No title

ADVANCES IN GENETICS

Edited by

JOHN G. SCANDALIOS

THEODORE R. F. WRIGHT

Deportment of Genetics North Carolina State University Raleigh, North Carolina

Department of Biology University of Virginia Charlottesville, Virginia

VOLUME 26 Edited by

JOHN G. SCANDALIOS Deportment of Genetics North Corolina State University Raleigh, North Carolina

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

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COPYRIGHT 0 1989 BY ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

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CONTRIBUTORS TO VOLUME 26 Numbers in parentheses indicate the pages on which the authors’ contributions begin.

A. EISENSTARK (991, Division of Biological Sciences and Department of Microbiology, University of Missouri, Columbia, Missouri 65211 INNAN. GOLUBOVSKAYA (1491, T h e N. I. Vavilov All-Union Institute of Plant Industry, Leningrad, 190000, Union of Soviet Socialist Republics HOSNIM. HASSAN (651, Departments of Food Science, Microbiology, and lkxicology, North Carolina State University, Raleigh, North Carolina 2 7695 DAVIDL. MULCAHY (11, Department of Botany, University of Massachusetts, Amherst, Massachusetts 01003 ERCOLE OTTAVIANO (11, Department of Genetics and Microbiohgy, University of Milan, 20133 Milan, Italy

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PREFACE

Advances in Genetics was the first serial publication devoted solely to the burgeoning field of genetics. The series was founded in 1946 by Dr. Milislav Demerec, then director of the Genetics Department at the Carnegie Institution of Washington in Cold Spring Harbor, New York. The stated purpose for the series was “that critical summaries of outstanding genetic problems, written by prominent geneticists in such form that they will be useful as reference material for geneticists and also as a source of information to nongeneticists, may appear in a single publication.” Over the years, the goals set forth initially have been more than fulfilled, and a lasting tradition of excellence has been established. In more recent years, our field has experienced some revolutionary developments emanating from the enormous technological advances that have occurred. Recombinant DNA and related molecular technologies now make possible the intricate manipulation of genetic information, in virtually every cell type and organism, that could not even have been imagined at the time when Advances in Genetics was initiated. These developments have led to an unparalleled information explosion. Because of the diversity of genetics as a science, Advances in Genetics has adhered to the policy of publishing a series of outstanding but largely unrelated articles in each volume, and it is felt that this policy should be maintained. However, the editors will, on occasion, depart from this format and review periodically a central topic in a special “topical” or “thematic” volume, as we feel this is essential in view of the extremely rapid developments in genetics. nYo such volumes (Volumes 22 and 24) have been published to date and have been well received by the scientific community; others are in preparation. Our purpose is not merely to inform but also to stimulate the reader, whether a beginning or an advanced scholar, to explore, question, and, whenever possible, test various hypotheses advanced herein. We hope that each volume covers some material of lasting value, in view of the very rapid developments in this field. ix

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PREFACE

The death of my friend and fellow editor, Professor Ernst W. Caspari, in 1988, has left a void and he shall be missed. I am, however, pleased to welcome Dr. Theodore R. F. Wright, University of Virginia, as my new co-editor. JOHN

G. SCANDALIOS

GENETICS OF ANGIOSPERM POLLEN Ercole Ottaviano* and David L. Mulcahyt *Department of Genetics and Microbiology, University of Milan, 20133 Milan, Italy tDepartment of Botany, University of Massachusetts, Amherst, Massachusetts 01003

I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. The Male Gametophytic Generation. . . . . . . . . . . . . . . . . 111.

IV.

V.

VI. VII.

A. The Developmental Stages of the Male Gameto B. Genetic Control of Pollen Development Garnetophytic (Haploid) Gene Expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Pollen Mutants and Distorted Segregation.. . . . . . . . . . . . . . . . . . . . . . . . . . . B. Experimental Gametophytic Selection .... ..... C. Isozyme and mRNA Analysis.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gene Expression and Gametophytic Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Gene Expression in Pre- and Postpollination Phases B. Genetic Overlap and Garnetophytic Specific Genes . . C. The Interaction between Pollen and Pistil.. . . . . . . . . . . . . . . . . . . . . . . . . . . Gametophytic Gene Expression and Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Pollen Competition in Natural Populations. . . . . . . . . . . . . . . . . . . . . . . . . . . B. Pollen Competition in Crop Plants. . . . . C. Evolutionary Rate under Gametophytic ................. D. Gametophytic Selection and Genetic Load.. . E. Angiosperm Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................................. Practical Applications. . . . . . . . . . A. Male Gametophytic Selection as a Breeding ............... B. Genetic Manipulations of the Gametophytic Conclusions References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. Introduction

The angiosperm life cycle consists of two alternating phases-a diploid, morphologically elaborate and conspicuous sporophyte and a much reduced, haploid gametophyte. The former serves, directly or indirectly, as the sustenance and, for much of the world, the shelter and fuel of our species, while the latter is generally unseen and even unknown except to specialists and hay-fever sufferers. Many individuals have commented on the resemblance of the microgametophyte, the 1 ADVANCES IN GENETICS, Vol. 26

Copyright (C 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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pollen, to microorganisms. The haploid genome should expose recessive mutations to selection and large population sizes should generate both rare alleles and improbable allelic combinations. Together, these qualities should allow a significant increase in the speed with which a n angiosperm is able t o evolve. This possibility was first clearly appreciated by Buchholz (1922). However, several considerations weighed against pollen selection playing a n active role in sporophytic adaptation: gametophyte and sporophyte differ so greatly in morphology, physiology, and function that adaptations in one phase of the life cycle might be irrelevant or even detrimental to the other. In fact, Brink and MacGillivray (1924) stated that “Chaos would certainly result if conditions were such that the style regularly functioned as a gametic sieve.” Haldane’s (1932) concerns were more explicit: “A gene which greatly accelerates pollen tube growth will spread through a species even if it causes moderately disadvantageous changes in the adult plant.” For this reason Haldane suggested that genes which function in one portion of the life cycle should not function in the other. Support for this suggestion was provided by the fact that, in Zea mays, one of the first pollen-expressed genes to be studied, Waxy, was not expressed in the sporophytic portion of the life cycle (at least, not beyond that transitional tissue, the endosperm). Haldane quoted a second observation which seemed to underscore the danger of haploid gene expression; in Drosophila, spermatozoa function perfectly well even if they lack a substantial part of the haploid genome (Muller and Settles, 1927, quoted in Haldane, 1932). Finally, a genetic syllogism, often suggested to the authors, seemed to argue against the expression of sporophytic genes in the pollen. If sporophytically expressed genes are expressed also in pollen, they should have a n effect on pollen tube growth rate or transmission. That is, fertilization should not be random. Fertilization generally is random. Therefore, sporophytically expressed genes are not generally expressed in the pollen. Each of these three arguments against genetic overlap between the two phases of the life cycle has been answered, or at least vitiated. Although the gene Waxy is expressed only in the gametophytic portion of the life cycle, many others, discussed below, are active in both. Furthermore, the pollen tube, as a microgametophyte, is not equivalent to a spermatozoan; in fact, it carries two sperm cells within it. The Muller and Settles observation is thus irrelevant to this issue. Finally, it was shown that the failure to detect significant deviations from Mendelian expectations might be due to the sizes of samples employed in most studies. The 7324 observations of round versus wrinkled peas reported by Mendel were sufficient to detect, with 90% certainty,

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deviations from random fertilizations of 1.97% or greater. Subsequent studies almost invariably used smaller samples and thus, expectedly, the only deviations from random fertilizations reported involved genes with sublethal or greater effects (Mulcahy and Kaplan, 1979). J u s t as the failure to detect nonrandom fertilization does not demonstrate that fertilization is indeed random, detecting errors in the arguments against pollen selection of sporophytically expressed genes does not prove that such expression exists. Only demonstrable sporophytic effects of pollen selection could do that. The first of these was perhaps the demonstration that, in the dioecious herb, Silene alba (Melandrium album), pollen grains carrying the Y chromosome grow more slowly than do those carrying the X chromosome (Correns, 1927). This was probably also the first report of stylar influence on pollen tube growth rates, since some pistillate parents produced nearly 100% pistillate offspring when excessively polIinated but others barely deviate from Mendelian expectations under the same circumstances. Different growth rates for X and Y chromosome-bearing pollen tubes occur in other species (reviewed in Mulcahy, 1967). Although these differences might be functionally significant, gene expression here certainly represents a highly atypical case. A more general demonstration of pollen selection effects on the sporophyte were the reports by Ter-Avanesian (1949, 1978) that, among progeny of Gossypium, Vigna, and Triticum, morphological variation was increased by applying limited amounts of pollen in crosses. Since the pollen in each case came from single pollen sources, the only explanation for these observations is that pollen tubes which grow slowly give rise to sporophytes which exhibit morphological extremes. Mulcahy (1971, 1974) reported significant and positive correlations between pollen tub growth rates and sporophytic qualities in 2. mays, but since pollen from different lines was being compared, the differences reported could have been determined largely, or even exclusively, by the sporophytic pollen source. However, varying the quantity of pollen (from a single plant) did have a n effect on sporophytic growth rate in Petunia hybrida, and, when the resulting F1 individuals were selfed (with no effort being made t o control pollen quantity in the selfing), the F2 progeny also differed significantly. Fz progeny from the F1 individuals that were produced with the most intense pollen competition were the most rapidly growing Fz progeny (Mulcahy et al., 1975, 1978). Recent investigations have concentrated on quantifying the extent to which genes are expressed in both parts of the life cycle, on basic and applied uses of this overlap, on the influence of the style on pollen tube growth, and on the significance of pollen competition in natural populations.

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II. The Male Garnetophytic Generation

A. THE DEVELOPMENTAL STAGES OF THE MALEGAMETOPHYTE The following discussion concerns the general ontogeny of pollen and, although the sources involve diverse species, the generalizations seem to be quite secure throughout the angiosperms. Emphasis will be on phenomena which may hold significance for gene expression in pollen. Prior to meiosis, sporogenous cells are interconnected by normal plasmodesmata, each approximately 250 A in diameter, and, in totality, representing less than 1%of the contact area between cells (J. Heslop-Harrison, 1971a,b, and personal communication). However, early in the meiotic prophase, that is, toward the close of leptotene, other and larger channels, up to 2.5 pm in diameter, develop between meiocytes. The diameters of these are sufficient to allow the movement of organelles between meiocytes, and in Lilium henryi these channels occupy over 10% of the area between meiocytes, nearly 24% in Dactylorchris fuchsii. Thus interconnected, the meiocytes then function as a single syncytium. Tapetal cells also become linked together by unusually large channels, but meiocytes and tapetum are not interconnected. During zygotene, there begins the deposition of callose (/3-1,3-glucan)in the walls of the individual meiocytes. Extending from the corners of individual cells, these impermeable and imperforate deposits eventually result in the complete isolation of meiocytes from each other and from all other cells. Channels between meiocytes persist, in L. henryi, until metaphase I. When the first meiotic division is accompanied by wall formation, as it is in the monocots, the phragmoplast is coated with callose at the end of telophase I, thus making the two daughter cells separate physiological units. In the dicots, delayed, simultaneous cytokinesis postpones this independence of the cytoplasts until the end of meiosis. In addition to its effects on the time of physiological independence, the timing of cytokinesis influences also the morphology of pollen tetrads. Successive cytokinesis in the monocots restricts the movement of the cytoplasts and thus, when monocots shed pollen in tetrads, e.g., in Typha latifolia, the grains are arranged in a single plane. With dicots, successive cytokinesis allows freedom of movement and the microspores assume the tetrahedral configuration typical of dicot tetrads. Determination of exine pattern occurs during prophase of meiosis and a t the level of the plasma membrane in young microspores of

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Lilium. Dickinson and Sheldon (1986) characterize this determination

as a “self-assembly” in which neither organelles nor the cytoskeleton appear to be directly involved. Instead, the presently unknown determinant of pattern appears to be synthesized during meiotic prophase (Dickinson, 1987). The position of the colpus, which can be modified by centrifugation of microspores, is linked with the orientation of the meiotic spindle (Heslop-Harrison, 197lc; Sheldon and Dickinson, 1983). The patterning of the exine, still sufficiently plastic during meiotic prophase to be modified by the spindle, is apparently presaged a t that time. A cellulosic microfibrillar network, the primexine, is generated by the cell undergoing meiosis. Radially oriented rods (the probacula) grow from the primexine, and subsequently developed connections between these rods appear both above and below the primexine. The product is a structure upon which sporopollenin of the sculptured exine will be deposited. Echlin (1971) suggested that sporopollenin deposition may begin while microspores are still bound within the tetrad, the precursors being provided by the microspore, but certainly most of it is derived from the tapetum (Heslop-Harrison, 1971a). With the completion of meiosis, callase (/3-1,3-glucanase) releases the microspores from tetrads, and once uncovered, they receive the bulk of exine sporopollenin according to the primexine pattern. The source of this sporopollenin is the anther tapetum, transfer being accomplished by Ubisch bodies, which are themselves coated with sporopollenin. These bodies first appear as an accumulation of sporopollenin-rich orbicules on the surface of the tapetum and migrate to the patterned primexine on the microspore surface (Chapman, 1987). Spores are nonvacuolate when p-1,3-glucanase releases them from the tetrad and, at that point, they undergo a rapid expansion, increasing in volume by a factor of two to three. The expansion is facilitated by the existence of pores which penetrate the exine (Miki-Hiroshige and Nakamura, 1983). Intine deposition begins (Knox and Heslop-Harrison, 1970; Knox, 1971) before the completion of exine growth and usually before a process of vacuolation, which occurs late in the spore expansion. Intine growth is most active during the vacuolate period, and it is during this rapid growth that enzymes-acid phosphatases, RNases, and esterases, among others-are incorporated into the intine wall. Vesicles associated with the production of polysaccharide precursors are produced by the dictyosomes of the microspore and move toward the plasmalemma. A t the same time, proteins are inserted into the wall by a variety of mechanisms. In Crocus, the plasmalemma projects finger-

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like columns into the developing wall; polysaccharides are deposited between these columns. The columns, rich in protein, are separated from the plasmalemma and remain embedded within the intine. In Cosmos, instead of these columns, multiple layers of scattered disklike packets of proteins are inserted within the pectocellulosic intine. Concentrations of intine-bound inclusions are greatest a t aperture sites, a fact which points to their presumed function in pollen germination and stigma penetration. Incorporation of these inclusions ceases before the growth of the intine, resulting in an inclusion-free layer of pectocellulosic material a t the innermost surface of the intine. Despite the fabled durability of the pollen exine it presents a biologically active surface (Tsinger and Petrovskaya-Boranova, 1961). In fact, the exine exhibits markedly different staining when adjacent to the generative cell as opposed to the vegetative cell cytoplasm (Rowley and Rowley, 1983, 1986). Once the exine is fully formed, tapetal secretions or, in the case of a n amoeboid tapetum, individual tapetal protoplasts are deposited upon the exine. Whether secretory or amoeboid, tapetal cells finally disintegrate and are deposited upon the microspores. This deposit, known variously as perine, tryphine, or pollenkitt, provides the adhesives, coloring, and aromas which characterize mature pollen grains (HeslopHarrison, 1971a; Chapman, 1987). More importantly, in the Cruciferae and the Compositeae, which exhibit homomorphic selfincompatibility, the exine holds components which play a n active role in the self-incompatibility reaction (Heslop-Harrison et al., 1974).

Genetic Significance of Pollen Ontogeny At least four features in the ontogeny of pollen hold important implications for genetic phenomena. These are the connections between meiocytes, the callose which separates microspores, the tapetal secretions and deposits, and the determination of the exine pattern. Barber suggested a function for meiocyte interconnections in the some Orchidaceae, where they are unusually persistent, functioning even after anthesis. He based this hypothesis on his observation that, in an Uvularia sp. (Liliaceae), heat shock will prevent cell wall formation between microspores, thus mimicking persistent cytoplasmic connections between them. Normally, microspores which carry unbalanced genomes abort, but when Barber disrupted wall formation, all interconnected microspores developed normally and synchronously, regardless of their genotype. Interpreting this observation in the context of naturally persistent interconnections, Barber concluded that these eliminated microgametophytic selection. I n the Ophydeae

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(Orchidaceae), several hundred grains were conjoined into a single packet (with many such packets per anther locule). Members of a packet are synchronized and, even when aberrant meiosis in Anacomptis pyramidalis ( 2 n = 18) results in a “tetrad” of five microspores, one of which might contain only two to four chromosomes, all microspores develop normally (Barber, 1942). Barber suggested that, since the Orchidaceae represent a highly specialized family, these the interconnections express the continuation of a long-term reduction in gametophytic function; that is, independent genetic expression of the gametophytic genotype would be curtailed by interconnections. But what function could be served in a vast majority of the species where the interconnections are severed before the completion of meiosis? One possible function of the interconnections is t o reduce asynchrony among meiocytes. In the absence of cytoplasmic interconnections, meiocytes within different positions of the anther locule would receive different nutrient levels and thus develop at different rates; those occupying favorable sites would be able to preempt resources normally consumed by others. This environmental variance would diminish the effect of genetic differences between microspores. Synchrony in meiosis, and thus the network of cytoplasmic interconnections, both enhance pollen selection. What function is served by isolating pollen grains in callose? Heslop-Harrison (1971a) suggested that the isolation of microspores allows them to assert their “genetic independence” (Heslop-Harrison, 1971b). This was in reference to independence from the sporophytic tissue and the separation of diplophase and haplophase development. However, when viewed in the light of Barber’s conclusions on how interconnections allow the survival of even unbalanced microspores, another possible function of microspore isolation becomes apparent. The isolation subjects each microspore t o a rigorous test, and those lacking balance genome abort. Certainly this represents a costeffective method of eliminating deleterious alleles and gene combinations. It is perhaps for this reason that many deleterious mutants fail to be transmitted through the pollen but succeed through the egg. The tapetal deposits provide a mechanism whereby the sporophytic genotype is able to determine pollen characteristics. In addition to the examples listed above, many other sporophytic determinants of pollen quality could be expressed through the same mechanism. The sporophytic control of exine pattern, expressed during and after meiosis, raises the question of what other gametophytic characteristics might be influenced by the sporophyte. The intine inclusions presumably represent products controlled by the gametophytic genotype, an

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assumption which is secure to the extent that microspore products are under gametophytic control. A reorganization of the cytoplasm at prophase of meiosis eliminates a large fraction of both ribosomes and extractable RNA from the cytoplasm, a process which might serve to impede the transmission of virus particles to gametes (Mather and Jinks, 1958; Mather, 19651, but a n additional possible function of this reorganizations is highly relevant here. The reorganization has been suggested to eliminate sporophytically produced messages and thus allow the expression of gametophytic genes (Heslop-Harrison, 197lc; Dickinson, 1987; see also Vaughan et al., 1980). Despite this reorganization, there is the sporophytic determination of exine pattern and, as discussed in Section II,B, a gene competition model which indicates the projection of premeiotic phenomena beyond meiosis. Despite these caveats, the assumption that many gametophytic qualities are determined by the haploid genome seems generally correct. OF POLLEN DEVELOPMENT B. GENETICCONTROL

Pollen characteristics are influenced by both sporophytic and gametophytic genotypes, as well as environmental factors, and, in some cases, it is possible to determine the relative contributions of these several influences. As is discussed in Section 11,3, exine sculpturing is under exclusively sporophytic control. No segregation in the exine pattern of pollen from a heterozygote is known, and even nonviable grains may exhibit typical exine patterns (Heslop-Harrison, 1971a). By means of the tapetum, the sporophyte also controls differences in pollen color, due to the deposit of flavonoids, as in white pollen ( w h p genotype) and in fluorescent pollen controlled by the bronze (bz)allele in maize (Larson and Coe, 1968, 1976; Sari Gorla et al., 1986). Because exine components are synthesized in sporophytic tissues and the control of exine pattern is sporophytically determined (HeslopHarrison, 1971c, 1972; Heslop-Harrison et al., 1973; Vithanage and Knox, 19761, gametophytic determination of morphological variability is limited to pollen size. Pollen size, in contrast, is determined by both sporophytic and gametophytic genotypes. In cases of pollen polymorphisms related to heteranthery (Pacini and Bellani, 1986; Anderson et al., 1986) or heterostyly (Glover and Barrett, 19861, control is exclusively sporophytic. Gametophytic determination of pollen size, however, is shown by the gene sp-1 (small pollen) in maize (Mangelsdorf, 1932) and by many other sp genes, most of which are probably minute deletions. Heterozygous (+ lsp-1) plants produce pollen which fits a

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bimodal distribution of sizes, whereas pollen from wild-type (+ / +) homozygotes is unimodal. The sp-1 homozygotes are nonviable. The sp-1 study is particularly significant in that the upper range of the bimodal distribution clearly exceeds that of the unimodal distribution. Since the pollen described by the upper mode is presumably the wild type, one would expect it to equal, rather than to surpass, the unimodal distribution from the wild-type ( + I +) homozygotes. However, wild-type pollen grains from the heterozygote are larger than are wild types from the homozygote. Comparable results were obtained by C. M. Johnson and D. L. Mulcahy (unpublished) in a study of pollen diameters in irradiated 2. mays. We found that the volumes of viable pollen grains produced by such plants were directly proportional to the percentage of sterile grains produced within an influorescence. Apparently, pollen grains within the anther locule can compete for limited resources of nutrients, water, and space. Resources not utilized by grains of reduced viability, or of limited competitive ability, can be preempted by more vigorous individuals. The possible significance by this intergametophytic competition is indicated by the finding that, when eight inbred lines ofZ. mays are compared, the in uitro pollen tube growth rate was significantly correlated with pollen diameter (Kumar and Sarkar, 1980). This effect was seen only beyond the initial period of germination. In uiuo, there was a nonsignificant correlation between pollen tube growth rate and pollen diameter. This latter observation may indicate that the influence of stylar genotype on pollen tube growth rate overwhelms any effects of pollen diameter. Combined sporophytic and gametophytic control has also been demonstrated in the determination of pollen size. Within a series of progressively inbred lines of 2. mays, F1 through F7,both the mean and the coefficient of variation for pollen diameter exhibit a statistically significant decrease (Johnson et al., 1976). The reduction in coefficient of variation presumably reflects the decreased heterogeneity among individual pollen grains as heterozygosity is reduced during inbreeding. This is a conservative conclusion since developmental homeostasis (Lerner, 1954; Govindaraju and Dancik, 1987) is associated with heterozygosity and should thus decrease with inbreeding. Reduced sporophytic developmental homeostasis would cause the variance of meiocytes, and thus the variance of pollen grains, to increase. Concomitantly, the inbreeding should reduce opportunities for genetic segregation and thus make the individual pollen grains with an individual more homogeneous in genotype; hence, pollen size variance which is controlled by gametophytic genetic variation is reduced. Despite a presumed loss in sporophytic homeostasis, there

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was a significant reduction in coefficient of variation in pollen diameter. That clearly indicates that the gametophytic genotype has a significant influence on this character. However, the decreased mean diameter presumably reflects the decreased sporophytic stature (and this very likely indicates a decrease in cell size) that characterizes inbred lines. Sari Gorla et al. (1975) found that pollen diameter and the in uitro pollen tube growth rate exhibited reduced variation in genetically homogeneous populations of pollen, indicating that the pollen tube growth rate is also influenced by the gametophytic genotype. The relationship between pollen diameter and pollen tube growth is fairly complicated (Kumar and Sarkar, 1980). Among eight inbred lines of 2. mays, the correlation between pollen diameter and rate of in uitro tube growth was not significant during the initial stages of germination, but, a t 3 hours after sowing, the average pollen tube length was significantly and positively correlated with initial pollen diameter. In uzuo, with the pollen labeled with 32P,there was no significant correlation between pollen tube growth rate and pollen grain diameter. No correlation exists between in vivo and in uitro growth (a commonly observed fact, indicating the significant influence of the style upon pollen tube growth rates). There was also a tendency for larger grains to have slower growth, although this was not statistically significant. Within the Polemoniaceae, there is a strong and statistically significant correlation between style length and pollen diameter (Plitmann and Levin, 1983), indicating that diameter correlates more with length than with rate. However, Yamada and Murakami (1983) demonstrated that pollen tubes from pollen of F1 hybrids of 2. mays grow through (nonself styles more rapidly than do tubes from pollen of inbred individuals. The precise extent to which this reflects the imprint of superior sporophytic vigor upon the F1 pollen grains is not known. However, Johnson et al. (1976) found that pollen from F1 plants of 2. mays had a significantly greater diameter than did pollen from inbred lines. The minimal conclusions at this point are that, between species, there is a correlation between pollen diameter and both style length and maximum length of pollen tubes, but, within species, size may indicate sporophytically and gametophytically determined vigor. This vigor may be associated with increased pollen tube growth rate. At what developmental stage does the sporophytic determination of pollen quality cease? The intine-held inclusions (see Section I1,B) are presumably under gametophytic control. However, the sporophytic determination of exine pattern as late as prophase of meiosis indicates that caution must be exercised in this conclusion. If exine patterns are

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determined by double-membraned inclusions synthesized during the prophase of meiosis, that is, still during sporophytic influence, we wonder what other sporophytically produced units could be contained within the meiocytes. One might assume that segregation or lack of it in heterozygous material would reveal whether determination is gametophytic or sporophytic, but even this is open to question. More to the point is a model of gene competition presented by Schwartz (1971). In seedlings and in endosperm, the alcohol dehydrogenase alleles, A d h l F and A d h l S , or their respective products are equally active in individuals homozygous for those alleles, but in heterozygotes, A d h l F is more active, according to densitometer readings of zygmograms. A d h l alleles are expressed in both the sporophyte and pollen. Because the protein product is a dimeric enzyme, heterozygotes exhibit A d h l F l A d h l F , A d h l F l A d h l S , and A d h l S I A d h l S dimers in diploid tissues. In pollen, postmeiotic translation precludes the formation of the A d h l F I A d h l S heterodimer. As in seedlings and in endosperm, the two alleles are equally active in pollen of homozygotes, but in heterozygotes, the A d h l F I A d h l F homodimer is considerably more active than is the A d h l S I A d h l S homodimer. The absence of the heterodimer proves that transcription and translation occurred premeiotically. Explaining this observation by assuming that, as is the case with lactate dehydrogenase, heterodimers cannot form is excluded because Schwartz (1971) and Frova et al. (1983) demonstrated ADH heterodimers in pollen containing duplicated A d h loci. The evidence suggests that the two alleles compete for some substance at the premeiotic stages and the outcome of this competition is not expressed until postmeiotic translation. The data suggest that sporophytic effects may be evident well into pollen development.

Ill. Gametophytic (Haploid) Gene Expression

A. POLLEN MUTANTS AND DISTORTED SEGREGATION Classical methods of detecting gene expression in the male gametophytic generation are based on the observation of genetic segregation of pollen characters. Although within-species morphological variability of the pollen is very limited, several instances concerning features other than morphological variability indicate that a very large set of genes is expressed during the male gametophytic generation. For example, male gametophytic selection against most chromosomal deficiencies is usually intense, showing that a large part of the genome

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is needed for normal microspore development (Stadler, 1933; Stadler and Roman, 1948). On the contrary, in the female gametophyte, characterized by a continuous dependence on sporophytic tissues, selection against deficiencies is relatively poor (McClintock, 1944; Burnam, 1962). Biochemical differences due to single-gene effects have been detected by specific staining techniques applied to single pollen grains produced by heterozygote plants. By this approach, segregating phenotypes have been shown in maize for two endosperm gene mutants, i.e., waxy (wx)(Demerec, 1924; Brink and McGillvrary, 1924) and amylose-extender (ae) (Moore and Creech, 1972), which control glycosyl transferase (Nelson, 1978) and branching enzyme IIb (Boyer and Preiss, 19811, respectively, and for A d h (Freeling, 1976). In Brassica oleracea, phenotypic segregation in pollen has been shown in plants heterozygous for P-galactosidase (Gallgal). Cases of pollen genetic sterility due to single gene effects have been reported for several species (Gottschalk and Kaul, 1974; Albertsen and Phillips, 1981; Madjodelo et al., 1966). However, most of them are the results of recessive alleles of genes expressed in sporophytic tissues. Gametophytic gene expression has been shown for Rf3, a maize fertility restoration factor specific for cms-S cytoplasm; Rf3lrf3 plants segregate normal and sterile pollen grains (Buchert, 1961; Laughnan and Gabay, 1973). Information about gene expression in the male gametophytic generation is also obtained from genetic analysis showing distorted Mendelian segregation due to genes affecting pollen development, pollen germination, or tube growth. The phenomenon of abnormal segregation also occurs in megasporogenesis in maize (Longley, 1945; Rhoades and Dempsey, 1966) and in Arabidopsis (Redei, 1965). However, most of the cases described concern microsporogenesis and pollen competitive ability. Jones (1928) describes such cases in maize, Melandrium, Datura, and Hordeum. In B. oleracea, distorted segregation is found for the gal locus, where the gal-carrying pollen grains are less competitive in effecting fertilization than are wild-type grains (Singh et al., 1985). In wheat, Loegering and Sears (1963) described a distorted inheritance for a gene conferring resistance to stem rust due to a linked factor pollen-killing, ( k i ) , which causes pollen abortion during microspore development in ki, ki plants. In Nicotiana tabacum the killing effect is produced by a genetic factor carried by an alien chromosome from Nicotiana plumbaginifolia (Cameron and Moav, 1957). The abortion is observed for microspores not carrying the alien chromosome and produced in n + 1 plants. As a consequence, the phenomenon leads to a preferential transmission of the extra chromosome. Rick (1965) described a gamete eliminator (Ge) factor producing abortion in female

GENETICS OF ANGIOSPERM POLLEN

13

and male gametophytes carrying Gc alleles when produced by heterozygote (Get, GeP) plants. In tomato, distorted segregation has been shown for a gene controlling resistance to Fusarium oxysporium (Kedar et al., 1967). It is produced by a male prepollination mechanism, probably microspore abortion, which favors the microspore carrying the allele for resistance. The distortion is supposed to arise from pleiotropic effects of I or from the effect of a gametophytic factor ( X ) closely linked to I (Pecaut, 1976; Rabinowitch et al., 1978). In Medicago falcata (Barnes and Cleveland, 1963a,b), preferential fertilization was observed as a result of a n unequal final length of the pollen tube and in lima beans (Bemis, 1959) as a consequence of unequal pollen efficiency. A number of chlorophyll mutants showing distorted segregation due to low viability and vitality of the microspores carrying the mutant allele have been reported by Doll (19671, who interpreted the effect as due to the chlorophyll mutant gene per se. However, as the material was produced by X-ray treatments, the reduced transmission through the male gametophyte could also arise from deleterious effects on linked chromosomal sites (see later discussion of gene transfer, Section VI). Similar results in Pisurn satiuum have been reported ( J a h r and Gottschalk, 1973). In maize, a n abnormal ratio (AR), in both female and male transmission, of genes controlling aleurone color was described by Sprague and McKinney (1966,1971). To explain the phenomenon they hypothesized the effect of a n extra nuclear particle, probably an infective virus, which produced alterations in the regulatory system in the a locus, whereas Samson et al. (1979) suggested that the inactivation could involve genes other than a but that are also required for aleurone color. Nelson (1985) has shown that the deviations from Mendelian ratios can be explained as due to a complementary factor and a linked lethal allele. In the same species, a slight differential gene transmission due to slower germination of recessive alleles carrying pollen grains has been detected for wx (Sprague, 1933) and opaque-2 (02) (Sari Gorla and Rovida, 1980). Prepollination differential transmission has been reported for a restorer gene specific for T cytoplasm (Josephson, 1962). Lethal embryo mutants in Arubidopsis (Meinke, 1982,1985; Meinke and Baus, 1986) and defective endosperm (de) mutants in maize (Jones, 1928; Ottaviano et al., 1987) showed male gametophytic expression affecting both pollen development and function. A particular category of genes affecting the reproductive system by their effect on pollen and style functions is represented by the gametophytic factors (Gal and self-incompatibility (s)genes. Gu factors typically exhibit normal female transmission and distorted male

(n

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ERCOLE OTTAVIANO AND DAVID L. MULCAHY

transmission. In maize at least nine different Ga loci have been detected (Mangelsdorf and Jones, 1926; Nelson, 1952; Schwartz, 1960; Bianchi and Lorenzoni, 1975). These loci shown gametophytic gene expression and the Ga allele confers high competitive ability on the pollen tube growth rate. Differential competitive ability between Ga and ga pollen is expressed in Ga Ga or Gaga styles, while ga pollen, in the absence of pollen carrying the dominant allele, performs normal fertilization. In the case of Ga-S, a gene located on chromosome 4, ga-carrying pollen grains are unable to function in Ga-S, Ga-S or in Ga-S, ga silks (Demerec, 1929; Schwartz, 19601, while alleles at different locus, symbolized Ga9-m, interact with Gal and show “resttricted” male gametophytic gene expression: the advantage of Gag-m pollen revealed in gal ,ga9lgal ,ga9 silks is not found in gal ,Ga9-ml galga9 silks (Jimenez and Nelson, 1965; Maletzky and Siritza, 1972). Gametophytic self-incompatibility is a mechanism by which pollen is unable to grow in the pistil of the plant. This is regulated by the action of S genes, which are expressed both in pollen grains and in the style and affecting pollen tube growth. The phenomenon is widespread in plants but is beyond the scope of the present review. Correlations between gametophytic and sporophytic traits, although important for the determination of interplant variability in pollen quality, do not discriminate between gametophytic and sporophytic control. Strong support for postmeiotic genetic effects as a component of gametophytic variability has been provided by analyses of pollen germination and tube growth in uitro and of inbreeding and heterosis effects. In maize, Sari Gorla et al. (1975) showed that pollen from F1 plants shows greater variability of in uitro tube growth rate than does pollen of their inbred parental lines. Moreover, the within-plant variability is reduced by inbreeding (Johnson et al., 1976) and pollen competitive ability is enhanced through successive generations of selfing (Johnson and Mulcahy, 1978). A more direct proof of postmeiotic gene expression has been given by Simon and Peloquin (1976). In potato, diploid pollen obtained by a first division restitution, which maintains much of the sporophytic heterozygosity, is more vigorous (in size and germinability) than is diploid pollen from a second division restitution, which carries homozygous diploid nuclei.

B. EXPERIMENTAL GAMETOPHYTIC SELECTION Gene expression in the male gametophytic phase can be proved by the response to selection applied to the heterogeneous genetic gameto-

GENETICS OF ANGIOSPERM POLLEN

15

phyte (developing microspores, mature pollen, and growing pollen tubes) populations produced by homogeneous heterozygote plants. Suggesting this approach are the observed correlations between male gametophytic and sporophytic traits. Mulcahy (1971, 1974) analyzed the variability among inbred lines of maize and showed a positive correlation between pollen tube growth rate and kernel and seeding weight. Ottaviano et al. (1980) also found this correlation in F1 hybrid combinations, and that pollen competitive ability can be used t o predict combining ability values of the inbred parental lines. Ar association with tolerance t o phytotoxins among in uitro germinating pollen and sporophytic tissues was found in Beta vulgaris for the herbicide ethofumerate (Smith, 19861, in maize for the pathotoxin produced by Helminthosporium maydis (Laughnan and Gabay, 1973), and in N . tabacum and P. hybrida for ozone (Feder and Sullivan, 1969; Feder, 1986). Searcy and Mulcahy (1985a,b) showed that zinc tolerance in Silene dioica and S . alba and copper tolerance in Mimulus guttatus are expressed in pollen (germination and tube growth) and that the expression parallels that of the sporophytic tissue (root growth) in the pollen sources. Pollen resistance to kanamycin has been demonstrated in tomato plants transgenic for a chimeric gene for resistance to this antibiotic; the experimental results also indicate that the gene is probably expressed during microspore development (Bino et al., 1987). A more general approach to the problem of postmeiotic gene expression is provided by male gametophytic selection (MGS) experiments; the application of selective forces to the gametophytic populations, produced by single or genetically homogeneous heterozygote plants (F1 progeny of inbred parents or vegetative clonal progeny), is expected to produce genetic changes in the sporophytic population if the variability of the character is controlled by genes which are equally expressed in the sporophytic and the gametophytic generations (gametophyticsporophytic genetic overlap). However, as the majority of the experiments reported in the literature are based only on the analysis of the sporophytic generation, they do not yield information about the genes specific for only the haploid phase. Two basic approaches have been used: (1)selection of pollen competitive ability a t different intensities of selection and (2) treatments producing environmental stresses during microspore development andlor during pollen function (germination and pollen tube growth). Two methods have been devised to achieve differential gametophytic selection: (1)pollinations with different numbers of pollen grains per flower (pollination intensity) to vary the pollen competition within each style and (2) variation of the distance that competing pollen tubes

16

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have to cover in the style (greater distance would increase the probability of the most competitive pollen reaching the ovule for fertilization). Response to gametophytic selection would be detected either as a change in the mean values and/or as a decrease of the genetic variance. A third approach is to mix pollen from a variety of pollen sources. The most rapidly growing pollen tubes from a mixture of five different pollen types would presumably exceed the fastest tubes from four out of five single-pollen types. However, this method is complicated by sporophytic influences on pollen quality (see Section I1,B) by differential interactions between pollen and pistil (Section IV,C) and by pollen-pollen influences (Section IV,C). Effects on variances and means have been detected by TerAvanesian (1949, 1978) in Vigna, cotton, and wheat for characters expressing plant vigor and seed production. Similar results on progeny means have been obtained in P. hybrida (Mulcahy et al., 1978). Effects on seedling height and weight due to excess pollination treatment have been demonstrated in Turnera ulmifolia, a neotropical weed, in a series of intermorph crosses (between thrum and pin flowers, which are short- and long-styled, respectively); response to selection was not associated with a gene controlling flower polymorphism (McKenna, 1986). In Cucurbita pep0 Stephenson et al. (1986) demonstrated that the progeny of fruits produced by high pollen loads are more vigorous than progeny of fruits produced by low and medium pollen loads. Moreover, poor seed set due to low pollen load was associated with fruit abortion. A more recent study confirmed this result in C. pep0 and Lotus corniculatus (Schlichting et al., 1987) and confirmed TerAvanesian’s reported relationship between pollen competition and sporophytic variance: increases in pollen load reduce the genetic variability in the progeny. In Cassia fasciculata the greater vigor of the progeny produced under high pollen competition is detected in competitive conditions: plants produced under high pollen load were more vigorous when in competition with those produced under low pollen load (Lee and Hartgerink, 1986). Variation of the distance that competing pollen tubes have to travel was first suggested by Correns (1928): high and low intensities of selection are obtained by placing the pollen on the stylar tip (long distance from the ovary) and on the stylar base (short distance). This method was applied in Dianthus chinensis where tip-pollinated flowers (intense pollen competition) produced a population progeny showing greater seedling weight and reduced variation in germination time (Mulcahy and Mulcahy, 1975). In the same species it was also found

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17

that MGS confers higher competitive ability a t the seedling stage (McKenna and Mulcahy, 1983). This method of MGS is easily applied in Anchusa officinalis, a species with distyle self-incompatibility flowers, in which intramorph crosses are fully fertile. The pin flowers, compared with the thrum flowers, produced more vigorous progeny in terms of seed weight and seed germinability (McKenna, 1986). An intriguing variation on this relationship between speed of pollen tube growth and position in the ovary occurs in the Cruciferae. Typically, the most rapidly growing pollen tubes enter the ovules nearest the ovary-style junction. Meinke (1982) reported that, in Arabidopsis thaliana (Cruciferae), approximately one-third of the deleterious developmental mutants he studied exhibited a nonrandom distribution in the fruit. Apparently these developmental mutants are expressed in both the sporophytic and the gametophytic phases of development. However, the mutants were more common in the apical portion of the fruit, suggesting that they had a beneficial effect upon pollen tube growth rates. This surprising result has recently been explained. In the Cruciferae, before reaching ovules, pollen tubes travel to the base of the ovary, growing between the septum which divides the fruit. The fastest pollen tubes enter the basal ovules, leaving apical positions for slower tubes. Furthermore, perforations in the septum are usually bypassed by rapidly growing tubes, but less often by the slower tubes. This also contributes to the nonrandom distribution (Hill and Lord, 1986, 1987). Maize has a very suitable structure for the application of the distance-related MGS method. At pollination time, silk length varies according to the position of the flowers on the ear, increasing from the top to the base. This feature was first used to study distorted segregation (Mangelsdorf and Jones, 1926) and to measure gametophytic competitive ability (Jones, 1928; Mulcahy, 1971; Ottaviano et al., 1980) in mixed pollination. It was used by Ottaviano et al. (1982,1988) as a device to apply high- and low-intensity selection for pollen competitive ability. Positive response for pollen competitive ability due to pollen tube growth rate and correlated responses for sporophytic traits (kernel weight, seedling weight, and root growth in uitro) were obtained when MGS was applied during selfing and in a study based on a recurrent selection scheme. Mixing pollen from a variety of sources was used to study Costus allenii, a neotropical herb. Schemske and Pautler (1984) employed mixtures of one, two, three, and five pollen types in crosses and compared the resultant fruits and offspring. Although the pure-stand yield of progeny from one-parent crosses was greater than that of

18

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progeny from the five-parent crosses, the latter were superior in mixtures. Neither seed number per fruit nor percentage of seed germination showed any significant trend from one- to five-parent crosses, except for a significant decrease in seed number with fiveparent crosses. However, seed weight (per fruit) was significantly higher in fruit from three- and five-parent crosses than from one- and two-parent crosses, and total dry weight per plant (in the next generation) from three- and five-parent crosses was significantly higher than from one-parent crosses. In addition to confirming the effectiveness of MGS in different plant species and the postmeiotic expression of genes controlling the pollen’s competitive ability, all of these studies indicate correlated sporophytic responses for traits representing expression of plant vigor. Competitive pollen tube growth rate and competitive plant growth depend on basic metabolic activities, such as those involved in energy production, starch synthesis, and wall building, the variability of which is likely to be controlled by genes equally expressed in sporophytic and gametophytic tissues (Ottaviano et al., 1980, 1982). Experimental results have been produced in studies of MGS for tolerance to environmental stresses. Detailed studies of lowtemperature tolerance in tomatoes have been carried out (Zamir et al., 1981, 1982, 1983, 1987) using a sensitive species, Lycopersican esculentum, and a tolerant species, Lycopersicon hirsutum. Under stress conditions in mixed pollination, the gametophyte from L. hirsutum is more competitive than those from the sensitive species. A positive response, detected by isozyme analysis in the progeny, was obtained when the stress was applied to the F1 microspores (during pollen development) and to pollen (during pollen function, while growing in the style), although the response was more intense during the independent phase of the gametophyte (pollen function). However, different results were obtained by Den Nijs et al. (1983) when MGS for low-temperature tolerance was applied to gametophytes produced by crossing cultivars of the same species, although the experimental data did not discriminate between pre- and postpollination events and the intensity of selection was not very high. Negative results have also been obtained in attempting to select, in uitro, for resistance to apple scab fungus (Venturia inaequalis Cke. Wint.) (Visser and van der Meys, 1986). When fusaric acid was applied to styles of Nicotiana langsodrfii, highly heterogeneous pollen growing through these styles did give rise to progeny which differed significantly from control progeny. However, the result was opposite that expected. Progeny from selected treatments were more sensitive to fusaric acid than were the

GENETICS OF ANGIOSPERM POLLEN

19

controls (Simon and Sanford, 1986). Similar results were obtained by Rowe et al. (19861, who exposed pollen of alfalfa (Medicagosativa L.) to crude extracts of the pathogen Fusarium oxysporum Schlecht f. sp. medicaginis (Weimer) Snyder and Hansen. While the selections with both fusaric acid and pathogen extracts are presently inexplicable, it should be noted that selection by pathotoxins is complex. Pollen of Avena satiua lines were surveyed for susceptibility or resistance to toxin from Helminthosporium victoriae, but no positive results were obtained (R. Popp, unpublished results). However, Rines and Luke (1985) were able to select for toxin-insensitive plants from tissue cultures with the same system. Popp suggests that a t least three factors could explain the lack of correlation between pollen and sporophytic response in Avena. Perhaps the genes for susceptibility or resistance are not expressed in the pollen. Perhaps the calcium contained in the pollen germination medium prevents the action of the toxin, as has been reported in several other systems (Pasternak, 1986). Perhaps, the lack of selection is due to the duration and concentration of toxin exposure. Damage from the toxin of H. victoriae is proportional to both of these factors (Goodman et al., 1986) and it may be that the extremely rapid germination of grass pollen would show effects of the toxin only with concentrations of toxin higher than those so far employed. A promising method of selecting for resistant genotypes is the use of synthetic stigmas (Bowman, 1984). With this procedure, stigmas are removed and replaced with agar containing a selective agent such as herbicide, salt, or pathotoxin, plus media to sustain pollen tube growth. This has the advantage of allowing a wide range of stresses to be employed with great speed. Positive results by means of MGS have been obtained for tolerance to salinity. Interspecific crosses between Solanum pennellii (salt tolerant) and Lycopersicum esculentum (salt sensitive) produced an excess of tolerant progeny when F1 plants were grown in hydroponic solution with an excess of NaCl (Sacher et al., 1983). The response was also proved by isozyme analysis, showing that the genome of the tolerant parent is preferentially transmitted under stress conditions (E. Ottaviano et al., unpublished). However, since stress was applied throughout the life cycle in the Sacher et al. study, selection could have taken place in megaspores, zygotes, and embryos, as well as in microspores or pollen. Heavy metal tolerance was studied in S . dioica and M . guttatus (Searcy and Mulcahy, 1985a,b). Plants grown in a toxic solution of zinc and copper produce flowers with a potentially toxic amount of the metals, providing a stress condition for pollen development and tube

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growth (prezygotic selection) and for early zygote abortion (postzygotic selection). Clear response to selection was detected for pollen development and an increase in the tolerant progeny was observed. No effect was found on the rate of pollen tube growth in treated pistils. However, in this case, a reduction of fertilization and of viable seeds was observed when pollen from nontolerant plants was used. Differential pollen transmission due to pollen storage effects was first studied in maize. Germinability of pollen grains carrying wx and sh is reduced when compared with pollen carrying the normal alleles (Pfahler, 19741. MGS due to pollen storage effects was not effective for plant vigor traits of the same species (Pfahler, 19861, was but highly so in P . hybrida (G. B. Mulcahy et al., 1982); wet-storage treatment produced significant responses in potato for quality of true potato seeds (Pallais et al., 19861.

C. ISOZYMEAND mRNA ANALYSIS The tapetal tissue has a central role in the regulation and nutrition of the microspores during their development (Section 11).It synthesizes the components of the outermost cell layers (exine) of the pollen grains, while the components of the internal wall layer (intine), such as hydrolytic enzymes controlling germination and tube penetration in the pistil, and storage substances derive from microspore metabolism (Heslop-Harrison, 1972). Many different proteins have been detected in germinating and in mature pollen grains (Mascarenhas and Bell, 1969; Mascarenhas and Mermelstein, 1981; Mascarenhas et al., 1984). A list of 80 enzymes detected in pollen has been reported by Stanley and Linskens (1974). Detailed accounts of isozymes in pollen have been given by Scandalios (19641, Makinen and MacDonald (19681, Weeden and Gottlieb (19801, Ortega and Bates (19801, Chandlee and Scandalios (19841, and Shivanna and Johri (19851. Moreover, there is information showing that these proteins are largely synthesized during microspore development, pollen germination, and early pollen tube elongation (for reviews see Mascarenhas, 1975; Shivanna and Johri, 1985). All these data strongly support the active role of the haploid genome. However, they are not direct proof of gametophytic (postmeiotic) gene expression. Premeiotic gene transcription producing stable mRNA, existing as RNA-protein complexes (Mascarenhas, 19751, and the physiological interaction of the developing microspores with the tapetal tissues could account for many of the metabolic activities. A direct approach proving postmeiotic gene expression is based on

GENETICS OF ANGIOSPERM POLLEN

21

the analysis of pollen grains produced by plants heterozygous for genes coding for enzyme activity, the phenotypic effect of which can be easily revealed by cytochemical staining techniques. Pollen produced by + and - alleles, where the latter is generally a null allele, shows different coloration. As discussed above, this technique has been used to detect the gametophytic gene expression of waxy, amylose extender, and alcohol dehydrogenase in maize, and of p-galactosidase in Brassica. Although this approach offers the possibility of powerful genetic analysis (Nelson, 1958; Freeling, 19761,it has been applied to a limited number of cases. The main limitation may reside in the availability of null viable mutants in which enzyme activity is controlled by a single genetic factor. A different approach to the analysis of segregation at the single pollen grain level has been attempted on the basis of unidirectional protein electrophoresis, both in microcolumns and in microslabs (Mulcahy et al., 1979; Gay et al., 1986). In interspecific crosses between Cucurbita species, segregation indicating postmeiotic gene expression has been detected for genes controlling acid phosphatase (Mulcahy et al., 1981). Gay et al. (1986) found no evidence of postmeiotic gene expression, presumably because they analyzed only intraspecific material. A simple method to discriminate between sporophytic and gametophytic gene expression which can be easily applied to a large set of genes is based on the electrophoretic analysis of dimeric or multimeric enzymes of a sample of pollen grains produced by plants heterozygous for alleles that code for polypeptides showing different electrophoretic mobilities. As was suggested (Brewbaker, 1971; Mascarenhas, 19751, if the enzyme is of sporophytic origin, pollen extracts from heterozygous fast/slow (F/S)plants display the same bands seen using sporophytic tissues (for a dimer, two homodimers and one heterodimer), whereas in the case of haploid transcription the pollen extract reveals only two parental homodimeric bands for a given enzyme, because the gene is transcribed in the phase in which only one allele is present. Causes other than haploid transcription, such as posttranscriptional or a posttranslational processing of the monomers, have been ruled out in maize by the use of partially diploid pollen, produced in plants heterozygous for a translocation between chromosome A and chromosome B (TB-A) and for alleles controlling electrophoretic mobility. When the gene is located in the duplicate segment, the pollen is heterozygous and the typical sporophytic pattern is observed (Frova et al., 1983). Although the method does not provide information for single pollen grain variability, it is a powerful system for analysis of the extent of

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gene expression in the gametophytic phase. Weeden and Gottlieb (1979) demonstrated the phenomenon for genes controlling phosphoglucoisomerase (PGI) in CZarkia dudleyana. In tomato Tanksley et al. (1981) assayed nine enzyme systems resulting from the expression of 28 structural genes and estimated that 62% of them are expressed in pollen. In maize Sari Gorla et al. (1986) considered 15 enzyme systems, including 34 isozymes and estimated that the extent of gene expression in pollen is 78.9%. InPopuZw species Rajora and Zsuffa (1986) assayed 15 enzyme systems for the expression of 45 to 51 structural genes and obtained results similar to those obtained in maize. This type of analysis deals only with a portion of genetic variability, since it does not measure differences for nonenzymatic proteins. However, it supports the idea that a large number of gene products operating in the pollen are gametophytically controlled, and that their expression involves a substantial portion of the plant genome. To the extent that one is correct in assuming that isozymes present in the pollen are the products of gametophytic transcription and translation-and the cytoplasmic reorganization which precedes meiosis (Section I1,C) indicates that the assumption may be correct-then it appears that also in Hordeum vulgare and Hordeum bulbosum there is this indirect evidence for substantial overlap between gametophytic and sporophytic genomes (Pederson et al., 1987). Moreover, those authors report that 15 isozymes specific to the sporophyte and all 5 isozymes specific to the gametophyte were unspecific for substrate. In contrast, nearly all the substrate-specific enzymes were expressed in both parts of the life cycle. Since substrate-specific isozymes are expected to participate in metabolic pathways, then functioning in both phases seems logical. Extensive information about gene expression in the male gametophytic generation has been obtained by means of mRNA analysis. Willing and Mascarenhas (1984) found that mature pollen of Tradescantia paludosa contains about 20,000 different mRNAs; these can be subdivided into three abundant classes, including 40,1400, and 18,000 different sequences, each represented by 26,000, 3400, and 100 copies per pollen grain, respectively. This method is based on the analysis of the kinetics of hybridization of L3H1cDNA with poly(A) RNA. Although the discrimination between pre- and postmeiotic transcription would require comparisons of the transcripts produced at different microspore developmental stages, data available (Tupy, 1982; Stinson et al., 1987) strongly support postmeiotic transcription. Moreover, these mRNAs have been shown to code proteins similar to those synthesized during germination and tube growth (Mascarenhas et al., 19841, indicating that many of the

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metabolic activities in these stages are controlled by the gametophytic genome. IV. Gene Expression and Garnetophytic Function

A. GENEEXPRESSION IN PRE-AND POSTPOLLINATION PHASES Most of the information concerning gene expression in the male gametophyte, as obtained by molecular methods (isozyme and mRNA analysis), refers to the mature pollen released from the anthers or to germinating pollen, representing a very early stage of the pollen function. Mature pollen is characterized by a relative dormancy, reached after partial dehydration, in which most of the metabolic processes are reduced to a minimal level. Indeed, with respect to several physiological and molecular features, mature pollen grains show a remarkable resemblance to dormant seeds (Pacini, 1986). The genetic quiescence of mature pollen is indicted by the fact that, in several monocot and dicot species, pollen will germinate and exhibit fully normal tube growth even after intense ionizing irradiation (Brown and Cave, 1954; Brewbaker and Emery, 1961; Den Nijs and van den Boom, 1983; Sniezko and Visser, 1987). This suggests that these phenomena are controlled by long-lived mRNA produced during pollen maturation. The irradiated pollen grains do sustain genetic damage, however, as indicated by the abortion of many, and, in some cases, all, zygotes resulting from fertilizations by the gametes of such pollen (Brown and Cave, 1954). Analysis of the genetic control of male gametophyte development and function can be efficiently accomplished by physiological and molecular analyses of gametophytic mutants. Due to the limited research in this area and to difficulties in devising efficient methods for mutant selection and characterization, suitable material for these studies is not plentiful. As mentioned above, most of the genetic (chromosomal genes) male-sterile mutations detected in various species affect microsporogenesis at different stages, from meiosis to later stages of pollen development (Gottschalk and Kaul, 1974). However, most of these mutants either are not characterized for their gametophytic or sporophytic genetic control or show that the microspore degenerative process is related to the tapetal tissue. Although mutations of the sporophytic tissue affecting microsporogenesis are required to analyze “signals” from the tapetum, mutations of genes expressed by the pollen genome are needed for the dissection of the male gametophyte development.

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To overcome the difficulties of detection and characterization of gametophytic mutants (a first screening should be made on the basis of within-anther segregation for viability and/or size), the problem has been indirectly approached by analysis of gametophytic gene expression in sporophytic mutants (Ottaviano et al., 1987). The idea is based on the observation that pollen development and function show positive correlation with endosperm development (see Section 111) and that the alleles determining defective endosperm in maize (Jones, 1928) and lethal embryos in Arabidopsis (Meinke, 1982; Meinke and Baus, 1986) are expressed in the male gametophyte. In the analysis of a large collection of defective endosperm (de) mutants in maize, Ottaviano et al. (1987) found that 65% of these mutants are also detected in the gametophytic stage. Genetic analysis of gametophytic competitive ability and pollen size allowed distinction of three different mutant classes: (1) de-ga mutants for genes affecting pollen development processes (pollen size, pollen sterility, and early stages of pollen tube growth), (2) mutants for pollen development and tube growth in early stages, and (3) mutants for pollen tube growth rate. These mutants provide the material to study single genes controlling pollen development, genes that are expressed in both gametophytic and endosperm tissues. However, pollen-specific genes remain unexplored and a different approach is required. Data describing gene expression in different stages of microsporogenesis are provided by the analysis of mRNAs and genetic differences revealed as enzyme activity or as isozyme variability. Stinson et al. (1987) studied two Tradescantia pollen-specific clones from mRNA not expressed in sporophytic tissues. The Northern blot analysis at different stages of pollen development revealed that transcription can be first detected after microspore mitosis; the mRNA continues to accumulate thereafter, reaching the maximum in immature pollen grains. No specific mRNAs are detected prior t o microspore mitosis; beginning just after meiosis, they increase until the late pollen interphase and then decrease. A correspondence with mRNA kinetics is shown by the analysis of P-galactosidase in Brassica campestris (Singh et al., 1985) and alcohol dehydrogenase in maize (Stinson and Mascarenhas, 1984). In both cases, enzyme activity detected in pollen grains carrying wild-type alleles ( Gal or A d h ) is compared with pollen grains with null mutant alleles(ga1 or Adh). Enzyme activity shows biphasic accumulation kinetics; it is not detected before microspore release from tetrads, increases until microspore mitosis, when a second period of increase occurs, and lasts until generative cell division. Similar observa-

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tions have been described in B . oleracea and in Helianthus annuus (Vithanage and Knox, 1976, 19791, although the gametophytic origin of the enzymes is not directly proved by mutant analysis. In the absence of null mutants and suitable single microspore enzyme assays, information about the timing of gene expression can be obtained by means of isozyme analysis, using the same approach described for mature pollen (see Section 111). In maize (Frova et al., 1987) the analysis of a set of pollen isozymes from heterozygous (FIS) plants revealed four typical patterns: (1)enzyme bands [for ADH1, aspartate aminotransferase (AAT), Glu-1, and glu-21 detected in the first-stage assay just after tetrad release and showing the same pattern all through microspore development until sheding; (2) genes expressed during development and not at maturation [catalase-4 (CAT-4) and AST-3](3) gene expression observed in later stages (CAT-1 appears at the 17-day assay, indicating that catalase is a highly regulated enzyme system, as has been described for sporophytic development) (Scandalios, 1979, 1983); and (4)enzymes showing in early developmental stages an isozyme pattern typical of the sporophytic tissue, indicating either sporophytic control (later replaced by gene products from haploid transcription) or migration of mRNA (and/or enzyme monomers) through the not-yet-completely closed cytoplasmic channels during the tetrad stage, with heterodimeric forms assembled in pollen grains. Finally, a quite different approach is used for gene products coded as consequences of gene activation under stress conditions. Protein synthesis induced by heat shock has been investigated in maize (Mascarenhas, 1984). These proteins have not been detected in germinating pollen of P. hybrida and Lilium longiflorum (Schrauwen et al., 1986) nor in Tradescantia (Mascarenhas and Altschuler, 19831, although a significant level of thermotolerance was induced by heat shock. However, analysis of developing microspores in maize has shown that in this stage the gametophyte responds to heat stock by synthesizing a set of proteins, some of which are specific for this tissue (Frova et al., 1986, 1987).

B. GENETICOVERLAPAND GAMETOPHYTIC SPECIFIC GENES The large extent of male gametophytic gene expression revealed by the studies reported in Section IV,A raises a further fundamental question. It concerns the proportion of the plant genome expressed in both the sporophytic and the gametophytic generation (gametophyticsporophytic genetic overlap (GSGO) and the proportion of genes

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expressed only in the gametophytic generation. The extent of GSGO, as is shown later, has important consequences in the evolution of higher plants and for practical applications. On the other hand, knowledge and detection of genes expressed only in the male gametophyte have a twofold interest. First, they are fundamental for understanding the genetic control of microsporogenesis and gametophyte development in the pistil. Moreover, genes of this type offer the possibility of using a very efficient system for studying tissue-specific gene regulation in higher plants. In fact, microspores are simple structures; a t maturity, they are formed by three haploid cells that can be easily handled for bioassays and for biochemical and molecular analysis in uiuo and in uitro. A clear demonstration of overlap is given by the above-mentioned classic examples of genes expressed in pollen and in endosperm (wx and ae) or in plant tissues (Adh and gal). Special cases involving only tissues (pollen and pistil) are represented by gametophytic factors (gal and s genes controlling self-incompatibility. Additional information is provided by distorted segregation, although some of the cases studied require more detailed analysis to distinguish between sporophyticgametophytic gene expression and the effect due to the linkage between genes that show distorted segregations at the sporophytic level and genes that affect viability and competitive ability of the pollen. Finally, the majority of male gametophytic selection experiments have revealed the significance of the phenomenon, which affects different important plant characters. Quantitative estimates of the overlap are thus provided by isozyme, mRNA, and endosperm mutant analyses. The isozyme approach has shown the extent of GSGO to be 58,72, and 77% in tomato, maize, and Populus species, respectively (Tanksley et al., 1981; Sari Gorla et al., 1986; Rajora and Zsuffa, 1986). Data in Tradescantza, based on heterologous hybridization between cDNA from sporophytic tissues and poly(A1 RNA from pollen and on the reciprocal hybridization, revealed that 54% of the sporophytic 30,000 sequences show GSGO (Willing and Mascarenhas, 1984). Maize colony hybridization, involving cloned cDNA from pollen and poly(A) RNA from roots and shoots, revealed 65% of genetic overlap (Willing et al., 1984). As described in Section VI,A, the extent of GSGO has been estimated also on the basis of de genes. The proportion of these endosperm genes, which proved to affect competitive ability of immature or functioning pollen grains, was 65%(Ottaviano et al., 1987). The approaches used in these studies allow the exploration of different categories of genes. Isozyme analysis mainly refers to genes controlling basic metabolic pathways; de analysis involves also genes

GENETICS OF ANGIOSPERM POLLEN

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controlling growth (Ottaviano et al., 1987); nucleic acid analysis relates to all types of DNA sequences coding for mRNA. The remarkable similarity of the results of these studies involving different plant species strongly supports the possibility that a large proportion of the genome is expressed in pollen. Considering the pattern of gene expression during microsporocyte development, which shows gene activities not found in mature pollen, the extent of the overlap may be even greater than these estimates. A number of genes, although representing only a small fraction of the expressed genome, are specific for the gametophytic generation (i.e., they are only expressed in the haploid phase). However, it is likely that this proportion is overestimated because in these studies not all the sporophytic tissues have been tested. With respect to gametophytic specific mutants, the literature reports only one welldocumented case, i.e., the restorer factor (Rf3) specific for the S m s cytoplasm. However, also in this case, transcription and/or translation of the gene in some sporophytic tissue cannot be excluded. C. THE INTERACTION BETWEEN POLLEN AND PISTIL Pollen germination and tube growth are highly sensitive to, and dependent on, the surrounding environment. With in uitro studies, the density and quality of neighboring grains are important, as are, in uiuo, the genotype and the condition of the pistil. Jennings and Topham (1971) diluted pollen of cultivated raspberry with heat-killed pollen and found that the percentage of pollen germinating is disproportionately reduced by the dilution. This result parallels the in uitro dependence of pollen germination on pollen density reported by Brink (19241, Brewbaker and Majumder (19611, and Brewbaker and Kwack (1963).The last study concluded that calcium, the effect of which could be enhanced by potassium, magnesium, or sodium, would greatly reduce the influence of pollen density. Presumably calcium “leaks” from pollen grains, and a high density of pollen or exogenous supplies of the ion, will minimize the effects of this leakage. However, in neither Phlox drummondii (Levin, 1975) nor Medicago (Miller and Schonhorst, 1968) was there any indication of the in uiuo density effect. The presence, in some cases, and absence in others, of an effect was paralleled by one data set within the Jennings and Topham (1971) study. In one cultivar, Malling Jewel, the percentage of drupelets developing was largely independent of the pollen dilution, but in Malling 69/139, the effect of dilution was highly significant. To extrapolate from the Brewbaker and Kwack data, it may be that stigmas of Malling Jewel were sufficiently rich in calcium or other

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critical substances so as to render pollen density unimportant. In other stylar genotypes, such as Malling 69/139, inadequate stigmatic nutrients might make pollen density a critical factor. The complexity of interactions between pollen grains was demonstrated in Iwanami (19701, who reported that in vitro pollen tube growth was directly proportional to population density in L . longiflorum, inversely proportional in Camelia sasanqua, and independent of density in Camelia japonica. Sown on agar in mixtures, C. sasanqua pollen inhibited pollen tube growth of L. longiflorum and C. japonica (Iwanami, 1970). Hodgkin and Lyon (1986) have used an elegant method of determining the effects of specific compounds on pollen. Extracts of tissues are separated on thin-layer chromatography plates; pollen, suspended in germination medium, is then sprayed onto the plates. After incubation under appropriate conditions, it was found that germination was enhanced in some areas and inhibited in others. This method is important, not only in its great potential for investigating factors that influence pollen germination and elongation, but also because it demonstrates that tissues contain multiple stimulatory and inhibitory factors. Genetic differences in either stigma content or pollen sensitivities could explain the great variety of responses reported by Iwanami and others. It has also been proposed that an abundance of one type of pollen could physically exclude or physiologically inhibit another type of pollen from the stigma (Wasser, 1978; Sukhada and Jayachandra, 1980; Thompson et al., 1981). This theory, investigated and reviewed by Shore and Barrett (19841, showed that, in heterostylous T . ulmifolia, this “stigma clogging” had a significant effect only when five anthers of incompatible pollen were applied to stigmas 1.5 or 3.0 hours before pollen of one anther was applied. This effect on seed set was observed only in the most extreme treatments and only with the long-styled form. Shore and Barrett suggest that absence of effect with the short-styled form might be due either to the fact that the shortstyled form received approximately 25% less pollen that did the long-styled form, or to the fact that, in heterostylous species, the inhibition of incompatible pollen occurs closest to the stigma in the shortest styled forms, farther toward the ovary in longer styles. Clogging may therefore occur not on the stigma, but within the style. They conclude that, in T . ulmifolia, stigma clogging is unlikely to be an important phenomenon in natural populations. However, we believe that pollen-pollen effects, either direct or mediated by the pistil, are certainly significant phenomena in natural populations.

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Pollen tubes growing in vitro will take up carbohydrates and amino acids (Kendall et al., 1971; Kwack, 1983; Mulcahy and Mulcahy, 1988) and, presumably, these and other nutrients are also supplied by the style, in vivo. The relationship between pollen and pistil is, however, not merely one of the growing entity and a passive nutrient substrate. Instead, it is a complex interchange between two highly responsive systems (discussed below). The precision of pollen-pistil relationships is illustrated by a n important finding among six inbred lines of 2. mays. Here, threonine content of pollen grains is seen to be inversely and closely correlated with the threonine content of styles (Linskens and Pfahler, 1977). Apparently, low threonine content of pollen is precisely compensated for by increased threonine in the style. A corollary of this observation is that the concentration of threonine must be particularly important for the growth of pollen tubes. Although concentrations in pollen and styles of other amino acids did not follow this intriguing relationship, it is clear that, for threonine, and presumably for some other critical substances, the relationship between pollen and style is very closely controlled. This should be particularly so with inbred lines of small populations, where time has allowed natural selection to adjust the content of pollen and styles. Once within the style, pollen tubes are subject to a variety of influences: other pollen tubes, inherent qualities of the style, and stylar qualities developed in response to these and other pollen tubes. In Epilobium canum, the probability of a pollen grain producing a pollen tube is independent of the number of grains on the stigma (at least, from 4 to 40 grains). However, the ratio of tubes to seeds is higher when there are 40 grains on the stigma than it is with either 4 or 20 grains. Since ovules are not limiting for 40 pollen grains, Snow (1986) concluded that the decline must be due to a reduction in pollen tube success with the higher pollen loads. A possible explanation for this reduction may be that some conditions or nutritive factors provided by the style become modified or limited with increased densities of pollen tubes, thus increasing the mortality of pollen tubes within the style. Not surprisingly, the style has a major effect upon pollen tube growth rate and success, but there are two aspects to this effect. The first is characterized by the absence of a statistically significant interaction between stylar and pollen genotypes. For example, Jennings and Topham (1971) found that, in one 4 x 4 diallele, the number of pollen tubes seen within a style was strongly dependent on the stylar genotype. Similarly, in Raphanus sativus, the relative competitive abilities of different pollen types were constant over different maternal

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plants (Marshall and Ellstrand, 1986). In the second category of stylar effects, significant interactions between style and pollen have been found. Pfahler (1967) found that the outcome of pollen tube competition was significantly influenced by stylar genotype, and, in raspberry, even though pollen genotype did not have a statistically significant effect on the number of pollen tubes per style, there were small but significant interactions between stylar and pollen genotypes (Jennings and Topham, 1971). Cases such as self-incompatibility and Ga (gametophytic) factors, discussed below, also represent specific style and pollen interactions, but these are special phenomena, distinct from more general interactions. When pollen mixtures are applied to stigmas, some fairly complex interactions occur. For example, in 2. mays, when a mixture of pollen from lines C123 and RNY is used in pollinations, the tubes from C123 outcompete tubes from RNY; in RNY and WF9 pollen mixes, RNY tubes outcompete WF9 tubes. On the basis of these data, it was predicted that pollen tubes from C123 would outcompete those from WF9, but, in fact, WF9 outcompeted C123 (Sari Gorla et al., 1975). This was logically interpreted as a n intergametophytic effect, but changes in competitive hierarchies may also indicate that different pollen gentoypes are limited by different characteristics (substances or conditions) within the style. That is, C123 may be superior to RNY in obtaining one substance, with the two genotypes equal in most other characteristics; RNY could be superior to WF9 in the same way, while C123 and WF9 could be equal in this regard but with WF9 superior to C123 in regard to the next limiting factor. Furthermore, the interactions between pollen of different genotypes could be even more involved because of pollen-influenced differential changes in the style. Pollination induces a general redistribution of organic compounds within the flower; the style receives compounds mobilized from the stamens, corolla, and calyx (Linskens, 1974). The effect is less pronounced when self-pollen, rather than nonself-pollen, is used. It is not known if all compatible pollen is equally effective in causing this distribution, but differences would certainly create different stylar conditions. These, in turn, could modify the relative competitive abilities of different pollen types. The possibility of indirect effects of one pollen type on another, mediated through a modified stylar environment, is well demonstrated in the mentor effect. Stettler mixed compatible but radiation-killed pollen of Populus deltoides with otherwise incompatible pollen of Populus alba. The mentor effect also was successful in overcoming self-incompatibility in Cosmos (Stettler, 1968; Howlett et al., 1975;

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Stettler and Ager, 1984; Knox et al., 1972, 1987). Another pollenpollen interaction, the pioneer pollen effect, is also mediated through the style. When pollen of apple was applied to compatible stigmas in two doses, with the second dose 1 or 2 days after the first, the second dose sired, in one series, 58% of the seeds and, in another, 70% of the seeds. Visser and Verhaegh (1980) referred to the first pollen application as the pioneer application because it appeared to promote, to its own detriment, the performance of the second pollen application. The pioneer pollen effect is not a universal one-Epperson and Clegg (1987) reported that, in Ipornoea purpurea, the first pollen applied to the stigma is the first to effect fertilization. This is not to trivialize the pioneer effect, since Visser also reported that both mentor and pioneer effects were subject to variables of environmental conditions and of genotype within species and between species. The basis for the mentor (and possibly also the pioneer) effectb) was first investigated by Knox et al. (19721, who found that a methanol extract of the mentor from P . deltoides contained a proteinaceous fraction which was itself somewhat effective as a mentor for P.alba pollen. Extracts were also effective as mentors in overcoming selfincompatibility, and a variety of explanations have been advanced (reviewed by Knox et al., 1987). A possible explanation for the mentor and pioneer effects may be the interaction between the pollen and ovary. In the simplest case, pollen of Petunia hybrids exhibits a biphasic pattern of growth. Growth is first slow and apparently autotrophic, that is, largely dependent on reserves carried by the pollen (Brewbaker and Kwack, 1963; Rosen, 1971; Mulcahy and Mulcahy, 1983). This is followed by a faster growth phase, presumably sustained by stylar inputs. One demarcation between these two phases is the occurrence of pollen tube mitosis and, in some species, perhaps also the expression of self-incompatibility (Brewbaker and Kwack, 1963). However, another phenomenon also coincides with the transition between the phases. When pollen touches the stigma, a signal is rapidly transmitted to the ovary (Jensen et al., 1977, 1983). Upon receipt of this signal, the ovary undergoes multiple changes, including synergid breakdown and polar nuclei fusion. Recent evidence indicates that a countersignal is emitted from the ovary to the approaching pollen tubes (Mulcahy and Mulcahy, 1987). The emission of this countersignal is blocked if the ovary is removed from the pistil. With intact pistils, the signal reaches the pollen tubes at about the time of transition from slow to rapid growth and may be a n essential component in this alteration. If it is, it would contribute to the mentor and pioneer effects. Compatible pollen would signal its

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presence to the ovary, initiating the emission of a pollen-stimulating signal. It is not known if incompatible pollen will also trigger the ovarin signal, but, if it does not, otherwise incompatible pollen could be stimulated by a signal emitted in response to compatible pollen that was applied either simultaneously (as in the mentor effect) or previously (as in the pioneer effect). Whether the coincidence between the expression of self-incompatibility and the arrival of the ovarian stimulation of pollen tube growth is significant is unknown. Should it be significant, it might indicate one mechanism whereby the selfincompatibility reaction is mediated. To the above-mentioned pistil-mediated pollen-pollen effects, it appears that a chemotropism for pollen tubes may also be added. Although these chemotropisms have been subjected to extensive and effective study (Mascarenhas, 19781, the behavior of pollen grains which were surgically placed within unpollinated styles casts a question over the significance of pollen tube chemotropism. Such grains appear to germinate normally, but the tubes which they produce are as likely to grow toward the stigma as toward the ovary (Buchholz et al., 1932; Y. Iwanami, quoted in Shivanna and Johri, 1985). Although these observations are not widely known, they indicate that pollen tube chemotropisms are not preformed within the style. It is perhaps for this reason that, although pollen tubes will respond to different attractants in uitro (Mascarenhas, 19781, a recent review (HeslopHarrison and Heslop-Harrison, 1986) of pollen tube chemotropisms indicates that the phenomenon is far from being understood. However, if intrastylar pollinations are accompanied by simultaneous stigmatic pollinations, the otherwise random directionality of pollen tube growth is replaced by a significantly basipetal tendency (Mulcahy and Mulcahy, 1987). In addition, only stigmatic pollinations significantly impeded the growth of intrastylar pollen tubes and did not affect those growing toward the ovary. Subsequent studies have shown that growth toward the ovary is significantly stimulated by stigmatic pollination (G. B. Mulcahy, 1989).

V. Garnetophytic Gene Expression and Evolution

A. POLLEN COMPETITION IN NATURALPOPULATIONS Feinsinger et al. (1986) studied pollen loads on the stigmas of four hummingbird-pollinated cloud forest species. Each species received a number of pollen grains equal to or greater than the number of ovules

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in a percentage of flowers sampled in three runs; for Besleria triflora (gesneriaceae) this was 4-16%; for Drymonia rubra (Gesneriaceae), 4-16%; for Hansteinia blepharorachis (Acanthaceae),50-74%; and for Razisea spicata (Acanthaceae), 15-30%. More than one pollen grain/ ovule is usually required for full seed set; thus, these data indicate that pollen competition in these species may have occurred sporadically and by no means unfailingly. Furthermore, they found that the frequency of adequate pollen loads differed significantly over the flowering reason for each species. In Epilobium canum (Onagraceae), another hummingbirdpollinated species, pollen is shed in tetrads and the stigmas are available for pollination over a period of 2-3 days. In two seasons of observation, 53 and 69% of all pollinated flowers received the 20 or more tetrads required for full seed set (Snow, 1986). Since >30 tetrads on a stigma would mean that 30% of the pollen tubes would be superfluous, it was estimated that a significant degree of pollen tube competition occurred in about 30% of all flowers examined. In more than half of these receiving >30 tetrads, >40 tetrads were deposited, indicating that intense pollen competition occurred in at least 15%of all flowers examined. Snow’s data are particularly valuable in that they indicate that the intensity of pollen tube competition is not simply a function of how great an excess of pollen a stigma receives. Snow found that the average individual pollen deposition consisted of 20-24 tetrads and also that 20-30% of the flowers received more than one deposit. Of those receiving >30 tetrads, 23% received pollen on two consecutive days. Since the first pollen deposit would very likely use most, but possibly not all, of the 18-37 (mean = 20) ovules per ovary, pollen of the second deposit would likely experience very intense competition for the remaining unfertilized ovules. Thus a 10% excess of pollen might indicate a very limited degree of pollen tube competition, when, in reality, 90% of the ovules was fertilized without pollen tube competition and the remaining 10% was fertilized under conditions of fairly intense competition. Snow (1986) has summarized data on the occurrence of pollen limitation in natural populations of 25 species and concluded that seed set was usually limited in 6 of these species and not limited in the remaining 19 species. Presumably these values correspond to the absence or presence, respectively, of pollen tube competition. Stanton (1987) found that seed set is not limited by the availability of pollen in R . satzuus, but reviewed many examples in which pollen is, in fact, limiting (see also Bertin, 1982). Pollen limitation in seed set is, perhaps not unexpectedly, common in species which flower in the early

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spring (Bierzychudek, 1981) or in other cold, damp conditions (Campbell, 1987). One approach for assessing the natural availability of pollen is to determine if supplemental pollinations enhance seed set. An affirmative result is often interpreted as indicating that seed set is pollen limited. However, maturing fruits compete for resources, and those carrying the greatest number of seeds are less likely to abort than are those containing only a few seeds (Lee, 1984; Stephenson and Winsor, 1986; Winsor et al., 1987). Lee has hypothesized that this pattern of fruit abortion benefits plants through the mechanism of pollen tube competition. Multiseeded fruits are very likely the result of intense pollen competition, whereas seeds with a less than full complement of seeds were produced without pollen tube competition. Differential abortion of the latter would result in genetically superior offspring because rapidly growing pollen tubes give rise to rapidly growing plants. Given that pollen competition occurs in some circumstances and species, how can the degree of competition be expressed? One approach is t o estimate the average growth rate of successful pollen tubes, that is, those reaching a n unfertilized ovule. If the growth rates of successful pollen tubes exceed the average of unselected pollen tubes, the difference should be a quantitative expression of pollen tube competition. This method has been used in a study of pollen tube growth and pollination in a natural population of Geranium maculatum. Six variables were included in that study: (1) time between pollinator visits, (2) average number of pollen grains left in one visit, (3) length of the style, (4) pollen tube growth rate, (5) variance in pollen growth rates, and (6) number of ovules available. Time between visits is important in that the first visit may deposit just enough pollen to fertilize all ovules, and, if a second pollinator visit comes much later, all ovules may be fertilized before that occurs. Analysis of the pollen load on the stigma would indicate that pollen competition had occurred, when, in fact, it had not. Obviously this situation will depend on the number of pollen grains left in each visit, the second variable. Pollen tubes from one visit may overtake those from an earlier visit if time between visits is short enough and the number of grains in the first visit is not too great. Otherwise, the fastest tubes of the first visit will occupy all available ovules. Competition between pollen tubes from different visits will be enhanced with longer styles, the third variable, or slower pollen tube growth rate, the fourth variable. Both of these will prolong the travel through the style, thus reducing the effect of time between visits. The sixth variable will also influence competition between pollen tubes of separate visits. The

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greater the variance, the greater will be the probability that rapidly growing pollen tubes from late arrivals will be able to overtake the slowest tubes of early deposits. The importance of the last variable is obvious. In G . maculatum, an unselected population of pollen tubes exhibits a n average growth rate of 3.2 mmlhour. Since flowers of that species each contain 10 ovules (only half of which yield seeds), 20 pollen tubes will probably suffice to effect complete seed set. Using estimates of the above variables, it was calculated that the first 20 tubes to reach the ovary would have a n average growth rate of 4.3 mm/hour, 34% greater than that of the unselected pollen tubes (Mulcahy et al., 1983; Mulcahy, 1983a). A recent study has demonstrated not only pollen tube competition in a natural population but also evidence that the resultant sporophytic generation is modified by that competition. Working with Aureolaria fiaua (Scrophulariaceae), Ramstetter (1987) controlled the level of pollen competition by varying the time and duration of allowable pollinator visits. Flowers of this species open for only a single day and, given a full day of exposure to pollinators, fruits produced a n average of 210.33 (SE = 10.79) seeds. This was taken as a standard by which all other pollination exposures were measured. Exposure of flowers until noon, for example, resulted in an average of 197.53 (SE = 11.91) seeds per fruit, 94% of the full day exposure. Other treatments (with the SE in parentheses) resulted in 189.27 (15.521, 124.53 (18.591, and 84.27 (18.05) seeds per fruit or, respectively, 90, 59, and 40% of the standard value. It is important to note that the standard errors of these means are fairly high and it is this which allows the occurrence of pollen competition even when the average seed set is less than 100%. The average seed set is thus a measure of the relative frequency and intensity of pollen tube Competition. Ramstetter found that seed set (and presumably pollen tube competition) was not correlated with average seed weight or percentage germination. However, seed set was significantly and positively correlated with percentage seedling survival, with number of leaves and rosette area at 70 and 92 days after planting, with root length, and with the frequency of rosettes producing shoots during this study period.

B. POLLEN COMPETKTION IN CROPPLANTS Cultivated plants generally interact with a special environment in crop fields and in breeding nurseries, which can greatly affect the parameters controlling male gametophytic fitness (Ottaviano and Mulcahy, 1986). Single species in crop fields, high population density,

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and compressed flowering time would increase the amount of pollen available per unit of field area; consequently, the pollen load per stigma is expected to be higher than in natural populations, with either wind or insect pollination. Chemical treatment and biotic (viral, bacterial, and fungal diseases) and abiotic stresses (high and low temperatures, drought, salinity, and pesticides) produce specific selection pressures. Although it is generally found that the number of pollen grains per plant (or per flower) greatly exceeds the number of ovules (Ter Avanesian, 1949; Stephenson and Bertin, 1983), information with regard to the pollen load per stigma (pollen grains per stigma and time interval of pollen deposition) is available only for a few species. In maize Goss (1968) estimated 21,000 pollen grains per ear and about 21 grains per kernel produced. Sadras et al. (1985) found that in 3 hours of pollen sheding, the average number of pollen per silk is about 12 grains and that a large proportion of silks receive 12-20 pollen grains. For crop plants, pollen competition arising from breeding practices deserves special consideration. Most of these species are subject to recurrent crossing to produce genetical variability. Controlled pollination is generally carried out with a very large amount of pollen. In maize a t least a million pollen grains are used for pollination of fewer than 1000 styles (E. Ottaviano, unpublished data), while in tomato about 10,000 pollen grains are applied to a single stigma (Zamir and Jones, 1981). Finally, studies of the relationship between fruit abortion and seed set, showing the increase of the probability of abortion in fruit with low seed set (Stephenson et al., 19861, indicate that in many plants the viable progeny is produced under pollen competition.

C. EVOLUTIONARY RATE UNDER GAMETOPHYTIC SELECTION The results presented above, showing that a significant proportion of genes is expressed in the male gametophytic generation and that most of these genes are also expressed in the sporophytic generation, suggest that selective forces act during the haploid phase of the life cycle; thus, they may play important roles in determining the genetic structure and evolution of plant populations. This situation contrasts with that found in animal species, where the haploid phase is confined to one cell generation; apart from the exception of meiotic drive, which leads to non-Mendelian segregation (see, for example, Hartl, 1970a; Peacock and Miklos, 19731, genes are generally repressed (Beatty, 1975).

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The definition of gametophytic selection in plants is important for the discussion of the evolutionary significance of the phenomenon. Sporophytic selection (SS) accounts for the change in gene frequency resulting in selection pressures in all diploid stages of the life cycle. Beginning with the zygote and ending with meiosis, gametophytic selection (GS) refers to the changes in gene frequency occurring between the parental sporophytic population and the population of gametes participating in fertilization (Harding, 1975; Ottaviano and Mulcahy, 1986). A more general terminology for both plants and animals refers to the phenomenon as gametic selection, prezygotic selection, or haplont selection (Hiraizumi, 1964; Hartl, 1970b). Selection can act on both male and female gametophytic generations. However, because of the large size of the male gametophytic population, the pollen grains’ independence of the maternal plant, their direct exposure to environmental stresses, and competition in the same style, male gametophytic selection is expected to be more effective than the selection operating on the female counterpart. Pfahler (1975) distinguished two main components affecting differential gene transmission in the male gametophytic generation, i.e., transmission opportunity and transmission ability. The former depends on the relative number of pollen grains per plant and on their physical distribution. This is largely controlled by plant development and vigor, and consequently is sporophytically determined. The latter, i.e., transmission ability, refers to the fitness of the gametophyte. It includes components affecting pollen development (growth, viability, and competition within the anther) and components affecting pollen function (germination, resistance to environmental stresses, pollen tube growth, and fertilization ability). It shows a large amount of genetic variability, arising from both sporophytic and gametophytic gene expression (Ottaviano et al., 1982, 1987). Although the independence of sporophytic and gametophytic genetic determination of the character is a n oversimplification of the phenomenon, it is useful to consider the sporophytic component of this variability as one of the features of sporophytic fitness. Consequently, male gametophytic selection will here strictly refer to that arising from the effects of genes expressed during the male gametophytic generation. With the limitations of this definition, male gametophytic selection reflects the most basic features of haploid selection in microorganisms, i.e., large population size and haploid state. Population size in the male gametophytic generation largely exceeds that of the sporophytic generation. For example, in maize a single

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plant produces from 14 x lo6 to 50 X lo6 pollen grains (Miller, 1982). Effective population size differs in pollen development and pollen function (postshedding) phases. In pollen development it includes all the microspores produced, and in pollen function it is largely reduced by the number of pollen grains required to obtain normal shedding and by the number of grains reaching the stigma. However, also in this phase the number of grains reaching the same stigma and competing in the same style would allow the selection to act at a n intensity higher than that operating in the postzygote phase. Apart from cases of pollen lethality, such as that produced by chromosomal aberration or by single lethal genes, male gametophyte selection due to pollen competition in the anther during microspore development and within the style due to germination time and pollen tube growth rate can be regarded as a type of soft selection, as defined by Wallace (1968). The second special feature of MGS is the haploid state, which has important consequences. Because the number of genotypic combinations in haploid progeny is much lower ( 2 4 for n genes with two alleles) than that of the sporophytic generation (3n), the possibility of selecting complex allele combinations is higher than in the sporophytic generation. Moreover, the haploid state in the gametophyte leaves the recessive allele uncovered and, consequently, the kinetics of the population under MGS is expected to show a higher evolution rate than in the case of sporophytic selection. The consequences of population size and haploidy point to the great importance of MGS in the evolution of higher plant populations. Although several models which take into account the effect of gametic selection have been elaborated, a comprehensive study which considers zygotic and prezygotic (gametic or gametophytic) selection, Malthusian parameters in both male and female generations, and the relative number of genotype combinations has not been produced. The topic was treated by Haldane (19241, showing that selection in the haplophase is much more efficient than in the diplophase. More recently, simple models involving two alleles have been used (Prout, 1953; Buck, 1957; Lewontin, 1958, 1968). Scudo (1967) developed a deterministic model and referred to the case in which haplont selection acts as distortion factor in heterozygote individuals. Hartl (1970a) presented a more general model which includes the effect of the meiotic drive, i.e., gametic selection under monogamy and under complete gamete mixing as occurs in cross-pollinated plants. Hartl (19751 extended the statistical model of selection to gametophytic selection. A statistical model and the experimental method for simultaneous estimation of male gametophytic selective values and of

GENETICS OF ANGIOSPERM POLLEN

39

outcrossing rate parameters were defined by Harding and Tucker (1969). Harding (1975) worked out a model for female and male gametophytic selection in connection with different types of mating systems; in particular, MGS is considered taking into account the early (prepollination) and the late (postpollination) stages. Jain (1975) studied the population dynamics of a gametophytic factor (locus Ga, showing differential selective advances in Ga and ga pollen grains) under mixed selfing and random mating, in different combinations of gametic-zygotic selection. Analytical comparisons of the effectiveness of GS versus SS, considering correlated andlor independent effects of the two types of selections, have been reported (Ottaviano and Sari Gorla, 1979; Pfahler, 1983). Experimental confirmation that GS is a common phenomenon in plant populations has been obtained by means of comparison of gametic and sporophytic gene frequencies in multiple census experiments. The information obtained from this type of experiment allowed estimation of parameters describing the mating system and components of selection in different stages of the life cycle. Estimations of pollen selective values have been obtained for populations of Clarkia exilis and Clarkia tembloriansis (Vasek and Harding, 19761, Phaseolus lunatus and 2. mays (Harding and Tucker, 19691, and barley (Clegg et al., 1978). In all these cases the values of the parameters were quite high; in some cases the variability was comparable to that of the net fitness. Although these results point to the importance of GS in population structure and the dynamics of higher plants, they did not discriminate between gametophytic transmission opportunity and ability, nor did they furnish information about sporophytic and gametophytic control of the latter. The effects of genes expressed in the haploid phase on the gametophytic fitness variability have been evaluated in a gametophytic selection experiment in maize (Ottaviano et al., 1988). A large genetic variability was found for parameters estimating gametophytic competitive ability of the early stage of pollen competition in the style (germination time and tube growth rate) and for the parameter representing competitive ability due to pollen tube growth in later stages. Considering that a significant response to within-plant gametophytic selection was detected only for the latter component, it appears that competitive ability in the early stages of pollen function is controlled by sporophytic tissues. These tissues control the amount of storage material contained in the grain, an amount which supports only the first stage of pollen tube growth (autotrophic development) (Heslop-Harrison et al., 19841, whereas the later growth rate, which depends on metabolites furnished by the style (heterotrophic develop-

40

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ment), is largely controlled by the gametophytic genome. Strong sporophytic components in the early stage of the pollen function can explain the results obtained by Yamada and Murakami (1983) regarding the superiority of pollen produced by hybrid combinations over that of inbred parents. In this study, early and later competitive abilities were not distinguished and consequently the observed heterosis could be the result of the effect of the sporophytic component. However, this does not mean that the haploid genome would affect only pollen fitness components due to later stages of the gametophytic phase. I n fact, deleterious mutants have been proved to affect both pollen development and function. Again, in maize, a species in which genetic distortions can be easily related to pre- and postpollination effects, the analysis of a large set of de (defectiue endosperm) mutants revealed that gametophytic gene action can significantly reduce the fitness values in both stages (Ottaviano and Mulcahy, 1986; Ottaviano et al., 1987).

D. GAMETOPHYTIC SELECTION AND GENETIC LOAD Data concerning abnormal segregation due to prezygotic selection, response to artificial M G S , genetic variability of gametophytic fitness as detected in a number of plant species, and the large extent of gene expression in the male gametophytic phase strongly support the idea that gametophytic selection is a widespread phenomenon in higher plant populations. This conclusion has a n important implication in the discussion of the possible role of GS as a factor controlling the amount of polymorphism and the genetic structure of populations. Kimura (1959) showed that prezygotic selection taking place among the gametes of a n individual can be responsible for a significant amount of the genetic load and that a stable equilibrium is maintained even if the implicated locus has a deleterious effect in diploid individuals. The slight effect of prezygotic selection increases the frequency of deleterious mutant genes and, consequently, the genetic load to a significant extent (Hiraizumi, 1964). Jain (1975) has showed that a gametophytic allele (Gal favored in GaGa or Gaga pistils can maintain a stable or transient polymorphism (for Ga and for linked factors) in a model involving balanced pressures of gametic and zygotic selection or heterozygote advantage of Ga and a t the linked loci, under mixed selfing and random mating. While the sporophytic average fitness change rate is not affected when prezygotic and postzygotic factors are uncorrelated (Hartl, 1970b), drive elements with better fitness than normals (positive correlation) reduce the substitutional load through the process of gene replacement and accelerate the accumulation of beneficial elements in a population (Hiraizumi, 1964).

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41

Most of the experimental results reported in the previous sections (gametophytic selection experiments; distorted segregations) indicate positive correlations between the sporophytic and the gametophytic effects of the genes expressed in both phases of the life cycle. Therefore, GS appears to be a n important factor for removing deleterious alleles and for obtaining high rates of evolution. A special role of GS can be postulated for the regulation of the genetic (variability of quantitative traits controlled by complex gene combinations (Ottaviano et al., 1987). For these characters, the large genetic load produced by recombination can be removed by the action of GS at a cost (loss of pollen genot~ypes)compatible with the size of the male gametophytic population. In this sense, GS is a more efficient mechanism than those provided by chromosomal rearrangements, which eliminate the product of the recombination. On the other hand, the special features of the gametophytic population (large size and haploidy) would allow a high evolutionary rate a t a cost compatible with the biological features of the species: the elimination of the genetic load needed for a high rate of evolution (Haldane, 1957, 1960; Maynard-Smith, 1971) can be obtained without great effects on the sporophytic fitness mean. E. ANGIOSPERM EVOLUTION Experimental results concerning gametophytic gene expression, gametophytic-sporophytic genetic overlap, response to GS selection, and theoretical population genetic analysis may provide a better understanding of evolution of the gametophytic generation of the angiosperms. Classical views of the problem attribute t o diploidy a meritorious condition (heterozygosity) and hence natural selection would lead to a gradual suppression of the haploid generation to a minimum, as is found in animal species (see Heslop-Harrison, 1979, for a review). Accordingly, a large extent of gametophytic screening may be deleterious for the sporophytic generations, because it would remove alleles which negatively affect pollen development and function, but positively affect the sporophytic fitness. However, the expected progressive suppression of gene expression and the establishment of an independent haploid domain have not been obtained in the angiosperms (Heslop-Harrison, 1979). Mulcahy (1979) proposed that gametophytic selection is one of the major factors of the evolutionary rise of angiosperms. In these species, insect pollination and the presence of the style result in a selective mechanism which produces a high degree of competition between pollen grains. The insect pollination allows a large number of pollen grains to reach the stigma simultaneously, so that the effect of

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ERCOLE OTTAVIANO AND DAVID L. MULCAHY

variability of pollen germination time is reduced. The competition of several pollen tubes (fast growing) in the same style allows the fittest gametophyte (fast growing) to reach the ovule for fertilization. Taking into account the large and positive gametophytic-sporophytic genetic overlap and the high evolutionary rate which can be attained in the male gametophytic generation, MGS should be considered a n important factor which contributes to angiosperm evolution. Moreover, if it is assumed that MGS is also a factor for the regulation of genetic variability in the population, mechanisms favoring MGS (gene expression, pistil acting as a sieve for the fittest gametophytes, and insect pollination) should evolve under the pressure of natural selection. In fact, two such mechanisms have recently been documented. With the first of these, in Leucaena leucocephala, pollen grains will not germinate on the stigma until a specific minimal number of grains is present (Ganeshaiah et al., 1986). The intriguing possibility that this represents a mechanism for intensifying pollen competition may, of course, be merely a fortuitous consequence of a more basic physiological process (see Section IV,C). However, in a second pollen-intensifying mechanism, it seems clear that a specific mechanism exists to intensify pollen competition. In Talinum mengesii (Portulacaceae), the stigma is available for pollination at anthesis. However, in one population, pollen will not germinate until 2 hours after anthesis. In another population, pollen grains germinated without delay. This suggests that the delay which characterizes the first population is not a secondary effect of other phenomena (Murdy and Carter, 1987). Instead, it is very likely a mechanism for intensifying pollen competition. Considering MGS in both pre- and postpollination phases, it is expected that natural selection would enhance pollen grain independence of both maternal (anther) and stylar sporophytic tissues; these tissues would fulfill the role of primary metabolic substrates (Frova et al., 1986). Trends of evolution in angiosperms would therefore show a n increase of the pollen selection due either to pollination mechanisms or to pollen competitive ability. As suggested by Hoekstra (19831, this situation is reached by a shift from the post- to the prepollination phase of several important biosynthetic processes important for pollen tube growth, and would be expressed as a reduction of the lag period of germination and as an increase of pollen tube growth rate. It has been found that quickly germinating pollen utilizes, during emergence and early tube growth, preformed proteins and mRNAs synthesized during microspore development in the anther (Hoekstra, 1983; Mascarenhas, 1984). These types of pollen would attain a more complete stage of

GENETICS OF ANGIOSPERM POLLEN

43

development and therefore would be characterized by a reduced lag phase and high competitive ability. On the evolutionary scale these species include many advanced angiosperms, among which there is the greatest competition between pollen grains for fertilization. As the evolutionary trend seems t o point toward rapid fertilization and to the anticipation of many metabolic activities by the developing microspores, it seems reasonable to hypothesize that the genetic and physiological bases of a n enhanced competitive ability expressed by the pollen in the postpollination stages are to be found in the course of the previous formation phase (Frova et al., 1986). Crop plants deserve a special comment in this discussion. As mentioned above, these species face special environmental conditions which are likely to affect the parameters of MGS. The high intensity of gametophytic selection would produce rapid and positive changes for several important agronomic traits (endosperm growth; resistance t o biotic and abiotic stresses). For this reason, it has been proposed that MGS plays a special role in the evolution of crop plants (Ottaviano and Mulcahy, 1986).

VI. Practical Applications

A. MALE GAMETOPHYTIC SELECTION AS A BREEDING TOOL The large extent of gene expression and of haplo-diploid genetic overlap, the special features of the male gametophytic generation (haploidy and large population sizes), and the experimental results obtained strongly support the use of MGS as a tool to add to the efficiency of conventional breeding procedures. In this respect, MGS has some useful features for in vitro culture selection: the male gametophyte has a simple three-cell structure and large populations can be easily handled in controlled environments. In addition, it offers important advantages for practical applications: 1. Genetic variability to be used under selection pressure is produced by genetic recombination and can be easily predicted on the basis of parental selection. Androgenetic doubled haploid lines (ForaughiWehr et al., 1986) from anther cultures are the only in vztro technique which combines the positive aspects offered by in vitro selection with the possibility of utilizing the genetic variability reassorted by genetic recombination. 2. Besides the possibility of selecting for sporophytic traits, pollen viability and function are important components of plant tolerance

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to environmental stresses (Stevens and Rudich, 1978; Halterlein et al., 1980; Hong-Qui and Croes, 1982; Mackill et al., 1982; Schoper et al., 1986; Qian et al., 1986; Herrero and Johnson, 1980). 3. MGS experiments generally do not require expensive equipment and sophisticated technologies. 4. MGS utilization is not conditioned by plant regeneration Efficient use of MGS in breeding programs requires setting up suitable selection procedures. The main technological aspect to be considered concerns the modalities for the application of the selective pressures. Selective forces can be easily applied when dealing with environmental stresses, which can be reproduced in growth chambers or with chemicals such as salt and heavy metals, which can easily be absorbed and then translocated either within the anther to affect developing microspores or within the pistil to select growing pollen tubes. Selection experiments may be more difficult t o set up when dealing with pathotoxins or herbicides, because the tissues supporting microspore development and tube growth are easily damaged by toxic chemicals and, consequently, gametophyte development would be arrested. Techniques which may help in these situations include in uitro cultures of developing microspores (Pareddy et al., 1987) and of germinating pollen (Hodgkin, 1987; Bowman, 1986). The evaluation of the efficiency of MGS is normally based on the response to selection. This evaluation may be a difficult task when dealing with complex genetic traits. For instance, tolerance to high and low temperatures or to salinity has a complex physiological determination and only some of the basic mechanisms are expressed at the cellular level. For these characters, selection studies require suitable experimental designs by which the effects of genes controlling mechanisms acting at the tissue or plant level are experimentally controlled. Another possibility for the efficient monitoring of the response to MGS can be based on the analysis of differential transmission of molecular markers as isozymes and restriction fragment length polymorphisms (RFLPs), which, being essentially neutral, can be used to detect, in selected progeny, chromosome segments carrying genes implied in the determination of the selected characters.

B. GENETICMANIPULATIONS OF THE GAMETOPHYTIC GENERATION Genetic manipulations affecting the microspore genome, recognition mechanisms in pollen-pistil interactions, and developmental control of microspores can be used to develop a number of methodologies useful

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in plant breeding. Included are procedures to obtain androgenetic double haploid lines (Snape, 1982; Bajaj, 1983; Davies and Hopwood, 19801, in uitro selection of cells and cultures derived from microspores (Foraughi-Wehr et al., 19861, use of meiotic mutants to change the ploidy in the sporophyte (Peloquin, 1983, 19861, efficient mutagenic techniques (Coe, 1966; Gavazzi et al., 1983), and use of male sterility (Duvick, 1965; Laughnan and Gabay-Laughnan, 1983; Edwardson, 1970). All these techniques have been extensively reviewed and therefore will not be discussed in this article. A growing area in pollen technology involves intra- and interspecific gene transfer. Intraspecific gene transfer of one or a few traits from one line to another is based on the observation that pollen treated with sublethal (10-20 kilorads) or lethal (100-150 kilorads) radiation dosages and therefore having heavily damaged chromosomes carries out a normal pollen function (tube growth and discharge of sperm nucleus into the embryo sac), although few viable seeds are recovered (Pfahler, 1975). In the following generation (Fz),the progeny genome is largely maternal, while a portion (changing with radiation dosage) of the treated male parent is lost. The phenomenon was first described by Pandey (1975) and analyzed in detail by Jinks et al. (3981) in crosses between lines of Nicotiana rustica. According to these authors, two basic mechanisms can explain the phenomenon: (1)“egg transformation,” i.e., high radiation dosages that pulverize pollen chromosomes; no normal biparental zygotes are formed, but parthenogenetic progeny are produced and show occasional pollen chromosomal traits inserted during early zygote chromosome replication (Pandey, 1980); and (2) differential gene transfer, i.e., sublethal levels of radiation that allow the formation of the biparental zygote, which occasionally expresses recessive maternal traits; in the subsequent generation, segregation distortion due to differential transmission of donor chromosomes may occur (Jinks et al., 1981; Caligari et al., 1981; Panday, 1983). The extensive research carried out in this field did not confirm the “egg transformation” hypothesis. Disagreement on this phenomenon clearly exists (Sanford et al., 1984; Pandey, 1983). In 2. mays, Sari Gorla et al. (1987) analyzed F2 and BC generations and showed loss of dominant markers but no case of true gene transfer. The significant excess of recessive maternal alleles could be explained as selection effects against the irradiated paternal genome. Studies using tomato (Zamir, 1983b), Pisum (Davies, 19841, Capsicum (Daskalov, 19841, Triticum (Snape et al., 1983), and Nicotiana (Cornish and Werner, 1985) reported cases of mutational loss due to irradiation, followed by selection of F1 gametophytes or in the resultant zygotes. It thus

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appears that although true transformation is not confirmed, radiation techniques, if based on sublethal treatments, might be used to reduce the number of generations required by classical recurrent backcrossing for gene introgression. Intraspecific barriers due to self-incompatibility and interspecific barriers in closely related species due to prezygotic isolation can be overcome to produce hybrids by the techniques referred to as “mentor pollen.” The phenomenon was first observed by Michurin (1950) and was extensively used to overcome incompatibility barriers. The efficiency of the method is increased if the compatible pollen is killed by high dosages of radiation or by chemical treatments: killed mentor pollen germinates and grows in the style, but is unable to perform normal fertilization, which occasionally is achieved by the incompatible pollen used in the mixture. Positive results in overcoming incompatibility have been reported in Populus species (Stettler, 1968; Pandey, 1975, 1977) and in Nzcotiana species (Sree Ramulu et al., 1979); self-incompatibility has been reported in apple (Dayton, 1974; Visser, 1981). A very promising approach to direct gene transfer by exogenous DNA is based on pollen transformation: pollen grains can transfer exogenous genetic material to the zygote, overcoming all the difficulties found with plant regeneration from in uitro cultures. The male gametophyte has a number of features which can be utilized either for direct transformation of pollen nuclei or for delivering exogenous DNA in the embryo sac. Exogenous DNA is absorbed by pollen grains when incubated in germination media (Hess, 1987). Totally marked DNA was first used by Hess (1980). Results (de Wet et al., 1986; Ohta, 1986) indicated that it may be possible to achieve transformation by this approach in maize. The technique was improved by the use of cloned genes (Negrutiu et aZ., 1986) and viruses (Hess, 1981,1988). Use of the Ti plasmid for transformation has been attempted (Jackson et al., 1980; Sanford, 1986). Although these studies provided encouraging results, no precise demonstration of stable genetic transformation has been obtained. DNA microinjection in sporophytic tissues communicating with archesporial cells or directly in pollen grains has been reported (de la Pena et al., 1987). A different method of pollen transformation can be developed by transferring exogenous DNA in isolated sperm cells to be used for in uitro fertilization. Techniques to obtain isolated sperm cells have been studied in wheat and maize and in Gerbera (Southworth and Knox, 1987).

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VII. Conclusions

At least four phenomena in the ontogeny of pollen impinge on the genetic determination of pollen quality: cytoplasmic interconnections which establish developmental synchrony among meiocytes, callose envelopes which isolate microspores from each other, tapetal deposits which coat the pollen exine, and sporophytic inputs which remain in the meiocyte cytoplasm. The first two of these enhance the expression of genetic differences between microspore and the latter two allow the sporophytic influence of pollen quality. Some pollen characteristics are determined exclusively by the sporophyte, as is the case with the exine. Others, such as Ga (gametophytic) factors, gametophytic self-incompatibility, and the presence of specific enzymes (alcohol dehydrogenase, P-galactosidase, etc.) are under gametophytic control. Still others, such as pollen volume, are clearly influenced not only by the sporophyte but also by the genotype of the individual pollen grain and also by competition from other microspores sharing the same locule. Despite the attractiveness of these several determinants of pollen quality, we have concentrated largely on those which involve postmeiotic gene expression. Gene expression in pollen is demonstrated by a variety of phenomena. In a small number of cases, segregating alleles induce visible differences in pollen [waxy, Adh, Rf3 (in conjunction with cms-S)]. More frequently, segregation is expressed in pollen tube growth rates, either monofactorially-as gametophytic self-incompatibility, Ga factors, and many deleterious mutants-or multifactorially in pollen grain diameters or in pollen tube growth rates. A particularly interesting demonstration of gene expression in pollen was the discovery of gametophytically determined hybrid vigor; meiotic mutants which produce heterozygous diploid pollen give rise to pollen grains which exhibit heterotic qualities, while homozygous diploid pollen from other mutants lack these. The most comprehensive investigations of gene expression in pollen have been those dealing with isozymes or homology of mRNA sequences from gametophytic and sporophytic generations. These indicate not only abundant gene expression in the pollen, but also a remarkably consistent and extensive sharing of genes by gametophyte and sporophyte. A promising and still largely unexplored field is the interaction between pollen and pistil. In uztro pollen germination and pollen tube growth rates provide intriguing hints of how pollen may be influenced

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ERCOLE OTTAVIANO AND DAVID L. MULCAHY

by both biotic and abiotic factors. However, these provide only the most tenuous indications of in vivo performance, because the pistil has a substantial influence on pollen behavior. Pistils have a general influence on pollen tube germination and pollen tube growth rate, some producing rapid pollen tube growth and others not. There are also significant pollen-pistil interactions, so that particular pollen genotypes are more favored in some styles than in other styles. There is also preliminary evidence of direct pollen-pollen interaction within the pistil and also of pollen-pollen interaction mediated by the pistil. The mentor effect is a classic example of the latter phenomenon but not the only one. Pollen is, in some cases, stimulated by the presence of other pollen tubes within the style; in other cases it is significantly impeded. Each of these phenomena, now clearly demonstrated, is susceptible to investigation and explanation. Pollen competition does occur in natural populations and available data indicate that it is a frequent but not invariant phenomenon. Its influence on the resulting sporophytic generation has recently been documented in at least one species, Aureolaria peducularia (Scrophulariaceae). The presence of at least one (possibly two) pollen competition enhancing systems indicates that it may be a significant adaptive mechanism. One system, seen in T . mengessii, is a mechanism which delays the germination of pollen. This would mean that the outcome of pollen tube competition would be determined more by pollen tube growth rate, and less by chance time of pollen arrival on the stigma. With delayed pollen germination, greater numbers of pollen grains are able to compete effectively than would be the case without that delay. The requirement in L. Zeucocephala, for a minimal number of pollen grains on the stigma before the pollen germinates may serve a comparable function of intensifying pollen tube competition. Pollen competition may be significant phenomenon in the evolution of crop species and also in the reduction of genetic load. Pollen competition may play a significant role in the adaptative capacities of the angiosperms, since this group possesses three characteristics which enhance pollen tube competition. These are heavy stigmatic loads of pollen, the simultaneous arrival of many pollen grains on the stigma (both consequences of insect pollination), and the requirement for extensive pollen tube growth (imposed by the closed carpels). Since -60% of the sporophytically expressed structural genes are exposed to selection also in the pollen, it seems highly unlikely that this syndrome of pollen competition-enhancing characteristics could be without evolutionary significance. A variety of practical applications for protein techniques have been

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suggested and are presently under study. For basic aspects of pollen selection, the occurrence and, in particular, the sporophytic consequences of pollen selection in natural populations need further study. Finally, direct and indirect in vivo interactions between pollen grains and particularly pollen-pistil interactions need evaluation.

REFERENCES Albertsen, M. C . , and Phillips, R. L. (1981). Developmental cytology of 13 male sterile loci in maize. Can. J . Genet. Cytol. 23, 195-208. Anderson, M. A,, Cornish, S. L., William, E. G., et al. (1986).Cloning ofcDNA for a stylar glycoprotein associated with expression of self-incompatibility in Nicotzana d a t a . Nature (London) 321, 38-44. Bajaj, Y. P. S. (1983). In uztro production of haploids. In “Handbook of Plant Cell Culture” (D. A. Evans, W. R. Sharp, P. V. Ammirato, and Y . Yamada, eds.), pp. 228-287. Macmillan, New York. Barber, H. N. (1942). The pollen grain division in the Orchidaceae. J . Genet. 42, 223-257. Barnes, D. K., and Cleveland, R. W. (1963a). Pollen tube growth of diploid alfalfa in uitro. Crop Sci. 3, 291-295. Barnes, D. K., and Cleveland, R. W. (196310).Genetic evidence for non-random fertilization in alfalfa as influenced by differential pollen tube growth. Crop Scz. 3, 295-297. Beatty, R. A. (1975). Genetics of animal spermatozoa. In “Gamete Competition in Plants and Animals” (D.L. Mulcahy, ed.), pp. 61-68. North-Holland Publ., Amsterdam. Bemis, W. P. (1959). Selective fertilization in lima beans. Genetics 44, 555-562. Bertin, R. I. (1982). Floral biology, hummingbird pollination, and fruit production in trumpet creeper (Campsis radicans). Am. J . Bot. 69, 122-134. Bianchi, A,, and Lorenzoni, C. (1975). Gametophytic factors in Zea mays. In “Gamete Competition in Plants and Animals” (D. L. Mulcahy, ed.). Elsevier, Amsterdam. Bierzychudek, P. (1981). Pollinator limitation of plant reproductive effort. Am. Nut. 117, 838-840. Bino, R. J., Hille, J., and Franken, J. (1987). Kanamycin resistance during in vitro development of pollen from transgenic tomato plants. Plant Cell Rep. 6 , 333-336. Bowman, R. N. (1984). Experimental non-stigmatic pollinations in Clarkia ungulata Lindl. (Onagraceae). Am. J . Bot. 71, 1338-1346. Bowman, R. N. (1986). Factors influencing artificial gametophyte selections using synthetic stigmas. In “Biotechnology and Ecology of Pollen” (D. L. Mulcahy, G. Mulcahy Bergamini, and E. Ottaviano, eds.), pp. 113-118. Springer-Verlag, New York. Boyer, D. C., and Preiss, J. (1981). Evidence for independent genetic control of multiple forms of maize endosperm branching enzyme and starch synthetases. Plant Physiol. 67, 1141-1145. Brewbaker, H. L. (1971). Pollen enzymes and isoenzymes. I n “Pollen Development and Physiology” ( J. Heslop-Harrison, ed.). Butterworths, London. Brewbaker, J. L., and Emery, G. C. (1961). Pollen radiobotany. Radiat. Bot. 1,101-154. Brewbaker, J. L., and Kwack, B. H. (1963). The essential role of calcium ion in pollen germination and pollen tube growth. Am. J . Bot. 50, 859-865.

50

ERCOLE OTTAVIANO A N D DAVID L. MULCAHY

Brewbaker, J. L., and Majumder, S. K. (1961).Cultural studies of the pollen population effect and the self-incompatibility inhibition. A m . J . Bot. 48, 457-464. Brink, R. A. (1924). The physiology of pollen, IV. Chemotropism; effects on growth of grouping grains; formation and function of callose plugs; summary and conclusions. A m . J . Bot. 11, 417-436. Brink, R. A., and MacGillivray, J. H. (1924).Segregation of the waxy character in maize pollen and differential development of the male gametophyte. A m . J . Bot. 11, 465-469. Brown, S. W., and Cave, M. (19541. Detection and nature of dominant lethals in Lilium. I. Effects of X rays on the development of the post-fertilization ovule. A m . J . Bot. 41, 451. Bruck, D. (1957). Male segregation ratio advantage as a factor in maintaining lethal alleles in wild populations of house mice. Proc. Natl. Acad. Sci. U.S.A.43, 152-158. Buchert, J . G. (1961). The stage of genome-plasmon interaction in the restoration of fertility to cytoplasmically pollen-sterile maize. Proc. Natl. Acad. Sci. U.S.A. 47, 1426-1440. Buchholz, J. T. (1922). Developmental selection in vascular plants. Bot. Gaz. 73, 249-286. Buchholz, J. T., Doak, C. C., and Blakeslee, A. F. (1932). Control of gametophytic selection in Datura through shortening and splicing of styles. Bull. Torrey Bot. Club 59,109-118. Burnham, C. L. (1962). “Discussion in Cytogenetics.” Burges, Minneapolis. Caligari, P. D. S., Ingram, N. R., and Jinks, J . L. (1981). Gene transfer in Nicotiana rustica by means of irradiated pollen. I. Unselected progeny. Heredity 47, 12-26. Cameron, D. R., and Moav, R. M. (1957). Inheritance in Nicotiana tabacum. XXVII. Pollen killer, an alien genetic locus inducing abortion of microspores not carrying it. Genetics 42, 326-335. Campbell, D. R. (1987). Interpopulation variation in fruit production: The role of pollination-limitation in the Olympic Mountains. A m . J . Bot. 74, 269-273. Chandlee, J . M., and Scandalios, J. G. (1984). Some properties of genetically defined proteins in maize. Maize Genet. Coop. N . L. 58, 172-177. Chapman, G. P. (1987). The tapetum in pollen: Cytology and development. Int. Rev. Cytol. 107, 111-126. Clegg, M. T., Kahler, A. L., and Allard, L. W. (1978).Estimation oflife cycle components of selection in an experimental plant population. Genetics 89, 765-792. Coe, E. H., J r . (1966). Liquid media suitable for suspending maize pollen before pollination. Proc. Mo. Acad. Sci. 3, 7-8. Cornish, M. A., and Werner, C. P. (1985).Gene transfer inNicotiana rustica by means of irradiated pollen. V. Quantitative characters. Heredity 55, 321-326. Correns, C. (1927). Der Unterschied in der Keimungsgeschwindigkeit der Mannchensamen und Weibchensamen bei Melandrium. Hereditas 9,33-44. Correns, C. (1928).Handb. Vererbungswiss. 2, 1-128. Daskalov, S. (1984). Pollen irradiation and gene transfer in Capsicum. Theor. Appl. Genet. 68, 135-138. Davies, D. R. (1984). Pollen irradiation and the transfer of maternal genes in Pisum sativum. Theor. Appl. Genet. 67, 245-248. Davies, D. R., and Hopwood, D. A. (1980). The plant genome. J . Znnes Symp. 4th; Int. Haploid Conf. 2nd. Dayton, D. F. (1974). Overcoming self-incompatibility in apple with killed compatible pollen. J . A m . SOC.Hortic. Sci. 99, 190-192.

GENETICS OF ANGIOSPERM POLLEN

51

de la Pena, A., Lorz, H., and Shell, J. (1987). Transgenic rye plants obtained by injecting DNA into young floral tillers. Nature (London) 325, 274-276. Demerec, M. (1924). A case of pollen dimorphism in maize. A m . J . Bot. 11, 461-464. Demerec, M. (1929). Cross-sterility in maize. Z.I.A.V. 50, 281-291. Den Nijs, A. P. M., and Van den Boom, A. (1983). Competition between ovules between irradiated and fresh pollen in Cucumis sativus L. I n “Pollen: Biology and Implications for Plant Breeding” (D. Mulcahy and E. Ottaviano, eds.), pp. 397-404. Elsevier, New York. De Nijs, A. P. M., Maisonneuve, B., and Hogenboom, N. G. (1986). Pollen selection in breeding glasshouse tomatoes for low energy conditions. I n “Biotechnology and Ecology of Pollen” (D. L. Mulcahy, G. Bergamini Mulcahy, and E. Ottaviano, eds.). Springer-Verlag, New York. de Wet, J. M. M., Bergquist, P. R., Harlan, J. R., Brink, D. E., Cohen, C . E., Newel, C. A,, and De Wet, A. E. (1986). Exogenous gene transfer in maize (Zea mays) using DNA-treated pollen. I n “Experimental Manipulation of Ovule Tissues” (G. P. Chapman, S. H. Mantell, and R. W. Daniels, eds.), pp. 197-209. Longmans, Green, New York. Dickinson, H. G. (1987). The physiology and biochemistry of meiosis in the anther. Int. Reu. Cytol. 107, 79-110. Dickinson, H., and Sheldon, J. M. (1986).I n “Pollen and Spores: Structure and Function” (S. Blackmore and T. K. Ferguson, eds.), pp. 2-17. Academic Press, London. Doll, H. (1967). Segregation frequencies of induced chlorophyll mutants in barley. Hereditas 58,464-472. Duvick, D. N. (1965). Cytoplasmic pollen sterility in corn. Adu. Genet. 13, 1-56. Echlin, P. (1971). Production of sporopollenin by the tapetum. In “Sporopollenin” ( J . Brooks, P. R. Grant, M. Muir, P. van Gijzel, and G. Shaw, eds.), pp. 220-247. Academic Press, London. Edwardson, J. R. (1970). Cytoplasmic male sterility. Bot. Rev. 36, 341-420. Epperson, B. K., and Clegg, M. T. (1987). First-pollination primacy and pollen selection in the morning glory Ipomoea purpurea. Heredity 58, 5-14. Feder, W. A. (1986). Predicting species response to ozone using a pollen screen. I n “Biotechnology and Ecology of Pollen” (D. Mulcahy, G . B. Mulcahy, and E. Ottaviano, eds.), pp. 89-94. Springer-Verlag, New York. Feder, W. A,, and Sullivan, F. (1969). Differential susceptibility of pollen grains to ozone injury. Phytopathology 59, 399. Feinsinger, P., et al. (1986). Floral neighborhood and pollination success in four hummingbird-pollinated cloud forest plant species. Ecology 67, 449-464. Foraughi-Wehr, B., Friedt, W., Schuchmann, R., Kohler, F., and Wenzel, G. (1986). In uitro selection for resistance. I n “Somaclonal Variation and Crop Improvement” ( J . Semal, ed.), pp. 35-44. Nijhoff, Dordrecht. Freeling, M. (1976). Intragenic recombination in maize: Pollen analysis methods and the effect of parental A d h l +iso-alleles. Genetics 83, 701-717. Frova, C., Sari Gorla, M., and Ottaviano, E. (1983). Haplo-diploid gene expression in maize and its detection. Biochern. Genet. 21, 923-931. Frova, C., Binelli, G., and Ottaviano, E. (1986). Male gametophyte response to high temperature in maize. I n “Biotechnology and Ecology of Pollen” (D. L. Mulcahy, G. Bergamini Mulcahy, and E. Ottaviano, eds.), pp. 33-38. Springer-Verlag, New York. Frova, C., Binelli, G., and Ottaviano, E. (1987). Isozyme and hps gene expression during male gametophyte development in maize. I n “Isozymes. Genetics, Development, and

52

ERCOLE OTTAVIANO AND DAVID L. MULCAHY

Evolution” (M. C. Rattazzi, J . G. Scandalios, and G. S. Whitt, eds.), pp. 97-120. Liss, New York. Ganeshaiah, K. N., Shaanker, R. U., and Shivashankar, G. (1986). Stigmatic inhibition of pollen grain germination-Its simplications for frequency distribution of seed number in pods of Leucaena leucicephala. Oecologia 70, 568-572. Gavazzi, G., Sanguineti, M. C., and Todesco, G. (1983). Pollen mutagenesis in maize. I n “Pollen: Biology and Implications for Plant Breeding” (D. L. Mulcahy and E. Ottaviano, eds.), pp. 351-357. Elsevier, New York. Gay, G., Kerhoas, C., and Dumas, C. (1986). Micro-isoelectric focusing of pollen grain proteins in Cucurbita pep0 L. I n “Biotechnology and Ecology of Pollen” (D. L. Mulcahy, G. Mulcahy Bergamini, and E. Ottaviano, eds.), pp. 496-498. SpringerVerlag, New York. Glover, D., and Barrett, S. C. H. (1986). Stigmatic pollen loads in populations of Pontederia cordata from southern US.Am. J . Bot. 73, 1607-1612. Goodman, M. M., and Stuber, C. W. (1983). Maize. I n “Isozymes in Plant Genetics and Breeding” (S. D. Tanksley and T. J. Orton, eds.), Part B, pp. 1-33. Elsevier, Amsterdam. Goodman, R. N., KJiraly, Z., and Wood, K. R. (1986). “The Biochemistry and Physiology of Plant Disease.” Univ. of Missouri Press, Columbia. Gottschalk, W., and Kaul, M. L. H. (1974). The genetic control of microsporogenesis in higher plants. Nucleus 17, 133-166. Govindaraju, D. R., and Dancik, B. P. (1987). Allozyme heterozygosity and homeostasis in germinating seeds of jack pine. Heredity 59,279-283. Gross, J . A. (1986). Development, physiology and biochemistry corn and wheat pollen. Bot. Rev. 34,333-358. Haldane, J. B. (1924).A mathematical theory of natural and artificial selection. Part 1. Trans. Cambridge Philos. Soc. 23, 19-41. Haldane, J. B. (1932). “The Causes of Evolution” Harper, New York. Haldane, J. B. (1957). The cost of natural selection. J . Genet. 55, 511-524. Haldane, J. B. (1960). More precise expressions for the cost of natural selection. J . Genet. 57,351-360. Halterlein, A. J., Clayberg, C. D., and Teare, D. (1980). Influence of high temperature on pollen grain variability and pollen tube growth in the styles of Phaseolus uulgaris. J . Am. Soc. Hortic. Sci. 105, 12-14. Harding, J. (1975). Models for gamete competition and self fertilization as components of natural selection in populations of higher plants. I n “Gamete Competition in Plants and Animals” (D. L. Mulcahy, ed.). North-Holland Publ., Amsterdam. Harding, J., and Tucker, C. L. (1969). Quantitative studies on mating systems. 11. Method for the estimation of male gametophytic selective values and differential outcrossing rates. Evolution 23, 85-95. Hartl, D. L. (1970a). Meiotic drive in natural populations of Drosophila melanogaster. IX. Suppressors of segregation distorter in wild populations. Can. J . Genet. Cytol. 12, 594-600. Hartl, D. L. (1970b). Population consequences of non-mendelian segregation among multiple alleles. Evolution 24, 415-423. Hartl, D. L. (1975). Stochastic selection of gametes and zygotes. I n “Gamete Competition in Plants and Animals” (D. L. Mulcahy, ed.), pp. 233-242. North-Holland Publ., Amsterdam. Herrero, M. P., and Johnson, R. R. (1980). High temperature stress and pollen viability of maize. Crop Sci. 20, 796-800.

GENETICS OF ANGIOSPERM POLLEN

53

Heslop-Harrison, J., ed. (1971a). The cytoplasm and its organelles during meiosis. “Pollen: Development and Physiology,” pp. 16-31. Butterworths, London. Heslop-Harrison, J. (1971b). Wall pattern formation in angiosperm microsporogenesis. Symp. Soc. Exp. Biol. 25, 277-300. Heslop-Harrison, J., ed. (1971~).The pollen wall: Structure and development. I n “Pollen: Development and Physiology,” pp, 75-98. Butterworths, London. Heslop-Harrison, J. (1972). Patterns in plant cell walls: Morphogenesis in miniature. Proc. R . Inst. Great Br. 45, 335-352. Heslop-Harrison. J. (1979). The forgotten generation: Some thoughts on the genetics and physiology of Angiosperm gametophytes. I n “The Plant Genome” (D. R. Davies and D. A. Hopwood, eds.), J . Innes Symp., 4th. Crowe, Norwich, England. Heslop-Harrison, J., Heslop-Harrison, Y . , Knox, R. B., and Howlett, B. (1973). Pollen wall proteins: Gametophytic and sporophytic fraction in pollen wall of Malvaceae. A n n . Bot. 37, 403-412. Heslop-Harrison, J., Knox, R. B., and Heslop-Harrison, Y. (1974). Pollen wall proteins: Exine held fractions associated with the incompatibility response in Cruciferae. Theor. A p p l . Genet. 44, 133-137. Heslop-Harrison, J., and Heslop-Harrison, Y. (1986). Pollen-tube chemotropism: Fact or delusion? I n “Biology of Reproduction and Cell Motility in Plants and Animals” (M. Cresti and R. Dallai, eds.), pp. 169-174. Univ. of Siena, Siena, Italy. Heslop-Harrison, Y., Reger, B. J., and Heslop-Harrison, J. (1984). The pollen-stigma interaction in the grasses. 6. The stigma (“silk”)ofZea mays L. as host to the pollens of Sorghum bicolor L. (Moench) and Pennisetum americanum L. (Leeke). Acta. Bot. Neerl. 227, 205-227. Hess, D. (1979). Genetic effects in Petunia hybrida induced by pollination with pollen treated with gal transducing phages. 2. Pflanzenphysiol. 93,429-436. Hess, D. (1980). Investigation on the intra- and interspecific transfer of anthocyanin genes using pollen a s vector. 2. Pflanzenphysiol. 98, 321-337. Hess, D. (1981) Attempts to transfer kanamycin resistance of bacterial plasmid origin in Petunia hybrLda using pollen as vectors. Biochern. Physiol. Pflanzen 176, 322-328.

Hess, D. (1988). Direct and indirect gene transfer using pollen as carriers of exogenous DNA. I n “Crop Plant Biotechnology. Biotechnology in Tropical Crop Improvement,” Proc. Int. Biotechnol. Workshop, ICRISA, pp. 11-26. Hyderabad, India. Hess, D. (1987). Pollen-based techniques in genetic manipulation. Int. Reu. Cytol. 107, 367-395.

Hill, J. P., and Lord, E. M. (1986). Dynamics of pollen tube growth in the wild radish Raphanus raphanistrum (Brassicaceae). I. Order of fertilization. Euolution 40, 1328-1334.

Hill, J., and Lord, E. M. (1987). Dynamics of pollen tube growth in the wild radish Raphanus raphanistrum (Brassicaceae). 11. Morphology, cytochemistry and ultrastructure of transmitting tissues, and path of pollen tube growth. A m . J . Bot. 74, 988-997.

Hiraizumi, Y. (1964). Prezygotic selection a s a factor in the maintenance of variability. Cold Spring Harbor Symp. Quant. Biol. 29, 51-60. Hodgkin, T. (1987). A procedure suitable for in vitro pollen selection in Brassica oleracea. Euphytica 36, 153-159. Hodgkin, T., and Lyon, G. D. (1986). The effect of Brussica olerucea estracts on the germination of B. oleracea pollen in a thin layer chromatographic bioassay. J . Exp. Bot. 37, 406-411.

54

ERCOLE OTTAVIANO AND DAVID L. MULCAHY

Hodgkin, T., and Macdonald, M. V. (1986). The effect of a phytotoxin from Alternaria brassicicola on Brassica pollen. New Phytol. 104, 631-636. Hoekstra, F. A. (1983). Physiological evolution in angiosperm pollen: Possible role of pollen vigor. In “Pollen: Biology and Implications for Plant Breeding” (D. L. Mulcahy and E. Ottaviano, eds.), pp. 35-42. Elsevier, New York. Hong-Qui, Z., and Croes, A. F. (1982). Protection of pollen germination from adverse temperature: A possible role for proline. Plant Cell Enuiron. 6, 471-476. Howlett, B. J., Knox, R. B., Paxton, J. H., and Heslop-Harrison, J . (1975). Pollen-wall proteins: Physiochemical characterization and role in self-incompatibility in Cosmos bipinnatus. Proc. R . SOC. London Ser. B 188, 167-182. Iwanami, Y. (1970). Researches of pollen. XX. Population effect and mixture effect in pollen culture. Bot. Mag. (Tokyo) 83, 364-373. Jackson, J . F., Verburg, B. M. L., and Linskens, H. F. (1980). On the inability of Agrobacterium tumefacens Ti plasmids to transform pollen of Petunia hybrida. Acta Bot. Neerl. 29, 277-283. Jahr, G., and Gottschalk, W. (1973). Das Pollenschlauch-Wachstum van PisumMutanten unter Beriicksichtigung der freien Aminosauren. Theor. AppE. Genet. 43, 281-290. Jain, S. K. (1975). Genetic selection in mixed selfing and random mating plant populations. In “Gamete Competition in Plants and Animals” (D. L. Mulcahy, ed.), pp. 265-278. North-Holland Publ., Amsterdam. Jennings, D. L., and Topham, P. B. (1971). Some consequences of raspberry pollen dilution for its germination and for fruit development. New Phytol. 70, 371-380. Jensen, W. A., Schulz, P., and Ashton, M. (1977). An ultrastructural study of early endosperm development and synergid changes in unfertilized cotton ovules. Planta 133, 179-189. Jensen, W. A,, Ashton, M., and Beasley, C. A. (1983). Pollen tube-Embryo sac interaction in cotton. In “Pollen: Biology and Implications for Plant Breeding” (D. Mulcahy and E. Ottaviano, eds.), pp. 67-72. Elsevier, New York. Jimenez, J . R., and Nelson, D. E. (1965). A new fourth chromosome gametophyte locus in maize. J . Hered. 56, 259-262. Jinks, J. L., Caligari, P. D. S., and Ingram, N. R. (1981). Gene transfer in Nicotiana rustica using irradiated pollen. Nature (London) 291, 586-588. Johnson, C. M., and Mulcahy, D. L. (1978). Male gametophyte in maize: 11. Pollen vigor in inbred plants. Theor. Appl. Genet. 51, 211-215. Johnson, C. M., Mulcahy, D. L., and Galinat, W. C. (1976). Male gametophyte in maize: Influences of the gametophytic genotype. Theor. Appl. Genet. 48, 299-303. Jones, D. F. (1928). “Selective Fertilization.” Univ. of Chicago Press, Chicago. Josephson, L. M. (1962). Gamete deletion in male-sterile crosses. Maize Genet. Coop. N.L. 36, 92-93. Kedar, N., Reting, N., and Katan, J . (1967). Non-random segregation of gene I for Fusarium resistance in the tomato. Euphytica 16, 258-266. Kendall, W.D., Lowe, R., and Taylor, N. (1971). Growth of red clover pollen. 111. Free amino acid composition in grains and supplements. Crop Sci. 11, 112-114. Kimura, M. (1959). Genetic load of a population and its significance in evolution. Jpn. J . Genet. 35, 7-33 (in Japanese with English summary). Knox, R. B. (1971). Pollen wall proteins: Localization, enzymic and antigenic activity during development in Gladiolus (Iridaceae). J . Cell Sci. 9, 209-217. Knox, R. B., and Heslop-Harrison, J. (1970). Pollen wall proteins: Localization and enzymic activity. J . Cell Sci. 6, 1-27.

GENETICS OF ANGIOSPERM POLLEN

55

Knox, R. B., Willing, R. R., and Ashford, A. E. (1972). Role of pollen-wall proteins a recognition substances in interspecific incompatibility in poplars. Nature (London) 237, 381-383. Knox, R. B., Gaget, M., and Dumas, C. (1987). Mentor pollen techniques. Int. Rev. CytoZ. 107, 315-332. Kumar, D., and Sarkar, K. R. (1980). Correlation between pollen diameter and rate of pollen tube growth in maize ((Zeamays L.). Indian J.Exp. Bot. 1242-1244. Kwack, B. H. (1983). Autoradiographic studies of methionine incorporation into and exclusion of pectin from pollen tubes of various ornamental species. Acta Hortic. 138, 23-27. Larson, R. L., and Coe, E. H. (1968). Enzymatic action of Bz l anthocyanin factor in maize. Proc. Int. Congr. Genet. 12th 1, 131. Larson, R. L., and Coe, E. H. (1976). Gene dependent flavonoid glucosyltransferase in maize. Biochent. Genet. 15, 153-156. Laughnan, J. R., and Gabay, S. J. (1973). Reaction of germinating maize pollen to Helminthosporium maydis pathotoxins. Crop Sci. 43, 681-684. Laughnan, J. R., and Gabay-Laughnan, S. (1983). Cytoplasmic male sterility in maize. Annu. Rev. Genet. 17, 27-48. Lee, T. D. (1984). Patterns of fruit maturation: A gametophytic competition hypothesis. A m . Nat. 123,427-432. Lee, R. D., and Hartergerink, A. P. (1986). Pollination intensity, fruit maturation pattern, and offspring quality in Cassia fasciculata (Leguminosae). In “Biotechnology and Ecology of Pollen” (D. L. Mulcahy, G. Bergamini Mulcahy, and E. Ottaviano, eds.), pp. 417-422. Springer-Verlag, New York. Lerner, I. M. (1954). “Genetic Homeostasis.” Oliver & Boyd, Edinburgh. Levin, D. A. (1975).Gametophytic selection in Phlox. I n “Gamete Competition in Plants and Animals” (D. L. Mulcahy, ed.), pp. 207-217. Elsevier, Amsterdam. Lewontin, R. C. (1958). A general method for investigating the equilibrium of gene frequency in a population. Genetics 43, 419-434. Lewontin, R. C. (1968). The effect of differential viability on the population dynamics oft alleles in the house mouse. Evolution 22, 262-273. Linskens, H. F. (1974). Translocation phenomena in the petunia flower after cross- and self-pollination. In “Fertilization in Higher Plants” (H.F. Linskens, ed.), pp. 285-292. North-Holland Publ., Amsterdam. Linskens, H. F., and Pfahler, P. L. (1977). Genotypic effects on the amino acid relationships in maize (Zea mays L.). Theor. Appl. Genet. 50, 173-177. Loegering, W. Q., and Sears, E. R. (1963). Distorted inheritance of stem-rust resistance of timstein wheat caused by a pollen-killing gene. Can. J. Genet. Cytol. 5, 65-72. Longley, A. E. (1945). Abnormal segregation during megasporogenesis in maize. Genetics 30, 100-113. McClintock, B. (19441. The relations of homozygous deficiency to mutations and allelic series in maize. Genetics 29, 478-502. McKenna, M., and Mulcahy, D. L. (1983a). Ecological aspects of gametophytic competition in Dianthus chinensis. In “Pollen: Biology and Implications in Plant Breeding” (D. L. Mulcahy and E. Ottaviano, eds.). Elsevier, New York. McKenna, M. A. (1986). Heterostyly and microgametophytic selection: The effect of pollen competition on sporophytic vigor in two distylous species. In “Biotechnology and Ecology of Pollen” (D. L. Mulcahy, G. Bergamini Mulcahy, and E. Ottaviano, eds.), pp. 443-448. Springer-Verlag, Berlin and New York.

56

ERCOLE OTTAVIANO AND DAVID L. MULCAHY

Mackill, D. J., Coffman, W. R., and Rutger, J . N. (1982). Pollen shedding and combining ability for high temperature tolerance in rice. Crop Sci. 22, 730-733. Madjodelo, S. D. P., Grogan, C . O., and Sarvella, P. A. (1966). Morphological expression of genetic male sterility in maize (Zea mays L.). Crop Sci.6, 379-380. Makinen, Y., and MacDonald, T. (1968). Isoenzyme polymorphism in flowing plants. 111. Pollen enzymes and isoenzymes. Physiol. Plant. 21, 477-486. Maletzky, S. I., and Siritza, B. N. (1972).Effect of pollination condition on the expression of the gametophyte locus of chromosome 4. Maize Genet. Coop. N.L. 46, 66-68. Mandelsdorf, P. C. (1932). Mechanical separation of gametes in maize. J . Hered. 23, 288-295. Mangelsdorf, P. C., and Jones, D. F. (1926). The expression of mendelian factors in the gametophyte of maize. Genetics 11, 423-455. Marshall, D., and Ellstrand, N. C. (1986). Sexual selection in Raphanus satiuus: Experimental data on nonrandom fertilization, maternal choice and consequences of multiple paternity. A m . Nut. 127, 446-461. Mascarenhas, J . P. (1975). The biochemistry of Angiosperm pollen development. Bat. Reu. 41, 259-314. Mascarenhas, J . (1978). Sexual chemotaxis and chemotropism in plants. I n “Receptors and Recognition” (G. L. Hazelbauer, ed.), pp. 171-203. Chapman & Hall, London. Mascarenhas, J. P. (1984). Molecular mechanisms of heat stress tolerance. I n “Applications of Genetic Engineering to Crop Improvement” (G. B. Collins and J . G. Petolino, eds.). Nijhoff/Dr. Junk, Dordrecht. Mascarenhas, J . P., and Altschuler, M. (1983). The response of pollen to high temperatures and its potential applications. I n “Pollen: Biology and Implications for Plant Breeding” (D. L. Mulcahy and E. Ottaviano, eds.). Elsevier, New York. Mascarenhas, J. P., and Bell, E. (1969). Protein synthesis during germination of pollen: Studies on polysome formation. Biochim. Biophys. Acta 179, 199-203. Mascarenhas, J . P., and Mermelstein, J . (1981). Messenger RNAs: Their utilization and degradation during pollen germination and tube growth. Acta Sac. Bat. Pol. 50, 13-20. Mascarenhas, N. T., Bashe, D., Eisenberg, A., Willing, R. P., Xiao, C . M., and Mascarenhas, J. P. (1984). Messenger RNA in corn pollen and protein synthesis during germination and pollen tube growth. Theor. Appl. Genet. 68, 323-326. Mather, K. (1965). Genes and cytoplasm in development. Encyclop. Plant Physiol. 15, 41-67. Mather, K., and Jinks, J . L. (1958). Cytoplasm in sexual reproduction. Nature (London) 182, 1188-1190. Maynard-Smith, J. (1971). What use is sex? J . Theor. Biol. 30, 319-335. Meinke, D. W. (1982). Embryo-lethal mutants of Arabidopsis thaliana: Evidence for gametophytic expression of the mutant genes. Theor. Appl. Genet. 63, 381-386. Meinke, D. W. (1985). Embryo-lethal mutants of Arabidopsis thaliana: Analysis of mutants with a wide range of lethal phases. Theor. Appl. Genet. 69, 543-552. Meinke, D. W., and Baus, A. D. (1986). Gametophytic gene expression in embryo-lethal mutants of Arabidopsis thaliana. I n “Biotechnology and Ecology of Pollen” (D. L. Mulcahy, G. Bergamini Mulcahy, and E. Ottaviano, eds.). Springer-Verlag, Berlin and New York. Michurin (English Editor) (1950). Selected works. Nauka, Moscow. Miki-Hiroshige, H., and Nakamura, S. (1983). Incorporation of label from myoinositol-23H by young anther of Lilium longiflorum.Phytomorphology 32, 85-94. Miller, D. P. (1982). Maize pollen: Collection and enzymology. I n “Maize for Biological Research” (F. Sheridan, ed.), pp. 279-293. Plant Mol. Biol. Assoc., Washington, D.C.

GENETICS OF ANGIOSPERM POLLEN

57

Miller, M. K., and Schonhorst, M. H. (1968). Pollen growth of alfalfa in vitro as influenced by grouping of grains on the medium and greenhouse versus field sources. Crop Sci. 8, 525-526. Moore, C. W., and Creech, R. G. (1972). Genetic fine structure analysis of amyloseextender locus in Zea mays L. Genetics 70, 611-619. Mulcahy, D. L. (1967). Optimal sex ratio in Silene a h . Heredity 22, 411-423. Mulcahy, D. L. (1971). A correlation between gametophytic and sporophytic characteristics in Zea mays L. Science 171, 1155-1156. Mulcahy, D. L. (1974). Correlation between speed of pollen tube growth and seedling weight in Zea mays L. Nature (London) 249,491-492. Mulcahy, D. L. (1979). The rise of the angiosperms: A genecological factor. Science 206, 20-23. Mulcahy, D. L. (1983a). Models of pollen competition in Geranium maculatum. In “Pollination Biology” (L. Real, ed.), pp. 151-161. Academic Press, New York. Mulcahy, D. L. (198313). Manipulation of gametophytic population. In “Efficiency of Plant Breeding” (L. Lange, A. C. Zeven, N. G. Hogenboom, eds.), Proc. Congr. EUCARPIA, loth, pp. 167-175. Pudoc, Wageningen, The Netherlands. Mulcahy, D. L., and Kaplan, S. M. (1979). Mendelian ratios despite nonrandom fertilizations? A m . Nut. 113, 419-425. Mulcahy, D. L., and Mulcahy, G. B. (1975).The influence of gametophytic competition on sporophytic quality in Dianthus chinensis. Theor. Appl. Genet. 46, 277-280. Mulcahy, G. B. (1989). In preparation. Mulcahy, G. B., and Mulcahy, D. L. (1983). A comparison ofpollen tube growth in bi- and trinucleate pollen. In “Pollen: Biology and Implications for Plant Breeding” (D. Mulcahy and E. Ottaviano, eds.), pp. 29-33. Elsevier, New York. Mulcahy, G. B., and Mulcahy, D. L. (1985). Ovarian influence on pollen tube growth, as indicated by the semiuitro method. A m . J . Bot. 72, 1078-1080. Mulcahy, G. B., and Mulcahy, D. L. (1987). Induced pollen tube directionality. A m . J . Bot. 74, 143-1459, Mulcahy, G. B., and Mulcahy, D. L. (1988).The effect of supplemented media, in uitro, on bi- and trinucleate pollen. Plant Sci. 55, 213-216. Mulcahy, D. L., Mulcahy, G. B., and Ottaviano, E. (1975). Sporophytic expression of gametophytic competition in Petunia hybrida. In “Gamete Competition in Plants and Animals” (D. L. Mulcahy, ed.). North Holland Publ., Amsterdam. Mulcahy, D. L., Mulcahy, G. B., and Ottaviano, E. (1978). Further evidences that gametophytic selection modifies the genetic quality of the sporophyte. SOC.Bot. Fr. Actual. Bot. 1-2, 57-60. Mulcahy, D. L., Mulcahy, G . B., and Robinson, R. W. (1979). Evidence for postmeiotic genetic activity in pollen of Cucurbita species. J . Hered. 70, 365-368. Mulcahy, D. L., Robinson, R. W., Ihara, M., and Kesseli, R. (1981). Gametophytic transcription for acid phosphatases in pollen of Cucurbita species hybrids. J . Hered. 72,353-354. Mulcahy, G . B., Mulcahy, D. L., and Pfahler, P. L. (1982). The effect of delayed pollination in Petunia hybrida. Acta Bot. Neerl. 31, 97-103. Mulcahy, D. L., Curtis, P. S., and Snow, A. A. (1983). Pollen competition in a natural population. In “Handbook of Experimental Pollination” (C. E. Jones and R. J . Little, eds.), pp. 330-337. Scientific and Academic Editions, New York. Murdy, W. H., and Carter, M. E. B. (1987). Regulation of the timing of pollen germination by the pistil in Talinum mengesii (Portulacaceae). A m . J . Bot. 74, 1888-1892. Negrutiu, K., Heberie-Bors, E., and Potrykus, I. (1986). Attempts to transform for

58

ERCOLE OTTAVIANO AND DAVID L. MULCAHY

kanamycin-resistance in mature pollen of tobaco. In “Biotechnology and Ecology of Pollen” (D. L. Mulcahy, G. Mulcahy Bergamini, and E. Ottaviano, eds.), pp. 65-70. Springer-Verlag, New York. Nelson, 0. E. (1952). Non-reciprocal cross sterility in maize. Genetics 37, 101-124. Nelson, 0. E. (1962). The waxy locus in maize. I. Intralocus recombination frequency estimates by pollen and by conventional analyses. Genetics 47, 737-742. Nelson, 0. E. (1978). Gene action and endosperm development in maize. In “Maize Breeding and Genetics” (D. B. Walden, ed.). Wiley, New York. Nelson, 0. E. (1985). Aberrant ratio revisited. In “Cellular and Molecular Biology of Plant Stress” ( J . L. Key and T. Kosuge, eds.), pp. 1-12. Liss, New York. Nybom, H. (1987). Pollen-limited seed set in pseudogamous blackberries (Rubus). Oecologia 72, 562-568. Ohta, Y. (1986). High-efficiency genetic transformation of maize by a mixture of pollen and exogenous DNA. Proc. Natl. Acad. Sci. U.S.A. 83,715-719. Ortega, E. I., and Bates, L. S. (1980). Enzymatic, isoelectric, and molecular-weight characterization of water-soluble maize-pollen proteins. Physiol. Plant. 48,37 1-374. Ottaviano, E., and Mulcahy, D. L. (1986). Gametophytic selection as a factor of crop plant evaluation. I n “The Origin and Domestication of Cultivated Plants” (C. Barigozzi, ed.). Elsevier, Amsterdam. Ottaviano, E., and Sari Gorla, M. (1979). Genetic variability of male gametophyte in maize. Pollen genotype and pollen-style interaction. Isr.-Ital. Joint Meet. Genet. Breed. Crop Plants. Monogr. Genet. Agrar. (Rome)4. Ottaviano, E., Sari Gorla, M., and Mulcahy, D. L. (1980). Pollen tube growth rate in Zea mays: Implications for genetic improvement of crops. Science 210, 437-438. Ottaviano, E., Sari Gorla, M., and Pe, E. (1982). Male gametophytic selection in maize. Theor. Appl. Genet. 63, 249-254. Ottaviano, E., Petroni, D., and Pe, M. E. (1987). Gametophytic expression of genes controlling endosperm development in maize. Theor. Appl. Genet. 75, 252-258. Ottaviano, E., Sari Gorla, M., and Villa, M. (1988). Pollen competitive ability in maize: Within population variability and response to selection. Theor. Appl. Genet. 76, 601-608. * Pacini, E. (1986). An approach to harmomegathy. In “Biology of Reproduction and Cell Motility in Plants and Animals” (M. Cresti and R. Dallai, eds.), pp. 125-134. Univ. of Siena, Siena, Italy. Pacini, E., and Bellani, L. M. (1986). “Lagerstroemia indica L. Pollen: Form and Function,” pp. 347-357. Linn SOC.,London. Pallais, N., Malagamba, P., Fong, N., Garcia, R., and Schmiediche, P. (1986). Pollen selection through storage: A tool for improving true potato seed quality? In “Biotechnology and Ecology of Pollen” (D. L. Mulcahy, G. Bergamini Mulcahy, and E. Ottaviano, eds.), pp. 153-158. Springer-Verlag, New York. Pandey, K. K. (1975). Sexual transfer of specific genes without gametic fusion. Nature (London) 256,310-313. Pandey, K. K. (1977). Mentor pollen: Possible role of wall-held pollen growth promoting substances in overcoming intra- and interspecific incompatibility. Genetica 47, 219-299. Pandey, K. K. (1980). Further evidence for egg transformation in Nicotiana. Heredity 45, 15-29. Pandey, K. K. (1983). Evidence for gene transfer by the use of sublethally irradiated pollen in Zea mays and theory of occurrence by chromosome repair through somatic recombination and gene conversion. Mol. Gen. Genet. 191, 358-365.

GENETICS OF ANGIOSPERM POLLEN

59

Pareddy, D. R., Greyson, R. I., and Walden, D. B. (1987). Fertilization and seed production with pollen from in-uitro-cultured maize tassels. Planta 170, 141-143. Pasternak, C. A. (1986). Virus, toxin, complement: Common actions and their prevention by Ca or Zn. BioEssuys 6, 14-19. Peacock, W. J., and Miklos, G. L. G. (1973). Meiotic drive in Drosophila: New interpretation of the segregation distorter-sex chromosome systems. Adu. Genet. 17, 361-410. Pecaut, P. (1976). Search of ili varieties, without the gametophytic factor X. Tomato Coop. Rep. 26, 10. Pederson, S., Simonsen, V., and Loeschcke, V. (1987). Overlap of gametophyte and sporophytic gene expression in barley. Theor. Appl. Genet. 75, 200-206. Peloquin, S. J. (1983). Genetic engineering with meiotic mutants. I n “Pollen: Biology and Implications for Plant Breeding” (D. L. Mulcahy and E. Ottaviano, eds.), pp. 311-316. Elsevier, New York. Peloquin, S. J. (1986). Chromosome engineering with meiotic mutants. In “Biotechnology and Ecology of Pollen” (D. L. Mulcahy, G. Mulcahy Bergamini, and E. Ottaviano, eds.), pp. 47-52. Springer-Verlag, New York. Pfahler, P. L. (1965). Fertilization ability of maize pollen grain. I. Pollen sources. Genetics 52, 513-526. Pfahler, P. L. (1967). Fertilization ability of maize pollen grains. 11. Pollen genotype, female sporophyte and pollen storage interactions. Genetics 57, 513-521. Pfahler, P. L. (1974). Fertilization ability of maize (Zea mays L.) pollen grains. IV. Influence of storage and the alleles a t the shrunken, sugary and waxy loci. I n “Fertilization in Higher Plants” (H. F. Linskens, ed.), pp. 15-25. North-Holland Publ., Amsterdam. Pfahler, P. L. (1975). Factors affecting male transmission in maize (Zeu mays L.). I n “Gamete Competition in Plants and Animals” (D. L. Mulcahy, ed.). North-Holland Publ., Amsterdam. Pfahler, P. L. (1983). Comparative effectiveness of pollen genotype selection in higher plants. I n “Pollen: Biology and Implication for Plant Breeding” (D. L. Mulcahy and E. Ottaviano, eds.). Elsevier, New York. Pfahler, P. L. (1986). Pollen storage effects on early seedling growth in maize. I n “Biotechnology and Ecology of Pollen” (D. L. Mulcahy, G. Bergamini Mulcahy, and E. Ottaviano, eds.), pp. 147-152. Springer-Verlag, New York. Plitmann, U., and Levin, D. A. (1983). Pollen-pistil relationship in the Polemoniaceae. Evolution 37, 957-967. Prout, T. (1953). Some effects of variations in the segregation ratio and of selection upon the frequency of alleles. Acta Genet. Stat. Med. 4, 148-151. Qian, C. M., Xu, A,, and Liang, G. H. (1986). Effects of low temperatures and genotypes on pollen development in wheat. Crop Sci. 26, 43-46. Rabinowitch, H. D., Reting, N., and Kedar, N. (1978). The mechanism of preferential fertilization in tomatoes carrying the I-allele for Fusarium resistance. Euphytica 27, 219-224. Rajora, 0. P., and Zsuffa, L. (1986). Sporophytic and gametophytic gene expression in Populus deltoides Marsh., P. nigru L., and P. maximowiczii Henry. Can J . Genet. Cytol. 28, 476-482. Ramstetter, J. (1987).Pollen competition in Aureolaria. PhD. dissertation, University of Massachusetts, Amherst. Redei, G . P. (1965). Non-mendelian megagametogenesis in Arabidopsis. Genetics 51, 857-872.

60

ERCOLE OTTAVIANO AND DAVID L. MULCAHY

Rhoades, M. M., and Dempsey, E. (1966). The effect of abnormal chromosome 10 on preferential segregation and crossing over in maize. Genetics 53,989-1020. Rick, C. M. (1965). Abortion of male and female gametes in the tomato determined by allelic interaction. Genetics 53, 85-96. Rines, H. W., and Luke, H. H. (1985). Selection and regeneration of toxin-insensitive plants from tissue cultures of oats (Auena satiua) susceptible to Helrninthosporiurn uictoriae. Theor. Appl. Genet. 71, 16-21. Rosen, W. G. (1971). Pollen-pistil interactions in Liliurn. In “Pollen: Development and Physiology,” pp. 239-254. Butterworths, London. Rowe, D. E., Stortz, D. L., and Gilette, D. S. (1986). Alfafa pollen and callus responses to Fusarium. In “Biotechnology and Ecology of Pollen” (D. Mulcahy, G. B. Mulcahy, and E. Ottaviano, eds.), pp. 100-106. Springer-Verlag, New York. Rowley, J. R., and Rowley, J. S. (1983). Modification of the exine during germination of pollen grains. In “Pollen: Biology and Implications for Plant Breeding” (D. L. Mulcahy and E. Ottaviano, eds.), pp. 173-182. Elsevier, New York. Rowley, J. R., and Rowley, J. S. (1986). Ontogenetic development of microspores of Ulrnus (Ulmaceae). In “Pollen and Spores: Structure and Function” (S. Blackmore and I. K. Ferguson, eds.), pp. 19-33. Academic Press, London. Sacher, R., Mulcahy, D. L., and Staples, R. (1983). Developmental selection for salt tolerance during self pollination of Lycopersicon x Solanum F1 for salt tolerance of F2. In “Pollen: Biology and Implications for Plant Breeding” (D. L. Mulcahy and E. Ottaviano, eds.). Elsevier, New York. Sadras,V. O., Hall, A. J., and Schlichter, T. M. (1985). Kernel set of the uppermost ear of maize. I. Quantification of some aspects of floral biology. Maydica 30, 37-48. Samson, R. G., Brakke, M. K., and Compton, W. A. (1979). Evidence for gene inactivation in the virus-induced aberrant ratio phenomenon in maize. Genetics 92, 1231-1239. Sanford, J. C. (1986). Attempted pollen-mediated transformation using Ti-plasmids. In “Biotechnology and Ecology of Pollen” (D. L. Mulcahy, G. Mulcahy Bergamini, and E. Ottaviano, eds.), pp. 77-82. Springer-Verlag, New York. Sanford, J. C., Chyi, Y. S., and Reish, B. I. (1984). Attempted “egg transformation” in Zea mays L., using irradiated pollen. Theor. Appl. Genet. 68,269-275. Sari Gorla, M., and Rovida, E. (1980). Competitive ability of maize pollen. Intergametophytic effects. Theor. Appl. Genet. 57,37-41. Sari Gorla, M., Ottaviano, E., and Faini, D. (1975). Genetic variability of gametophytic growth rate in maize. Theor. Appl. Genet. 46, 289-294. Sari Gorla, M., Frova, C., Binelli, G., and Ottaviano, E. (1986). The extent of gametophytic-sporophytic gene expression in maize. Theor. Appl. Genet. 72,42-47. Sari Gorla, M., Villa, M., and Ottaviano, E. (1987). Pollen irradiation and gene transfer in maize. Maydica 32,239-248. Scandalios, J. G. (1964). Tissue-specific isozyme variations in maize. J . Hered. 55, 281-285. Scandalios, J. G. (1979). Control of gene expression and enzyme differentiation. In “Physiological Genetics” (J. G. Scandalios, ed.), pp. 63-107. Academic Press, New York. Scandalios, J. G. (1983). Molecular varieties of isozymes and their role in studies of gene regulation and expression during eukaryote development. In “Isozymes: Current Topics in Biological and Medical Research” (M. C. Rattazzi, J. G. Scandalios, and G. S. Whitt, eds.), pp. 1-31. Liss, New York. Schemske, D. W., and Pautler, P. (1984). The effects of pollen composition on fitness components in a neotropical herb. Oecologia 62,31-36.

GENETICS OF ANGIOSPERM POLLEN

61

Schlichting, C. D., Stephenson, A. G., Davis, L. E., and Winsor, J. A. (1987). Pollen competition and offspring variance. Euol. Trends Plants 1, 35-39. Schoper, J . B., Lambert, R. J., and Vasilas, B. L. (1986). Pollen viability, pollen shedding and combining ability for tassel heat tolerance in maize. Crop Sci. 27, 27-31. Schrauwen, J. A. M., Reijnen, W. H., and van Herpen, M. M. A. (1986). The influence of heat stress on protein synthesis in germinating pollen of Petunia hybrida and Lilium longiflorum. I n “Biology of Reproduction and Cell Motility in Plants and Animals” (M. Cresti and R. Dallai, eds.). Univ. of Siena, Siena, Italy. Schwartz, D. (1960). The analysis of a case of cross-sterility in maize. Proc. Natl. Acad. sci. U.S.A. 36,719-724. Schwartz, D. (1971). Genetic control of alcohol dehydrogenase-A gene competition model for regulation of gene action. Genetics 67, 411-425. Scudo, F. M. (1967). Selection on both haplo- and diplophase. Genetics 56, 693-704. Searcy, K. B., and Mulcahy, D. L. (1985a). Pollen selection and the gametophytic expression of metal tolerance in Silene dioicu (Caryophyllaceae) and Mimulus guttatus (Scrophulariaceae). Am. J . Bot. 72, 1700-1706. Searcy, K . B., and Mulcahy, D. L. (198513).The parallel expression of metal tolerance in pollen and sporophytes of Silene dioica (L.) Clairv., S. albu (Mill.) Krause and Mimulus guttatus DC. Theor. Appl. Genet. 69, 597-602. Sheldon, J. M., and Dickinson, H. (1983). Determination of patterning in the pollen wall of L i h m henryz. J . Cell s c i . 63,191-208. Shivanna, K. R., and Johri, B. M. (1985). “The Angiosperm Pollen. Structure and Function.” Wiley, New Delhi. Shore, J. S., and Barrett, S. C. H. (1984). The effect of pollination intensity and incompatible pollen on seed set in Turneru ulmifolia (Turneraceae). Can. J . Bot. 62, 1298- 1303. Simon, C . J., and Sanford, J . C. (1986). Induction of gametic selection in situ by stylar application of selective agents. I n “Biotechnology and Ecology of Pollen” (D. Mulcahy, G. B. Mulcahy, and E. Ottaviano, eds.), pp. 107-112. Springer-Verlag, New York. Simon, P. W., and Peloquin, S. J. (1976). Pollen vigor as a function of mode of 2n gamete formation in potatoes. J . Hered. 67, 204-208. Singh, M. B., O’Neill, P. M., and Kinox, R. B. (1985). Initiation of postmeiotic ,R-galactosidase synthesis during microsporogenesis in oilseed rape. Plant Physiol. 77, 225-228. Smith, G. A. (19861. Sporophytic screening and gametophytic verification of phytotoxin tolerance in sugarbeet (Beta uulguris L.). I n “Biotechnology and Ecology of Pollen” (D. L. Mulcahy, G. Bergamini Mulcahy, and E. Ottaviano, eds.), pp. 83-88. Springer-Verlag, New York. Snape, J. W. (1982). The use of double haploids in plant breeding. “Induced Variability in Plant Breeding” Int. Symp. Section Mutat. Polyploidy Eucarpia, Wageningen, pp. 52 -58. Centre for Agricultural Publishing and Documentation, Wageningen, The Netherlands. Snape, J . W., Parker, B. B., Simpson, E., Ainsworth, C. C., Payne, P. I., and Law, C. N. (1983). The use of irradiated pollen for differential gene transfer in wheat (Trzticum estiuurn). Theor. Appl. Genet. 65, 103-111. Sniezko, R., and Visser, T. (1987). Embryo development and fruit-set in pear induced by treated and irradiated pollen. Euphytica 36, 287-294. Snow, A. (1986). Pollination dynamics in Epilobiurn canum (Onagraceae): Consequences for gametophytic selection. Am. J . Bot. 73, 139-151. Southworth, D., and Knox, R. B. (1987). Methods of isolating spermcells from pollen. In

62

ERCOLE OTTAVIANO AND DAVID L. MULCAHY

“Plant Sperm Cells as Emerging Tools for Crop Biotechnology” (C. J . Keijzer and H. J. Wilms, eds.). Pudoc, Wageningen, The Netherlands. Sprague, G. F. (1933).Pollen tube establishment and deficiency of waxy seeds in certain maize crosses. Proc. Natl. Acad. Sci. U.S.A. 19, 838-841. Sprague, G. F., and McKinney, H. H. (1966). Aberrant ratio: An anomaly in maize associated with virus infection. Genetics 54, 1287-1296. Sprague, G. F., and McKinney, H. H. (1971).Further evidence on the genetic behavior of AR in maize. Genetics 67, 533-542. Sree Ramulu, K., Bredemeijer, G. M. M., and Dijkhuis, P. (1979).Mentor pollen effects on gametophytic incompatibility in Nicotiana, oenothera and lycopersicum. Theor. Appl. Genet. 54, 215-218. Stadler, L. J. (1933). On the genetic nature of induced mutation in plants. 11. A haplo-viable deficiency in maize. Res. Bull. Mo. Agric. Exp. Stn. 204, 29. Stadler, L. J., and Roman, H. (1984). The effect of X-rays upon mutation of a gene A in maize. Genetics 33, 273-303. Stanley, R. G., and Linskens, H. F. (1974). “Pollen: Biology. Biochemistry. Management.” Springer-Verlag, Berlin. Stanton, M. (1987). Reproductive biology of petal color variants in wild populations of Raphanus sativus: 11. Factors limiting seed production. A m . J.Bot. 74, 188-196. Stephenson, A. G., and Bertin, R. I. (1983).Male competition, female choice, and sexual selection in plants. In “Pollination Biology” (L. Real, ed.), pp. 109-149. Academic Press, Orlando, Florida. Stephenson, A. G., and Winsor, J. A. (1986). Lotus corniculatus regulates offspring quality through selective fruit abortion. Evolution 40, 453-458. Stephenson, A. G., Winsor, J . A,, and Davis, L. E. (1986). Effects of pollen load size on fruit maturation and sporophyte quality in zucchini. In “Biotechnology and Ecology of Pollen” (D. L. Mulcahy, G. Mulcahy Bergamini, and E. Ottaviano, eds.), pp. 429-434. Springer-Verlag, New York. Stettler, R. F. (1968). Irradiated mentor pollen: Its use in remote hybridization of black cottonwood. Nature (London)219, 746-747. Stettler, R. F., and Ager, A. A. (1984). The mentor effect. In “Cellular Interactions” (H. F. Linskens and J . Heslop-Harrison, eds.), pp. 609-623. Springer-Verlag,Berlin and New York. Stevens, M. A., and Rudich, J . (1978). Genetic potential for overcoming physiological limitations on adaptability, yield, and quality in the tomato. HortScience 13, 673-678. Stinson, J., and Mascarenhas, J . P. (1984). Onset of alcohol dehydrogenase synthesis during microsporogenesis in maize. Plant Physiol. 77, 222-224. Stinson, J. R., Eisenberg, A. J., Willing, R. P., Pe, M. E., Hanson, D. D., and Mascarenhas, J . P. (1987). Gene expressed in the male gametophyte of flowering plants and their isolation. Plant Physiol. 83, 442-447. Sukhada, D. K., and Jayachandra. (1980).Pollen allelopathy-A new phenomenon. New Phytol. 84, 739-746. Tanksley, S. D., Zamir, D., and Rick, C. M. (1981). Evidence for extensive overlap of sporophytic and gametophytic gene expression in Lycopersicon esculentum. Science 213,453-455. Ter-Avanesian, D. V. (1949). The role of the number of pollen grains per flower in plant breeding. Bull. Appl. Bot. Plant Breed. 28, 19-33 (in Russian). Ter-Avanesian, D. V. (1978).The effect of varying the number of pollen grains used in fertilization. Theor. Appl. Genet. 52, 77-79.

GENETICS OF ANGIOSPERM POLLEN

63

Thompson, J. D., Andrews, B. J., and Plowright, R. C. (1981). The effect of a foreign pollen on ovule development in Diervilla lonicera (Caprifoliaceae).New Phytol. 90, 777-783. Tsay, H. S., Miao, S . H., and Widholm, J. M. (1986). Factors affecting haploid plant regeneration from maize anther culture. J . Plant Physiol. 126, 33-40. Tsinger, V. N., and Petrovskaya-Boranova, T. P. (1961). The covering of a pollen grain-A living physiological active structure. Dokl. Akad. Nauk SSR 138, 446469. Tupy, J. (1982). Alterations in polyadenylated RNA during pollen maturation and germination. Biol. Plant. 24, 331-340. Vasek, F. C., and Harding, J. (1976). Outcrossing in natural populations. V. Analysis of outcrossing, inbreeding and selection in Clarkia exilis and Clarkia tembloriensis. Evolution 30, 403-411. Vaughn, K. C., et a!. (19801. Organelle alteration as a mechanism for maternal inheritance. Science 208, 196-198. Visser, T. (1981). Pollen and pollination experiments. IV. “Mentor pollen” and “pioneer pollen” techniques regarding incompatibility and incongruity in apple and pear. Euphytica 30, 363-369. Visser, T., and van der Meys, Q. (1986).In vitro reaction between apple pollen and apple scab fungus (Venturia inaequalis Cke. Wint.). In “Biotechnology and Ecology of Pollen” (D. Mulcahy, G. B. Mulcahy, and E. Ottaviano, eds.), pp. 119-124. Springer-Verlag, New York. Visser, T., and Verhaegh, J. (1980). Pollen and pollination experiments. I. The contribution of stray pollen to the seed set of depetalled hand-pollinated flowers of apple. Euphytica 29, 379-383. Vithanage, H. I. M. V., and Knox, R. B. (1976). Pollen wall proteins: Quantitative cytochemistry of the origin of intine and exine enzymes in Brassica oleracea. J . Cell Sci. 21, 95-106. Vithanage, H. I. M. V., and Knox, R. B. (1979). Pollen development and quantitative cytochemistry of exine and intine enzymes in sunflower, Helianthus annuus L. Ann. Bot. 44, 95-106. Wallace, B. (1968). Polymorphism, population size, and genetic load. In “Population Biology and Evolution” (R. C. Lewontin, ed.), pp. 87-108. Syracuse Univ. Press, Syracuse, New York. Wasser, N. (1978). Competition for hummingbird pollination and sequential flowering in two native Colorado wildflowers. Ecology 59, 934-944. Weeden, N. F., and Gottlieb, L. D. (1980). Isolation of cytoplasmic enzymes from pollen. Plant Physiol. 66, 400-403. Willing, R. P., and Mascarenhas, J. P. (1984). Analysis of complexity and diversity of mRNAs from pollen shoots of Tradescantia. Plant Physiol. 75, 865-868. Willing, R. P., Eisenberg, A., and Mascarenhas, J. P. (1984). Genes active during pollen development and the construction of cloned cDNA libraries to mRNAs from pollen. Plant Cell Zncomp. Newslett. 16, 11-12. Winsor, J. A,, Davis, L. E., and Stephenson, A. G. (1987). The relationship between pollen load and fruit maturation and the effect of pollen load in offspring vigor in Cucurbita pepo. A m . Nut. 129, 643-656. Yamada, M., and Murakami, K. (1983). Superiority in gamete competition of pollen derived from F1 plant in maize. In “Pollen: Biology and Implications for Plant Breeding” (D. L. Mulcahy and E. Ottaviano, eds.). Elsevier, New York. Zamir, D. (1983a). Pollen gene expression and selection: Applications in plant breeding.

64

ERCOLE OTTAVIANO AND DAVID L. MULCAHY

In “Isozymes in Plant Genetics and Breeding” (S. D. Tanksley and J . J. Orton, eds.). Elsevier, Amsterdam. Zamir, D. (1983b). Pollen irradiation in tomato: Minor effects on enzymic gene transfer. Theor. Appl. Genet. 66, 147-151. Zamir, D., and Gadish, I. (1987). Pollen selection for low temperature adaptation in tomato. TAG 74, 545-548. Zamir, D., and Jones, R. A. (1981).Estimates of the number of pollen grains applied to a stigma in a single pollination. Tomato Genet. Coo. 31, 21. Zamir, D., and Vallejos, E. C. (1983). Temperature effects on haploid selection of tomato microspores and pollen grains. In “Pollen: Biology and Implication for Plant Breeding” (D. L. Mulcahy and E. Ottaviano, eds.). Elsevier, New York. Zamir, D., Tanksley, S. D., and Jones, R. A. (1981).Low temperature effects on selective fertilization by pollen mixtures of wild cultivated tomato species. Theor. Appl. Genet. 59, 235-238. Zamir, D., Tanksley, S. D., and Jones, R. A. (1982).Haploid selection for low temperature tolerance of tomato pollen. Genetics 101, 129-137.

MICROBIAL SUPEROXIDE DISMUTASES Hosni

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Departments of Food Science, Microbiology, and Toxicology, North Carolina State University, Raleigh, North Carolina 27695

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A. Cloning.. . . . B. Isolation of S

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. Introduction

The presence of oxygen in the environment presents both advantages and a threat to all forms of life. The use of oxygen as a final electron acceptor provides more energy than that afforded by anaerobic fermentation. Oxygen is also useful in many biosynthetic reactions. The threat comes from the fact that oxygen is toxic and causes many deleterious effects. The potential toxicity of oxygen was first recognized and appreciated in 1861 when Louis Pasteur found that certain microorganisms would not grow in air. It is now recognized that the toxicity of oxygen is a common phenomenon among all forms of life. The toxicity of oxygen is related to the partially reduced and dangerously reactive intermediates generated during the univalent reduction of oxygen to water, namely, the superoxide anion radical ( 0 2 - 1 , hydrogen peroxide (H202), and the hydroxyl radical (OH.). The gradual oxygenation of the biosphere, as a consequence of photosynthesis, must have constituted a great selective pressure to the early primitive cells that resulted in the evolution of defense and repair mechanisms for protection against oxygen toxicity. It is obvious that 65 ADVANCES IN GENETICS, Val. 26

Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved

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the first and best line of defense would be to avoid the generation of these reactive intermediates. Indeed, this is the case. Most of the oxygen consumed by aerobic organisms is tetravalently reduced to water by the cytochrome oxidase system without significant release of partially reduced oxygen intermediates. Nevertheless, small amounts of both 0 2 - and H202are normal products of oxygen reduction, which resulted in the evolution of antioxidant enzymes as the second line of defense against oxygen toxicity. The superoxide radical is eliminated by superoxide dismutases (SODs), while hydrogen peroxide is removed by hydroperoxidases. These two lines of defense are designed to prevent damage to cellular constituents. To ensure aerobic survival, postdamage repair mechanisms have also evolved. These include inducible DNA repair mechanisms that are specific for the repair of oxidatively damaged DNA and inducible proteases that turn over and prevent the accumulation of oxidatively damaged proteins. The objective of this article is to review the diversity, function, genetics, and regulation of superoxide dismutases in unicellular microorganisms. For complete accounts of SODs in other organisms the reader should consult recent reviews by Steinman (1982a1, Fridovich (1985, 19861, and Touati (1988b). II. Types and Distribution of Superoxide Dismutases

Superoxide dismutases (EC 1.15.1.1.) are found in oxygenconsuming organisms (McCord et al., 19711, a n aerotolerant anaerobes (Tally et al., 1977; Rolf et al., 1978; Gregory et al., 1978), and in some obligate anaerobes (Hewitt and Morris, 1975; Morris, 1976). SODs are unique in that their substrate ( 0 2 - 1 is a free radical. All SODs are metalloenzymes that catalyze the conversion of 02-to H202 and 0 2 at about the same rate, 2 x lo9 M-' sec-'. Superoxide dismutases, isolated from a wide range of organisms, fall into three classes, depending on the metal found in their active center. In 1969, McCord and Fridovich were the first to describe the superoxide-dismuting ability of a green copper-containing protein that was isolated some 30 years earlier and thought to be a copper storage protein (Mann and Keilin, 1938). This enzyme is now known to contain both copper and zinc (CuZnSOD), where the zinc plays a structural role. Two other SODs were soon discovered: one contains manganese (MnSOD) (Keele et al., 1970) and the second contains iron (FeSOD) (Yost and Fridovich, 1973). The distribution of these three isozymes is distinctly different, and may provide a n interesting evolutionary

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scheme. The CuZnSODs are typically found in the cytosol of eukaryotes, while the FeSODs are found in prokaryotes. On the other hand, MnSODs are found in prokaryotes and in mitochondria. There are, however, some exceptions to this apparently simple evolutionary scheme. Puget and Michelson (1974) were the first to isolate and characterize a CuZnSOD from a prokaryote, Photobacterium leiognathi. This luminescent marine organism lives in symbiotic association within the ponyfish, Leiognathus splendens. This finding led Fridovich (1978) to propose the possibility of gene transfer from the eukaryotic host to the bacterium. This hypothesis was strengthened by finding that the amino acid composition of the bacterial enzyme more closely resembles that of the amino acid composition of fish CuZnSODs than the CuZnSODs from any other species (Martin and Fridovich, 1981). Recent studies based on similarities between the active-site residues of the fish and the bacterial enzymes (Bannister and Parker, 19851, as well as secondary-structure predictions made from the amino acid compositions using computer graphics (Cornish-Bowden, 1985), have supported the gene transfer hypothesis. However, Leunissen and deJong (1986) used computer analysis to compare the complete amino acid sequence of CuZnSOD from P. leiognathi with the sequence from eight eukaryotic species and concluded that gene transfer from the fish lineage to the bacterium is most unlikely. They also concluded that bacteriocupreins most likely separated from the eukaryotic enzyme family before the divergence of fungi and animals and the separation between prokaryotes and eukaryotes. The hypothesis of gene transfer was further challenged especially after the finding of CuZnSODs in free-living bacteria. Vignais et al. (1982) found CuZnSOD in Paracoccus denitrificans. Steinman (1982b) purified and characterized CuZnSOD from Caulobacter crescentus. Steinman (1985) also found CuZnSOD in two strains of the genus Pseudomonas. The presence of FeSOD, a typical prokaryotic enzyme, in some higher plants is another exception to the simple evolutionary scheme for SODS. Salin and Bridges (1980, 1982) isolated FeSOD from mustard leaves (Brussica campestris) and from water lilies (Nuphar luteurn). Duke and Salin (1985) isolated FeSOD from the ginko trees (Ginko biloba). The enzyme was found only in the stroma of chloroplasts, and showed amino acid sequence homology to the FeSODs from Nuphar, Brassica, and Escherichia coli (Duke and Salin, 1985). Bridges and Salin (1981) examined 43 other plant families and found that FeSOD is not widely distributed. FeSOD is also found in a eukaryotic alga, Euglena gracilis (Kanematsu and Asada, 1979). The

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evolutionary route for FeSOD, a typical prokaryotic enzyme, in eukaryotes raises several interesting questions for which no answers are yet available. Amino acid sequence data show that the three types of SODs segregate into two distinct phylogenetic families, the CuZnSOD and the Fe-MnSOD. There is no sequence homology or secondary structure similarity between the two families (Harris and Steinman, 1977; Harris et al., 1980). The FeSODs and MnSOD show a high degree of amino acid sequence and structural homology, while they are completely unrelated to CuZnSODs (Steinman and Hill, 1973; Stallings et al., 1984). Thus, it seems that these two families of SODS have evolved independently in response to a common selection pressure, i.e., the threat of oxygen toxicity. This also suggests that there is no common ancestral enzyme that evolved into the extant Fe-MnSODs and CuZnSODs (Bridgen et al., 1975; Asada et al., 1980). A. COPPER-ZINCSODs Copper-zinc superoxide dismutases have been isolated from a wide range of eukaryotic organisms. The enzymes are characteristically found in the cytosol of eukaryotic cells. CuZnSODs isolated from different sources have very similar amino acid compositions and a high degree of structural homology. The enzyme is a homodimer with a molecular weight of about 32,000 Da, and contains one atom of both copper and zinc per subunit. The enzyme is remarkably stable and remains active in the presence of 8.0 M urea or in 4% sodium dodecyl sulfate (Forman and Fridovich, 1973; Malinowski and Fridovich, 1979a). However, dialysis of the native enzyme at low pH against EDTA causes loss of both copper and zinc, with a concomitant loss of activity. Reconstitution studies revealed that addition of copper alone to the apoprotein restores its full enzymatic activity (Beem et al., 1974; Fee and Briggs, 1975). Attempts to replace the copper with other metals have always resulted in loss of enzymatic activity. On the other hand, the zinc may be replaced by a number of other metals without loss of activity (Forman and Fridovich, 1973). Replacement of zinc by mercury yields a n active enzyme that is more stable than the native enzyme (Forman and Fridovich, 1973). These studies clearly established that copper plays a catalytic role, whereas zinc plays a structural role and contributes to the stability of the enzyme. Johansen et al. (1979) and Steinman (1980) reported the complete amino acid sequence of CuZnSOD from Saccharomyces cereuisiae. The yeast enzyme was found to have 55% homology with the sequence of

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the bovine enzyme previously reported by Steinman et al. (1974). CuZnSODs from human, horse, housefly, and other sources also show a high degree of sequence homology, which indicates that CuZnSODs are highly conserved. It is interesting to note that the degree of homology and sequence conservation is even higher (i.e., >85%) around the active-site channel (Tainer et al., 1982, 1983). Hybridization studies (Tegelstrom, 1975) have also indicated that the subunit interface is highly conserved among the CuZnSOD family. Immunological cross-reactivity studies show that the forms of CuZnSOD from different human organs are immunologically identical (Hartz et al., 1973).No immunological cross-reactivity is seen between more distant relatives (Baret et al., 1979).Martin and Fridovich (1981)also reported that antibody to CuZnSOD from P. lezognathi does not react with CuZnSOD isolated from its host, the ponyfish. These data suggest that surface antigenic domains (hydrophobicity, charge, etc.) are less conserved than the catalytic domains. [For more details on the structure of CuZnSOD, see Steinman (1982a) and Fridovich (19861.1 Specific inhibitors of CuZnSOD are limited in number, but they have been useful in many biochemical studies. Thus, CuZnSODs are reversibly inhibited by cyanide (Rotilio et al., 1972)-the carbon portion of cyanide binds to the copper in the enzyme (Haffner and Coleman, 1973). Azide is also a reversible inhibitor of the enzyme and binds to the copper (Beem et al., 1977). Higher concentrations of azide (-32 mM) are required to achieve 50% inhibition, as compared to 50 p M of cyanide (Misra and Fridovich, 1978). Hydrogen peroxide irreversibly inactivates CuZnSODs (Hodgson and Fridovich, 1975) in a reaction associated with the loss of one histidine residue per subunit (Bray et al., 1974). Hydrogen peroxide reduces Cu2+ to Cu', which slowly reduces oxygen to 02-.The rate of inactivation by HzO2 increases with increasing pH, which suggests that H02-, rather than H202, is the species causing inactivation of the enzyme (Blech and Borders, 1983). Finally, the copper-chelating agent diethyldithiocarbamate (DDC) has been shown to inactivate CuZnSODs by removing the copper (Heikkila et al., 1976; Misra, 1979). DDC has been used to diminish the level of CuZnSOD in uiuo to show that animals treated with DDC are more susceptible to the lethal effects of hyperoxia (Frank et al., 1978). B. MANGANESE-IRON SODS MnSOD (Keele et al., 1970) and FeSOD (Yost and Fridovich, 1973) were first isolated from E . Cali. These enzymes have since been isolated from a wide range of organisms. The Fe-MnSODs are generally found

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in prokaryotes. MnSODs are also found in the matrix of mitochondria. The Mn-FeSODs show a great deal of amino acid sequence (Steinman, 1978; Harris et al., 1980) and structural (Stallings et al., 1984) homology. These findings suggest that the two enzymes have evolved from a common ancestral protein. MnSODs and FeSODs have a subunit molecular weight of about 20,000 and contain one atom of metal per subunit. The bacterial enzymes are most often homodimers. However, tetrameric forms of MnSODs have been isolated from Thermus thermophilus (Sato and Nakazawa, 1978), Thermus aquaticus (Sato and Harris, 19771, and Mycobacteria (Kusunose et al., 1976a,b). Also, tetrameric forms of FeSODs have been isolated from Mycobacterium tuberculosis (Kusunose et al., 1976a) and Rhodococcus bronchialis (Ichihara et al., 1980). Complete amino acid sequences have been reported for MnSODs from E . coli (Steinman, 19781,Bacillus stearothermophilus (Brock and Walker, 19801, T . thermophilus (Sato et al., 19871, and S. cerevisiae (Ditlow et al., 19821, and for FeSOD from Pseudomonas ovalis (Isobe et al., 19871,P. leiognathi (Barra et al., 19871, and E. coli (Schinina et al., 1987). Partial amino-terminal sequence data are available for many Fe-MnSODs. It is interesting to note that MnSODs from E . coli and B . stearothermophilus show about 60% homology (Brock and Walker, 1980). This degree of identity is only 5% higher than that reported for CuZnSODs from S. cerevisiae and bovine erythrocytes, which are phylogenetically more distant than the two bacterial species. These data may suggest that the amino acid sequences of the prokaryotic Mn-FeSODs are less conserved than are the CuZnSODs among eukaryotes. This could be due to the short generation time characteristic of prokaryotes, thus allowing more mutation events per unit time. In spite of the high degree of sequence homology among FeMnSODs, they are immunologically distinct from each other. Thus, antibody to the MnSOD from E . coli does not react with FeSOD from the same organism (Touati, 1983; Schiavone and Hassan, 1988; H. M. Hassan and D. A. Clare, unpublished). Also, antibody to the FeSOD from E . coli does not react with MnSOD (H. M. Hassan and D. A. Clare, unpublished). Antisera against the MnSOD from Streptococcus faecalis cross-reacts with the enzyme from other S. faecalis strains but not with other Streptococcus species nor with species from other genera (Britton et al., 1978). No information is available about the subunit interactions between MnSODs and FeSODs. However, in E . coli, a hybrid enzyme is present (Hassan and Fridovich, 1977a; Dougherty et al., 1978). The hybrid enzyme is a dimer consisting of one subunit each of the Mn- and the

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Fe-enzyme with only iron (0.8 g-at./mol of dimer) in the active center. More recently, however, the hybrid enzyme was found to contain both functional iron and manganese in the active center (Clare et al., 1984; Hassan and Moody, 1987). This clearly indicates that Fe-MnSODs have some structural homology at their surfaces. 1. Metal Coordination-Substitution Although the complete amino acid sequences are known for several MnSODs, assignment of the manganese ligands is not possible due to lack of X-ray crystallographic data (Stallings et al., 1981). On the other hand, the three-dimensional structures for FeSODs from E. coli (Stallings et al., 1983) and P. oualis (Ringe et al., 1983) have been determined. The four iron ligands were assigned to residues 26, 69, 148, and 152 of the E . coli enzyme and to residues 26,69, 151, and 155 of the P. oualis FeSOD. Stallings et al. (1985) determined the X-ray structure of MnSOD from T. thermophilus at a 2.4-A resolution and tentatively assigned His2', Hisa3, and His169as ligands t o the manganese. The exact ligand assignment in FeSODs from E . coli and P. ovalis has not been reported. Metal substitution studies are valuable tools in understanding the structure and function of enzymes and the catalytic role of the native metal. Removal of the metal from SODS results in loss of activity. Attempts at metal replacement using FeSOD and MnSOD from different bacterial species have demonstrated strict metal cofactor specificity of these enzymes (Kirby et al., 1980).Similar studies with E . coli Fe-MnSODs have demonstrated that substitution of Fe for Mn, or vice versa, results in inactive enzymes (Ose and Fridovich, 1976, 1979). These data suggest that the two enzymes, in spite of their relatedness, must have diverged in a way such that only the native metal can restore their catalytic activity. Recent studies, however, have demonstrated that FeSODs from anaerobic organisms can be reconstituted or modulated during biosynthesis to contain manganese in place of iron. Thus, the FeSOD from Bacterozdes fragilis can be reconstituted as a Mn-containing enzyme (Gregory and Dapper, 1983). Furthermore, anaerobically grown B. fragilis contains FeSOD, but oxygen-stressed cells apparently use the same apoenzyme to make an active MnSOD (Gregory, 1985). Also, Propionibacterium shermanii is able to synthesize either FeSOD or MnSOD, using the same protein, depending on the metal supplied in the growth medium (Meier et al., 1982). Martin et al. (1986) also found that S. mutans uses the same apoprotein to produce either FeSOD or MnSOD, depending on the availability of either iron or manganese

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in the growth medium. They suggested the term “Cambialistic enzymes,’ to describe this class of SODS. The evolutionary significance of this class of enzymes is not clear. However, it is interesting to note that most of the “Cambialistic enzymes” are from anaerobic cells, from cells that do not use oxygen as an electron acceptor and only possess the FeSOD gene. Accordingly, one may propose that organisms that use oxygen as a terminal electron acceptor or possess the MnSOD gene or both Fe-MnSOD genes may not be able to exchange their metal cofactors and remain active. This possibility needs to be tested. Also, complete amino acid sequences and X-ray crystallographic data for these “Cambialistic enzymes” are needed in order to be able to compare the ligand fields of this unique class of enzyme with “normal” FeMnSODs. 2 . Inhibitors of Mn-FeSODs The list of specific inhibitors for Fe-MnSODs is very short. Unlike CuZnSODs, Fe-MnSODs are resistant to cyanide. Therefore, the use of cyanide has been a convenient tool for distinguishing the two families. Both Fe-MnSODs are inhibited by azide, where FeSODs are more susceptible than MnSODs (Misra and Fridovich, 1978). A 50% inhibition of FeSODs and MnSODs is seen in the presence of 4 and 20 mh4 azide, respectively. Hydrogen peroxide irreversibly inactivates FeSODs, but has no effect on MnSODs (Asada et al., 1975).This property has been used as a convenient test to distinguish FeSODs from MnSODs (Britton et al., 1978). Inactivation of FeSODs from P . ovalis by H202 has been correlated with losses of tryptophan, histidine, and cysteine residues (Yamakura, 1984). Recently, the inactivation of FeSOD from E . coli by H202 was correlated with losses of tryptophan and some of the iron (i.e., no loss of histidine or any other amino acid was noted) (Beyer and Fridovich, 1987). Inactivation of FeSOD by H202 depends on the presence of iron at the active site, since treatment of Mn-substituted enzyme or the apoenzyme with H2Oz resulted in no loss of activity after reconstituting the treated protein with iron and assaying for activity (Beyer and Fridovich, 1987). These data seem to suggest that H202 reacts with the iron a t the active site to generate OH. or a potent oxidant capable of destroying tryptophan residues.

3. Survey of Fe-MnSODs in Prokaryotes There appears to be no distinct correlation between the type of SOD present and the Gram reaction (Britton et al., 1978). In general, there are no well-defined parameters that allow us to predict which types of

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SOD will be present in a given organism or species. A very broad generalization would be that anaerobic organisms, if they exhibit SOD activity, usually have the iron enzyme. Most obligate aerobes have MnSODs, and facultative anaerobes appear to be intermediate in that they generally contain both MnSODs and FeSODs, with some (i.e., the Enterobacteriaceae family) having a hybrid form of these two enzymes as well (Schiavone and Hassan, 1987). FeSODs are found in Aeromonas, Bacteroides, Bdellovibrio, Beneckea, Methanobacteria, Neisseria, Photobacteria, Pseudomonas, Spirochaeta, Thiobacillus, Treponema, and Vibrio genera with the exceptions of Bacteroides bivis and Bacteroides oralis, which are reported to have no SODs (Gregory et al., 1978). Neisseria ouis and Neisseria caviae contain MnSODs instead of FeSODs (Norrod et al., 1981), and a strain of P. aeruginosa is reported to contain both Fe- and MnSOD instead of FeSOD only (Britton et al., 1978). The two Neisseria species reported to have MnSODs are presently not thought to be true Neisseria (Vedros, 1984). Four strains of Neisseria gonorrhoeae were reported to have no SOD activity but to have very high catalase activity (Norrod and Morse, 1979). MnSODs are found in Thermus, Streptococcus, Staphylococcus, Paracoccus, R hodopseudomonas, Micrococcus, Erwinia, Bacillus, and Mycobacteria with the exception of M. tuberculosis, Bacillus cereus, Bacillus subtilis, and Staphylococcus aureus, which contain FeSODs instead of MnSODs, and Streptococcus intermedius, a strict anaerobe, which is reported to have no SOD (Moody, 1985). Bacillus megaterium (Anastasi et al., 1979) and Bacillus polymyxa are reported to have both Mn-FeSODs. All Acholeplasma reported seem t o have some SOD, but only one report (Reinards et al., 1984) indicates that MnSOD is the type found in this genus. There appears to be no species conformity among the Corynebacteria, i.e., some contained FeSODs and others contained MnSODs; Corynebacterium uaginale, a n obligate anaerobe, had no SOD. The obligately anaerobic genera, Clostridia, Desulfovibrio, Eubacteria, and Fusobacteria are reported to have no SODs with the exception of Clostridium perfringens, Desulfovibrio desulfuricans, Desulfovibrio gigas, Desulfovibrio vulgaris, and Methanobacterium bryantii (Moody, 1985). Some lactic acid bacteria are reported to have no SODs while others are reported to contain the enzyme. This erratic distribution is probably due to the fact that Lactobacillus plantarum and other lactic acid bacteria accumulate very high intracellular manganese concentrations, which can stoichiometrically remove Oz-, thus sparing these

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organisms the need for SODs (Gotz et al., 1980; Archibald and Fridovich, 1981). The Mycoplasma are parasitic, obligate aerobes which are reported t o have no SODs or hydroperoxidases but they are spared the need for these antioxidant enzymes by utilizing the protective enzymes of their host organism (Lynch and Noble, 1980). The facultative anaerobes, Enterobacter, Klebsiella, Proteus, and Serratia, were found to contain both Fe and Mn superoxide dismutases, and Citrobacter, Escherichia, and Salmonella have a hybrid form (i.e., consisting of one subunit each from the iron and manganese enzymes) in addition to Fe- and MnSODs (Schiavone and Hassan, 1987). The presence of CuZnSODs in some prokaryotes was discussed in Section 11. Ill. Evolution of Superoxide Disrnutases

The evolution of three types of SODS that catalyze the same reaction, the dismutation of 02-, clearly demonstrates the vital role of these enzymes in aerobic survival. Our present knowledge about amino acid sequences, DNA sequences, and X-ray crystallographic data of the three types of SODs, as well as knowledge about the geochemical history of Earth, permits us to draw the following plausible scenario for the evolution of SODs (Hassan and Schiavone, 1988). The primitive Earth's atmosphere was reduced and oxygen free. It is believed that the first organisms were anaerobic heterotrophs living in the deep ocean and thus escaping the damaging effect of UV radiation. It is also believed that ionizing radiation might have caused photolytic dissociation of water and the release of small amounts of oxygen. The concentration of oxygen liberated increased as a result of the evolution of photosynthetic organisms, i.e., cyanobacteria followed by green plants. The concentration of oxygen in the biosphere, however, must have increased very slowly because of the presence of huge reservoirs of reduced elements (i.e., Fe2+)that reacted with oxygen. The gradual increase in oxygen concentration presented a challenge and a threat to the early anaerobic organisms; this probably resulted in the evolution of the electron transport chain and the SODs as defense mechanisms against oxygen toxicity. The first prototype of SOD, most likely, was an iron-containing enzyme, because of the abundance and the solubility of Fez+(Asada et al., 1980).Indeed, this is the case; superoxide dismutase present in obligate anaerobes and facultative anaerobes is an ironcontaining enzyme. MnSODs have, most likely, evolved from FeSODs as a result of either gene duplication (Hassan and Schiavone, 1988) or

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unavailability of soluble Fez+,due to its oxidation to the insoluble Fe3+, and the appearance of soluble Mn3+ that replaced the iron (Ochiai, 1983; Kwiatowski, 1987). The possibility of gene duplication stems from the fact that the genes for MnSOD and FeSOD are located about 180” apart on the E . coli chromosome; i.e., MnSOD is at 87.5 minutes (Touati, 1983) and FeSOD is at -37 minutes (Nettleton et al., 1984). These physical map locations seem to support the current views that during evolution, the E . coli genome underwent two duplications (Zipkas and Riley, 1975). Following gene duplication, subtle nucleic acid-amino acid changes must have taken place that favored the insertion of manganese in place of iron. Furthermore, the presence of “Cambialistic SODs” (see Section II,B,l) in some anaerobes may represent a n evolutionary intermediate class of SODs that are less fastidious with respect to the type of metal required to maintain a catalytically active enzyme. Comparative studies about the ligand fields of these “Cambialistic” forms of the enzyme should prove to be very fascinating and valuable. CuZnSODs evolved independently of the Fe-MnSOD family, as is evident by the complete lack of amino acid sequence, DNA sequence, and structural homologies between members of the two families (Steinman, 1982a, 1983). It is generally believed that CuZnSODs must have evolved independently but in response to the same evolutionary pressure that caused the evolution of Fe-MnSODs. However, at the time CuZnSODs evolved, the biosphere was oxygenated and copper was present in the more soluble-oxidized state, Cu2+(Ochiai, 1983). The presence of MnSOD in the organelles of eukaryotic cells has been taken as evidence for the endosymbiotic association between prokaryotes and eubiots that led to present-day eukaryotes (Fridovich, 1974). On the other hand, the presence of FeSODs in plants and CuZnSODs in bacteria remains a scientific mystery, and needs further investigation.

IV. Catalytic Mechanism of Superoxide Dismutases

All known types of superoxide dismutases catalyze the dismutation of 02-according to the following reaction: 02-+ 02-+ 2H’

*

HzOz + O2

The rate constant for the enzyme-catalyzed reaction is 2 x lo9 M-’ sec-’ at 25”C, which is apparently diffusion limited, i.e., the rate of the

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reaction is inversely affected by the viscosity of the solvent. All evidence available to date indicates that the dismutation of 02-is the only known biological function of SODs. The principal mechanism of the enzymatic dismutation of 0 2 - has been elucidated primarily by pulse radiolysis (Klug et al., 1972; Pick et al., 1974; Lavelle et al., 1977).Studies with CuZnSOD revealed cyclical changes in the valence of the copper brought about by 0 2 - (Klug et al., 1972; Rotilio et al., 1972; Klug-Roth et al., 1973). Thus, the absorbance of the enzyme at 680 nm, which is due to Cu2+,is bleached by a pulse of 0 2 - . The bleaching effect is due to reduction from the cupric state to the cuprous state at the active site. Furthermore, when the enzyme is first reduced to the Cu+ state by hydrogen peroxide and then exposed to a pulse of 02-,a partial restoration of absorbance at 680 nm is observed. These studies indicate that the copper undergoes cycles of reduction and reoxidation during its successive encounters with 0 2 - . Similar findings have been obtained with MnSODs and FeSODs (Pick et al., 1974; McAdam et al., 1977; Lavelle et al., 1977). A generalized mechanism for catalysis is presented in the following equations: E-M" + 0 2 E-M"-' + 0 2 + 0 2 - + 2H+ + E-M" + H202 -+

E-M"-' Overall effect:

02-+ 02-+ 2H'

-+

O2 + H202

where E is SOD and M is the metal in the active site. Thus, the copper in CuZnSODs oscillates between the cupric and cuprous states, while the iron and manganese in FeSODs and MnSODs oscillate between the trivalent and the divalent states. Electrostatic channelling was proposed to play an important role in directing the negatively charged substrate ( 0 2 - 1 to the active site of SODs (Koppenol, 1981). Indeed, this is the case. Cudd and Fridovich (1982) reported that increasing the ionic strength decreased the activity of bovine CuZnSOD, and acylation of .+amino groups of lysine residues abolished this response. Similar data were obtained with Mn-FeSODs from E . coli (Benovic et al., 1983). These results indicate that the lysine residues play an important role in facilitating the enzymatic dismutation of 02-.Recent X-ray crystallographic data (Tainer et al., 1983; Getzoff et al., 1983) confirmed the presence of a long and deep solvent channel leading to the Cu2+site. These studies also confirmed the important role of arginine 141 in the catalytic activity of CuZnSOD, as previously suggested by Mailnowski and Fridovich (1979b). Similar data about the active sites of Fe-MnSODs are not available.

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V. Biological Function of Superoxide Disrnutases

Oxygen is toxic (Haugaard, 1968; Gottleib, 1971; Fridovich, 1978) and mutagenic (Fenn et al., 1957; Grifford, 1968; Bruyninckx et al., 1978; Moody and Hassan, 1982; Farr et al., 1986). The toxic effect of oxygen in microorganisms can be manifested in terms of growth inhibition or loss of viability. The outcome and the degree of oxygen toxicity are dependent on the concentration of oxygen, the organism, the suspending medium, and the cellular concentrations of superoxide dismutases. In E . coli, 4.2 atm of oxygen inhibits growth (Brown, 19721, while 20 atm O2 causes loss of viability (Gregory and Fridovich, 1973b; Hassan and Fridovich, 1977a, 1978). Exposure of E. coli to redox-active compounds, which cause a n increase in the intracellular flux of 02-,can be bacteriocidal (Hassan and Fridovich, 1978; Kitzler and Fridovich, 1986) or bacteriostatic (Fee et al., 1981; Kitzler and Fridovich, 1986) depending on the concentration used and on the composition of the suspending medium which affects the permeability of cells toward these compounds (Kitzler and Fridovich, 1986). Also, lack of cellular permeability and uptake of these redox-active compounds (e.g., paraquat) provides E . coli with another mechanism for resisting the toxicity of these intracellular oxyradical generators (Kao and Hassan, 1985a,b). The bacteriostatic effects of oxygen, or of redox-cycling compounds, are correlated with inhibition of branched-chain amino acid biosynthesis (Boehme et al., 1976; Brown and Yein, 1978; Brown and Seither, 19831, inhibition of NAD biosynthesis (Brown and Song, 19801, and decrease in thiamin content (Brown et al., 1981). Recent studies (Kuo et al., 1987) demonstrated that one of the enzymes essential for branched-chain amino acid biosynthesis in E. coli, a,@-dihydroxyisovalerate dehydratase, is extremely sensitive to increasing intracellular fluxes of 0 2 - caused by the presence of redox-active compounds (paraquat and plumbagin) in the growth medium. The inactivation is oxygen dependent, and the enzyme is protected by SOD but not catalase. These findings indicate that 02-inactivates the enzyme and may explain the bacteriostatic effects of moderate hyperoxia and sublethal concentrations of parquat (Fee et al., 1981; Brown and Seither, 1983). Several lines of evidence are available which support the superoxide theory of oxygen toxicity and the physiological role of superoxide dismutases (Fridovich, 1975, 1976). McCord et al. (1971) found that aerobic organisms contain more SOD than do aerotolerant organisms, while anaerobes have no detectable SOD activity. Later studies of

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several species of obligately anaerobic bacteria demonstrated that some anaerobes contain SOD (Hewitt and Morris, 1975; Carlsson et al., 1977; Kirby et al., 1981; Fulghum and Worthington, 1984). However, a positive correlation is found between the levels of SOD in the different anaerobes and their degree of tolerance to oxygen (Tally et al., 1977). The presence of low levels of SOD in many anaerobes indicates their vulnerability to the superoxide radical, which they encounter during transient exposures to oxygen in route from one anaerobic niche to another. Therefore, the presence of SOD in some anaerobes is an evolutionary response to ensure their survival. Further proof of this point is seen in the fact that some anaerobes are able t o induce higher levels of SOD when exposed to reduced levels of oxygen (Privalle and Gregory, 1979). Increased levels of MnSOD in E . coli, induced by a variety of physiological conditions, impart increased resistance against oxygen toxicity (Hassan and Fridovich, 1977b-d, 1978, 1979a-d, 1980, 1982) and against the mutagenicity of oxyradicals (Moody and Hassan, 1982; Hassan and Moody, 1982). The physiological role of SODS in protecting E. coli against oxygen toxicity and mutagenicity has been further explored and confirmed in mutants lacking MnSOD (sodA),FeSOD (sodB), or both (Farr et al., 1986). Conversely, the presence of a plasmid overproducing either FeSOD or MnSOD reduces the mutagenic level seen in the double mutant (sodA so&) to that of the wild type. The oxygen-dependent enhancement of mutagenesis is RecA-independent but is dependent on the presence of a functional exonuclease I11 (Farr et al., 1986). Recent advances in molecular biology and the isolation of SOD genes and SOD cDNAs have facilitated more direct investigations into the physiological function of superoxide dismutases. Thus, the human copper-zinc superoxide dismutase (h-CuZnSOD) complements SOD deficiency in E . coli (Natvig et al., 1987). Normally, the s o d sodB mutant is unable t o grow aerobically in minimal medium, and growth in rich medium is inhibited by the presence of paraquat or hydrogen peroxide; however, the expression of h-CuZnSOD in this mutant converts it back to the wild-type phenotype. Eliasson et al. (1986) have demonstrated that SOD is essential for the activity of E . coli ribonucleotide reductase. They proposed that SOD protects a n oxidation-sensitive intermediate formed during the reduction/oxidation of the reductase iron center, which leads to oneelectron oxidation of tyrosine 122 of the reductase to form a tyrosine radical. It is not clear how this tyrosine radical is formed anaerobically, and if SOD is required.

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These findings clearly support the notion that oxyradicals are toxic and that superoxide dismutases provide, i n uiuo, cellular protection by virtue of their ability to catalytically scavenge 02-.Recently, however, Bloch and Ausuble (1986) and Scott et al. (1987) reported that strains of E . coli containing a plasmid overproducing MnSOD or FeSOD were more sensitive to paraquat toxicity than were the wild-type strains. At the present time, there is no reasonable explanation for this apparent anomaly; furthermore, we have not been able to repeat these observations (H. M. Hassan and L. T. Whritenour, unpublished; D. Touati and A. Carlioz, personal communication). VI. Molecular Genetics of Superoxide Dismutases

This area of SOD research is very young, but great strides have been made as a result of the recent advances in molecular biology. This newer approach is expected to contribute t o our better understanding of the biology and function of superoxide dismutases. A. CLONING The genes for eukaryotic MnSOD and CuZnSOD have been isolated from different sources (i.e., human, rat, maize, and Drosophila) (see Touati, 1988b), and will not be included in this review. The CuZnSOD from P. leiognathi (Steinman, 19871, and S. cereuisiae (Gralla et al., 1988) has been cloned and sequenced. The nucleotide sequence of P. leiognuthi predicts a protein containing a 151-aminoacid of sequence identical to a previously published amino acid sequence (Steffens et al., 1983). The coding sequence was also found to include a 22-residue amino-terminal sequence characteristic of a leader peptide signal (Steinman, 1987). These data suggest that the enzyme may be located in the periplasinic space of this unique organism, a conclusion that awaits further testing. The possibility that the gene for bacterial CuZnSODs (bacteriocupreins) may be widely present, but silent, in other prokaryotes is being tested using the cloned bacteriocuprein gene from P. Zeiognathi as a probe (H. M. Steinman, personal communication). This type of approach may shed light on the evolutionary origin of bacteriocupreins. In S. cereuisiae, MnSOD is located in the mitochondria1 matrix. It is synthesized in the cytoplasm as a precursor protein with a larger molecular weight, which is processed during its translocation into the mitochondria (Autor, 1982). The yeast MnSOD gene has been cloned using a mRNA hybridization technique (Marres et al., 1985). Indeed,

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the nucleotide sequence of the cloned gene shows a n amino-terminal extension of 27 amino acids, consistent with the previous findings of Autor (1982). The nucleotide sequence data also revealed the presence of an upstream conserved sequence similar to the sequence implied in the regulation by heme and oxygen of the iso-l-cytochrome c gene of S . cereuisiae (Marres et al., 1985). The genes for the E . coli MnSOD (sodA) (Touati, 1983; Takeda and Avila, 1986) and FeSOD (so&) (Sakamoto and Tiuati, 1984; Nettleton et al., 1984) have been isolated, and their sequences have been published (Takeda and Avila, 1986; Carlioz et al., 1988). The amino acid sequences, deduced from the nucleotide sequences of MnSOD and FeSOD, were found to be identical to previously published amino acid sequences of the mature proteins. These findings indicate the absence of leader peptide signals and confirm previous findings showing their cytoplasmic location (Britton and Fridovich, 1977). The DNA sequence for MnSOD indicates that the gene constitutes a single operon having two possible promoters, but only one of them appears active under normal aerobic growth conditions (Takeda and Avila, 1986). The DNA sequence also demonstrates the presence of a n almost perfect 19-base palindrome a t the - 35 region, which represents a potential binding site for a repressor molecule. Takeda and Avila (1986) also found that in vitro transcription is more efficient when a linearized pSOD-1 DNA is used than when the supercoiled form is used. These data may suggest that initiation of transcription is affected by the degree of supercoiling of the gene. The degree of supercoiling is also influenced by topoisomerases, gyrases, and oxyradicals that are known to cause DNA single-strand breaks. Studies on the regulatory role of DNA supercoiling in MnSOD biosynthesis will prove to be very valuable. The DNA sequence for the sodB gene (Carlioz et al., 1988) shows lack of regulatory element(s), which is consistent with the current view that FeSOD in E . coli is a constitutive enzyme. B. ISOLATION OF SOD-NEGATIVE MUTANTS Mutants of E . coli lacking MnSOD, FeSOD, or both activities have been isolated using transposon insertions in the cloned structural genes, sodA and sodB (Carlioz and Touati, 1986). The mutated genes, carried on plasmids, were exchanged with the chromosomal genes. The double mutant sodA sodB was constructed by P1 transduction and was shown to lack SOD activity. The SOD-double mutant is unable to grow aerobically on minimal medium but can grow normally on amino

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acid-rich medium or on minimal medium in the absence of oxygen. The double mutant is also hypersensitive to paraquat (Carlioz and Touati, 1986). The molecular basis for its inability to grow aerobically on minimal medium remains unknown, but Fee et al. (1988) isolated several mutants with a suppressor mutation(s) that allows this SODdouble mutant to grow aerobically on minimal medium. These suppressor mutants were shown to still lack SOD activity and remained hypersensitive to paraquat. The nature of these suppressor mutations is not known at the present time, but they should provide useful information in the near future. The SOD-deficient mutants have, indeed, proved to be very valuable tools in studying the oxyradical theory of oxygen toxicity and the biological role of superoxide dismutases (see Section V). In a similar vein, a mutant of S. cereuisiae lacking mitochondria1 MnSOD was isolated (Van Loon et al., 1986) and was found to be unable to grow in air in liquid medium containing nonfermentable carbon source. Furthermore, aerobic growth was impaired in a medium containing glucose. The mutant, however, grows normally under reduced oxygen concentrations. C. BIOSYNTHESISAND REGULATION Microorganisms exhibit the broadest range of diversity with respect to oxygen tolerance, which makes them a n ideal model system for studying the regulation of superoxide dismutases. The induction of SOD in unicellular eukaryotes and in prokaryotes will be dealt with in this section. The biosynthesis of SODS is under rigorous control. Microorganisms are unique in their ability to grow fast and to readjust their cellular composition to meet the fast changes in their environment. Cellular modulation is the key for achieving the best cellular economy. This is usually accomplished through induction, repression, derepression, activation, etc. The same mechanisms apply to the modulation of SOD biosynthesis. Indeed, the level of SOD in a given organism is fine tuned to protect the cells against oxidative damages. Exposure to high concentrations of oxygen induces SOD biosynthesis in E . coli (Gregory et al., 1973; Hassan and Fridovich, 1977a; Yano and Nishie, 19781, S. faecalis (Gregory and Fridovich, 1973a), B. fragilis (Privalle and Gregory, 19791,Bdellovibrio stolpii Won Stein et al., 19821, Streptococcus sanguis (DiGuiseppi and Fridovich, 1982), S. cerevisiae (Gregory et al., 1974; Lee and Hassan, 1986a), Rhizobium japoincum (Stowers and Elkan, 19811, and many other organisms. The presence of increased

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concentrations of ozone also induces SOD biosynthesis in E . coli (Whiteside and Hassan, 1987). Saccharomyces cerevisiae has been used as a model system to study the biosynthesis of SODS in eukaryotic microorganisms. Under aerobic conditions, a cytochrome c-deficient mutant was found to make twice as much SOD as its isogenic wild-type strain (Lee and Hassan, 1986a). The increased synthesis of SOD is due to increased intracellular flux of 0 2 - caused by the lack of a functional electron transport chain, as depicted from the increased rate of cyanide-insensitive oxygen uptake (Lee and Hassan, 1986a). Addition of paraquat caused a n increase in the rate of cyanide-insensitive respiration and in the synthesis of MnSOD and CuZnSOD. The presence of copper further stimulated the synthesis of CuZnSOD (Lee and Hassan, 1986b). Similar findings were reported for S. cereuisiae growing in chemostat cultures (Lee and Hassan, 1987). The effect of copper on SOD biosynthesis was also found in Dactylium dendroides (Shatzman and Kosman, 1978). When grown in a copper-deficient medium the organism makes less of the CuZnSOD but more of the MnSOD, so that the total SOD in the cells remains unaffected by the lack of copper. These studies clearly indicate that the organism requires a certain level of SOD for survival in a given environment, and that one type of SOD can substitute for another. In S . cereuisiae, the gene C Y C f seems to control the biosynthesis of SOD, and a mutation in this gene results in constitutive expression of SOD as well as of two other oxygen-inducible genes (Lowry and Zitomer, 1984). The regulation of SOD biosynthesis is better understood in prokaryotes. Most of the work that has led to our present knowledge of the regulation of SOD and its biological role in protection against oxygen toxicity has come from studies using the facultative anaerobe E . coli. This organism possesses three isozymic forms of SOD: MnSOD, FeSOD, and a hybrid SOD which consists of one subunit each of the iron and manganese isozymes. The FeSOD is present in both aerobically and anaerobically grown cells and is considered to be a constitutive enzyme (Hassan and Fridovich, 1977a). On the other hand, the manganese-containing enzyme and the hybrid form are not made anaerobically but are rapidly induced upon exposure to air. Growth of E . coli in a glucose-limited chemostat culture maintained under constant and abundant aeration demonstrated that the rate of MnSOD biosynthesis is proportional to the specific rate of growth and the rate of respiration (Hassan and Fridovich, 197713). These results indicate that the inducer for MnSOD can not be molecular oxygen, per se, but rather a unique product of its metabolism. These results also advanced the hypothesis that the concentration of MnSOD in the cells is

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modulated to correlate with the intracellular flux of 0 2 - generated during aerobic growth. It was also noted that when E . coli is grown in a rich medium containing limited amounts of glucose (trypticase-soyyeast extract; TSY), the cell's content of MnSOD remains low while utilizing glucose but increases after glucose is exhausted (Hassan and Fridovich, 1977~). These findings indicate that the flux of 0 2 - in E . coli is low during glucose fermentation, but increases during oxidative metabolism, and that the cells are capable of modulating their content of SOD to meet the changing demand to scavenge 0 2 - generated during their active metabolism. Most fascinating was the finding that increasing the intracellular flux of 02-,via the addition of redoxcycling compounds such as methyl viologen (paraquat), causes induction of MnSOD in E . coli (Hassan and Fridovich, 1977c,d, 1979a). The induction of MnSOD by redox-cycling compounds is prevented by inhibitors of transcription or of translation, but not by inhibitors of replication (Hassan and Fridovich, 1979a). Recent studies have demonstrated that the regulation of MnSOD in E . coli is independent of the inducible DNA repair system (SOS) (Hancock and Hassan, 1985), the oxidative stress regulons (oxyR) (Touati, 1988a; Bowen and Hassan, 1988), and the heat-shock regulon (htpR) (Touati, 1988a; Hassan and Lee, 1989). Recent findings have shown that ferrous iron plays an important regulatory role in the biosynthesis of MnSOD (Hassan and Moody, 1984; Moody and Hassan, 1984; Pugh and Fridovich, 1985). A model has been proposed suggesting the synthesis of MnSOD in E . coli is regulated by a negative-control operon, where the repessor protein (RP) contains iron (Moody and Hassan, 1984). This iron-containing repressor may exist in either the ferric (RP-Fe3+)or the ferrous (RP-Fe2+)state (Fig. 1)depending on the redox state of the cell. It is

/

RP

/

Fez' L

RP-Fez+

FIG. 1. Schematic model for the regulation of MnSOD by oxygen, superoxide radical, and iron chelators. RG, Regulatory gene; P, promoter; 0, operator; SG, structural gene (MnSOD); RP, aporepressor protein (inactive); RP-Fe3+,ferric repressor (inactive); RP-Fez+,ferrous repressor (active). [From Moody and Hassan (19841,with permission.1

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proposed that the active repressor contains Fez+,while the inactive form of the repressor contains either Fe3+or no iron. Accordingly, in the absence of oxygen the active form of the repressor will be predominant and no MnSOD will be made. On the other hand, conditions known t o oxidize Fez+to Fe3+(i.e., oxygen or oxyradicals) or to remove Fe2+ from the cells (i.e., iron chelators) will generate the inactive forms of the repressor and will result in the synthesis of the enzyme, even in the absence of oxygen. A similar study confirmed the fact that iron chelators induce MnSOD in E . coli, but the data were explained by another hypothetical model (Fig. 2) (Pugh and Fridovich, 1985). In this model, both FeSOD and MnSOD are viewed as autogenously regulated catalysts whose conversion from an inactive apoprotein to a catalytically active holoenzyme depends on the availability of the appropriate metal cofactor and upon an unknown A key feature of this model is that the two SODS concentration of 02-. are thought to be in dynamic equilibrium with their respective metal cofactors. Thus, under anaerobic conditions the apo-MnSOD is made but the active site is occupied by iron and is inactive. Aerobically, Mn2+is oxidized to Mn3+,which is a strong competitor for the active site and generates active MnSOD (Pugh and Fridovich, 1985). In this Autogen ou s Mn-m-apo (Active)

AMINO ACIDS

Fe-m-apo (Inactive)

\

7

Mn-f-apo (Inactive)

b a p o Autogenous repression Mn(l1) + 02-+ 2H' Fe(II1) + 02-

Fe-f-apo (Active) -

-

Mn(IIIj + H202

-

-

Fe(IIj + O2

FIG. 2. Effects of metals on the biosynthesis of SODS-a proposal. m-apo, Apoprotein of MnSOD; f-apo, apoprotein of FeSOD. The valences of the metals are not shown. It is assumed that Fe(I1) binds to both m-apo and f-apo more avidly than Mn(II), but not more avidly than Mn(II1). The reactions illustrated a t the bottom indicate why 02-increases the availability of Mn(II1). [From Pugh and Fridovich (1985),with permission.]

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model iron chelators are thought t o remove the wrong metal (iron) from the inactive iron-containing MnSOD and allow the right metal (Mn2+)to compete successfully for the active site. This model also proposes autogenous repression by the inactive enzyme. Fortunately, both regulatory models are testable. Recent studies have demonstrated that 59Febinds to all the dismutase isozymes of E . coli, whereas 54Mn is more specific for the manganese and the hybrid isozymes (Hassan and Moody, 1987). In the presence of paraquat, more 59Feis incorporated into MnSOD, whereas no 54Mn is incorporated into FeSOD. These data are the opposite to what is predicted by the Fridovich model. Furthermore, the inactive form of the MnSOD (i.e., the one containing 59Fe)appears to have no autoregulatory function, since its abundant presence does not prevent the induction of MnSOD by paraquat (Hassan and Moody, 1987). Preliminary evidence was also presented for the presence of two unique iron proteins (Hassan and Moody, 1987) that may qualify for the repressor function proposed in the transcriptional repression model (Moody and Hassan, 1984). Furthermore, the presence of a multicopy plasmid carrying the sodA gene was shown to cause the anaerobic expression of MnSOD, presumably by neutralizing the limited number of repressor molecules found in the cell (Hassan and Moody, 1987; Touati, 1988a). The most convincing arguments in support of the negatively controlled operon come from the following findings: (1) anaerobically grown E . coli cells do not contain MnSOD-antigen (Carlioz and Touati, 1986; Schiavone and Hassan, 19881, thus ruling out the possibility of an inactive MnSOD being present anaerobically; (2) the nucleotide sequence of sodA shows a region of diad symmetry a t the -35 region, which may serve as a potential binding site for a regulatory protein (Takeda and Avila, 1986); (3) P-galactosidase is induced by paraquat in a strain of E . coli carrying so& ::LacZ protein fusion (Touati, 1988a), thus showing that the sodA gene is controlled at the transcriptional level and that manganese ions play no regulatory role; (4) MnSOD is induced anaerobically by isopropyl p-Dthiogalactoside (IPTG) in a ptuc ::sodA operon fusion strain (Touati, 1988a1, thus showing that manganese ions of the proper valence are available anaerobically to allow the synthesis of active MnSOD; (5) MnSOD is induced by oxygen in a strain of E . coli overproducing FeSOD (Nettleton et al., 19841, thus suggesting that superoxide radicals are not directly involved in the regulation of MnSOD; and (6) MnSOD is induced anaerobically by potassium ferricaynide and other oxidants capable of positively changing the redox potential

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of the cells (Schiavone and Hassan, 1988).In toto, the data support the proposed model (Moody and Hassan, 1984) of a negatively controlled operon where the repressor molecule is now envisioned as an allosteric redox-sensing protein (Schiavone and Hassan, 1988). Recent findings by Touati (1988a1, support the negatively controlled transcriptional model, but also suggest the presence of a positive transcriptional control by 0 2 - and an autogenous control that does not require the entire sodA gene product. Regulatory mutants of MnSOD have been isolated (Hassan and Touati, 1989) and found to express the sodA gene anaerobically and to be uninducible by paraquat or iron chelators. Indeed, work with these regulatory mutants will allow better understanding of the regulation and genetics of MnSOD.

VII. Conclusion

The past two decades have witnessed a revolution in our understanding of oxygen biology. Superoxide radicals are normal and common by-products of aerobic existence. Superoxide dismutases are indispensable for protection against the toxicity of superoxide radicals. The evolution of three types of SODs to accomplish the same reaction in different organisms attests to their vital role in aerobic survival. Despite the great progress in research on SODs and oxyradicals, there are still many questions to be answered and many more to be raised. The structure of the active centers of Mn-FeSOD is currently unknown, but research is in progress and we will soon know the details. The differences between the ligand binding sites of the fastidious types of Fe-MnSODs nd the promiscuous “Cambialistic” SODs should shed light on their evolution. The mysterious presence of FeSODs in several plants and CuZnSODs in some bacteria is being examined by several investigators, and the results should be enlightening. Recent develompents in the regulation of MnSODs and the success in cloning FeSOD and MnSOD genes of E . coli and the isolation of regulatory mutants will certainly open new research avenues to better understand the regulation of these enzymes. The knowledge gained from E . coli will make it possible to explore the regulation of SODs in other organisms. We look forward to active and exciting developments in SOD research.

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ACKNOWLEDGMENTS This work was supported by DMB-8609239 from the National Science Foundation and 86-G-00719 from the North Carolina Biotechnology Center. This is paper number 11245 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27695-7601. The use of trade names in this publication does not imply endorsement by the North Carolina Agriculture Research Service of the products named. nor criticism of similar ones not mentioned.

REFERENCES Anastasi, A,, Bannister, J. V., and Bannister, W. H. (1979). Isolation and characterization of iron superoxide dismutase from Bacillus megaterium. Int. J . Biochem. 7, 541-546. Archibald, F. S., and Fridovich, I. (1981). Manganese, superoxide dismutase and oxygen tolerance in some lactic acid bacteria. J . Bacteriol. 146, 928-936. Asada, K., Yoshikawa, K., Takahashi, M., Maeda, Y., and Emanji, K. (1975). Superoxide dismutase from a blue green alga, Plectonema boryanum. J . Biol. Chem. 250, 2801-2807. Asada, K., Kanematsu, S., Okaka, S., and Hayakawa, T. (1980). Phylogenetic distribution of three types of superoxide dismutase in organisms and cell organelles. I n “Chemical and Biochemical Aspects of Superoxide and Superoxide Dismutase” (J.V. Bannister and H. A. 0. Hill, eds.), pp. 136-153. Elsevier, Amsterdam. Autor, A. P. (1982). Biosynthesis of mitochondrial manganese superoxide dismutase in Saccharomyces cereuisiae: Precursor form of mitochondrial superoxide dismutase made in the cytoplasm. J . Biol. Chem. 257, 2713-2718. Bannister, J . V., and Parker, M. W. (1985). The presence of a copper/zinc superoxide dismutase in the bacterium Photobacterium leiognathi: A likely case of gene transfer from eukaryotes to prokaryotes. Proc. Nutl. Acad. Sci. U.S.A. 82,149-152. Baret, A,, Michel, P., Imbert, M. R., Morcellet, J. L., and Michelson, A. M. (1979). A radioimmunoassay for copper containing superoxide dismutase. Biochem. Biophys. Res. Commun. 88,337-345. Barra, D., Shinina, M. E., Bannister, W. H., Bannister, J . V., and Bossa, F. (1987). The primary structure of iron-superoxide dismutase from Photobacterium leiognathi. J . Biol. Chem. 262,1001-1009. Beem, K. M., Rich, W. E., and Rajagopalan, K. V. (1974). Total reconstitution of copper-zinc superoxide dismutase. J . Biol. Chem. 249, 7298-7305. Beem, K. M., Richardson, D. C., and Rajagopalan, K. V. (1977). Metal sites of copper-zinc superoxide dismutase. Biochemistry 16, 1930-1936. Benovic, J., Tillman, T., Cudd, A,, and Fridovich, I. (1983). Electrostatic facilitation of the reaction catalyzed by the manganese-containing and iron-containing superoxide dismutases. Arch. Biochem. Biophys. 221, 329-332. Beyer, W. F., and Fridovich, I. (1987). Effect of hydrogen peroxide on the iron-containing superoxide dismutase of Escherichia coli. Biochemistry 26, 1251-1257. Blech, D. M., and Borders, C. L. (1983). Hydroperoxide anion, HOz-, is an affinity reagent for the inactivation of yeast Cu, ZnSOD: Modification of one histidine per subunit. Arch. Biochem. Biophys. 224, 579-586.

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Bloch, C. A., and Ausubel, F. M. (1986). Paraquat-mediated selection for mutations in the manganese-superoxide dismutase gene so&. J . Bacteriol. 168, 795-798. Boehme, D. E., Vincent, K., and Brown, 0. R. (1976).Oxygen and toxicity inhibition of amino acid biosynthesis. Nature (London)262, 418-420. Bowen, S., and Hassan, H. M. (1988).Induction of the manganese-containing superoxide dismutase in Escherichia coli is independent of the oxidative stress (oxyRcontrolled) regulon. J . Biol. Chem. 263, 14808-14811. Bray, R. C., Cockle, S. A,, Fielden, E. M., Roberts, P. B., Rotilio, G., and Calabrese L. (1974). Reduction and inactivation of superoxide dismutase by hydrogen peroxide. Biochem. J . 139, 43-48. Bridgen, J., Harris, J. I., and Northrop, F. (1975). Evolutionary relationships in superoxide dismutases. FEBS Lett. 49, 392-395. Bridges, S. M., and Salin, M. L. (1981). Distribution of iron-containing superoxide dismutase in vascular plants. Plant Physiol. 68, 275-278. Britton, L., and Fridovich, I. (1977). Intracellular localization of the superoxide dismutases of Escherichia coli: A reevaluation. J . Bacteriol. 131, 815-820. Britton, L., Malinowski, D. P., and Fridovich, I. (1978). Superoxide dismutase and oxygen metabolism in Streptococcus faecalis and comparisons with other organisms. J . Bacteriol. 134, 229-236. Brock, C. J., and Walker, J. E. (1980). Superoxide dismutase from Bacillus stearothermophilus: Complete amino acid sequence of manganese enzyme. Biochemistry 19, 2873-2882. Brown, 0. R. (1972).Reversible inhibition of respiration in Escherichia coli. Microbios 5, 7-16. Brown, 0. R., and Seither, R. L. (1983).Oxygen and redox-active drugs: Shared toxicity sites. Fundam. Appl. Toxicol. 3, 209-214. Brown, 0. R., and Song, C. S. (1980). Pyridine nucleotide coenzyme biosynthesis: A cellular site of oxygen toxicity. Biochem. Biophys. Res. Cornmun. 93, 172-178. Brown, 0. R., and Yein, F. (1978). Dihydroxyacid dehydratase: The site of hyperbaric oxygen poisoning in branched-chain amino acid biosynthesis. Biochem. Biophys. Res. Commun. 85, 1219-1224. Brown, 0. R., Heitkamp, M., and Song, C. S. (1981). Niacin reduces paraquat toxicity in rats. Science 212, 1510-1512. Bruyninckx, W. J., Mason, H. S., and Morse, S. A. (1978). Are physiological oxygen concentrations mutagenic? Nature (London)274, 606-607. Carlioz, A., and Touati, D. (1986).Isolation of superoxide dismutase mutants in E . coli: Is superoxide dismutase necessary for aerobic life? EMBO J . 5, 623-630. Carlioz, A,, Ludwig, M. L., Stallings, W. C., Fee, J. A,, Steinman, H. M., and Touati, D. (1988). Iron superoxide dismutases: Nucleotide sequence of the gene from E . coli K12 and correlations with crystal structures. J . Biol. Chem. 263, 1555-1562. Carlsson, J., Wrethen, J., and Beckman, G. (1977).Superoxide dismutase in Bacteroides fragilis and related Bacteroides species. J . Clin. Microbiol. 6, 280-284. Clare, D. A., Blum, J., and Fridovich, I. (1984). A hybrid superoxide dismutase containing both functional iron and manganese. J . Biol. Chem. 259, 5932-5936. Cornish-Bowden, A. (1985). The amino acid sequences of the copper/zinc superoxide dismutases from swordfish and Photobacter leiognathi confirm the predictions made from the compositions. Eur. J . Bzochem. 151, 333-335. Cudd, A., and Fridovich, I. (1982).Electrostatic interactions in the reaction mechanism of bovine erythrocyte superoxide dismutase. J . Biol. Chem. 257, 11443-11447.

SUPEROXIDE DISMUTASES

89

DiGuiseppi, J., and Fridovich, I. (1982). Oxygen toxicity in Streptococcus sanguis: The relative importance of superoxide and hydroxyl radicals. J . Biol. Chem. 257, 4046-4051. Ditlow, C., Johansen, J. T., Martin, B. M., and Svendsen, I. B. (1982). The complete amino acid sequence of manganese-superoxide dismutase from Saccharomyces cereuisiae. Cartsberg Res. Commun. 47, 81-91. Dougherty, H. W., Sandowski, S. J., and Baker, E. E. (1978). A new iron-containing superoxide dismutase from Escherichia coli. J. Biol. Chem. 253, 5220-5223. Duke, M. V., and Salin, M. L. (1985). Purification and characterization of an ironcontaining superoxide dismutase from the eucaryote, Ginlto biloba. Arch. Biochem. Biophys. 243, 305-314. Eliasson, R., Jornvall, H., and Reichard, P. (1986). Superoxide disrnutase participates in the enzymatic formation of the tyrosine radical of ribonucleotide reductase from Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 83, 2373-2377. Farr, S. B., D’Ari, R., and Touati, D. (1986). Oxygen-dependent mutagenesis in Escherichia coli lacking superoxide dismutase. Proc. Natl. Acad. Sci. U.S.A. 83, 8268-8272. Fee, J. A,, and Briggs, R. G. (1975). Studies on the reconstitution of bovine erythrocyte superoxide disrnutase. V. Preparation and properties of derivatives in which both zinc and copper sites contain copper. Biochim. Biophys. Acta 400, 439-450. Fee, J. A., Lees, A. C., Bloch, P. L., Gilliland, P. L., and Brown, 0. R. (1981). Oxygen stasis of bacterial growth: Analogy between the stasis of E . coli by hyperbaric oxygen and by aerobic paraquat. Biochem. Znt. 1, 304-311. Fee, J. A., Niederhoffer, E. C., and Naranjo, C. (1988). First description of a variant o f E . coli lacking superoxide dismutase activity yet able to grow efficiently on minimal oxygenated medium. In “Trace Element Metabolism in Man and Animals” (L. Hurley, C. Keen, B. Lonnerdal, and R. Rucker, eds.), pp. 239-243. Plenum, New York. Fenn, W. O., Gerschman, R., Gilbert, D. L., Terwilliger, D. E., and Gothran, F. V. (1957). Mutagenic effects of high oxygen tensions on Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 43, 1027-1032. Forman, H. J., and Fridovich, I. (1973). On the stability of bovine superoxide dismutase: The effect of metals. J . Biol. Chem. 248, 2645-2649. Frank, L., Wood, D. L., and Roberts, R. J. (1978). Effect of diethyldithiocarbamate on oxygen toxicity and lung enzyme activity in immature and adult rats. Biochem. Pharmacol. 27, 251-254. Fridovich, I. (1974). Evidence for the symbiotic origin of mitochondria. Life Sci. 14, 819-826. Fridovich, I. (1975). Superoxide dismutases. Annu. Rev. Biochem. 44, 147-159. Fridovich, I. (1976). Oxygen radicals, hydrogen peroxide, and oxygen toxicity. In “Free Radicals in Biology” (W. A. Pryor, ed.), Vol. 1, pp. 239-277. Academic Press, New York. Fridovich, I. (19781. The biology of oxygen radicals. Science 201, 875-880. Fridovich, I. (1985). Superoxide dismutases: Regularities and irregularities. Harvey Lect. 79, 51-75. Fridovich, I. ( 1986). Superoxide dismutases. Adu. Enzymol. 58, 62-97. Fulghum, R. S., and Worthington, J . M. (1984). Superoxide dismutase in ruminal bacteria. Appl. Environ. Microbiol. 48, 675-677. Getzoff, E. D., Tainer, J . A., Weiner, P. K., Kollman, P.A., Richardson, J . S., and

90

HOSNI M. HASSAN

Richardson, D. C. (1983). Electrostatic recognition between superoxide and copper, zinc superoxide dismutase. Nature (London) 306, 287-290. Gottleib, S. F. (1971). Effects of hyperbaric oxygen on microorganisms. Annu. Rev. Microbial. 25, 111-152. Gotz, F., Elstner, E. F., Sedewitz, B., and Lengfelder, E. (1980). Oxygen utilization by Lactobacillus plantarum. Arch. Microbiol. 125, 215-220. Gralla, E. B., Bermingham-McDonogh, O., Valentin, J. S., Change, E., Kreuger, T., and Kosman, D. (1988). Cloning and biological activity of the CuZnSOD gene of S. cereuisiae. In “Oxyradicals in Molecular Biology and Pathology” (P. A. Ceruti, I. Fridovich, and J. M. McCord, eds.), pp. 173-182. Liss, New York. Gregory, E. M. (1985). Characterization of the 02-induced manganese-containing superoxide dismutase from Bacteroides fragilis. Arch. Biochem. Biophys. 238, 83-89. Gregory, E. M., and Dapper, C. H. (1983). Isolation of iron-containing superoxide dismutase from Bacteroides fragilis: Reconstitution as a Mn-containing enzyme. Arch. Biochem. Biophys. 220, 293-300. Gregory, E. M., and Fridovich, I. (1973a). The induction of superoxide dismutase by molecular oxygen. J . Bacteriol. 114, 543-548. Gregory, E. M., and Fridovich, I. (1973b). Oxygen toxicity and the superoxide dismutase. J . Bacteriol. 114, 1193-1197. Gregory, E. M., Yost, F. J., and Fridovich, I. (1973). Superoxide dismutases of Escherichia coli: Intracellular localization and functions. J . Bacteriol. 115, 987-991. Gregory, E. M., Goscin, S. A., and Fridovich, I. (1974). Superoxide dismutase and oxygen toxicity in a eukaryote. J . Bacteriol. 117, 456-460. Gregory, E. M., Moore, W. E. C., and Holdeman, L. V. (1978). Superoxide dismutase in anaerobes: Survey. Appl. Enuiron. Microbiol. 35, 988-991. Grifford, G. D. (1968). Mutation of a n auxotrophic strain of Escherichia coli by high pressure oxygen. Biochem. Biophys. Res. Commun. 33, 294-298. Haffner, P. H., and Coleman, J. E. (1973). Cu(I1)-carbon bonding in cyanide complexes of copper enzymes: 13Csplitting of the Cu(I1) electron spin resonance. J . Biol. Chem. 248, 6626-6629. Hancock, L. C., and Hassan, H. M. (1985). Regulation of the manganese-containing superoxide dismutase is independent of the inducible DNA repair system in Escherichia coli. J . Biol. Chem. 260, 12954-12956. Harris, J. I., and Steinman, H. M. (1977). Amino acid sequence homologies among superoxide dismutases. In “Superoxide and Superoxide Dismutases” (A. M. Michelson, J. M. McCord, and I. Fridovich, eds.), pp. 225-230. Academic Press, New York. Harris, J. I., Auffret, A. D., Northrop, F. D., and Walker, J. E. (1980). Structural comparisons of superoxide dismutase. Eur. J . Biochem. 106, 297-303. Hartz, J. W., Funakoshi, S., and Deutsch, H. F. (1973). The levels of superoxide dismutase and catalase in human tissues as determined immunochemically. Clin. Chim. Acta 46, 125-132. Hassan, H. M., and Fridovich, I. (1977a). Enzymatic defenses against the toxicity of oxygen and of streptonigrin in Escherichia coli.J . Bacteriol. 129, 1574-1583. Hassan, H. M., and Fridovich, I. (1977b). Physiological function of superoxide dismutase in glucose-limited chemostat cultures ofEscherichia coli. J . Bacteriol. 130,805-81 1. Hassan, H. M., and Fridovich, I. (1977~). Regulation of superoxide dismutase synthesis in Escherichia coli: Glucose effect. J . Bacteriol. 132, 505-510. Hassan, H. M., and Fridovich, I. (1977d). Regulation of the synthesis of superoxide

SUPEROXIDE DISMUTASES

91

dismutase in Escherichia coli: Induction by methyl viologen. J . Biol. Chem. 252, 7667-7672. Hassan, H. M., and Fridovich, I. (1978). Superoxide radical and the oxygen enhancement of the toxicity of paraquat in Escherichia coli. J . Biol. Chem. 253,8143-8148. Hassan, H. M., and Fridovich, I. (1979a). Intracellular production of superoxide radical and of hydrogen peroxide by redox active compounds. Arch. Biochem. Biophys. 196, 385-395. Hassan, H. M., and Fridovich, I. (1979b). Superoxide, hydrogen peroxide, and oxygen tolerance of oxygen-sensitive mutants of Escherichia coli. Reu. Infect. Dis. 1, 357-367. Hassan, H. M., and Fridovich, I. (1979~). Superoxide dismutase and its role for survival in the presence of oxygen. I n “Strategy of Microbial Life in Extreme Environments” (M. Shilo, ed.), pp. 179-193. Verlag Chemie, New York. Hassan, H. M., and Fridovich, I. (1979d). Paraquat and Escherichia coli: Mechanism of production of extracellular superoxide radical. J . Biol. Chem. 254, 10846-10852. Hassan, H. M., and Fridovich, I. (1980). The mechanism of the antibiotic action of pyocyanine. J . Bacteriol. 141, 156-163. Hassan, H. M., and Fridovich, I. (1982). Exacerbation of oxygen toxicity by redox active compounds. I n “Oxidases and Related Redox Systems” (T. E. King, H. S. Mason, and M. Morrison, eds.), pp. 151-167. Univ. Park Press, Baltimore, Maryland. Hassan, H. M., and Lee, F.-J. (1989). Effect of temperature and htpR on the biosynthesis of superoxide dismutase in Escherichia coli. FEMS Microbiol. Lett. 58, 133-138. Hassan, H. M., and Moody, C. S. (1982). Superoxide dismutase protects against the toxicity and mutagenicity of oxygen free radicals in Salmonella typhimurium. Can. J . Physiol. Pharmacol. 60, 82-92. Hassan, H. M., and Moody, C. S. (1984). Induction of the manganese-containing superoxide dismutase in Escherichia coli by nalidixic acid and by iron chelators. FEMS Microbiol. Lett. 25, 233-236. Hassan, H. M., and Moody, C. S. (1987). Regulation of manganese-containingsuperoxide dismutase in Escherichia coli: Anaerobic induction by nitrate. J . Biol. Chem. 262, 17173-17177. Hassan, H. M., and Schiavone, J. R. (1989). The role of oxygen free radicals in biology and evolution. I n “Metazoan Life without Oxygen” (C. Bryant, ed.), Croom-Helm, Australia. Hassan, H. M., and Touati, D. (1989). In preparation. Haugaard, N. (1968). Cellular mechanisms of oxygen toxicity. Physiol. Reu. 48,311-373. Heikkila, R. E., Cabbat, F. S., and Cohen, G. (1976). I n uiuo inhibition of superoxide dismutase in mice by diethyldithiocarbamate. J . Biol. Chem. 251, 2182-2185. Hewitt, J., and Morris, J . G. (1975). Superoxide dismutase in some obligately anaerobic bacteria. FEBS Lett. 50, 315-318. Hodgson, E. K., and Fridovich, I. (1975).The interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: Inactivation of the enzyme. Biochemistry 14, 5294-5299. Ichihara, K., Kasaoka, I., Kusunose, E., and Kusunose, M. (1980). A manganese and iron-containing superoxide dismutase from Rhodococcus bronchialis. J . Gen. Appl. Microbiol. 26, 387-393. Isobe, T., Fang, Y. I., Muno, D., Okuyama, T., Ohmori, D., and Yamakura, F. (1987). Amino acid sequence of iron-superoxide dismutase from Pseudmonas oualis. FEBS Lett. 221, 87-90. Johansen, J . T., Overballe-Petersen, C., Martin, B., Hassemann, V., and Svendsen, I.

92

HOSNI M. HASSAN

(1979). The complete amino acid sequence of copper, zinc, superoxide dismutase from Saccharomyces cereuisae. Carlsberg Res. Commun. 44,201-217. Kanematsu, S., and Asada, K. (1979). Ferric and manganic superoxide dismutases in Euglena gracilis. Arch. Biochem. Biophys. 195, 535-545. Kao, S. M., and Hassan, H. M. (1985a). Isolation of paraquat-resistant mutants of Escherichia coli: Lack of correlation between resistance and the activity of superoxide dismutase. FEMS Microbiol. Lett. 28, 93-97. Kao, S. M., and Hassan, H. M. (1985b). Biochemical characterization of a paraquattolerant mutant of Escherichia coli. J . Biol. Chem. 260, 10478-10481. Keele, B. B., McCord, J. M., and Fridovich, I. (1970). Superoxide dismutase from Escherichia coli B: A new manganese-containing enzyme. J . Biol. Chem. 245, 6176-6181. Kirby, T., Blum, J., Kahane, I., and Fridovich, I. (1980). Distinguishing between Mn-containing and Fe-containing superoxide dismutases in crude extracts of cells. Arch. Biochem. Biophys. 201,551-555. Kirby, T. W., Lancaster, J. R., and Fridovich, I. (1981). Isolation and characterization of the iron-containing superoxide dismutase of Methanobacterium bryantii. Arch. Biochem. Biophys. 210, 140-148. Kitzler, J., and Fridovich, I. (1986). Effects of salts on the lethality of paraquat. J . Bacteriol. 167, 346-349. Klug, D., Rabani, J., and Fridovich, I. (1972). A direct demonstration of the catalytic action of superoxide dismutase through the use of pulse radiolysis. J . Biol. Chem. 247,4839-4842. Klug-Roth, D., Fridovich, I., and Rabani, J. (1973). Pulse radiolytic investigation of superoxide catalyzed disproportionation: Mechanisms for bovine superoxide dismutase. J . Am. Chem. SOC.95, 2786-2790. Koppenol, W. H. (1981). The physiological role of the charge distribution on superoxide dismutase. In “Oxygen and Oxy-radicals in Chemistry and Biology” (M. A. J. Rodgers and E. L. Powers, eds.), pp. 671-674. Academic Press, New York. Kuo, C. F., Mashino, T., and Fridovich, I. (1987). a$-Dihydroxyisovalerate dehydratase: A superoxide-sensitive enzyme. J. Biol. Chem. 262, 4724-4727. Kusunose, E., Ichihara, K., Noda, Y., and Kusunose, M. (1976a). Superoxide dismutase EC 1.15.1.1 from Mycobacterium tuberculosis. J . Biochem. (Tokyo) 80, 1343-1352. Kusunose, M., Noda, V., Ichihara, K., and Kusunose, E. (1976b). Superoxide dismutase from Mycobacterium species, strain Takeo. Arch. Microbiol. 108, 65-73. Kwiatowski, J. (1987). Comparison of chloroplast and cytosolic CuiZn superoxide dismutase isozymes from tomato in relation to superoxide dismutase evolution. In “Isozymes: Current Topics in Biological and Medical Research” (M. C. Rattazzi, J. G. Sandalios, and G. S. Whitt, eds.), Vol. 15, pp. 121-129. Liss, New York. Lavelle, F., McAdam, M. E., Fieiden, E. M., Roberts, P. B., Puget, K., and Michelson, A. M. (1977). A pulse-radiolysis study of the catalytic mechanism of the ironcontaining superoxide dismutase from Photobacterium leiognathi. Biochem. J . 161, 3-11. Lee, F . J . , and Hassan, H. M. (1986a). Biosynthesis of superoxide dismutase and catalase in Saccharomyces cereuisiae: Effects of oxygen and cytochrome c deficiency. J . Zndust. Microbiol. 1, 187-193. Lee, F.-J., and Hassan, H. M. (1986b). Biosynthetic of superoxide dismutase in Saccharomyces cereuisiae: Effects of paraquat and copper. Free Radical Biol. Med. 1, 319-325. Lee, F.-J., and Hassan, H. M. (1987). Biosynthesis of superoxide dismutase and catalase

SUPEROXIDE DISMUTASES

93

in chemostat culture of Saccharomyces cereuisiae. Appl. Microbiol. Biotechnol. 27, 72-74. Leunissen, J . A. M., and deJong, W. W. (1986).Copperizinc superoxide dismutase: How likely is gene transfer from ponyfish to Photobacterium leiognathi? J . Mol. Euol. 25, 250-258. Lowry, C. V., and Zitomer, R. S. (1984). Oxygen regulation of anaerobic and aerobic genes mediated by a common factor in yeast. Proc. Natl. Acad. Sci. U.S.A. 81, 6129-6133. Lynch, R. E., and Noble, B. C. (1980). Mycoplasma pneumoniae: A procaryote which consumes oxygen and generates superoxide but which lacks superoxide dismutase. Biochem. Biophys. Res. Commun. 96, 98-105. McAdam, M. E., Fox, R. A., Lavelle, F., and Fielden, E. M. (1977). A pulse-radiolysis study of the manganese-containing superoxide dismutase from Bacillus stearothermophilus. A kinetic model for the enzyme action. Biochem. J . 165, 71-79. McCord, J . M., and Fridovich, I. (1969). Superoxide dismutase: An enzymatic function for erythrocuprein (hemocuprein).J . Biol. Chem. 244, 6049-6055. McCord, J. M., Keele, B. B., and Fridovich, I. (1971). An enzyme-based theory of obligate anaerobiosis: The physiological function of superoxide dismutase. Proc. Natl. Acad. Sci. U.S.A.68, 1024-1027. Malinowski, D. P., and Fridovich, I. (1979a). Subunit association and sidechain reactivities of bovine erythrocyte superoxide dismutase in denaturing solvents. Biochemistry 18, 5055-5060. Malinowski, D. P., and Fridovich, I. (1979b). Chemical modification of arginine a t the active site of the bovine erythrocyte superoxide dismutase. Biochemistry 18, 5909-5917. Mann, T., and Keilin, D. (1938). Hemocuprein and hepatocuprein, copper-protein compounds of blood and liver in mammals. Proc. R . SOC.London Ser. B 126, 303-3 15. Marres, C. A. M., Van Loon, A. P. G. M., Oudshoorn, P., Van Steeg, H., Grivell, L. A., and Slater, E. G. (1985). Nucleotide sequence analysis of the nuclear gene coding for manganese superoxide dismutase of yeast mitochondria, a gene previously assumed to code for the Rieske iron-sulphur protein. Eur. J . Biochem. 147, 153161.

Martin, J. P., and Fridovich, I. (1981). Evidence for a natural gene transfer from the ponyfish to its bioluminescent bacterial symbiont Photobacter leiognathi. The close relationship between bacteriocuprein and the copper-zinc superoxide dismutase of teleost fishes. J . Biol. Chem. 256, 6080-6089. Martin, M. E., Byers, B. R., Olson, M. 0.J., Salin, M. L., Arceneaux, J. E. L., and Tolbert, C. (1986). A Streptococcus tnutans superoxide dismutase that is active with either manganese or iron a s a cofactor. J . Biol. Chem. 261, 9361-9367. Meier, B., Barra, D., Bossa, F., Calabrese, L., and Rotilio, G. (1982).Synthesis of either Fe- or Mn-superoxide dismutase with a n apparently identical protein moiety by a anaerobic bacterium dependent on the metal supplied. J . Biol. Chem. 257, 1397713980. Misra, H. P. (1979). Reaction of copper-zinc superoxide dismutase with diethyldithiocarbamate. J . Biol. Chem. 254, 11623-11628. Misra, H. P., and Fridovich, I. (1978).Inhibition of superoxide dismutases by azide. Arch. Biochem. Biophys. 189, 317-322. Moody, C. S. (1985). Regulation of superoxide dismutase biosynthesis in Escherichia coli. Ph.D. thesis, North Carolina State University, Raleigh.

94

HOSNI M. HASSAN

Moody, C. S., and Hassan, H. M., (1982). Mutagenicity of oxygen free radicals. Proc. Natl. Acad. Sci. U.S.A.79, 2855-2859. Moody, C. S., and Hassan, H. M. (1984). Anaerobic biosynthesis of the manganesecontaining superoxide dismtuase in Escherichia coli. J . Biol. Chem. 259, 1282112825. Morris, J. G . (1976).Oxygen and the obligate anaerobe. J . Appl. Bacteriol. 40,229-244. Natvig, D. O., Imlay, K., Touati, D., and Hallewell, R. (1987). Human copper-zinc superoxide dismutase complements superoxide dismutase deficient E . coli mutants. J . Biol. Chem. 262, 14697-14701. Nettleton, C. T., Bull, C., Baldwin, T. O., and Fee, J. A. (1984).Isolation ofEscherichia coli iron superoxide dismutase gene: Evidence that intracellular superoxide concentration does not regulate oxygen-dependent synthesis of the manganese superoxide dismutase. Proc. Natl. Acad. Sci. U.S.A.81, 4970-4973. Norrod, P., and Morse, S. A. (1979).Absence of superoxide dismutase in some strains of Neisseria gonorrhoeae. Biochem. Biophys. Res. Commun. 90, 1287-1294. Norrod, E. P., Bartenstein, L., and Morse, S. A. (1981). Comparison of the superoxide dismutase of Branhemella catarrhalis, Neisseria ovis, and Neisseria caviae. Int. J . Syst. Bacteriol. 31, 392-395. Ochiai, E. I. (1983). Copper and biological evolution. Biosystems 16, 81-86. Ose, D. E., and Fridovich, I. (1976). Superoxide dismutase: Reversible removal of manganese and its substitution by cobalt, nickel, or zinc. J . Biol. Chem. 251, 1217-1218. Ose, D. E., and Fridovich, I. (1979). Manganese-containing superoxide dismutase from Escherichia coli: Reversible resolution and metal replacement. Arch. Biochem. Biophys. 194, 360-364. Pick, M., Rabani, J., Yost, F., and Fridovich, I. (1974). The catalytic mechanism of the manganese-containing superoxide dismutase of Escherichia coli studies by the pulse radiolysis. J . A m . Chem. SOC.96, 7329-7332. Privalle, C. T., and Gregory, E. M. (1979). Superoxide dismutase and O2 lethality in Bacteroides fragilis. J . Bacteriol. 138, 139-145. Puget, K., and Michelson, A. M. (1974).Isolation of a new copper-containing superoxide dismutase, bacteriocuprein. Biochem. Biophys. Res. Commun. 58, 830-838. Pugh, S. Y. R., and Fridovich, I. (1985). Induction of superoxide dismutases in Escherichia coli B by metal chelators. J . Bacteriol. 162, 196-202. Reinards, R., Ohlenbush, H. D., and Altdorf, R. (1984). Purification and properties of a manganese containing superoxide dismutase from Acholeplasma laidlawii. HoppeSeyler’s 2. Physiol. Chem. 365, 549-554. Ringe, D., Petsko, G. A., and Yamakura, F. (1983). Structure o f iron superoxide dismutase from Pseudomonas ovalis a t 2.9A resolution. Proc. Natl. Acad. Sci. U.S.A. 80,3879-3883. Rolf, R. D., Hentges, D. J., Campbell, B. J., and Barrett, J. T. (1978).Factors related to the oxygen tolerance of anaerobic bacteria. Appl. Enuiron. Microbiol. 36,306-313. Rotilio, G . , Bray, R. C., and Fielden, E. M. (1972).A pulse radiolysis study of speroxide dismutase. Biochim. Biophys. Acta 268, 605-609. Sakamoto, H., and Touati, D. (1984). Cloning of the iron superoxide dismutase gene (sodB) in Escherichia coli K-12. J . Bacteriol. 159, 418-420. Salin, M. L., and Bridges, S. M. (1980). Isolation and characterization of an ironcontaining superoxide dismutase from a eukaryote, Brassica campestris. Arch. Biochem. Biophys. 201, 369-374.

SUPEROXIDE DISMUTASES

95

Salin, M. L., and Bridges, S. M. (1982). Isolation and characterization of a n ironcontaining superoxide dismutase from water lily Nuphar luteum. Plant Physiol. 69, 161-165. Sato, S., and Harris, J. I. (1977). Superoxide dismutase from Thermus aquaticus: Isolation and characterization of manganese and apoenzymes. Eur. J . Biochem. 73, 373-381. Sato, S., and Nakazawa, K. (1978). Purification and properties of superoxide dismutase from Thermus thermophilus HB8. J . Biochem. (Tokyo)83, 1165-1171. Sato, S., Nakada, Y., and Nakazawa-Tomizawa, K. (1987). Amino-acid sequence of a tetrameric, manganese superoxide disrnutase from Thermus thermophilus HB8. Biochim. Biophys. Acta 912, 178-184. Schiavone, J. R., and Hassan, H. M. (1987). Biosynthesis of superoxide dismutase in eight prokaryotes: Effects of oxygen, paraquat and an iron chelator. FEMS Microbiol. Lett. 42, 33-38. Schiavone, J. R., and Hassan, H. M. (1988). The role of redox in the regulation of manganese-containing superoxide dismutase biosynthesis in Escherichia coli. J . Biol. Chem. 263,4269-4273. Schinina, M. E., Maffey, L., Barra, D., Bossa, F., Puget, K., and Michelson, A. M. (1987). The primary structure of iron superoxide dismutase from Escherichia coli. FEBS Lett. 221, 87-90. Scott, M. D., Meschnick, S. R., and Eaton, J. W. (1987). Superoxide dismutase-rich bacteria: Paradoxical increase in oxidant toxicity. J . Biol. Chem. 262, 3640-3645. Shatzman, A. R., and Kosman, D. J. (1978).The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides. Biochim. Biophys. Acta 544, 163-179. Stallings, W. C., Pattridge, K. A,, Powers, T. B., Fee, J. A,, and Ludwig, M. L. (1981). Characterization of crystals of tetrarneric manganese superoxide dismutase from Thermus thermophilus HB8. J . Biol. Chem. 256, 5857-5859. Stallings, W. C., Powers, T. B., Pattridge, K. A., Fee, J. A., and Ludwig, M. L. (1983). Iron superoxide dismutase from Escherichia coli at 3.1A resolution: A structure unlike that of copperizinc protein a t both monomer and dimer levels. Proc. Natl. Acad. Scl. U.S.A. 80, 3884-3888. Stallings, W. C., Pattridge, K. A., Strong, R. K., and Ludwig, M. L. (1984). Manganese and iron superoxide dismutase are structural homologs. J . Biol. Chem. 259, 10695-10699. Stallings, W . C., Pattridge, K. A,, Strong, R. K., and Ludwig, M. L. (1985).The structure of the manganese superoxide dismutase from Thermus thermophilus HB8 a t 2.4-A resolution. J . Biol. Chem. 260, 16424-16432. Steffens, G. J., Bannister, J. V., Bannister, W. H., Flohe, L., Gunzler, W. A., Kim, S. M. A., and Otting, F. (1983).The primary structure of CuZn superoxide dismutase from Photobacterium leiognathi: Evidence for a separate evolution of CuZn superoxide disrnutase in bacteria. Hoppe Seyler’s 2. Physiol. Chem. 364, 675-690. Steinman, H. M. (1978). The amino acid sequence of rnangano superoxide dismutase from Escherichia coli B. J . Biol. Chem. 253, 8708-8720. Steinman, H. M. (1980). The amino acid sequence of copper-zinc superoxide dismutase from bakers’ yeast. J . Biol. Chem. 225, 6758-6765. Steinman, H. M. (1982a). Superoxide dismutases: Protein chemistry and structurefunction relationships. In “Superoxide Dismutases” (L. W. Oberley, ed.), Vol. 1, pp. 11-68. CRC Press, Boca Raton, Florida.

96

HOSNI M. HASSAN

Steinman, H. M. (198213). Copper-zinc superoxide dismutase from Caulobacter crescentus CB15. A novel bacteriocuprein form of the enzyme. J . Biol. Chem. 257, 1028310293. Steinman, H. M. (1983). Chemical aspects of structure, function, and evolution among superoxide dismutases: The general scenario and the bacteriocuprein exceptions. In “Oxy Radicals and Their Scavenger Systems, Molecular Aspects” (G. Cohen and A. Greenwald, eds.), Vol. 1, pp. 167-177. Elsevier, New York. Steinman, H. M. (1985). Bacteriocuprein superoxide dismutases in Pseudomonads. J . Bacteriol. 162, 1255-1260. Steinman, H. M. (1987). Bacteriocuprein superoxide dismutase of Photobacterium leiognathi: Isolation and sequence of the gene and evidence for a precursor form. J . Biol. Chem. 262, 1882-1887. Steinman, H. M., and Hill, R. L. (1973). Sequence homologies among bacterial and mitochondrial superoxide dismutases. Proc. Natl. Acad. Sci. U S A . 70,3725-3729. Steinman, H. M., Naik, V. R., Abernethy, J . L., and Hill, R. L. (1974). Bovine erythrocyte superoxide dismutase. Complete amino acid sequence. J . Biol. Chem. 249, 73267338. Stowers, M. D., and Elkan, G. H. (1981),. An inducible iron-containing superoxide dismutase in Rhizobium japonicum. Can. J . Microbiol. 27, 1202-1208. Tainer, J . A,, Getzoff, E. D., Beem, K. M., Richardson, J. S., and Richardson, D. C. (1982). Determination and analysis of the 2A structure of copper-zinc superoxide dismutase. J . Mol. Biol. 160, 181-217. Tainer, J. A,, Getzoff, E. D., Richardson, J . S., and Richardson, D. C. (1983). Structure and mechanisms of copper, zinc superoxide dismutase. Nature (London) 306, 284-287. Takeda, Y., and Avila, H. (1986). Structure and gene expression of the E . coli Mn-superoxide dismutase gene. Nucleic Acids Res. 14, 4577-4589. Tally, F. P., Goldin, B. R., Jacobus, N. B., and Gorbach, S. L. 11977). Superoxide dismutase in anaerobic bacteria of clinical significance. Infect. Zmmun. 16, 20-25. Tegelstrom, H. (1975). Interspecific hybridization in vitro of superoxide dismutase from various species. Hereditas 81, 185-198. Touati, D. (1983). Cloning and mapping of the manganese superoxide dismutase gene (so&) of Escherichia coli K-12. J . Bacteriol. 155, 1078-1087. Touati, D. (1988a). Transcriptional and posttranscriptional regulation of manganese superoxide dismutase biosynthesis in Escherichia coli, studied with operon and protein fusions. J . Bacteriol. 170, 2511-2520. Touati, D. (198813). Molecular genetics of superoxide dismutases. Free Radical Biol. Med. 5, 393-402. Van Loon, A. P. G. M., Pesold, B., and Schatz, G. (1986). A yeast mutant lacking mitochondrial superoxide dismutase is hypersensitive to oxygen. Proc. Natl. Acad. Sci. U.S.A. 83, 3820-3824. Vedros, N. A. (1984). Genus I. Neisseria. I n “Bergey’s Manual of Systemic Bacteriology” (N. R. King, ed.), pp. 296. Williams & Wilkins, Baltimore, Maryland. Vignais, P. M., Terech, A., Meyer, C. M., and Henry, M. F. (1982). Isolation and characterization of a protein with cyanide-sensitive superoxide dismutase activity from the prokaryote Paracoccus denitrifans. Biochim. Biophys. Acta 701,305-317. Von Stein, R. S., Barber, L. S., and Hassan, H. M. (1982). Synthesis of oxygendetoxifying enzymes in Bdellovibrio stolpii. J . Bacteriol. 152, 792-796. Whiteside, C., and Hassan, H. M. (1987). Induction and inactivation of catalase and

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superoxide dismutase of Escherichia coli by ozone. Arch. Biochem. Biophys. 257, 464-471. Yamakura, F. (1984). Destruction of tryptophan residues by hydrogen peroxide in iron-SOD. Bzochem. Biophys. Res. Commun. 122, 635-641. Yano, K., and Nishie, H. (1978). Superoxide dismutase in facultatively anaerobic bacteria: Enzyme levels in relation to growth conditions. J . Gen. Appl. Microbtol. 24, 333-339. Yost, F. J., and Fridovich, I. (1973). An iron-containing superoxide dismutase from Escherichia coli B. J . Biol. Chem. 248,4905-4908. Zipkas, D., and Riley, M. (1975). Proposal for mechanism of evolution of the E . coli genome. Proc. Natl. Acad. Sci. U.S.A. 72, 1354-1358.

BACTERIAL GENES INVOLVED IN RESPONSE TO N EAR-ULTRAVIOLET RADIATION A. Eisenstork Division of Biological Sciences and Department of Microbiology, University of Missouri, Columbia, Missouri 6521 1

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Significance , . . . . . . . . B. Model for NUV Action . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . s (Photoreceptors) of NUV

. . . . . . . . . . . . . .. . . . . . . . . . . . ,

.

......

........ .

tRNA . . . . . . , C. Porphyrins and Other M ........... D. Amino Acids and Polypeptides.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Photoprotective Chromophores 111. Oxidative Photoproducts of NUV. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Hydrogen Peroxide . . . . . . . . . . . . . .

C. Hydroxyl Radic

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

A. Catalase . . . ... . . .. . . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . .. .. . ... B. Superoxide Dismutase C. Exonuclease I11 and Endonuclease IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C. Membrane Effects . . . . . . . . .

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

100 100 101 103 103 105 107 108 110 111 112 113 114 115 115 119 119 122 123 124 126 126 126 128 129 129 131 132 132 132 134 136

99 ADVANCES IN GENETICS, Vol 26

Copyright Q 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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I. Introduction

A. SIGNIFICANCE Solar near-ultraviolet radiation (NUV; 290-400 nm), because of its abundance, is perhaps the most mutagenic agent to which organisms are exposed. In addition to its mutagenic effect, NUV influences biological systems in unique and often subtle ways. Many microbial, plant, and animal biochemical reactions may be induced, cued, and modulated by NUV in the organism’s developmental, growth and behavioral activities. Solar NUV may have deleterious effects on bacteria; therefore, bacteria must constantly cope with fluctuating solar NUV conditions (Calkins, 1982; Gameson and Gould, 1975).This is particularly true of Escherichia coli, which lives in both aerobic and anaerobic environments, as well as in both sunlight and darkness as the organism cycles between hosts. The genetic mechanisms that have evolved in bacteria to cope with solar NUV stress are the focus of this article. There is increasing concern that additional NUV (particularly UV-B; 290-320 nm) may impinge on the earth’s surface as a result of depletion of the ozone filter in the stratosphere; this depletion may result from use of fluorocarbon aerosols, nitrogen oxide pollution, and supersonic flights, as well as from other products and procedures of modern technology. Recently, even greater focus on this problem has resulted from the finding that the stratospheric ozone screen may be lost over the Antarctic; the effect of this excess NUV on zooplankton and other organisms in the food chain is not yet known (Calkins, 1982; Helene et al., 1982). Faced with the possibility of this altered global environment, it is appropriate that we seriously assess the mutagenic and toxic nature of NUV. In addition to natural radiation, many humans receive excessive NUV from artificial illumination, such as sun lamps and therapeutic lamps. Also, photosensitizing molecules, with 290- to 400-nm absorbance, abound in natural substances and may affect human health (Ames, 1983; Blum, 1941; Straight and Spikes, 1985). One of the effects of NUV may be t o generate excess active oxygen species; these have been implicated in a wide variety of environmental and health effects (Adelman et al., 1988; Halliwell, 1987; Marx, 19871, including premature aging (Bissett et al., 19871, circulatory diseases, rheumatoid arthritis, and induction of cancers and cataracts (Adelman et al., 1988; Ames, 1983; Petkau, 1986; Touati, 1988a). Critical microbial experiments may be traced to the late Alexander

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Hollaender (1943), whose pioneering paper stimulated UV research. This was followed by numerous NUV research and review papers (Eisenstark, 1971, 1985, 1987; Helene et al., 1982; Kubitschek et al., 1986; Webb, 19771, plus a book by Jagger (1985). Answers to major questions have emerged recently, particularly as to the identification of NUV chromophores, targets, toxic photoproducts and their subsequent quenching action, the specific nature of DNA lesions (mutagenic and nonmutagenic), induced synthesis of protective molecules, and repair of DNA damage following NUV radiation. This review will focus on NUV lethal and mutagenic effects on bacteria and phages, and will attempt to identify some decisive experiments that might resolve unanswered questions. Effects on higher organisms have been reviewed (Calkins, 1982; Helene et al., 1982; Jagger, 1985). Also, the chemistry of the generation of toxic oxygen species, which are photoproducts of NUV, both with and without added photosensitizers, may be found in excellent reviews (Jagger, 1985; Sies, 1985; Sohal and Allen, 1986).

B. MODELFOR NUV ACTION The complex mechanism for coping with NUV may be divided into two parts: (1) detoxification of reactive molecules that result from photooxidation (e.g., H202, 0 2 - , OH., and singlet oxygen) and (2) repair of DNA damage and resynthesis of damaged tRNA. Because there are multiple responses to NUV, it is difficult to follow a single set of events; much depends on the initial photoreceptor and subsequent reactions. Some of these reactions occur simultaneously. 1. By analogy with far-ultraviolet radiation (FUV) effects, the simplest reaction would be that NUV photons directly alter DNA and produce mutations or other deleterious effects, and that the cell copes with the changes to return to a “nonstressed” state. Although the frequency of such lesions may be considerably less than for lesions produced by indirect photo action, they still may be significant. 2. Instead of (or in addition to) direct photo action, the DNA damage could be indirect, via a n endogenous photosensitizer (photodynamic action) (Straight and Spikes, 1985). There are abundant heme, flavin, and other cellular molecules that could serve as photoreceptors, with energy transfer causing damage to DNA. Neither the specific DNA lesion nor the recovery from indirect action need be the same as from direct photo action.

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3. H202 and other reactive oxygen species generated by NUV may (a) peroxidize lipids and other molecules, (b) damage DNA, and (c) signal a cascade of coping events, including the induction of several gene products, some under control of the oxyR regulon. The oxidative damage to the DNA could produce a lesion that might differ from the lesions of 1and 2, above, and could perhaps induce a different regulon. 4. Even at low fluences, there are profound biological consequences, such as when thiolated tRNA molecules are inactivated by NUV (Favre et al., 1985; Kramer et al., 1988); these include abrupt cellular growth delay and reduction of cell size. This effect on biomolecules could be independent of H202. The triggering of guanosine tetraphosphate (ppGpp) synthesis or other nucleotides (alarmones) is a n intriguing aspect of the recovery process, since mutants lacking thiolated tRNA (nuu) or mutants defective in the stringent response (relA)are sensitive to low fluence of NUV (Favre et al., 1985; Kramer et al., 1988). Also, dihydroxy acid dehydratase (DHAD), a key enzyme in the amino acid biosynthetic pathway, is sensitive t o NUV (Wilke, 1988) and could trigger a protective response. 5. One of the earliest biological effects a t very low fluences occurs a t the membrane level and is detected by sharp exclusion of exogenous molecules. One of the intriguing aspects here is that the presence of catalase hydroperoxidase I (HPI) overcomes this exclusion. 6. Initially, there is a derepression of a regulatory gene(& followed by derepression of appropriate stress genes and synthesis of proteins necessary to cope with the stress. These inductions include detoxifying molecules (e.g., catalase HPI and Mn superoxide dismutase), ppGppinducible proteins, and DNA repair enzymes (xthA, dam, and polA genes are involved in repairing DNA damage, but they are not inducible by NUV). Along with synthesis of some new proteins, NUV shuts off synthesis of many other proteins. Also, other gene products, not yet identified, may be involved. 7. One of the effects of NUV is nitrogenous base destruction. After removal of the base, the AP site is recognized by exonuclease I11 (exoIII) (xthA gene product), producing nicks 5’ to the base-free deoxyribose. The polA gene product, polymerase I (polI), could remove free sugar (5’ to 3’). The product of the d a m gene may also be involved in the recovery process (Yallaly, 1988). The precise role of ligase in the final step is obscure (Zig mutants are not NUV sensitive). 8. Following DNA damage, several pathways might be available for repair or bypass, depending on the specific nature of the lesion. The SOS pathway may be involved indirectly, but other enzymatic actions also occur. Among SOS gene products, the recombination function of

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the product of recA may be important for repair or bypass of NUV lesions. Glucosylase is probably not involved in recovery (ung-I mutants are not NUV sensitive). 9. Whether NUV mutagenesis is the same as 02-mutagenesis remains unresolved. However, in both cases, excess catalase HPI and absence of ex0111 are antimutagenic. 10. The product of the katF gene has a role in protection against NUV. It is a regulatory gene that is necessary for the production of catalase HPII and ex0111 (Sak et al., 1989). 11. There may be two distinct types of pathways of damage and recovery, one type occurring a t low NUV fluence rates (Kramer and Ames, 1987; Lang et al., 1986) or H202doses (Linn and Imlay, 1987) and other pathways occurring at higher fluence rates and doses. The lower fluences and doses may involve iron (Fenton chemistry). 12. The model must also account for the need of a delicate quantitative balance of some of the enzymes (excess may have a deleterious effect), as well as quantitative and changing ratios between different enzymes and radical scavengers a t different steps in a pathway.

II. Chromophores (Photoreceptors) of NUV

A. DNA Studies on effects of ultraviolet radiation are usually performed with germicidal lamps that emit radiation maximally at 254 nm (farultraviolet radiation). At this wavelength, DNA (A,, = 260 nm) is the major chromophore, and the mechanisms of DNA mutagenic and lethal events have been described in some detail. Thymine dimers, as well as other DNA photoproducts, are formed and the cell responds accordingly. However, studies of NUV effects show that these differ from FUV, and they are much more complex (Ferron et al., 1972); this would be expected since cells have both DNA and a number of non-DNA photoreceptors with A,, in the range 290-400 nm. Although DNA may still absorb a small quantity of radiation (e.g., -0.1%, at 320 nm, of that which is absorbed at 260 nm) (Jagger, 19851, this may not be sufficient t o account for all NUV-induced mutations (Cabrera-Juarez, 1981; Favre et al., 1985; Kubitschek et al., 1986; Moody and Hassan, 1982; Turner and Webb, 1981). However, even a t low absorbance, long exposures to NUV could account for accumulated DNA damage. Therefore, despite several decades of study, the role of

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DNA as a critical NUV chromophore still needs to be sorted out from the effects on non-DNA chromophores. While DNA may not be the critical chromophore for NUV, it is a main target of NUV, resulting in death, mutation inhibition of DNA synthesis (Parsons and Hayward, 19851, and biochemical alteration. “Target” is defined as the damaged molecule that results in death, mutation, or other physiological alteration. As noted, DNA may be both a chromophore and a target for NUV (1)via direct photon action on DNA similar to FUV, although the lesions differ; (2) photodynamically by striking an absorbing chromophore (e.g., hematin) that transfers the energy to adjacent DNA; (3)via photooxidation to generate toxic oxygen species (e.g., H202, Oa-, OH., and singlet oxygen); (4) via destruction of critical enzymes that assist in DNA lesion formation (e.g., catalase, ribonuclease reductase, and dihydroxy acid dehydratase); and (5)via destruction of thiolated tRNA and 2-thiouracil photosensitization (Peak et al., 1987b, 1988). Thus DNA alterations may occur via several distinctly different routes; step 1 assumes that DNA is the photoreceptor, but steps 2-5 assume that DNA is the target, with energy transferred from another molecule. In addition to physical measurements that show 290- to 320-nm NUV is absorbed by DNA, there is biological evidence of direct DNA alteration. Perhaps the most supportive evidence comes from studies of photoreactivation (see Section II,E), in which DNA damaged by FUV is partially repaired by NUV directly, even without the photoreactivating enzyme. This indicates that the DNA bases, although altered by FUV, can absorb NUV directly. Nonenzymatic photoreactivation and other photoreversal effects occur in cells devoid of photoreactivating enzymes ( p h r mutants) and under stringent anoxic conditions; this should preclude effects via intermediate reactive oxygen species, and indicates that the NUV action is directly on DNA. Another direct way to determine whether DNA is a n effective NUV chromophore is to irradiate transforming DNA under anoxic conditions and to assay for mutations and other destructive effects (Cadet et al., 1986; Cabrera-Juarez, 1964, 1981; Cabrera-Juarez and Setlow, 1976). Despite some debate about whether other molecules might tenaciously remain as part of a DNA preparation and act as photosensitizers, it is clear that DNA may directly absorb NUV photons, resulting in genetic damage [Cabrera-Juarez, 1964, 1981; CabreraJuarez and Setlow, 1976; see Jagger (1985) for a n excellent discussion of the photochemistry and photobiology involved]. Further evidence that DNA may be a direct antenna for NUV

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photons is that careful action spectra studies always show an abrupt 330- to 340-nm break in the slope for lethality, mutagenesis, and single-strand DNA breaks (SSBs) (Ananthaswamy et al., 1979; Jagger, 1985; Kubitschek et al., 1986; Mackay et al., 1976; Peak et al., 1983) for both phages and bacteria, even under strict anoxic conditions. Whether the 330- to 340-nm chromophore is a n unidentified configuration of the DNA or a non-DNA molecule is not fully resolved. The ability to detect mutations a t the base sequence level (Miller, 1985) might be a conclusive way of determining whether DNA can be a photoreceptor. By treating appropriate lac1 cells with NUV, both under aerobic and anaerobic conditions, l a d dmutants can be collected. By recombination with F1 phage, the lacl region can be sequenced to determine the specific base changes produced by NUV. Another technique for identification of direct DNA lesions by NUV might be by antibody reaction (Katcher and Wallace, 1983; Kow and Wallace, 1985). Although direct damage of DNA by NUV cannot be ignored, it is likely that oxygen in a n excited or radical state is responsible for most of the DNA damage (Breimer and Lindahl, 1985; Jagger, 1985; Martin et al., 1984; Nishida et al., 1981; Quintiliani, 1986; Sestili et al., 19861, since a number of quenching agents can protect transforming DNA against NUV. Although transforming DNA is altered more readily in the presence than in the absence of 0 2 (Cabrera-Juarez, 1981; Jagger, 1985; Nishida et al., 19811, there is a possibility that the excited or radical oxygen comes from DNA. Since xthA and sodAB mutants are sensitive to both NUV and H202, it is likely that part of the DNA damage occurs by altering the sugar moiety (Fenton reaction), which ultimately leads to base damage. Oxygen is required for most (but not all) biological reactions by NUV. In these photooxidations, if the target is the DNA or the membrane, this could have drastic consequences. The subject of photodynamic action has been extensively reviewed ( Jagger, 1985; Straight and Spikes, 1985), especially with regard to psoralens. It should be kept in mind that cells contain numerous photodynamic endogenous photosensitizers.

B. THIOLATED tRNA Another NUV chromophore reaction that affects DNA is the absorption of 334 nm by thiolated tRNA, as studied in E . coli by Favre and by Jagger and their associates (Blanchet et al., 1984; Caldeira de Araujo and Favre, 1986; Favre et al., 1985; Hajnsdorf and Favre, 1986; Jagger,

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1985; Salet et al., 1985; Peak et al., 1988) and in Salmonella typhimurium by Kramer et al. (1988). The primary biological effect is one of growth delay (see Section V1,A). NUV produces a cross-link of the 4-thiouracil with cytosine, thus inactivating the acetylation capacity of this tRNA. There is an enormous increase (100-fold) in ApppGpp and ppGpp, which may signal the induction of other proteins [this increase does not occur in nuu mutants (Kramer et al., 1988)I. Growth is resumed upon synthesis of new thiolated tRNA. It is intriguing to note that the altered tRNA may also initiate a signal that is involved in stopping normal protein synthesis and DNA metabolism, leaving numerous single-strand DNA gaps as part of the normal DNA replication process. However, it is hard to distinguish DNA gaps as a result of growth delay from direct NUV effects. The relationship between the rate of chromosome synthesis and NUV sensitivity is not clear-cut, and use of nuu mutants that lack the thiolated tRNA may be a way to distinguish between direct and indirect actions of NUV. Cells with no (or slowed) DNA synthesis (fewer chromosomal growing forks) are more resistant to NUV than are rapidly growing cells with several growing forks (Eisenstark, 1982; Hartman and Eisentstark, 19781, yet 4-thiouridine mutants that do not exhibit DNA growth delay (and thus have more growing forks when cells are in log growth phase) are more resistant t o high fluences of NUV than are the wild-type bacteria (Favre et al., 1985; Hajnsdorf and Favre, 1986; Jagger, 1985).This is an apparent paradox, since the growing cells should be more sensitive as a result of more chromosomal growing forks. However, the following, observations may help to resolve this inconsistency. At much lower fluences, results are quite different; Kramer et al. (1988) compared nuu' and nuu- mutants and found that the nuumutants, lacking thiouridine, is actually more sensitive. They argue that thiolated tRNA molecules in E . coli act as photoreceptors and initiate alarmone synthesis, e.g., ppGpp (Favre et al., 1985; Hajnsdorf and Favre, 1986) or AppppA (Bochner et al., 1986; Lee et al., 19831, which signals numerous protective, enzymatic activities. Although NUV fluences that cause growth delay are not lethal, if these fluences are maintained for several hours, cells may not recover. In nature, such low fluences may still have lethal and mutagenic effects on bacteria, especially if the bacteria are stressed by low (normally nonlethal) doses of other agents (Kramer and Ames, 1987). As noted, the action spectrum for inactivating T7 phage (or bacteria) fits neither the absorption spectrum of DNA nor that of protein. Further, if phage particles are irradiated in a nonlethal concentration

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of H202,the NUV effect is greatly amplified, with a peak of 340 nm for this synergistic effect (Ananthaswamy et al., 1979). Since phages contain no thiolated bases, there is obviously another (non-tRNA) effect of NUV a t -340 nm, which could be either direct DNA damage or DNA-protein cross-linking. AND OTHERMOLECULES C. PORPHYRINS OF THE RESPIRATORY CHAIN

Porphyrin components of the respiratory chain in E . coli are chromophores that may be involved in endogenous photodynamic effects on either DNA or cell membranes (Jagger, 1985; Kramer and Ames, 1987; Peters, 1977; Tuveson and Summartano, 1986). Some of the evidence is based on the observation that a hem mutant, which is defective in the ability to make porphyrin, is resistant to NUV; other mutants that accumulate heme are sensitive to NUV (Peak et al., 1987). The photodynamic behavior of porphyrins has been reviewed (Sies, 1985). Tuveson and Summartano (1986) point out that, since photodynamic action requires oxygen, additional components of the respiratory chain might be involved (Aliabadi et al., 1986; Gennis, 1987; Ingeldew and Poole, 1984; Jagger, 1985). Indeed, there are a number of molecules associated with the respiratory chain that absorb in the NUV range [e.g., riboflavin = (Amm = 375 nm), menaquinone (A = 330 nm), pyridoxal phosphate (A = 390 nm), and porphyrins (A = 380 nm)]. Of particular interest is the sensitivity of a mutant that lacks NADH dehydrogenase ( n d h ) to low concentrations of H202 (Imlay et al., 1988) and to NUV. Note also that heme-containing catalase may be a photosensitizer, as observed by increased sensitivity when cells contain increased catalase (Eisenstark and Perrot, 1987; Kramer and Ames, 1987). Also, NUV can inactivate catalase both in uitro and in uiuo (Cheng et al., 1981; Freierabend and Engel, 1986; Wilke, 1988). It should be noted that, although riboflavin synthesis and porphyrin biosynthesis involve different pathways, if the two were coordinately regulated, riboflavin might be an important endogenous photosynthesizer (Tuveson and Summartano, 1986). Indeed, it is an excellent exogenous photosensitizer, probably yielding 0 2 - and/or singlet oxygen. Kramer and Ames (1987) noted that cells that are ahp' (flavincontaining alkyl hydroperoxidase) are more NUV resistant than are ahp-. Storz et al. (1989) have analyzed mutant- and plasmidcontaining strains of ahp and found that one ahp gene is under oxyR control, but another one is not.

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D. AMINOACIDSAND POLYPEPTIDES Early in our studies, we proposed that most NUV damage, in contrast to FUV damage, might be the result of a secondary effect rather than of a direct hit on DNA (McCormick et al., 1976; Yoakum et al., 1975). Tryptophan was found to be a chromophore for NUV and yielded H202 as a photoproduct (McCormick et al., 19761, as well as N-formylkynurenine (A,, = 318 nm). H202, in turn, was assumed to produce lesions in DNA (Ananthaswamy and Eisenstark, 1976, 1977; Ananthaswamy et aZ.,1979; Hartman, 1986; Hartman and Eisenstark, 1978,1980; Hartman et al., 1979). H202 also may be a photoproduct of cysteine irradiation (Greenberg and Demple, 1986; McCormick et al., 1976, 1982; Owens and Hartman, 1986). We have since found that Hz02, in the presence of certain wavelengths of NUV, synergistically kills bacteria and phages (Eisenstark et al., 1980; Hartman and Eisenstark, 1978, 1980, 1982; Hartman et al., 1979). This synergistic action may be the result of NUV conversion of H202 to 0 2 - (Ahmad, 19811, and this action may generate a new NUV photoreceptor (McCormick et al., 1982). Another possibility is that NUV generates 02- to give a Fenton reaction ( 0 2 - + H202 + Fe2+),yielding OH.. Further support that HzO2 may be a critical photoproduct of NUV comes from the knowledge that certain mutations lead to greater sensitivity to both NUV and H202 (Eisenstark, 1985; Eisenstark and Perrot, 1987; Sammartano and Tuveson, 1983; Sammartano et al., 1986; Tyrrell, 1985). Studies of phage T7, which contains only DNA and protein, emphasize a paradox that could be explained by a polypeptide acting as a photoreceptor; peak absorption is 254 nm for DNA and 280 nm for protein, but action spectra for killing of phage T7 (Ananthaswamy et al., 1979) and S. typhimurium (Eisenstark, 1971; Mackay et al., 1976) show distinct shoulders a t -334 nm. The action spectrum for inactivating phage T7 (or bacteria) fits neither the absorption spectrum of DNA nor of protein. In a search for a NUV chromophore, we observed that the sulfhydryl in peptide-bound cysteine, in the presence of oxygen (or H202), is photochemically altered (McCormick et al., 1982). Treatment of reduced glutathione (a cysteine-containing tripeptide) with 2.5 mM H202 results in the formation of a NUV chromophore having a maximal absorption 25 nm above the absorption of the initial glutathione. From examination of related compounds, it is apparent that the N-acylcysteinamide of the peptide residue is the key element required for generation of the new chromophore. Although we have not

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yet determined the structure of the new chromophore, we do know that it is not simply the oxidized cysteinyl residue. Glutathione is a useful model molecule to show that a cysteine-containing polypeptide can be altered to generate a new chromophore, one that can absorb NUV and possibly result in biological damage (Greenberg and Demple, 1986; Owens and Hartman, 1986). There are a number of enzymes with absorption in the 290- to 400-nm range, but in only a few cases is i t known whether NUV (or a reactive oxygen molecule) will produce a critical biological change. In the case of one enzyme in particular, dihydroxy acid dehydratase, its sensitivity to 02-has been well characterized (Brown and Seither, 1983; Kuo et al., 1987). It is also sensitive to NUV; the amount of its activity in E . coli cells can be reduced by 50% with a fluence that will inactivate only 5% of population (Wilke, 1988). Although the absorption specturm of E . coZi DHAD has not been determined, DHAD isolated in the oxidized state from spinach has its major absorption between 290 and 450 nm (Flint and Emptage, 1988). The enzyme contains a n iron-sulfur cluster (which may account for its particular absorption spectrum); a shift from the oxidized to the reduced state greatly reduces enzyme activity and greatly reduces its absorption from about 370 to 450 nm. Primary studies with E . coli DHAD indicate that it has a similar iron-sulfur cluster (Flint and Emptage, 1988). In addition to its reputation of being the enzyme most sensitive to 0 2 - , DHAD inactivation may be important in triggering the stringent response (Brown and Seither, 1983; Cashel and Rudd, 1987). This sensitivity of DHAD is supported by the observation that sodAB mutants grow very poorly on minimal medium (Touati, 1988b). Since 02- inactivates DHAD (an important enzyme in amino acid biosynthesis), this failure could be due to starvation of branched-chain amino acids. DHAD catalyzes a step in the valine-isoleucine biosynthetic pathway. While there are mutants (ilvD)that accumulate this enzyme, it is difficult to study its NUV sensitivity resistance because the presence of branched amino acids reduces the DHAD in the cell and valine needs to be added to the medium for growth of the mutant to occur. In vivo NUV inactivation ofE. coli ribonucleotide reductase has been reported (Peters, 1977). Its role in coping with NUV deserves further study. Other examples of proteins that could be inactivated directly by NUV are noted in Section 111. Figure 1shows a schematic summary of the photoreceptors present in cells, as well as other key molecules involved in NUV damage and recovery.

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J

P

FIG. 1. (See the text, Section 1,B.) The photoreceptors are the outer and inner membrane, molecules in the periplasmic space, thiolated tRNA, the two catalases (HPI and HPII), dihydroxy acid dehydratase (DHAD), DNA, flavin- and hemecontaining molecules, and other molecules that absorb in the 300-to 400-nm range. Hydrogen peroxide, superoxide anion, hydroxy radical, and singlet oxygen are toxic photoproducts, a s well as DNA with photo alterations. Two superoxide dismutase enzymes (Mn and Fe) detoxify the superoxide anion.

E. PHOTOPROTECTIVE CHROMOPHORES When cells have been damaged by FUV, they may be rescued from death by NUV. This photoprotection is due to one of several different biological reactions, and deliniation of these may give us some understanding of NUV cellular chromophores and targets. There is a photoreactiving (PR) enzyme (DNA photolyase) with a n absorption peak of 380 nm; it complexes with the pyrimidine dimers produced by FUV, and NUV rapidly splits the complex, leaving pyrimidine monomers (Sancar et al., 1983).This is another example of a protein chromophore for NUV. The PR enzyme is -49 kDa (Sancar and Sancar, 1984)and has a flavin adenine dinucleotide component as a chromophore. If this enzyme action were the only explanation of photoprotection, a mutant lacking this enzyme should not display photoprotection. Such mutants and deletions of phr have been studied carefully, but even these strains are capable of some degree of dimer cleavage by NUV (Hussain and Sancar, 19871, even under conditions that would indicate involvement of a nonenzymatic reaction. It is possible that a C-C (6-4) dimer is the direct NUV photoreceptor, resulting in restoration of the

BACTERIAL GENES INVOLVED IN RESPONSE TO

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111

monomer state upon irradiation. This is important, since it would further support the view that DNA configurations can be photoreceptors of NUV, as noted in Section II,A. It would also indicate that NUV may be an antimutagen, since such C-C dimers (6-4) are mutagenic (Glickman et al., 1986). Ill. Oxidative Photoproducts of NUV

There is considerable evidence that H202 is a photoproduct of NUV and that other reactive oxygen molecules are subsequently generated (McCormick et al., 1976). Also, it is possible that 0 2 - could be a direct photoproduct of NUV. At least two sets of enzymes, catalases and superoxide dimutases, are known to detoxify these reactive molecules; numerous endogeneous radical scavengers, such as glutathione, are also known. However, as the roles of these enzymes are characterized in stress experiments, it is obvious that they operate in a highly sophisticated manner, and these enzymes and radical scavengers may have functions in addition to being general detoxifiers in cells, such as involvement in metal ion metabolism. It would be expected that reactive oxidative species (and NUV) might be more damaging a t certain precise locations than at others, and that catalase and/or SOD might be expected to be located at these sites. Furthermore, it should be kept in mind that these enzymes could have effects at precise times that are critical in growth and development of cells. An example is the striking difference in the need for catalase at different stages of vegetative growth and sporulation in Bacillus subtilis (Dowds et al., 1987). As another example of a specific role for catalase, it is interesting to note that the katG gene product is located at the cytoplasmic membrane but the katE gene product is located in the cytoplasm (Heimberger and Eisenstark, 1988). Moreover, the role of catalase HPI in restoration of membrane transport is intriguing and raises a question of its importance in all transport systems (Farr et al., 1988). Additionally, HPI is important in overcoming the mutations in sodA sodB strains and thus is a n antimutagen (J. Hoerter and A. Eisenstark, unpublished). Another possible role for SOD, in addition to quenching 02-and involvement in Fe/Mn metabolism, might be to activate or inactivate enzymes, thus serving a n interesting cellular regulatory role. As noted

112

A. EISENSTARK

above (Section I1,D) an example is dihydroxy acid dehydratase, which is inactivated by 02-(Brown and Seithers, 1983) and NUV (Wilke, 1988). The special role of the SOD/02- balance may be to modulate enzyme activity, as well as to signal the stringent response in E . coli, with the abrupt cessation of protein synthesis. Other examples include the 3’ to 5’ exonuclease activity of phage T7 that specifically inactivated by molecular oxygen (Tabor and Richardson, 1987). Of particular interest is a possible role of SOD in the activation of ribonucleotide reductase in E.coli (Peters, 1977). Flavin reductase generates superoxide radicals, which would inactivate ribonucleotide reductase (Fontecave et al., 1987). There are also examples in organisms other than E . coli, such as the oxidation of rhodanase in algae that renders them susceptible to proteolysis (Horowitz and Bowman, 19871, and a possible superoxide-dependent peptidyl deaminase for making citrulline in mouse bone marrow (Kamoun et al., 1988). A. HYDROGEN PEROXIDE There is evidence that the toxic effect of HzOz is primarily to damage DNA; this is attributed to a Fenton reaction that generates OH. from H202,DNA-bound iron, and a source of reducing equivalents (Imlay and Linn, 1988). In our earlier studies on the effect of NUV on bacteria, we found that a photoproduct of NUV was HzOz, which, in part, might have a role in NUV lethality. Since that time, numerous observations have been made of the similarities between Hz02 and NUV effects (Ahmad, 1981; Ananthaswamy and Eisenstark, 1976; Eisenstark, 1985; Hartman, 1986; Roth, 1981; Sammartano and Tuveson, 1983; Tyrrell, 19851, consistent with the view that biologically relevant quantities of HzOz may be generated in sztu following NUV irradiation of cells. But, again, we face a paradox. It would be expected that mutants that are devoid of catalase would be very sensitive to NUV; they are not. Although Sammartano et al. (1986) showed that katF mutants that lack catalase activity are sensitive to NUV, we now know that katF is a regulatory gene for katE and xthA and not a structural gene for catalase. The sensitivity of katF mutants to NUV and H202 is due to the lack of ex0111 enzyme and not the lack of catalase HPI (Sak et al., 1989). Consistent with the NUV/H202 relationship is the observation that small doses of either H202or NUV often induce proteins that protect against challenge doses of either (see Section V1,B).However, it should be noted that there are some exceptions to reciprocity of induction

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113

(Eisenstark and Perrot, 1987; Kramer and Ames, 1987). In addition, the fact that catalase mutants (katE and katG and even the katE katG double mutant) are void of enzyme activity but are not sensitive to NUV (Eisenstark, 1982) shows that a NUV/H202relationship is not simple. Also, the amount of endogenous catalase can be physiologically manipulated; when this is done t o reduce catalase drastically, cells actually become more resistant, rather than sensitive. The amount of catalase in a cell can also be increased via a multicopy plasmid with the katG gene. Cells with excess HPI are not more resistant to H202; they actually become more sensitive to NUV (Eisenstark and Perrot, 1987; Wilke, 1988). Upon comparison of new proteins that are synthesized and shut off following induction by H202 and NUV, there are numerous differences (Kramer et al., 1988; Pierceall, 19881, further supporting nonidentical effects. However, the oxyR regulon is involved in both HzOz and NUV stress, since a deletion of the oxyR gene results in hypersensitivity to both (Eisenstark and Perrot, 1987; Kramer and Ames, 1987). Also, mutations in glutathione reductase ( g s h ) and alkyl hydroperoxide reductase (ahp),both regulated by oxyR, result in sensitivity to NUV (Kramer and Ames, 1987; Storz et al., 1989). These observations, plus toxicity differences in mutants defective in porphyrin (Kramer and Ames, 1987; Tuveson and Sammartano, 1986) and tRNA synthesis (Favre et al., 19851, further indicate that H202 and NUV differ in their modes of killing cells. This further is supported by the synergistic (and not additive) action by NUV and H202 (Ahmad, 1981; Ananthaswamy and Eisenstark, 1976; Ananthaswamy et al., 1979; Hartman and Eisenstark, 1978, 1980; Hartman et al., 1979). Yet, there are important overlaps, particularly in the roles of enzymes regulated by the oxyR gene. From these comparisons of NUV and H202 toxicity, it would appear that H202 is only indirectly involved in NUV killing, but still may be a source of oxygen radicals. B. SUPEROXIDE ANION NUV irradiation results in 0 2 - increase (Ahmad, 1981) (see Sections I11 and V,B for discussion of superoxide dismutase). Not only can this be observed chemically, but the fact that sodA sodB strains are highly sensitive to NUV (A. Eisenstark, unpublished) would indicate that 0 2 - is produced. Perhaps a better explanation is that H202 is generated by NUV and this reacts with 0 2 - to yield OH., which damages DNA.

114

A. EISENSTARK

Before considering the effects of SOD deficiencies in mutants, it is of interest to note the effect of overproduction, since one of the explanations of human Down's syndrome is that excess SOD is synthesized as a result of triploid chromosome 21. Excess MnSOD in E. coli does not lead to increased sensitivity to NUV, but cells with plasmid FeSOD are more sensitive (A. Eisenstark, unpublished). Scott et al. (1987) reported that bacterial cells with excess FeSOD are more sensitive to paraquat and attributes this to the increased HzO2 (and perhaps subsequent hydroxy radicals) that results from the SOD action. The fact that the sodA sodB double mutant is hypersensitive to both NUV and H202 could result from generation of hydroxyl radicals through the iron-catalyzed Haber-Weiss reaction: Fez++ HzOz + H' + Fe3+ + HO. + H20 HO. + H202 -+ HO, + HZ0 HOi + Fe3+-+ O2 + Fez++ H' 2H202 + 0

2

+ 2Hz0

The sodA sodB double mutant has metabolic problems (e.g., it cannot grow on minimal medium) and is highly mutagenic. It is interesting to note that all of these deficiencies can be overcome by the addition of a plasmid containing either of the two E . coli SOD genes, or by the human Cu/Zn SOD plasmid (Natvig et al., 1987; Touati, 1988a). Also, it should be noted that mutagenesis is oxygen dependent and requires the ex0111 repair enzyme t o transform the premutational DNA lesion into a mutation. In assessing any correlation between the quantity of SOD and protection against oxidative damage, there is disagreement in the literature. Experiments utilize paraquat to generate 02- and to determine its effect on sod mutants and plasmid strains. Perhaps anomalous observations could be the result of other affects of paraquat, and not directly the result of 0 2 - . Another viewpoint is that the role of SOD may not be primarily for quenching 02-;rather, SOD is more important in metallic ion metabolism in cells (Fee et al., 1989; Niederhoffer et al., 1989).

C. HYDROXYL RADICAL The hydroxyl radical (OH.) reactivity is so great that it will react almost immediately with whatever molecules are in its vicinity. By the use of OH. scavengers, experiments have shown that excess of this radical may have critical consequences (Billen, 1984). Rowley and Hallewell (1982) showed, using thiol compounds that readily react with OH., that thiol compounds did not prevent 02--dependent formation of

BACTERIAL GENES INVOLVED IN RESPONSE TO

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115

the OH radical in the presence of iron ions. The thiol compounds could produce the radical themselves. They also showed that addition of only OH. scavengers prevented DNA strand scission, and the addition of SOD and catalase prevented strand scission. They concluded that both 0 2 - and HzOz are needed for strand scission but the OH. is the actual attacking molecule. D. SINGLET OXYGEN Singlet oxygen ('Oz),the lowest electronically excited energy state of molecular oxygen, has a relatively long lifetime and the potential to react with a variety of biological substrates. There is disagreement, however, as to its biological significance. Dahl et al. (1987, 1988) state that singlet oxygen is highly toxic but not mutagenic. DecuyperDebergh et al. (1987), however, found that it produces a high frequency of mutations in the lac2 region upon exposure of RF (double-stranded) DNA of phage M13 mp19. Photosensitizing systems are used to implicate singlet oxygen involvement in biological effects such as bacteriophage 4x174 inactivation (Houba-Herin et al., 1982). Dahl et al. (1987, 1988) recently demonstrated directly that exogenous pure singlet oxygen is over 10,000-foldmore toxic to bacteria on a molar basis than is H202?but that it is not mutagenic. Biological sources of singlet oxygen may include photosensitization reactions involving endogenous sensitizers, decomposition or interconversion of other active oxygen species, electron transport systems, and some enzyme systems. In order to cope with adverse effects of singlet oxygen, living systems may use at least two defense strategies: (1) interception of singlet oxygen either by quenchers (Ames et al., 1981; Dahl et al., 1987, 1988) and/or (2) prevention of singlet oxygen formation by direct quenching of excited-state sensitizers (Dahl et al., 1987, 1988). Dahl et al. (1988) investigated the abilities of several biomolecules at physiologically relevant concentrations and neutral pH to protect against oxidation of a target substrate by singlet oxygen. Dahl et al. (1989) noted that bacterial species that contain carotenoids are more resistant to killing by singlet oxygen than those that are void of carotenoids. IV. DNA Damage

The critical DNA lesions produced by NUV and HzOz differ from

FUV lesions, despite some qualitative similarities in photoproducts

116

A. EISENSTARK

(Ferron et al., 1972). The evidence is as follows: (a) analyses of DNA damage by the different agents yield different profiles (Ananthaswamy and Eisenstark, 1976; Caimi and Eisenstark, 1986; Hartman and Eisenstark, 1982, 1987; Mitchell and Clarkson, 1984; Tuveson et al., 1983); (b) defense against these agents involves different regulons (Aliabadi et al., 1986; Christman et al., 1985; Eisenstark, 1985; Morgan et al., 1986) with induction of different proteins; (c) different sets of mutants are hypersensitive to these agents (Eisenstark, 19851, although some mutants are hypersensitive to both; (d) DNA repair and mutagenesis of FUV damage are influenced by u m u and muc genes, but not after NUV irradiation (Eisenstark, 1983); and (e) numerous physiological effects are different (Jagger, 1985) (Table 1). There is strong evidence that a NUV lesion is a substrate for exonuclease 111. The DNA lesion may be a single-strand break with a 3’-end-blocking group (phosphoglycoaldehyde ester), which may be activated by exonuclease I11 to allow synthesis by polymerase I (Demple et al., 19861. This is consistent with the observations that xthA (exoIII) and polA (pol11 mutants are sensitive to both H202 and NUV. Endonuclease IV may also be involved in the process by initiating the repair of ruptured 3’-dexoxyribose (Demple et al., 1986). Further support, although indirect, comes from the observation that, when ex0111 acts on DNA damaged by 02-,mutation frequency is increased (Farr et al., 1986). NUV is one of the ways that 02-might be generated (Ahmad, 1981; Fridovich, 1986; Kramer and Ames, 1987), thus the similarity with regard to exoIII. We have examined the possibility that an initial lesion might be a DNA-protein cross-link produced by NUV. DNA-protein cross-links occur particularly in phage (Hartman et al., 1979; Casas-Fimet et al., 1984) and mammalian cells (Eisenstark et al., 1982), where the DNA and protein are packaged geometrically in close quarters. Evidence with bacteria is far less striking, but only a small fraction of DNA and protein is tightly bound in these cells. The observation of DNAprotein cross-links as a critical NUV lesion may not conflict with the observation of DNA strand breaks (Ananthaswamy and Eisenstark, 1977; Caimi and Eisenstark, 1986; Demple et al., 1986; Eisenstark et al., 1982; Hartman and Eisenstark, 19801, since these single-strand and double-strand DNA breaks could be secondary consequences of photochemical damage, following the initial cross-link. Note that the number of DNA breaks is not correlated with the number of lethal NUV events (Ananthaswamy and Eisenstark, 1976, 1977; Hartman and Eisenstark, 1980; Tuveson et al., 1983). By comparison, the number of DNA-protein cross-links of phage (Hartman and Eisen-

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117

TABLE 1 Some Distinct Differences between Far- and Near-UV Damage in Bacteria and Phages Effect DNA degradation in recA mutants Mutation enhancement Growth inhibition (division delay) Use of SOS regulon Log cells more sensitive than stationary Sensitivity of katF mutant Sensitivity of r t h A mutant Sensitivity of nfo mutant Sensitivity of polA mutant Sensitivity of sodA sodB double mutant Production of DNAphage protein links Block phage DNA injection Sensitivity of Deinococcus radiodurans Enhancement of resistance and mutation by plasmid pKMlOl Synergistic action with nonlethal doses of HZO2 Weigel reactivation of A phage Oxygen demand for lethality Enzyme induction inhibition Liquid holding recovery

Far-UV

NUV

References

Yes

No

Ferron et al. (1972)

High Low

Limited High

Favre et al. (1985) Jagger (1985)

Yes

No

Slight

High

Turner and Eisenstark (1984) Eisenstark (1982)

Low

High

Eisenstark and Perrot (1987)

Low

High

Low

High

Sammartano and Tuveson (1983) A. Eisenstark (unpublished)

Low

High

Eisenstark and Perrot (1987)

No

Yes

A. Eisenstark (unpublished)

Low

High

Eisenstark et al. (1982)

No

Yes

Resistant

High

Hartman and Eisenstark (1982) Caimi and Eisenstark (1986)

High

No

Eisenstark (1983)

No

Yes

Ananthaswamy and Eisenstark 11976)

Yes

No

Low

High

Turner and Eisenstark (1984) Ferron et al. (1972)

No

Yes

Yes

No

Jagger 11985);Turner and Eisenstark (1984) Jagger (1985)

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A. EISENSTARK

stark, 1980; Tuveson et aZ.,1983) correlated very well with the number of phages inactivated, but DNA breaks did not show this correlation. One way to rationalize these conflicting results is t o consider that NUV damage may involve a cascade of events, including cross-links, followed by an alkylated base, with subsequent breakage of one DNA strand and then the other strand. Further disenchantment with SSBs as direct lethal events comes from knowledge that xthA and poZA mutants are very HzOzand NUV sensitive, but fewer SSBs occur than expected when compared to the number of SSBs in wild-type strains (Demple et al., 1983; Miguel and Tyrrell, 1986).The number of SSBs by NUV is much lower in E . coli than in Deinococcus radiodurans (Caimi and Eisenstark, 1986), an organism that is very sensitive to NUV but notoriously resistant to FUV. There are features of DNA damage that are common to both NUV and Hz02, but there are also some features that are unique to NUV. This may be due not only to a direct action of H202 on DNA, but also to an indirect photoeffect on DNA, perhaps by way of membrane (Klamen and Tuveson, 1982; Kelland et al., 1984; Kramer and Ames, 19871, tRNA (Blanchet et al., 1984; Cadet et aZ., 1986; Favre et al., 1985; Hajnsdorf and Favre, 1986; Klamen and Tuveson, 1982; Kelland et al., 1984; Peak et al., 1987a), and/or other photoreceptors (Jagger, 1985; Spitzer and Weiss, 1985; Tuveson and Summartano, 1986). The biological similarities between the DNA damage and repair produced by NUV and Hz02 could be accounted for if some DNA damage by NUV is the generation of H202 (and subsequent reactive oxygen molecules) (McCormick et al., 1976; Yoakum et al., 1975). To verify this, the DNA lesions after H2O2 treatment should be compared in detail with NUV lesions, both at low and high doses (Imlay and Linn, 1986; Kramer and Ames, 1987; Linn and Imlay, 19871, as well as by sequencing genes that have been mutated by the two agents. A t high concentrations of HzOz, it has been shown by alkaline sucrose sedimentation experiments that DNA is cleaved (Ananthaswamy and Eisenstark, 1977). In the presence of a low concentration of ferric chloride and low concentrations of H202, 4x174 supercoiled DNA is incised (Van Rijin et al., 1985). The H202 photoproduct of NUV inhibits replication gap closure (Yoakum et al., 1975) and also stimulates DNA repair synthesis (Hagensee and Moses, 1986);this repair synthesis requires polymerase I (polA) (Ananthaswamy and Eisenstark, 1977), exonuclease I11 (xthA) (Weiss and Cunningham, 19851, and probably polymerase I11 (poZC) (Hagensee and Moses, 1986). It also has been observed that all four of the bases may be released from the backbone (Breimer and

BACTERIAL GENES INVOLVED IN RESPONSE TO

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119

Lindahl, 1985). Hagensee and Moses (1986) reported that cells with a temperature-sensitive polymerase I11 mutation are sensitive to H202, but cannot synthesize DNA after H202treatment, unlike WT cells. They suggest that pol111 may either be required for a small (but undetectable) amount of synthesis or may provide a terminus modification function for subsequent DNA polyI synthesis. The observation that certain mutants (i.e., xthA and poZA) are sensitive indicates that NUV lesions become substrates for the appropriate DNA repair enzymes. However, since the r t h A gene product may have four different enzymatic activities, there is some uncertainty as to the nature of the lesion. Analyses of DNA lesions by sequencing of appropriate mutated genes (Ito et al., 1988; Miller, 1985) are informative. Base-specific damage was determined by 4-thiouridine (4TU) photosensitization with 334-nm radiation in M13 phage DNA (Ito et al., 1988). Whether the same damages would occur i n vivo without 4TU is not known. Identification of endonuclease cleavage sites (Wei et al., 1986) andlor the use of monoclonal antibodies against NUV- and H202-damagedDNA (Mitchell and Clarkson, 1984) might be another approach to identify specific modifications. V. NUV-Sensitive Mutants

Table 2 lists genes that may be involved in cellular responses to NUV. Mutations in several of these genes render cells sensitive to NUV (see Section 111for discussion of effects of oxidative photoproducts of NUV). A. CATALASE Throughout our studies, we have considered catalse as a logical “NUV-recovery enzyme,” since NUV yields H202 (McCormick et al., 1976; Yoakum et al., 1975). However, as noted in Section III,A, the role of catalase is complex and may be minor in the capability of the cell to deal with NUV damage (Eisenstark and Perrot, 1987). Indeed, the H202 photoproduct of NUV may have only a minor role in the lethal process, particularly when compared to alkyl hydroperoxidases and other photoproducts (Kramer and Ames, 1987). The fact that katF (catalase) mutants are sensitive to NUV would support the argument that H202is a photoproduct of NUV (Eisenstark and Perrot, 1987; Samartano et al., 19861, since katF+ is necessary for the synthesis of HPII. katF+ is also a regulatory gene for ex0111

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A . EISENSTARK

TABLE 2 Genes of Escherichia coli That May Influence NUV Responses Gene

Map position

Phenotype

NUV sensitive

ahp

13

Flavin-containing hydroperoxidase

Yes

CYd

17

Cytochrome

Yes

darn gsh

74 58

DNA adenine methylase Glutathione reductase

Yes Yes

Heme synthesis

See the text

Dihydroxy acid dehydratase; isoleucine-valine synthesis Catalase activity

See the text

Regulatory gene for katE synthesis

Yes

Catalase activity

Yes

No

hemA -H

8-90

iluD

85

katE

38

katF

59

katG

7

led

92

ndh

22

Resistance or sensitivity to X-rays and UV; repressor of SOS proteins NADH dehydrogenase

nfo

60

Endonuclease IV

Yes

nrdl3

49

Ribonucleotide reductase

Yes

nth

36

Endonuclease I11

Yes

Near-ultraviolet radiation growth delav

Yes

nuuA

9

Yes

Yes

References Kramer et al. (1988); Storz et al. (1989) Sammartano and Tuveson (1987) Yallaly (1988) Kramer and Ames (1987) Tuveson and Sammartano (1986); Bachmann (1987) Bachmann (1987)

Loewen and Triggs (1984) Loewen and Triggs (1984); Sak (1989) Loewen and Triggs (1984); TriggsRaine and Loewen (1987); Triggs-Raine et al. (1988) Walker (1987)

Imlay and Linn (1988) Saporito et al. (1988) A. Eisenstark (unpublished); Fontecave et al. (1987) Weiss and Cunningham (1985) Favre et al. (1985)

BACTERIAL GENES INVOLVED IN RESPONSE TO

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TABLE 2 (Continued) Gene

Map position

Phenotype

nuuC

45

oxyR

89

Phr

16

polA

87

recA

58

recB -C

61

relA

60

SOdA

88

sodB

37

umuC-D uurA

26 92

XthA

38

General recombination, repair of radiation damage; induction of phage A Recombination and repair of radiation damage; exonuclease V subunits Regulation of RNA synthesis; stringent factor; ATP : GTP 3' pyrophosphotransferase Mn superoxide dismutase Fe superoxide dismutase Induction of mutation Repair of UV damage to DNA; excision nuclease Exonuclease I11

gal-halt deletion (unknown gene)

16

NUV sensitivity

a

Near-ultraviolet radiation growth delay Regulator of oxidative stress Deoxypyrimidine photolysase; photoreactivation DNA polymerase I

NUV sensitive

References

Yes

Favre et al. (1985)

See the text No

Kramer and Ames (1987) Husain and Sancar (1987)

Yes

Yes

Sancar et al. (1983); Ananthaswamy and Eisenstark (1977) Walker (1987)

No

Walker (1987)

Yes

Kramer et al. (1988)

No"

Touati (1988a)

No"

Touati (1988a)

No Yes

Walker (1987) Turner and Webb (1981)

Yes

Eisenstark and Perrott (1987) Husain and Sancar (1987)

Yes

The sodA sodB double mutant is NUV sensitive (A. Eisenstark, unpublished data)

(product of xthA) (Sak et al., 1989; Schellhorn and Hassan, 19881, which is the basis for sensitivity of katF mutants. The lack of correlation between catalase content and NUV sensitivity is emphasized in experiments in which the amount of intracellular

122

A. EISENSTARK

catalase is manipulated in a number of ways (Eisenstark and Perrot, 1987): (1) by the use of mutant and plasmid strains with altered endogenous catalase; (2) physiologically, by the addition of glucose; and (3) by induction of catalase synthesis with oxidizing agents. Not only is there no correlation between NUV resistance and catalase activity, but in some cases the correlation is inverse. As noted above, mutants other than katF (i.e., katE, IzatG, and double mutant katE katG) that are defective in catalase activity (Loewen, 1984; Loewen et al., 1983, 1985a; Loewen and Triggs, 1984) are not particularly sensitive to NUV (Eisenstark and Perrot, 1987; Wilke, 1988). Also, a strain that carries the KatG plasmid (Loewen et al., 1983) and overproduces catalase is actually more sensitive to NUV (Eisenstark and Perrot, 1987). It thus appears that while catalase may decompose H202 produced by NUV, it may also act as a photosensitizer in the cell when it is present in excess (Wilke, 1988). It is possible that catalase may have a physiological role only under very special conditions of stress, since its protective role is less important than certain other factors (Carlsson and Carpenter, 1980; Eisenstark and Perrot, 1987; Sammartano et al., 1986). Despite the lack of sensitivity of kat mutants, a role in protection against NUV cannot be completely ignored, since bovine catalase in plating media will reduce the lethal effect of NUV (Kramer and Ames, 1987; Sammartano and Tuveson, 1984; and our own observations). Interestingly, we have found that if cells are stressed by a number of agents (including heat), catalase in the medium will give higher colony counts (unpublished); H202 may be a common product of generalized cellular stress (Ames, 1983; Hartman, 1986; Richter and Loewen, 1981; Tyrrell, 1985; Vassilyadi and Archibald, 1985; Winguest et al., 1984). As discussed elsewhere in this review, the observations that catalase HPI may be antimutagenic and that this enzyme has a role in permeation indicate that it has specific functions that are not visible in tests for survival alone.

B. SUPEROXIDE DISMUTASE Judging by the number of recent publications dealing with superoxide dismutase and 4ts effects on 0 2 - , perhaps no other enzyme has received more attention. The numerous theories of the role of 0 2 - in various disease and aging processes, together with searches for therapeutic uses of SOD, may be found in several reviews (Touati, 1988; Hassan, this volume; Fridovich, 1986; Petkau, 1986; Rotilio, 1986; Sies, 1985). A breakthrough in our ability to understand the mechanism of SOD

BACTERIAL GENES INVOLVED IN RESPONSE TO NUV

123

action came when Touati (1983; Sakamoto and Touati, 1984) first isolated SOD genes in E . coli. She succeeded in isolating these from a cosmid bank, of which clones were individually tested for SOD overproduction. Since that time, Touati and associates have obtained mutants that are defective in two SODSin E . coli, a n inducible MnSOD (sodA), and a constitutive FeSOD (sodB). MnSOD is present only under aerobic growth, whereas FeSOD is expressed both aerobically and anaerobically. The MnSOD DNA sequence (Carlioz et al., 1988), together with a detailed study of MnSOD regulation (Touati, 1988b), suggests two possible promotors, perhaps one that is active in normal aerobic growth and another that is active under oxidative stress conditions. Superoxide dismutase is a logical candidate as a NUV-recovery enzyme (Ahmad, 1981; Farr et al., 1986; Touati, 1983; Touati and Carlioz, 1986).We have found that the presence of a plasmid carrying an inducible Mn superoxide dismutase (sodA) (Bloch and Ausubel, 1986; Carlioz and Touati, 1986; Tajeda and Avila, 1986; A. Eisenstark, unpublished) endows the cell with some resistance to NUV, but not t o FUV. Although the amount of protection is small, it does implicate 02-as a lethal radical. This strain carries a low-copy-number plasmid in a cell that already has a n Mn sod gene. A plasmid containing the FeSOD gene (sodB)(Nettleton et al., 1984; Sakamoto and Touati, 1984) does not endow the cell with NUV resistance (Scott et al., 19871, although excess SOD is produced. We have tested sodA and sodB strains (Carlioz and Touati, 1986; Farr et al., 1986; Touati, 1983, 1988a,b; Touati and Carlioz, 1986), but found them t o be only slightly more sensitive than the wild type under aerobic conditions; however, the sodA sodB double mutant is very sensitive t o NUV. This is compatible with the results of Carlioz and Touati (1986), who found that among such mutants only the double mutant was sensitive to paraquat, a generator of superoxide anion. They conclude that the role of SOD was to handle 02-only when the cell is under special stresses. These double mutants have a number of metabolic problems, but an additional (suppressor) mutation can restore the ability to grow on minimal medium (Fee et al., 1989). This mutation might be involved in the branched-chain amino acid pathway, since DHAD could be depleted in the double mutants. Also, such double mutants probably contain specific DNA lesions, which, when acted on by exoIII, yield a higher mutation frequency (Farr et al., 1986) (see Section VIII). C. EXONUCLEASE I11 AND ENDONUCLEASE IV The observations that x t h A mutants are more sensitive to NUV but no more sensitive to FUV than are the wild type, and that there are

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fewer single-strand DNA breaks in the xthA mutant, support the view that a major role of xthA (ex0111 enzyme) may be to recognize apurinic or apyrimidinic sites and to nick the DNA at the 3’ side of such sites (Demple et al., 1983, 1985, 1988; Kow and Wallace, 1985; Wallace, 1988; Weiss and Duker, 1986,1987).Since xthA mutants are sensitive to both NUV and H202, a logical conclusion might be that both produce apurinic sites. However, there are other enzymatic activities of the xthA gene protein, and thus other lesions may also be recognized. DNA protein cross-links might interfere with action by ex0111 enzyme; thus, there could be two actions in the case of a DNA-protein cross-link, proteinase action on the attached polypeptide, leaving a n exposed altered base, and direct action by ex0111 at the cross-link site. There are two mutations (nfo and nth) that influence the sensitivity of xth mutants. The nfo mutation increases sensitivity to H202 and NUV; the nfo gene codes for endonuclease IV. However, the addition of a third mutation (nth, coding for endonuclease 111) gives only small protection to the double mutant (Cunningham et al., 1986; our observations). EndoIV enzyme is induced by paraquat and other 0 2 - generating agents (Chan and Weiss, 1987). Although strains with xthA mutations are sensitive to NUV, strains that carry a plasmid with the xthA gene and thus overproduce the enzyme are not more NUV resistant than are the wild type. One possible mode of action on NUV lesions is that the enzyme (exoIII) recognizes the urea moiety that results from alteration of thymine in DNA (Katcher and Wallace, 1983; Kow and Wallace, 1985; Taylor and Weiss, 1982). Since mutants that lack the enzyme are sensitive to NUV, it would appear that NUV may create this urea moiety, which may be lethal to the cell unless it is repaired. In the absence of the ex0111 enzyme in the cell, this nick obviously would not take place. Ex0111 may have an important role in the mutational process (see Section VIII). D. OTHERNUV-SENSITIVEMUTANTS 1. polA. Strains with the polA mutation are sensitive to both H202 and NUV (Ananthaswamy and Eisenstark, 1977); these strains are deficient in the 5’ to 3’ exonuclease (but not in 3’ to 5‘) activity. The polA gene product is not induced by NUV (A. Eisenstark, unpublished). Also, a strain with a polA-bearing plasmid (an overproducer of the enzyme) is not resistant to NUV. The fact that polA cells are sensitive to NUV indicates that, following the production of the

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urea moiety by NUV action, there may be no ex0111 repair, but the gap that is left requires the polA gene product (Kow and Wallace, 1985). 2. dam. The product of the dam gene is a DNA adenine methylase which methylates deoxyadenosine in -GATC- sequences in doublestranded DNA (Marinus, 1987; Szyf et al., 1986). Strains with dam mutations are sensitive to NUV (Yallaly, 1988). Since the dam gene enzyme is not required for viability, it may be that NUV damage within this sequence may be lethal unless removed by the dam gene enzyme. Increasing the levels of dam gene enzyme in plasmid-carrying strains reduces this sensitivity slightly, as might be expected. However, increasing the levels of enzyme much above wild-type levels results in a sharp increase in the sensitivity of the cell. A possible explanation is that for wildtype levels of methylase, only one of the two DNA strands is methylated, as needed for appropriate activity, but excess methylation leads t o undesirable methylation of adenines on the other strand as well. 3. recA. This protein has a role in NUV recovery (Ferron et al., 1972; Miguel and Tyrrell, 19861, but whether the entire SOS system is involved is difficult to assess, because of some physiological factors that are invovled (see Section VII,A). Although NUV lesions do not lead directly to SOS induction, it should be emphasized that constitutive levels of certain SOS proteins, particularly recA recombinase, may still function to repair NUV damage (Ennis et al., 1985; Tessman et al., 1986; Turner and Eisenstark, 1984). Thus, recombinational repair may be critical for recovery from NUV damage. It is important to note that while recA mutants are very sensitive to NUV in rapidly growing cells, they are not particularly sensitive when cells are in stationary phase. 4. uvr. Cells that lack excision repair capacity are slightly more sensitive to killing by NUV, but become very sensitive to mutation. The double mutant recA uurA is hypersensitive t o NUV. It would be interesting to test mutagenicity in this strain,since recA strains are “mutation proof” t o FUV. 5. hem. Based on the theory that hematins in porphyrins are photoreceptors, and that NUV acts photodynamically to injure cells, it would be expected that a mutation that results in excess of these photoreceptors would render them sensitive and that a mutation that depletes these photoreceptors would make the cells more resistant. Indeed, this is the case for such hem mutants (Sammartano and Tuveson, 1987; Kramer et al., 1988). Probably for the same reasoning, flavin mutants are also sensitive (Kramer et al., 1988). 6. Deletion of gal-hatt. Cells that have the deletion gal-att are

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sensitive to NUV; the reason is not yet known, but this region contains both hem and chl genes and appropriate mutants need to be tested. 7. nuuA and reZA. Mutations that lead to the absence of 4thiouridine in tRNA (nuvA)were found to be sensitive when irradiated with NUV at a low fluence rate (Kramer et aZ., 19881, but Favre et al. (1985) found such mutants to be more resistant than wild type, using higher fluence rates and monochromatic illumination at 334 nm. His explanation was that such mutants did not undergo growth delay, and thus were physiologically healthier. 8. ahp. As noted in Section II,C, a mutation in the flavin hydroperoxidase gene makes the cell sensitive to NUV. 9. nrdB. Mutants that are defective in the synthesis of the B subunit of ribonucleotide reductase are very sensitive to NUV (A. Eisenstark, unpublished). This subunit of the enzyme contains an organic free radical at position tyrosine-122 of its polypeptide chain (Fontecave et al., 19871, has strong absorption in the NUV region of the spectrum, and has been shown to be inactivated by NUV (Peters, 1977). VI. Physiological Effects of NUV

At NUV fluences far below that which show lethal or mutagenic effects, dramatic physiological changes may be observed: (a) as little as 15 kJ/@ of NUV blocks induction of P-galactosidase synthesis by FUV in a recA ::lac fusion, and (b) uptake of [3Hlproline is blocked by the same dosage. The basis for such sharp changes with so little energy has not been fully resolved, but may rest on knowledge of the precise mechanism whereby the inactivation of a thiolated tRNA stimulates synthesis of an alarmone. A. GROWTH DELAY One of the striking effects of a nonlethal dose of NUV is the abrupt cessation of cell division, reduction in cell size, and cessation of protein synthesis. This growth delay phenomenon has been studied by Favre and associates and Jagger and associates. These cellular changes are triggered by photon action on thiolated tRNA as described in Section II,B.

B. NUV INDUCTION OF NEWPROTEINS Upon gel electrophoresis, a number of new [35Slmethionine-labeled proteins are observed following Hz02 and NUV stresses (Pierceall, 1988; Kramer et al., 1988; Christman et al., 1985; Morgan et al., 1986;

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Peters and Jagger, 1981; Van Bogelen et al., 19871, but the newly induced proteins are not identical for the two stresses. In both E . coli and S. typhimurium there is a positive regulator (oxyR) of H202inducible proteins (Christman et al., 1985). A mutation in this gene results in the constitutive synthesis of 30 proteins, including catalse HPI, alkyl hyperoxidase reductase, and the heat-shock protein, dnaK. Mutation in oxyR makes cells resistant to NUV and H202, but a deletion of this regulator gene makes them hypersensitive (Eisenstark and Perrot, 1987). Among the inducible genes that katG (catalase) (Demple and Halhrook, 1983) and sodA, which are induced by both H202 (Gregory and Fridovich, 1973; Morgan et al., 1986) and NUV (Eisenstark and Perrot, 1987); however, the s o d 4 gene may not be under oxyR control (Touati, 1988a). Neither agent induces synthesis of polA, sodB, or xthA gene products. There are distinct differences between NUV and H202 with regard to protein synthesis patterns, particularly during the first 10 minutes of H202 treatment (Pierceall, 1988). After that time, no differences were noted among the NUV and H202 protein patterns. The differences could he explained if (1)the damages are the same, hut NUV generates H202slowly (or, alternatively, NUV inactivates catalase, allowing an accumulation of metabolically generated H202); or (2) the two agents induce different proteins initially and may be under the control of two different regulons, but only for a short period of time. In answering these questions, it should be noted that NUV causes sharp growth delay, but the growth delay by H202is for a much shorter time (Pierceall, 1988). Experiments have been hampered by the fact that NUV normally stops protein synthesis (growth delay) as well as producing membrane changes that diminish the uptake of [35S]methionine.The method was improved by using the nuuA mutant, which lacks thiolated tRNA (Favre and Hafnsdorf, 1983; A. Eisenstark, unpublished observations) and does not have “growth delay.” J. Hoerter and A. Eisenstark (unpublished) observed that synthesis of numerous polypeptides (-44% of the 509 spots that can be clearly identified on a two-dimensional electrophoresis gel) is completely shut off after NUV treatment. Even fewer polypeptides could be seen after treatments with 1 mM H202.Thus, synthesis of numerous polypeptides continues after NUV treatment but are shut off after Ha02 treatment. Synthesis of 30 polypeptides (6%)that are shut off by NUV reappear after 10 minutes of NUV, even if NUV is continued; synthesis of 54 polypeptides (11%) that are turned off during NUV reappear within 10 minutes following discontinuation of the NUV treatment. Upon comparison of the polypeptides that remain after NUV with the polypeptides that remain after H202 treatment, only a few polypep-

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tides were found to migrate to the same position on the gels. This further supports the view that the polypeptides needed in the recovery process are different in the two treatments. There is a paradox with regard to induced protection by small doses of H202and NUV. Several reports would lead to the conclusion that there is complete reciprocity with regard to challenging doses and induction of resistance of both H202 and NUV (Brawn and Fridovich, 1985; Eisenstark and Perrot, 1987; Sammartano and Tuveson, 1985; Tyrrell, 1985). However, interpretation of numerous experiments performed in our laboratory leads to a different conclusion. We found that nonlethal doses of H202 sensitize (rather than protect) bacterial cells and phages following irradiation with NUV (Ananthaswamy and Eisenstark, 1976; Anasthaswamy et al., 1979; Hartman and Eisenstark, 1978,1980). We have also found that NUV may make cells more sensitive to H202 (Hartman and Eisenstark, 1978, 1980, 1982; Hartman et al., 19791, a n observation also made by Kramer and Ames (1987). Since NUV irradiation is carried out over 0-60 minutes in our studies of NUV-H202 synergistic effects, there would have been ample time for induction (Hartman and Eisenstark, 1978, 1980, 1982; Hartman et al., 1979). Thus, in our studies of the synergistic action of NUV and H202, not only did we fail to observe interchangeable induction by the two agents, but there is strong indication that NUV and H202 produce different kinds of damages, and that each of the damages sensitizes the cell to the other agent. C. MEMBRANE EFFECTS The NUV effects ofE. coli membranes are rather drastic (Farr et al., 1988; Klamen and Tuveson, 1982; Chamberlin and Moss, 1986; Kelland et al., 1984). The main questions are (1) whether nonlethal membrane changes are involved in lethality and mutagenesis, and (2) whether there is a relationship between growth delay and membrane changes (Pizzaro and Orce, 1988). The evidence that membrane damage may contribute less than DNA damage is based on the observation that certain mutants that are very sensitive to NUV (xthA, recA, polA, and darn) are all involved in DNA metabolism. It might be worthwhile to examine mutants with membrane protein defects for sensitivity to NUV. To weigh the relative importance of membrane damage to DNA damage, it should be determined whether cell death occurs before one can see a single DNA lesion. If membrane damage were more important than DNA damage, then death and severe membrane damage should occur at fluences that show no DNA lesions. It should be noted that membrane damage by NUV can be amplified greatly by salts in

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minimal media used for plating (Klamen and Tuveson, 1982; Kelland et al., 1984). In a careful action spectrum study by Kelland et al.(19841, considerable membrane damage was observed at wavelengths above 310 nm, peaking a t 334 nm. They also showed that this membrane damage could contribute to lethality in repair-proficient strains. However, in repair-deficient uurA or uurA recA strains, lethality due to membrane damage was not apparent. This further indicates that DNA damage overrides the membrane damage and thus is a more important contributor to lethality. Membrane damage can be monitored by growing cells in “RB’ as a substitute for K + and looking for Rb leakage after NUV (there is little or no leakage at wavelengths below 305 nm). Leakage occurs at fluences equal to or slightly less than fluences causing inactivation at wavelengths above 305 nm (Klamen and Tuveson, 1982). VII. Regulons Involved in Response to NUV Stress

A regulon has been defined as one or more operons under the control of a common regulatory protein (Neidhardt, 1987; Neidhardt and von Bogelen, 1987). Thus, a single mutation in a regulatory gene (e.g., recA, htp, oxyR, or rel) can alter synthesis of a battery of enzymes upon environmental stress (e.g., blockage of DNA synthesis, heat shock, excess oxidation, or starvation of a required amino acid). For each regulon, there may be a key molecule, known as a n “alarmone,” such as ppGpp, which is synthesized and triggers the reaction. When cells are stressed by NUV, some of the enzymes controlled by each of the above regulatory genes may be involved, but NUV stress may stimulate still another regulon, the characteristics of which are not yet known. There is evidence that response to NUV stress may involve genes that are under the control of a t least four known regulons, and perhaps another yet to be determined regulon. Oxygen-related stimulons have also been described (Aliabadi et al., 1986; Jamison and Adler, 1987). Also, a regulon for iron metabolism ( f u r ) has been described (Nettleton et al., 1984; Niederhoffer et al., 1989)that could be involved in NUV responses, but there is no information as yet. A. SOS REGULON When bacterial DNA is damaged by FUV, or when DNA synthesis is blocked by other means, recA protein cleaves the lexA repressor, which sits at about 20 promotor sites, and a battery of gene products are synthesized (Walker, 1987).

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Recovery from NUV stress involves some of the genes under SOS control, but there is also considerable evidence that there is not a complete overlap between the two. For example, if there were a n overlap, a n inducing dose of FUV (or nalidixic acid) should protect the cell against a challenge dose of NUV, since the SOS proteins would have been induced. Experiments show that there is no protection. When one looks at individual genes that are induced by NUV, there is evidence that SOS may not be operating. For example, plasmid pKM101, whose mucA and mucB genes endow cells with enhanced mutation frequency and enhanced resistance to FUV (Walker, 19841, has no influence on these properties when cells are damaged by NUV. Thus, NUV lesions do not induce SOS repair nor subsequent expression of mucA and mucB genes on plasmid pKM101. Further, when cells are preirradiated with NUV and subsequently irradiated with FUV, there is blockage of SOS repair, including the repair normally controlled by genes on pKMlOl (Eisenstark, 1983; Turner and Eisenstark, 1984). Note that this blockage may not occur in nuu mutants (Caldeira de Araujo and Favre, 1986; see next paragraph). When E. coli cells in which the recA promoter is fused to a lac structural gene Mud[(Ap,lac) ::recA1 are irradiated with selected monochromatic wavelengths (245, 313, 334, and 365 nm), only the 254-nm wavelength induces high yields of P-galactosidase, but there is no induction by any of the NUV wavelengths (Turner and Eisenstark, 1984). Also, A prophage induction and Weigle reactivation are observed with FUV but not with NUV. Further, prior exposure of the cells to the selected monochromatic NUV wavelengths inhibits (1)the induction of P-galactosidase by subsequent 254-nm radiation, (2) subsequent 254-nm induction of A prophage, (3) Weigle reactivation of FUV-damaged phage, and (4) increase in mutation frequency. These observations are consistent with the hypothesis that NUV blocks subsequent recA protease action, although other possibilities are not yet ruled out. Caldeira de Araujo and Favre (1986) presented an alternative explanation for the observed blockage of the SOS response by NUV. Their experiments support the view that the transient cessation of growth and protein synthesis produced by NUV prevents the expression of the inducible SOS response; in mutants (nuu)cells that escape this growth delay effect, NUV triggers the SOS response as assayed by induction of a n sfi ::lac2 fusion strain (sfi is one of the SOS genes). It is obvious that further tests are needed to see if recovery from NUV damage is completely under SOS control. The recA protein has multiple activities, one of which is genetic recombination (Tessman et al., 1986). NUV damage does not block

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recA recombinase activity (Turner and Eisenstark, 1984). To test whether the recombination function is influenced by NUV, host C600 cells were irradiated with a selected wavelength or a combination of wavelengths (254, 365, or 365 nm, then 254 nm) and were then infected with Xbio-11, a phage that requires a functional recA+ recombinase activity to form plaques. Plaque formation was observed after such treatment. Thus, recA recombinase activity is not blocked by FUV or NUV; indeed, recombination is stimulated (Turner and Eisenstark, 1984). Many of these tests for SOS response have been performed for H20z induction (Imlay and Linn, 1987); results differ somewhat from NUV induction. They found that very low (Mode 1) doses of H202 induce synthesis of the recA protein, but mutations in genes in the SOS pathway did not make cells more sensitive. Further, H202 failed to show Weigle recovery of treated phage, nor did it show mutagenesis via urnuCD. Since both are identified with the SOS response, this indicates that HzOZ (like NUV) does not involve all of the steps associated with the SOS response. There is a further matter that is yet to be clarified on a molecular basis. Certain mutants (e.g., recA) are more sensitive than wild type to NUV (Carlsson and Carpenter, 1980; Eisenstark, 1971; Eisenstark et al., 1980). Since inducible SOS does not account for NUV repair in wild-type bacteria, resistance to NUV is assumed to be due to the approximately 1000 molecules of constitutive recA recombinase protein present in each noninduced cell (Tessman et al., 1986). Also, recA cells are highly sensitive to NUV when in log phase, but not when in stationary phase (Peak et al., 1983; Tuveson et al., 1983); it is assumed that SOS induction is necessary for repair of log cells, but constitutive recA recombinase is sufficient for repair of stationary cells with completed chromosomes and no growing forks (Sharma and Smith, 1985; Smith and Sharma, 1987). Various roles of the recA protein can now be tested by use of the Tessman mutants of recA, one class of which has lost protease activity only and another class that has lost recombinase activity only (Tessman et al., 1986; Ennis et al., 1985).

B. OXIDATIVE STRESS REGULON( o x y R ) The oxidative stress regulon is controlled by the oxyR gene; a single mutation in oxyR will result in a shift from induced to constitutive synthesis of a battery of proteins, and a deletion in oxyR will result in absence of these (Christman et al., 1985; Kramer and Ames, 1987; Kramer et al., 1988). The oxyR regulon is a positive regulator. The oxyR regulates at least nine proteins involved in defense against

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oxidizing agents, i.e., HzOz. Some of the genes involved are katG, which codes for HPI; ahp, which codes for alkylhydroperoxidase reductase; and gshA, which codes for glutathione reductase. The E . coli oxyR2 mutant constitutively overexpresses 50-fold HPI and 20-fold alkylhydroperoxidase reductase. The oxyR deletion mutants are hypersensitive to killing by NUV. A number of mutants have been studied that affect sensitivity to oxygen ( Jamison and Adler, 1987), but the relationship of these to oxyR has not been reported. It should be emphasized that the induction of the oxyR regulon by NUV is not identical to induction by H202 (Kramer et al., 1988; Pierceall, 1988). Although there is some overlap, different sets of proteins are induced.

C. STRINGENT REGULON The stringent response is defined as a shutdown in the synthesis of rRNA and ribosomal protein operon expression during starvation for amino acids. The stringent response is mediated by guanosine tetraphosphate, ppGpp. ppGpp is a n alarmone, which is a regulatory molecule that alerts cells to the onset of oxidative stress and perhaps other stresses. Stringency is a regulatory response to unloaded tRNAs acting as the triggering device. This results in a sudden and complete shutoff of stable RNA synthesis followed by the cessation of protein synthesis. The cessation of protein synthesis leads to growth delay. The triggering device is a n 8-13 adduct in tRNAs, as discussed above. The relationship of the stringent response to NUV effects has been described (Hajnsdorf and Favre, 1986). D. HEAT-SHOCK REGULON Heat shock is regulated by the HtpR protein, which is a positive sigma-32 factor. Escherichia coli induces 17 proteins under the heatshock response. The induction occurs transcriptionally and requires RNA synthesis. The inducer could again be a small nucleotide, i.e., AppppA. Overlap between proteins induced by heat shock and by NUV has been noted (J. Hoerter and A. Eisenstark, unpublished; Pierceall, 1988). VIII. Mutation by NUV

Compared to FUV, NUV is relatively nonmutagenic on a per-lethalhit basis (Turner and Webb, 1981). Either NUV produces fewer mutagenic-type DNA lesions relative to FUV or a large portion of

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NUV lethality is due to non-DNA damage. Indeed, the frequency and the specificity of mutation (Eisenstadt, 1987; Levin and Ames, 1986) by NUV is an area that is poorly understood and in need of attention, especially considering the abundant exposure of organisms to these wavelengths of radiation. There are a number of reports of NUV mutagenesis, even under anoxic conditions (Cabrera-Juarez, 1964, 1981; Peak et al., 1983; Webb, 1977), although the frequency is much less than under aerobic conditions. Mutations by NUV must occur by a route different from FUV mutation, since only FUV mutation involves the umuDC gene products or that of their analog mucAB on the pKMlOl plasmid. It also requires recA protein in its activated form. When cells with mucAB genes are induced by an agent that turns on SOS repair, mutation frequency rises substantially. However, DNA damage by NUV fails to give this increased frequency of mutations in strains containing the mucAB plasmid (Eisenstark, 1983). Since NUV mutations may be via oxidative damage of DNA, then the SOS repair may not be involved (Farr et al., 1986).Note the lack of involvement of umu genes in H202 mutagenesis (Imlay and Linn, 19871, similar to the case with NUV. Kubitschek et al. (1986) presented a precise action spectrum for genetic mutations in E. coli, which was corrected for finite slit width when irradiating with a monochrometer attached to a light source. Their data verify that there is a plateau at -334 nm for mutations as well as for lethality, further supporting a unique aspect of mutagenesis by NUV. Assuming that 0 2 - is a product of NUV, NUV would be expected to have mutagenic effects similar to those observed following oxidative damage to DNA (Greenberg and Demple, 1988; Storz et al., 1987). 0 2 was found t o be mutagenic by Farr et al. (19861, but only in sodAB strains with ex0111 activity. The triple mutants, sodAB &A, lacked ex0111 activity and did not yield mutations. Although the evidence is only indirect, 0 2 - as a mutagen produced by NUV must be seriously considered (Farr et al., 1986; Greenberg and Demple, 1988; Fridovich, 1986; Moody and Hassan, 1982). It would be interesting to compare mutations in NUV-irradiated cells with and without a plasmid that overproduces exoIII. It should also be noted that endonuclease IV is induced by methyl viologen, a generator of 02-,and that this enzyme may have a role in mutation processing (Chan and Weiss, 1987; Cunningham et al., 1986). Some SOS mutation repair following irradiation at 365 nm has been observed in experiments with DNA-repairless mutants (Turner and Webb, 1981). However, distinct differences among the 254- and 365nm effects were seen and can be accounted for by proposing that there

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is selective damage a t 365 nm that inhibits error-free and error-prone repair systems. The fact that recA mutants are nonmutable at 365-nm radiation makes the SOS role confusing (Turner and Webb, 1981). Since the pKMlOl plasmid does not protect cells from NUV, and since it does not increase mutation frequency (Eisenstark, 1983) following NUV, one would assume that SOS is not involved. However, since the absence of recA proteins knocks out NUV mutagenesis, this indicates that the recA protein has some role (Turner and Webb, 1981). It may be that recA recombinase but not recA protease or another gene product of SOS induction is still necessary for NUV mutations. Also, preirradiation with NUV (sublethal dose) blocks both the SOS response and subsequent tryptophan reversions by FUV (Turner and Eisenstark, 1984). It should also be noted that NUV can be antimutagenic (see Section I1,E) by breaking C-C, 6-4 dimers. Additional examples of NUV mutagenic and antimutagenic effects remain a puzzle. For example, Leonard0 et aZ. (1984) showed that some enrichment in plating media is necessary for mutants to show up after NUV irradiation. Also, the number of mutations by NUV is higher in uvrA strains (Turner, 19841, as is the case for FUV and spontaneous mutations (Sharma and Smith, 1985). IX. Summary

A model of the possible pathways of activities following NUV treatment was presented in Section I and in Fig. 1. Some of the components are firmly established, some are speculative, and many are difficult to evaluate because of insufficient experimental information. Perhaps the most relevant experiments, especially concerning ozone depletion, would be t o determine the mutational specificity of NUV. By selecting ZacI mutants after exposing cells to NUV, and sequencing the bases of this gene, this is now feasible. There are some problems, however. The mutation frequency is normally so low that it might be difficult to distinguish NUV mutants from spontaneous mutants. However, by irradiating cells having a uvrA or uvrB mutation, the frequency of mutation above background can be increased considerably. There remains the problem as t o what fraction of the observed mutations results from oxidative damage. Some of this could be clarified by comparing mutation spectra of cells treated with NUV and cells subjected to excess oxidative damage and determining what fraction results from other avenues of lesion formation in DNA.

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Different species of reactive oxygen could cause different kinds of DNA lesions, and, fortunately, use of appropriate mutants should allow us to sort out any differences in specificity of lesions. Also, by appropriate manipulation of quantities of endogenous photosensitizers, it might be possible to sort out the specific mutations that are caused by photodynamic action. Another avenue of research is t o explore the pathways by which NUV lesions are repaired, and whether such repair is error prone or error free. Again, the use of mutants such as xthA, uur, and polA should assist in our understanding of the specificity of the mutational events. There are now a number of examples of global control mechanisms whereby cells abruptly shift their protein synthesis pattern under environmental stress. It is important to understand whether NUV stress results in induction of one or more of the known regulatory genes, or whether another regulon might be involved. One particular aspect of regulation that remains unsolved is the role of the katF gene, which is known to regulate the xthA and katE, but it may also regulate other genes as well. A number of striking physiological events occur even a t very low fluences of NUV irradiation of cells. In part, this may be related to regulon induction. However, some of these events are in need of special exploration, such as changes a t the membrane level. Also, there is a need to understand the requirement for balance among some of the enzymes involved in response to NUV stress. As was the case for sorting out various avenues of mutation by FUV, it would be important to identify which physiological events are due to each of the various reactive oxygen molecules, and which events are the result of direct photoreaction. Most intriguing is the ability of very low fluences of NUV to produce abrupt membrane changes. The results of many of these explorations may assist in understanding many metabolic processes (both normal and diseased), and perhaps in understanding certain development problems. There is obviously a triggering of certain events by modulation of reactive oxygen species that may result from NUV irradiation.

ACKNOWLEDGMENTS Research by the author was supported in part by the National Science Foundation (DMS-85027-08), by the University of Missouri Institutional Biomedical Research Support Grant RFR 07053 (National Institutes of Health), and by the National Institute of Environmental Health (DHHS ES04889).Assistance by Susan Bradford and Barb Owen, who typed several revisions of the manuscript, is gratefully acknowledged.

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REFERENCES Adelman, R., Saul, R. L., and Ames, B. N. (1988). Oxidative damage to DNA: Relation to species metabolic rate and life span. Proc. Natl. Acad. Sci. U.S.A. 85, 2706-2708. Ahmad, S. I. (1981). Synergistic killing of coliphage T7 by near-ultraviolet radiation plus hydrogen peroxide: Possible role of superoxide radicals. Photobiochem. Photobiophys. 2, 173-180. Aliabadi, Z., Warren, F., Mya, S., and Foster, J. W. (1986). Oxygen-related stimulons of Salmonella typhimurium identified by Mud (Ap lac) operon fusions. J . Bacteriol. 165, 780-786. Alonso-Moraga, A. A., Bocanegua, J . M., Torres, J., Lopez-Barea, J., and Peuyo, C. (1987). Glutathione status and sensitivity to GSH-reacting compounds of Escherichia coli strains deficient in glutathione metabolism and/or catalase activity. Mol. Cell. Biochem. 73, 61-68. Ames, B. N. (1983). Dietary carcinogens and anticarcinogens. Science 221, 1256-1264. Ananthaswamy, H. N., and Eisenstark, A. (1976). Near UV-induced breaks in phage DNA: Sensitization by hydrogen peroxide (a tryptophan photoproduct). Photochem. Photobiol. 24, 439-442. Ananthaswamy, H. N., and Eisenstark, A. (1977). Repair of H202-indueed single strand breaks in E . coli DNA. J . Bacteriol. 130, 187-191. Ananthaswamy, H. N., Hartman, P. S., and Eisenstark, A. (1979). Synergistic lethality of phage T7 by near-UV radiation and hydrogen peroxide: An action spectrum. Photochern. Photobiol. 29, 53-56. Anderson, R. (1983). Alkylamines: Novel lipid constituents in Deinococcus radiodurans. Biochim. Biophys. Acta 743, 266-268. Anderson, R., and Hansen, K. (1985). Structure of a novel phosphoglycolipid from Deinococcus radiodurans. J . Biol. Chem. 260, 12219-12223. Anderson, R., Daya, M., and Reeve, J. (1987). An evaluation of the contribution of membrane lipids to protection against ultraviolet radiation. Biochim. Biophys. Acta 905,227-230. Bachmann, B. (1987). Linkage map of Escherzchia coli K12. I n “Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology” (F. C. Neidhardt et al., eds.), 7th Ed. Am. SOC.Microbiol., Washington, D.C. Barbado, C. M., Ramirez, M. A., Blanco, J., Lopez-Barea, J., and Pueyo, C. (1983). Mutants of Escherichia coli sensitive to hydrogen peroxide. Curr. Microbiol. 8, 251-253. Billen, D. (1984). The role of hydroxyl radical scavengers in preventing DNA strand breaks induced by X irradiation of toluene-treated Escherichia coli.Radiat. Res. 97, 626-629. Bissett, D. L., Hannon, D. P., and Orr, T. V. (1987).An animal model of solar-aged skin: Histological physical, and visible changes in UV-irradiated hairless mouse skin. Photochem. Photobiol. 46, 367-378. Blanchet, T., Hajnsdorf, E., and Favre, A. (1984). Metabolism of tRNA in nearultraviolet-illuminated Escherichia coli. The tRNA repair hypothesis. Eur. J . Biochem. 139,547-552. Bloch, C. A,, and Ausubel, F. M. (1986). Paraquat-mediated selection for mutations in the manganese-superoxide dismutase gene sodA. J . Bacteriol. 168, 795-798. Blum, H. F. (1941). “Photodynamic Action and Diseases Caused by Light.” Van Nostrad-Reinhold, Princeton, New Jersey (reprinted in 1964 with an updated appendix by Harper, New York). Bochner, B. A,, Zylicz, M., and Georgopoulos, C. (1986). Escherzchia coli DnaK protein

BACTERIAL GENES INVOLVED IN RESPONSE TO

NUV

137

possesses a 5’-nucleotidase activity that is inhibited by AppppA. J . Bacteriol. 168, 931-935. Brawn, M. K., and Fridovich, I. (1985). Increased superoxide radical production evokes inducible DNA repair in Escherichia coli. J . Biol. Chem. 260, 922-925. Breimer, L. H., and Lindahl, T. (1985). Enzymatic excision of DNA bases damaged by exposure to ionizing radiation or oxidizing agents. Mutat. Res. 150, 85-89. Brown, 0. R., and Seither, R. L. (1983). Oxygen and redox-active drugs: Shared toxicity sites. Fundam. Appl. Toxicol. 3, 209-215. Cabrera-Juarez, E. (1964). “Black light” inactivation of transforming DNA from Haemophilus influenzae. J . Bacteriol. 87, 771-778. Cabrera-Juarez, E. (1981). Near-ultraviolet mutation of transforming DNA irradiated in uiuo. Mutat. Res. 83, 301-306. Cabrera-Juarez, E., and Setlow, J . K. (1976). Mutation of Haemophilus influenzae transforming DNA in uitro with near-ultraviolet irradiation: Action spectrums. Mutat. Res. 35, 199-206. Cadet, J., Berger, M., Decarroz, C., Wagner, J . R., Van Lier, J . E., Ginot, Y. M., and Vigny P. (1986). Photosensitized reactions of nucleic acids. Biochimie 68, 813-834. Caimi, P., and Eisenstark, A. (1986). Sensitivity of Deinococcus radiodurans to near-ultraviolet radiation. Mutat. Res. 162, 145-151. Caldeira de Araujo, A., and Favre, A. (1986). Near ultraviolet DNA damage induces the SOS response in Escherichia coli. EMBO J . 5, 175-179. Calkins, J., ed. (1982). “The Role of Solar Ultraviolet Radiation on Marine Ecosystems,” p. 724. Plenum, New York. Carlioz, A., and Touati, D. (1986). Isolation of superoxide dismutase mutants in E . coli:Is superoxide dismutase necessary for aerobic life? EMBO J . 5,623-630. Carlioz, A,, Ludwig, M. L., Stallings, W. C., Fee, J. A., Steinman, H. M., and Touati, D. (1988). Iron superoxide dismutase: Nucleotide sequence of the gene from Escherichia coli K12 and correlations with crystal structures. J . Biol. Chem. 3, 1555-1562. Carlsson, J., and Carpenter, V. S. (1980). The recA’ gene product is more important than catalase and superoxide dismutase in protecting Escherichia coli against hydrogen peroxide toxicity. J . Bacteriol. 142, 319-321. Casas-Fimet,J. R., Toulme, J . J., Cazenave, C., and Santus, R. (1984). Role of tryptophan and cysteine in binding of gene 32 protein from phage T4 to single-strand DNA. Modification of crucial residues by oxidation with selective free-radical anions. Biochemistry 23, 1208-1213. Cashel, M., and Rudd, K. E. (1987). The stringent response. Zn “Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (F. C. Neidhardt et al., eds.), 7 Ed., pp. 1410-1438. Am. SOC.Microbiol., Washington, D.C. Chamberlin, J., and Moss, S. H. (1986). Lipid peroxidation and other membrane damage produced in E . coli K1060 by near-UV radiation and deuterium oxide. Photochem. Photobiol. 45, 625-630. Chan, E., and Weiss, B. (1987). Endonuclease IV o f E . coli is induced by paraquat. Proc. Natl. Acad. Sci. U.S.A. 84, 3189-3194. Cheng, L., Kellog, E. W., and Packer, L. (1981). Photoinactivation of catalase. Photochem. Photobiol. 34, 125-129. Christman, M. F., Morgan, R. W., Jacobson, F. S., and Ames, B. N. (1985). Positive control of a regulon for defenses against oxidative stress and some heat-shock proteins in Salmonella typhimurium. Cell 41, 753-762. Clare, D. A., Duong, M. N., Darr, D., Archibald, F., and Fridovich, I. (1984). Effects of molecular ozxygen on the detection of superoxide radical with nitroblue tetrazolium and an activity strain for catalase. Anal. Biochem. 140, 532-637.

138

A . EISENSTARK

Coulondre, C., and Miller, J . H. (1977). Genetic studies of the lac repressor. IV. Mutagenic specificity in the l a d gene ofEscherichia coli. J . Mol. Biol. 117,577-606. Cunningham, R. P., Saporito, S. M., Spitzer, S. G., and Weiss, B. (1986).Endonuclease IV (nfo)mutant of Escherichia coli. J . Bacteriol. 168, 1120-1127. Dahl, T. A., Midden, W. R., and Hartman, P. E. (1987). Pure singlet oxygen cytotoxicity for bacteria. Photochem. Photobiol. 46, 345-352. Dahl, T. A., Midden, R. W., and Hartman, P. E. (1988). Some prevalent biomolecules as defenses against singlet oxygen damage. Photochem. Photobiol. 47, 357-362. Dahl, T. A,, Midden, R. W., and Hartman, P. E. (1989). Comparison of killing of gram-negative and gram-positive bacteria by pure singlet oxygen. J . Bacteriol. 17, 2188-2194. Davies, K. J., Delsignore, M. E., and Lin, S. W. (1987). Protein damage by oxygen radicals: 11. Modification of amino acids. J . Biol. Chem. 262, 9902-9907. Decuyper-Debergh, D., Piette, J., and Van de Vorst, A. (1987). Singlet oxygen-induced mutations in M13 lac2 phage DNA. EMBO J . 6, 3161. Demple, B., and Halbrook, J . (1983). Inducible repair of oxidative DNA damage in E. coli. Nature (London)304, 466-468. Demple, B., Halbrook, J., and Linn, S. (1983). E . coli rth mutants are supersensitive to hydrogen peroxide. J . Bacteriol. 153, 11079-11082. Demple, B., Daikh, Y., Greenberg, J., and Johnson, A. (1985). Alkylation and oxidative damages to DNA: Constitutive and inducible repair systems. I n “Antimutagenesis and Anticarcinogenesis: Mechanisms” (D. M. Schenkel, P. E. Hartman, T. Kada, and A. Hollaender, eds.), pp. 205-217. Plenum, New York. Demple, B., Johnson, A., and Fung, D. (1986). Exonuclease I11 and endonuclease IV remove 3’ blocks from DNA synthesis primers in HzOz-damagedEscherichia coli. Proc. Natl. Acad. Sci. U S A . 83, 7731-7735. Dowds, B. C. A,, Murphy, P., McConnell, D. J., and Devine, K. M. (1987). Relationship among oxidative stress, growth cycle, and sporulation in Bacillus subtilis. J . Bacteriol. 169, 5771-5775. Droffner, M. L., and Yamamoto, N. (1983). Anaerobic cultures of Salmonella typhimurium do not exhibit inducible proteolytic function of recA gene and recBC function. J . Bacteriol. 156, 962-965. Droffner, M. L., and Yamamoto, N. (1985). Isolation of thermophilic mutants of B. subtilis and B . pumilus and transformation of the thermophilic trait to meosphilic strains. J . Gen. Microbiol. 131, 2791-2794. Eisenstadt, A. (1987). Analysis of mutagenesis. I n “Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology” (F. C. Neidhardt et al., eds.), 7th Ed., pp. 1016-1033. Am SOC.Microbiol., Washington, D.C. Eisenstark, A. (1971). Mutagenic and lethal effects of visible and near-ultraviolet light on bacterial cells. Adu. Genet. 16, 167-198. Eisenstark, A. (1982). Sensitivity to UV-B irradiation as related to bacterial life cycles. In “The Role of Solar Ultraviolet Radiation in Marine Ecosystems” ( J . Calkins, ed.), pp. 389-391. Plenum, New York. Eisenstark, A. (1983). Genetic damage in Salmonella typhimurium by near-ultraviolet radiation: Lack of repair by plasmid pKMlO1. Mutat. Res. 122, 267-272. Eisenstark, A. (1985). Recovery from near-ultraviolet radiation damage in bacteria. I n “Molecular Biology of Bacterial Growth” (M. Schaechter, F. C. Neidhart, J . L. Ingraham, and N. 0. Kjeldgaard, eds.), pp. 243-255. Jones & Bartlett, Boston, Massachusetts. Eisenstark, A. (1987). Mutagenic and lethal effects of near-ultraviolet radiation (290400 nm) on bacteria and phage. Enuiron. Mol. Mutagen. 10, 317-337.

BACTERIAL GENES INVOLVED IN RESPONSE TO NUV

139

Eisenstark, A., and Perrot, G. 119871. Catalase has only minor role in protection against near-ultraviolet radiation damage in bacteria. Mol. Gen. Genet. 207, 68-72. Eisenstark, A., Buzard, R. L., and Hartman, P. S. (1980). Inactivation of phage by near-ultraviolet radiation and hydrogen peroxide. Photochem. Photobiol. 44,603606. Eisenstark, A., Kovacs, S., and Terry, J. (1982). Cell damage by near-ultraviolet radiation. Role of DNA-protein cross-links. Natl. Cancer Inst. 60, 177-181. Ennis, D. G., Fisher, B., Edmiston, S., and Mount, D. W. (1985).Dual role forEscherichia coli RecA protein in SOS mutagenesis. Genetics 82, 3325-3329. Ewing, D. (1983).Radiation sensitization ofE. coli B/r by nitrous oxide. Radiat. Res. 96, 275-283. Farr, S. B., DAri, R., and Touati, D. (1986). Oxygen-dependent mutagenesis in Escherichia coli lacking superoxide dismutase. Proc. Natl. Acad. Sci. U.S.A. 83, 8268-8272. Farr, S. B., Touati, D., and Kogoma, T. (1988). Effects of oxygen stress on membrane functions in Escherichia coli: Role of HPI catalase. J . Bacteriol. 170, 1827-1842. Favre, A., and Hajnsdorf, E. (1983). Photoregulation of E. coli growth and the near ultraviolet photochemistry of tRNA. I n “Molecular Models of Photoresponsiveness” (G. Montagnoli and B. F. Erlanger, eds.), pp. 75-94. Plenum, New York. Favre, A,, Hajnsdorf, E., Thiam, K., and Caldeira de Araujo, A. (1985). Mutagenesis and growth delay induced in Escherchia coli by near-ultraviolet radiation. Biochimie 67, 335-342. Fee, J. A., Niederhoffer, E. C., and Naranjo, C. (1989). First description of a variant ofE. coli lacking superoxide dismutase activity yet able to grow efficiently on minimal, oxygenated medium. I n “Trace Element Metabolism in Man and Animals” (L. Hurley, C. Keen, B. Lonnerdal, and R. Rucker, eds.), pp. 239-243. Plenum, New York. Ferron, W. L., Eisenstark, A,, and MacKay, D. (1972). Distinction between far- and near-ultraviolet light killing of recombinationless (recA)Salmonella typhimuriurn. Biochim. Biophys. Acta 277, 651-658. Flint, D. H., and Emptage, M. H. (1988). Dihydroxy acid dehydratase from spinach contains a [ 2 Fe-2Sl cluster. J . Biol. Chem. 263, 3558-3564. Fontecave, M., Grusland, A., and Reichard, P. (1987). The function of superoxide dismutase during the enzymatic formation of the free radical of ribonucleotide reductase. J . Biol. Chem. 262, 12332-12336. Freierabend, J., and Engel, S. (1986).Photoinactivation of catalase in vitro and in leaves. Arch. Biochem. Biophys. 251, 567-576. Fridovich, I. (1986).Superoxide dismutases. Adu. Enzymol. 58, 61-85. Gameson, A. L. H., and Gould, D. J. (1975). Effects of solar radiation on the mortality of some terrestrial bacteria in sea water. I n “Discharge of Sewage from Sea Outfalls” (A. L. H. Gameson, ed.), pp. 209-219. Pergamon, New York. Gennis, R. B. (1987). The cytochromes of Escherichia coli. Microbiol. Reu. 46, 387399. Glickman, B. W., Schaaper, R. M., Haseltine, W. A., Dunn, R. L., and Brash, D.E. (1986). The C-C (6-4) UV photoproduct is mutagenic in Escherchia coli. Proc. Natl. Acad. SCZ. U.S.A. 83, 6945-6949. Greenberg, J. T., and Demple, B. (1986).Glutathione inEscherchia coli is dispensable for resistance to HzOzand gamma radiation. J . Bacteriol. 168, 1026-1029. Greenberg, J. T., and Demple, B. (1988). Overproduction of peroxide-scavenging enzymes in Escherichia coli suppresses spontaneous mutagenesis and sensitivity to redox-cycling agents in oxyR- mutants. EMBO J . 7, 2611-2617.

140

A. EISENSTARK

Gregory, E. M., and Fridovich, I. (1973).Induction of superoxide dismutase by molecular oxygen. J . Bacteriol. 114, 543-548. Hagensee, M. E., and Moses, R. E. (1986). Repair response of Escherichia coli to hydrogen peroxide. J . Bacteriol. 168, 1059-1065. Hajnsdorf, E., and Favre, A. (1986). Metabolism of tRNAs in growing cells ofEscherichia coli illuminated with near-ultraviolet light. Photochem. Photobiol. 43, 157-164. Halliwell, B. (1987). Oxidants and human disease: Some new concepts. FASEB J . I, 358-364. Hartman, P. S. (1986).In situ hydrogen peroxide production may account for a portion of NUV (300-400 nm) inactivation of stationary phase. E . coli. Photochem. Photobiol. 43, 87-89. Hartman, P. S., and Eisenstark, A. (1978). Synergistic killing of E. coli by near-UV radiation and hydrogen peroxide: Distinction between recA-repairable and recAnonrepairable damage. J . Bacteriol. 131, 769-779. Hartman, P. S., and Eisenstark, A. (1980). Killing of Escherichia coli K12 by nearultraviolet radiation in the presence of hydrogen peroxide: Role of double-strand DNA breaks in the absence of recombinational repair. Mutat. Res. 72, 31-42. Hartman, P. S., and Eisenstark, A. (1982). Alteration of bacteriophage attachment capacity by near-UV irradiation. J . Virol. 43, 529-532. Hartman, P. S., and Eisenstark, A. (1987). Synergistic killing of E . coli by near-UV radiation and hydrogen peroxide. Distinction between recA-repairable and recAnonrepairable damage. J . Bacteriol. 131, 769-779. Hartman, P. S., Eisenstark, A., and Pauw, P. G. (1979). Near-ultraviolet radiation plus Hz02 inactivation on phage T7: DNA-protein crosslinks prevent DNA injection. Proc. Natl. Acad. Sci. U.S.A. 76, 3228-3232. Hassan, H. M. (1984). Exacerbation of superoxide radical formation by paraquat. In “Methods in Enzymology” (L. Palker, ed.), Vol. 105, pp. 523-532. Academic Press, New York. Hassett, D. J., Britigan, B. E., Svendsen, T., Rosen, G. E., and Cohen, M. S. (1987). Bacteria form intracellular free radicals in response to paraquat and streptonigrin. J . Biol. Chem. 262,13404-13408. Heimberger, A., and Eisenstark, A. (1988). Compartmentalization of catalases in Escherichia coli. Biochem. Biophys. Res. Commun. 15, 392-397. Helene, C., Charlier, M., Montenay-Garestier, T., and Laustriata, G., eds. (1982). “Trends in Photobiology,” p. 820. Plenum, New York. Hollaender, A. (1943). Effect of long ultraviolet and short visible radiation (3500-4900 & on E . coli. J . Bacteriol. 46, 531-541. Horowitz, P. M., and Bowman, S. (1987). Oxidation increases the proteolytic susceptibility of a localized region in rhodanase. J . Biol. Chem. 262, 14544-14548. Houba-Herin, N., Calberg-Bacq, C. M., Piette, J., and VandeVorst, A. (1982). Mechanisms for dye-mediated photodynamic action: Singlet oxygen production, deoxyguanosine oxidation, and phage inactivation efficiencies. Photochem. Photobiol. 36, 297-308. Husain, I., and Sancar, A. (1987). Photoreactivation in phr mutants of Escherichia coli. J . Bacteriol. 169, 2367-2372. Imlay, J. A., and Linn, S. (1986). Bimodal pattern of killing of DNA-repair-defective or anoxically grown Escherichia coli by hydrogen peroxide. J . Bacteriol. 166,519-527. Imlay, J. A., and Linn, S. (1987). Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide. J . Bacteriol. 169, 2967-2976. Imlay, J. A., and Linn, S. (1988). DNA damage and oxygen radical toxicity. Science 240, 1302-1309.

BACTERIAL GENES INVOLVED IN RESPONSE TO

NUV

141

Imlay, J. A., Chin, S. M., and Linn, S. (1988).Toxic DNA damage by hydrogen peroxide through the Fenton reaction i n vivo and in uitro. Science 240, 640-642. Ingledew, W. J., and Poole, R. K. (1984). The respiratory chain of Escherichia coli. Microbiol Rev. 48, 222-271. Ito, A., Robb, F. T., Plak, J. G., and Peak, M. J . (1988). Base-specific damage induced by 4-thiouridine photosensitization with 334-nm radiation in M13 phage. Photochem. Photobiol. 47, 231-240. Jagger, J. (1985). “Solar-UV Actions on Living Cells.” Praeger, New York. Jamison, C. S., and Adler, H. I. (1987). Mutations in Escherichia coli that effect sensitivity to oxygen. J . Bacteriol. 169, 5087-5094. Kamoun, P. P., Schneider, E., and Dy, M. (1988). Superoxide induced diimination of arginine in hematopoietic cells. FEBS Lett. 226, 285-286. Katcher, H. L., and Wallace, S. S. (1983). Characterization of the Escherchia coli X-ray endonuclease, endonuclease 11. Biochemistry 22, 4071-4081. Kelland, L. R., Moss, S. H., and Davies, D. J . G. (1984). Leakage of “RB+ after ultraviolet irradiation of E . coli K12. Photochem. Photobiol. 39, 329-335. Kirkman, H. N., and Gaetani, G. F. (1984). Catalase: A tetrameric enzyme with four tightly bound molecules of NADPH. Proc. Natl. Acad. Sci. U.S.A. 81, 4343-4347. Kitzler, J., and Fridovich, I. (1986).Effects of the lethality of paraquat. J . Bacteriol. 167, 346-349. Klamen, D. L., and Tuveson, R. W. (1982). The effect of membrane fatty acids composition on the near-UV (300-400 nm) sensitivity of Escherichia coli K1060. Photochem. Photobiol. 35, 167-173. Kogoma, T., Farr, S. B., Joyce, K. M., and Natvig, D. 0. (1988).Isolation of gene fusions ( s o i : :lacZ) inducible by oxidative stress in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 85,4799-4803. Kow, W. Y., and Wallace, S. S. (1985). Exonuclease I11 recognizes urea residues in oxidized DNA. Proc. Natl. Acad. Sci. U.S.A. 82, 8354-8358. Krabbenhoft, K. L., Anderson, A. W., and Elliker, P. R. (1967). Influence of culture media on the radiation resistance of Micrococcus radiodurans. Appl. Microbiol. 15, 178- 185. Kramer, G. F., and Ames, B. N. (1987). Oxidative mechanisms of toxicity of lowintensity near-UV light. J . Bacteriol. 169, 2259-2266. Kramer, G. F., Baker, J. C., and Ames, B. N. (1988). Near-UV Stress in Salmonella typhinurium: 4-Thiouridine in tRNA, ppGpp, and ApppGpp as components of an adaptive response. J . Bacteriol. 170, 2344-2351. Kubitschek, H. E., Baker, K. S., and Peak, M. J. (1986). Enhancement of mutagenesis and human skin cancer rates resulting from increased fluences of solar ultraviolet radiation. Photochem. Photobiol. 43, 443-447. Kuo, C. F., Mashino, T., and Fridovich, I. (1987). a,P-Dihydroxy-isovaleratedehydratase: A superoxide-sensitive enzyme. J . Biol. Chem. 262, 4724-4727. Lancy, P., and Murray, R. G. E. (1978). The envelope of Micrococcus radiodurans: Isolation, purification, and preliminary analysis of the wall layers. Can. J . Microbiol. 24, 162-176. Lang, H., Riesenberg, D., Zimmer, C. H., and Bergter, F. (1986). Fluence-rate dependence of monophotonic reactions of nucleic acids in vitro and in uiuo. Photochem. Photobiol. 44, 565-570. Laspia, M. F., and Wallace, S. S. (1988). Excision repair of thymine glycols, urea residues, and apurinic sites in E . coli. J . Bacteriol. 170, 3359-3366. Lee, P. C., Bochner, B. R., and Ames, B. N. (1983). AppppA, heat shock stress in cell oxidation. Proc. Natl. Acad. Sci.U.S.A. 80, 7496-7500.

142

A . EISENSTARK

Leonardo, J. M., Reynolds, P. R., and Tuveson, R. W. (1984). Mutation induction by 365-nm radiation and far-ultraviolet light in E . coli strains differing in near- and far-ultraviolet light sensitivity. Mutat. Res. 126, 1-8. Levin, D. E., and Ames, B. N. (1986). Classifying mutagens as to their specificity in causing the six possible transitions and transversions: A simple analysis using the Salmonella mutagenicity assay. Enuiron. Mutagen. 8, 9-28. Lewis, N. F., Alur, M. D., and Kumta, U. S. (1973). Role of carotenoid pigments in radio-resistant micrococci. Can. J . Microbiol. 20, 455-459. Lin, W. S., Wong, F., and Anderson, R. (1987). Role of superoxide in radiation-killing of Escherichia coli and in thymine release from thymidine. Biochem. Biophys. Res. Commun. 147, 778-786. Linn, S., and Imlay, J. A. (1987). Toxicity, mutagenesis, and stress responses induced in Escherichia coli by hydrogen peroxide. J . Cell Sci. (Suppl.)6, 289-301. Loewen, P. E. (1984). Isolation of catalase-deficient Escherichia coli mutants and genetic mapping of katE, a locus that affects catalase activity. J . Bacterial. 157, 622-626. Loewen, P. C., and Triggs, B. L. (1984). Genetic mapping of katF, a locus that with katE affects the synthesis of a second catalase species in Escherichia coli. J . Bacterial. 160, 668-675. Loewen, P. C., Triggs, B. L., Klassen, G. R., and Weiner, J. H. (1983). Identification and physical characterization of Col E l hybrid plasmid containing a catalase gene of Escherichia coli. Can J . Biochem. Cell Biol. 61, 1315-1321. Loewen, P. C., Switala, J., and Triggs-Raine, L. (1985a). Catalases HPI and HPII in Escherichia coli are induced independently. Arch. Biochem. Biophys. 243,144-149. Loewen, P. C., Triggs, B. L., George, C. S., and Hrabarchuk, B. E. (1985b). Genetic mapping of katG, a locus that affects synthesis of the bifunctional catalaseperoxidase hydroperoxidase I in Escherichia coli. J . Bacteriol. 162, 661-667. McCormick, J. P., Fisher, J. R., Pachlatko, J. P., and Eisenstark, A. (1976). Characterization of a cell-lethal tryptophan photoxidation product: Hydrogen peroxide. Science 191,468-469. McCormick, J. P., Klita, S., Terry, J., Schrodt, M., and Eisenstark, A. (1982). Formation by hydrogen peroxide or 254 nm radiation of a near-UV chromophore from peptide-bound cysteine. Photochem. Photobiol. 36, 367-369. Mackay, D., Eisenstark, A., Webb, R. B., and Brown, M. S. (1976). Action spectra for lethality in recombinationless strains of S. typhimurium and E . coli. Photochern. Photobiol. 24, 337-343. Marinus, M. G. (1987). Methylation of DNA. I n “Escherichia coli and Salmonella typhirnurium: Cellular and Molecular Biology” (F. C. Niedhardt et al., eds.), 7th Ed., pp. 697-702. Am. SOC.Microbiol., Washington, D.C. Martin, M. E., Strachan, R. C., Aranha, H., Evans, S. L., Salin, M. L., Welch, B., Arceneaux, J. E. L., and Byers, B. R. (1984). Oxygen toxicity in Streptococcus: Manganese, iron and superoxide dismutase. J . Bacteriol. 159, 745-749. Marx, J. L. (1987). Oxygen free radicals linked to many diseases. Science 235,529-530. Meir, E., and Yagil, E. (1984). Catalase-negative mutants of Escherichia coli. Curr. Microbiol. 11, 13-18. Miguel, A. G., and Tyrrell, R. M. (1986). Repair of near-ultraviolet (365nm)-induced strand breaks in E . coli DNA: The role of the recA andpolA gene products. Biophys. J . 49,485-491. Miller, J. H. (1985). Mutagenic specificity of ultraviolet light. J . Mol. Biol. 131,223-248. Mitchell, D. L., and Clarkson, J. M. (1984). Induction of photoproducts in synthetic polynucleotides by far and near ultraviolet radiation. Photochem. Photobiol. 40, 735-741.

BACTERIAL GENES INVOLVED IN RESPONSE TO

NUV

143

Moody, C. S., and Hassan, H. M. (1982). Mutagenicity of oxygen free radicals. Proc. Natl. Acad. Sci. U.S.A.79, 2855-2859. Morgan, R. W., Christman, M. F., Jacobson, F. S., Storz, G., and Ames, B. N. (1986). Hydrogen peroxide-inducible proteins in Salmonella typhimurium overlap with heat shock and other stress proteins. Proc. Natl. Acad. Sci. U.S.A. 83,8059-8063. Moseley, B. E. B. (1963). The variation in X-ray resistance of Micrococcus radioduruns and some of its less-pigmented mutants. Int. J . Radiat. Biol. 6, 489. Moseley, B. E. B., Mattingly, A., and Copland, H. J. R. (1972). Sensitization to radiation by loss of recombination ability in a temperature-sensitive DNA mutant of Micrococcus radiodurans held a t its restrictive temperature. J . Gen. Microbiol. 72, 329-338. Murphy, P., Dowds, B. C. A., McConnell, D. J., and Devine, K. M. (1987). Oxidative stress and growth temperature in Bacillus subtilis. J . Bacteriol. 169, 5766-5770. Natvig, D., Imlay, K., Touati, D., and Hallewell, R. A. (1987). Human copperhinc superoxide dismutase complements superoxide dismutase-deficient E . coli mutants. J . Biol. Chem. 262, 14697-14701. Neidhardt, F. C. (1987). Multigene systems and regulons. In “Escherichia coli and Salmonella typhirnurium: Cellular and Molecular Biology” (F. C. Neidhardt et al., eds), 7th Ed., pp. 1313-1317. Am. SOC. Microbiol., Washington, D.C. Neidhart, F. C., and van Bogelen, R. A. (1987). Heat shock response. In “Escherichia coli and Salmonella hyphimuriurn: Cellular and Molecular Biology” (F. C. Neidhardt et Microbiol., Washington, D.C. al., eds.), 7th Ed., pp. 1334-1345. Am. SOC. Nettleton, C. J., Bull, C., Baldwin, T. O., and Fee, J . A. (1984). Isolation of the Escherichia coli iron superoxide dismutase gene: Evidence that intracellular superoxide concentration does not regulate oxygen-dependent synthesis of the manganese superoxide dismutase. Proc. Natl. Acad. Sci. U.S.A. 81, 4970-4973. Niederhoffer, E. C., Naranjo, C. M., and Fee, J. A. (1989). Relationship of the superoxide dismutase genes, sodA and SodB, to the iron uptake ( f u r )Regulon in Escherichia coli K12. I n “Metal Ion Homeostasis: Molecular Biology and Chemistry” (D. Winge and D. Hamer, eds.), pp. 149-158. Liss, New York. Nishida, K. I., Takagi, M., and Yano, K. (1981). Escherichia coli inactivation by superoxide anion and hydrogen peroxide. J . Gen Microbiol. 27,447-485. Owens, R. A,, and Hartman, P. E. (19861. Glutathione: A protective agent in Salmonella typhimurium and Escherichia coli as measured by mutagenicity and by growth delay assays. Enuiron. Mutagen. 8, 659-673. Parsons, P. G., and Hayward, I. P. (1985). Inhibition of DNA repair synthesis by sunlight. Photochem. Photobiol. 42, 287-293. Peak, J . G., Peak, M. J., and Tuveson, R. W. (1983). Ultraviolet action spectra for aerobic and anaerobic inactivation of Escherichia coli strains specifically sensitive and resistant to near ultraviolet radiations. Photochem. Photobiol. 38, 541-543. Peak, M. J., Johnson, J . S., Tuveson, R. W., and Peak, J. G. (1987a). Inactivation by monochromatic near-UV radiation of a n Escherichia coli hemA8 mutant grown with and without aminolevulinic acid: The role of DNA vs membrane damage. Photochem. Photobiol. 45, 473-478. Peak, M. J., Ito, A., Foote, C. S., and Peak, J . G. (1987b). Photosensitized inactivation of DNA by monochromatic 334-nm radiation in the presence of 2-thiouracil: Genetic activity and backbone breaks. Photochem. Photobiol. 47, 809-814. Peak, M. J., Midden, R., Babasick, D. M., and Haugen, D. A. (1988). Photochemistry of 4-thiouridine and thymine, Photochem. Photobiol. 48, 229-232. Peters, J. (1977). In uiuo photoinactivation of E . coli ribonucleotide reductase by near-ultraviolet light. Nature (London)267, 546-548.

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Peters, J., and Jagger, J . (1981).Inducible repair of near-UV radiation lethal damage in E . coli. Nature (London) 289, 194-195. Petkau, A. (1986). Scientific basis for the clinical use of superoxide dismutase. Cancer Treat. Rev. 13, 17-44. Pierceall, W. (1988). Comparison of induced proteins synthesized in hydrogen peroxideand near-ultraviolet-treated bacterial cells. M.A. thesis, Univ. of Missouri, Columbia. Pizarro, R. A., and Orce, L. V. (1988). Membrane damage and recovery associated with growth delay induced by near-UV radiation in Escherichia coli K-12. Photochem. Photobiol. 47, 391-397. Quintiliani, M. (1986).The oxygen effect in radiation inactivation ofDNA and enzymes. Int. J . Radiat. Biol. 4, 573-594. Rebeyrotte, N., Rebeyrotte, P., Maviel, M. J., and Montaudon, D. (1979). Lipides et lipopolyosides de Micrococcus radiodurans. Ann. Microbiol. (Inst. Pasteur). 130B, 407-414. Richter, H., and Loewen, P. C. (1981). Induction of catalase in E . coli by ascorbic acid involves hydrogen peroxide. Biochem. Biophys. Res. Cornmun. 100, 10391046. Roth, D. (1981). DNA-denaturing effect of irradiated hydrogen peroxide. Photochem. Photobiol. 33, 251-252. Rotilio, G. (1986). “Superoxide and Superoxide Dismutases in Chemistry, Biology and Medicine.” Elsevier, Amsterdam. Rowley, D. A., and Hallewell, B. (1982). Superoxide dependent formation of hydroxyl radicals in the presence of thiol compounds. FEBS Lett. 138, 33-36. Sak, B. (1989).Exonuclease I11 and catalase HP-I1 in Escherichia coli are both regulated by the katF gene product. M.A. dissertation. Univ. of Missouri, Columbia. Sak, B. D., Eisenstark, A,, and Touati, D. (1989). Exonuclease 111 and hydroperoxide I1 in Escherichia coli are both regulated by the katF gene product. Proc. Natl. Acad. Sci. U.S.A.86 (in press). Sakamoto, H., and Touati, D. (1984). Cloning of the iron superoxide dismutase gene (so&) in Escherichia coli K-12. J . Bacteriol. 159, 418-420. Salet, C., Bazin, M., Moreno, G., and Favre, A. (1985).4-Thiouridine as a photodynamic agent. Photochem. Photobiol. 41, 617-619. Sammartano, L. J., and Tuveson, R. W. (1983). Escherichia coli rthA mutants are sensitive to inactivation by broad-spectrum near-ultraviolet (300-400 nm) radiation. J . Bacteriol. 156, 904-906. Sammartano, L. J., and Tuveson, R. W. (1984). The effects of exogenous catalse on broad-spectrum near-UV (300-400 nm) treated Escherichia coli cells. Photochem. Photobiol. 40, 607-612. Sammartano, L. J., and Tuveson, R. W. (1985).Hydrogen peroxide induced resistance to broad-spectrum near-ultraviolet light (300-400 nm) inactivation in Escherichia coli. Photochem. Photobiol. 41, 367-370. Sammartano, L. J., and Tuveson, R. W. (1987). Escherichia coli strains carrying the cloned cytochrome d terminal oxidase complex are sensitive to near-UV inactivation. J . Bacteriol. 169, 5304-5307. Sammartano, L. J., Tuveson, R. W., and Davenport, R. (1986). Control of sensitivity to inactivation by HzOz and broad-spectrum near-UV radiation by the Escherichia coli katF locus. J . Bacteriol. 168, 13-21. Sancar, A., and Sancar, G. B. (1984).Escherichia coli DNA photolyase is a flavoprotein. J . Mot. Biol. 172, 223-227. Sancar, G. B., Smith, F. W., and Sancar, A. (1983).Identification and amplification ofthe E . coli phr gene product. Nucleic Acids Res. 11, 6667-6677.

BACTERIAL GENES INVOLVED IN RESPONSE TO

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145

Saporito, S. M., Smith-White, B. J., and Cunningham, R. P. (1988).Nucleotide sequence of the xth gene of Escherichia coli K12.J.Bacteriol. 170,4542-4547. Sato, N., Ohnishi, T., Tano, K.,Yamamoto, K.,and Nozu, K.(1985).Induction of umuC gene expression in E . coli irradiated by near-ultraviolet light. Photochem. Photobiol. 42, 135-139. Schaaper, R. M., Danforth, B. N., and Glickman, B. W. (1985).Rapid repeated cloning of mutant lac repressor genes. Gene 39, 181-189. Schellhorn, H. E., and Hassan, H.M. (1988).Transcriptional regulation of kutE in Escherichia coli K12.J . Bucteriol. 170,4286-4292. Scott, M. D., Meshnick, S. R., and Eaton, J. W. (1987).Superoxide dismutase-rich bacteria. J. Biol. Chem. 262, 3640-3645. Sestili, P., Piedimonte, G., Flaminio, C., and Cantoni, 0. (1986).Induction of DNA breakage and suppression of DNA synthesis by the OH radical generated in a Fenton-like reaction. Biochem. Znt. 12,493-501. Setlow, J. K.,and Dugan, D. E. (1964).The resistance of Micrococcus radiodurans to ultraviolet radiation. I. Ultraviolet-induced lesions in the cell’s DNA. Biochim. Biophys. Acta 87, 664-668. Sharma, R. C., and Smith, K.C. (1985).A minor pathway of postreplication repair is independent of the recB, recC, and recF genes. Mutat. Res. 146,169-176. Sies, H., ed. (1985).“Oxidative Stress.” Academic Press, London. Slonczewski, J. L., Gonzalez, T. N., Bartholomew, F. M., and Holt, N. J. (1987).Mu d-directed lucz fusions by pH in Escherichia coli. J.Bacteriol. 169,3001-3006. Smith, K. C., and Sharma, R. C. (1987).A model for the recA-dependent repair of excision gaps in UV-irradiated E. coli. Mutat. Res. 183, 1-9. Sohal, R. S.,and Allen, R. G. (1986).Relationship between oxygen metabolism, aging and development. Adv. Free Radical Biol. Med. 2, 117-160. Spitzer, E. D., and Weiss, B. (1985).dfp gene of Escherichia coli K-12,a locus affecting DNA synthesis, codes for a flavoprotein. J.Bucteriol. 164, 994-1003. Storz, G., Christman, M. F., Sies, H., and Ames, B. N. (1987).Spontaneous mutagenesis and oxidative damage to DNA in Salmonella typhimurium. Proc. Natl. Acad. Sci. U.S.A.84,8917-8921. Storz, G.,Jacobson, F. S., Tartaglia, L. A,, Morgan, R. W., Silveira, L. A., and Ames, B. N. (1989).An alkyl hydroperoxide reductase induced by oxidative stress in Salmonella typhimurium and Escherichia coli: Genetic characterization and cloning of ahp. J. Bucteriol. 171,2049-2055. Straight, R. C., and Spikes, J . D. (1985).Photosensitized oxidation of biomolecules. Zn “Singlet Oxygen” (A. A. Frimer, ed.), Vol. 4,pp. 91-143. CRC Press, Boca Raton, Florida. Sweet, D. M., and Moseley, B. E. B. (1976).The resistance ofMicrococcus radioduruns to killing and mutation by agents which damage DNA. Mutat. Res. 34, 175-186. Szyf, M.,Meisels, E., and Razin, A. (1986).Biological role of DNA methylation: Sequence-specific single-strand breaks associated with hypomethylation of GATC site of Escherichia coli DNA. J . Bacteriol. 168, 1487-1490. Tabor, S.,and Richardson, C. (1987).Selective oxidation of the exonuclease domain of bacteriphage T7 DNA polymerase. J. Biol. Chem. 262, 15330-15333. Tajeda, Y.,and Avila, H. (1986).Structure and gene expression of the E. coli Mnsuperoxide dismutase gene. Nucleic Acids Res. 14,4577. Taylor, A. F., and Weiss, B.(1982).Role of exonuclease I11 in the base excision repair of uracil-containing DNA. J.Bacteriol. 151, 351-357. Tessman, E. S., Tessman, I., Peterson, P. K., and Forestal, J. D. (1986).Roles of RecA protease and recombinase activities of Escherichia coli in spontaneous and UVinduced mutagenesis and in Weigle repair. J.Bucteriol. 168, 1159-1164.

146

A . EISENSTARK

Thompson, B. G., Anderson, R., and Murray, R. G. E. (1980). Unusual polar lipids of Micrococcus radiodurans stain Sark. Can J . Microbiol. 26, 1408-1411. Thornley, M. J., Horne, R. W., and Glauert, A. M. (1965). The fine structure of Micrococcus radiodurans. Arch. Mikrobiol. 51, 267-289. Touati, D. (1983). Cloning and mapping of the maganese superoxide dismutase gene ( s o d ) of Escherichia coli K-12. J . Bacteriol. 155, 1078-1087. Touati, D. (1988a).Molecular genetics of superoxide dismutases. Free Radical Biol. Med. 5, 393-402. Touati, D. (1988b). Transcriptional and posttranscriptional regulation of manganese superoxide dismutase biosynthesis in E. coli, studied with operon and protein fusions. J . Bacteriol. 170, 2511-2520. Touati, D., and Carlioz, A. (1986).Superoxide dismutase mutants of Escherichia coli. I n “Superoxide and Superoxide Dismutase in Chemistry, Biology and Medicine” (G. Rotilo, ed.). Elsevier, Amsterdam. Triggs-Raine, B. L., and Loewen, P. C. (1987). Physical characterization of katG, encoding HPI of Escherichia coli. Gene 52, 121-128. Triggs-Raine, B. L., Dable, B. W., Mulvey, M. R., Sorby, P. A,, and Loewen, P. C. (1988). Nucleotide sequence of katG encoding catalase HPI of Escherichia coli. J . Bacteriol. 170,4415-4419. Turner, M. A,, and Eisenstark, A. (1984). Near-ultraviolet radiation blocks SOS responses to DNA damage in Escherichia coli. Mol. Gen. Genet. 193, 33-37. Turner, M. A., and Webb, R. B. (1981). Comparative mutagensis and interaction between near-ultraviolet (313-405 nm) and far-ultraviolet (254 nm) radiation in E. coli strains differing in repair capabilities. J . Bacteriol. 147, 410-417. Tuveson, R. W., and Sammartano, L. J. (1986).Sensitivity of hemA mutant Escherichia coli cells to inactivation by near-ultraviolet light depends on the level of supplementation with delta-aminolevulic acid. Photochem. Photobiol. 43, 621-626. Tuveson, R. W., Peak, J. G., and Peak, M. J. (1983).Single-strand DNA breaks induced by 365 nm radiation in E . coli strains differing in sensitivity to near- and far-UV. Photochem. Photobiol. 37, 109-112. Tyrrell, R. M. (1985). A common pathway or protection of bacteria against damage by solar UVA (334 nm, 365 nm) and oxidizing agents (HzO,). Mutat. Res. 145,129-136. Van Bogelen, R. A,, Kelly, P. M., and Neidhardt, F. C. (1987).Differential induction of heat shock, SOS, and oxidative stress regulons and accumulation of nucleotides in E . coli. J. Bacteriol. 169, 26-32. Van Rijin, K., Mayer, T., Blok, J.,Verberne, J. B., and Loman, H. (1985). Reaction rate of OH radicals with 4x174 DNA: Influence of salt and scavenger. Int. J . Radiat. Biol. 47,309-317. Vassilyadi, M., and Archibald, F. (1985). Catalase, superoxide dismutase, and the production of 02-sensitive mutants of Bacillus coagulans. Can. J . Microbiol. 31, 994-999. Walker, G. C. (1984). Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol. Rev. 48, 60-93. Walker, G. C. (1987). The SOS response of Escherichia coli. In “Escherichia coli and Salmonella typhimuriurn: Cellular and Molecular Biology” (F.C. Neidhardt et al., eds.), 7th Ed., pp. 1346-1357. Am. SOC.Microbiol., Washington, D.C. Wallace, S. S. (1988).AP endonucleases and DNA glycosylases that recognize oxidative DNA damage. Enuiron. Mol. Mutagen 12, 431-477. Webb, R. B. (1977). Lethal and mutagenic effects of near-ultraviolet radiation. Photochem. Photobiol. Rev. 2, 169-261.

BACTERIAL GENES INVOLVED IN RESPONSE TO

NUV

147

Wei, K., Tomita, S., and Suzuki, K.(1986). Block of cleavage of ultraviolet-irradiated DNA with restriction endonucleases. J . Radiat. Res. 27,247-253. Weiss, B., and Cunningham, R. P. (1985). Genetic mapping of nth, a gene affecting endonuclease I11 (thymidine glycol-DNA glycosylase) in Escherichia coli K-12. J . Bacterial. 162, 607-610. Weiss, R. B., and Duker, N. J. (1986). Photoalkylated DNA and ultraviolet-irradiated DNA are incised at cytosines by endonuclease 111. Nucleic Acids Res. 14,6621-6631. Weiss, R.B.,and Duke, N. J. (1987). Endonucleolytic incision of UVB-irradiated DNA. Photochem. Photobiol. 45, 763-768. Wilke, A. (1988). Effects of increased endogenous catalase on resistancehensitivity to near-ultraviolet radiation in E . coli. M.A. thesis, Univ. of Missouri, Columbia. Winguest, L., Rannung, U., Rannug, A., and Ramel, C. (1984).Protection from toxic and mutagenic effects of Hz02by catalase induction in Salmonella typhimurium. Mutut. Res. 141, 145-147. Yallaly, P. (1988). Influence of DNA adenine methylase on sensitivity of E . coli to near-ultraviolet radiation and hydrogen peroxide. M.A. thesis, Univ of Missouri, Columbia. Yamamoto, N., and Droffner, M. L. (1985). Mechanisms determining aerobic or anaerobic growth in the facultative anaerobe Salmonella typhimurium. Proc. Natl. Acad. sci.U.S.A.82,2077-2081. Yamamoto, N., Droffner, M. L., and Yamamoto, S. (1987). Conditional mutations of Salmonella typhimurium that are suppressed by anaerobic or aerobic environments. FEMS Microbiol. Lett. 42, 249-252. Yoakum, G.,Eisenstark, A., and Webb, R. B. (1975). Near-UV photoproduct(s) of L-tryptophan: Aninhibitor of medium-dependent repair of X-ray-induced singlestrand breaks in DNA which also inhibit replication-gap closures in Escherichia coli DNA. “Molecular Mechanisms for Repair of DNA, Part B” (P. C. Hanawalt and R. B. Setlow, eds.), pp. 453-458. Plenum, New York.

MEIOSIS IN MAIZE: mei GENES AND CONCEPTION OF GENETIC CONTROL OF MEIOSIS lnna N. Golubovskaya The N. I. Vavilov All-Union Institute of Plant Industry, Leningrad, 190000, Union of Soviet Socialist Republics

I. Introduction ... ..... .... ... 11. Brief Description of the Maize mei Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. An ameiotic Mutation Controlling the Initiation of Meiosis in Maize.. . B. The afd Mutation Substituting the First Division of Meiosis for Mitosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. The Blockage of Meiosis after Pachytene: An Effect of the MeiO25 Gene . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . D. rnei Genes of Maize: Impairing the Pairing of Homologous Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Genes Controlling Segregation of Homologous Chromosomes . . . . , . . . . . F. A mei Mutation, elongate (el),Controlling the Second Division of Meiosis.. . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . G. A mei Mutation, uariable (ua),Impairing Cytokinesis .... ... H. mei Mutations ( p a m l and p a d ? ) Causing Nonspecific ....... Multiple Abnormalities of Meiosis. . . . . . . . . . . . I. Precocious Postmeiotic Mitosis: A polymitotic ( P O Gene with a Series of Alleles (ms6 and ms4) . , . . . . . . . . . . . . . . . . . . . . . . . J. Summary 111. Cytogenetic Evidence for the Genetic Control of Meiosis.. . . . . . . . . . . . . . . . . A. The Choice of mei Mutations as Experimental Models B. Independent Action of Genes Controlling C. Consequent Activation of rnei Genes in Meiosis.. . . . . . . . . . . . . . .. . . . . . D. Hierarchy among mei Genes . . . . . . . . . . . . . . . . . IV. Speculation about the Possible Pathways of Geneti V. Conclusion: Theoretical and Applied Aspects of Meiosis Genetics References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . I

149 153 153 158 160 164 167 168 169 169 173 173 174 174 176 177 181 183 185 186

I. Introduction

Knowledge of the genetic control of meiosis may provide a key to understanding the complex and important processes involved in sexual reproduction. In this era of biotechnology and genetic engineering, which provides great possibilities for the construction of new plant and animal genotypes, meiosis should be in the forefront of biological 149 ADVANCES IN GENETICS, Val. 26

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investigations. Creation of new organisms and their fate will depend on whether the gametes can pass through the “sieve” of meiotic selection and can leave viable progeny. In this case, meiosis carries out an important evolutionary function as the barrier in the way of inviable chromosomal or genic combinations (White, 1973). Studies in the field of meiotic biochemistry (Stern and Hotta, 1973, 1974) and ultrastructure of meiotic prophase (Moses, 1956; Bogdanov, 1975; Westergaard and von Wettstein, 1972) stimulated investigations of the genetics of meiosis in the 1970s. These early studies showed that there were enzymes that could take part in meiotic recombination, in DNA repair after damage by UV rays in somatic cells, and in the degree of enzymatic mutagenic sensitivity (Boyce and HowardFlanders, 1964; Kushev, 1972). The necessity for direct study of meiotic genetics stems from cytogenetic investigations of common wheat and its distant hybrids with the subtribe Triticinae. The cytogenetic stability of such hybrids, obtained in vivo and in vitro, appears to depend on genes which control chromosome behavior in meiosis (Riley and Chapman, 1958; Riley et al., 1966; Golubovskaya, 1973; Golubovskaya et al., 1966). Thus, from the general question, whether meiosis is controlled genetically, the following questions derive: the number and identity of the genes involved in meiosis, the genetic mechanisms that occur for the initiation of meiosis, how the main meiotic events (for example, pairing of homologous chromosomes or meiotic recombination) are controlled, and how transition from one event to another is genetically controlled. These and many other questions require answers. Therefore, an essential methodology for investigating such questions was developed. First, mei mutants were collected from Saccharomyces cerevisiae, Neurospora crassa, Drosophila melanogaster, Pisum sativum, and Zea mays. The peculiarities of the genetic control of meiosis in yeast, particularly the question of how the initiation of meiosis is controlled, were investigated with the help of the Saccharornyces and Neurospora mei mutants (Esposito and Esposito, 1969; Klapholz and Esposito, 1980). The mei mutants of D. melanogaster were obtained in order to study the genetic control of meiotic recombination in higher eukaryotes (Baker and Carpenter, 1972; Carpenter and Sandler, 19741,and to examine the relationship of the mei genes to genetic systems controlling DNA repair processes after UV irradiation (Smith, 1973; Smith et al., 1980; Baker et al., 1976a). In addition, the effect of genes controlling meiotic recombination and mutagenesis induced by transposable genetic elements was studied (Green, 1978; Eeken and Sobels, 1981). There exists a n enormous gap between the general concepts relative

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to the genetic control of meiosis and the actual understanding of the intricate peculiarities of such control. Our initial task was to try to understand some of the major peculiarities of the genetic control of meiosis in maize (Golubovskaya, 1975). The basic premise was that meiosis is a universal process for all sexually reproducing organisms. This principle permitted us t o ignore species-specific aspects of meiosis, and allowed us to utilize all available data about all types of mei mutations which have appeared in the scientific literature for classifying mei mutations. To simplify the classification of mei mutations, we compared their effects on separate cytogenetic events of meiosis (i.e., initiation of meiosis, pairing of homologous chromosomes, meiotic recombination, chiasma formation, segregation of chromosomes, the second meiotic division, and cytokinesis). Systematization of mei mutations has revealed that similar types of mei mutations concerning one or several of the above-mentioned meiotic events occur in organisms ranging from unicellular algae and yeast to higher plants, and in animals ranging from nematodes and Drosophila t o humans. A detailed classification of mei mutations was summarized in previous reviews (Golubovskaya, 1975, 197913). In this review the emphasis will be on distinct types of mei mutations, those which show initial cytological effects. On comparing these with each other, we were able to uncover the following main effects they have on the genetic control of meiosis: 1. The seven key cytogenetic steps of meiosis are under strict genetic control, each being controlled by a group of genes acting relatively independently from each other. 2. At least three hierarchical levels of mei genes are noted: there are genes controlling key blocks of meiotic events, those controlling elementary steps in meiosis, and peculiarities of individual chromosomal behavior, including species-specific characteristics of meiosis. 3. The principle of stepwise switching on of mei mutations during meiosis was formulated. Every logical argument demands concrete experimental evidence. For this purpose, it was necessary to have a collection of different rnei mutants affecting the key cytogenetic steps of meiosis. Maize (2.mays L.) was selected for this purpose, because it is well studied genetically and convenient for observing meiotic events (Fig. 1). For the cytogenetic model experiments, nine mei mutants, induced by chemical mutagens, and some mei mutants provided by the Maize Genetics Stock Center (Urbana, Illinois), were used (Table 1).

FIG. 1. The meiotic cytological behavior of chromosomes in normal maize plants. (a-e) Prophase I of meiosis with normal pairing of homologous chromosomes. (a) Zygotene. (b and c) Pachytene. (d) Diplotene. (e) Diakinesis. (f) In metaphase I, 10

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II. Brief Description of the Maize mei Mutants

The mei mutations do not influence vegetative development of plants and do not change their phenotypes. The mei mutations in plants can be revealed only during tassel inflorescence. As a rule, mei mutants display complete or partial male and female sterility, but in some cases they have either male or female sterility only. Special attention to cytological characteristics will be paid throughout the description of maize mez mutants. The genetics of mei mutants are summarized in Tables 2 and 3.

A. AN ameiotic MUTATION CONTROLLING THE INITIATION OF MEIOSISIN MAIZE A recessive ameiotic (am) mutation (chromosome 5 , short arm) was isolated (Rhoades, 1956) and cytologically studied (Palmer, 1971). Homozygous amlam plants exhibit complete male and female sterility. Cytological analyses of homozygotes have revealed that the last premiotic mitosis proceeded regularly, but that the subsequent meiosis did not occur in the meiocytes. Instead of meiosis, there are two to three synchronous mitoses and subsequent degradation of chromatin in the cells. Among the dividing cells of the anther, all stages of the meiotic cell cycle, from interphase to teleophase, are present. The pattern of meiosis in ameiotic mutants grown in Krasnodar (USSR) was similar to that described by Palmer (1971). In my opinion, there was a single exclusion in Palmer’s cytological observation: only the first mitotic division in ameiotic homozygotes was synchronized, and therefore, in the next mitotic cell cycles, only rare cells were seen at different stages of mitosis. To be sure that meiosis was completely omitted in this mei mutant, the prophase of ameiotic mitosis was examined by electron microscopy. Synaptonemal complex (SC) formation was absent in the cells (Golubovskaya and Khristoljubova, 1985). That is, the single mutational action converts the whole meiotic process into mitosis, as shown by the ameiotic mutant. It is reasonable to suppose that the product of the normal allele of the ameiotic gene is responsible for the initiation of meiosis, and that the gene has the highest hierarchical level, i.e., switching on all steps bivalents are seen. ( g ) In anaphase I, 10 chromosomes pass to each opposite pole. (h-n) A second division of meiosis. (h-i) Prometaphase. (j)Metaphase 11. (k-1) In anaphase 11, centromere sister chromatids separate. (m) Telophase 11. (n) Tetrads.

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TABLE 1 List of Maize Meiotic Mutants Cytological effect of mei gene

Symbol for mei gene

Location

References

Comment

Substitution of meiosis by mitosis Substitution of first meiotic division by mitosis

ameiotic (am)

5s

Rhoades (1956); Palmer (1971)

afd W23

6L

Stickiness of chromosomes

sticky (st)

4s

Golubovskaya and Mashnenkov (1975); Golubovskaya (1979a, 1987) Meiotic arrest Beadle (1937) after pachytene Mashnenkov and Golubovskaya (1980) Nelson and Clary (1952) Beadle (1930)

MeiO25

Desynapsis of chromosomes

desynaptic (dy)

asynaptic (as) dsy 1 , dsy 2, dsy 3, dsy 4

Abnormal chromosome segregation

Abnormalities of second meiotic division

1s non-lS, lL, 6L

ms43 A344

non-lS, 5s,5L, 6L, 7L 9s 8L

ms28A344

1s

elongate (el)

8L

diuergent (du)

Golubovskaya and Mashnenkov (1976); Golubovskaya and Urbach (1980) Central spindle Clark (1940) apparatus is not formed Curtis (1983) Golubovskaya and Parallel spindle Sitnicova orientation a t second divi(1980); sion is abGolubovskaya sent and Distanova (1986) Golubovskaya and Delayed Sitnikova depolymerization of (1980); spindle fibers Golubovskaya (1987) Rhoades (1956); Curtis (1983)

155

MEIOSIS I N MAIZE

TABLE 1 (Continued) ~~

Cytological effect of mei gene

Symbol for mei gene

Irregular cytokinesis Supernumerary mitoses during pollen grain formation

variable (ua) polymitotic (PO),ms6

Nonspecific abnormalities of meiosis

pan1 A344, pam2 W64A

Location

References

7L

Beadle (1932a)

6s

Beadle (1928,

Comment -

ms6, and ms4 are allelic Precocious Golubovskaya and Urhach postmeiotic mitoses (1980) Golubovskaya and Mapping is Mashnenkov needed to (1976) verify cytology PO,

1933)

ms4 6L

TABLE 2 Inheritance of Eight New Maize mei Mutations as Defined by Pattern of Meiosis and Fertility Segregation in F 2 mei mutant

dsy dsy2 ms43 ms28 ms4

Cross combination Fz(pamlpam x A344) pam2Ii @ afdl+ 0" Fz(afd1afd x W23) dsyl+ @ FZ(dsyl+ X A344) dsy2l+ @ F2(dsy2Idsy2 X A344) ms431+ @ ms43lms43 x ms43/+ F2(ms431ms43 x A344) ms281+ 0" ms28lms28 x m s 2 8 l t Fz(ms28/ms28 X A344) ms4l+ @ F9(ms41ms4 X A3441

' @, Self-pollination.

* Not significant.

Normal fertile

Mutant sterile

Expected ratio

75 43 127 112 132 65 64 36 83 58 102 41 65 18 48 11

22 10 48 39

3:1 3:1 3:l 3:1 3:l 3:1 3:1 3:1 3:l 1:l

42

20 29 5 6 28 21 8 43 5 17 6

3:l 3:1 1:l

3:l 3:1 3:l

x2

value

0.28 1.06 0.55 0.05 0.07 0.09 1.89 3.58 15.82' 10.46' 4.12 1.96 4.48* 0.13 0.15 0.96

156

INNA N. GOLUBOVSKAYA

TABLE 3 Allelic Relationships of the mei Mutations as Defined by Pattern of Meiosis and Fertility ~~

Fz segregation

F, segregation Cross combination and meiotic events Homologous pairing dsy2Idsy2 x dsyl+ aslas x d s y l t aslas x a f d l t dsy2Idsy2 x a s i t aslas x dsy2/+ afdli x dsylt Segregation ms43lms43 x ms28l-t ms28lms28 X ms431 t ms43lms43 x d u l t ms28lms28 x d v l t Differentiation pamlpam x p a m 2 I i ualva X p a m l t amlf x afdlt aslas x m s 4 3 l t ms4lms4 x pol+

Normal fertile

Mutant sterile

41 4 24 23

0 0 0 0 0 0

17

40 39

20 27 17

2 8

21 20 8

0 0 0 0 0 0 0 0 2

x2 value Fertile

Sterile

9: 7

-

97 I5 35

1.56

0.33

0.5 5.0

0.25

0.7

107

105 42 20

-

1 :1

-

18

179

141

0.01

4.5

-

-

-

-

-

-

-

-

-

-

-

50

39

0.15

1.4

36

35 116 21 -

0.90

0.01

0.25

2.25 2.28

140 32 -

0.37 -

-

of meiosis simultaneously. Experimental proof for this last statement will be discussed below. If the mutation of a single gene causes conversion of meiosis to mitosis, the principal irreversible product determining the pathway of meiosis must also exist. From this viewpoint, it is interesting to consider the biochemical data, which indicate a meiosis-specific protein, the leptotene protein (L-protein) (Hotta et al., 1984). This 73-kDa protein has been isolated and purified from the nuclear membranes of preleptotene, leptotene, and zygotene cells of lily. The protein has been found to be highly specific in its inhibitory activity, supressing the replication of zygotene DNA (zyg DNA)sequences until the initiation of zygotene. The L-protein has also the capacity to nick the bound DNA in the presence of ATP, and is considered to be responsible for the irreversible commitment of cells to meiosis after entering the premeiotic S phase.

MEIOSIS IN MAIZE

157

Initiation of Meiosis and mei Genes in Other Organisms The yeasts S. cereuisiae and Schizosaccharomyces pombe are most convenient for studying the mechanisms of initiation of meiosis. Significant factors for the cells to enter into meiosis in these species, which represent lower eukaryotes, are the heterozygous state of mating sites and nitrogenous starvation. There is a specific point in the GI phase of the mitotic cell cycle, called “start,” which must necessarily be blocked for transition of the cell from mitosis to meiosis. The transition in S. cereuisiae has been shown to be strictly genetically controlled and a function of the normal alleles of the genes of the cell division cycle (cdc genes); namely, cdc’28, cdc+25,cdc+35, tru’3, etc., are needed (Hereford and Hartwell, 1974). The functions of cdcf28 and tra’3 genes were found to be important for entering into meiosis, and the cdc’25 and cdc’35 genes were demonstrated t o be responsible for the choice between meiosis and mitosis and to depend on the components of the nutrient medium. In addition, two genes, CYR 3 and cyr 1, are involved in controlling meiosis and both genes initiate meiosis independent of nutrient components in the medium (Matsumoto et al., 1983; Uno et al., 1982). Biochemical studies of these mutants and their comparison with other cdc genes have allowed the authors to conclude that the initiation of meiosis depends on the repression of CAMPproducts and inactivation of CAMP-dependent protein kinase (Matsumoto et al., 1983). Based on genetic, biochemical, and molecular investigations of cdc mutants in S . cereuisiae, a model of initiation of the cell cycle has been suggested. According to this model, the decreased level of protein kinase enzymatic activity is significant for the beginning of meiosis, and the GI phase of the cell cycle is critical for initiation of meiosis. Recent genetic data obtained using S. pombe have also supported this model (Iino and Yamamoto, 1985a,b). The patl gene is considered to be very important for initiation of meiosis. Investigations of the interaction of the patl gene with a gene defective in the mating system (meil gene), and of the mei2 and mez.3 genes needed for initiation of premeiotic DNA synthesis, have shown that the patl product releases a negative control in meiotic initiation. Additional evidence for the essential role of the loss of protein kinase activity in initiation of meiosis in both yeast species is available from the studies of cdc genes (Shimodo and Uehira, 1985).Recent results of cloning of cdc2 in S.pombe (Hindley and Phear, 1984) have permitted the isolation of the protein product of the cdc2 gene (Simanis and

158

INNA N. GOLUBOVSKAYA

Nurse, 1986). It is a 34-kDa phosphoprotein with kinase activity. The protein plays a significant role in entering the “start” point and in initiating the mitotic cycle. When mutant cells are placed on poor nutrient medium, phosphorylation of the cdc2 protein and loss of protein kinase activity occur. They are the basic factors which determine the entry of cells into meiosis (Simanis and Nurse, 1986). The identity and conservation of genetic and molecular mechanisms of cell division initiation in two species of yeast were examined by identifying the protein product encoded by both the cdc’28 gene (5’. cerevisiue) and the cdc+2 gene (5’.pombe) (Lee and Nurse, 1987). The conservation of genetic regulatory mechanisms of the cell division cycle is widespread throughout the evolutionary ladder. A human homolog of the cdc2 gene has been isolated and cloned from a cDNA library of S.pombe. The protein sequence of the human homologue is very similar to that of the cdc2 gene in fission yeast. The CDC2 H s gene encodes a 34-kDa phosphoprotein with kinase activity. The yeast and human proteins have 63% homology in amino acid sequences. These experimental data indicate that the factors by which the cell cycle is controlled are likely to be conserved among yeast and humans (Lee and Nurse, 1987). B. THE ufd MUTATION SUBSTITUTING THE FIRST DIVISIONOF MEIOSISFOR MITOSIS The ufd mutation is another phenomenon in which the entire pathway of meiosis is altered by a single mutation that is induced by a chemical mutagen in maize (Golubovskaya and Mashnenkov, 1975). The recessive ufd gene responsible for the mutant phenotype is located on the long arm of chromosome 6 by B-A translocation stocks (Golubovskaya, 1987). In ufd homozygous plants, typical stages of prophase I, such as leptotene, zygotene, pachytene, and diplotene, are omitted. At a stage which can conditionally be called diakinesis, the 20 chromosomes of maize are represented by univalents, arranged in a disorderly manner in the cells, and a nucleolus is clearly seen. At metaphase I, 20 univalents are lined up in an orderly manner on the plate of spindle. At anaphase I, 20 chromatids move t o the opposite pole as a result of division and separation of centromeres of sister chromatids, as a rule at the first meiotic division instead of the second. Thus, the first division of meiosis in ufd mutants proceeds like mitosis. A t anaphse-telophase of the second division of meiosis, chromatids are distributed irregularly between the two poles. As a result, about 98%

MEIOSIS I N MAIZE

159

abnormal tetrads are formed in mutant plants. Homozygotes for the afd gene have male and female sterility. The first meiotic division, being similar t o mitosis, proceeds abnormally. There is pulverization of chromosomes at anaphase I, resulting in 67% abnormal chromosomes. Indirectly, this indicates the defects in the repair process, which normally accompanies meiotic recombination. Meiotic recombination, as experimentally proved (Stern and Hotta, 1974; Hotta et al., 19791, occurs only in the case of intimate pairing of homolgous chromosomes. But the question arises as to how, in the absence of cytologically visible pairing of chromosomes in the afd mutant, events connected with meiotic recombination are possible. Electron microscopic studies of the meiotic chromosomes at prophase I help to understand this contradiction. The short SC fragments were found to appear at the early stage of prophase I, and the morphology is correct in one case and defective in another case. The total length of the SC in the afd mutants is only 12% of that in normal plants (Golubovskaya and Khristoljubova, 1985; Golubovskaya et al., 1980). The formation of the SC is not complete and its short fragments are quickly destroyed and disappear. Whether the afd mutation is due to defects in the pairing of homologous chromosomes or to a systematic mutation induced by changes in the whole meiotic process is not clear. However, in my view the last alternative is more likely, based on a comparison of the afd mutants with asynpatic and ameiotic mutants in maize, as described above.

Comparisons between the afd and the asynaptic and ameiotic Mutants Some asynaptic mei mutants have been described: the c3G gene in D . melanogaster (Gowen, 1933; Gowen and Gowen, 1922; King, 1970; Smith and King, 1968),the asynpatic gene of Triticum durum (Martini and Bozzini, 1966; LaCour and Wells, 19701, the rad6-1 gene of S . cerevisiae (Kundu and Moens, 19821, the syn gene of Secale cereale (Fedotova et al., 1987), and the case of male sterility in man associated with total blocking of the SC formation at prophase I (Vidal et al., 1982). The asynpatic mutations of these different organisms have the same general peculiarities, such as the total blockage of SC formation (in some cases, for example, in rye, durum wheat, and humans, only lateral elements are formed), inhibition of crossing over, and anomalies of chromosomal segregation as a result of the univalent state a t metaphase I. It is necessary to emphasize that meiosis in asynaptic

160

INNA N. GOLUBOVSKAYA

mutants proceeds in two divisions and that centromeres of sister chromatids segregate, as a rule, at the second division of meiosis. The difference between the umeiotic mutants and the ufd mutant is that the first division of meiosis of ufd is substituted for mitosis. In the ufd mutants, the second division takes place as in normal plants, but the abnormalities are observed because the function of the second division is carried out in the first division. On the other hand, the existence of the afd mutant indicates that the total reversion of mitosis in the cells irreversibly committed to meiosis (after they enter into the zygotene stage, the ability to form the SC can be considered) is excluded. Also, the behavior of centromeric regions and homologous chromosomal pairing are independent events. An indirect proof for this is the appearance of mei mutants, determining the precocious separation of centromeric regions at the first division of meiosis. Other examples are the pc mutation of Lycopersicon esculentum (Clauberg, 1959) and the ord gene of D . melanoguster (Mason, 1976; Goldstein, 19801, which show common genetic control of disjunction of centromeric regions in chiasmatic meiosis of females and in achiasmatic meiosis of males in D . melanoguster (the ord and the mei-S332a genes) (Sandler et ul., 1968; Davis, 1971; Goldstein, 1980). Hence, it is not surprising that the premature disjunction of sister centromeres can accompany some mutations [the dy mutant in maize (Nelson and Glary, 1952) and the 2982 mutant ofpisumsutiuurn (Klein, 1969)l.It has been suggested by Maguire that it is the dy gene that is responsible for the behavior of centromere regions in maize (Maguire, 1978a,b). OF MEIOSISAFTER PACHYTENE: C. THE BLOCKAGE AN EFFECTOF THE MeiO25 GENE

1 . Inheritance of MeiO25 In 1979, six sterile mutant MeiO25 plants were pollinated with the inbred line of maize W64A (Table 4).Cytological analysis of segregation in the F1 progeny suggested that (1)in general, the segregation pattern of meiosis among F1 hybrids confirmed a 1: 1ratio (26 normal and 30 MeiO25) and the anomalies of meiosis that occur in the first progeny are likely caused by a dominant gene; (2) male sterility is not a reliable criterion for the detection of the mei mutation, as some of the plants of F1 progeny exhibit a Mei025-type meiosis (18 of 30) and are fertile; and (3) a variation of segregation ratio (normal : mutant) ranges from 10: 1 to 3 : 10, indicating that there are differences in

161

MEIOSIS IN MAIZE

TABLE 4 Character of Meiosis and Fertility in Maize F1 Progeny from Crossing Sterile Me2025 Mutants with Original Inbred W64A Stock Pattern of meiosis and fertility

Cross (1) 32st x W64A ( 2 ) 32-5st X W64A (3) 3 2 - 7 ~Xt W64A (4) 32-9st X W64A (5) 23st X W64A (6) F1(5) X W64A

Total Normal :Mei

Normal meiosis, fertile

Abnormal meiosis (Mei025) Fertile

Sterile

Total

4

4

3

2 4 5

0 3 2

5 1 1

8 8 10 13 11 6

12

56

4

3 10 2

26 26

0 3 18

30

segregation ratio among individual crosses. It is probable that these differences are due t o the presence of a modifier gene (m factor) which normalizes meiosis in some mutant plants. The m-factor action is dose dependent in such a way that in homozygotes and heterozygoes for m factor, the pattern of meiosis is normalized (Table 4). It has been confirmed cytogenetically that the Mei025 mutation is dominant and has complete expression in homozygous and heterozygous states with respect to meiotic patterns and is incompletely dominant in relation to fertility.

2 . Cytological Analysis of MeiO25 Cytologic analysis of the Me2025 mutants has shown that meiosis proceeds correctly until metaphase I; then the chromosomes lose their contours and ability to move and cling together in a dense cluster. In pycnotic conditions chromatin remains until interkinesis. At this stage, a decondensation of pycnotic chromatin was found, but at metaphase I1 the chromatin in the cells clustered again. As a result of such meiotic anomalies, the pollen grains formed by Me2025 mutants were incapable of fertilization (Golubovskaya, 1979a; see Fig. 4). The occurrence of the modifier genes in the W64A inbred line of maize influences the expression of the Me2025 gene and has led t o the study of the main peculiarities of the latter gene without (or with weaker) action of the m gene. For this purpose mutant plants were crossed with other inbred lines (A344 and W23).Analysis of characteristics of meiosis in the segregating mutants in these crosses has

INNA N. GOLUBOVSKAYA

FIG. 2. The pattern of meiosis in Me2025 mutant segregation in F1 progeny of the individual crosses (Me2025 X A344 inbred line). (a-c) Prophase I. (a) In Pachytene, a regular pairing of homologous chromosomes is seen. (b) Arrest of meiosis after pachytene. (c) At diakinesis. (d and e) A degradation of chromatin is seen in cells. (d) Pycnosis. (el Early stage of lysis. (f 1 Cytokinesis in the cell with pycnotic chromatin. (g) Arrest of meiosis and pulverization are seen. (h) Arrest of meiosis after prophase I. (i) Pollen envelopes are formed around the cell with arrest of meiosis.

demonstrated the same pattern of meiosis as in previous studies, but there seem to be some essential differencies. The observed abnormalities, such as pycnosis of chromatin, were displaced at the pachytene stage, and meiosis in some cells with pycnosis did not proceed further, i.e., meiosis was blocked at the pachytene stage in such cells (Fig. 2).

MEIOSIS IN MAIZE

163

This observation suggests that the cytological effect of the MeiO25 gene may be associated with the blockage of meiosis after pachytene. These types of mei mutants are widespread in all eukaryotes examined. 3. mei Mutations Arrest Meiosis after Pachytene in Different Species

The recessive mutation me13 (group I linkage) in N . crassa has been described. It has been found that there is complete arrest of meiosis during early stages (Newmeyer and Galeazzi, 1974; Perkins and Barry, 1977). Two mutations, meil and mei2, were isolated in Podospora anserina (Simonet and Zickler, 1972). Both mutants have the same cytological features: meiosis is blocked at the pachytene stage and chromosomes cling together in dense clusters. The isolation of different alleles of the mei2 gene has permitted study of the influence of this gene regarding the frequency of crossing over. The mei2 gene has been found to cause increased frequency of meiotic recombination close to the centromere region and decreased frequency a t the distal region of the chromosomes (it is demonstrated for the three linkage groups) (Simonet and Zickler, 1972). The m e 8 gene of P. anserina causes chromosomal stickiness at the early displotene stage. In humans, a mei mutation led to the sterility of three sons in one family (Cantu et al., 1981). The reason for the sterility was blockage of meiosis after pachytene. I n the yeast cdc5 mutant (8.cereuisiae), meiosis proceeds correctly at 25"C, but when cultures are transferred to 34"C, meiosis stops at pachytene and remains a t this stage until the yeast culture is returned to 25°C. Electron microscopy of prophase I of meiosis has demonstrated that, in the presence of temperature arrest of meiosis, the SC maintains, and as a result, the frequency of meiotic recombination is increased (Simchen et al., 1981). In the cdc4-3 mutants of yeast, meiosis is also arrested a t pachytene a t 34°C. There is also an increase in meiotic recombination, and reduplicated polar bodies of spindle do not pass to opposite poles but rather lag in the center of the cell (Byers and Goetsch, 1982). The arrest of meiosis at pachytene due to temperature and prolonged retention of the SC in the cells increases the rate of crossing over and nondisjunction of doubled pole structures. The result of these irregularities is that disjunction of homologous chromosomes does not begin (Simchen et al., 1981; Byers and Goetsch, 1982). These facts throw light on the mechanisms of the anomalies in mei mutants, which arrest meiosis at prophase I. Moreover, the data help to interpret these mutations as mutations that act in meiosis inversely in comparison of desynaptics, which lead to the precocious destruction of the SC.

164

INNA N. GOLUBOVSKAYA

4. Blockage of Meiosis at Prophase I and the Phenomenon of

Chromosomal Stickiness in sticky Mutants Consideration of the above-mentioned phenomena has led to the conclusion that the sticky mutations described for different species of plants [ Z . mays (Beadle, 1937); S . cereale (Sosnichina, 1970); Allopecurus (Johnson, 1944); and Collznsia tinctoria (Mehra and Rai, 197211 likely act by blocking meiosis at prophase I and that stickiness of chromosomes is the only phenotypic display. Convincing arguments may be as follows: (1) that the meil and mei2 mutations of P. anserina cytologically affect meiosis, causing stickiness of chromosomes after pachytene (and in the case of the me13 mutation, after diakinesislthus, cytologic phenotypes of these mutations are similar to those we observed in plants homozygous for the sticky mutations; and (2) that stickiness of chromosomes, being a primary meiotic abnormality for sticky mutants, is probably due to chromosomal degradation in meiocytes, which is observed cytologically as pycnosis and pulverization of chromatin. The last phenomenon is typical for both sticky and Mei0.25 mutants (Fig. 2). In oogenesis of different animal species, natural arrest of meiosis is observed. Prophase I, metaphase-anaphase I, metaphase 11, and the female pronucleus are the stages in which reversible arrest of meiosis takes place. In mammals, two blockages of meiosis are possible: the first is at prophase I and the second is a t metaphase I1 (Dyban and Baranov, 1978). A study of causes of parthenogenetic development of egg cells in a n inbred line (LTISu) of mouse (Stevens and Varnum, 1974) has demonstrated that natural blockages of meiosis are under genetic control. Use of biochemical markers has shown that parthenogenesis is a n effect of removing the natural blockage of meiosis in some oocytes. Completion of the first and second divisions of maturation following initiation of cleavage division leads to parthenogenesis and the appearance of ovarian teratomas in mice (Stevens and Varnum, 1974; Epping et al., 1977). The genes described in the following section cause reversible blockage of meiosis. Mutations of these genes lead either to irreversible blockage of meiosis, as was previously described for humans (Cantu et al., 1981) or to the reversal of natural blockage of meiosis, as observed in some ovule cells (LTISv). D. mei GENESOF MAIZE:IMPAIRING THE PAIRING OF HOMOLOGOUS CHROMOSOMES One of the first meiotic mutations was a desynaptic mutation (asynaptic or as; chromosome l), isolated and studied by Beadle

MEIOSIS IN MAIZE

165

(1930). A second gene, desynapsis (dy),was also isolated (Nelson and Clary, 1952). One important cytogenetic distinction of these mutants associated with their crossing over should be emphasized, albeit without a detailed description of the cytology of the mutants. The as gene increases the percentage of crossing over in close centromeric and distal regions of chromosomes without changing the total length of the genetic chromosomal map (Dempsey, 1959; Rhoades, 1947; Beadle, 1930; Rhoades and Dempsey, 1949). It was found to be connected with redistribution of chiasmata, mostly located in centromeric and distal regions of chromosomes (Miller, 1963). The analysis of crossing over frequency in the intercalary zone of chromosome 3 has demonstrated some decreased recombination frequency, thus confirming Miller’s cytological observations (Nel, 1979). The dy gene does not influence meiotic recombination frequency, and distribution of chiasmata is unaltered. At prophase I, normally developed SC is seen, but it is disturbed a t pachytene (Maguire, 1978a,b). Univalent chromosomes of this mutant undergo the equational segregation in the first meiotic division. The comparison of these two desynaptic mutations in maize allows us to demonstrate the special function of the maintenance of chiasmata independent of crossing over. Possibly the dy gene is responsible for this function. The only question under discussion is whether the function of cohesiveness of sister chromatids in the first division of meiosis should be attributed to the SC (Maguire, 1978a,b, 1979, 1981, 1982). From this position, it is difficult to explain the reductional disjunction of chromatids in the asynuptic mutant of durum wheat (Martini and Bozzini, 1966; LaCour and WelIs, 1970). Mutations of desynuptic genes of maize (dsyl and dsy2) were induced by chemical mutagens (Golubovskaya and Mashnenkov, 1976; Golubovskaya, 1979a; Fig. 3). Two genes, nonallelic and independent of the as gene, were identified, but have not been assigned to a specific choromosome. Ultrastructural analysis of these two mei-mutants was similar, and the normally developed SC was observed at the pachytene stage of meiosis. The total length of SC per cell in desynaptic mutants did not exceed 50% of the SC length in normal plants (Golubovskaya et al., 1980; Golubovskaya and Khristoljubova, 1985). Centromeres of sister chromatids in both mutants divided at anaphase 11. Two additional nonallelic genes of desynapsis (dsy3 and dsy4) were isolated and were shown to be independent of the dsyl and dsyZ genes. Only rare bivalents were observed in cells, and chromosomes were mainly represented by univalents. The pairing of homologous chromosomes is the main event of meiosis. To provide correct pairing, specific for meiotic cells, the SC

166

INNA N . GOLUBOVSKAYA

FIG. 3. Comparison of the character of chromosomal disjunction and the shape of the spindle apparatus in normal plants and two mei mutants (dv and ms28) a t the first meiotic division in maize. (a-d) A normal plant: a pattern of chromosome disjunction and spindle shape is observed in pollen mother cells (PMCs) fixed in Wada fluid. (a) In metaphase I, regularly oriented bivalents and a normally formed spindle apparatus are seen. (b and c) Subsequent stages of chromsome segregation a t anaphasetelophase I. Spindle fibers connecting opposite poles to each other and spindle fibers running from each kinetochore toward a pole are well demosntrated. (e-h) A divergent mei mutants, a characteristic of chromosome disjunction observed in PMCs fixed in Newcomber (e and f ) and in Wada (g and h ) fluids. (e) All 10 bivalents lie randomly in the cell; a disorderly orientation of bivalents a t the metaphase plate takes place. ( f ) Disorderly segregation of chromosomes a t anaphase I. (g and h) Chromosomal spindle fibers run radially from cell center to periphery; spindle fibers connecting two poles t o each other are absent. (i-1) A ms28 mei mutant. (i and j ) Incomplete cytokinesis as a result of delaying disassembly of the spindle fibers (k and 1) after the first meiotic division.

167

MEIOSIS IN MAIZE

structure is formed. This structure should function in the cell strictly up to a certain time, and the parameters of time are also controlled by genes (normal alleles of desynaptic genes). If premature SC destruction leads to defects in pairing and to the appearance of univalents in cells, then delaying SC destruction will also be observed to cause chromosomal anomalies, for example, arrest of meiosis.

E. GENES CONTROLLING SEGREGATION

OF

HOMOLOGOUS CHROMOSOMES

The three types of mei mutations with segregation and spindle abnormalities (divergent or dv, ms43, and ms28) were studied. A common characteristic of all three genes is that they do not influence the pairing of homologous chromosomes. 1. The divergent (du) mei Mutant The divergent mutation isolated by Clark (1940) was shown to be responsible for determining the divergent shape of the spindle. Attempts to map the du gene with the help of B-A translocations have been made (Curtis, 19831, and the six arms of maize chromosomes lS, 5L, 5S, 6S, 7L, and 9 s are excluded. Analysis of meiotic stages in homozygotes for the dv gene demonstrated the absence of ordered orientation of the homologous chromosomes between opposite poles at metaphase I (Fig. 3e and f ) , perhaps due t o partial or completely blocked metakinesis. The defect in the shape and function of the spindle apparatus has been assumed to be in the du mutants. It seems there is no normal two-pole spindle apparatus in the du mutants: a central spindle apparatus is absent and only chromosomal spindle fibers run radially from the center to the periphery of the cell (Fig. 3; compare a-d and g and h). Thus, the dv gene impairs the spindle structure. 2. T h e ms43 Mutant The ms43 gene, located in the short arm of chromosome 8, is the second mei gene that induces irregular disjunction of homologous chromosomes (Golubovskaya, 1979, see Fig. 5; Golubovskaya and Distanova, 1986). In spite of detailed cytologic analysis of meiosis in this mutant, mechanisms of the irregular segregation of chromosomes remain unclear. Analysis of the spindle morphology in the first meiotic division and analysis of a birefringence of the spindle fibers did not

168

INNA N. GOLUBOVSKAYA

show distinctive differences in either criteria in the ms43 mutant and in normal plants (Shamina et al., 1981; Shamina and Gruzdev, 1987). Careful analysis of the spindle structure at the first and second divisions of meiosis in the ms43 mutants, fixed in Wada fluid, has shown that the ms43 gene mainly disturbs the orientation of two spindles relative to each other in the second division of meiosis, and the orientation of spindle fibrils within the spindle apparatus. As a result, the two spindles in mutant plants dislocated each other under oblique angles or formed a joint (common) spindle (Fig. 4A and B). A type of mei mutation that changes the orientation of the cell division spindle during meiosis has been described in the potato (parallel spindle or ps gene) (Mok and Peloquin, 1975) and in the sugar beet (Maluta, 1980). In these two cases, the two spindle apparatuses a t the second division of meiosis are situated parallel to each other instead of their normal location a t an angle of 60". In maize, the situation is just the opposite: the normal parallel disposition of the two spindle apparatuses is changed such that the spindles are at different angles.

3. The ms28 mei Mutant The ms28 mei gene is the third gene that effects abnormal segregation of chromosomes in meiosis in maize (Golubovskaya and Sitnikova, 1980); the gene maps to the short arm of chromosome 1(Golubovskaya, 1987). In ms28 homozygous mutants, all anomalies of chromosomal segregation and cytokinesis are dependent upon a delay of spindle fibril depolymerization, which in normal meiosis begins promptly after anaphse I (Fig. 3i-1). In Aspergillus, a recessive mutation, benA33, has been demonstrated to induce hyperstabilization of microtubules in meiotic spindles and results in structural changes of p-tubulin (Oakley and Morris, 1981). In this mutant, similar to the ms28 mutant, the spindle assembly process occurs normally but the disassembly process is either eliminated o r strongly delayed. elongate (el), CONTROLLING THE F. A mei MUTATION,

SECOND DIVISIONOF MEIOSIS

This mutation was obtained and studied by Rhoades (1956). The el gene has been mapped a t the long arm of chromosome 8 (Curtis, 1983). The genetic effect of the mei gene is manifested in the appearance of numerous unreduced egg cells, resulting in presentation of the polyploids in the progeny of the mutant plants. Cytogenetic investigations of microsporogenesis in mutant plants have demonstrated that

MEIOSIS IN MAIZE

169

the el gene has no influence either on chromosomal pairing or on crossing over (Rhoades and Dempsey, 1966; Nel, 1975). It is probable that the el gene is efficient at the second division of meiosis and causes defective cytokinesis. An additional effect of the gene is an elongated process of chromosome spiralization in meiosis. Alexander (1957) utilized the el gene to obtain polyploids in maize. A recessive mutation with the same cytologic effect (the elongated chromosomes) was obtained in diploid S. cereale (Sosnichina, 1970).

G . A mei MUTATION, variable (ua), IMPAIRING CYTOKINESIS A recessive ua mutation was isolated by Emerson and was investigated and mapped at chromosome 7 by Beadle (1932a). Cytologic analysis of mutant plants has revealed abnormalities in cytokinesis, accompanying both the first and the second divisions. This mutation has different degrees of expression and penetrance of sterility; therefore, portions of the anthers are thrown out and some of the pollen grains can be differentiated. The percentage of sterile pollen grains in different plants ranges from 30 to 90%. In Drosophila a recessive autosomal mutation with the same cytologic and genetic phenotypes was induced by ethyl methanesulfonate (EMS) and was called (ms 2R)(Romrell et al., 19721.

H. mez MUTATIONS ( p a m l AND p a m 2 ) CAUSING NONSPECIFIC MULTIPLE ABNORMALITIES OF MEIOSIS This type of mutation has not been previously discussed in the literature (but see Golubovskaya and Mashnenkov, 1976; Golubovskaya, 1979a). It seems that all events of meiosis are impaired. There are several characteristics of meiosis in the mutant plants: 1. Among uninuclear cells, there are multinuclear microsporocytes (cenocytes) (14%) that contain 2-14 nuclei and divide autonomously and synchronously; there are also polyploid cells (6%). 2. Meiosis, as a rule, begins in all cells synchronously and proceeds asynchronously after pachytene. 3. The degradation of chromatin by pycnosis and lysis is observed in uninuclear and multinuclear cells (19.2%). 4. In different cells of the same anther, meiosis occurs according to its own program: normal cells comprise 39% of the total number; in other cells, either desynapsis is observed, meiosis is substituted for mitosis, or cytokinesis does not occur.

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FIG. 4. The shape of the spindle apparatus a t the first (A) and second (B) meiotic divisions in the ms43 mei mutant (Wada fluid). (A) The first meiotic division. (a-g) Stepwise stages of chromosomal disjunction through metaphase I to interkinesis. Normal spindle apparatus shapes are seen (compare Fig. 3a-d). (B) The second meiotic division (a-i). (a and b) Abnormal segregation of chromosomes in the PMCs without cytokinesis after the first meiotic division. (c) Tripolar spindle in the cell. (d and e) Lack of normal parallel orientation of the two spindle apparatuses a t metaphase 11. (f-h) The two spindle apparatuses approach each other (f and g) and complete fusion of two spindles (h). (i) A two-nucleus cell as a result of meiotic abnormalities.

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FIG. 4B.

It is clear that the pam-type mutations concern genetic systems that regulate meiosis. Activation of these systems proceeds in premiotic mitoses, otherwise it is difficult to explain the existence of multinuclear masses of cenocytes at prophase I. The pam mutant looks as if different types of mei genes (ameiotic, desynaptic, ms43, and ua) are joined together in the same plant. This

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list could be supplemented with a mu gene, which causes the appearance of cenocytes and was described in barley (Smith, 1942). Only amphidiploids of distant hybrids, for example, Triticale, have the same pattern (but more weak) of meiosis with different types of abnormal meiocytes as p a m mutants. I. PRECOCIOUS POSTMEIOTIC MITOSIS: A polymitotic ( P O ) GENEWITH A SERIESOF ALLELES( m s 6 AND m s 4 ) Beadle (1928) obtained a recessive mutation, termed polymitotic, and located it on chromosome 6. The ms6 gene, allelic to the PO gene, was found among genes that determine nuclear male serility in maize (Beadle, 1928, 1933). Other alleles of the PO gene (rns4)were induced with N-nitrosomethylurea. In mutants homozygous for the PO gene, two postmeiotic mitoses that normally accompany pollen grain formation apparently begin prematurely at the tetrad stage and thus the replication of DNA needed for these process does not occur. It is this event that is responsible for the pattern of anomalies in the meiosis of mutants (Fig. 5 ) and for the formation of sterile pollen grains. Three allelic mutations of the polymitotic gene in maize, isolated in different independent genetic experiments, have indicated that the transition from meiosis to postmeiotic mitosis is controlled by a single gene with a series of alleles. This type of mei mutation is interesting not only in itself but also as a mutation forestalling a sequence of events in meiosis. Probably, a great role in establishing meiosis as a developmental process is played by the mutation providing the precocious events.

J. SUMMARY The types of mei mutants currently known in maize are summarized in Table 1. Similar data for rice mei mutants have been obtained

FIG. 5. A comparison of normal processes of the first postmeiotic mitosis and processes in the polymitotic rnei mutant. (a-d) The first postmeiotic mitosis during the process of maturation of a pollen grain through prophase to telophase in maize. (e-h) The abnormalities caused by the polyrnitotic gene. It is clear that the precocious postmeiotic mitosis induced a t the tetrad stage of meiosis is a primary function of the P O gene. Abnormal segregation of chromosomes during precocious postmeiotic mitosis is a result of a delayed or omitted reduplication of chromosomes. The first postmeiotic mitosis is complete with the formation of octads ( i ) . If the process is incomplete, tetrads separate into definite sporads (i and k).

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(Kitada et al., 1983). Among 281 mutants with nuclear male sterility induced with NMM, 25 appeared to be meiotic; 19 of them influence the pairing of homologs and the other 6 rnei mutations have a defect in cytokinesis and disjunction of homologous chromosomes. The meiotic abnormalities of the MM23 mutant in rice are similar to that of the ms28 maize mutant, and the MM24 mutation in rice causes multiple defects of meiosis like the pam mutation in maize. Let us estimate the percentage of mei mutations among the total number of mutations causing nuclear male sterility. In our experiment the total number of induced visible mutations was 856. Among them, 93 are mutations with nuclear male or both male and female sterility. Of lines with nuclear male sterility, 52 have been studied cytologically, and 9 mutations appeared to be meiotic. If its is considered that half of the mutations of the total number are allelic, the percentage of mutations causing male sterility of the total number of visible mutations is 10.8% (46.6: 428); hence the percentage of the mei mutations among the nuclear ms mutations is 19.3%(9 :46.5) (Mashnenkov and Golubovskaya, 1980). These data are similar to the data obtained for D. melanogaster. Among 600 induced male-sterile mutations, 50 were cytologically analyzed, with 20% consisting of mei mutations (Lifschytz and Meyer, 1977).

Ill. Cytogenetic Evidence for the Genetic Control of Meiosis

A. THE CHOICEOF mei MUTATIONS AS EXPERIMENTAL MODELS The mei-mutants can be utilized to obtain experimental proof that meiosis is under genetic control. Double mei mutants were chosen as an experimental model. To prove that there is independent action of the mei genes that control different cytogenetic events during meiosis, it was necessary to utilize such mutants. Independent expression of the two genes is expected in double mei mutants; i.e., in the progeny of self-pollinated double heterozygotes, the four classes of meiotic patterns were expected in a ratio of 9 :3 :3 : 1.The pattern of last class of plants, comprising 1/ 16 and represented by double homozygotes, was easily revealed cytologically. In conforming with the idea of sequential switching on of genes, meiosis should follow the pathway of the gene switched on first in double homozygous mutants. In genetic terms, this would mean that each mutation is epistatic to the ones aligned next in the ordered series

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of the mei mutations. In this case the segregation ratio in Fz progeny will be 9 : 4 : 3 instead of 9 : 3 : 3 : 1,because double mei mutants (1l16) will be added to the class of plants homozygous for the epistatic gene. It is important that the mei genes used should have been located on different chromosomes; in the opposite case, the linkage effect will superimpose on a n interaction of these mei genes. Moreover, mei genes specific for meiosis during microsporogenesis or macrosporogenesis should have been excluded from model experiments. The phenomenon of unisexual male sterility caused by rnei genes (ms28 and ms43, for example) is very mysterious for maize, a monoecious diclinous species. It is important to exclude a specific action of these genes on meiosis in microsporogenesis.

The tassel seed and meiotic Genes The problem of excluding mei genes having specific actions in microsporogenesis can be solved genetically with the help of mutations that determine the sex of the maize flowers. Mutations transforming sex are widely used in genetic analyses of Drosophila to reveal the specific action of lethal genes in males or females (Belote and Lucchesi, 1980). The transformer (tra) and intersex (ix) mutations, which transform genotypic females (XX) into phenotypic males, were used to prove a specific action of the SR (sex ratio) factor on genotypes having Y chromosomes (Miyamoto and Oishi, 1975). For our purposes, the tassel seed mutations, which cause in maize the transformation of male flowers into female flowers, resulting in the development of ovules and silks in the tassel instead of the anthers, are the most efficient. If nongenetic factors are responsible for male sterility without female sterility in some rnei mutants of maize, a n expression of the mez mutations in female meiosis of double homozygotes (tslts meilmez) and the segregation of sterile plants among the tassel seed forms in Fz progeny is expected. If male sterility is determined by a specific action of rnei genes during meiosis in microsporogenesis or of ms genes during gametogenesis after meiosis, all tassel ears of the double mutants will be fertile and the class of tassel seed plants in F2 progeny will be exclusively fertile. In our experiments, the tassel seed (ts2) mutation, the mei afd mutation (responsible for the substitution of the first meiotic division into mitosis and resulting in bisexual sterility), and mutations causing unisexual male sterility were used. The last class is formed by two types of mutations: two mei mutations that induce irregular chromosome disjunction (ms28 and ms43), and the ms2 mutation, which has

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no effect on meiosis but induces abnormalities during the process of pollen grain maturation. The mei ufd mutation and the ms2 mutation were used as controls for ms28 and ms43. Results of genetic analysis for two characteristics-the tassel morphology and sterility in F2 progeny of these combinations of crossesare represented in Table 5. The results of segregation in F2 progeny from crosses between tassel seed mutants and three mei mutants (ufd, ms28, and ms43) correspond with the expected ratio 9 :3 : 3 : 1,and are distinguishable from the pattern of segregation of a cross between ts2 and ms2 mutants. In this last cross, the segregation is 9 :4 : 3, because the tassel seed plants among the F2 progeny are represented by the fertile plants. Thus, the specific action of ms43 and ms28 genes is refuted by these data, and these rnei genes could be used in cytologic experiments despite the fact that both of them caused unisexual male sterility.

B. INDEPENDENT ACTIONOF GENESCONTROLLING DIFFERENT MEIOTICEVENTS For this experiment, we chose two mei genes, the desynuptic gene (us) responsible for the pairing of homologous chromosomes and the TABLE 5 Expression of ms28 and ms43 on the Background of Homoeotic tassel seed Mutation" Fz segregationb

Cross ms43lms43 x ts2lts2 ts2lts2 x ms43lt ms28lms28 x ts2lts2 ts2lts2 x ms281+ ts2Its2 x afdl+ ts2lts2 x afdl+ ms2lms2 x ts2lts2

Genotype of F1 as defined by selfing

a

b

a

b

Total

ts2/+ ms43/+ t s 2 I t ms43/+ ts2l+ t / + ts2/+ ms28/+ ts2/+ ms28/+ ts2I+ +I+ ts2/+ +lafd ts2/+ +I+

61 39 67 64 60 23 72 110

5 3 0 9 21 0 19 0

13 10 23 14 25 5 26

2 4 0 3 5 0 9 0

81 56 90 90 111 28 118 136

ts2/+ +lafd ts2/+ ms2/+

104 181

31 63

25 79

7 0

167 323

Normal

ts2lts2

18

a An experiment with the ms2 gene was made in another year for comparison with the rest of the mei mutants, because the afd mutant was used as a control. a, Fertile plants; b, sterile plants.

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ms43 gene controlling the segregation of chromosomes. A cross was made in the next scheme: P ms43/ms43 x 6 a s / + . In the progeny of double heterozygotes, segregation into four types of patterns of meiosis was obtained: 32 plants had normal meiosis, 7 had the as type, 8 had the ms43 type, and 6 had the as ms43 type, joining the characteristics of the two mei mutations. This ratio corresponds to the expected 9 :3 :3 :1 ratio (x2 = 3.69, df = 3, 0.5 > p > 0.25). The cytology of meiosis in double homozygotes as compared with the original single mutants (Table 6) has shown that the first division of meiosis proceeds like that of the desynaptic (as)mutant, with impaired homologous pairing. The effect of the second mei gene is added and defects of chromosomal segregation and cytokinesis are observed. The independent action of two mei genes responsible for different events of meiosis is proved by the data. This means that the as gene product is not needed for the occurrence of the events controlling the ms43 gene.

C. CONSEQUENTACTIVATION OF rnei GENESIN MEIOSIS The stepwise activation of mei genes, from the initial steps of meiosis through the steps involved in the pairing of homologous chromosomes, has been analyzed. The primary elementary events of this complex process have become clear as a result of investigations and comparisons of different types of mei mutations. These events are shown in

TABLE 6 Meiotic Lesions in Single and Double mei Mutants Affecting Pairing and Segregation of Chromosomes Metaphase I: Number of bivalents (cell %) Genotype aslas +I+

+I+ ms43lms43

aslasms43Jms431

10

9-7

6-4

3-1

0

Total cells

3.8 75.0 0

1.8 24.5 0

16.2 0.5 6.8

49.9 0 27.2

28.3 0 66.0

106 171 132

Meiosis 11: Type of tetrads (cell %) ~

With With Cells at Normal micronucleus macronucleus Polyads M1, A l , A2 aslas +I+

+ I + ms43lms43 aslas ms43lms43

26.5 26.2 9.3

26.5 9.5 22.7

47.0 0 8.0

0 29.9 34.8

0 34.3 25.2

530 695 337

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,,,

INNA N.GOLUBOVSKAYA

PAIRING OF HOMOLOGOUS CHROMOSOMES

+ Initiation

of meiosis

am, Pam

I

of + Alignment homologs

?

+

as

Initiation of SC formation

+

afd

Completion of SC formation

+

Destruction of formed SC

dSY

FIG. 6 . Elementary steps at the start of meiosis and the rnei genes controlling these steps. This sequence and the genes have been established by investigating different types of mei mutants and by comparing them to each other.

Fig. 6. This logical scheme enables determination of the time of action of the known mei genes controlling the initial events of meiosis: am++ as+ + afd+-+dsy’

If this gene chain is correct, then the ameiotic mutation is epistatic over the asynaptic gene and the others following it, the asynaptic gene is epistatic over the afd gene and the others following it, and the afd gene is epistatic over the dsy gene. Unfortunately, the asynaptic gene completely blocking the SC formation is absent in mei mutation in maize. There are only three types of mei mutations-the ameiotic, which completely blocks meiosis; the afd, which converts the first meiotic division into mitosis; and the desynaptic mutations (dsy and as), which cause premature destruction of the SC and, as a result, defective pairing of homologous chromosomes. Results of epistatic interactions between three pairs of mei genes are demonstrated in Table 7. 1. Interactions between am and afd As expected, four plant genotypes in a ratio of 1 : 1 : 1 : 1 were obtained in the progeny from the first cross shown in Table 7 (A). Only double heterozygotes were of interest to us, because, in their selfing, progeny could be segregated from the double homozygotes. There are three types of meiotic patterns among the progeny: plants with normal meiosis, with the a m type of meiosis, and with meiosis of the afd type in a ratio of 9 : 4 : 3 (x2 = 0.44,df = 2, 0.9 > p > 0.75).

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TABLE 7 Epistatic Relationships of mei Genes Controlling the Sequential Steps of Meiosis

Fz segregation" Cross, genotype, and ratio in the F2 progeny as defined by selfing

Number of family

(A) afdl+ x aml+ 114 +I+ +I+ 1 / 4 a m / + +I+ 114 + I + afdl+ 114 a r n l f afdl+

(B) dsyl+ x afdl+

114 +I+ +I+ 114 +I+ dsyl+ 1 / 4 a f d l + +I+ 114 a f d l f d s y l i

(C) aslas x afdl+ 112 + I + ad+ 112 afdl+ asl+

3 1

Fertility a

b

Type of meiosis C

d

46 0 34 15 52 14 140 116 - 9:7

46 34 52 140

343 0 84 25 346 106 179 141 - 9:7

60 84 64 179

am 0 15 0 64 9:4:3 afd 0 0 23 77

96 33 105 75 - 9:7

26 50

9:4:3 afd 0 24 9:4:3

Total afd 0 0 14 52

46 49 66 256

dSY 0 25 0 64

60 109 89 320

as

7 13

-

33 87

a, Fertile plants; b, sterile plants; c, normal meiosis; d, abnormal meiosis.

This means that the a m gene has recessive epistasis over the afd gene, or, in other words, the presence of the am+ gene product is necessary for initiating the events controlled by the afd+ gene. The am+ and afd+ genes control the same meiotic pathway, although the am+ gene switches on earlier than the ufd' gene during meiosis; i.e., the afd+ gene or its product remains inactive until the product of the am+ gene appears.

2. The Pair afd-dsy The same result has been obtained with other pairs of mei genes [see Table 7 (B)]. Segregation of the meiotic pattern among the progeny of selfed double heterozygotes corresponds well with the expected ratio of 9 : 4 : 3, with epistasis of the afd gene over the dsy gene. To determine whether the interaction of the two last types of mei mutations is a rule and not an exception, the other desynaptic gene (as)

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INNA N. GOLUBOVSKAYA

was paired with the afd gene. In this case [Table 7 (C)], afd is epistatic over as (x2 = 0.5, df = 2 , p > 0.3). These data confirm the previous conclusions concerning the necessity of the afd' product action for realizing the events controlled by desynuptic genes. Analysis of epistatic groups of genes is widely used in genetics for establishing genetic pathways. The common genetic pathway controls complex cellular processes such as sensitivity to radiation and to chemical mutagens. Analysis of double mus mutants in yeast has allowed establishment of three independent genetic pathways for repair of damage caused by UV irradiation and X rays (Haynes, 1978). Genetic analysis of double mutants and isolation of the epistatic groups has shown that there are concrete stages in the genetic regulation of the DNA repair process in yeast (Game and Cox, 1972). The characteristics of interaction of mei mutations with mus mutations in D . melanogaster have been studied (Smith, 1976; Smith et al., 1980). Recombination-defective mutants are known at the X chromosomal loci in Drosophila (mei9 and mei41, for example). In addition, by decreasing the frequency and altering the distribution of exchanges along the chromosome length during meiosis in females, these mutations produce elevated frequencies of nondisjunction of all chromosome pairs. The recombination deficiency and strong mutagen sensitivity of the mei41 and mei9 genes suggest that these loci specify the function essential for both meiotic recombination and the repair of damage caused by mutagens in somatic cells. A demonstration of allelic relationships between mus loci and the mei41 locus suggests the previous conclusion. Analysis of UV sensitivity in the double mutant, with joined mei9 and mei41 loci, has shown that mei9 and mei41 interact synergistically, with increasing UV sensitivity, and suggest that these loci function in alternative pathways for the repair of UV-induced damage. These genetic data have been proved biochemically. It seems that the mei9 locus reduces the rate of both repair replication and pyrimidine dimer excision and that it is defective in a postincision step of excision repair. The mei41 locus is defective in a pathway of postreplication repair (Baker et al., 1976a; Boyd et al., 1976; Smith et al., 1980; Boyd and Setlow, 1976). In the double mutant mei9 m ~ s 2 0 5two ~ , loci interacting epistatically are defective in the process of excision of pyrimidine dimers. The use of double mutants proved to be effective in genetic control of different steps in yeast cell division cycle pathways. Isolation of epistatic groups of cdc genes, genes having additive interactions and independent abilities in S. cereuzsiue, helped to determine the genetic

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code for control of different cell cycle steps (Hartwell, 19781, and to understand the function of cdc genes in the process of chromosomal disjunction (Murray and Szostack, 1987) and the function of centromeric regions (Cumberledge and Carbon, 1987). In our study, double mei mutants were used for investigation of the basic characteristics of genic control during meiosis in higher plants (Golubovskaya, 1979a; Golubovskaya and Urbach, 1980; Golubovskaya et al., 1980). Direct cytogenetic evidence for a postulated chain of rnei genes that activates the initial steps of meiosis and for a conforming, sequential switching off the mei genes has been obtained from studies of double mei mutants in maize. D. HIERARCHY AMONG mei GENES If the highest hiearchical level in mei genes occurs then genic function is required for initiation of several independent meiotic events simultaneously. The occurrence of a gene with such a function in meiosis would require u priori that the locus be involved in both pairing and segregation of homologous chromosomes and also in the control of other meiotic steps. The wild allele of the ameiotic gene, known from the mei genes in maize, could be proposed for this role. The experimental data presented in Table 7 demonstrate that the am+ locus is necessary for initiation and completion of homologous chromosome pairing. To prove that the am+ gene is capable of inducing the disjunction of chromosomal pairs, the double mutants amlam ms43lms43 were used. The ms43 gene is required for disjunction of chromosomes. The disjunction defect in ms43 mutants is manifested as impaired spindle orientation in meiotic cells (Fig. 4B). In addition, it has been demonstrated that the disjunction defect of the ms43 gene shows up independently from the desynuptic gene (as) in double rnei mutants (aslas ms43lms43). If the function of the a m gene is required for meiotic disjunction, it should be expected that a m is epistatic over ms43. In the opposite case, the defect of disjunction induced by ms43 on the background of ameiotic mitosis should be expected, because in ameiotic mitoses the tissue and cell barriers are removed. The results of segregation in F2 progeny in the relative pattern of meiosis seen experimentally clearly demonstrate the epistatic effect of the a m gene over ms43. The segregation of 127 fertile plants and 87 sterile plants corresopnds to the ratio of 9 : 7 (x2 = 0.85). Cytologic analyses of 105 out of 214 plants have indicated that 57 plants have normal meiosis, 27 have meiosis of the a m type, and 19 have meiosis of

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INNA N.GOLUBOVSKAYA

the ms43 type. This segregation is in accordance with the ratio of 9 : 4 :3 (x2 = 0.4). A detailed study of meiosis in ameiotic plants did not reveal any anomalies of chromosome disjunction. All 29 am segregants underwent meiosis typical for ameiotic mutants (Fig. 7). There were no anomalies in the segregation of chromosomes in 574 cells counted at the metaphase-anaphase stages. Therefore, the ms43 mutations are not expressed in ameiotic mitosis. In other words, the am+ gene product is necessary for realizing the events controlled by the ms43 gene. Hence, the wild allele ameiotic function is required for both pairing and disjunction of homologous chromosomes. The use of tusseZ seed mutations and analysis of the nature of the interaction of the mei genes (especially am and ms43) poses a problem

FIG. 7. The nature of cell division of the ameiotic mutants segregated in the Fz progeny in the cross rns43ims43 x a m / + . (a-e) Stages of mitotic cycle in the ameiotic plant, There are neither abnormalities of chromosome segregation nor of spindle shape.

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regarding the specific action of mei genes. Experiments with the tassel seed gene have shown that the mei genes ms28 and ms43 appear to fail to specify function as required for male meiosis. Female sterility occurred only when the male flowers of the tassel were transformed to female (a situation in double mutants tslts mslms).This gives rise to a question about the occurrence of specific genes for meiosis. On the one hand, establishment of the specific meiotic SC cytological structure and the genes controlling formation and function of the SC is most important as evidence for specificity of the genic system in meiosis. On the other hand, in Drosophila and yeast it has been proved that the same recombinant-defective genes are required for both meiotic recombination and DNA repair processes in somatic cells (Baker et al., 1976a; Gatti et al., 1980; Zimmering and Thompson, 1987). Whether the overlapping function exists exclusively for recombination-defective mutations and how the disjunction-defective genes behave are unclear. Till now it was unknown whether the same genes causing defective disjunction of homologous chromosomes could be effective in both meiosis and mitosis if tissue and cell barriers are removed. Double mutants with the am gene can help to answer these questions because the ameiotic gene induces mitotic division in the meiocytes, i.e., the cells are committed to the meiotic process throughout whole pathways of biochemical events. Hence, the ameiotic gene removes specific tissue and cell barriers and there are many possibilities for the expression of mei genes that control disjunction of chromosomes, if the genes have, in part, a common function in meiosis and mitosis. Epistasis of the arneiotic gene over the ms43 gene is evidence that meiotic chromosme disjunction is controlled by specific mei genes that are not effective in mitotic division. In conclusion, one more revision could be accomplished by mutation, i.e., controlling the assembly of the spindle apparatus in meiosis (the divergent gene in maize, for example). IV. Speculation about the Possible Pathways of Genetic Control of Meiosis

It follows from cytological analyses that the general events of meiosis from beginning to end are controlled by a group of epistatic genes, acting in sequence, for example, t o realize the events of the second division of meiosis. An early example supporting this is the dyad gene of Datura stramonzum (Satina and Blakeslee, 1935). Among the genes initiating the key steps of meiosis, the gene that switches on

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INNA N. GOLUBOVSKAYA

the previous steps should be epistatic over the subsequent genes oppressed. This is the principle of cascade regulation of meiosis. A great number of nonallelic cytologically similar mei mutants influencing the same event of meiosis have been identified, indicative of different pathways controlling the same meiotic event. It is interesting t o investigate independent pathways controlling the same meiotic event, for example, pairing of homologous chromosomes. The nullisomic state of two different chromosomes (3B and 5B) in common wheat (Kempanna and Riley, 1962; Riley and Law, 1965) is a case where the 3B chromosome is responsible for chromosomal pairing in such a way that the loss of 3B leads to the desynaptic effect in meiosis. The effect of 5B in meiosis of hexaploid wheat is determined by the Ph gene located on the long arm of this chromosome. The P h gene is responsible for permitting the homoeologous chromosome pairing. The deficiency of the P h locus (in nullisomics, 5B) or the presence of the recessiveph allele in the homozygous state permits the pairing of both homologous and homoeologous chromosomes and, as a result, some multivalents are observed in cells at metaphase I. In double nullisomic plants without 3B and 5B, two types of pollen mother cells are simultaneously present in one anther. There are pollen mother cells (PMCs) with only univalent chromosomes (16%; nullisomic state for 3B is realized) and the PMC with multivalent chromosome configurations (84%; nullisomic effect for 5B chromosome is realized). It is possible that these cytological data can be interpreted as an independent effect of two mei genes controlling the same event in meiosis and acting at the same time during meiosis. At present, it is known that the P h gene does not have any effect on the ability of chromosomes to associate and form the SC. The Ph gene effect could control the rate of pairing (Gillies, 1987). The desynuptic gene, as described above, determines the time of formation of the SC at prophase of meiosis. From studies of double mei mutants it is possible to assign such genes to specific epistatic groups in order to understand the genetic basis of meiotic pathways in higher plants and to indicate the groups of genes controlling the steps of similar pathways in meiotic cells. Initial studies of double mei mutant combinations in maize indicate that the range of interaction observed with cdc yeast mutants and n u s and recombination-deficiency mutants in Drosophila is also present in higher plant system (Hartwell, 1978; Murray, 1987; Smith et al., 1980; Baker et al., 1976b). In direct experiments with double mei mutants, the chain of mei genes a m afd dsy (as) controlling the initial steps of meiosis has

-

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185

been established. The independent action of the desynaptic (as) and ms43 genes impairing chromosomal segregation and the epistatic interaction between the ameiotic and ms43 genes have also been proved in cytogenetic experiments. This suggests that the as and ms43 genes are involved in parallel chains of rnei genes and, hence, the a m gene simultaneously switches on the two gene chains in meiosis: a m + afd -----+

+ dsy

(as) ms43

Thus, the occurrence of both cascade and fan actions of mei genes in switching on meiosis is likely in effect. V. Conclusion: Theoretical and Applied Aspects of Meiosis Genetics

In addition to “canonical meiosis,” as charcterized for maize and other organisms, variations of meiosis have been observed among virtually all sexually reproducing organisms. There are species with achiasmatic meiosis; species with semimeiosis (Davison, 1984) involving only the first meiotic division and omitting the pairing of homologous chromosomes and crossing over; holocentric species in which the first division of meiosis is equational, i.e., centromeres of sister chromatids divide at the first meiotic division; apomictic plant species in which various transformations from regular meiosis to mitosis take place; and parthenogenetic animal species in which different mechanisms of blockage of chromosome pairing and crossing over occur (see in detail John and Lewis, 1965; Raikov, 1975; Noda, 1975; Oakley and Morris, 1981; Gustafsson, 1946; Rasmussen, 1977; Rasmussen and Holm, 1981). The various types of meiotic processes are interesting in several aspects: (1) in understanding different modes of meiotic division; (2) in the possibilities of several variants of meiosis existing as a result of fixation of meiotic mutations in the evolutionary process; and (3) in the opportunity for predicting the existence of new types of mei mutants. The general peculiarities of mei mutants include the formation of gametes with unreduced diploid chromosome sets, gametes with noncrossover chromosomes, and gametes with aneuploid chromosome sets. All these characters can be used successfully for applied genetic programs. The ability of the elongate mei mutants of maize to produce unreduced egg cells was used to provide tetraploid maize (Alexander, 1957). The same ability of parallel spindle mei mutants in sugar beet and potato was used to obtain polyploids and fertile hybrids in distant crosses (Maluta, 1980; Peloquin, 1983).

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The ability of mei mutants to produce aneuploid gametes can be used to obtain a series of aneuploid stocks in different plant species. Thus, a series of aneuploids in common wheat was obtained by Sears with the help of 3B nullisomics, which have a desynuptic effect in meiosis. The series of aneuploids can be used to obtain addition, substitution, and translocation lines, if they are involved in interpsecies and intergeneric crosses. The mei mutants can be used for genetic construction of apomictic plants. The mei mutants may play a significant role in the creation of a polyploid series of chromosomal sets in the evolution of angiosperms, because they are not only donors of urneduced gametes, but are also testers for the unreduced gametes and they have the ability to give fertile progeny in cases of fertilization of egg cells with unreduced pollen cells.

REFERENCES Alexander, D. E. (1957). The genetic induction of autotetraploidy. A proposal for its use in corn breeding. Agron. J . 49,40-43. Baker, B. S., and Carpenter, A. T. C. (1972). Genetic analysis of sex chromosomal meiotic mutants in Drosophila melanogaster. Genetics 71, 255-286. Baker, B. S., Boyd, J. B., Carpenter, A. T. C., Green, M. M., Nguyen, T. D., Ripoll, P. D., and Smith, P. D. (1976a). Genetic control of meiotic recombination and somatic DNA metabolism in Drosophila melanogaster. Proc. Natl. Acad. Sci. U.S.A. 73, 4140-4144. Baker, B. S., Carpenter, A. T. S., Esposito, M. S.,Esposito, R. E., and Sandler, L. (1976b). The genetic control of meiosis. Annu. Rev. Genet. 10, 53-135. Beadle, G . W. (1928). A gene for supernumerary mitosis during spore development in Zea mays. Science 70,406-407. Beadle, G. W. (1930). Genetical and cytological studies of mendelian asynapsis in Zea mays. Mem. Cornell Uniu. Agric. Exp. Sta. 129, 1-28. Beadle, G. W. (1932a). Genes in maize for pollen sterility. Genetics 17, 413. Beadle, G . W. (193213). A gene in Zea mays for failure of cytokinesis during meiosis. Cytologia 3, 142-155. Beadle, G. W. (1933). Polymitotic maize and the precocity hypothesis of chromsome conjugation. Cytologia 5, 118-130. Beadle, G. W. (1937). Chromosome aberration and the gene mutation in sticky chromosome plants of Zea mays. Cytologia (Fujii Jubilee Vol.),43-56. Belote, J. M., and Lucchesi, J. C. (1980). Male specific lethal mutation of Drosophila melanogaster. Genetics 96, 165-186. Bogdanov, Yu. F. (1975). Ultrastructure of chromsomes into meiosis and synaptonemal complex. In “Cytology and Genetics of Meiosis” (V. V. Khvostova and Y. F. Bogdanov, eds.), pp. 58-137. Nauka, Moscow (in Russian). Boyce, R. P., and Howard-Flanders, P. (1964). Release of ultraviolet light-induced thymine dimers from DNA in E . coli K-112. Proc. Natl. Acad. Sci. U.S.A. 51, 293-300.

MEIOSIS I N MAIZE

187

Boyd, J. B., and Harris, P. V. (1981). Mutants partially defective in excision repair at five autosomal loci in Drosophila melanogaster. Chromosoma 82, 249-257. Boyd, J. B., and Bellow, R. B. (1976). Characterization of postreplication repair in mutagen sensitive strains of Drosophila melanogaster. Genetics 84, 507-526. Boyd, J. B., Golino, M. D., Nguyen, T. D., and Green, M. M. (1976). Isolation and characterisation of X-linked mutants of Drosophila melanogaster which are sensitive to mutagens. Genetics 84, 485-506. Boyd, J. B., Harris, P. V., Osgood, C. J., and Smith, K. S. (1980). Biochemical characterization of repair-deficient mutants of Drosophila. In “DNA Repair and Mutagenesis in Eucaryotes” (W.M. Generoso, M. D. Shelby, and F. J. de Serres, eds.), pp. 209-221. Plenum, New York. Byers, B., and Goetsch, L. (1982). Reversible pachytene arrest of Succharomyces cerevisiae a t elevated temperature. Mol. Gen. Genet. 187, 47-53. Cantu, J. M., Rivas, F., HernandezJauregue, P., Diaz, M., Cortes-Gallegos, V., Vaca, G., Velazques, A,, and Ibarra, B. (1981). Meiotic arrest a t first spermatocyte level: A new inherited infertility disorder. Hum. Genet. 59, 380-385. Carpenter, A. T. C., and Baker, B. S. (1974). Genic control of meiosis and some observations on the synaptonemal complex in Drosophila melanogaster. Mech. Recomb. Proc. Biol. Diu. Res. Conf.,27th, pp. 365-375. Carpenter, A. T. C., and Sandler, L. (1974). On recombination-defective meiotic mutants in Drosophila melanogaster. Genetics 76, 453-475. Clark, F. L. (1940). Cytogenetic studies of divergent meiotic spindle formation in Zea mays. A m . J . Bot. 27, 547-559. Clauberg, C. D. (1959). Cytogenetic studies of precocious meiotic centromere division in Lycopersicon esculentum Mill. Genetics 44, 1335-1347. Coe, E. H., and Neuffer, M. G. (1977). The genetics of corn. “Corn and Corn Improvement,” pp. 111-224. Madison, Wisconsin. Cumberledge, S., and Carbon, J . (1987). Mutation analysis of meiotic and mitotic centromere function in Saccharomyces cereuisiae. Genetics 117, 203-212. Curtis, Ch. (1983). Mapping of dv and el. Maize Genet. Coop. News Lett. 57, 32. Davis, B. K. (1971). Genetic analysis of a meiotic mutant resulting in precocious sister centromere separation in Drosophila melanogaster. Mol. Gen. Genet. 113, 251-272. Davison, J. A. (1984). Semi-meiosis as a n evolutionary mechanism. J . Theor. Biol. 111, 725-735. Dempsey, E. (1959). Analysis of crossing over in haploid gametes in asynpatic plants. Maize Genet. Coop. News Lett. 33, 54. Dyban, A. P., and Baranov, V. S. (1978). “Cytogenetics of Mammalian Development.” Nauka, Moscow. Eeken, J. C. J., and Sobels, F. H. (1981). Modification of MR-factor activity in a repair deficient strain of Drosophila melanogaster. Mutat. Res. 83, 191-203. Epping, J. J., Kozak, L. P., Eicher, E. M., and Stevens, L. C. (1977). Ovarian teratosomas in mice are derived from oocytes that have completed the first meiotic division. Nature (London) 269, 517-518. Esposito, M. S., and Esposito, R. E. (1969). The genetic control of sporulation of Saccharomyces. I. The isolation of temperature-sensitive sporulation deficient mutants. Genetics 61, 79-89. Fedotova, Y. S., Kolomietz, 0. L., and Bogdanov, Yu. F. (1987). Electron-microscopic study of asynaptic mei-mutants of rye. “Molecular Mechanisms of Genetical Process.” Y. All-Union Symp., Moscow (in Russian). Game, J. C., and Cox, B. S. (1972). Epistatic interaction between four rad loci in yeast. Muat. Res. 16, 353-362.

188

INNA N. GOLUBOVSKAYA

Gatti, M., Pimpinelli, S., and Baker, B. S. (1980). Relationships among chromatid interchanges, sister chromatid exchanges, and meiotic recombination in Drosophila melanogaster. Proc. Natl. Acad. Sci. U.S.A. 77, 1575-1579. Gilles, C. B. (1987). The effect ofPh gene alleles on synaptonemal complex formation in Triticum aestivum x T. kotschii hybrids, Theor. Appl. Genet. 74,430-438. Goldstein, L. S. B. (1980). Mechanism of chromosome orientation revealed by two meiotic mutants in Drosophila melanogaster. Chromosoma 78, 79-111. Golubovskaya, I. N. (1973). Desynapsis-The main cause of irregularity of meiosis in incomplete wheat-Agropyron amphidiploids. Genetika (Moscow)9, 64-77. Golubovskaya, I. N. (1975). Genetical control of meiosis. I n “Cytology and Genetics of Meiosis” (V. V. Khvostova and Y. F. Bogdanov, eds.), pp. 312-343. Nauka, Moscow (in Russian). Golubovskaya, I. N. (1979a). Meiotic mutations in maize induced chemical mutagens. M.N.L. 53,66-70. Golubovskaya, I. N. (1979b). Genetical control of meiosis. Znt. Rev. Cytol. 58, 247-290. Golubovskaya, I. N. (1987). Mapping two mei-genes in maize by A-B translocation stocks. Genetika (Moscow)23, 698-706. Golubovskaya, I. N., and Distanova, E. E. (1986). Mapping mei-gene ms 43 by B-A translocation stocks. Genetika (Moscow) 22, 1173-1180. Golubovskaya, I. N., and Kristoljubova, N. B. (1985). Cytogenetic evidence of the gene control of meiosis. UCLA Symp. Mol. Cell. Biol. New Ser. 35, 723-738. Golubovskaya, I. N., and Khvostova, V. V. (1973). Symposia about meiotic problems. Ontogenesis (Moscow)4, 637-640. Goiubovskaya, I. N., and Mashnenkov, A. S. (1975). Genetical control of meiosis. I. Mei-mutation in maize afd I . Genetika (Moscow) 11, 11-17. Golubovskaya, I. N., and Mashnenkov, A. S. (1976). Genetical control of meiosis. 11. Desynaptic mutant in maize. Genetika (Moscow) 12, 7-14. Golubovskaya, I. N., and Mashnenkov, A. S. (1981). Genetic control of chromosome segregation during the first meiotic division. M.N.L. 55, 78-80. Golubovskaya, I. N., and Sitnikova, D. V. (1980). Three mei-mutations in maize impairing the segregation of homologous chromosomes. Genetika (Moscow) 16, 656-665. Golubovskaya, I. N., and Urbach, V. G. (1980). Allelic relationships between meiotic mutations with similar disturbances of meiosis. M.N.L. 55, 80-81. Golubovskaya, I. N., Nikoro, Z. S., and Khvostova, V. V. (1966).Analysis of selection on high fertility in constant 56-chromosome wheat-Agropyron hybrids. Genetika (Moscow)4, 86-97. Golubovskaya, I. N., Safonova, V. T., and Khristoljubova, N. B. (1980). Stepwise switching of mei-genes into meiosis. Dokl. Acad. Sci. USSR 250, 280-284. Gowen, J. W. (1933). Meiosis as a genetic character in Drosophila melanogster. J . Exp. ZOO^. 65, 83-106. Gowen, M. S., and Gowen, J. W. (1922). Complete linkage in Drosophila melanogaster. Am. Nut. 56,286-288. Green, M. M. (1978). The genetic control of mutation in Drosophila. Stadler Symp. 10, 95-104. Gustafsson, A. (1946). Apomixis in higher plant. I. The mechanisms of apomixis. Lunds Univ. Arskr., 1-66. Hartwell, L. H. (1978). Cell division from a genetic perspective. J . Cell Biol. 77,627-637. Haynes, R. H. (1978). Workshop summary: DNA repair in lower eucaryotes. I n “DNA Repair Mechanisms” (P. C. Hanawalt, E. C. Freidberg, and C. F. Fox, eds.), pp. 405-411. Academic Press, New York.

MEIOSIS IN MAIZE

189

Hereford, L. M., and Hartwell, L. H. (1974). Sequential gene function in the initiation of Saccharomyces cerevisiae DNA synthesis. J . Mol. Biol. 84, 445-461. Hindley, J., and Phear, G. A. (1984). Sequence of the cell division gene cdc 2 from S. pombe patterns of splicing and homology to protein kinases. Gene 31, 129-134. Hotta, Y., Bennett, M. D., Toledo, L. A,, and Stern, M. (1979). Regulation of R-protein and endonuclease activities in meiocytes by homologous chromosome pairing. Chromosoma 72, 191-201. Hotta, Y., Tabota, S., and Stern, H. (1984). Replication and nicking of zygotene DNA sequences control by a meiosis specific protein. Chromosoma 90, 243-253. Iino, Y., and Yamamoto, M. (1985a). Mutants of Schizosaccharomyces pombe which sporulate in the haploid state. Mol. Gen. Genet. 198, 416-421. Iino, J., and Yamamoto, M. (198513). Negative control for the initiation of meiosis in Schizosaccharomyces pombe. Proc. Natl. Acad. Sci. U.S.A.82, 2447-2451. John, B., and Lewis, K. R. (1965). The meiotic systems. Protoplasmatologiu 6, 202. Johnsson, H. (1944). Meiotic aberrations and sterility in Alopecurus myosuroides Huds. Hereditas 30, 469-566. Kempanna, C., and Riley, R. (1962). Relationships between the genetic effects of deficiencies for the chromosomes I11 and V on meiotic pairing in Triticum aestivum. Nature (London) 195, 1270-1273. King, R. (1970). The meiotic behavior of the Drosophila oocyte. Znt. Rev. Cytol. 28, 125-168. Kitada, K., Kubata, N., Saton, H., and Omura, T. (1983). Genetic control of meiosis in rice, Oryza sativa L. I. Classification of meiotic mutants induced by MNU and their cytogenetical characteristics. Jpn. J . Genet. 53, 231-241. Klapholz, S., and Esposito, R. E. (1980). Recombination and chromosome segregation during the single division meiosis in spo-12-Z and spo-13-I diploids. Genetics 96, 589-611. Klein, M. D. (1969). Desynaptishe Mutanten mit Unterschieden in Univalenten Verhalten. Genetica 40, 566-576. Korochkin, L. I. (1977). “The Relationships of Genes in Development.” Nauka, Moscow (in Russian). Kundu, S. C., and Moens, P. B. (1982). The ultrastructural meiotic phenotype of radiation sensitive mutant rad6-Z in yeast. Chromosoma 87, 125-132. Kushev, V. V. (1972).Zn “Mechanisms of Genetical Recombination” (S. E. Bressler, ed.), pp. 246. Nauka, Leningrad (in Russian). La Cour, L. F., and Wells, B. (1970). Meiotic prophase in anthers of asynaptic wheat. Chromosoma 29,419-427. Lee, M. G., and Nurse, P. (1987). Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc 2 . Nature (London)327, 31-35. Lifschytz, E. L., and Meyer, G. F. (1977). Characterization of male meiotic-sterile mutations in Drosophila melanogaster. The genetic control of meiotic divisions and gametogenesis. Chromosoma 64, 371-392. Maguire, M. P. (1978a). A possible role for the synaptonemal complex in chiasma maintenance. Exp. Cell Res. 112, 297-308. Maguire, M. P. (1978b). Evidence for separate genetic control of crossing over and chiasma maintenance in maize. Chromosoma 65, 173-183. Maguire, M. P. (1979). An indirect test for a role of the synaptonemal complex in the establishment of sister chromatid cohesiveness. Chromosoma 70,312-321. Maguire, M. P. (1981). A search for the synaptonemal adjustment phenomenon in maize. Chromosoma 81,717-725. Maguire, M. P. (1982). Evidence for a role of the synaptonemal complex on provision for

190

INNA N. GOLUBOVSKAYA

normal chromosome disjunction at meiosis I1 in maize. Chromosoma 84, 675686. Maluta, E. I. (1980).Mei-mutation induced formation of unreduced gametes in diploid sugar beet. In “Induced Mutagenesis and Apomixis” (D. F. Petrov, ed.),pp. 102-108.

Nauka, Novosibirsk (in Russian). Martini, G., and Bozzini, A. (1966).Radiation induced asynaptic mutations in durum wheat (Triticum durum Desf.). Chromosoma 20,251-266. Mashnenkov, A. S.,and Golubovskaya, 1. N. (1980).Mei-mutations in maize induced by N-nitroso-methylurea. Genetika (Moscow) 16,1632-1640. Mason, J. M. (1976).Orientation disruptor (or& a recombination-defective and disjunction-defective meiotic mutant in Drosophila melanogaster. Genetics 84, 545-

572.

Matsumoto, K., Ino, I., and Ishikawa, T. (1983).Initiation of meiosis in yeast mutants defective in adenylate cyclase and cyclic AMP-dependent protein kinase. Cell 32,

417-423.

Mehra, R. C., and Rai, K. S. (1972).Cytogenetic studies of meiotic abnormalities in Collinsia tinctoria. 11. Desynapsis. Can. J. Genet. Cytol. 14, 637-644. Miller, 0. L. (1963).Cytological studies in asynaptic maize. Genetics 43, 1445-1467. Miyamoto, Ch., and Oishi, K. (1975).Effects of SR-spirochete infection on Drosophila melamgaster carrying intersex genes. Genetics 79,55-61. Mok, D.W.S.,and Peloquin, S. J. (1975).Three mechanisms of 2n pollen formation in diploid potatoes. Can. J. Genet. Cytol. 17, 217-225. Moses, M. J. (1956).Chromosomal structures in crayfish spermatocytes. J . Biophys. Biochem. Cytol. 2,215-217. Murray, A. W.(1987).Cell cycle control. Nature (London)327, 14-16. Murray, A. W., and Szostak, J. W. (1985). Chromosome segregation in mitosis and meiosis. Annu. Rev. Cell Biol. 1, 289-315. Nel, P. M. (1975).Crossing over and diploid egg formation in the elongate mutant of maize. Genetics 79,435-450. Nel, P. M. (1979).Effects of the asynaptic factor on recombination in maize. J . Hered. 70,

401-406.

Nelson, 0. E., and Clary, G. B. (1952).Genetic control of semi-sterility in maize. J. Hered. 43,205-210. Newmeyer, D., and Galeazzi, D. R. (1974).A genetic factor which causes deletion of duplication in Neurospora. Genetics 74,48. Noda, S. (1975).Achiasmate meiosis in the Fritillariajaponica group. I. Different modes of bivalent formation in the two sex mother cells. Heredity 34,373-380. Oakley, B. R., and Morris, N. R. (1981).a-Tubulin mutation in Aspergillus nidulans that blocks microtubule function without blocking assembly. Cell 24, 837-847. Oakley, H.A., and Jones, G. H. (1982).Meiosis in Mesostoma ehrenbergii (Turhlluria rhubdocoela).I. Chromosome pairing, synaptonemal complexes and chiasma localization in spermatogenesis. Chromosoma 85, 311-323. Palmer, R. G. (1971).Cytological studies of ameiotic and normal maize with reference to premiotic pairing. Chromosoma 35, 233-246. Peloquin, S. J. (1983).Genetic engineering with meiotic mutants. In “Pollen: Biology and Implications for Plant Breeding” (D. L. Mulcany and E. Ottaviano, eds.), pp. 311-316. Elsevier, New York. Perkins, D. D., and Barry, E. G. (1977).The cytogenetics Neurospom. Adu. Genet. 19,

134-285.

Petrov, D. F. (1964).“Genetical Regulated Apomixis,” pp. 169.Nauka, Novosibirsk (in Russian). Rajkov, I. B. (1975).The problem of origin and evolution of meiosis. “Cytology and Genetics of Meiosis,” pp. 344-371. Nauka, Moscow (in Russian).

MEIOSIS IN MAIZE

191

Rasmussen, S. W. (1977). The transformation of the synaptonemal complex into the “elimination chromatin” in Bombyr mori oocytes. Chromosoma 60, 205-221. Rasmussen, S. W., and Holm, P. B. (1981). Mechanisms of meiosis. Hereditas 93, 187-217. Rhoades, M. M. (1947). Crossover chromosomes in unreduced gametes of asynaptic maize. Genetics 32, 101. Rhoades, M. M. (1956). Genetic control of chromosomal behaviour. Maize Genet. Crop News Lett. 30, 38-42. Rhoades, M. M., and Dempsey, E. (1949). Maize Genet. Crop News Lett. 23, 56-57. Rhoades, M. M., and Dempsey, E. (1966). Induction of chromosome doubling a t meiosis by the elongate gene in maize. Genetics 54, 505-522. Riley, R., and Chapman, V. (1958). Genetic control of cytologically diploid behaviour of hexaploid wheat. Nature (London) 182, 713-715. Riley, R., and Law, 0. N. (1965). Genetic variation in chromosome pairing. Adu. Genet. 13,57-75. Riley, R., Chapman, V., and Belfield, A. M. (1966). Induced mutation affecting the control of meiotic chromosome pairing in Triticum aestiuum. Nature (London)211, 368-369. Romrell, L. J., Stanley, H. P., and Bowman, J. T. (1972). Genetic control of spermiogenesis in Drosophila melanogaster. An autosomal mutant msl213R demonstrating failure of meiotic cytokinesis. J . Ultrastruct. Res. 38, 563-577. Sandler, L., Lindsley, L. L., Nicoletti, B., and Trippo, C. (1968). Mutants affecting meiosis in natural populations of Drosophila melanogaster. Genetics 60, 525558. Satina, S., and Blakeslee, A. L. (1935). Cytological effects of a gene in Datura which cause dyad formation in spclrogenesis. Bot. Gaz. 96, 521-532. Shamina, N. V., and Gruzdev, A. D. (1987). A study of birefringence of cell division spindles in the meiotic mutants of Zea mays. Cytologia 24, 104-110 (in Russian). Shamina, N. V., Golubovskaya, I. N., and Gruzdev, A. D. (1981). Morphological irregularities of meiosis in some met-mutants in maize. Cytologia 23, 275-279. Shimodo, C., and Uehira, M. (1985). Cloning of the Schzzosuccharomyces pombe mei-3 gene essential for initiation of meiosis. Mol. Gen. Genet. 201, 353-356. Simanis, V . , and Nurse, P. (1986). The cell cycle control gene cdc2 of fission yeast encodes a protein kinase potentially regulated by photoregulation. Cell 45, 261268. Simchen, G., Kassir, Y., Horesh-Cabilly, O., and Friedman, A. (1981). Elevated recombination and pairing structures during meiotic arrest in yeast of the nuclear division mutant cdc 5 . Mol. Gen. Genet. 184, 46-51. Simonet, J. M. (1973). Mutations affecting meiosis in Podospora anserina 11. Effect of mei-2 mutants on recombination. Mol. Gen. Genet. 123, 263-281. Simonet, J. M., and Zickler, D. (1972). Mutations affecting meiosis in Podospora anserina. I. Cytological studies. Chromosoma 37, 327-351. Smith, L. (1942). Cytogenetics of a factor for multiploid sporocytes in barley. Am. J . Eot. 29,451-456. Smith, P. (1973). Mutagen sensitivity of recombination in Drosophilu melanogaster. I. Isolation and preliminary characterization of a methyl-methanesulfonate sensitive strain. Mutat. Res. 20, 215-220. Smith, P. D. (1976). Mutagen sensitivity in Drosophila melanogaster. 111. X-Linked loci governing sensitivity to methyl-methanesulfonate. Mol. Gen. Genet. 149, 7387. Smith, P., and King, R. (1968). Genetic control of synaptonemal complexes in Drosophila rnelanogaster. Genetics 60, 335-351. Smith, P. D., Snyder, R. D., and Dusenbery, R. L. (1980). Isolation and characterization

192

INNA N. GOLUBOVSKAYA

of repair-deficient mutants of Drosophila melanogaster. In “DNA Repair and Mutagenesis in Eucaryotes” (W. M. Generoso, ed.), pp. 175-187. Plenum, New York. Sosnichina, S. P. (1970). Genetical control of meiosis in rye. 11. Genetika (Moscow) 9, 21-30. Stern, H., and Hotta, Y. (1973). Biochemical control of meiosis. Annu. Rev. Genet. 7, 37-67. Stern, H., and Hotta, Y. (1974). DNA metabolism during pachytene in relation to crossing over. Genetics 78, 227-235. Stevens, L. C., and Varnum, D. L. (1974). The development of teratomas from partheno-genetically activated ovarian mouse eggs. Dev. Biol. 37, 369-380. Uno, I., Matsumoto, K., and Ishikawa, T. (1982). Characterization of cyclic AMPrequiring yeast mutants altered in the regulatory subunit of protein kinase. J . Biol. Chem. 257,14110-14115. Vidal, F., Templado, C., Navarro, J., Brusadin, S., Marina, S., and Egozcue, J. (1982). Meiotic and synaptonemal complex studies in 45 subfertile males. Hum. Genet 60, 301-304. Westergaard, M., and von Wettstein, D. (1972). The synaptonemal complex. Annu. Rev. Genet. 6, 71-110. White, J. (1968). Chromosomal rearrangements and speciation in animals. Annu. Rev. Genet. 2, 75-97. White, M. J. D. (1973). “Animal Cytology and Evolution.” Cambridge Univ. Press., London and New York. Wood, J. S., and Hartwell, L. H. (1982). A dependent pathway of gene functions leading to chromosome segregation in Saccharomyces cerevisiae. J . Cell Biol.94, 7 18-726. Zimmering, S., and Thompson, E. D. (1987). Mutagenesis with ethyl-nitrosourea (ENU) in oogonia of repair-deficient mus(2)201 Dl Drosophila females (MTRL 040). Mutat. Res. 192, 55-58.

INDEX

A N-Acylcysteinamide,in peptide, NUV absorption, 108-109 Alcohol dehydrogenase, in developing pollen, maize, 21,24 isozyme analysis, 25 Anacomptis pyramidalis, meiocyte interconnections in pollen ontogeny, 7 Anchusa officinalis,MGS, experimental, 17 Apple, self-incompatibility overcoming mentor pollen, 46 pioneer pollen effect, 31 Arabidopsis thaliana, pollen lethal embryo mutant expression, 13 MGS, experimental, 17 Aspartate aminotransferase, isozymes in pollen during development, maize, 25 Aspergillus nidulans, benA33 mutant, meiotic spindle disassembly delay, 168 Aureolaria spp. pollen competition in natural populations, 35, 48 Avena sativa, pollen and sporophytic response to Helminthosporium victoriae, 19 Azide, inhibition of SODS, 69,72

B Bacillus stearothermophilus, MnSOD, 70 Bacteriophage T7 NUV absorption by peptide, 108-109 oxygen-inactivated 3' to 5 ' exonuclease, SOD effects, 112

Bacteroides fragilis, metal substitution in Fe-MnSODs, 71 Besleria trifloru, pollen competition, 33 Beta vulgaris, ethofumerase tolerance in pollen and sporophyte, 15 Brassica campestris leaves, FeSOD, 67 pollen, pgalacotosidase, 24 Brassica oleracea, pollen, distorted segregation for gal locus, 12

C Callase, see P-1,3-Glucanase Callose, microspore isolation, 7 Cassia fasciculata, MGS, excess pollination and, 16 Catalase deficiency in mutants not sensitive to NUV, E. coli, 102, 112-113, 119, 121-122 detoxification of NUV-induced HzOz, 111, 122 inactivation by NUV in vitro and in vivo, 107 isozymes, in pollen during development, maize, 25 Caulobacter crescentus, CuZnSOD, 67 Chemotropism, pollen tubes in vitro and in vivo, 32 Chromosomes 3B nullisomics, desynaptic effect in meiosis, wheat, 184, 186

193

194

INDEX

in mei mutants pairing impairment, maize, 164-167 segregation abnormalities, maize, 167-168 stickiness, plants, 164 X and Y in pollen tubes, role in growth rate, Silene alba, 3 Clarkia dudleyana, pollen, phosphoglucoisomerase-encoding genes, 22 Collinsia tinctoria, sticky mutants, meiosis blockage, 164 Copper induction of CuZnSOD, 82 tolerance in plants MGS and, 19-20 in pollen and sporophyte, 15 Cosmos, pollen ontogeny, 6 Costus allenii, MGS, experimental, 17-18 Crocus, pollen ontogeny, 5-6 Cucurbita pepo, MGS, excess pollination and, 16 Cucurbita spp., pollen, acid phosphataseencoding genes, 21 Cyanide irreversible inhibition of CuZnSOD, 69 resistance in Fe-MnSODs, 72 Cyclic AMP-dependent protein kinase, inactivation in meiosis initiation, yeast, 157-158

D Dactylium dendroides, SOD biosynthesis, copper-induced, 82 Dactylorchris fuchsii, pollen ontogeny, 4 Datura stramonium, meiosis, dyad gene and, 183 Deinococcus radiodurans, NUV-sensitive, FUV-resistant, 118 Dianthus chinensis, MGS, experimental, 16-17 Diethyldithiocarbamate, inactivation of CuZnSOD, 69 cqPDihydroisovalerate dehydratase, protection by SOD, E. coli, 77 Dihydroxy acid dehydratase, inactivation by NUV, E. coli, 102,109,111 DNA exogenous, direct gene transfer in pollen, 46 FUV-induced SOS repair, 129-130

NUV effects, bacteria damage and protection, models, 101-103 direct absorption, alterations and, 103-105 distinction from FUV effect, 115-117 DNA-protein cross-links, 116, 118 lesions as substrate for exonuclease 111, 116, 118 similarity to HzOz effects, 118-119 SOS repair blockage, 129-131 DNA photolyase, photoprotection against NUV, 110-111 Drosophila melanogaster, mei mutants, 150 asynaptic (as), 159 genetic analysis using sex-transforming mutations, 175 interaction with mus mutations, 180 male sterility and, 174 ord gene, 160 variable (va), 169 Drymona rubra, pollen competition, 33

E Electrophoresis, pollen protein and isozyme analysis, 21-22 Endonuclease IV, response to NUV and, 124 Epilobium canum, pollen competition in natural populations, 33 germination, style effect, 29 Escherichia coli catalase-deficient mutants, NUV resistance and, 102,112-113, 119, 121-122 genes for FeSOD and MnSOD isolation, 80, 122-123 lacking in mutants, 78,80-81 location on chromosomes, 75 sequencing, 80 hybrid between Mn- and Fe-containing SODS, 70-71,74 iron-containing SOD biosynthesis during aerobic and anaerobic growth, 82, 123 regulation by metals, 84-85 isolation, 67 three-dimensional structure, 71 manganese-containing SOD amino acid sequences, 70

195

INDEX biosynthesis during aerobic growth, 02--induced, 82-83, 123 iron regulatory role, 83-86 catalytic mechanisms, 78 isolation, 69 NUV effects absorption by dihydroxy acid dehydratase, 102, 109,111 porphyrins, 107 ribonucleotide reductase, 109 thiolated tRNA, 102, 105-106 mutagenic, 132-134 physiological growth delay, 106, 126 membrane damage, 128-129 protein synthesis induction, 126-128 sensitivity in mutants gal-Xatt deletion, 125-126 hem (hematins), 125 katE: katE, katG (catalase), 112, 119, 121-122 nuuA (thiolated tRNA), 106, 126,127 polA (5’to 3’ exonuclease), 124-125 RecA (recombinase), 125, 130-131 uur, 125, 129 xthA (exonuclease III), 123-124 oxygen toxicity, protection by SOD branched-chain amino acid biosynthesis and, 77 or,/?-dihydroisovalerateactivity and, 77 ribonucleotide reductase activity and, 78, 112 sodA sodB double mutant construction and properties, 80-81 human CuZnSOD plasmid effect, 78,114 hypersensitivity to NUV and H202, 114, 123 Euglena gracilis, FeSOD, 67 Evolution in angiosperms, pollen selection role, 41-43; see also Male gametophytic selection superoxide dismutases of three types, 68,74-75 Exine pattern, formation during pollen ontogeny, 4-6 sporophytic control, 7-8, 10-11, 47

Exonuclease I11 deficiency in xthA mutants, sensitivity to NUV and, 123-124 protection against NUV, 112 DNA repair synthesis and, 116,118

F Far-ultraviolet radiation (FUV) damage different from NUV action, 115-117 mutagenicity, 132-134 SOS response induction, 129-130 Fusarium oxysporum resistance Z gene-controlled, tomato, 13 MGS and, alfalfa, 19

G &Galactosidase, in pollen during develop ment, Brassica campestris, 24 Gametophyte, male, see Pollen selection, see Male gametophyte selection Gametophytic-sporophytic genetic overlap, 25-27 Hordeum spp.. 22 maize, 26 Tradescantia, 26 Genes Adhl (alcohol dehydrogenase), plants alleles, expression in sporophyte and pollen, 11 gametophytic control of expression, 47 cdc (cell division cycle), yeast isolation and function, double mutants, 180-181 meiosis initiation control, 157 mutations, 163 sequence homology with human gene, 158 copper-zinc-containing SOD-encoding cloning and sequencing, Ph. leiognathi, S. cerevisiae, 79 human, complementing SOD deficiency in E. coli, 78, 114 de (defective endosperm), pollen mutants, maize, 13,24,26, 40 Ga (gametophytic factors), alleles expressed in pollen, maize, 13-14,40 gal (P-galactosidase), Brassica oleracea gametophytic control, 47

196

INDEX

gametophytic-sporophytic genetic overlap, 26 phenotypic segregation in pollen mutants, 12 I (resistance to Fusarium oxysporium), distorted segregation, tomato, 13 iron-containing SOD-encoding, sodB from E. coli, sequencing, 80 manganese-containing SOD-encoding from S. cerevisiae, cloning and sequencing, 79-80 sodA, E. coli anaerobic expression, 85-86 NUV resistance induction, 123 sequencing, 80, 85 mei (meiotic), see also Meiotic mutants chain controlling intitial steps,

184-185 consequent activation, 177-178 hierarchy, 181-183 independent action, 176-177 in NUV response, see also Escherichia coli, NUV effects, sensitivity in mutants W E , katE katG (catalase), 103,112, 119-122 oxyR (oxydative stress), 126-127, 131-132 phenotypes and map positions, 119-120 (table) xthA (exonuclease III), 112, 118, 119, 123-124 Rfe (restorer factor), gametophytespecific, 27 S (self-incompatibility), expression in pollen and style, maize, 13-14 sp-1 (small pollen), pollen size gametophytic determination, maize, 8-9 tassel seed, interaction with mei genes, 175-176,182-183 transfer in pollen, 45-46 Way,maize expression in pollen and endosperm, 2 gametophytic-sporophytic genetic overlap and, 26 segregating phenotypes in mutants, 12 Geranium maculatum, pollen competition, quantitative expression, 34-35 p-1,3-Glucanase, microspore release from tetrads, 5

H Hansteinia blepharorachis, pollen competition, 33 Heat-shock proteins in E. coli, response to NUV and, 132 in pollen during development, maize, 25 Helminthosporium maydis, tolerance in pollen and sporophyte, maize, 15 H.victoriae, tolerance, MGS role, Auena sativa, 19 Hordeum spp., gametophytic-sporophytic genetic overlap, 22 Humans Down’s syndrome, SOD excess and, 114 gene for CuZnSOD, effect on E. coli sodA sodB double mutant, 78,114 mei mutations, male sterility and, 159,163 Hydrogen peroxide induction by NUV, 101, 111 irreversible inhibition of copper-zinc-containing SOD, 69 iron-containing SOD, 72 manganese-containing SOD unaffected by, 72 oxygen toxicity and, 65-66 toxicity, comparison with NUV effect catalase role in protection, 111-113, 122 DNA damage, 118-119 protein synthesis, 113 Hydroxyl radical, NUV-induced, toxicity, 114-115

I Iron, ferrous, MnSOD biosynthesis regulation, E. coli, 83-85 Irradiation, sublethal, effects on pollen genes in intraspecific crosses, 45-46 Isozymes, in pollen, postmeiotic gene expression, 20-22

L Leucuena leucocephala, pollen, competition-intensifying mechanisms, 42,48 Lilium henryi, pollen ontogeny, 4-5

INDEX Lotus carniculatus, MGS, excess pollination and, 16 Low-temperature tolerance, MGS, tomato, 18 Lycopersicon esculenturn rnei mutants in pc gene, 160 MGS, mixed pollination cross with Lycopersicon hirsutum, lowtemperature tolerance and, 18 cross with Solanum pennelii, salinity tolerance and, 19 pollen competition, 36 distorted phenotype segregation in mutants, 13 enzyme systems, gene expression and. 22

M Maize, meiosis in rnei mutants, see Meiotic mutants, maize in normal plants, 152, 166, 172-173 Maize, pollen alcohol dehydrogenase during develop ment, 21, 24-25 competition, 36 in mixture from two lines, style effect, 30 de mutants, pre- and postpollination effects, 13,24,26, 40 distorted phenotypic segregation in mutants, 12, 13 enzyme systems, gene expression and, 22 gametophytic mutants, 12-14, 24 germinating in uitro, tolerance to pathotoxin, 15 growth rate correlation with kernel and seedling weight, 15 effect on sporophyte qualities, 3 variability, inbreeding and, 14 heat-shock protein synthesis, 25 irradiation, effects on crosses between lines, 45 MGS, experimental, 17 sporophyte role, 39-40 size control gametophytic, s p l gene study, 8-9

197

pollen tube growth and, 10 sporophytic, 9-10 threonine content, inverse correlation with content in styles, 29 Wazy gene expression, 2, 12,26 Male gametophytic selection (MGS) in breeding programs advantages, 43-44 efficiency evaluation, 44 evolutionary significance angiosperm evolutionary rise and, 41-43 effectiveness comparison with sporophytic selection, 39 models, 38-39 genetic load and, 4 0 4 1 gene transmission opportunity and ability, 37 haploid state and, 36, 38,41 large population size and, 37-38, 41 pollen selective values, 39 experimental distance-relating for competing pollen, 15-17 excess polination treatment, 15, 16 mixing pollen from various sources, 16-18 sporophyte role, maize, 39-40 storage effect, 20 tolerance t o environmental stresses, 18-20 Manganese, competition with iron in MnSOD biosynthesis, 84-85 Medicago satiua, tolerance to Fusarium, MGS and, 19 Meiosis elementary steps, control by rnei genes, 177-178 genetic control pathways, 177-178, 183-185 in mei mutants abnormalities cytokinesis impairments, 169 nonspecific multiple, 169-173 second division control, 168-169 blockage after pachytene, 160-163 a t prophase I, 164 first division substitution for mitosis, 158-160

198

INDEX

initiation control maize, 153, 156 yeast, 157-158 in normal plants, maize, 152, 166, 172-173 Meiotic (mei) mutants, maize afd, first meiotic division substitution for meiosis crosses with am, 178-179 crosses with dsy, 179-180 cytology, 158-159 allelic relationships, 156 (table) ameiotzc ( a n ) ,meiosis blockage biochemistry, 156 crosses with afd, 178-179 cytology, 153 in double mei mutants, 181-183 asynaptic (us), crossing over increase, 164-165 crosses with ms43, 176-177 characteristics, 154-155 (table) desynaptic (dsy),abnormal chromosomal disjunction and segregation, 160, 165-167 crosses with afd, 179-180 divergent (du),spindle shape and function defect, 166, 167 double am gene epistasis over ms43, 181-183 segregation ratio, 174-175 with tassel seed mutants, 175-176, 182-183 elongate fen, second division control, 168-169 tetraploid mutant production, 185 inheritance, 155 (table) male sterility and, 174 Mei025, meiosis blockage after pachytene crosses with inbred W64A line, 160-161 cytological analysis, 161-163 sticky mutations and, 164 ms28, spindle fibril depolymerization delay, 166, 167-168 ms43, disturbance in two spindle orientation, 167-168 crosses with as, 176-177 in double mei mutants, 181-183 p a m l , p a d , multiple abnormalities, 169, 171, 173

polymitotic (po),precocious postmeiotic mitosis, 172-173 variable (ua), cytokinesis impairment, 169 Mentor pollen incompatiblity overcoming technique, 46 style indirect effect, 30-31 Mimulus guttatus, copper tolerance in germinating in uitro pollen and growing roots, 15 MGS and, 19-20 Mouse, inbred line, meiosis blockage under genetic control, 164

N Near-ultraviolet radiation (NUV) absorption by N-acetylcysteinamide-containingpeptide, phage, T7, 108-109 dehydroxy acid dehydratase, 102, 109, 111 DNA, 103-105 heme-containing porphyrins and catalase, 107 riboflavin, 107 ribonucleotide reductase, 109 thiolated tRNA, effect on DNA, 102, 105-107 DNA damage, 115-119 distinction from FUV lesions, 115-117 induction and repair, models, 101-103 similarity to HzOz effect, 118-119 mutagenicity, 132-135 oxidative photoproducts detoxification by catalase and SOD, 111-112 hydrogen peroxide, 112-113 singlet oxygen, 115 superoxide anion radical, 114-115 photoprotection by DNA photolyase, 110-11 1 physiological effects, E. coli growth delay, 106, 126 membrane damage, 128-129 protein synthesis induction, 126-128 regulons involved in response to, 129-131; see also Regulons

INDEX

sensitivity in mutants, 119-126; see also Escherichia coli, NUV effects, sensitivity in mutants sources, natural and artificial, 100-101 Neurospora crassa, mei mutants, 150 meiosis arrest after pachytene, 163 Nicotiana langsdorfii, resistance to apple scab fungus, MGS and, 18 N. rustica, pollen sublethal irradiation, effects on crosses between lines, 45 N. tabacurn, ozone tolerance in pollen and sporophyte, 15 Nuphar luteum, iron-containing SOD, 67 NUV, see Near-ultraviolet radiation

0 Ovary, interaction with germinating pollen, Petunia hybrids, 31 Oxygen high concentration, SOD induction in bacteria and yeast, 81 toxicity bacteriostatic effects, 77-78 superoxide radical production and, 65-66, 77; see also Superoxide anion radical Ozone depletion in stratosphere, NUV excess and, 200, 134 tolerance, in pollen and sporophyte, 15

P Paracoccus dinitrificans, copper-zinccontaining SOD, 67 Paraquat copper-zinc-containing SOD biosynthesis induction, S. cereuisiae, 82 endonuclease IV induction, 124 manganese-containing SOD biosynthesis induction in E. coli, 85 in S. cerevisiae, 82 Pathotoxin tolerance, MGS and, 18-19 Petunia hybrida and hybrids ozone tolerance in pollen and sporophyte, 15 pollen -ovary interaction, 31

199

selection, effect on sporophytic growth, 3 Photobacterium leiognathi copper-zinc-containing SOD isolation, 67 gene for CuZnSOD, cloning and sequencing, 79 iron-containing SOD, amino acid sequences, 70 Phytotoxin tolerance, in in vitro germinating pollen and sporophyte tissues, 15 Pioneer pollen effect, style role, apple, 31 Pistil, effects on pollen behavior, 47-48; see also Stigma, Style Podospora anserina, mei mutants chromosome stickiness, 164 meiosis arrest after pachytene, 163 Pollen, see also specific plants competition in natural populations crop plants, 35-36 intensifying mechanisms, 42 wild species, 32-35, 48 gametophytic gene expression distorted phenotype segregation in mutants, 12-13 gametophytic factors and, 13-14 overlap with sporophytic genes, 22, 25-27; see also Gametophyticsporophytic genetic overlap postmeiotic, 14 timing during development, 23-25 gene transfer, practical application, 45-46 genome, comparison with that of sporophyte, 2 interaction with pistil, 27-32 isozyme analysis, 20-22 mentor, see Mentor pollen mRNA analysis, 22-23 ontogeny developmental stages, 4-6 exine pattern, sporophyte control, 7-8, 10-11,47 meiocyte interconnections, Orchidaceae, 6-7 microspore isolation in callose, 7 pioneer, see Pioneer pollen properties during germination, correlation with those of sporophyte, 15 selection, see also Male gametophytic selection (MGS) application i n breeding, 43-44

200

INDEX

effects on sporophyte, 3 evolutionary significance, 36-43 angiosperm evolutionary rise and, 4143 effectiveness, 38-39 genetic load and, 40-41 large population size and, 37-38,41 experimental, 16-20 Pollen tube chemotropism in uitro and in uiuo, 32 growth rate correlation with kernel and seedling weight, maize, 15 difference between X and Y chromosome-bearers, Silene alba, 3 effect on sporophytic qualities, maize, 3 in natural populations, 34-35 in uitro, correlation with pollen size, maize, 10 variability, inbreeding effect, maize, 14 Populus, pollen, enzyme-encoding gene expression, 22 F! deltoides, pollen as mentor for F! alba, 30,31,46 Porphyrins deficiency in hem mutants, resistance to NUV and, 107,125 NUV absorption, E. coli, 107 Potato, mei mutants, parallel spindle (ps), 168 Propionibacterium shermanii, metal substitution in Fe-MnSODs, 71 Protein synthesis, NUV-induced, bacteria comparison with HzOz effect, 126-128 onyR mutations and, 126-127,131-132 Pseudomonas, copper-zinc-containing SOD, 67 I? oualis, iron-containing SOD, 70,71

R Raspberry, pollen germination in uitro, density effect, 27 in uiuo stigmatic nutrient effects, 27-28 stylar and pollen genotype role, 30 Razisea spicata, pollen competition, 33 Regulons, in bacterial response to NUV heat-shock control and, 132

oxidative stress control (oxyR) and, 113, 126-127, 131-132 SOS control and, 129-131, 134 stringent response control and, 132 Riboflavin, NUV sensitivity and, 107 Ribonucleotide reductase, E. coli activation by SOD,78,112 inactivation by NUV, 109 RNA messenger (mRNA), in pollen long-lived, production during maturation, 23 postmeiotic transcription, 22 thiolated tRNA, bacterial deficiency in nuuA mutants, sensitivity to NUV and, 106, 126-127 NUV absorption, 102, 105-107 growth delay and, 106, 126

S Saccharomyces cereuisiae cdc genes epistatic group, cell cycle step control, 180-181 meiosis initiation control, 157, 158 mutant cdc5, meiosis arrest after pachytene, 163 copper-zinc-containing SOD amino acid sequences, 68-70 inhibitors, 69 double mus mutants, 180 gene for CuZnSOD, cloning and sequencing, 79 manganese-containing SOD amino acid sequences, 70 biosynthesis, induction by oxygen, 81-82 localization in mitochondria1 matrix, 79 mei mutants, asynaptic (as), 159 Salinity tolerance, MGS role in interspecific crosses, plants, 19 Salmonella thyphimurium, NUV effects absorption by thiolated tRNA, 106 protein synthesis induction, 126 Schizosaccharomyces pombe, meiosis initiation, cdc gene control, 157-158 Secale cereale, mei mutants asynaptic (as), 159

201

INDEX elongate (el), 169 sticky, 164 Silene alba, pollen bearing X and Y chromosomes, growth rate difference, 3 germinating in uitro, zinc tolerance, 15 S. dioica zinc tolerance in in uitro germinating pollen and growing roots, 15 MGS and, 19-20 Singlet oxygen, NUV-induced, toxicity, 115 Sporophyte effect on exine pattern, 7-8, 10-11, 47 MGS, maize, 39-40 pollen size, 9-10 growth, pollen selection effect, 3 selection, comparison with MGS, 39 Sporopollenin, deposition on exine, 5 Stigma nutrients, effects on pollen germination raspberry, 27-28 Turnera ulmifolia, 28 pollen loads, hummingbird-pollinated forest species, 32-33 Streptococcus mutans, metal substitution in Fe-MnSODs, 71-72 Style effects on pollen germination, 3, 29-30 mediation of pollen-pollen interaction chemotropism for pollen tubes and, 32 mentor effect and, 30-31 pioneer pollen effect and, 31 pollen mixture from two lines and, 30 Sugar beet, mei mutants, parallel spindle (Ps), 168 Superoxide anion radical elimination by SOD, 66, 74, 77-79, 111-112, 122-123 catalytic mechanisms, 75-76 mutagenicity, 133 NUV-induced, toxicity, 111, 113-114, 116, 122-123 SOD effects, 111, 114, 123 SOD induction during aerobic growth, E. coli, 83-84, 86 Superoxide dismutases (SOD) biosynthesis i n microorganisms oxygen-induced, 81-82

superoxide-induced, E. coli, 82-86 metal cofactor availability and, 84-85 negatively controlled operon and, 83-86 copper-zinc-containing (CuZnSOD), eukaryotic amino acid sequences, 68-70, 79 distribution in bacteria, 67 induction by copper and Oz-, 82 specific inhibitors, 69 in yeast, see Saccharomyces cerevisiae evolution, 68, 74-75 genes coding for, 79-80, 85-86, 122-123 manganese-iron-containing (Mn-FeSODs), procaryotic, see also Escherichia coli amino acid sequences, 68-70 Cambialistic forms, 72, 75 distribution in bacteria, 72-74 iron-containing SOD in plants, 67 inhibitors, 72 metal substitution, 71-72 mutants defective for, E. coli, 78, 80-81, 114, 123 ribonucleotide reductase activation, E. coli, 112 superoxide radical elimination, 66, 74, 77-79,111-112,122-123 catalytic mechanisms, 75-76 protection against NUV, 111, 114, 123

T Talinum mengesii, pollen, competitionintensifying mechanisms, 42, 48 Therm us ther moph ilus, manganesecontaining SOD, 70, 71 Threonine, in pollen and styles, inverse correlation, maize, 29 Tomato, see Lycopersicon esculentum Tradescantia, gametophytic-sporophytic genetic overlap, 26 'I: paludosa, pollen, mRNA classes, 22, 24 Triticum durum, mei mutants, asynaptic (as), 159 Tryptophan, NUV absorption, 108

202

INDEX

Turnera ulmifblia, heterostylous incompatible pollen inhibition, 28 MGS,excess pollination and, 16 n p h a latifolia, pollen ontogeny, 4

W Wheat, meiosis, genetic control, 184

aneuploid gamete production and, 186

Z Zinc tolerance, expression in pollen, 15 MGS and, 19-20