives on biocommerce, information management and project planning. Similarly, the availability of more advanced postgraduate modules as part of higher degrees (encompassing topics such as finance, market research, innovation and entrepreneurship) would be a useful adjunct to purely technical research. A logical extension of this would be the creation of multi-disciplinary research projects 279

Entrepreneurship in Plant Science b a s e d a r o u n d p r o p r i e t a r y lead b i o t e c h n o l o g y p r o d u c t s / s e r v i c e s , identified b y the h o s t university for r e v e n u e g e n e r a t i n g potential, and b r i n g i n g t o g e t h e r r e s e a r c h e r s f r o m science, b u s i n e s s m a n a g e m e n t / a d m i n i s t r a t i o n , m a r k e t i n g , i n f o r m a t i o n t e c h n o l o g y and law, a m o n g s t others.

E u r o p e m u s t b e g i n to test and d e v e l o p n e w m o d e l s for b i o e n t r e p r e n e u r s h i p w h i c h will

p r o v i d e a strong basis for e c o n o m i c d e v e l o p m e n t in the l o n g - t e r m .

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AUTHOR INDEX Abad. Pierre ..................................................................... Andr6. Bruno ................................................................... Arcioni. S ......................................................................... Aristizibal. F. A ............................................................... Arthur. Eddie ................................................................... Bachem. Christian ........................................................... Bennett. Malcolm J .......................................................... Bleve. Teresa .................................................................... Blok. Vivian ..................................................................... Bonnel. Eric ..................................................................... Borkhardt. Bernhard ........................................................ Bottalico. A ...................................................................... Bowler. Chris ................................................................... Bowra. Steve .................................................................... Burssens. Sylvia .............................................................. Bush. Max ........................................................................ Casadoro. Giorgio ............................................................ Castresana. Carmen ......................................................... Chatot-Balandras. Catherine ........................................... Chen. Xinwei ................................................................... Concilio. Luigi ................................................................. . . Darmani. F. ....................................................................... De Martinis. Domenico ................................................... de Vries. Sacco ................................................................ de Vries. Gert E ............................................................... Dean. Caroline ................................................................. del Campo. F. F................................................................ Delseny. Michel ............................................................... Dijkwel. Paul ................................................................... Dix. Phil J ........................................................................ Doeswijk-Voragen. Chantal ............................................ Dopico. Berta ................................................................... Doyle. Owen .................................................................... Duroux. Meg .................................................................... Edwards. Keith J .............................................................. Fenoll. Carmen ................................................................ 283

159 225 257 177 29 223 149 159 159 81 245 195 109 29 13 245 133 157 81 81 81 257 123 113. 141 11 115 177 77 115 59 245 245 3 115 91 159. 177

Frommer. Wolf B ............................................................. 225 Gatehouse. John A ........................................................... 159 Gavaghan. Helen .............................................................. 105 Gebhardt. Christiane ........................................................ 81 115 Gendall. Tony ................................................................... Gheysen. Godelieve ......................................................... 159 Gojon. Alain ..................................................................... 225 Gray. J.C. ......................................................................... 59 Grundler. Florian M.W. ................................................... 159. 169 225 Harling. Hinrich ............................................................... Helder. Johannes .............................................................. 159 Herreros. E....................................................................... 177 Heselmans. Marianne ...................................................... 221 Hesselbach. Josef ............................................................. 81 Hobbs. Douglas ............................................................... 29 Hollricher. Karin .............................................................. 75 Hutchison. Claire ............................................................. 115 .. Ivanova. Hue .................................................................... 215 Johanson. Urban .............................................................. 115 189 Kalantidis. Kriton ............................................................ 59 Kavanagh. T.A. ................................................................ 215 Keerberg. Hille ................................................................ Keerberg. Olav ................................................................. 215 Kema. G.J.H. ................................................................... 195 235 Kossmann. Jens ............................................................... Labrador. Emilia .............................................................. 245 Lange. Lene ..................................................................... 245 Lerbs.Mache. S ................................................................ 59 115 Levy. Yaron ...................................................................... Lindsey. Keith .................................................................. 159 Lister. Clare ..................................................................... 115 Logrieco. A ...................................................................... 195 Macknight. Richard ......................................................... 115 Magnien. Etienne ............................................................. 27 Manousopoulos. J ............................................................ 189 Marchant. Alan ................................................................149 Made. Andrew ................................................................ 183 McCann. Maureen ........................................................... 245 284

Medgyesy. P..................................................................... Medley. Terry L ............................................................... Mengiste. Tesfaye ............................................................ Merrick. Mike .................................................................. Metzlaff. Karin ................................................................ Metzlaff. Michael ............................................................ Miller. Anthony ................................................................ Mordhorst. A.................................................................... Morvan. Claudine ............................................................ Muiioz. Francisco ............................................................ 0’Sullivan. Dona1 M ....................................................... Oberhagemann. Pea ......................................................... Ohl. Stephan .................................................................... Oomen. Ronald ................................................................ Paolocci. F........................................................................ Parnik. Tiit ....................................................................... Paszkowski. Jerzy ............................................................ Patriarca. Eduardo ........................................................... Pavanello. Anna ............................................................... Peltier. G.......................................................................... Pereira. Andy ................................................................... Perrone. G ........................................................................ Peugnet. Isabelle .............................................................. Puzio. P.S. ........................................................................ Regierer. Babette ............................................................. Reiss. Bernd ..................................................................... . . . Ritieni. A .......................................................................... Ritter. Enrique .................................................................. Rudolph. Brian ................................................................ Sagen. Kristina ................................................................ Salamini. Francesco ......................................................... Sanz-Alfkrez. S ................................................................ Schafer. C ......................................................................... Schafer.Preg1. Ralf .......................................................... Schell. Jeff ....................................................................... Schols. Henk .................................................................... Shields. Robert ................................................................ Simpson. Gordon ............................................................. 285

59 43 47 225 7 67 225 59 245 245 91 81 159 245 257 215 47 225 133 59 101 195 245 169 235 45. 225 195 81 245 159 81 177 59 81 17 245 159 115

Sivadon. Pierre ................................................................. Smart. Bonita ................................................................... Speulman. Elly ................................................................ Spolaor. Silvia .................................................................. Springer. F........................................................................ Swarup. Ranjan ................................................................ Sorensen. Susanne ........................................................... Tappeser. Beatrix ............................................................. Torney. Keri ..................................................................... Trainotti. Livio ................................................................. Tsaftaris. A ....................................................................... Tsagris. M........................................................................ Turchetti. V....................................................................... Tzortzakaki. S.................................................................. Uijtewaal. B ..................................................................... Ulvskov. Peter .................................................................. Uribe, X ........................................................................... van Loon. L.C. ................................................................. Van Montagu. Marc ......................................................... Vincken. Jean-Paul .......................................................... Visser. Richard ................................................................. von Wirkn. Nicolaus ........................................................ Voragen. Alphons ............................................................ West. Joanne .................................................................... Williams. Gwilym ...........................................................

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1 15 115 101 133 235 149 245 37 115 133 189 189 257 189 59 245 177 203 13 245 245 225 245 115 265

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