No title

International Review of

CytoIogy VOLUME 145

SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander Jonathan Jarvik

1949-1988 1949-1 984 19671984-1 992 1993-

ADVISORY EDITORS Aimee Bakken Eve Ida Barak Howard A. Bern Robert A. Bloodgood Dean Bok Stanley Cohen Rene Couteaux Marie A. DiBerardino Donald K. Dougall Charles J. Flickinger Nicholas Gillham Elizabeth D. Hay Mark Hogarth Keith E. Mostov

Audrey Muggleton-Harris Andreas Oksche Muriel J. Ord Vladimir R. Pantii: M. V. Parthasarathy Lionel 1. Rebhun Jean-Paul Revel L. Evans Roth Jozef St. Schell fliroh Shibaoka Wilfred Stein Ralph M. Steinman M. Tazawa Alexander L. Yudin

Edited by

Kwang W. Jeon Department of Zoology The University of Tennessee Knoxville, Tennessee

Jonathan Jarvik Department of Biological Sciences Carnegie Mellon University Pittsburgh, Pennsylvania

VOLUME 145

Academic Press, Inc. Harcourt Brace & Company San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. @ Copyright 0 1993 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.

Academic Press, Inc. 1250 Sixth Avenue, San Diego, California 92101-4311 United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NW1 7DX

Libraly of Congress Catalog Number: 52-5203 International Standard Book Number: 0-12-364548-4

PRINTED IN THE UNITED STATES OF AMERICA 9 3 9 4 9 5 9 6 9 7 9 8

BB

9 8 7 6 5 4 3 2 1

CONTENTS

Contributors ........................................................................................

ix

NMR and the Study of Pathological State in Cells and Tissues Jesljs Ruiz-Cabello and Jack S. Cohen I. 11. Ill. IV.

Introduction ................................................................................. NMR Techniques for Studies of Cells and Tissues ........ ....... Examples of Applications of NMR to Pathological Studies ................................. Conclusion .................................................................................. .... ............ ...................... References

1

13 34 46 51

Roles of Urease in Plant Cells Joseph C. Polacco and Mark A. Holland I. 11. Ill. IV. V.

VI.

Introduction ................................ ................... Metabolic Origins of Urea in Plants ........................................................ Elimination of Urease Activity: Consequences for the Plant ............................... Biochemical Genetics of Soybean Urease Production ..................................... Ureaand Nickel Metabolism: Two Points of Interactionbetween Soybean and a Commensal Bacterium ................................................................................... Summary and Prospects for Continued Research on Plant Urea Metabolism ............. References ................................. .....................

V

65 66 79 84 92 97 99

CONTENTS

vi

Aspects of Amphibian Metamorphosis: Hormonal Control Sakae Kikuyama, Kousuke Kawamura, Shigeyasu Tanaka, and Kazutoshi Yamamoto Introduction .............................. ............................................. Thyroid Hormone ........................................................................... Adrenocortical Hormones ................................................................... Prolactin ....................................................................................

105 105 119 128

V. Growth Hormone ........................................................................... VI. Conclusion .................................................................................. References .................................................................................

137 140 141

I. II. 111. IV.

Control of Metabolism and Development in Higher Plant Plastids M. J. Emes and A. K. Tobin I. II. 111. IV.

Introduction ................................................................................. Physical and Molecular Structure of Plastids ............................................... Metabolism of Higher Plant Plastids ........................................................ Summary ................................................................................... References ........................... .................................

149 150 167 204 204

Biomineralization and Eggshells: Cell-Mediated Acellular Compartments of Mineralized Extracellular Matrix Jose L. Arias, David J. Fink, Si-Qun Xiao, Arthur H. Heuer, and Arnold I. Caplan 1. II. 111. IV. V.

Introduction ................................................................................. Structural Organization and Composition of the Avian Eggshell .................... Fabrication of Eggshells ........................... .............................. Eggshells as Models of Biomineralization .................................................. Concluding Remarks ...................................... ............................. References .................................................................................

217

240

244 245

CONTENTS

vii

Regulation of lntracellular Movements in Plant Cells by Environmental Stimuli Reiko Nagai I. Introduction ................................................................................. I1 . Light-Induced lntracellular Movement ...................................................... 111. Responses of Streaming Cytoplasm to Low Temperatures ................................ IV. Wound-Induced Movement of the Nucleus and the Cytoplasm ............................ V. Responses of the Cytoplasm to Various Chemicals ....................................... VI. Concluding Remarks ....................................................................... References .................................................................................

251 252 288 290 297 298 302

Index ............................................................................................

311

This Page Intentionally Left Blank

CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors' contributions begin.

Jose L. Arias (217), Department of Animal Biological Sciences, Faculty of Veterinary Sciences, Universw of Chile, Casilla 2, Santiago, Chile Arnold I. Caplan (217), Skeletal Research Center, Department of Biology, Case Westem Reserve University, Cleveland, Ohio # 106 Jack S. Cohen (I), Department of Pharmacology, Georgetown University Medical School, Washington, D.C. 20007 M. J. Emes (149), Plant Metabolism Research Group, Department of Cell and Structural Biology, School of Biological Sciences, University of Manchester, Manchester M13 9PL, United Kingdom David J. Fink (217), CollaTek, Inc., Columbus, Ohio 43201 Arthur H. Heuer (217), Department of Materials Science and €ngineering, Case Western Reserve University, Cleveland, Ohio 44 106 Mark A. Holland (65), Department of Biochemistry and Interdisciplinary Plant Group, University of Missouri, Columbia, Missouri 65211 Kousuke Kawamura (105), Department of Biology, School of Education, Waseda University, Tokyo 169-50, Japan Saka6 Kikuyama (105), Department of Biology, School of Education, Waseda Universdy, Tokyo 169-50,Japan Reiko Nagai (251), Department of Biology, College of General Education, Osaka University, Toyonaka, Osaka 560, Japan Joseph C. Polacco (65), Departmentof Biochemistry and Interdisciplinary Plant Group, University of Missouri, Columbia, Missouri 6521 1 ix

X

CONTRIBUTORS

Jesus Ruiz-Cabello (1), Departamento Quimica-Fisica Farmaceutica, Universidad Complutense, Ciudad Universitaria, Madrid 28040, Spain Shigeyasu Tanaka (105),Institute of Endocrhology, Gunma University,Maebashi371, Japan

A. K. Tobin (149), Plant MetabolismResearch Group, Departmentof CellandStructural Biology, School of Biological Sciences, University of Manchester, Manchester M13 9PL, UnitedKngdom Si-Qun Xiao (217), Department of Materials Science and Engineering, Case Western Reserve University, Cleveland, Ohio 44106 Kazutoshi Yamamoto (105),Department of Biology, School of €ducation, Waseda University, Tokyo 169-50,Japan

NMR and the Study of Pathological State in Cells and Tissues Jesus Ruiz-Cabello' and Jack S. Cohen Department of Pharmacology, Georgetown University Medical School, Washington, D.C. 20007

1. Introduction

Nuclear magnetic resonance (NMR) has been used for metabolic studies of cells and living tissues for several years (Daly and Cohen, 1989). The number of applications of NMR has rapidly increased in the last few years and it is expected that this advance will continue. For pathological studies, NMR techniques are very useful tools, despite their inherent low sensitivity (Radda and Taylor, 1985). They have also become of increasing interest in clinical medicine. Clinical applications in the imaging field are broadly recognized, and an emerging clinical spectroscopy has significant prospects. With the development of localization techniques for spectroscopy, monitoring by NMR of tumor biology may open a new perspective in the pathological sciences and clinical applications.

A. Basic Concepts in NMR

The NMR process can be described from either a classical or a quantum mechanical point of view. The classical approach is used for visualization of experiments and elucidation of questions, particularly considering isolated spins. Quantum mechanical descriptions are necessary for detailed explanations in more complicated systems. We intend to orient this chapter to those with no experience in this field, to make it as accessible as possible. For this reason, we prefer to use concepts related to both models. The magnetism of some compounds (organic and inorganic), when they are placed in a strong magnetic field, arises from the magnetic moments

'

Present address: Departamento Quimica-Fka Farmacehtica, Facultad de Farmacia, Universidad Complutense. Ciudad Universitaria, Madrid 28040, Spain. Inlernarionul Reukw of Cytology. Vol. 145

1

Copyright 0 1993 by Academic Press. Inc. All rights of reproduction in any form reserved.

JESUS RUIZ-CABELLO AND JACK S. COHEN

2

of nuclei in the compound. Any rotation of a charge has associated with it a magnetic field and an angular momentum. In the case of nuclei and electrons, this angular momentum is quantized in units of h / 2 r , p =

Ih 2T

- 9

where h is the Planck constant. The constant of proportionality I (0, +,1, 2 , . . .) is called the nuclear spin. Many nuclei are observable by NMR (see Table I). The ‘H isotope is the most abundant nucleus in the body and it provides more information because of the large number of molecules (such as water) that contain it. It is the basis of imaging measurements, although this is currently extended to other nuclei. To observe the proton spectrum requires in many cases the suppression of unwanted resonances, such as those of water and fat, the molecules of which are present in very high concentrations. I3Cis the nonabundant iostope of carbon. This is one reason why carbon spectroscopy is less sensitive than proton spectroscopy. 13Ccan give valuable metabolic information. 31Pis the major nucleus of phosphorus, but, even so, it is less sensitive than IH-NMR (one-fifth less, because of the difference in gyromagnetic ratio y , see Eq. ( 2 ) ) . Phosphorus spectroscopy is being used to study the metabolism of ATP, creatine phosphate, phospholipid metabolites, and inorganic phosphate. TABLE I NMR Properties of Some Visible Nuclei and Applications to Pathological Studies of Cells and Tissues

Isotope

Natural Magnetogyric ratio Spin abundance ( x lo7 rad T-’ s - I )

‘H I3C

99.985 1.108

26.752 6.728

3lP

2 1

100

10.841

I9F

t

100

25.181

14N

1

99.630

1.934

I5N

d

2H

1

0.370 0.015

10.137 4.107

0.037

- 3.628

170

100

10.13 0.145 93.1

7.080 - 1.639 - 1.8025

1.250

Applications to pathological studies Unlimited; clinical applications Metabolic studies; possible clinical applications Energetic and phospholipid metabolism; clinical applications Pharmacologic; detection of tumors; possible clinical applications Characterization of tumor models; possible clinical applications Protein structure determination Tumor heterogeneity by perfusion; possible clinical applications Oxygen consumption; possible clinical applications Biochemistry of cells and tissues Biochemistry of cells and tissues Biochemistry of cells and tissues Biochemistry of cells and tissues

USE OF

NMR IN CELLiTlSSUE PATHOLOGY

3

Other nuclei provide additional information, such as I9F, which is not a natural component of molecules in uiuo, but compounds containing it may be directed to monitor in uiuo or in uitro effects (Prior et al., 1990), or to detect tumors (Ratner et al., 1988). 23Naand 39K are involved in many regulatory processes, so that their spectroscopies and images are a potential source of information in processes of great biological interest (Bryant, 1970; Delayre e t a l . , 1981 ;Moonen et al., 1987; Boulanger and Vinay, 1989; Kohler et al., 1991; Blum and Osbakken, 1991). ’H is used in biological applications as a method of detecting tumor heterogeneity via quantitative determination of tumor blood flow (Kim and Ackerman, 1988; Larcombe McDouall and Evelhoch, 1990). I5N and 14N are not very common in biological studies but they have some interest because ammonium compounds may be markers of breakdown in tumors (Gamcsik et al., 1991). ”0 has recently been used in oxygen consumption measurements (Pekar et al., 1991). Finally, the measurement of intracellular concentrations of cations such as calcium and magnesium are of interest, because of their implication in the control of key biological processes. Some applications have been described using indirect NMR observation (see below). 43Caand 25Mg,when directly observed have the inherent problem of all quadrupolar nuclei (with I > 4) and low sensitivity. The nuclei with I = 0 are not magnetic. Nevertheless, not all the nuclei with I f 0 are “visible” by nuclear magnetic resonance techniques. Other requirements are high molecular mobility and absence of paramagnetic materials in the sample. The NMR active nuclei (I # 0) experience a torque (pB,) when they are placed in a magnetic field B,, where p is the magnetic moment. The result is precession or spinning of the magnetic moments about B, with a frequency, called the Larmor frequency, w = YB,,

(2)

where y is an intrinsic property different for each nucleus called the gyromagnetic ratio (units of rad/T.sec). In the absence of a magnetic field (external field), the nuclear magnets do not have preferred orientation. In its presence, the nuclei align with the magnetic field rather than against it because this is a lower energetic state. If we consider the magnetic field along the z axis, the projection of the nuclear spin angular moment along the magnetic field, I ; , may have only discrete values, which are multiples of Planck’s constant h divided by 2 ~ ; I

=-

- - 7

mlh 2rr

(3)

where m, is an integer or half-integer ( - I , -I + 1 , . . . , I).Thus, for example, the nuclei with I = t , m, are t and - 8. The difference of population

4

JESUS RUIZ-CABELLO AND JACK

s. COHEN

between these two states (m, = 4 and -i) is small. The fractional excess, ANIN. in the lower energetic level (m, = -3) is

AN 2mB, -N

kT '

(4)

where k is the Boltzman's constant, B, is the magnetic field, and T is the temperature. In a magnetic field of 14,000 G (unit of magnetic field strength) this excess is about at room temperature. Also, this excess is proportional to the inducible signal in the detector. NMR is, then, a very insensitive technique. The energetic difference between these two states is in the radiofrequency range. Next, let us explain how the NMR signal is produced. In the presence of an external magnetic field B,, the sum of the resonance signals of different Larmor frequencies forms the macroscopic magnetization parallel to the external field B,, represented by M,. If a radiofrequency field, called B,, is applied along the x axis through the coil of the radiofrequency transmitter, the macroscopic magnetization is moved away from the z axis (this is the preferred orientation) depending on the length and power of the radiofrequency pulse. In NMR jargon, a 90", 180" flip angle is used to describe the radiofrequency field, which shifts the vector M 90" or 180" from equilibrium. This magnetization vector M rotates in the x , y plane, inducing a voltage in the detector or receiver coil (normally in the y axis). This is the NMR time signal or free induction decay (FID).A mathematical operation on the FID known as a Fourier transform (FT) produces the NMR frequency signal or spectrum (Fig. 1) (Ernst, 1966; Ernst and Anderson, 1966). The FID is a sinusoidal or cosinusoidal wave, or a complex interferogram, depending on whether there are one or more components of frequency.

Radio-frequency signal

,

FIG. 1 The NMR spectrum is the Fourier transform (FT) of the free induction decay (FID); FID is a plot of signal intensity versus time, whereas FT transforms it into an NMR spectrum, which is a presentation of frequency (Hz) versus chemical shift (ppm).

5

USE OF NMR IN CELLiTlSSUE PATHOLOGY

From Eq. (3), all the nuclei will precess with identical Larmor frequency if the Bo experienced by all these nuclei is the same. Fortunately the nuclei are sensitive to the other nuclei and bonds joined to them. This causes the net magnetic field felt by each nucleus, or group of magnetically equivalent nuclei, to be different. Since these changes are very small, it is necessary to use a very homogenous magnetic field. These differences are useful for NMR spectroscopy in order to identify different nuclear groups. The most important point of these experiments is the radiofrequency field applied to excite the nuclei, also called the radiofrequency pulse that corresponds to the magnetic field B , , perpendicular to the primary field B, (the axis of the transmitter coil is therefore perpendicular to B,, and the axis of the receiver should be perpendicular to both Bo and B , ) . The experimenter can control the length, phase, and power, as well as the pulse shape. Pulses produce power across a range of frequencies. Figure 2 represents the frequency domain equivalent F(v)of a short pulse in the time domainflt). Sometimes it is necessary to produce selective excitation of one frequency or a small range of frequencies. These pulses are known as selective pulses. This can be achieved by lengthening the pulse, although this procedure is not ideal (Fig. 2). A better alternative is to employ radiofrequency pulse shaped with, for instance, the Gaussian or sinc envelopes. In other cases, there are several pulses in the same sequence to achieve echoes of magnetization (see below), or to obtain information of groups chemically and/or spatially bonded. The time between some of the pulses, or evolution time, is essential and introduces the concept of multidimensional NMR spectroscopy: 2D, 3D, and even 4D NMR. This idea is central, for instance, in macromolecule structure elucidation experiments, which will not be described herein, but since we will discuss some applications of 2D NMR in biological studies, (0)

frequency (S.9

FIG. 2 A pulse in the time domain and its equivalent (after Fourier transformation) in the frequency domain. The chemical-shift differences for protons are of the order of 16 Hz, so that a pulse ofduration 7 = IOpsec gives an effective range offrequencies. The experimenter can control the length ( 7 ) . power ( B , ) ,and shape (in this case. rectangular). Lengthening the pulse may produce selective excitation of a resonance with ( I h ) very small.

6

JESUS RUIZGABELLO AND JACK S. COHEN

it is necessary to give a brief introduction. We record the free induction decay during the acquisition time tz as usual and we can even repeat this sequence to average the data and improve the signal-to-noise ratio. For each of these acquisitions, we increment the evolution time t , by the same amount a given number of times depending on the required resolution and available time and computational memory. A new dimension or several (if we repeat the same with another period between pulses) dimensions are introduced, which can be Fourier transformed and produce novel information. This information is contained in the cross peaks due to cross magnetization and magnetization transfer between coupled spins. In a certain way, the spectrum carries a calendar of events, which occurred before the acquisition time, and provides additional information in a single experiment. The principle of magnetic resonance imaging is similar. However, in this case magnetic field inhomogeneity is deliberately imposed across the sample volume in order to obtain spatial information (for instance, a linear magnetic field gradient: 6B1/6x = constant). If the magnetic field varies in space, the angular frequency of precession will reflect the same spatial dependence. This produces the excitation of those nuclei located in the sample volume where the value of the magnetic field gradient satisfies the resonance condition at the frequency of the selective pulse. Since each frequency corresponds to one strength of magnetic field, and each location has a certain field, frequency and position are equivalent parameters. Then, different projections are obtained from the object, each time changing the direction of the gradient. Lauterbur (1973) proposed this procedure, but, currently, there are other methods for reconstructing a plane or volume from NMR. Generally, the experimental procedure consists of two phases. The first is the preconditioning period, which determines the image contrast based in different parameters, such as spin density, longitudinal and transverse relaxation times (see next paragraph), the concentration of individual chemical components, or flow velocity of blood. The second is the image formation (determining image resolution). Since magnetic resonance spectroscopy detects signals that are in low concentration with respect to water, this experiment requires many repetitions of single experiments (scans) to obtain an acceptable signal-to-noise ratio (proportional to %' number of scans). The period of time between scans depends on the relaxation time of the protons in the molecules(s) (see below). There are also some instrumental differences: magnetic resonance spectroscopy needs excellent B, homogeneity so that chemical shift information is not lost (see next section). Magnetic field gradient coils are necessary in imaging instruments to obtain spatial localization. Currently gradient coils are also used in spectrometers to reduce experimental time

7

USE OF NMR IN CELLiTlSSUE PATHOLOGY

and to improve water suppression (van Zijl and Moonen, 1990,1992). This is particularly true for multidimensional (2D, 3D) experiments (Bax et al., 1980; Barker and Freeman, 1985; Hurd, 1990) and, as in imaging, for confining the signal to a desired volume or obtaining spatial localization in NMR spectroscopy (MRS) in uiuo (Bottomley, 1987). However, the requirements for localization are different: whereas MRS requires a homogeneous B, field for signal reception, magnetic resonance imaging (MRI) acquires the signals in the presence of a B, gradient.

B. Observed Parameters in NMR

1. Chemical Shift We have stated that the external magnetic field B, applied by the permanent, resistive, o r superconducting magnet to a sample is not the same for each nucleus. The differences derive from the interactions with their microenvironments, i.e., are a function of the electrons immediately surrounding the nucleus and by electrons of adjacent bonds. Equation (2) is better expressed as w =

@O(I

- u),

(5)

where m is called the screening or shielding constant. The values of m will vary depending on the position of the atom in the molecule. Therefore, each nucleus o r group of chemically equivalent nuclei (usually as a result of molecular symmetry) presents one distinct position in the NMR spectrum, which is known as the chemical shift. This property is essential in spectroscopy and allows the spectroscopist to detect a variety of individual protons from a given protein, or different phosphate signals from ATP, or many carbon signals of metabolic intermediates. In contrast, magnetic resonance imaging does not use chemicalshift information and often seeks to suppress it. However, the frontier between imaging and spectroscopy is becoming less clear with time; magnetic resonance spectroscopic imaging (Brown et al., 1982; Maudsley et a/., 1983; Mueller and Beckman, 1985; Luyten et al., 1990; Moonen et al., 1992), which is an image based on the signal from a particular metabolite in each voxel, is a clear example of this. The chemical shift definition is

where Y,td and vsampleare the standard and sample frequencies observed, respectively. This is divided by V,td to make it independent of the external

a

JESUS RUIZ-CABELLO AND JACK s. COHEN

magnetic field strength to be comparable among measurements from different spectrometers. The standard is different depending on the type of nucleus for spectroscopy. Thus, for 'H and I3C it is tetramethylsilane (TMS), for ,lP, methylenediphosphonate, etc. Usually, the values of chemical shift are referred to the value in one solvent, because these vary from one solvent to another. Because of the factor lo6, the units are expressed in parts per million (ppm) (Eq. (6)). 2. Spin Coupling and Spin Decoupling The signal splitting of some NMR signals is another source of information. Qualitatively speaking, spin coupling can be understood because for each spin state of one nucleus, there are two states of the contiguous nuclei. The splitting arises due to the transfer of the nuclear spin of magnetically equivalent nuclei through the bonding electrons to the coupled nuclei. This term is also known as multiplicity and it is spoken of as doublet, triplet, or, generally, multiplet splitting. The splitting can give information about the adjacent group. Figure 3 plots the 'Hspectrum of ethanol. This molecule has three types of hydrogen atoms (CH,, CH2, and

r!

-C-H

5

3

1

Chemical shift (ppm)

FIG. 3 The 'Hspectrum of ethanol at 1.5 T (tesla, magnetic field strength). Chemical shifts are relative to TMS. The cumulative intensity or integration of each peak in the spectrum is also presented. Ethanol has three groups of chemically nonidentical protons, hence the three peaks in the spectrum. The splitting of the peaks and their integration give information about the structure and bonds of the molecule.

USE OF NMR IN CELLiTlSSUE PATHOLOGY

9

OH), so that the spectrum should have three peaks. Instead of that, the spectrum consists of eight peaks.Fortunately, this gives additional information according to how each kind of nucleus is bound in the molecule. The value of the coupling is represented by JABand is called the spin-spin coupling constant between nuclei A and B (homo- or heteronuclear). The value is independent of the external magnetic field and is expressed in units of frequency (hertz or cycles per second). This value permits one to distinguish in many cases among different chemical groups. Decoupling is a related, and very important, concept in pulse experiments. Although we have said that signal splitting due to the nuclear coupling is a source of chemical information, sometimes it is convenient to run an experiment to remove it; this is called decoupling. Two radiofrequencies are needed, one to observe signals and another to irradiate strongly the resonance of the nucleus to be decoupled. The advantage is not immediately obvious, but in the first place the signal-to-noise ratio is improved. A common example is the I3C spectrum. This isotope only represents 1.1% of natural abundance (meanwhile other nuclei with spin +,such as ‘H, ”P, and 19F,represent almost 100%). This and other reasons (yC = f yH) explain the lower sensitivity of 13C spectroscopy versus ‘H spectroscopy. Therefore, the I3Cspectrum is sometimes obtained by applying a broadband radiofrequency pulse (to cover all the ‘H signals). This results in the collapse of multiplets (due to C-H coupling) into single peaks and improves the signal-to-noise ratio. The nuclear Overhauser effect (NOE) is another factor that explains the improvements in the I3C spectrum (Noggle and Shirmer, 1971). To understand this effect completely, it is necessary to introduce concepts beyond the scope of this review. From our perspective, it is a change in the integrated NMR absorption intensity of a nuclear spin when the NMR absorption of another spin in close proximity is saturated, resulting in a cross-relaxation effect (see below). 3. Relaxation Times As we have indicated, at resonance, the radiofrequency field B , causes a spin transfer from the lower to the upper magnetic state. The equilibrium distribution of the spins in the static field B, is disturbed. Following any disruption, the nuclear spins relax back to equilibrium with their surroundings (called the “lattice”). The relaxation time related to this mechanism is designated Tl the spin-lattice relaxation time or, otherwise, the longitudinal relaxation time. It also represents the rate of conversion of magnetic to heat energy. There is a second type of relaxation process that, as the former, is a first-order exponential and it is characterized as T2, the spin-spin relaxation time or the transverse relaxation time. Whereas TI spreads over

10

JESUS RUIZ-CABELLO AND JACK s. COHEN

to lo4 sec, T2is smaller or equal to T,. This is because T2reduces the lifetime of a nucleus in a particular spin state due to spin-spin interactions leading to spin dephasing. T2 is related to the linewidth of a signal at half-maximum height (neglecting field inhomogeneity). Sometimes, it is or effective transverse relaxation to include the decay represented by of the signal as a consequence of both field inhomogeneities and relaxation processes (Fig. 4). If the radiofrequency power is too high and/or the time between pulses is too short, relaxation cannot compete with the disruption of the equilibrium of spins. The population difference ANIN decreases to zero, and so does the intensity of the absorption signal. This is known as saturation. Saturation pulses are sometimes applied to reduce the intensity of an unwanted resonance, frequently water, that is in much greater concentration than the desired peaks. Experimentally, this pulse is applied onresonance; i.e., the transmitter carrier is placed at the water frequency. The relaxation properties of water 'H nuclei are the fundamentals of the contrast obtained by most of the nuclear magnetic resonance imaging techniques. A combination of TI, T2,proton density of body tissues, and the velocity at which these protons are moving determines how strong a signal that tissue emits when stimulated into magnetic resonance. The variations in relaxation rates for water proton generate image contrast among different tissues and pathologies, depending on how the NMR

FIG.4 Representation of the transverse magnetization decay versus time (FID).The envelope theoretically describes T? relaxation, but the actual decay is often much shorter because of magnetic field inhomogeneities. TT relaxation includes the effect of magnet inhomogeneities, and therefore it is shorter than Tz.

USE OF NMR IN CELLITISSUE PATHOLOGY

11

image is collected. Attempts to characterize pathologically altered tissues on the basis of differences in relaxation times have been presented (Damadian, 1971;Kiricuta and Simplaceanu, 1975; Mountford et al., 1986; Mariappan er al., 1988; Larsson et al., 1989; Srejic et al., 1990). Some of these results were interpreted as illustrating the degree of perturbation of some structure that can accompany malignant transformation and/or by differences in the degree of ordering of intracellular water. However, currently it is believed that most tissue water is free in solution and that only a small fraction is immobilized by macromolecules (Bottomley et al., 1984a; Bryant e t a l . , 1991; Fullerton, 1992). It is possible to obtain different signal intensities, and hence tissue contrast, since different tissues have different rates of relaxation. However, whether these measurements can be used as a diagnostic tool has yet to be demonstrated (Bottomley et al., 1987b).

4. Peak Intensity The area under each resonance is linearly proportional to the number of chemically equivalent nuclei (Fig. 3) and the concentration in the sample, when the spectrum is acquired under certain conditions. These are ( a )that the time interval between pulses be long enough to avoid saturation effects (accepted as S O T , )and ( b ) that the volume of the sample (cells, tissue) contributing to the NMR signal be known. The spectrum of ethanol in Fig. 3 represents the cumulative intensity of each peak, one proton for OH, two for CH,, and three for CH,.

5. Spin Echo The spin-echo sequence is a useful pulse combination consisting of a train of alternating 90" and 180" radiofrequency pulses (Fig. 5). As we indicated above, the 90" rotates the magnetization into the x-y plane. Then, the magnetization vector returns toward equilibrium alignment parallel to z as in the static magnetic field B,, depending on T , . Just as the 90" pulse is shut off, the spins are coherently resonating, but from then on, they start to lose their coherence because of T2 interactions and inevitable inhomogeneities in the static field. This latter effect produces unwanted loss of the transverse component of magnetization. The spin-echo sequence corrects for this loss of coherence. A 180" pulse is applied in the middle of the sequence to rephase the nuclei that fell out of coherence with each other because of magnetic field irregularities. It is important that the time between the 90" and 180" pulses and from this to the top of the echo be equal (half of time of echo on 3 TE); the time between repetition of experiments is designated TR (see Fig. 5). Therefore, after T E the

12

JESUS RUIZ-CABELLO AND JACK S. COHEN 900

puke

900

1800

puke

echo

188

900

188

B

180"

I/ I/ 1st echo

tt=

2nd echo

-

TEl TE2 = 2 TE1

--I --I

TR

FIG. 5 The spin-echo sequence. (A) Single sequence with a 90"pulse followed at time 4 TE by an 180" pulse, with an echo signal recorded after another TE. (B) Multiple sequence with successive 180" pulses to record various echoes. The time from the beginning to the repeat is known as repetition time (TR).

+

chemical shifts have been refocused, and it is now possible to record an NMR signal, known as an echo.

6. Resolution and Peak Shape Analysis of NMR spectra sometimes requires calculation of linewidth at half-height or sharpness of the peaks, which depends of the homogeneity of the applied magnetic field, homogeneity of the sample itself, and the relaxation time T2.All of these factors are included in the definition of (Fig. 4) and explain why the linewidth is broader than the theoretical value. This term is also known as resolution. The resolution normally required depends on the nucleus and there are two limiting factors to reach a particular resolution: sample and magnet homogeneity, and y of the nucleus. Total homogeneity is optimized by the use of shim coils, which are conducting loops carrying small variable currents to compensate for the local gradients in the sample. The symmetry of the peaks may be used as a reference to judge the homogeneity.

USE OF NMA IN CELLITISSUE PATHOLOGY

13

II. NMR Techniques for Studies of Cells and Tissues

In this section we will summarize some NMR techniques, particularly those applicable to the study of cells and tissues. We have divided these into two main topics, spectroscopy and imaging, and in addition we have included a section on a specialized NMR experiment, magnetization transfer, which can measure the rate of chemical-biological reactions (Alger and Shulman, 1984; Koretsky and Weiner, 1984; Brindle and Campbell, 1987). A. Spectroscopy

1. Cell Studies There are three possible approaches to cell studies; cellular extraction, cellular suspensions, and perfusion of intact cells. In the last two cases, because NMR is an insensitive method, it is necessary to have a large number of cells within the coil (107-109) and, therefore, this limits its use to cell lines that can be grown readily in culture conditions. Nevertheless, the ease in selecting analytical conditions to gain information from a wide range of endogenous metabolites and xenobiotics makes NMR a powerful technique. Studies with cells have the advantage that one can control the experimental conditions; however, in some cases, caution has to be adopted because the concentrations of metabolites are very sensitive to culture conditions. Perfused intact cells may represent the best ex uiuo approach to the noninvasive study of metabolism (Kaplan er al., 1992). When grown in culture conditions, the cells are homogeneous; thus these studies are advantageous in elucidating the in uiuo situation. Likewise they are metabolically stable, and thus much better than cell suspensions. However, data obtained from extracts are essential to evaluate the results of N.MR studies of cells and tissues. Finally, these techniques can be applied with many of the magnetic nuclei, such as 31P,IH, 13C, 23Na,and I9F. There are some differences in the extraction procedures of tissues and cells (Munch-Petersen et al., 1973; Barani and Glonek, 1982); the major difference between both studies is the participation of other tissues and blood vessels in the tissue extracts. The degree of participation of these tissues should be known for a correct interpretation of the data (Smith et al., 1991a). There are some advantages of studying extracts. In the first case, the improved resolution enables assignments of proximate signals. Additionally, data can be accumulated during longer time periods, and then compounds present at low concentrations may be observed and

14

JESUS RUIZ-CABELLO AND JACK S. COHEN

quantitated (Glonek etal., 1982; Burt et al., 1983; Evanochko etal., 1984a; Graham et af., 1987; Merchant et al., 1988; Kasimos et al., 1990; Smith et al., 1991a,b,c). Finally, the samples are lyophilized and redissolved in an appropriate solvent to avoid suppression of unwanted solvent resonances in the spectrum. However, data obtained from extracts should be evaluated with caution. The problems associated with the extraction are (a) insolubility or less extraction of some compounds by a particular procedure (it is recommended that different methods be used) and (b) molecular changes during the extraction (caution should be adopted to minimize this source of error). Cellular suspension studies have been used widely in the last decade (Shulman et af.,1979; Cohen et al., 1988), although currently it is preferable to carry out the experiments with perfusion techniques. Some drawbacks of suspension experiments can be noted. Because of the dense cell population needed, lack of oxygen, nutrients, and accumulation of waste toxic products complicate these studies. In addition, sedimentation of suspended cells aggravates the situation. Several techniques are available to overcome cell sedimentation, but in some cases, such as bubbling with air, this affects the field homogeneity. Moreover, only a small number of cells are normally in suspension; therefore, it is highly questionable whether reliable results are obtained from these studies. However, sometimes, immobilization techniques have been found to modify the functional properties of cells. For instance, the perfusion of kidney tubules in microfibers altered the net transport of ions, probably by interfering with adequate stirring in the lumen of the tubules (Boulanger and Vinay, 1986). In this case, a better solution was proposed by agitation in suspension and oxygenation (Ammann ef al., 1989). Nuclear magnetic resonance studies of perfused isolated cells constitute a very meaningful model for metabolic studies and monitoring physiological processes. A very good overview of the different perfusion methods has been published recently (Kaplan et al., 1992). We summarize the more interesting details of each technique in Table 11, directing the reader to other reviews (Egan, 1987; Kaplan et af., 1992) for more detailed information. There are several factors that have to be presented in comparing different methods. Technical factors, for both the magnet and the perfusion apparatus, such as the need to have a special probe, use of reactors, good resolution, and sensitivity, limit the ability to repeat experiments many times and tend to produce markedly variable results. Maximum longevity of the experiment in each system, sterility, and penetration of perfusate molecules are also features that experimenters use in differentiating and choosing a particular system. Some methods are appropriate only for anchorage-dependent cells, whereas in others anchorage-independent cells can also be studied. Cells sequestrated in some of these methods do not multiply, and thus studies of cellular growth cannot be performed.

15

USE OF NMR IN CELLiTlSSUE PATHOLOGY TABLE II Perfusion Techniquesfor NMR Studies of Cells

Technique

Advantage

Microcarrier beads

Effect of large compounds; simple; long experiment

Gel agarose threads

Simple; 24- to 36-hr experiment; good SIN ratio and homogenization Simple; long experiment; good SIN ratio and homogenization

Matrigel

Disadvantage

Reference

Bad SIN and homogeneity; only anchoragedependent cells studies Limited penetration of large molecules: harvesting cells before experiment Needs to be checked in terms of diffusion of molecules and be validated for hormone studies Less field homogeneity

Ugurbil et al. (1981)

Agarose or alginate capsules

Simple, good SIN ratio

Hollow fibers and membranes

Long experiment

Technical difficulties: few publications

Spheroids

More similarity to in uiuo tumor heterogeneity

Not uniform perfusion

Foxall and Cohen (1983); Foxall el al. (1984) Daly et al. (1988)

Vogel and Brodelius (1984); Lim and Moss (1981) Gonzalez-Mendez et al. (1982): Boulanger and Vinay (1986) Deen et al. (1980); Freyer (1988); Ronen and Degani ( 1989)

The first published technique with microcarrier beads (Ugurbil et al., 1981) is very simple and may be used to investigate large molecule effects. The preparation is metabolically stable for prolonged experiments. Bad signal-to-noise ratio and magnetic field homogeneity reduce the extension of these studies in many cases. Moreover, it can only be employed with anchorage-dependent cells. There are a variety of methods using gels as matrices for restraining cells in NMR studies. In these, the porosities of the matrix are of critical importance when diffusion of large molecules is monitored. In this case, both anchorage-dependent and -independent cells can be studied. The use of agarose threads (Foxall and Cohen, 1983; Foxall el al., 1984) is one of the most widely used methods. It is a very simple and easily performed technique (see Fig. 6). Relatively long experiments (24 hr) can be monitored and good sensitivity and signal-to-noise ratios are achieved. The need for cell trypsinization prior to the experiment is the main disadvantage of this method. An alternative method with matrigel or basement membrane, with similar characteristics, was also described

16

JESUS RUIZ-CABELLO AND JACK S. COHEN

A

Teflon tube (0.5mm i.d.)

Air pressure from pump

seal cap Water bath

37” Liquid gel cell mixture Extruded gel+3ll thread

B Oxygen electrodes

1

L Waste

Tube FIG.6 Ex uiuo studies with agarose. (A) Diagram of the apparatus used to embed cells within agarose gel threads. A mixture of cells in medium is extruded through a fine Teflon capillary tube in chilled ice. The gel thread is then extruded directly into medium in the 10-mm screw cap N M R tube. (B) Schematic of the perfusion system, showing the arrangement of the polyethylene insert (for details, see Foxall and Cohen, 1983; Foxall et al., 1984; Cohen et al., 1988).

USE OF NMR IN CELLiTlSSUE PATHOLOGY

17

by Cohen's group (Daly et al., 1988). In this case, the harvesting process is not necessary and the cells can proliferate in the gel, as shown in Fig. 7. However, this method must be checked in terms of molecular diffusion and validated for hormone or growth factor investigations (Kaplan et al., 1992), since matrigel contains traces of growth factors. A method of encapsulation in gel has been described with agarose (Vogel and Bordelius, 1984) and alginate capsules (Lim and Moss, 1981). The magnetic field homogeneity achieved with capsules is inferior to the procedure with agarose threads. The methods with hollow fibers (GonzalezMendez et al., 1982) and dialysis membranes (Boulanger and Vinay, 1986) serve for prolonged metabolic experiments. Nuclear magnetic resonancecompatible hollow-fiber bioreactors are available for exhaustive control of the experimental variables (Gillies et al., 1989). Technical difficulties seem to be the main drawback of these systems. Finally, spheroids approximate many characteristics of in uiuo tumors and have potential as an efficient experimental model (Deen et al., 1980; Freyer, 1988). However, the cells are not uniformly perfused, leading to variable metabolic status and necrosis in the innermost cells. This has been seen as an advantage in mimicking in uiuo tumor heterogeneity (Ronen and Degani, 1989). Nevertheless, caution must be taken in interpreting results from this experimental model.

2. Nuclear Characteristics Due to the focus we have adopted in this review, a detailed analysis of each magnetic nucleus is not appropriate. However, it is worthwhile to introduce some concepts, including information derived from, and troubleshooting involved with, a particular nucleus, especially for ex uiuo studies. Perhaps, the most important nucleus is proton ('H), because a broad number of natural molecules contain it and are implicated in interesting metabolic phenomena (see Table I). It also has high NMR sensitivity, and although the range of chemical shift information is reduced to about 15 ppm, its spectroscopic observation is more versatile than any other technique. However, dynamic range problems (ability of the analog-todigital converter to digitize weak signals faithfully in the presence of strong signals, such as water) and severe signal overlaps accompany proton spectra. A very large peak for water and broad lipid resonances occupy much of the 'H-NMR spectrum. In extracts, this problem is not encountered, since they are usually lyophilized and are redissolved in D20. This molecule contains a NMR visible nucleus (see Table I) but the resonance frequency for it is different from that of the proton and does not appear in the spectrum. Cellular extracts also have the advantage of improved resolution, which enables assignments of proximate signals. Nevertheless,

18

JESUS RUIZ-CABELLO AND JACK S. COHEN

A

(T)Media

\=

WThreads

B DAY 1

DAY 4

DAY 8

FIG. 7 Ex uiuo studies with a basement membrane gel. (A) Schematic representation of perfusion system for studying cells in basement membrane gel or matrigel threads. (B) Micrographs of a breast cancer cell line (MDA-MB 231) growing in matrigel threads in petri dishes at 37°C under a 95% air/5% C 0 2atmosphere. Each thread is 500 pm in diameter. [From Daly et al. (1988).]

USE OF NMR IN CELLiTlSSUE PATHOLOGY

19

with the present technology, most proton NMR studies can be performed in biological fluids, cell suspensions, or intact cells (Agris and Campbell, 1982; Hore, 1983a,b; Brindle and Campbell, 1987; Fabry, 1987; Mountford and Tattersall, 1987; Rabenstein et al., 1988; Mountford and Wright, 1988; van Zijl et al., 1991). 13C-NMRis useful in the observation of changes in cellular metabolism occurring in different physiological and disease states (Rothman et al., 1991). Although I3C is not the naturally abundant isotope (only 1.1%, Table I), it has been widely used in living cells to observe the metabolism of 13C intermediates from labeled substrates (Cohen, 1987a). The main difficulty in "C-NMR spectroscopy is the low natural abundance and its low gyromagnetic ratio, which explain why it is much less sensitive than proton spectroscopy (1 39%). Several methods are available for improving the signal-to-noise ration (Yeung and Swanson, 1989; Swanson et al., 1990) using distortionless sensitivity enhancement by polarization transfer (DEPT), which obtains a factor of 4 enhancement in the I3C signal strength (Doddrell et al., 1982; Bendall and Pegg, 1983). It is also possible to detect I3C via protons, and all of these methods rely on the J-coupling that exists between carbon and proton (Sillerud et al., 1988; Knuttel et al., 1990a,b). I3C chemical shifts provide more direct information because of the wider chemical-shift range (250 ppm instead of 15 ppm for proton). A prolific field is that of 31P-NMRspectroscopy, of which the chemicalshift range of endogenous phosphorus compounds is ca. 30 ppm. The study of the metabolism of ATP, creatine phosphate, inorganic phosphate, and phospholipid can be delineated in cells and tissues (Avison et al., 1986; Wehrle and Glickson, 1986). 31P-NMRof extracts is now broadly used to confirm and quantitate in uiuo results, although these techniques can also be applied as analytical method to compare different cells and tissues (Merchant et al., 1988; Kaplan et al., 1990a, 1991). Despite the fact that 31Pis 100% abundant, its NMR sensitivity is only 6.6% with respect to proton. NMR measurements of intracellular pH have been effected by phosphorus NMR. These methods rely on the observation of the positions of resonance lines whose chemical shifts are pH dependent. Chemical shift may also be affected by other electrolytes, so that accurate determination of pH is difficult. Moon and Richards (1973) primarily used red blood cells, and other groups have used it widely (see Gadian et al., 1982, for a review). A similar procedure is used to measure the concentration of intracellular free Mg2+ and the proportion of ATP complexed with Mg. This has been used to measure free Mg2+ concentrations in glycolizing erythrocytes (Gupta et al., 1978) and more recently in normal human brain and brain tumors by localization techniques (Taylor et al., 1991). However, the more interesting utility of "P-NMR is in studying the processes in cells and tissues that alter the concentration of phosphorus metabolites, such as response of tumors to different therapies (Steen, 1989).

20

JESUS RUIZ-CABELLO AND JACK s. COHEN

19Fis another common NMR nucleus with applications for pathological studies of cells and tissues. The sensitivity of this nucleus is only 83% with respect to proton and the spectrum of I9Fchemical shifts is over 500 ppm in width. For this reason, this nucleus is very attractive, although almost negligible endogenous fluorine compounds exist in uiuo. This eliminates interfering background signals and dynamic range problems and allows one to study the metabolism of a drug following administration of perfluorcarbon compounds. This has been reported by in uiuo spectroscopy methods (Stevens et al., 1984; Wolfet al., 1987,1990; Vervoort et al., 1991) or imaging studies (Shimizu et al., 1987; Ratner et al., 1987; Eidelberg et al., 1988; Nakada et al., 1988; Maxwell et al., 1989, 1991). In order to be detected, metabolites or drugs should be present in high enough concentration M for in v i m or M for in uiuo). pH intracellular measurements have been described (Taylor and Deutsch, 1983,1988; Deutsch and Taylor, 1987, 1989) and indirect I9F methods of measuring cations have also been presented (Ca2+:Metcalfe et al., 1985; Bachelard et al., 1988; Marban et al., 1988; Ca2+and Mg2+:Kirschenlohr et al., 1988; Na+: Smith et al., 1986). 23Nais also the natural form of sodium. It is a relatively sensitive nucleus (9.25% of 'H) and there are high concentrations of Na+ ion in tissues (5-150 mM). However, not enough resolution is reached to distinguish between intracellular or extracellular Na+ concentrations. The use of shift reagents was introduced, allowing a separation of these cellular compartments (Gupta and Gupta, 1982). Table I shows that Na+ has spin 4. Unlike nuclei with spin 4, such quadrupolar nuclei give rise to three different resonances, unless the nucleus is freely rotating in solution. For this reason, in most cases the observed intensity is equal to or less than 40%. Some of the applications of the 23Na-NMRtechnique is to follow changes in the sodium concentration of biological tissues, to determine the presence of tissue damage, and to study tumoral tissue in which there is a 300-400% increase in sodium levels compared with normal tissue (Boulanger and Vinay, 1989). Unfortunately, the sodium amounts determined by NMR can be underestimated. 39Kis a related nucleus and can be used with similar objectives. Although the 39K-NMR sensitivity is much less than that of sodium, acquisition of double-quantum-filtered potassium spectra in situ are still feasible (Lyon et a f . , 1991). 3. In Vivo Tissue Studies One of the most important aspects of these studies is the spectral localization (Ordidge et al., 1987). Because our space is too limited to explain the different pulse sequences in detail, we will discuss various features of several popular methods for obtaining spectra from a particular localized

USE OF NMR IN CELLlTlSSUE PATHOLOGY

21

region of interest (Table 111). Some pertinent reviews will be indicated. A singular aspect of in uiuo methods is that of quantification of absolute metabolite concentrations, which will be discussed in this section. Other aspects are equivalent to the spectroscopy of cells discussed above, such as the inherent problems associated with each nucleus that have been monitored in in uiuo and ex uiuo models (suppression of unwanted resonances, particular sensitivity of each nucleus, etc.). The simplest localization technique is the use of surface coils (Ackerman et af., 1980). The localization is based in the distribution of radiofrequency from a coil and it excites and detects those spins at a particular field strength. They provide an optimal sensitivity for superficial tissues. By appropriate choice of coil dimensions, coil position, and pulse width, it is possible to monitor tissue, for example, the tumor, selectively, without exciting spins in adjacent normal tissues. However, the application of surface coil to different tissues and small tumors is complicated because it excites deeper layers in the sample, depending on the exact position of the coil. To generate a sensitive volume deep within an object, implanted coils have been used (Koretsky et af., 1983). Clearly, this technique is opposite to the noninvasive character of in uivo NMR and therefore has not been frequently utilized (Aue, 1986). Other experiments that produce useful measurements in volumes deeper in the object have been proposed. So-called depth pulses (Bendall and Gordon, 1983; Bendall and Aue, 1983; Bendall, 1984) use a phase cycling procedure to suppress signals from the surface. A combination of methods with composite pulses that invert spins only in a narrow range of B , (Levitt and Freeman, 1979; Tycko et a / . , 1985) and long or short phase cycles (as in depth) have also been documented, but although they show little offset dependence, they are not widely used (Shaka and Freeman, 1984). An alternative method of localization is to use a selective radiofrequency pulse in the presence of a gradient to excite only a small part of the sample. There are several methods that are effective in localizing a single species of spin in a particular region, but we will discuss only some of them here (see also Aue, 1986; Bottomley, 1987; Ordidge et af., 1987; Matson and Weiner, 1992). Depth resolved surface spectroscopy (DRESS) provides a one-dimensional localization spectrum of the region of interest and is very easily implemented (Bottomley et af., 1984b). Stimulated echo acquisition mode (STEAM) realizes three-dimensional localization in a single scan, allowing adjustment of the magnetic field homogeneity or shimming (see above) on the volume of interest and use of optimum receiver dynamic range (Frahm et af., 1985, 1987). Point resolved spectroscopy (PRESS) also achieves three-dimensional localization in a single-shot and has an advantage over STEAM in that it avoids the potential loss of half of the signal (Bottomley, 1984; Ordidge et a / . , 1985). Both are nonsensitive to

22

JESUS RUIZ-CABELLO AND JACK

s. COHEN

TABLE Ill Comparison of Localization Techniques for in Vivo Spectroscopy Studies

Technique

BI gradient Surface coil Implanted surface coil

E l gradient, phase cycling Depth pulses

BI gradient, combinations

Advantage

Disadvantage

Reference

Good sensitivity in Signals of tissues far Ackerman et al. tissue near the (1980) from the coil not detected surface; simple; produce a sensitive Invasive; surgery Koretsky et al. (1983) deeper volume required

Select a deeper Bendall and Gordon Poor localization volume through a (1983) with one coil; reduced phase cycle Bendall and Aue procedure and the (1983); sensitivity for shape depends on Bendall (1984) tissues far from the B , distribution coil around surface coil Same than depth Same than depth pulses; less Shaka and Freeman pulses distortion of signal ( 1984)

BI gradient, rotating frame

No eddy current; high sensitivity for volumes close to the coil; no loss for component with short T2

No obvious applicability; efficient destgn of components for good result

Bo gradient Static field Produce a region of Sophisticated; few high field gradient or publications topical magnetic homogeneity resonance Slice selective All of these Different techniques localize compounds at regions different simultaneously resonances are localized to distinct regions in space DRESS Use of surface coil One dimensional approach STEAM Simple; proton Loss of potential studies; good half-signal water suppression PRESS Less sensitive to Worse water motion suppression; T2s limit

Hoult (1979); Haase etal. (1983); Ganvood et al. (1984)

Gordon et al. (1982)

Bottomley (1987)

Bottomley et al. (1984b. 1985a) Frahm et al. (1985, 1987, 1989a.b) Bottomley (1984); Ordidge et al. (1985) (continued)

23

USE OF NMR IN CELLlTlSSUE PATHOLOGY TABLE 111 (confinued)

Technique

Advantage

Disadvantage

Reference

ISIS

No losses due to Motion sensitive Ordidge et al. (1986) short TI; phosphorus studies Chemical shift Very flexible; easy Normal artifacts due Brown et al. (1982); positioning of the to gradient pulse Maudsley et a / . imaging (CSI) or following the (1983) sensitive volume; phase encoding radiofrequency signals from all the techniques; volume elements excitation; this there are three are acquired broadens signals; classes that simultaneously; large volume differ in the does not have the tissue; no high number of dimensions: ID, resolution with same problems as 2D. and 3D CSI slice selective present methods, due to technology; large short gradients; amounts of data results represented are produced in a single examination in spectral or image format

motion but STEAM offers advantages for observation of coupled metabolites with short T2 because it is possible to use a shorter echo time (TE) and effective water suppression sequences can be implemented without longer echo times (Moonen et al., 1989). Image selected in uiuo spectroscopy (ISIS) involves the addition and subtraction of different acquisitions in combination with phase cycles, so that it is very sensitive to motion (Ordidge et al., 1986). For this reason, it is widely used for phosphorus spectroscopy, where sizes of localized regions are large enough to minimize this problem. Finally, an alternative method of localization is based in the phase encoding of the positions of the spin with a phase-encoding gradient (similar to MRI) (Brown et al., 1982). Unlike some imaging techniques, the signal intensity in each voxel in magnetic resonance spectroscopic imaging arises from the signal of a particular metabolite in the voxel, rather than the signal from the water. This technique is known as spectroscopic or chemical shift imaging (CSI). Since the concentration of metabolites is less than 1/1000 that of water, the resolution and signal-to-noise ratio of the metabolite images are lower than that of water images. Acquisition can be accompanied by the use of surface coils, which do not give an accurate localization but are used to optimize sensitivity (Bourgeois et al., 1991).

24

JESUS RUIZ-CABELLO AND JACK s. COHEN

Quantification of metabolites is very important in obtaining physiological information by in uiuo spectroscopy. Although the results may be presented as the ratios of different metabolites, this method is not accurate because every resonance may have different radiofrequency properties relative to each other. Quantification of absolute metabolite concentration depends on the type of in uiuo experiment. Thulborn and Ackerman (1983) discussed the quantification from spectra acquired with a surface coil, which requires caution because of the coil's inhomogeneous B , field. They suggested using the water resonance as the internal standard (water concentration is relatively constant from one tissue to another), although they never developed the idea into a practical method. The procedure was described to quantitate 31Pmetabolites, although it is applicable to any nucleus of interest. Other authors stated that this method was impracticable because of the need to keep the ratio of coil quality factors at the two frequencies (phosphorus, for the sample, and proton, for the reference) constant (Shine et al., 1987). Instead, they used an injected internal 3'P concentration standard (methylphosphonic acid) to quantitate absolute concentrations of 31Pin the kidney, although these factors may be corrected (Tofts and Wray, 1988). Sometimes, the concentration of one of the metabolites can be established by other methods, and then the absolute quantification of other resonances is direct (Tofts and Wray, 1985). However, many times this is not available, so that Thulborn and Ackerman's method was implemented (Tofts, 1988). Other compounds may be utilized with this objective; for instance, N-acetylaspartate or creatine plus phosphocreatine has been used as an internal concentration standard for 'HNMR studies of brain (Williams et al., 1988; Pan et al., 1991). In any case, several factors should be measured for reliable quantification. These factors are due to the particular sequences used for in uiuo experiments: sequences to suppress the unwanted water and fat resonances and to localize the region of interest (Pan et a / . , 1991). External standards have also been used (Bottomley and Hardy, 1989). This requires accurate knowledge of the sample volume, and thus it is executed with imaging techniques for measuring the areas of metabolitebearing tissue in selected sections through the subject. A calibration procedure, which assumes a homogeneous distribution of metabolites, was also presented by Luyten et al. (1989a) for ISIS, although the procedure can be applied to 3'P-NMR spectra obtained by other localization schemes. It is necessary to ensure by prior imaging that the signal arises from a homogeneous volume. Finally, a related concept is that of spectral fitting, especially for 'Hspectroscopy, where overlapping of signals is critical. Methods for approaching this problem and other resulting distortions from the effects of the N M R pulse sequence on the spins have been described (de Graaf and BovCe, 1990).

USE OF NMR IN CELLiTlSSUE PATHOLOGY

25

B. Imaging After the first magnetic resonance image produced by Lauterbur (1973), MRI has evolved into a clinically useful imaging tool in diagnostic medicine. Many imaging sequences are now available, all with different objectives, making an in-depth discussion outside the scope of this review. Most of them have pathological applications and may be found in specialized imaging books. The different chemical compositions of tissues are responsible for their intrinsic magnetic properties: TI, T2, spin density. These parameters are also affected by pathology, which explains the application of MRI in pathologic studies. It is easy to acquire TI-or T,-weighted images and proton density images by modifying the experimental conditions. Depending on the problem, one sequence or another can be used. Thus, muscle tissues may be imaged with TI- or T2-weighted sequences. Basic pathologies (edemas, necrosis, neoplasias) can also be imaged with these sequences. Hemorrhages may be studied and it is possible to obtain significant information. Proton density images are valuable for the central nervous system. These experiments are quite well explained in all the general imaging manuscripts, and specialized books on pathology and anatomy can be found. On the other hand, conventional NMR imaging is not sensitive to rapid changes in tissues caused by ischemia or hypoxia. Nevertheless, less known sequences used for attaining physiologic or functional information have been described (Moonen et al., 1990). We consider these to be very interesting for pathological studies, and in consequence we will dedicate special attention to them. When necessary, we will comment on some features of the spin-echo techniques from which macroscopic structure information and anatomic information have been derived, avoiding complicated mathematical treatments. More specialized overviews can be found elsewhere (Mansfield, 1982; Morris, 1986; Emst, 1987; Mansfield, 1988).

1. Magnetic Resonance Angiography or Flow Imaging The principle of these methods is to generate images that are sensitive to flow. They represent a very important development to investigate noninvasively vascular dysfunction and studies of the brain substance. There are two mechanisms to explain the loss of the signal in an aqueous material flowing under the influence of a magnetic field gradient: (a) time of flight or transport of spins from one portion of the voxel (imaging volume) to another during the acquisition and ( 6 )dephasing of the moving spins induced by gradients. This gives rise to two varieties of NMR angiography: (a) methods that depend on the signal amplitude of the moving spins (Dixon et al., 1986; Nishimura, 1990; Wehrli, 1990a)and ( b )methods

26

JESUS RUIZ-CABELLO AND JACK S. COHEN

that depend on the phase information of the moving spins (Macovski, 1982; Moran, 1982; Axel and Morton, 1987; Dumoulin et al., 1989). The former gives information on geometry and state of a blood vessel, whereas in the latter case, vessel appearance depends on the direction as well as the velocity of flow. Time-of-flight procedures are 30-40% faster than phase-based methods (for comparable imaging parameters). They depict all directional components of the flow in a single acquisition and are very easy to implement. These methods have the disadvantage that signal amplitude is also affected by proton density (not only velocity). However, in phase-based angiography, the phase shifts of the moving spins directly depend on flow velocity and are independent of spin density. In addition, they are less sensitive to saturation problems, which is a special convenience for slow-moving spins. 2. Imaging of Diffusion Many physiological phenomena are reflected in respiratory motion, cardiac motion, vascular flow, incoherent displacements (i.e., microscopic random pattern or Brownian motion), etc. Magnetic resonance can be made very sensitive to motion by the application of a pair of pulsed magnetic field gradients (Stejskal and Tanner, 1965). The first gradient of the pair disperses the complete signal (So),which is (when molecules move coherently) regained by application of a compensating gradient after time A. However, diffusion of some component exponentially attenuates the signal (S), In (S/So) = --y2G2a2(A - N3)D

=

-bD,

(7)

where y is the gyromagnetic ratio (see Eq. (2)), G is the gradient strength (in units of T/m), 6 is the length of the gradient (in sec), D is the diffusion constant of one particular component (units of m2 sec-I), and b is the diffusion-weighting factor (units of rad.sec/m2). Diffusion measurements may differentiate freely diffusing water molecules in fluid compartments from water molecules with restricted mobility. A spectroscopic method separating intracellular and extracellular information in NMR spectra of perfused cells has been outlined recently (van Zijl et al., 1991). Figure 8 shows the comparison spectra of agarose plus breast cancer cells with different diffusion-weighting factors and the spectra of gel. Clearly, the different diffusion-weighting measurements permit one to distinguish between both compartments. On the other hand, diffusion methods by MRI allow accurate control of the diffusion direction and may help in finding insights in the macromolecular structure of tissues and understanding the processes for tissue breakdown (Hazlewood et al., 1991; Schmalbrock and Chakeres, 1992). Moseley et al. (1990a) presented

27

USE OF NMR IN CELLlTlSSUE PATHOLOGY A)

6)

b = 0.028 x 1010 s/m2

medium

+

gel

b

medium

=

+

0.770x 1010 Slm2

gel

cholines

medium + gel + cells (Adr)

I triglycerides

I " 6

1,

I " 4

'

I

'

2

/

'

0

PPM

6

4

2

0

FIG.8 Comparison of 200-MHz proton NMR spectra (TE/TR = 1212440 msec; line broadening, 4 Hz) for perfusion experiments with gel only (top) and with gel and cells (bottom), using different diffusion-weighting factors: (A) b = 0.028 x 10" sec/m*; (B) b = 0.770 x 1O'O sec/m2 At high diffusion weighting, no signal appears when perfused gel is used alone, whereas the complete intracellular spectrum appears when cells are present. Note that (A) (512 scans) and (B) (128) scans are displayed at different scales for optimum display. Adr, adriamycin resistant. [From van Zijl et al. (199U.l

an example with diffusion techniques to show extra- and intracellular spinal white matter organization, not achieved with standard imaging sequences (spin-echo images). In another study, they also declared that diffusionweighted imaging may be used to evaluate early infarcts, because the diffusion contrast in the stroke region is lowered (Moseley et al., 1990b). Figure 9 shows an example of the potential of this technique with an experimental stroke model (occlusion of the middle cerebral artery). Nevertheless, the diffusion effects can be visualized if enough gradient strength is available. However, the motion sensitivity is increased when gradient strength is also enhanced. For this reason, these measurements are very difficult. Clinical instruments, generally, do not have strong enough gradient strengths to do diffusion-weighted imaging. Techniques such as intravoxel incoherent motion (IVIM) imaging (Le Bihan et al., 1986, 1988) generate images from two equal spin-echo sequences with different diffusion gradient strengths. The IVIM images represent the apparent diffusion coefficient (ADC) obtained from such a pair of sequences on a voxel-by-voxel basis. Elimination of all other

28

JESUS RUIZ-CABELLO AND JACK S. COHEN

FIG. 9 Transverse image of cat brain following occlusion of the middle cerebral artery. (a) Conventional image, where the boundaries of the affected region are not clearly outlined. (b) The diffusion image shows the apparent diffusion constant for each voxel. The area with the lowered diffusion coefficient corresponds accurately with the ischemic region. [From Moonen et al. (1990). Copyright by the AAAS.]

USE OF NMR IN CELLiTlSSUE PATHOLOGY

29

resonance characteristics is now possible. The major disadvantage of these techniques is the long scan times required for multiple acquisitions. Quicker alternatives are also accessible. Fast steady-state free precession sequences (SSFP) (Lee and Cho, 1988) have been used for measurement of diffusion coefficients (Le Bihan, 1988). Snapshot-fast low-angle shot sequences (FLASH) (Haase, 1990) that reduce experimental time from 10-30 min to 30 sec versus the standard cine-FLASH (Frahm et al., 1986), fast acquisition in steady state or Fourier acquired steady state (FAST) (Gyngell, 1988), and echo planar imaging (EPI) (Mansfield, 1977) have also been used for diffusion imaging (Merboldt et al., 1989a,b, 1991; Turner and Le Bihan, 1990; Turner et al., 1991; Moseley et al., 1991). 3. Imaging of Perfusion Perfusion is assumed to have effects on images similar to those of diffusion, since both are characterized by a random motion of the spins, but it is more complex to generate perfusion-weighted images. There are many potential applications for these measurements related to the efficiency with which blood provides oxygen and nutrients to the tissue. Evaluation of brain ischemia, kidney function, and myocardial infarcts are examples of the possible applications. There is discussion about whether NMR methods based on motion-encoding gradients can measure tissue perfusion, at least in the classical sense (Henkelman, 1990), although if perfusion is thought of as pouring through, the fractional flow in a tissue as measured by IVIM (Le Bihan et al., 1986) is a type of perfusion (Dixon, 1991). Recently, it was shown that the perfusion-related parameters derived by Le Bihan’s method (Le Bihan et al., 1988) could not be obtained with sufficient accuracy (King et al., 1992; Pekar et al., 1992). However, the perfusion-weighted images appear to contain valuable information (King et al., 1992). Nuclear magnetic resonance techniques have been used as a detector of classical perfusion measurement with exogenous tracers, in both deuterium uptake and washout experiments to detect tumor heterogeneity (Ackerman et al., 1987), and in fluorine uptake experiments o r gadolinium inflow measurements with EPI (Rosen et al., 1989, 1990). The same kind of information obtained with the delivery of contrast agent is provided by an imaging experiment without invasive tracers (Detre et al., 1992). In this experiment, two images of the region of interest (R01) are acquired, one with and another without saturating upstream tissue. The difference between both images demonstrates spins accumulated due to blood flow. 4. Imaging of Magnetization Transfer

Recently, Wolff and Balaban (1989, 1990) have described a novel form of NMR image contrast. This method is based on the saturation transfer

30

JESUS RUIZ-CABELLO AND JACK

S. COHEN

method originally presented by Forsen and Hoffman (1963, 1964), and used profusely to measure chemical exchange rates in intact tissue. We will explain these experiments below. Magnetization transfer contrast (MTC) imaging is introduced by application of an off-resonance radiofrequency pulse of sufficient power to saturate the energy level populations for nuclei from one pool (‘H in immobile water), immediately before an excitation pulse. Exchange of this saturated magnetization with nuclei of other pools (“free” water) thus affects the signal intensity observed in a subsequent NMR image. The mechanisms of exchange are still not completely understood but many kinds may be involved. The more important point is that magnetization transfer can be used to exploit both the available longitudinal magnetization and the T , of normal and abnormal tissues. Thus, additional contrast is achieved, and this may have importance in clinical imaging (Hajnal et al., 1992). In addition, these experiments may provide new information about the complex state of water in tissues and add to the understanding of processes related to tissue damage.

5. Imaging of Metabolites Compared to other localization techniques (see Section II,A,3), spectroscopic imaging may provide an overview over larger tissue volumes. Both techniques are therefore complementary. One of the advantages of this method is that the data may be displayed in an image format as well as in a spectral format. Thus, it is analogous to conventional MRI, except that the signal intensity in each voxel depends on the concentration from a particular metabolite in each voxel, rather than the signal from the water (40 M water in most tissues, whereas other metabolites, such as lactate, may be 10-30 mM). Thus, this technique is much less sensitive than waterbased imaging (Arnold et al., 1991). We will discuss this new method in more detail in Section 111. 6. Receptor Imaging

We cannot include all aspects of imaging techniques, but in our attempt to present new applications we will need to introduce some further concepts. Many times, the proper selection of imaging sequences does not render enough contrast. Contrast agents can provide more efficacy in diagnostic imaging. They can also provide additional information by the dynamics of their organ distribution. It is possible to classify substances according to the type of effect that the magnetic field has on them: paramagnetic, diamagnetic, and ferromagnetic (Saini et al., 1988). Paramagnetic substances increase the local alternating magnetic fields. They contain un-

USE OF NMR IN CELLiTlSSUE PATHOLOGY

31

paired electrons and are used as contrast agents because they facilitate the relaxation of protons. Although very difficult to quantitate, perfusion techniques with paramagnetic contrast agents are also a useful method for detecting tumor heterogeneity (see Section II,B,3). The local magnetic field of a paramagnetic molecule increases the magnetic relaxation rates of surrounding protons, depending on many variables, and then they may have numerous diagnostic applications. Of special interest are those that give direct assessment of tissue function. A problem can arise when these paramagnetic agents are targeted to tissues with a rapid phagocytic system, such as liver and spleen. Even with conventional coating to protect, these agents are still phagocytosed by these organs. A concept recently introduced, and applied to the design of both diagnostic and therapeutic drugs, is to target specific receptors, coating particles with a high affinity component of these receptors. This strategy has been used for detection of liver cancer in rats (Reimer et al., 1990,1991). This has provided a new modality of imaging known as receptor imaging, which provides new directions in the development of tissue-specific contrast enhancement. 7. Cardiovascular System Images

Magnetic resonance imaging of the cardiovascular system is different from any other organ imaging. We will give a brief discussion of how MRI techniques eliminate motion-related artifacts. These artifacts derive from the asynchrony between the motion and the phase-encoding steps for data acquisition. They must be removed from the image for optimum results, although some authors have shown good quality images without triggering (Choyke et al., 1984; Frahm et al., 1990). This is especially important in evaluating patients with severe arrhythmias. Electrocardiographictriggered MRI correlates radiofrequency pulses to the heartbeat (other devices may also be used to monitor the cardiac phase, such as Doppler ultrasound). Data are acquired when the heart is at a particular phase along the electrocardiogram. Data acquisition is triggered during cardiac diastole and effective pulse TR is determined by the heart rate. Images can also be obtained during cardiac systole. Other modalities can be used to establish the flowing blood characteristics and are independent of the exact cardiac phase (Lenz et al., 1989; Wehrli, 1990b). These capabilities are important for the diagnosis of cardiovascular function.

8. Magnetic Resonance Imaging Microscopy As magnetic resonance evolved, volume acquisitions with microscopic resolution have been investigated. This is particularly important for pathological studies, and this technique is emerging as a potential substitute for

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JESUS RUIZ-CABELLO AND JACK S. COHEN

the complex sample preparation used in conventional histology and as a method for detecting tumor heterogeneity. As we discussed, differences in magnetic resonance. properties of water, such as relaxation times and spin density, are the basis for contrast and can be used to characterize tissues. If we add that magnetic resonance imaging techniques can attain spatial resolution, it appears that magnetic resonance microscopy will, in the future, represent a powerful tool (Johnson et al., 1986; Eccles and Callaghan, 1986; Aguayo et al., 1986). Good quality gradients for spatial encoding have enabled signal-to-noise ratio problems to be overcome. Currently it is possible to obtain volume elements as small as 3.4 x lop4 mm3, whereas in conventional water magnetic resonance imaging these sizes are 25,000 times larger for the same signal-to-noise ratio (Suddarth and Johnson, 1991). Apart from the resolution limits, MRI microscopy has shown applicability as a diagnostic tool for comparison with fixed pathological brain specimens (Johnson et af., 1987). 9. Other Imaging Modalities

Recently, new applications of nuclear magnetic resonance imaging have appeared. Measurements of cerebral oxygen consumption and blood flow in brain using I7O magnetic resonance imaging agree reasonably well with values obtained using classic procedures (Pekar et af., 1991). Injection of an arterial bolus tracer enriched with H2I7Oand inhalation of enriched I7O2 was used in these experiments. Independently, quantification of cerebral blood flow has also been reported using I9F of trifluoromethane (Branch et al., 1991) or proton NMR imaging using H2170 (Kwong et al., 1991), although further improvements in signal-to-noise ratio are needed. These measurements could allow future clinical diagnosis of perfusion abnormalities. Magnetic resonance imaging of water can sometimes be used for measurement of physiological events. Oxygenation level-sensitive contrast in water images has been presented as an indirect reflection of brain physiology (Ogawa et af.,1990). The contrast is produced by the susceptibility difference at the boundary of two materials, in this case between deoxygenated blood and surrounding tissue. This degrades the field homogeneity and it is visible in high field images with high spatial resolution. Contrast agents are also employed in these hemodynamic studies. The focus is on the accurate measurement of cerebral blood volume and cerebral blood flow using echo EPI techniques, which provide temporal resolution, and contrast agents, which affect the signal intensity from tissues (Rosen et af., 1989; 1990, 1991; Worthington and Mansfield, 1990; Belliveau et al., 1990, 1991a).

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C. Magnetization Transfer

These techniques have relevance to investigations of metabolic pathways and studies of cellular and tissue kinetics. The study of the rate of chemical reactions noninvasively may provide a good system for an examination of cell and tissue injury. Therefore, we have included in this section a technique that allows the measurement of chemical reaction rates and that was also discussed previously as an imaging procedure. Nuclear magnetic resonance spectroscopy can be used to monitor the rate of chemical reactions in cells and intact tissues through monitoring the changes of magnetization in a compound labeled with a magnetic nucleus. Our purpose in this section is to discuss some aspects of magnetization transfer methods and indicate various applications. Technical details, limitations of the method, and review of this topic are addressed in more specialized manuscripts (Jeener ef al., 1979; Balaban ef al., 1983; Alger and Shulman, 1984: Koretsky and Weiner, 1984; Koretsky ef al., 1985; Ugurbil, 1985a,b; Garlick and Turner, 1985; Rydzy ef al., 1990). As indicated above, these studies involve magnetic labeling of one or several peaks and subsequently observation of the chemical transfer of the label to other peaks. In saturation transfer, one peak is selectively saturated (elimination of magnetization). In inversion transfer, the magnetization is inverted (this is equivalent to the application of a 180" pulse). Measurements of fluxes need to be compensated for nonspecific and direct saturation effects. The area of the resonance exchanging with the saturated site is compared to that obtained with control irradiation. Measurements of the fractional magnetization after saturation transfer and intrinsic T , in the absence and in the presence of appropriate saturating radiation at the exchanging site are used to derive pseudo-first-order rate constants (Shoubridge et al., 1982). Fluxes are now determined as the product of the rate constant and the estimated intracellular concentration of the appropriate metabolite. Finally, there is another type of experiment using two-dimensional techniques (Balaban ef al., 1983). These experiments do not require selective irradiation and the exchanges are observed in a convenient two-dimensional plot in the form of cross peaks (see Section 1). The only limitations for these experiments are signal-to-noise ratio and experimental time. Thus, magnetic resonance techniques also are available for studying in uiuo enzymology. It can be used to define the kinetics of intracellular reactions under steady-state conditions (in the order of sec-I). The technique has been applied to many biological systems both in vivo and ex uiuo, and primarily through 3'P-NMR spectroscopy (Table IV).

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s. COHEN

TABLE IV NMR Studies of Cellular and Tissue Kinetics

Study

Cell or tissue

Creatine kinase

Perfused heart

Creatine kinase Creatine kinase

Perfused skeletal muscle In vivo skeletal muscle

Creatine kinase

In vivo brain

ATP synthesis

Perfused heart

ATP synthesis ATP synthesis ATP synthesis

Perfused liver Perfused kidney In vivo, kidney

ATP ATP ATP ATP

In vivo, limb In vivo, heart In uivo, malignant tumors

synthesis synthesis synthesis synthesis

Adenylate kinase

Reference Brown et al. (1978); Kobayashi et a/. (1982); Matthews et a/. (1982, 1983); Seymour et al. (1983); Kupriyanov et al. (1984); Ugurbil et a/. (1984); Ugurbil (1985a) Gadian et al. (1981) Shoubridge and Radda (1984); (Shoubridge et a/. (1984) Shoubridge et al. (1982)

Saccharomyces cerevisiae

Matthews et al. (1981); Koretsky et al. (1983, 1986); Ugurbil (198Sa); Ugurbil et al. (1986, 1987); Kingsley-Hickman et al. (1986, 1987); Brindle and Radda (1987) Thoma and Ugurbil (1987) Koretsky et al. (1983, 1986) Yahaya et al. (1984); Freeman et al. (1986) Brindle et al. (1989) Robitaille et al. (1990) Okunieff et al. (1989, 1991) Alger et al. (1982)

Red blood cell

Gupta (1979)

111. Examples of Applications of NMR t o Pathological Studies

A. Ex Vivo NMR Spectroscopy of Cells and Tissues We want to overview the in uitro, or better, the ex uivo, systems that are used to monitor NMR changes in cells. Likewise, NMR studies of body fluids and cells and tissue extracts give rise to certain information, not easily accessible by other techniques. Data obtained from cellular extracts are essential for the interpretation of results of intact cells and tissues. Initially, 3'P-NMR studies of extracts of tissues (muscles) were investigated (Hoult et al., 1974; Burt et al., 1983). Subsequently, the profiles of

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35

metabolites of many organs were also described using 'H- and 13C-NMR. Many studies of extracts focused on brain (Glonek et al., 1982;Behar et al., 1983; Cerdan et al., 1985; Fan et al., 1986; Ogawa et al., 1986; Pettegrew et al., 1987; Petroff et al., 1988, 1989; Nakada et af., 1989; Gill et al., 1989) and metabolic sequences of pathological states such as hypoglycemia (Behar et al., 1985), ischemia (Peeling et al., 1989), and neoplasia (Ross et al., 1988). The use of extracts can be partly explained by the lack of experimental models of perfused brain. Other organ, tissue, and tumor extracts studied by NMR include liver (Cohen, 1983, 1987b; Cerdan et al., 1988) and the effects of ethanol (Ling and Brauer, 1990), heart (Kopp, 1983; Malloy et al., 1988), kidney (van Waarde et al., 1989; Jans et al., 1989), pancreas (Kaplan et af., 1987; Morris et al., 1989),muscle (Arus et al., 1984; Venkatasubramanian et al., 1986), eye (Greiner et al., 1985; Apte et af., 1989; Merchant et af., 1991), reproductive organs (Navon et af., 1985), lung cancer (Onodera et af., 1986), colon cancer (Kasimos et al., 1990), sarcoma (Evanochko et al., 1984a), and normal and neoplastic breast tissues (Merchant et af., 1988; Barzilai et al., 1991; Smith et af., 1991a,b,c). These extract studies are mainly used to confirm and quantitate in uiuo NMR spectroscopy results (see Section 111,B). Extract studies of cells also help in following the changes monitored with perfusion or suspension. Navon et al. (1977) studied transformed Ehrlich ascites tumor cells. Further studies demonstrated substantial qualitative and quantitative differences in 31Pspectra of various mammalian cell lines (Navon et af., 1978), including human HeLa cells (Evans and Kaplan, 1977). Other cell lines have been investigated: breast carcinoma (Cohen et al., 1986; Kaplan et al., 1990a, 1991), melanoma (Corbett et al., 1987), neuroblastoma (Navon et al., 1983) retinoblastoma (Miceli et al., 1988), and colon carcinoma (Desmoulin et al., 1986). Combined studies (for instance 31P-'H) facilitate the determination of concentrations or' the phosphorus-containing metabolites and allow calculation of their precursor levels (Kaplan et al., 1990a). Figure 10 shows the phosphorus and proton spectra of an extract of a breast cancer cell line. Logically, the information content is extended, and it is particularly important to obtain information from overlapping resonances of metabolites that appear in both spectra, such as for creatine (and phosphocreatine) and choline (and phosphocholine). There is a vast literature on cellular suspension studies. We do not intend to summarize all of this information, since cellular perfusion methods are now much preferred, principally for prolonged experiments. However, as we indicated in Section II,A, 1, sometimes perfusion alters the biological properties. In this case, a better solution could be a method for agitation in suspension and oxygenation (Ammann et al., 1989). Suspensions have also been employed with isolated rat liver mitochondria respiring on succi-

36

JESUS RUIZ-CABELLO AND JACK S. COHEN

I

1

I

I

I

I

I

5

0

-5

-10

-15

-20

I

4

I

I

3

I

I

2

I

I

1

I

I

0

Chemical shift (ppm) FIG. 10 Example of extract spectra. (A) "P-NMR spectra at 162 MHz of extracts of a breast

cancer cell line (wild-type MCF-7). (B) 'H-NMR spectrum at 400 MHz of the extract of (A). The assignments of all the peaks are according to published data (Behar et a / . , 1983). For 'H studies the extracts were dissolved in I.O ml of DzO. A 3-sec repetition time and 90" flip angle were used, and 1200 transients were accumulated with 16K data points. For phosphorus studies, I ml was added and spectra were recorded with a 10-sec repetition time, 60" flip angle, 2400 transients, and 4K data points. Data were processed with a line broadening of 3 Hz for phosphorus and I Hz for proton. Peak identities are PE, phosphoethanolamine; PC, phosphocholine; Pi, inorganic phosphate; GPC, glycerophosphocholine; GPE. glycerophosphoethanolamine; PCr, phosphocreatine; NAD(P), nicotinamide dinucleotide (phosphate); UDPG, uridine diphosphoglucose plus nicotinamide dinucleotide (phosphate); Cr, creatines. [Adapted from Kaplan et al. (1990a).l

nate and glutamate (Hutson er al., 1992). This study was done to determine the location of the NMR-invisible intracellular phosphate in perfused or in uiuo studies. Nuclear magnetic resonance studies of body fluids, such as urine, human cerebrospinal fluid, and plasma, are currently used in biochemical pathol-

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ogy (Bell et al., 1989; Nicholson and Wison, 1989). Proton studies in urine showed increased amino acid concentration associated with hepatic failure (Bales et af., 1988). The same techniques were used to relate the spectra of human cerebrospinal fluid with several clinical conditions: meningitis, diabetes, hepatic coma (Bell et af., 1987). These authors found relationships between the presence of urea in urine and trimethylamine-N-oxide, which may indicate that this compound is closely related to the degree of renal failure (Bell et al., 1991). Human amniotic fluid has also been analyzed by 3'P-NMRfor assessing fetal pulmonary status by phospholipid quantification. This method correlated with an independent measure of fetal lung maturity and with gestational age (Pearce et af., 1991). The application of NMR as a tool to continuously follow biochemical events in intact cells expands its range. It is imperative that the cells are perfused and furnished with nutrients during the experiment. Table I1 summarizes the methods developed to restrain cells and perfuse them inside the NMR tube. Most of these studies have been done with phosphorus and carbon NMR, which do not need the suppression of unwanted solvent resonances. The microcarrier bead technique was the first cell perfusion technique used in NMR studies (Ugurbil et al., 1981),in which anchorage-dependent cells are attached to dextran beads. There are also beads of other composition: polystyrene, polyacrylamide, agarose, and polyacrolein. Effects of growth factors, drugs, and phospholipid metabolism have been shown using 3'P-NMR and demonstrate the applicability of this method for studying metabolic processes of cellular stimulation and hormonal agents, and differentiation (Kaplan et al., 1992). Glucose utilization and glycolysis products were monitored by using labeled compounds and in response to hormonal treatment in human breast cancer cells (Neeman and Degani, 1989a). Unlike other groups, this work showed an application with significantly smaller samples of cells. Gel procedures using agarose threads, matrigel, and alginate have been widely applied. The method using agarose was introduced by Foxall and Cohen (1983) and Foxall et af. (1984) and it is a very quick and inexpensive technique. Most studies with agarose threads have used cancer cells. Thus, bioenergetics and glycolysis (Lyon et af., 1988a), phospholipid pathways (Daly et al., 1987, 1990), drug resistance (Cohen et al., 1986; Kaplan et a/., 19!91), effects of drugs and antimetabolites (Kaplan et al., 1990b; Jaroszewski et al., 1990; Berghmans et af., 1992), and growth factors (Kaplan et al., 1990c) have been investigated in recent years by our group. Other authors have also used this method for the study of metabolism of cyclophosphamide in lymphoma cells (Boyd et al., 1986), energy metabolism, and control of substrate utilization in HeLa, murine hybridoma, and myeloma cells (Sri-Pathmannathan et af., 1990), metabolism of

38

JESUS RUIZ-CABELLO AND JACK S. COHEN

cultured human retinal epithelial and human retinoblastoma cells (Miceli ef al., 1987, 1988), and glucose metabolism of retinal epithelial cells (Miceli et al., 1990). We have recently introduced a modification of this perfusion method to adapt it for proton spectroscopy (van Zijl et al., 1991). Figure 11 shows the schematic for the 5-mm perfusion tube. Unlike the previous one, this has the novelty of perfusion from the bottom. In NMR experiments a radiofrequency coil is positioned around the vial, which is then placed in the magnet. This arrangement was employed to obtain the proton

OUT

FIG. 11 Schematic of the perfusion vial that contains gel threads. Fresh medium enters the

vial through Teflon tubing, which is held in position by tubing seal (3). containing an 0 ring (1). The medium is distributed for equal perfusion over the surface using a polyethylene filter

(70 pm) (2). An analogous filter in the cap prohibits the gel from flowing out. The simple construction consists of a screw cap (4) with 0 rings and a flask (5). The top (5A) and bottom (5B)of the flask are glued together. In N M R experiments a radiofrequency coil is positioned around the vial, which is then placed in the magnetic field. [From van Zijl et al. (1991).1

USE OF NMR IN CELLiTlSSUE PATHOLOGY

39

spectrum in Fig. 8. This new system requires much less (around lo7 cells) than the 10-mm tube (ca. 2 x lo8 cells). One of the disadvantages of perfusion studies with agarose threads is the limited proliferative activity inside the threads. This aspect was improved with the use of a basement membrane matrix or matrigel (Daly et al., 1988) and was used in studies of phospholipid pathways in human cancer cells (Daly et al., 1990). The system used included the insertion of large capacity filters which ensured sterility for long experiments (we have used them for up to 1 week). Figure 12 shows an example with a breast cancer cell line (MDA-MB 231) comparing perfusion with agarose and matrigel, and the spectrum in uiuo of the same cell line subcutaneously implanted in a nude mouse (Lyon et al., 1988b). Agarose or alginate capsules is another very simple method that can be used for prolonged experiments (up to 72 hr). This was adapted to NMR studies of normal and tumor human cells by Narayan et af. (1990). We have used this method to study lymphocyte metabolism by 3'P-NMR (Kaplan and Cohen, 1991). Hollow fibers and dialysis membrane methods are based in the separation of the cells from the flowing perfusion solution by permeable membranes, allowing diffusion of oxygen, nutrients, and waste materials, but restraining the cells in the NMR probe (Gonzales-Mendez et al., 1982). The major drawback of these procedures is the need for a special probe. An improvement was presented by Hrovat et al. (1985) to use in a conventional 15-mm vertical NMR probe. High-density cultures can be achieved and prolonged experiments can be performed. Finally, spheroids approximate many characteristics of in uiuo tumors and are potentially an efficient experimental model. Information on cell function and evaluation on cell necrosis was obtained by Ronen and Degani (1989) and Ronen et al. (1990). This group has presented studies of phospholipid metabolism in a human breast cancer cell line (Ronen et af.,1991), metabolic changes following treatment in human breast cancer cell lines (Neeman and Degani, 1989a,b), and melanoma cells (Degani et al., 1991). B. In Vivo NMR Spectroscopy of Tumors

Despite the significant limitation due to cellular heterogeneity in tumors, in uiuo experiments are being pursued, since these are closer to the clinical situation than in uitro tests or extract observations. However, it should be possible to perform a pathological examination of the tissue in order to determine its heterogeneity and therefore its possible influence on the spectrum. In any case, a comparison with extracts is recommended. Different nuclei have been used in these experiments, depending on the kind of information desired.

40

JESUS RUIZCABELLO AND JACK S. COHEN

PE PC

agarose

Pc

5 -5 -15 Chemical shift (ppm) FIG. 12 "P spectra of human breast cancer MDA-MB 231 cells, in uiuo (subcutaneously implanted in a nude mouse and with two different modalities of ex uivo perfusion methods: matrigel and agarose). The in vivo spectrum was collected for 100 scans using a 90"flip angle pulse width (adapted from Lyon et al., 1988b) and the ex uivo experiments were acquired with 1200 scans, duration 1 hr, using a 60" flip angle pulse width, with a Varian 10-mm highresolution probe at 37°C. The repetition time was 32 sec for in uivo and 4 sec for ex uiuo. A line broadening of 10 Hz was the same for all three cases. Peak identities are indicated in Fig. 10.

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41

In uiuo NMR experiments have been used to study tumor physiology and biochemistry, for prediction and detection of therapeutic response, and finally for pharmacologic properties (Daly and Cohen, 1989; Steen, 1989).Animal transplantable tumors, either syngeneic transplants or xenografts, are experimental models that have been used frequently. This is a disadvantage compared with the spontaneous tumors found in clinical applications. Other differences can be found with clinical tumors: vascular differences, infiltration grade within normal tissue, and reproducibility grade. The last factor often determines the choice of transplant. In model studies many factors have to be controlled: effects of anesthesia (Wehrle et al., 1987; Okunieff et al., 1988); localization of tumor implantation; and elimination of signals arising from surrounding tissues, blood supply, and perfusion (Cataland e t al., 1962; Chapman, 1984). For this reason, models, as is the case with perfusion of cells, allow one to investigate particular problems that otherwise would be very difficult, for instance, the mechanism of action of drugs. However, they cannot mimic exactly the clinical situation. We are going to discuss in this section some observations of in uiuo experiments with tumor models and extend them to clinical applications. NMR spectroscopic changes have been used to characterize the physiologic state of untreated tumor growth and in assessing the effects of antineoplastic therapy. Direct data can be obtained from the spectrum, such as tumor bioenergetic status, alterations in membrane composition, and/or fluidity associated with phospholipid metabolism, obtained by phosphorus NMR. Correlations with other NMR measurements, such as proton MRI (Furman et al., 1991) and functional MRI (see above), tissue extract spectra, tumor blood flow using deuterium NMR (Ackerman et af., 1987; Evelhoch et al., 1988), or in uiuo proton spectroscopy are necessary to facilitate the evaluation. Correlations with other physiologic findings are used by investigators because clear interpretation of magnetic resonance changes in tumors with and without treatment is usually not available. Measurements with oxygen electrode, pH electrode, and histology (Sostman et af.,1988) are still necessary to validate the results. In response to therapy, correlations with in uitro NMR experiments and physiological assays are also necessary: TD,, controls, growth delays, and clonogenic assays. However, more sensitive localization techniques and emerging functional magnetic resonance imaging are allowing measurements that are uniquely accessible by NMR methods. Such approaches provide different information, such as that provided by fluorine- 18 fluorodeoxyglucose positron emission tomography (PET) and localized spectroscopy. Positron emission tomography offers measurement of metabolic rate, whereas MRS provides information about metabolite equilibrium concentrations (Heiss et al., 1990; Alger et al., 1990), although the latter can in principal theoretically measure concentration variations in time.

42

JESUS RUIZ-CABELLO AND JACK S. COHEN

Most of the actual NMR information on tumor models is due to phospho-

rus studies. Many of these studies have been addressed to superficial tumors to correlate 3'P-NMR parameters with growth during untreated and treated growth. We are not going to discuss the appearance of these parameters under treatment, since good reviews cover this aspect (Steen, 1989). During untreated growth of tumors, the levels of energy-rich compounds (ATP and phosphocreatine, PCr) have been found to tend to decline relative to other spectral resonances (Ng e t al., 1982), possibly because a large mass of tumor cells became anoxic or hypoxic during tumor growth (Cataland et al., 1962; Chapman, 1984). In this sense, the PCrhorganic phosphate (Pi) ratios of large tumors were reduced compared with those of small tumors of the same histology (Evanochko et al., 1984b; Okunieff et al., 1986, 1987, 1988; Glickson et al., 1987; Ross et al., 1988). PCr is rapidly degraded during ischemia (Hoult et al., 1974) and probably represents a sensitive index of tissue metabolic state (Ackerman et al., 1980; Griffiths et al., 1983). The changes during growth are frequently associated with an acidic shift in tumor tissue. Growing tumors may show some acidification (Ng et al., 1982; Evanochko et al., 1983; Koeze et al., 1984; Irving et a!., 1985; Adams ef af., 1985; Evelhoch et al., 1986; Okunieff et al., 1986). However, observations of high pH in neoplasms have also been presented (Oberhaensli et al., 1986; Cadoux-Hudson et al., 1989). An increase in phosphomonoesters (phosphocholine, PC, and phosphoethanolamine, PE) has also been observed during untreated growth of different tumors (Lilly et al., 1984; Maris et al., 1985; Sijens et al., 1986; Steen et al., 1988). Differences in phosphomonoester contents have also been cited to distinguish between malignant and benign growths, or those from normal tissue (Koutcher and Damadian, 1977; Degani et al., 1986; Cadoux-Hudson et al., 1989). Malignancy has generally been associated with high phosphomonoester concentration (Degani et al., 1986; Merchant et al., 1988). These peaks are related to the phospholipid metabolism and they may be an indicator of cancer cell function (Ruiz-Cabello and Cohen, 1992). Spatial localization techniques are revolutionizing the field of applications of NMR. In order to convert it to a clinically useful tool, the application of spectroscopy has to be extended to different tissues, not just superficial ones. Phosphorus magnetic resonance studies of human tumors (mainly brain tumors) have been presented in situ and demonstrate its potential applicability. Segebarth er al. (1987) used proton imaging to define the region of interest and a P-3 1 head coil instead of a surface coil, to locate the ROI with accuracy. The spectra of different brain neoplasms showed a low level of PCr and a high level of phosphomonoester. The result is in accordance with the observation in different patients, although some variations occur (Evanochko e t al., 1984b; Oberhaensli et al., 1986).

USE OF NMR IN CELLiTlSSUE PATHOLOGY

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Clinical measurements have been presented in normal and various pathological states in heart. Bottomley (1985) and Blackledge et al. (1987) were the first to apply different localization techniques. Blackledge et al. (1987) also observed similar results measuring the PCr/ATP molar ratio with the rotating frame-depth selection technique (see Table 111) and spectroscopic imaging. The rotating frame technique was also used to study different cardiopathies (Rajagopalan et af., 1987). Reductions in PCr/P, ratio were observed in endocardially or transmurally derived magnetic resonance spectra from patients with myocardial infarction, when compared with normal patients (Bottomley et al., 1987a). Other organs and tissues have been studied, such as liver and kidney (Matson et al., 1988). Localized proton magnetic resonance spectroscopy has also been demonstrated as a probe of tumor metabolism (Gadian, 1991).Clinical applications have been addressed to brain tumor studies. Alterations from the spectra of normal brain tissue have been presented. Patients with astrocytomas and other tumors (gliomas, meningiomas, neurilemoma, etc.) showed markedly reduced resonances of N-acetylaspartate (NAA) (and sometimes creatine), probably because it originates from neuronal cells (Bruhn et af., 1989; Sauter et al., 1990; Segebarth et al., 1990; Spielman et af., 1991; Ross et al., 1992). Additional information about abnormal metabolism can be obtained from lactate, choline, and alanine signals. An increase in the number of accessible metabolites will facilitate a better characterization of tumor metabolism (Frahm et al., 1991). Thus, proton MRS under certain conditions may be an earlier indicator of pathology than is T,-weighted 'H-MRI (Segebarth et al., 1990). C. Imaging of Metabolites

It is too early to know whether this technique will be useful for routine clinical diagnosis. As we discussed previously, spectroscopic or CSI methods can be presented in an image format, as well as a spectrum containing metabolic information for any designated ROI within the field of view. In the former case, instead of collecting a spectrum, the ROI is phaseencoded to produce images of the various metabolites. This new dimension, chemical shift, allows the identification of metabolites with MRI methods by their chemical-shift differences. Thus, there is another source of contrast to monitor those diseases where the magnetic field properties of water do not change (Moonen et al., 1990). Spectroscopic imaging in an image format has the advantage of enhancement in the ability to correlate multiple types of information (Nelson et al., 1991). Imaging methods consist of slice-selection, phase-encoding, and readout gradients for obtaining spatial information. Details of gradient methods are beyond the scope of this article, but we recommend any general book

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on MRI. The main feature of spectroscopic imaging is to substitute the readout gradients for two or even three phase-encoding gradients (Brown et al., 1982; Maudsley et al., 1983). Then, spectroscopic information may be observed in a spatially selective manner over a wide field of view. Metabolite images representing distribution of proton and phosphorus metabolites have been presented in several tissues to identify tumors and to study various disorders, and some carbon experiments have also been presented (Swanson et al., 1990). Such images correlate spatial information with anatomical features and may become a very important tool for clinical studies. 31Pspectroscopic images were obtained when the following technical problems were solved (Bottomley et al., 1988):( a )line broadening produced by eddy-current fields (due to variable rapid gradient switching) and (b) low signal-to-noise ratios compared to surface coil experiments. Bailes et al. (1987, 1988), Lenkinski et al. (1988), and Tropp et al. (1988) showed the first 3D chemical-shift images with clinical applications for liver, brain, and limb. Since then, there have been numerous studies directed to these and other tissues. Heart applications present additional problems (Bottomley et al., 1990), because of ( a ) motion from the heart beating, ( 6 )greater anatomical complexity, and (c) small spectral variation that cannot be observed because of the large volumes (40-100 cm3) needed for adequate sensitivity. These problems could be overcome using cardiac-gated imaging sequences and a surface coil set. These measurements could confirm that 3'Pspectroscopic imaging specifically detects spatial variations in metabolites in myocardial ischemia and infarction. 31Pimages of human forearm muscle were obtained to study the response of high-energy phosphorus metabolites with exercise in skeletal muscle (Nelson et al., 1991). Clear increases in inorganic phosphate and decreases in PCr were presented in the exercised muscles. They use an improved method to determine the peak areas or other variable of interest from the spectra in an automatic fashion, based on a previous method (Nelson and Brown, 1987, 1989a,b). Finally, a recent advance uses a dual-tuned resonator to apply radiofrequency decoupling power at the proton resonant frequency while observing phosphorus (Luyten et al., 1989b; Murphy-Boesch et al., 1990). This, as we indicated in a previous section, collapses all the multiplets into a single line if sufficient proton irradiation power is applied. In addition to an increase in sensitivity due to the elimination of the residual proton-phosphorus J coupling, the increase in the phosphorus magnetization is due to the NOE effect (see Section I,B,2). This has allowed the observation of voxel sizes less that 8 cm3in a reasonable time (- 100 min), although with less spatial requirement data may be obtained in only 20-30 min (Vigneron et al., 1990). Direct overlays of these metabolite images on proton images showed correlation with anatomy.

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Despite these advances, the large volume that is still necessary to obtain satisfactory signal-to-noise ratio 3'P-NMR limits some studies. Partial volume effects may raise questions about the heterogeneity of the tissue. Proton NMR is a more sensitive technique. The spectral information provided by 'H-NMR is complementary to that provided by phosphorus. Whereas phosphorus may provide direct evidence of tissue energy metabolism, tissue pH, and phospholipid metabolism (Ruiz-Cabello and Cohen, 1992), proton allows one to obtain information on glycolysis (lactate), amino acid production (NAA, glutamate, taurine, etc.), neurotransmitters (GABA, glutamate), tricarboxylic acid cycle (glutamate), intracellular signal transduction (inositols), and energetic (creatine) and phospholipid (choline) metabolism. However, in order to see the metabolites of interest in a proton experiment, water and lipid resonances must be suppressed, especially from outside the ROI, and improvement in spatial resolution must be attained (Frahm er al., 1991). Lipid suppression is accomplished by limiting the excited ROI, avoiding those areas richer in fat (for instance, in brain, signals arisingfrom subcutaneous fat surrounding the skull). Previous to volume selection, water suppression sequences are included. Presaturation of the water resonance has been commonly used preceding the volume-excitation pulse sequence (Hore, 1983a; Haase et al., 1985). Water suppression can also be achieved by selective inversion of the water resonance prior to volume selection and adjustment of the time between the water inversion pulse and the start of the volume selection sequence to correspond to the zero crossing time of water (Patt and Sykes, 1972). Other authors incorporate the water suppression into the volume selection part of the excitation, although parallel suppression of the creatine/phosphocreatine peak was also obtained (Spielman et a / . , 1991). Adiabatic pulses can be used for better water signal inversion (Luyten er al., 1989a). Additional water suppression can be improved with a data-shift accumulation for overcoming dynamic range problems (Roth et al., 1980). Nevertheless, recent advances with techniques employing magnetic field gradients can revolutionize water suppression approaches (for a review, see van Zijl and Moonen, 1992). Thus, localization sequences, such as STEAM, with three gradient-based water suppression sequences before and two to three in the middle of the sequence can give suppression factors of over 15,000 (Fig. 13). Images of NAA, lactate, and other proton metabolites have been presented, mostly for brain studies (de Graaf et al., 1988; Luyten et al., 1990; van Vaals et al., 1991; Lampman et al., 1991; Spielman et al., 1991; Moonen et al., 1992), perhaps because whole brain is one of the few organs with larger scale homogeneity relative to the magnetic homogeneity. The magnetic field homogeneity over a large volume (as in spectroscopic imaging) is more difficult to adjust than that over

46

JESUS RUIZ-CABELLO AND JACK S. COHEN NAA Giu Gln

Cr GABA

NAA

I 8

"

'

I

"

6

'

I

"

'

I

4

"

'

I

' 2

0

PPM

FIG. 13 In uiuo proton spectrum of a 700-pI nominal volume localized in the cerebral cortex of cat brain in parts of the parietal and frontal lobes. (TE = 12 msec; TM = 60 msec; TR = 2.0 msec) The STEAM sequence used (2048 scans) had three water suppression sequences at the beginning of the sequence and three more in the middle. The assignments are according to published data (Behar et al., 1983). Abbreviations: NAA, N-acetylaspartate; Cho, cholines; Cr, creatines; lac, lactate; Ino, inositols; Glu, glutamate; Gln, glutamine; Glc, glucose derivatives; GABA, y-amino butyric acid; Asp, aspartate; Gly, glycine; ala, alanine; Tau, taurine; Ade, adenosines; prot, protein.

small volumes. Arnold et al., (1991), in an attempt to monitor temporal metabolite changes in demyelinating lesions, showed an initial increase in the lactate concentration, possibly associated with edema. Posteriorly, a rise in choline and a decrease in NAA were observed with the beginning of symptoms. Some of these changes were reversible, but other such as NAA did not resolve, although MRI continued showing normal patterns. Figure 14 shows as example the NAA, creatine, choline and lactate images acquired in a patient with a glioma (Moonen et al., 1992). IV. Conclusion Magnetic resonance imaging and spectroscopy may have considerable value in pathological studies to differentiate benign and malignant neo-

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b

4 3 2 1 0 4 3 2 1 0 4 3 2 1 0 4 3 2 1 0 4 3 2 1 0 4 3 2 1 0 4 3 2 1 0

FIG. 14 Imaging of metabolites of the human brain in transverse direction (slice thickness 15 mm, in-plane resolution 7.5 x 7.5 mm, and T E = 272 msec). (a) Gradient-recalled echo MR image (TR = 600 msec, TE = 30 msec, and 30" flip angle). (b) A 7 x 3 array spectra from the tumor region [indicated by the grid in image (a)]. (c) NAA image; (d) creatine image: (e) total choline image; (f) lactate (plus lipid) image. The distribution of these metabolites was plotted using a mask showing intensity only for tissue within the skull boundaries. Image intensity is linear from zero to maximum level. Note the changes in signal intensity of NAA (c) and total choline (e) in the area corresponding to the lesion in (a). Total creatine is enhanced also, but this may be due to overlap with the large choline resonance. Note the loss of metabolite intensity in the area of the lateral ventricles and near the brain midline, accurately corresponding with the anatomical image (a). Large signal losses are found for all metabolites close to the anterior part, probably as a result of susceptibility gradients arising from air/tissue interfaces in the sinuses. Note that no lactate could be detected [image (f) gives mainly noise level]. The high signal close to the borders of image (f) probably originates from the fat signal of the skull due to bleeding. [From Moonen et al. (1992).]

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FIG. 14 (continued)

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USE OF NMR IN CELLiTlSSUE PATHOLOGY

FIG. 14 (continued)

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s. COHEN

plasms from other tissues and in assessing the effects of antineoplastic therapy (Sostman et al., 1988). The tumor physiological environment is one of the least understood and most important factors in determining the response of solid tumors to cancer therapy. To examine some of these characteristics, there are several NMR techniques available. Nuclear magnetic resonance tissue characterization was attempted in the past with relaxation times measurements. It was established that malignant tissues have longer relaxation times, T , and T2,than normal control tissues (Damadian, 1971). Concepts such as “malignancy index” (Goldsmith et al., 1978; Koutcher et al., 1978) were based on the relationship between various malignant tissues and their relaxation parameters. Other attempts to characterize biochemical “fingerprints” of malignancy, or characteristics such as multidrug resistance by NMR techniques, have been fruitless (Graham et al., 1987; Kaplan et al., 1991). Although this may limit clinical applications of MRS, research applications in specific areas are still possible. Differences in intrinsic relaxation parameters are present in tissues and are the basis for MRI contrast. Thus, it has been suggested that it is possible to differentiate edemas from chronic gliosis by these differences (Barnes et al., 1988). Magnetic resonance images have also been the basis for classifying tissue in benign and malignant tumor types. As we indicated above, differences in relaxation properties of water ‘H nuclei determine the contrast obtained by most of the magnetic resonance imaging techniques. This determination would have a very important applicability on present diagnostic and surgical procedures for malignant tumor diseases. However, results are contradictory; some groups have failed to discern tumors (Gohagan et al., 1987; Just et al., 1988), whereas other have presented promising results (Pedrosa et al., 1989; Taxt et al., 1992). These water-based images provide anatomical details of high image quality and are capable of distinguishing a variety of pathological conditions. Nevertheless, they cannot measure metabolic reactions among reactants at small concentrations (mM) and changes in tissues caused, for instance, by ischemia or hypoxia. Functional magnetic resonance imaging adds a new dimension to the application of magnetic resonance to medicine (Moonen et al., 1990). It offers the combination of precise anatomical localization of function and complete 3D imaging of many tissues and organs. The development of a standardized functional anatomy will broaden our understanding of the unique structure-function relations (Belliveau et al., 1991b). Today, major efforts are placed on in uiuo spectroscopy for specific tissue correlation, although ex uiuo spectroscopy of cells and extracts may help to elucidate the results. Finally, magnetic resonance spectroscopic imaging is demonstrating metabolic changes for noninvasive chemico-pathological evalua-

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tion of lesions that appear normal in MRI. For these reasons, we believe that magnetic resonance techniques should provide the scientific community with new insights into both normal and pathological processes. Acknowledgments J.R.C. thanks the Spanish Ministerio de Educacion y Ciencia and Fulbright Commission for funding a postdoctoral fellowship. We thank Maria Jose Subiela for assistance and for creating Figs. 1-5.

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Joseph C. Polacco and Mark A. Holland Department of Biochemistry and Interdisciplinary Plant Group, University of Missouri, Columbia, Missouri 6521 1

1. Introduction

Urease (urea amidohydrolase, EC 3.5.1.5) has been both on the mainstream and a perplexing curiosity on the fringe of plant and general biology. Its substrate, urea, was the first organic molecule synthesized in the laboratory, an accomplishment that brought into question vitalist theories on the origin of organic compounds (Wohler, 1828). In 1926, Sumner reported the crystallization of urease from jackbean seeds; this feat struck the first major blow against hypotheses that enzymes were nonprotein catalysts. However, in 1975 Blakely, Zerner, and co-workers (see Dixon et al., 1975, 1980a,b,c) found that nickel was an essential active site component of urease. This not only was the first example of a biological role for nickel but obviously corroborates what has been learned since 1926, namely that not all enzymes are all protein. Despite the abundance of urease in some tissues, namely seeds of some members of the families Fabaceae (Leguminosae)and Cucurbitaceae, and its ubiquity in virtually all plants (Hogan et al., 1982) (and in all tissues of soybean, the best studied example) little has been revealed of the roles of urease. Indeed, as discussed in this chapter, many instances of chemically or genetically blocking urease activity or its synthesis are not overtly deleterious. A perception shared by many is that urease plays no major role because its substrate, urea, is not a major plant metabolite; urea is an excretory form of excess nitrogen in animals and plants do not excrete nitrogen. Another conundrum, revealed if not solved by studying plant ureases, is the nutritional relationship between plants and the commensal bacteria with which they associate. Under some conditions these bacteria make a significantcontribution to the “plant” urease profile and the plant (specifically soybean), in turn, appears to provide activated nickel essential for InIernaIional Reuiew of Cytology. Vol. 145

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synthesis of active bacterial urease. In addition we will explore the possibility that bacteria participate in turnover of the “plant” urea pool. The major conclusions of this chapter are that considerable amounts of plant nitrogen flow through urea, which can be recycled only by urease action. This recapture could have significant quality impact on proteinrich crops and appears to have an important role in germination of proteinpoor seeds. The abundant seed ureases, however, appear to be nonassimilatory and likely serve a plant defense function. Urea is derived both from arginine and from ureides. However, it appears that in soybean urea generation from ureides (which are important transporters of fixed nitrogen) is the result of the action of commensal bacteria associated with the plant. These bacteria contribute to the plant’s urease profile and, in turn, they are dependent on the plant for activating nickel for their own urease synthesis.

II. Metabolic Origins of Urea in Plants Mammals synthesize urea via the Krebs-Henseleit cycle (also called the arginine, ornithine, or urea cycle) as a nontoxic form ofjettisoned ammonia (Krebs and Henseleit, 1932). Plants usually have the opposite problem, i.e., how to conserve nitrogen, which, after carbon, is the most limiting element in plant nutrition (Schubert, 1986). This contention is consistent with the presence of urease in plants [and in most bacteria and fungi (Mobley and Hausinger, 1989)] and its absence in mammals. Whereas in the latter, urea is a nontoxic “waste” form of excess ammonia, in the former, ureolytic activity is necessary to recycle urea nitrogen (urea is 47% nitrogen). We discuss in this section the metabolic and tissue origins of plant urea, and the quantity of nitrogen flux through urea. A. The Arginine Angle

Arginine, by the action of arginase, is the immediate precursor of urea in the mammalian arginine (urea) cycle. Although an active functional arginine cycle in plants has been debated (Thompson, 1980), it is clear that arginase (EC 3.5.3.1) is widespread in the plant world. Plant arginases resemble the animal enzyme in their high pH optima (approximately 9.7) and Mn2+requirement (Kang and Cho, 1990; Splittstoesser, 1969; Cheema et al., 1969; Boutin, 1981; Wright et a / . , 1982). As arginase is widespread so is its substrate abundant: arginine is a major nitrogenous transport and storage compound in plants. It is the major nitrogen transport compound

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of deciduous (Tromp and Ovaa, 1979) and coniferous (Bidwell and Druzan, 1975) trees and a major component of underground storage organs [bulbs, roots, tubers (Schubert and Boland, 1990)l. It was shown to be one of the predominant amino acids in the seeds of 379 angiosperms (VanEtten et al., 1967). We recalculated (in mol%) the reported average seed amino acid composition of these 379 species. Arginine accounted for 7.7% of seed amino acids and its “N-weighted” contribution was 21.1% of total amino acid nitrogen, the highest contribution of any amino acid, with glutamine a close second (18.6% based on the assumption that half of the glutamate in protein hydrolysate came from glutamine) (VanEtten et al., 1967). In soybean, arginine contains 18% of seed protein-bound nitrogen (Micallef and Shelp, 1989a). At least 50% of free amino acid N in developing seeds of pea (de Ruiter and Kolloffel, 1983) and soybean (Micallef and Shelp, 1989a) is in the arginine pool. In addition, many legume seeds are exceedingly rich in free canavanine (VanEtten et al., 1963), an arginine analog. Half of arginine nitrogen (and two-thirds of canavanine nitrogen), in the guanidino moiety, is convertible to urea by arginasekanavinase action.

1. Seed Metabolism A seed germinating in water produces no net increase in seed protein during early development. Proteins degraded in the cotyledon must provide amino acids for those synthesized in the expanding axis. Thus, high levels of seed arginine require that it be catabolized during germination as part of the overall reconfiguration of free and storage protein-bound amino acid profiles to that of seedling “metabolic” protein. Whereas arginine constitutes an average of 7.7% of seed protein-bound amino acids in 379 species (VanEtten et al., 1967), codon usage frequencies for a variety of cloned plant genes (many of which code seed storage proteins) translate to arginine frequencies ranging from 2.6%for wheat to 5.6%for rice (Wada et al., 1991). Although it is obvious, then, that arginine undergoes net degradation during germination, the extent of arginase participation in arginine turnover is not well characterized. For instance, a competing reaction is arginine decarboxylation to agmatine in the polyamine biosynthetic pathway. Arginine decarboxylase is induced in germinating oat (Smith, 1983), Larhyrus satiua (Ramakrishna and Adiga, 1975), and peanut (Savithramma and Swamy, 1989), for example. However, consistent with an arginase role in germination is the increase in its activity in germinating soybean axes (Kang and Cho, 1990) and in germinating pumpkin (Splittstoesser, 1969) and Viciafaba [broad bean (Kolloffel and van Dijke, 1975)l. Whereas arginine decarboxylase activity increases in the embryonic axis of peanut

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JOSEPH C. POLACCO AND MARK A. HOLLAND

seedlings (Savithramma and Swamy, 1989), arginase increases in cotyledons (Desai and Sindhu, 1986),the arginine storage organ for the seedling. A physiological role for the arginase found in high levels in germinating pea is suggested by the rapid generation of I4C0, from L-[guanidin~-'~C]arginine injected into cotyledons (de Ruiter and Kolloffel, 1983); this 14C0, is apparently released as a result of combined arginasehrease action. The pumpkin seed is an excellent example of the importance of arginase action on arginine in seedling nitrogen nutrition (Chou and Splittstoesser, 1972). Within the globulin fraction (93% of seed protein) arginine constitutes about 30% of amino acid nitrogen. At 7 days after germination arginine nitrogen constitutes 51% of the free amino acid nitrogen pool, and its arginase-catalyzed breakdown product, ornithine, constitutes another 1.5%. Importantly, among the amino acids transported out of the cotyledon to the developing axis, arginine is a minor component (1.7% of amino acid nitrogen), whereas the ornithine nitrogen component has risen to 22%. y-Aminobutyrate, a putative ornithine derivative (Abdul-Baki and Anderson, 1973; Micallef and Shelp, 1989a), constitutes another 2.5% of this pool. Thus, in the pumpkin seed, arginine is extensively catabolized in the cotyledon before delivery of its nitrogen to the axis and arginase appears to play the major role in this catabolism. (Massive de nouo ornithine synthesis appears unlikely since proline levels did not vary greatly.) The authors presented no data on the fate of urea and urea-derived ammonia liberated from arginine. Other reports on the composition of cotyledon exudate in mung bean (Kern and Chrispeels, 1978) and in castor bean (Stewart and Beevers, 1967) also show a reduction in transported arginine versus its level in reserve protein, although the differences are not as dramatic as those for pumpkin. For example, in castor bean arginine was 15% of exported amino acid nitrogen, whereas it was 24% of endosperm reserve protein [our calculation, based on the data of Stewart and Beevers (1967) assuming that half of the resultant aspartate/glutamate in endosperm protein hydrolysate was originally present as amides]. In these systems, however, much of the delivered arginine is probably further catabolized in the elongating axis.

a. Compartmentation of Arginine and Urea Metabolism Where investigated, plant arginases have a mitochondria1 or particulate location [e.g., in soybean, jack bean (Downum et al., 1983), pea (Taylor and Stewart, 1981), broad bean (Kolloffel and van Dijke, 1975), Jerusalem artichoke tubers (Wright et al., 1981), and iris bulbs (Boutin, 1982)l. Thus, models in which arginase plays a role in arginine turnover must include the active transport of arginine into the mitochondrion. Utilization of the urea derived from arginase action requires that it leave the mitochondrion since

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plant ureases appear to be cytoplasmic (Faye et al., 1986, and Section IV,C,3,b). The other arginase product, mitochondrial ornithine, appears to be translocated to growing points in the axis in pumpkin (Chou and Splittstoesser, 1972). That which remains in the mitochondrion can be converted to glutamate by a mitochondrial (Taylor and Stewart, 1981) ornithine aminotransferase (OAT). Glutamate can apparently serve in transaminations or generate new carbon skeletons upon entry into the Kreb’s cycle as a-ketoglutarate or succinate, the latter derived from yaminobutyrate, a product of glutamate decarboxylase (Abdul-Baki and Anderson, 1973) (Fig. 1). Ornithine is also a precursor of proline but this synthesis likely requires movement of arginine-derived ornithine, or its derivatives, to the plastid, which may be a destination of ornithine delivered from the cotyledon to the axis. Mitochondrial, i.e., catabolic, ornithine is separated from ornithine destined for arginine biosynthesis. The “anabolic” ornithine pool appears to be both cytoplasmic and plastidic and derived from acetylornithine (Shargool et al., 1988), whose production is subject to arginine inhibition (Morris and Thompson, 1977). Plastidic ornithine activates carbamyl phosphate synthase, which provides the second reactant for the ornithine transcarbamylase (OTC) reaction in plastids of soybean (Shargool et a / . , 1978) and pea (Taylor and Stewart, 1981). [OTC and the other “biosynthetic” enzymes of the arginine cycle, i.e., those catalyzing the conversion of ornithine to arginine, are present in soybean (Shargool et al., 1978) as are the metabolic intermediates (Micallef and Shelp, 1989a,b)]. This route of arginine biosynthesis has long been recognized in plants (Thompson, 1980).

b. Separation of Arginine Synthesis and Breakdown in Pre- and Postdormancy Seeds Arginase appears to break down significant quantities of arginine in cotyledons of germinating pumpkin (Chou and Splittstoesser, 1972)and pea (de Ruiter and Kolloffel, 1983). The latter workers observed into I4CO2apparently as conversion of injected ~-[guanidino-’~C]arginine a result of arginasehrease action during germination. However, they observed little 14C02release or urea accumulation from ~-[guanidino-’~C]arginine injected into developing cotyledons, this despite high levels of both arginine and arginase during the developmental interval studied. In contrast to pea, labeling studies in developing soybean cotyledons led to the conclusion that approximately 20% of the pools of delivered and in situ synthesized arginine was routed to urea and ornithine (Micallef and Shelp, 1989~). To resolve these apparently conflicting results between developing pea and soybean embryos we assessed urea accumulation in embryos of a soybean mutant lacking all urease activity [eu3-eZleu3-eZ, Table I1 and

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JOSEPH C. POLACCO AND MARK A. HOLLAND

ARG

DEVELOPING COTYLEDON

GAB 4 ORN 4 UREA 4 ARG 4-

COTYLEDON AFTER GERMINATION

GAB d storage

ARG

ORN

nt.4,.

'

EMBRYONIC AXIS AFTER GERMINATION

\

(cytopl=m)

FIG. 1 Arginine synthesis and utilization as a function of tissue and development. Abbreviations: mt, mitochondrion; cp, chloroplast; pb/v, protein body or vacuole; ARG, arginine; CIT, citrulline; ORN, ornithine; AcORN, acetylornithine; GAB, y-aminobutyrate; vsp, vegetative storage protein.

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Meyer-Bothling er al. (1987) and Polacco er al. (1989)l. Developing embryos of this mutant accumulate considerable urea during development as evidenced by the high levels of urea in their mature seeds [50 pmoVg, a 250-fold increase in seed urea levels over that of the progenitor cultivar (Stebbins er a / . , 1991)l. Whereas at first glance this observation would tend to corroborate models (Micallef and Shelp, 1989c) that there is an active arginase reaction in developing seeds, it does not distinguish between urea derived in sifufrom that delivered to the embryo from maternal tissues. Nor, for that matter, does it indicate the urea precursor (see the ureide discussion below). To determine their relative contributions to urea levels in developing embryos we independently manipulated maternal and embryonic genotypes (Stebbins er al., 1991). Urease-negative embryos accumulated urea only when developed on urease-negative (and urea-accumulating) plants. We conclude that all embryonic urea is imported from maternal tissues and that arginase, therefore, does not “see” the arginine pool in the developing embryo. To reconcile our results with the conclusions of Micallef and Shelp (19894 we suggest that their injection of ~-[guanidino-’~C]arginine mechanically disrupted mitochondria resulting in interaction between released arginase and the injected labeled arginine. We propose that during embryo development none of the arginine pool is routed to arginase (Fig. 1). Upon breaking of embryo dormancy, i.e., seed germination, seed protein reserves are hydrolyzed and their arginine component is extensively catabolized, as described above. The possible extent of arginase participation in this process can be determined by measuring the accumulation of urea in germinating urease-negative eu3-elleu3-el seeds harvested from urease-positive plants. Since these seeds accumulated no urea during development (Stebbins er al., 1991), any seedling urea must, of course, have been derived from germination-associated metabolism. By the third day after germination (germination is defined as the protrusion of the radicle through the seed coat) cotyledon and radicle have accumulated 83 and 16 pmol uredg dry wt, respectively (Stebbins et al., 1991). These increases, approximately 40- to 150-fold over urease-positive progenitor seedlings, are probably underestimates since no corrections were made for urea lost to efflux from roots. In conclusion, arginine contributes significantly to the seedling urea pool (Fig. 1). As will be discussed in Section II,B,2, this argument is based partially on inhibition of urea accumulation by arginase inhibitors such as P-guanidinopropionate. To address whether plants have an active urea (arginine) cycle, we ought to question the value of an apparently futile cycle whereby energy is expended to convert NH,, COz, and aspartate to urea and succinate only to have an active urease convert urea back to NH,

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and C02. It appears that the enzymes and/or reactants of a urea cycle are separated so that arginine synthesis and degradation are temporally and developmentally separated. In soybean, at least, there is no urea cycle in the developing seed since no urea is generated during embyrogenesis (Stebbins et al., 1991), notwithstanding the considerable amounts of arginine synthesized within (Micallef and Shelp, 1989b)and delivered to (Rainbird et al., 1984) the developing embryo. However, the soybean embryo generates considerable urea upon germination (Stebbins et al., 1991) and at least half of this accumulation is blocked by arginase inhibitor, pguanidinopropionate (Section 11,B,2). Extensive degradation of arginine is well documented in germinating pumpkin (Chou and Splittstoesser, 1972) and pea (de Ruiter and Kolloffel, 1983) to name two species. A test of the operation of a cycle is to assess whether putative cycle intermediates play a catalytic role; i.e., does addition of intermediates lead to a large nonstoichiometric increase in ‘‘products”? Lovatt and Cheng (1984) applied NaH14C03to excised roots from 2-day squash seedlings and assessed the recovery of label in urea and arginine. Whereas addition of unlabeled 5 mM ornithine (a urea cycle intermediate) increased labeled arginine levels >20-fold, incorporation into urea increased only 2-fold. Although there appears to be a low-activity urea cycle operative in excised squash seedling roots, the data suggest that the urea cycle enzymes have an arginine biosynthetic function (which may be a response of excised roots deprived of nitrogen flux from the cotyledon). We emphasize, however, that it would be worthy to repeat these studies in other organs and developmental stages of a variety of species and to examine the regulatory role of NH4+,which both depressed arginine synthesis and increased urea production from NaHI4CO3(Lovatt and Cheng, 1984). 2. Nonseed Metabolism (Trees, Bulbs, etc.) We have focused on arginine metabolism in seeds because this is the realm of our experience and because most of our knowledge of arginine metabolism in plants has focused on seed development and germination. However, arginine is known to be the major nitrogen transport compound of deciduous (Tromp and Ovaa, 1979)and coniferous (Bidwell and Durzan, 1975) trees and a major component of underground storage organs (bulbs, roots, tubers). The reader is urged to see the review by Schubert and Boland (1990), which cites work on the underground storage roles of arginine. That arginine in these nonseed storage organs is convertible to urea is suggested by the high levels of arginase found in Jerusalem artichoke tubers (Wright et al., 1981) and iris bulbs (Boutin, 1982), to name two examples.

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UREASE IN PLANT CELLS

6 . The Case for and against Purines

Purine degradation can provide “recaptured” nitrogen in such senescing tissues as seedling cotyledons and old leaves of maturing plants. In addition, de nouo synthesized purines appear to provide ureides (allantoinand allantoic acid; Fig. 2) as nitrogenous transport compounds in soybean seedlings (Polayes and Schubert, 1984) and it is now firmly established that 70 to 90% of the nitrogen transported out of actively fixing soybean nodules is in the form of the purine-derived ureides, allantoin and allantoic acid (Matsumoto et al., 1977; McClure and Israel, 1979; McClure er al., 1980; Atkins et al., 1982; McNeil and LaRue, 1984). Soybean and other

PURINE RING

ALLANTOIN

ALLANTOIC ACID

CITRULLINE

UREWLYCOLATE

UREA

FIG. 2 The structures of the purine ring and “ureide” derivatives allantoin, allantoic acid, and ureidoglycolate. Strictly speaking, only allantoin, with its cyclic ureido group, qualifies as a ureide. In this discussion all purine-derived ureido compounds are considered ureides. Citrulline, by our loose definition, could be considered a ureide derivative of arginine. Also shown in the figure is the structure of urea.

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tropical legumes (e.g., cowpea, winged bean, mung bean) appear to transport ureides, whereas N,-fixing legumes of temperate origins (e.g., pea and lupine) transport the amide amino acids, asparagine and glutamine (Reynolds et al., 1982). A possible advantage to ureide transport in warm weather C, plants is the C/N ratio of 1 for ureides as opposed to 2 (asparagine) or 2.5 (glutamine) for amides. The ureides, therefore, are much more conserving of photosynthate in C, plants with stomates partially closed to prevent water loss. The generation of urea from degradation of ureides in plants is still controversial. The controversy is that between the amidinohydrolases and the amidohydrolases (Fig. 3). Amidinohydrolases catalyze the release of urea from allantoic acid and its ureidoglycolate product while amidohydrolases release ammonia and CO, directly from the ureides (Fig. 3). In plants the generation of urea from ureides is an attractive hypothesis since yeast (Cooper et al., 1979) and many, if not most, bacteria (Vogels and van der A. Amidinohvdrolases

B. Amidohvdrolases

("microbial")

Urea + Urea +

1/2 SERiNE

FIG. 3 Alternative (A) urea and (B) non-urea-generating pathways of ureide degradation. These reactions are catalyzed as follows: ( I ) allantoinase, (2) allantoate amidinolamido hydrolase, (3) ureidoglycolate amidino/amido hydrolase, (4) aminotransferase, (5) glycine decarboxylase, (6) seine hydroxymethyltransferase.

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Drift, 1976) employ arnidinohydrolases (“allantoicases” and “ureidoglycolases”) to hydrolyze allantoate to two molecules of urea and glyoxylate, and since allantoin-fed plants have been reported to accumulate some urea (Mothes, 1961;Atkins et al., 1982).It is obvious, however, from nutritional studies in which urease synthesis was inhibited biochemically or genetically that ureides are not converted exclusively to urea and glyoxylate in soybean. When synthesis of active urease in soybean cell culture was inhibited by citrate complexation of available nickel, urea-supported growth was blocked but growth with allantoin nitrogen source was not (Polacco et a / . , 1982). N2-fixingmutant plants (eu3-elleu3-el),lacking all urease activity, showed little diminution of growth relative to KN0,-fed plants (Stebbins et al., 1991, 1993). In the latter case, if 70 to 90% of the fixed nitrogen were funneled through ureides to urea, a urease-negative plant would be severely growth restricted with N2as sole nitrogen source. For example, yeast mutants unable to hydrolyze urea cannot utilize allantoin (Cooper et al., 1979). The question considered here, then, is whether some of the ureide pool is metabolized to urea by amidinohydrolases in soybean and other plants. We will try to present the cases for and against ureide-derived urea in higher plants and, finally, to explain the conflicting observations (here and in Section V). 1. Evidence Against Ureide-Derived Urea

Acetohydorxamate, employed as an inhibitor of urease in soybean leaf pieces floated on allantoin (Shelp and Ireland, 1985), stimulated accumulation of urea. Unfortunately, because there were no allantoin-less controls and [2-’4C]allantoatewas not employed, a nonureide (e.g., arginine) source of this urea could not be eliminated. Acetohydroxamate, however, was found to inhibit NH, and I4CO2evolution from [14C]ureaas well as from [2-’4C]allantoate, suggesting that allantoin was degraded via a urea intermediate. However, Winkler et al. (1985) found that acetohydroxamate was an effective inhibitor of allantoate amidohydrolase, Mn2+-dependent activity in soybean embryo extracts that catalyzes the direct release of C 0 2 and NH, from allantoate. Inhibition of allantoate amidohydrolase by acetohydroxamate is not related to its urease inhibition since the more potent and specific urease inhibitor phenylphosphorodiamidate (PPD) (Held et a / . , 1976; Liao and Raines, 1985; Kobashi et al., 1985; Zerner, 1991) had no effect on allantoate amidohydrolase (Winkler et al., 1985). Our studies of extracts of developing seeds (embryos as well as seed coats) revealed no urea production during allantoate breakdown ( Winkler et al., 1985). Although other studies (Shelp and Ireland, 1985) showed urea accumulation in leaf pieces floated on allantoin, the source of this urea

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was not conclusively identified. However, three objections can be raised to a generalization that urea is not a ureide degradation product in soybean: ( a ) It is possible that some of the urea evolution that Shelp and Ireland (1985) observed was indeed from ureides. (6) It is possible that we employed the wrong assay conditions or the wrong tissue to detect a ureideto-urea activity. (c) Yet another caveat is that allantoic acid can potentially generate urea even if the allantoate to ureidoglycolate conversion completely bypasses a urea liberation step (Fig. 3B). The resultant ureidoglycolate could be enzymatically converted to urea and glyoxylate (Fig 3A), as occurs in yeast and bacteria (Vogels and van der Drift, 1976). We tested the hypotheses raised in objections a to c, above, namely that whole leaf tissue, if not extracts of developing seeds, can evolve one or more urea from allantoin and allantoic acid. We used phenylphsophorodiamidate as a specific and avid urease inhibitor in leaf tissue and followed the fate of allantoin labeled either in the ureido ([2,7-'4C]allantoin) or nonureido (central) carbons ([4,5-'4C]allantoin). Phenylphosphorodiamidate did not inhibit release of I4CO2from [2,7-'4C]allantoin at concentrations that inhibited 299.9% of 14C02release from [14C]urea. The only detectable products from [2,7-'4C]allantoinwere 14C02and [14C]allantoate, whereas [4,5-'4C]allantoin was catabolized to allantoate, CO,, glyoxylate, glycine, and serine. These results are consistent with the NMR studies of Coker and Schaefer (1983, who found that in uirro cultured developing soybean cotyledons metabolized ['3C-15N]allantoin so that all C-N bonds were broken and that 50% of the internal two-carbon fragment and all of the ureido carbons were lost (presumably as CO,), whereas the remaining internal carbon was reincorporated as a methylene carbon. The results of Winkler er al. (1985, 1987) and of Coker and Schaefer (1985) have led us to propose the non-urea-generating route of ureide assimilation shown in Fig. 3B (Winkler et al., 1987). Since no urea was generated from [2-'4C]allantoin, the model predicts a ureidoglycolate amidohydrolase. This prediction was confirmed by identification of a ureidoglycolate amidohydrolase in soybean (Winkler er al., 1988) and in developing French bean (Phaseolus uulgaris L.) pods (Wells and Lees, 1991). Based on the loss of one internal carbon as CO, and the labeling of both glycine and serine, our model indicates that the resultant glyoxylate is metabolized via the photorespiratory pathway (Givan et al., 1988). This is consistent with the reported glyoxosomal location of French bean ureidoglycolate amidohydrolase (Wells and Lees, 1991). The pathway shown in Fig. 3B was confirmed in soybean cell suspension cultures (Stahlhut and Widholm, 1989). In this study the assimilation of urea was shown to be sensitive to nickel deprivation or to the urease inhibitor phenylphosphorodiamidate. However, cells utilizing allantoin were not growth restricted by nickel deprivation or by phenylphosphorodi-

UREASE IN PLANT CELLS

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amidate nor did they accumulate urea when utilizing allantoin in the presence of phenylphosphorodiamidate. So what of the reported accumulation of urea in plants fed ureides? Usually, these studies have not employed labeled precursors to identify the source of urea (e.g., Shelp and Ireland, 1985; Mothes, 1961). Atkins et al. (1982) did report ['4C]urea production in cowpea (Vigna unguiculata L. Walp.) leaf tissue fed [2-'4C]allantoin. However, they found urea in a Dowex 50 (H +) fraction where neutral urea should not be found, suggesting (to us) that urea was derived from acidic hydrolysis of allantoate. Urea found in phloem samples taken from feeding aphids may have been a product of aphid metabolism.

2. Evidence for Ureide-Derived Urea A chapter on the roles of urease in plant cells would not be the place to discuss ureide catabolism if the arguments against urea production from ureides were airtight and substantiated beyond doubt. However, "reasonable doubt" prevails when considering ureide degradation in tissues other than leaves or seed coats (not to mention in plants less studied than soybean). For example, what is the origin of nonureide xylem nitrogen [mainly amide amino acids (Peoples et al., 1985a)I in N,-fixing, ureidetransporting plants? Does it represent the product of ureide degradation, which in roots and/or nodules goes through a urea intermediate? Another tissue worthy of further study is the pod wall. Nitrogenous compounds translocated up the xylem in N,-fixing soybeans are transferred to both leaf and developing fruits, whereas nitrogen derived from NO,- is primarily incorporated into protein of leaves and roots before being utilized for seed development (Ohyama, 1983). The pod wall is a likely site for active ureide catabolism since 40% of its soluble nitrogen is ureide (Ohyama, 1984) and no ureides are delivered by the seed coat to the developing soybean embryo (Rainbird et al., 1984). In N,-fixing cowpea the composition of xylem from the root system is similar to that of the peduncle, which feeds the developing fruit, and both are rich in ureides (Peoples et al., 1985a). Phloem, collected from cowpea pods by a cryopuncture technique (Pate et al., 1984), or from seed coat exudate (Peoples et al., 1985b), contained little ureide, again indicating active ureide degradation in the pod wall or seed coat before delivery of amino acids to the developing embryo. We raise these questions on tissue-specific ureide degradation because nonleaf tissues for which urea generation from ureides seems to have been established are seedling axes and cotyledons (Fujihara and Yamaguchi, 1978). Allopurinol, a potent inhibitor of xanthine dehydrogenase from nodules of navy bean (Boland, 1981) and soybean (Triplett et al., 1980),

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JOSEPH C. POLACCO AND MARK A. HOLLAND

inhibited the accumulation of allantoin, allantoic acid, and urea in soybean seedlings [3 days after germination (Fujihara and Yamaguchi, 1978)l. The authors reported urea levels (8.6 and 18.6 pmoY10 g fresh weight (fw) cotyledon and axis, respectively) in untreated soybean seedlings at least 10 times those we observed (Stebbins et al., 1991). However, our allopurinol treatments, discussed below, tend to corroborate their conclusion that urea is derived from ureides in seedlings. Importantly, genetic inactivation of urease leads to even higher levels of accumulated urea than those seen by Fujihara and Yamaguchi (1978). Azaserine is an inhibitor of de nouo purine biosynthesis; it is a glutamine analog that inhibits the transamidation reaction leading to formation of the purine precursor formyglycinamide ribonucleotide (Webb, 1966). Fujihara and Yamaguchi (1978) reported that azaserine did not inhibit ureide accumulation in axes of 3-day seedlings. Although this would suggest that ureides in 3-day seedlings are derived from breakdown of cotyledonary nucleic acids, and not from de nouo synthesized purines, Polayes and Schubert (1984) extended these observations to the cotyledons in which they found that ureide accumulation was inhibited by azaserine (which, again, did not depress ureide accumulation in the axis). By the eighth day after germination the cotyledon had lost this azaserine sensitivity, whereas ureide accumulation in the axis was increasingly azaserine-sensitive. Thus, germinating soybeans are generators of ureides both from de nouo purine synthesis and from nucleotide breakdown. We employed eu3-elleu3-el soybean mutant seedlings lacking all urease (Table 11) activity to assess the effects of allopurinol on urea accumulation in the absence of complications associated with urea hydrolysis. Allopurino1 ( 1 mM) inhibited urea accumulation by about 50% while inhibiting ureide accumulation by 85% in radicles of 3-day seedlings. Fujihara and Yamaguchi (1978) reported 100% inhibition of urea accumulation by allopurinol. We were able, however, to eliminate all urea accumulation by applying a combination of allopurinol and 20 mM P-guanidinopropionate, an arginase inhibitor (Wright et al., 1981). Combined, these compounds reduced urea levels to those observed in control wild-type seedlings (Stebbins er al., 1993). In our hands 0-guanidinopropionate displayed a K j of 3 mM toward axis arginase (K,,, for arginine = 1 1 m w ) . These results indicate that urea is essentially from two sources in soybean seedlings, ureides and arginine. Eskew et al., reported that a urease-negative phenocopy can be induced in soybean (1983, 1984) and cowpea (1983) plants by nickel deprivation. Nickel-starved plants accumulated considerable urea, to the extent that. leaf tips became necrotic, containing 2.5% urea by dry weight. The authors reported that leaf tip necrosis was more severe in soybean plants relying completely on N, than in plants fed NH4+ and NO,- (Eskew er al., 1983). Although they presented no data on relative total urea levels, they

UREASE

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suggested that N,-fixing plants accumulated more urea because of their higher xylem ureide levels. Our observations tend to confirm this contention: Leaf and stem urea levels are fivefold higher in eu3-elleu3-ef mutant plants relying on N, as opposed to those utilizing 5 mM KNO, (Stebbins et al., 1993). Since there was little or no urea in extracts of wild-type N,-fixing plants, urea was not generated nonenzymatically from ureides during extraction and analysis (Stebbins et al., 1993). Callus is another tissue in which ureides may be degraded to urea. Whereas Stahlhut and Widholm (1989) found no urea generation from allantoin in soybean suspension cultures, we observed urea accumulation in eu3-eZleu3-eZ callus grown with allantoin as sole nitrogen source. Urea accumulated at even higher levels in cells fed arginine, whereas KNO,/ NH,NO,-grown cells accumulated no urea.

3. A Resolution In summary, the arguments for ureide-derived urea are ( a ) the increased levels of urea in nitrogen-fixing soybean plants (our results) and the increased leaf tip necrosis when Ni-deprived plants are dependent on fixed nitrogen (Eskew et al., 1983); ( 6 )allopurinol inhibition of urea accumulation in soybean seedlings (our results; Fujihara and Yamaguchi, 1978);and (c) urea accumulation in soybean callus utilizing allantoin nitrogen source (our results). The arguments against ureide-derived urea are ( a ) lack of inhibition of 14C02evolution from [2,7-'4C]allantoin in uitro (developing soybeans) (Winkler et al., 1985) or in uiuo (soybean leaf discs) (Winkler et al., 1987) by specific and avid urease inhibitors, and no detection of urea accumulation or diminution of I4CO2label by unlabeled urea in these systems; and (b) lack of generation of urea in soybean suspension cultures employing allantoin as sole nitrogen source (Stahlhut and Widholm, 1989). We believe that these conflicting results within soybean can be resolved by considering the metabolic contributions by commensal bacteria (discussed in Section V). For example, we ascribe the discrepancies between our allantoin-supported callus and the allantoin-supported suspension cultures of Stahlhut and Widholm (1989) to covert of commensal bacteria present in our cultures (Holland and Polacco, 1992)and absent from theirs ( J.M. Widholm, personal communication, confirmed by our own assays). These bacteria degrade allantoin exclusively via the urea-generating amidinohydrolase pathway (Fig. 3A and Section V,D). 111. Elimination of Urease Activity: Consequences for the Plant

In higher plants it appears that urea can be assimilated only by urease action. Urease-negative plants and cultures, induced genetically (Meyer-

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JOSEPH C. POLACCO AND MARK A. HOLLAND

Bothling et af., Polacco et a f . , 1989) with urease inhibitor (Krogmeier et al., 1989; Stahlhut and Widholm, 1989) or by nickel deprivation (Polacco, 1976, 1977a; Eskew et af., 1983, 1984), have been observed either to accumulate urea or to be blocked in the ability to employ urea as a nitrogen source. All plant (Polacco, 1977b) and bacterial (Mobley and Hausinger, 1989) ureases are probably nickel metalloenzymes. Seed ureases from jackbean (Dixon et af., 1975; Fishbein et a f . , 1976) and soybean (Polacco and Havir, 1979) have been shown to contain nickel. Duckweed plants (Gordon e f al., 1978) and callus of soybean, rice, and tobacco (Polacco, 1977a,b) are dependent on nickel for maximal growth with urea as sole nitrogen source. Urease appears to be the only nickel-requiring enzyme in plants since, as indicated below, nickel-deprived soybean plants have the same phenotype as those genetically blocked in urease synthesis (Stebbins et af., 1991). Thus, higher plants appear to lack the ATP-dependent urea amidohydrolase reported in algae and yeast (Roon and Levenberg, 1968, 1970; Thompson and Muenster, 1971; Whitney and Cooper, 1972). This biotin-containing carboxylase/hydrolase appears not to be a nickel metalloenzyme and has a urea-assimilatory function (e.g., Whitney et a f . , 1973) in these urease-negative lower eukaryotes. In an interesting example of the potential of urease to provide nitrogen for the plant, Meier-Greiner et a f . (1991) developed transgenic tobacco plants engineered for resistance to cyanamide. The resistance gene, from soil fungus Myrothecium verrucaria, codes a cyanamide hydratase that converts cyanamide to urea. Although urea levels are raised in such plants, the endogenous urease apparently hydrolyzes much of the liberated urea. A. Effect on Protein Deposition and Embryo Development

Given that urease is the plant’s only means of assimilating urea, the next question is the metabolic version of “If you’re so smart how come you ain’t rich?” When applied to urease it would read, “If you’re so important how come the plant survives without you?” Indeed, completely ureasenegative mutant soybean plants develop to maturity and produce a relatively good yield of seeds that germinate at normal frequency to propagate another generation. However, if the role of urease is to recycle urea nitrogen generated from arginine (and possibly ureide) degradation, then a protein-rich plant such as soybean may provide a “suppressing” background for a urease defect. Soybean has indeed been intensively bred for large and protein-rich seeds. However, even in soybean we question the dispensability of urease. Urease-negative mutant plants [eu3-el/eu3-el and eu4/eu4, Table I1 (Stebbins et al., 1991) and nickel-deprived wild type (Eskew et a f . , 1983, 1984)] exhibit necrotic leaf tips, apparently due to

UREASE IN PLANT CELLS

81

urea “burn.” Similar observations were made in nickel-deprived tomato (Shimada ef al., 1980; Shimada and Ando, 1980). More important, perhaps, than the deleterious effects of leaf burn is the loss of significant quantities of nitrogen in a urea dead-end, lost nitrogen that could have a significant negative impact on seed protein deposition during pod fill. The assessment of the agronomic impact of a urease-negative phenotype on soybean performance requires extensive field testing of isogenic o r nearly isogenic paired urease-positive and urease-negative lines, preferably in multiple environments. To obtain these lines we are currently engaged in the long process of backcrossing, generation advance, selection for uniformity in maturity group and plant architecture, etc., and amplification of seed stocks (Cianzio and Polacco, 1993). Our prediction that total seed proteidplant will decrease is suggested by Eskew et al. (1984), who reported a correlation between seed yield and nickel content per seed. However, there was too much variation in their material to obtain a statistically significant difference. We observed that at the time of flowering completely urease-negative soybean mutants accumulated approximately 100 pmol uredg dry wt of total leaf [green plus necrotic tip (Stebbins ef al., 1991)]. Assuming that 10% of the leaf dry weight is protein (16% nitrogen), much of which is destined to provide amino acids during pod fill, accumulated urea (47% nitrogen) represents 18% of the nitrogen in leaf protein. The developing soybean embryo does not generate urea (Stebbins et al., 1991). Thus, other than recycling maternally derived urea, urease appears to play no direct role in embryo metabolism. However, it is possible that in monocots a urease role is more critical in embryo development. Brown et al. (1987) reported that nickel is essential for development of viable barley embryos. Postdormancy grains could not be rescued by nickel, whereas developing grains could be rescued by the feeding of nickel to the maternal plant. The role of nickel in barley embryo development is not known and may be unrelated to urease. (Urease is the only nickel metalloenzyme yet identified in plants.) However, it is possible that a loss of urease activity under nickel-deprivation conditions leads either to urea poisoning or to nitrogen starvation of the embyro. It would be informative to study barley embryo development in uitro, where both nickel availability and nitrogen source can be manipulated.

B. Effects on Germination What is the consequence of blocking urease action during germination when large of amounts of arginine and ureides (see above) are mobilized and generate urea? In soybean we observed a 7- to 8-hr delay in germina-

82

JOSEPH C. POLACCO AND MARK A. HOLLAND

tion of its protein-rich seeds imbibed in the potent urease inhibitor (Held et al., 1976; Liao and Raines, 1985; Kobashi et al., 1985; Zerner, 1991) phenylphosphorodiamidate(PPD) (Zonia, 1992). Protein-poor Arabidopsis thaliana seeds imbibed in water with 50 p M PPD did not germinate at all. That PPD acts specifically on urease and brings about inhibition of germination by nitrogen limitation is indicated by parallel dose-response curves for germination and urease inhibition, and by reversal of inhibition of germination with added nitrogen sources, NH,NO, (5mM), or caseinhydrolysate (1 mg/ml) (Zonia, 1992). Zonia (1992) observed similar responses to a cyclotriphosphazatriene urease inhibitor (Savant et al., 1988). Within A . thaliana, large seeds germinated in distilled water tend to produce seedlings that survive longer than those from small seeds (Krannitz et al., 1991). Phenylphosphorodiamidatemay effectively reduce seed size by depriving the seedling of that portion of its nitrogenous reserves catabolized to urea. Hydrolyzed A. thaliana seed meal contains 5.5 g arginine per 16 g total nitrogen (VanEtten e f al., 1967). Thus, 1.76 g or 11% of seed nitrogen (free plus that bound in protein and nucleic acids) is in arginine and, potentially, 5.5% (or more, if arginine is proportionally higher in “reserves”) seed nitrogen can be converted to urea by arginase. Seed nitrogen in nucleic acids is another generator of urea; greater than 40% of urea generated in 6-day Arabidopsis seedlings is eliminated by allopurinol (Zonia, 1992). In other species, e.g., Lupinus texensis (Schaal, 1980) and wild radish (Stanton, 1984), larger seeds tend to germinate at a higher frequency than small seeds. We have observed a crude correlation, across species, between seed size and resistance to PPD inhibition of germination and will extend studies of relative PPD sensitivity to largeseeded soybeans vs selected small-seeded sister lines (LeRoy et al., 19911, when these lines are made available to the public. C. Loss of Chemical Protection

As described in the next section, soybean contains two distinct urease isozymes: the ubiquitous urease is made in all tissues examined, whereas synthesis of the embryo-specific urease is confined to the developing embryo and is retained in the mature seed where its specific activity is roughly 1000-fold greater than that of the ubiquitous urease in any tissue (Polacco and Sparks, 1982; Polacco and Winkler, 1984). Since mutants lacking the embryo-specific urease do not exhibit any of the abnormalities associated with loss of the ubiquitous urease [necrotic leaf tips, accumulation of urea in leaves or seeds, retarded germination (Stebbins et al., 1991)l we conclude that this enzyme has no essential physiological function. In uitro culture of developing cotyledons of pea (Lea et al., 1979) and soybean

UREASE IN PLANT CELLS

83

(Thompson et al., 1977; Stebbins et al., 1991) indicates that ureases play little or no role in embryo nutrition since urea was an extremely poor nitrogen source. Indeed, urea is not normally generated within the developing soybean cotyledon in uiuo (Stebbins et al., 1991), in agreement with the lack of ureide delivery to the legume embryo from maternal tissues (Rainbird et al., 1984; Peoples et al., 1985b). The obvious question from the observations of the previous paragraph is why would the developing soybean embryo [and those of jackbean, watermelon, and many other members of the Fabaceae (Leguminosae) and Cucurbitaceae (Bailey and Boulter, 1971)l invest in a very active ureolytic activity when it never “sees” urea. [Although much urea is generated upon germination (Stebbins et al., 1991) and although much of the embryo-specific urease is retained in seedling cotyledons and roots (Torisky and Polacco, 19901, the loss of the embryo-specific urease causes no discernible increase in seedling urea levels over those of wild type (Stebbins et al., 1991).] We suggest, therefore, that the embryo-specific urease plays no urea assimilatory role but rather one in seed chemical defense. To draw two parallels, at least, with the pathogenic effects of bacterial urease on vertebrates, active seed urease could cause either hepatic coma by subversion of the urea cycle or peptic ulceration by localized increases in NH,+and OH-ions (urea + 3H20 + 2NH4+ + HC0,- + OH-). A microaerophilic, bacterium, Helicobacter pylori, can colonize gastric epithelium because its active urease creates a more basic microenvironment in this acidic milieu (Eaton et al., 1991; Ferrero and Lee, 1991). Arguments summarized by Cussac et al. (1992) link bacterial urease to ulceration of the gastric mucosa either by the direct cytotoxicity of ammonia or by its prevention of proton flux from gastric glands to the gastric lumen resulting in a back-diffusion of protons. Hepatic coma results when intestinally derived nitrogenous compounds, e.g., ammonia, bypass the liver and reach the brain. Administration of urease inhibitors has proven effective in reducing hyperammonemia (Summerskill et al., 1967; Fishbein and Daly, 1970). It is easy to visualize an active seed urease mimicking these bacterial effects [the bacterial and plant seed ureases have >50% amino acid identity (Mulrooney and Hausinger, 1990)], especially urease aided by other cytotoxic components in the seed [protease inhibitors, lectins that disrupt intestinal brush borders (Pusztai et al., 1979), etc.]. Another postulated chemical defense for seed urease is its induction of a hostile environment upon microbial, and perhaps insect, attack. By this second model, wounding or infection of the immature embryo will lead to arginase release from ruptured mitochondria. Cytoplasmic arginase would generate urea from the large pool of arginine [which is at least 50% of free amino acid nitrogen (de Ruiter and Kolloffel, 1983; Micallef and Shelp,

04

JOSEPH C. POLACCO AND MARK A. HOLLAND

1989a)l and cytoplasmic urease would rapidly convert urea to ammonia. Indeed, we have observed that cultured cotyledons containing the embryospecific urease commit suicide (probably by the combined effects of ammonia toxicity and medium alkalinization) in the presence of urea (20 mM), whereas those containing only the ubiquitous urease isozyme survive and utilize this urea, albeit poorly (Stebbins et al., 1991). The assessment of pest resistance and herbivore avoidance of soybean cultivars lacking the embryo-specific urease requires extensive field testing of isogenic lines exposed to a variety of pathogens and pests. These studies await material from our current backcrossing program to introduce ureasenegative mutations into high-performance cultivars (Cianzio and Polacco, 1993). In addition to the abundant storage proteins, seeds contain several moderate- to high-abundance proteins with enzymatic or other biological activity. It is easy (at least for us) to invoke a plant protection role for many of these proteins: phytohemagglutinins (lectins) (cited in Liener, 1979), lipoxygenases (Gardner, 1979), ribosomal inactivators (Gatehouse et al., 1990) and inhibitors of amylase (Weselake et al., 1983), and animal proteases (Gatehouse and Boulter, 1983). The lack of an essential physiological role for many of these proteins in planta is suggested by the relatively high frequency of cultivars and varieties lacking one of them [urease (Polacco et al., 1982), lipoxygenases (Hildebrand and Hymowitz, 1982; Davies and Nielsen, 1986; Kitamura et al., 1983), lectin (Pull et al., 1978), etc.]. Lack of an essential physiological role for the seed (embryospecific) urease is further suggested by the high prevalence of seed urease nulls in Japanese populations of Glycine soja (Mervosh and Hymowitz, 1987), a sexually compatible close relative of cultivated soybean (G. max).

IV. Biochemical Genetics of Soybean Urease Production

A. Structural Genes

Our current understanding of urease expression in soybean is summarized in Fig. 4. Soybean produces two urease isozymes (Table I) (Holland et al., 1987): Ubiquitous urease is synthesized in all organs, but it appears to be most active in young tissues (Holland et al., 1987; Polacco and Winkler, 1984; Polacco et al., 1985). Embryo-specijic urease is synthesized exclusively in the developing embryo, although roots of young soybean plants retain considerable embryo-specific urease derived from the embryonic axis (Torisky and Polacco, 1990). It accumulates at 0.2 (Polacco and Havir, 1979) to 0.4% (Winkler et al., 1983) of total dry seed protein and has a specific activity from r100- to >500-fold that of the ubiquitous

85

UREASE IN PLANT CELLS

Eul (clone J 3 1)

p'ocessing functions:

Eu4 (clone LC4)

Eu2, Eu3

Nickel

~~

MATURE URJZASES EMBRY0-SPECIFIC

UBIQUITOUS

FIG.4 Current model of the genetic basis of soybean-encoded ureases. Mutation in any of four genes eliminates one or both ureases. Each structural gene (Eul and Eu4) encodes an ca. 91-kDa subunit. For simplicity, the multimeric holoenzymes are not indicated. The ubiquitous urease is trimeric (Polacco and Havir, 1979; Polacco et a / . , 1985), whereas the embryo-specific can exist in the 3n or 6n state and the propensity to make the 6n to 3n transition is controlled by E d alleles (Polacco and Sparks, 1982; Kloth and Hymowitz, 1985).

urease in developing (Polacco and Winkler, 1984) and mature (Polacco et al., 1982; Polacco and Winkler, 1984) embryos, respectively. Each urease isozyme appears to be encoded by a single structural gene (Fig. 4 and Table 11): Eul and Eu4 for the embryo-specific and ubiquitous ureases, respectively. Mutation at each affects only one isozyme (MeyerBothling and Polacco, 1987; Polacco et al., 1989). A structural role for Eul is based on the phenotypes of five allelic Eul mutations (Table 11) that collectively affect the level of embryo-specific urease mRNA and the nature (temperature sensitivity, activity, level) of its protein product (Kloth et al., 1987;Meyer-Bothling and Polacco, 1987).In addition ureasepositive Eul alleles affect the electrophoretic mobility (aggregation state) of the embryo-specific urease (Table I ) (Buttery and Buzzell, 1971; Kloth and Hymowitz, 1985). Homozgous eu4 plants have 52% wild-type urease activity in expanded leaves (Polacco et al., 19891, and similarly reduced activity in roots and hypocotyls (Torisky and Polacco, 1990).We extended observations of eu4-induced loss of ubiquitous urease activity to seed coats and eul-sunleul-sun embryos, indicating that Eu4 is active in all soybean tissues. Consistent with its role as the ubiquitous urease structural

86

JOSEPH C. POLACCO AND MARK A. HOLLAND

TABLE I Urease Enzyme Profile of Soybean

Ubiquitous urease Tissue source Crude seed extract specific activitp Subunit sizeb Subunit structureC.d pH optimap Km

Embryo-specific urease

Embryo, seed coat, leaf, hypocotyl, root, cell culture pmol/minmg

Embryo

91 kDa 3n 5.5, 8.8 0.8 m M p

91 kDa 6n ( E d - a )or 3n ( E d - 6 ) 7.5 2.9-476 mmf

1 pmoVminmg

Holland et al. (1987). Sizes of 95 and 93.5 kDa were inferred from migration of the ubiquitous and embryospecific ureases, respectively, on SDS-PAGE (Polacco et al., 1985). However, both LC4 and LSl, the structural genes for ubiquitous and embryo-specificureases, respectively (Fig. 4), contain ORFs of 840 amino acids (Torisky et al., 1993). This is the number of residues in completely sequenced jackbean seed urease (Riddles et al., 1991; Takishima et al., 1988), which has a computed subunit size of 91 m a . Polacco et al. (1985). Kloth and Hymowitz (1985). Kerr et al. (1983). Values for jackbean seed urease. Its K,,, varies with investigator and assay condition. The value of 2.9 mM from Zerner’s group (Zerner, 1991) is reliable. Talsky and Kunkler (1%7, cited in Reithel, 1971) reported literature K,,, values ranging from 19 to 476 m M .

gene in these tissues is the cosegregation of eu4 with an RFLP detected with a subclone of urease genomic clone LC4 (Torisky et al., 1993). In addition to genetic data, i.e., cosegregation of an LC4 restriction polymorphism with eu4, there is increasing molecular evidence that LC4 codes the ubiquitous urease. Its nucleotide and deduced amino acid sequences match those of leaf (i.e., ubiquitous urease) urease cDNA and protein, respectively, much more closely than the nucleotide sequence of a second heterologous clone, LS1. Clones LS1 and LC4 align perfectly in their 20 ,N-terminal deduced amino acids with sequences determined for the embryo-specific and ubiquitous urease proteins, respectively. The two isozymes, in turn, differ from each other in 2 of the 20 N-terminal residues (Torisky et al., 1993) (Fig. 5).

B. Accessory Genes Mutation at Eu2 or Eu3 eliminates the activities of both ureases but has little effect on their protein levels (Holland et al., 1987; Meyer-Bothling

TABLE II Phenotypic Properties of Soybean Urease Mutants

Embryo-specific urease" Class

Genotype

Progenitor (Williams) Structural Embryo-specific urease

Eul, Eu2, Eu3, Eu4 -

eul-n6 (ts) eul-n7

++ + + + +

eu4 eu2 eu3-I Eu3-e3

Specific activity (%) 100

eul-sun eul-n4

eul-nd Structural Ubiquitous Accessory

mRNA

0 0 0.09 0 0.7 100 0.7

0 0.1

antigen (%) 100

0 0 (5) 0 (0.5) 100 100 100

Ubiquitous urease ActivJAggreg. antigen (%) state 100

3n

Activityb

Antigen'

100

100

100

(100) (2)

6n

(100) (100)

(100)

3n

(100)

6n

6n 6n

52 0 0 0.16

38 38

90

Note. All mutant alleles are recessive except for Eu3-e3, which is codominant with the progenitor Eu3 allele (Meyer-Bothling er a / . , 1987). The euln6/eul-n6 mutant produces a temperature-sensitive embryo-specific urease (Meyer-Bothling and Polacco, 1987). The Eul, Eu2, Eu3, and Eu4 loci show no linkage (Meyer-Bothling er al., 1987; Polacco er al., 1989; J. C. Polacco and S.R. Cianzio, unpublished data). " eu4 specific activity values are from Polacco er a / . (1989). All others are from Meyer-Bathling and Polacco (1987) and Meyer-Bothling er a / . (1987). Antigen values in parentheses are estimates from intensities of Western blots. Aggregation state was determined by Western blot analysis of native gels. Ubiquitous urease values in parentheses were determined on leaves of varying developmental stages and varied from 25 to 225% of Williams leaves. Urease levels in Williams leaves vary over a 2.5-fold range depending on degree of expansion and leaf node (Holland et a/., 1987). Ubiquitous urease antigen values are from ELISA assays of extracts of leaves from 2-week plants (Polacco et a / . , 1989).

'

08

JOSEPH C. POLACCO AND MARK A. HOLLAND

LC4 deduced Ubiquitous Urease Protein

MKLSPREIEKLDLHNAGYLA

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

MKLSPREIEKLDLHNAGYLA

*-* LS1 deduced

MKLSPREVEKLGLHNAGYLA

Embryo-specific Urease protein

MKLSPREVEKLGLHNAGYLA

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

Jackbean Urease (cDNA and Protein) MKLSPREVEKLGLHNAGYLA FIG. 5 Sequences of the 20 N-terminal amino acids of the soybean embryo-specific and ubiquitous urease isozymes compared to deduced amino acid sequences of two soybean urease genomic clones (LSI and LC4). The jackbean seed urease sequence is based on both nucleotide (Riddles et al., 1991) and amino acid (Takishimaet a / . , 1988) sequence analyses. Asterisks denote identity between sequences; *, denote two differences between the seed and the upiquitous isozymes. One of these differences, I + V, is a conservative change, whereas the other, D + G, is not (Torisky ef al., 1993).

et al., 1987; Table 111. We suspect that these pleiotropic mutations affect the nickel activation function(s) necessary for maturation of each urease isozyme (Fig. 4). The defect is not simply in nickel uptake or transport (Cataldo et al., 1978a,b): Uptake of 63Ni2+is normal in mutant plants; wild-type scions grafted on urease-negative stocks produce seeds with normal urease activity (Holland and Polacco, 1992);fertilization of mutant flowers with wild-type pollen yields fully urease-positive heterozygous seeds that obviously had been fed sufficient nickel by mutant maternal tissue (Stebbins et al., 1991; Meyer-Bothling et al., 1987). The next section, however, discusses hydrogenase- and urease-less phenotypes of commensal bacteria isolated from eu2 and eu3 plants which suggest that mutant host plants cannot properly activate Ni*+ordeliver it to active sites of apoenzymes (Holland and Polacco, 1992). Homozygous plant with lesions at Eu2, Eu3, or Eu4 share acommon phenotypic trait, i.e., lack of the ubiquitous urease, and in addition exhibit necrotic leaf tips. These are likely due to accumulated urea: The three genotypes have at least 50 x the wild-type leaf urea level (Stebbins et al., 1991). Further, low-urease phenocopies induced by nickel deprivation also produce leaves that accumulate urea and have urea-rich necrotic tips (Eskew et al., 1983, 1984). The Eul, Eu2, Eu3, and Eu4 loci are unlinked (MeyerBothling et al., 1987; Polacco et al., 1989; Torisky et al., 1993).

C. Gene Products 1. Structural Genes The basis for electrophoretic allelomorphs (Buttery and Buzzell, 1971) at the Eul locus of the seed (embryo-specific) urease appears to be due to

UREASE IN PLANT CELLS

89

differences in aggregation states, the fast form being a trimer and the slow a hexamer (Polacco and Havir, 1979: Polacco et al., 1985). That the seed urease hexamer can form a trimer in uitro (Polacco and Havir, 1979; Polacco and Sparks, 1982) was confirmed by Kloth and Hymowitz (1983, who reexamined the dominance relationships at the EuZ locus and determined that the “genetic isozymes” [fast (trimeric) and slow (hexameric)] of seed urease were codominant only when seed extracts were examined in high salt buffers, which stabilized the genetic hexamer in the presence of the genetic trimer. The “genetic” trimer, however, has not been observed to dimerize in uitro to the hexameric form. A variety that produced the hexameric seed urease produced a ubiquitous urease that was trimeric, as it appears to be in all varieties so far tested (Polacco et al., 1985). Earlier reports on multiple aggregation states were made by Fishbein et al. (1975) for jackbean seed urease and by Delisle (1977) for a mycoplasma urease. The complete, chemically determined amino acid sequence of jackbean seed urease reveals a 90.8-kDa protein of 840 amino acids (Takishima et al., 1988). The open reading frame of two contiguous urease cDNA clones from jackbean seed yields a deduced 840 amino acid protein that differs from the chemically determined sequence by one conservative amino acid change (Riddles et al., 1991). It would be informative to determine whether the varieties yielding these two sequences contain urease electrophoretic allelomorphs, i.e., 3n vs 6n. Jackbean and soybean seed ureases are serologically related and their subunits migrate identically on SDS-PAGE gels (Polacco and Havir, 1979). Partial sequence analysis of a soybean urease genomic clone (LC4, Fig. 4) (Krueger et al., 1987) revealed a deduced amino acid run of 130 residues of which 108 (83%) were identical to the corresponding jackbean sequence. Sequence conservation of ureases is extended across kingdoms, i.e., between plants and bacteria. The a, p. and y subunits of the Klebsiella aerogenes urease, when aligned with the single subunit of jackbean urease, show 59, 52, and 60% identity, respectively, to the plant enzyme (Mulrooney and Hausinger, 1990). 2. Regulation of Structural GenedGene Products Why is the urease subunit so large and why does it form multimers? [In plants it has a 3n, 6n, or higher degree of aggregation, whereas the K. aerogenes and other bacterial ureases have an a2p4y4 structure (Todd and Hausinger, 1987, 1989).] Large size and multimeric structure suggest allosteric control. However, there are no known effectors on the plant ureases. In fact, they exhibit, in uiuo, an uncontrolled hydrolysis of urea to the extent that urease-positive soybean callus is killed in the presence of 225 mM urea, presumably due to excessive ammonia or OH- derived from urea hydrolysis (Meyer-Bothling et al., 1987; Holland et al., 1987; Polacco et al., 1989). Urease-negative callus showed no ill-effects in 25 to

90

JOSEPH C. POLACCO AND MARK A. HOLLAND

250 mM urea (Meyer-Bothling et al., 1987). Soybean callus contains the ubiquitous urease, the isozyme likely having an assimilatory function (Stebbins et al., 1991). We suggest that plants, which have evolved under chronic nitrogen-need conditions, do not have elaborate mechanisms of dealing with conditions of nitrogen excess. Yeast, an organism capable of urea hydrolysis (Whitney et al., 1973), is not growth inhibited in 200 mM urea provided either as sole nitrogen source or in conjunction with proline as a nitrogen source (J. C. Polacco, unpublished observations). There have been a few studies on induction of plant ureases by urea. In barley, Chen and Ching (1988) presented evidence for multiple isozymes of urease. Two of these appeared to be urea-inducible. The earlier literature has several reports of the inducibility of urease by urea [e.g., in rice (Matsumoto et al., 1966), in potato (Mokronosov et al., 1966), and in soybean suspension culture (Polacco, 1976)l. Skokut and Filner (1980) reported a slow, reversible adaptive response of cultured tobacco cells to urea as sole nitrogen source. This response was characterized by continuous increases in urease activity and urea-supported growth rate and appeared to be too rapid and reversible to result from selection of highurease mutant cells. We believe, as explained in the next section, that virtually all cases of urease induction by urea can be explained either by induction of urease in commensal bacteria in the plant tissue or by proliferation of high-urease bacterial subpopulations. Urease transcript, protein, and activity are stimulated in parallel during germination of A. thaliana (Zonia, 1992), a pattern consistent with a nitrogen assimilatory role. Increases in urease activity during germination have been reported for pea (de Ruiter and Kolloffel, 1983). 3. Nickel Activation and the Subcellular Location of Plant Ureases

This is not the place for a detailed discussion on urease structure and mechanisms of catalysis. However, two other features of urease are germane to our discussion: the subcellular location of plant ureases and the insertion of essential nickel into the urease active site. The cellular location of the ureases is of obvious importance in developing models, presented above, of their roles in metabolism (Fig. 1) and chemical defense. Nickel insertion is important, because as discussed in the next section, plant nickel activation functions may serve both the plant and commensal bacteria. The contribution of the latter to plant nitrogen metabolism may be considerable. Plant ureases appear to be cytoplasmic. Faye et al., (1986) showed that newly synthesized jackbean seed urease was not in organellar fractions [rough endoplasmic reticulum (ER), Golgi, mitochondria] but was associ-

UREASE IN PLANT CELLS

91

ated with soluble cellular fractions, which include cytosolic proteins and soluble proteins from the protein body. A protein body location is unlikely, however, because the seed urease does not undergo signal peptide cleavage; the N-terminal amino acid sequence of mature urease (Takishima er al., 1988)matches that beginning from the initiator methionine of the open reading frame (Riddles et al., 1991). Faye et al. (1986) showed that lack of entry of urease into the cellular secretory pathway was consistent with evidence that it is not a glycoprotein (lack of incorporation of [3H]glucosamine and concanavalin A binding or reduction in size with tunicamycin). These observations are confirmed by the deduced (Riddles et al., 1991) and chemically determined (Takishima et al., 1988) sequences, neither of which contain glycosylation sites (Asn-X-Ser/Thr) on which high mannose chains can attach. The N-terminal 20 residues of soybean seed urease, determined either chemically or deduced from the nucleotide sequence (in clone LSl, Fig. 3, both begin with methionine and are identical (Torisky et al., 1993). The ubiquitous urease, for which the evidence indicates a metabolic function, also appears to be cytoplasmic. In unpublished cell fractionation studies we have observed that this isozyme is cytoplasmic. In addition, the N terminus of the open reading frame of clone LC4 appears to be identical to the amino acid sequence of ubiquitous urease, and both begin with methionine (Fig. 5 ) (Torisky et al., 1993). 4. Accessory Gene Function and Products The seed (embryo-specific) ureases of jackbean (Dixon et al., 1980a) and soybean (Polacco and Havir, 1979) contain two nickel atoms per subunit. Inactivation of urease by PPD and other phosphoramides likely occurs through their binding of active-site nickel (Dixon et al., 1980c; Zerner, 1991). The proper emplacement of two nickel atoms at the urease active site appears to require participation by accessory functions. Nickel-free embryo-specific urease in seeds of nickel-deprived soybean plants could not be reactivated by nickel in uitro. However, imbibition in Ni2+resulted in almost complete activation of urease (in a tissue that no longer synthesizes the embryo-specific urease) even in the presence of cycloheximide (Winkler et al., 1983) or electron transport inhibitors ( J . C. Polacco and R. G. Winkler, unpublished observations). Cycloheximide did not block nickel activation of urease in intact algal (Rees and Bekheet, 1982) or cultured soybean (Polacco and Winkler, 1984) cells deprived of nickel. However, despite efforts by several groups, especially those of Zerner and Blakeley (see Zerner, 1991), we know of no successful in uitro nickel activation of a plant apourease, either synthesized in nickel-free tissue (Winkler et al., 1983 and unpublished experiments) or chemically stripped

92

JOSEPH C. POLACCO AND MARK A. HOLLAND

of nickel (Dixon et al., 1980b; Zerner, 1991). It has similarly been observed that nickel can activate bacterial ureases in uiuo but not in uitro. Mulrooney and Hausinger (1990) examined the inactive urease made in Escherichia coli containing a plasmid with the K. aerogenes urease operon lacking the urease accessory genes. This urease had 0.25 mol of Ni rather than the normal 4 per mole of a2p4y4multimer and could not be reactivated in uitro with Ni. However, Lee et al. (1990) reported that the apourease produced by nickel-deprived K. aerogenes cells (containing urease accessory genes) was activated by nickel in uiuo, even in cells treated with the protein synthesis inhibitor spectinomycin. A temporal separation between urease synthesis and nickel activation may explain why the appearance of urease activity lags that of urease urease antigen in developing soybean embryos (Polacco and Sparks, 1982). Although the urease accessory genes of several bacteria have been cloned, little has been inferred of their function. A C-terminal histidinerich region in Klebsiella accessory gene UreE suggested a divalent cation binding site (Mulrooney and Hausinger, 1990). However, the homologous gene necessary for urease expression in H. pylori lacks this histidine-rich region (Cussac et al., 1992). What seems likely though is that at least some accessory genes are involved in nickel metabolism or urease insertion. Also likely is that the soybean accessory genes Eu2 and Eu3 are involved in nickel activation since commensal bacteria living on these mutants lack the activities of two nickel metalloenzymes, urease and hydrogenase (Holland and Polacco, 1992),as discussed in the next section.

V. Urea and Nickel Metabolism: Two Points of Interaction betweeen Soybean and a Commensal Bacterium A. A ”Plant” Urease Is Bacterial

In addition to the embryo-specific and ubiquitous ureases, specified by the Mendelian genes Eul and Eu4, respectively (Fig. 4), soybean also contains a “background” urease. In fully expanded eu4/eu4 leaves this activity is 1 2 % that of progenitor (Eu4/Eu4)leaves. However, in expanding unifoliates and callus cultures of eu4/eu4 it reaches 20 to 40% of progenitor activity in whole tissue assays (Polacco et al., 1989). In tissue extracts the pH profile of the background urease was found to be closer to that of the embryo-specific than the ubiquitous urease, suggesting that it may represent a “trickle” level (<1%) of expression of Eul in nonembryogenic tissue. However, double-mutant (euZ-sun/eul-sun,eu4/eu4) unifoliates and callus retained the background urease (Holland and Polacco, 1992).

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Although it is formally possible that the background urease in eu4/eu4 results from incomplete in activation of Eu4 expression, evidence suggests that most or all of it is bacterial. A common commensal bacterium in plants is the pink-pigmented, facultative methylotroph (PPFM) assigned to the genus Methylobacterium (Green and Bousfield, 1983).These bacteria have been reported from a large number of plant species (Corpe, 1985; Corpe and Basile, 1982). In young soybean leaves we have observed up to 45 x 10’ cfu/g fw, whereas the titer drops to lo5 cfu/g fw in mature leaves. We cannot remove PPFMs from seeds or leaves by surface sterilization and they are found in all of our callus and suspension cultures although they do not overgrow the cultured cells (Holland and Polacco, 1992). Others have reported covert bacterial “contamination” of plant cell cultures (Horsch and King, 1983; Leifert et a/., 1991). By the criteria of pH preference, recognition by antibodies to the embryo-specific urease, and borate sensitivity, the background urease in leaf and callus extracts of eu4/eu4 tissues more closely resembled urease from free-living PPFMs than the ubiquitous urease [from mature Eu4/Eu4 (wild-type) leaves]. A more convincing demonstration of the bacterial origin of the background urease was the reduction in its activity concomitant with the removal of bacteria from eu4/eu4 tissues. Thus, cefotaxime (100 pg/ml) treatment over a 3-week period reduced both the PPFM titer and the background urease of eu4/eu4 callus by 30%, while having little effect on Eu4/Eu4 callus urease or on the growth (with NH,+/N03- nitrogen source) of either callus genotype. Seeds of eu4/eu4 were heat-treated [dry seeds at 45”C/48 hr or imbibed seeds at 52”C/10 min (Rodrigues Pereira et al., 1972)] and yielded 4-week-old, soil-grown plants with no detectable PPFMs in 70 to 88% of trifoliates [assayed by “press-plating” washed trifoliate leaves on selective mineral salts with methanol carbon source (Corpe, 1985)l. Leaves from these plants showed a concomitant 80% reduction in background urease compared to levels in leaves from untreated eu4/eu4 plants (Holland and Polacco, 1992). B. Some Resident Bacteria Require Plant Nickel Activation Functions

Surprisingly, expanding leaves and callus of mutants in the putative urease accessory genes, Eu2 and Eu3, did not exhibit background urease (Polacco et a/., 1989) although they harbored normal numbers of PPFMs. Bacteria newly isolated from eu2/eu2 or eu3-eZleu3-eZ plants were urease“negative” in free-living overnight liquid culture unless nickel supplements were provided. Bacteria isolated from eu4/eu4 or wild-type plants did not require nickel supplements to express maximal urease levels. We

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extended these observations to hydrogenase; many of the bacterial uptake hydrogenases require active-site nickel for activity (Ankel-Fuchs and Thauer, 1988; Walsh and Orme-Johnson, 1987). As was the case for urease production, there was much higher hydrogenase activity in PPFMs recently isolated from wild-type and eu4/eu4 plants than in isolates from eu2/eu2 and eu3-elleu3-el plants unless nickel had been added to the growth medium (Holland and Polacco, 1992). The observations of the previous paragraph can be explained by a block in nickel uptake/transport in eu2 and eu3-el mutants. Cataldo er al. (1978a, b) observed saturable nickel uptake in soybean. Its kinetics and competition suggested that Ni2+,Cu2+and Zn2+shared the same carriers; during vegetative growth leaves accumulated 90% of label from 63Ni2+andat senescence >70% of shoot label was mobilized to the developing seeds. We found that 63Ni2+ root uptake and translocation to the leaf were indistinguishable among normal, eu2, and eu3-el plants. Normal nickel uptake and translocation were confirmed by the fully urease-positive phenotype of seeds borne by wild-type scions grafted on mutant stocks (Holland and Polacco, 1992). [Seed urease activity is much more sensitive to nickel limitation than is leaf activity (Eskew er al., 1983), probably because the leaf sequesters trace nickel before it can be mobilized to the developing seed.] Heterozygous embryos developing on homozygous eu2 and eu3-el plants (Meyer-Bothling er al., 1987; Stebbins er al., 1991) apparently receive enough Ni from maternal tissues to be fully urease-positive. Thus, it appears that nickel activation and not movement is controlled by Eu2 and Eu3 and that the resident PPFMs require this activation function. The dependence of commensal bacterium on plant-activated nickel in order to synthesize a “plant” (i.e., background) urease suggests that other aspects of “plant” metabolism are a resultant of both microbial and plantencoded functions. At the least, commensal bacterial nutrition is mainly or wholly dependent on plant products. For example, methylotrophic PPFMs may consume (and perhaps detoxify) methanol, which is found in their cuticular location (Corpe and Rheem, 1989). Methanol may be released from methylated pectin uronic groups that undergo changes in degrees of esterification during elongation [e.g., in the germinating maize coleoptile (Kim and Carpita, 1992)l. It is possible that PPFMs participate in pectin turnover (we find them at highest levels in soybean growth zones). In the next section we explore the possibility that PPFMs are involved in plant ureide and urea metabolism. C. Bacterial Urease Roles in Developing and Germinating Seeds

In eu4/eu4 leaves, which contain only the background/bacterial urease, urea accumulates to roughly the same level as in eu3-elleu3-el leaves

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lacking both ubiquitous and background urease (about 100 pmollg dry wt). Thus, the background urease is unable to prevent urea accumulation in leaves (and its spillover into developing seeds, detected by completely urease-negative “indicator” embryos developing on eu4leu4 plants). However, when the background urease is the sole ureolytic activity in developing embryos (i.e., eu4/eu4,eul-sun/eul-sun), it prevents virtually all urea accumulation in the developing seed. Urease from the bacteria may even benejit the developing seed: when cotyledons containing only the backgroundlbacterial urease are cultured in uitro with urea as sole nitrogen source, they synthesize more protein than cotyledons (eu3-ell eu3-el)lacking all ureases (Stebbins et al., 1991). Large quantities of mobilized nitrogen are funneled through urea during soybean germination. Completely urease-negative (eu3-elleu3-el)seedlings, lacking embryo-specific, ubiquitous, and background ureases, accumulate >80 pmol urealg dry wt radicle by the third day of germination. Assuming that 10% of radicle dry weight is protein (16% N), the urea (47% N) not utilized at Day 3 (83 pmollgm dry wt, a minimal value since it does not account for urea lost to efflux) represents 15% of the nitrogen incorporated into protein. Surprisingly, eu4/eu4,eul-sunleul-sun embryos containing only the background urease accumulated 85% less urea (Stebbins et al., 1991). This can be ascribed either to efficient urea hydrolysis by resident bacteria in eu4/eu4,eul-sunleul-sunseeds or to generation of urea by urease-negative bacteria in eu3-elleu3-el seeds. As described in the next section we favor the latter explanation.

D. Bacteria as Generators of Urea from Ureides In soybean, both in uitro (Winkler et al., 1985, 1988) and leaf disc (Winkler et al., 1987) studies revealed that the ureide nitrogen of allantoin and allantoic acid was converted to utilizable ammonia without the generation of a urea intermediate. These conclusions were confirmed in cultured soybean cells whose utilization of urea but not allantoin was blocked by the urease inhibitor PPD. Cells growing with allantoin as sole nitrogen source did not accumulate urea in the presence of PPD (Stahlhut and Widholm, 1989). Although these convincing observations lead to the comfortable conclusion that urea is not ureide-derived in soybean, two observations tend to shake our faith: (a) Fujihara and Yamaguchi (1978)reported that allopurinol(1 mM), which blocks ureide accumulation by its inhibition of xanthine dehydrogenase (Boland, 1981; Triplett et al., 1980), inhibited the accumulation of allantoin, allantoic acid, and urea in soybean seedlings (3 days after germination). We have found similar allopurinol effects on urealureide accumulation in completely urease-negative, eu3-eZleu3-el seedlings. (b) Leaf and stem urea levels are five fold higher in eu3-elleu3-

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e l mutant plants relying on N, as opposed to those utilizing 5 mM KNO, (Stebbins et al., 1993). We believe that the ureidolytic activities of commensal PPFM bacteria may be responsible for ureide-derived urea in soybean. Our argument is that these bacteria degrade ureides to urea in free-living culture and that their (partial) removal from plant tissue lowers the level of ureide-derived urea. In free-living culture these PPFMs degrade ureides by the pathway shown in Fig. 3A. The evidence is as follows: phenylphosphorodiamidate completely blocks growth with either urea or allantoin as sole nitrogen source but has no effect on NH4C1-supported growth (Fig. 6). Although phenylphosphorodiamidate causes no urea accumulation in NH4CIsupported cells, the addition of urea-free allantoin to the medium results in intracellular accumulation of urea (our unpublished results). We point out, in addition, that arginine does not serve as a nitrogen source for PPFMs (Fig. 6) and thus these bacteria probably do not contribute to the arginine-derived urea pool in planta. However, do PPFM bacteria contribute to a ureide-derived urea pool in planta? To test this we assessed the effect of removal of bacteria from germinating seeds on their allopurinol-sensitive production of urea. To remove bacteria we followed the heat-curing protocol of Rodrigues Pereira PPFMs on Nitrogen Sources +I- PPD

-&-Urea

20

30

+ PPD

40

50

60

HOURS

70

80

90

100

FIG. 6 Phenylphosphorodiamidate(PPD) growth sensitivity of a soybean PPFM isolate supported by nitrogen sources urea and NH&I and potential urea generators arginine and allantoin. All nitrogen sources were fed at 5 meq nitrogen per liter. Each nitrogen source tested was the sole nitrogen source for the culture. Growth is expressed as an increase in the optical density (OD 550) of the culture at 550 nm.

97

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k

I

cr

5

" CONlRoL

COMRoL+AP

HEAT

HEAT + AP

TREATMENT

Effect of heat-curing (Holland and Polacco, 1992) on allopurinol (AP)-sensitive urea production in 3-day soybean seedlings. Solid bars indicate urea production.

FIG. 7

et ul. (1972) for seeds of Ardisiu species. Heat-cured soybeans produced 2-week plants lacking detectable PPFMs in 70 to 80% of trifoliates (Holland and Polacco, 1992). Heating dry seeds 24 hr at 50°C resulted in a 90 to 95% reduction of PPFM colony-forming titer in 3-day seedlings. Germination of eu3-el leu3-el seeds in 1 mM allopurinol reduced urea accumulation by 50% compared to germination in water (Fig. 7). Seedlings from heattreated seeds of the same genotype germinated in water showed the same reduction in urea accumulation. Significantly, the combination of these treatments (i.e., germination of heat-treated seeds in 1 mM allopurinol) resulted in no additional reduction in urea accumulation (Fig. 7). We conclude that since heat curing and allopurinol have identical, nonadditive effects, seedling urea derived from ureide breakdown is a product of bacterial activity.

VI. Summary and Prospects for Continued Research on Plant Urea Metabolism

In summary there are several indications that urease is important for efficient nitrogen assimilation. The urease substrate urea is derived from

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arginine and from ureides. Arginine, is the richest nitrogen repository among the amino acids of seed storage proteins. Ureides are not only significant sources of nitrogen in nucleic acid turnover but are also the predominant transport from of fixed nitrogen in soybean and other “tropical” legumes. Urease-negative plants accumulate substantial, nonutilizable urea in both maternal and embryonic tissue. During germination of urease-negative seeds, further urea accumulates as a dead end in nitrogen metabolism. Although this accumulation may not be a lethal defect for large protein-rich seeds like soybean, small or protein-poor seeds, such as Arubidopsis, may be severely retarded or blocked in germination by the lack of an active urease. The better known, abundant seed ureases, for example, Sumner’s jackbean urease, may play a chemical defense role, similar to those postulated for some ureases of pathogenic bacteria. All of the ureases characterized to date, both from bacteria and plants, resemble each other in primary structure and in their requirement for accessory genes. Although the functions of the accessory genes have not yet been fully characterized they are undoubtedly involved in insertion of a nickel cofactor at the urease active site. A study of urease has revealed what may be a universal phenomenon in plant biochemistry: that enzyme profiles may be a composite of both plant and microbial activities. Indeed, the microbial contributions may be physiologically significant to the plant; in soybean we have observed that bacteria are responsible for generating urea from ureides and mitigating urea accumulation in some ureasenegative mutants. In the future, there is a real need to quantify nitrogen fluxes through urea and to assess in the field and greenhouse the effects of urease inactivation on germination, seedling vigor, protein deposition, etc. Such studies are best performed in isogenic plant lines differing in the presence or absence of urease. Soybean is so far the best experimental subject in light of its battery of urease mutants, its importance as a protein crop, and knowledge of i t s physiology. The ability to eliminate the abundant embyrospecific urease, exclusively, will allow us to focus on its possible defense roles, especially when the seed urease-negative trait is combined with those eliminating lectins (Pull et u f . , 1978), protease inhibitors (Orf and Hymowitz, 1979), and other putative defense factors. At the molecular genetic level a firmer correlation needs to be established between putative structural gene loci and cloned ureases. “Nail in the coffin” evidence requires that mutants be corrected with reintroduced cloned genes. Obviously, the accessory genes need to be isolated to help us understand their function, not only from sequence algorithms but also from their potential to correct analogous defects in transgenic bacteria or fungi (Mackay and Pateman, 1980). Comparison of accessory genes (not yet isolated) from PPFMs (M. mesophificum) and from soybean may lead

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to some insight into how plant eu2 and eu3 mutations lead to effectively nickel-deprived PPFMs in planta. Again, accessory gene exchanges (performed by the experimentalist) between mutant plant and bacteria should improve our understanding of at least two important areas: ( a ) urease activation specifically and metal metabolism in general; ( b ) the nature of the PPFM-soybean relationship that appears to represent a general plant-bacterial association. Obviously, improved understanding of the extent of plant-bacterium interdependence requires that we successfully cure plants of the bacterium. It is not clear whether this has been achieved, even in plants regenerated from cell culture. Finally, we need to understand the nature of controls on urease. Are the signals leading to its elevation in germinating pea (de Ruiter and Kolloffel, 1983)and Arubidopsis (Zonia, 1992) related to the jasmonic acid induction of vegetative storage proteins (Staswick et al., 1991)? Is urease part of an ensemble of N assimilatory enzymes whose expression is coordinately controlled?

Acknowledgments The authors thank J. Dunleavy and W. A. Corpe for sharing insights about the PPFMs, R. Torisky and J. D. Griffin for sharing unpublished data, and D. Blevins for helpful comments on the manuscript. The work described herein was supported in part by Grants NSF DCB 8718314, NSF DCB 8804778, and NSF IBN 91 1768 from the National Science Foundation.

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Polacco, J. C., and Havir. E. A. (1979). J. Biol. Chem. 254, 1707-1715. Polacco, J. C., and Sparks, R. B., Jr. (1982). Plant Physiol. 70, 585-591. Polacco, J. C., and Winkler, R. G. (1984). Plant Physiol. 74, 800-803. Polacco, J. C . , Thomas, A. L., and Bledsoe, P. J. (1982). Plant Physiol. 69, 1233-1240. Polacco, J. C., Krueger, R. W., and Winkler, R. G. (1985). Plant Physiol. 79, 794-800. Polacco, J. C.. Judd, A. K., Dybing, J. K.. and Cianzio. S. R. (1989). Mol. Gen. Genet. 217, 257-262.

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Savithramma, N., and Swamy. P. M. (1989). Ann. Bot. (London) 64, 337-341. Schaal, B. A. (1980). A m . J . Bor. 67, 703-709. Schubert, K. R. (1986). Ann. Rev. Plant Physiol. 37, 539-574. Schubert, K. R., and Boland, M. (1990). In “The Biochemistry of Plants” (B. J. Miflin and P. J. Lea, eds.), Vol. 16, pp. 197-282. Academic Press, San Diego. Shargool, P. D., Steeves, T., Weaver, M., and Russell, M. (1978). Can. J. Biochem. 56, 273-279; errata: 56, 926. Shargool, P. D., Jain, J. C., and McKay, G. (1988). Phyrochemistry 27, 1571-1574. Shelp, B. J., and Ireland, R. J. (1985). Plant Physiol. 77, 779-783. Shimada, N., and Ando, T. (1980). Nippon Dojo Hiryogaku Zasshi 51, 493-496. Shimada, N., Ando, T., Tomiyama M., and Kaku, H. (1980). Nippon Dojo Hiryogaku Zasshi 51,487-492.

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Aspects of Amphibian Metamorphosis: Hormonal Control Sakae Kikuyama, Kousuke Kawamura, Shigeyasu Tanaka,* and Kazutoshi Yamamoto Department of Biology, School of Education, Waseda University, Tokyo 169-50, Japan; and *Institute of Endocrinology, Gunma University, Maebashi 371, Japan

1. Introduction

Almost eight decades have passed since Gudernatch (1912) first demonstrated the role of the thyroid gland in amphibian metamorphosis. During that period, a number of investigators have tried to elucidate the endocrine mechanisms underlying amphibian metamorphosis, and in almost every decade, studies on the hormonal control of amphibian metamorphosis have been extensively reviewed (Allen, 1938; Bounhiol, 1942; Etkin, 1955, 1964; Dodd and Dodd, 1976; White and Nicoll, 1981; Rosenkilde, 1985; Norris and Dent, 1988). Based on present knowledge, several pituitary hormones and adrenocortical hormones, in addition to thyroid hormone, are considered to be involved in metamorphosis. Recently, efforts have been made to quantify these hormones in the circulation, their receptors in target organs, and the gene expression of these hormones and of their receptors in metamorphosing larvae. In this chapter, recent studies on metamorphosis are reviewed from the viewpoint of hormonal control. Readers are also referred to other reviews on amphibian metamorphosis, focusing on biochemical and cell-biological (Galton, 1983; Yoshizato, 1989) and morphological (Fox, 1984) aspects. For convenience, the developmental stages of larvae of representative species are given in Tables I and 11. II. Thyroid Hormone A. Thyroid Hormone and its Receptors

On the basis of a series of experiments using thyroidectomized tadpoles, Etkin (1968) concluded that thyroid hormone levels need to be low in Internotional Reuiew of Cytology. Vol. 145

105

Copyright 8 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

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TABLE I Comparison of Embryonic and Prefeeding Stages in the Normal Tables of Xenopus laevis and Rana pipiens

X . laevis Specialized features

R . pipiens NF' stages

Common characteristics

1-9

Fertilized egg-blastula Gastrulationdevelopment of a recognizable head Tail fins clearly seen

Shumwayb stages Specialized features

~

10-24

Spontaneous movements start

Stomodeum development beginsC

Mouth breaks through

26

1-9 10-16

16

29-30

Tailbud stage

18

31-32

Visceral arches develop Heart starts to beat

19

33-34

Spontaneous movements start

20

37-38 39 40

Gill circulation begins

21 22 23

Tail circulation begins

operculumC

Feeding starts, cloaca opens 45 46 47

I

1

24

TK stage I 25

TK stage I1 Feeding starts

Note. Table modified from Dodd and Dodd (1976). Stages of Nieuwkoop and Faber (1956). Stages of Shumway (1940). Arrows indicate stages at which particular changes begin and end.

order for premetamorphic tadpoles to grow without showing metamorphic changes. In the subsequent prometamorphic period, hormone levels gradually increase, and as a consequence some metamorphic changes such as hind limb growth occur. For the final phase of metamorphosis, known as climax, higher levels of thyroid hormones are required, since in thyroidec-

HORMONAL CONTROL OF AMPHIBIAN METAMORPHOSIS

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tomized tadpoles with relatively low concentrations of thyroid hormone, no tail shortening occurs. This situation has been partially confirmed by radioimmunoassay (RIA) for thyroid hormones. The RIA data have been obtained mainly from Rana catesbeiana tadpoles (Miyauchi et al., 1977; Regard et al., 1978; Mondou and Kaltenbach, 1979; Suzuki and Suzuki, 1981), from which it is easy to collect blood samples because of their relatively large body size. But, plasma hormone concentrations in other amphibian species, such as Xenopus laeuis (Leloup and Buscaglia, 1977), R. calmitans (Weil, 1986), Eurycea bislineata (Alberch et al., 1986), Ambystoma gracile (Eagleson and McKeown, 1978), A . tigrinum (Larras-Regard et al., 1981; Norman et a f . , 1987), and Hynobius nigrescens (Suzuki, 1987), have also been measured. Recently, thyroid hormone has been extracted from the whole tissue of small tadpoles such as those of Bufo japonicus and measured by RIA (Niinuma et al., 1991b). Data on R. catesbeiana tadpoles are somewhat inconsistent among investigators, but it is evident that the hormone levels are high during climax. It is surprising that in several instances, thyroid hormone levels during prometamorphosis and even at the onset of climax still remain undetectable. However, even if the thyroid hormone levels at these stages are low, initiation of metamorphosis could still be feasible if an increase in receptors for thyroid hormone and elevation of plasma concentrations of adrenal corticoids, which are known to augment the activity of thyroid hormone (III,A), occur. It is generally known that triiodothyronine (T,) is biologically more active than thyroxine (T,) and that T, is generated mainly by deiodination of T, in peripheral tissues. In fact, T, is several times as potent as T, in inducing tail regression in R. catesbeiana (Kistler et al., 1977), Xenopus (Robinson et al., 1977) and B. japonicus (Kikuyama et al., 1983). Galton and Hiebert (1988) examined the ontogeny of iodothyronine 5 ' deiodinase activity, which generates T, from T,, and 5-deiodinase activity, which converts T, to reverse T, in major tissues of R. catesbeiana tadpoles. They showed that during prometamorphosis, 5'-deiodinase activity is undetectable in liver, tail, heart, and kidney, minimal in brain and gut, and present in skin. During climax, 5'-deiodinase activity is undetectable in liver, heart, and kidney but present in tail tissue, and is increased more than fivefold in skin and gut. Conversely, 5-deiodinase is detectable in most tissues during prometamorphosis and hardly detectable during climax. It was concluded that at preclimax stages, accumulation of T, generated from T4 in the tadpole is minimal due to the predominance of 5-deiodinase activity, and that during climax accumulation of T, is possibly due to the increase in 5'-deiodinase activity and the decline of 5-deiodinase activity. The significance of conversion of T, to T, in metamorphosis was well demonstrated in an experiment conducted by Buscaglia et al. (1985).

TABLE II Comparison of Larval Stages in the Normal Tables of Larvae of Xenopus laevis and Rana pipiens

X. laevis

R . pipiens

NP Main diagnostic features Premetamorphosis' Embryonic nonfeeding stages

z

First thyroid follicles recognizable

stages 1-45 46 47-48 49-50

(See Table I )

I

(See Table I )

I1 I11 IV V VI VII VIII IX

51

52 53

Hind leg length, 2.0 mrn

TK~ stages

Common characteristics

Foot paddle stages

54

Main diagnostic features

A

Prometamorphosis' Hind leg length, 3.0 mm

55

56 56 +

Hind limb stages

XI XI1 XI11 XIV

51

xv

51 +

XVI XVII

Foot stages

Cloaca1 tailpiece maximal

Front limbs erupt

58

Pharynx shrinkage starts

59 59 + 60

Tadpole at maximum length TIB > 2 Total length reduces slowly

Operculum narrows

61

Tail length not reduced

62

TIB 2 or > 2 Rapid tail resorption begins

ClimaxC Tail fins shrink Tail shrinkage starts; still used in swimming

XVII + XVIII

Cloacal tailpiece begins to shrink Cloacal tailpiece lost

XIX

Skin window seen Mouthparts larval

xx

Front limbs erupt Larval mouthparts lost Mouth widens Tail shrinkage starts Gill resorption starts Operculum narrows

XXI Tail no longer used in swimming

63 63 + 64 65 66

Note. Table modified from Dodd and Dodd (1976). Stages of Nieuwkoop and Faber (1956). Stages of Taylor and Kollros (1946). Terminology used by Etkin (1968).

TIB 1.5 TIB 1 . 1 TIB 0.3 Tail stump Slow resorption of stump Miniature adult

XXII XXIII XIV

xxv

110

SAME KIKUYAMA ET AL.

They used Xenopus larvae, in which endogenous thyroid biosynthesis and secretion were inhibited by perchlorate. In such tadpoles, metamorphosis was reinduced with threshold doses of either T4 or T,. Thyroxine 5’deiodination was inhibited using iopanoic acid, a potent inhibitor of deiodinase. When the Xenopus larvae were immersed in water containing iopanoic acid, T,-induced metamorphosis was blocked and the larvae became giant larvae, whereas T,-induced metamorphosis was completed. It is well understood that binding to a nuclear site in the target cell is the first step in the biological action of thyroid hormones. Yoshizato and Frieden (1975) first described the nuclear receptor for thyroid hormone in amphibian tissue. They observed that the nuclear receptor for T, in the tail fin cells of bullfrog tadpoles shows a twofold increase in maximum binding capacity with a slight increase in Kd value during metamorphosis from TK (Taylor and Kollros, 1946) stage X to the climax stage (XXII). They concluded that the apparent increase in T3receptors may result from stimulation by the gradually increasing thyroid hormone levels. More recently, Moriya et al. (1984) reported that the number of binding sites for T, in larval-type red blood cell nuclei increases fourfold during the same period of metamorphosis. They also showed that treatment of prometamorphic tadpoles with T, induces an increase in both the number and the K d value of the binding sites for T,. Similar results have been obtained by Galton (1984). These findings suggest that the increase in binding sites for T, during spontaneous metamorphosis is induced by the increasing level of endogenous thyroid hormone around the onset of climax, although the function of thyroid hormone in red blood cells has not been clarified. With regard to the localization of thyroid hormone on and in amphibian red blood cells, Kaltenbach (1982) pointed out that T, is distributed mainly in the nucleus and around the plasma membrane on the basis of observations of blood smears from bullfrog tadpoles by indirect immunofluorescence staining. Recently, Yamauchi et al. (1989) demonstrated the presence of T,-specific binding sites on the plasma membrane. They concluded that T, binds initially to specific binding sites on the cell surface and then enters the cell by receptor-mediated endocytosis. Red blood cells could be a good model for studies of thyroid hormone receptors because pure and intact cells are easily obtainable by simple centrifugation procedures. Kistler et al. (1975, 1977) studied nuclear receptors in tail fin and liver cells from bullfrog larvae in uitro. According to their data, T, has twice the number of binding sites as T,, although the & values for T, and T, are comparable. A competition experiment revealed that T, binds to two sites, whereas T, binds to only one. Accordingly, the greater biological activity of T, may be due to the higher number of binding sites available for it rather than higher affinity of receptors for the hormone.

111

HORMONAL CONTROL OF AMPHIBIAN METAMORPHOSIS

Recently, thyroid hormone receptors in the amphibia have been studied using a molecular endocrinological approach. Yaoita et al. (1990) have cloned and characterized the cDNAs encoding the thyroid hormone a and p receptors from X. laeuis. They studied the expression of the thyroid hormone receptor genes in Xenopus larvae (Yaoita and Brown, 1990) and found that the level of thyroid hormone a-receptor mRNA in the whole body increases throughout the premetamorphic stage, reaches maximum by prometamorphosis, and declines after metamorphic climax. On the other hand, the level of thyroid hormone @-receptor mRNA remains very low during prometamorphosis and reaches a peak at metamorphic climax in parallel with plasma thyroid hormone levels (Fig. 1). The close correlation between thyroid hormone P-receptor mRNA and endogenous thyroid hormone formation in tadpoles suggests that expression of the thyroid hormone @-receptorgene is regulated by thyroid hormone. In fact, treatment of premetamorphic tadpoles with T, produces marked elevation of the level of thyroid hormone receptor @ mRNA and moderate elevation of that of thyroid hormone receptor a mRNA. The T,-induced elevation of thyroid hormone receptor p mRNA is attenuated by withdrawal of T,. Comparison of the levels of thyroid hormone receptor a and P mRNAs in the hind limbs and tail at N F (Niewkoop and Faber, 1956) stages 58 and 63 revealed that both mRNAs are elevated in the hind legs when they are growing (stage 58) and in the tail when it is regressing (stage 63). Kawahara et al. (1991) observed a similar pattern of increase in the expression of thyroid hormone receptor a and P genes during metamorpho-

I

I

I

I

I

~

10

l

l

l

l

,

20

l

l

l

l

,

l

l

l

l

,

l

l

l

l

~

30 40 50 Developmental stages

l

l

l

l

,

"

'

l

60

FIG. 1 Developmental changes of TRa and TRP genes throughout embryogenesis, tadpole growth, and metamorphosis of Xenopus laeuis. The levels of TRP cDNA in tadpoles upregulated by exogenous T3during premetamorphosis are also shown with the plasma levels of T3 (Leloup and Buscaglia, 1977). [Modified from Yaoita and Brown (1990).]

112

SAME KIKUYAMA ET AL.

sis of Xenopus. Moreover, they studied the localization of expression of the thyroid hormone a-receptor gene by in situ hybridization and found that the concentrations of thyroid hormone receptor transcripts were highest in the central nervous system of N F stage 44 tadpoles and in the hind limb buds of tadpoles at stage 55 when those organs become discernible. Of interest was that between stages 44 and 54, accumulation of thyroid hormone receptor mRNAs was seen in the tail and gut, which would undergo degeneration at future stages but were still actively growing when examined. This in situ hybridization experiment could demonstrate the localization of mRNA for thyroid hormone receptor a but not p. Information about the tissue distribution and developmental variations in the expression of mRNAs for thyroid hormone receptor p is awaited. Thyroid hormone mRNA concentrations decrease in most of the tissues that undergo substantial remodeling. In the developing ovary of the juvenile toad, however, high levels of thyroid hormone a-receptor mRNA are detected. The accumulation of thyroid hormone receptor transcripts in oocytes is sustained and continues to increase throughout further development (Kawahara er al., 1991). Recently, the presence of thyroid hormone in teleost unfertilized eggs and embryos has drawn a considerable amount of attention from endocrinologists and developmental biologists (see Bern, 1990). The hormone, which disappears as the yolk is absorbed, is presumed to be derived from the mother and transported into the egg during vitellogenesis. The presence of T4 in the eggs and early embryos of bullfrogs has also been reported (Fujikura and Suzuki, 1991). Thyroid hormone concentrations decrease during early embryogenesis as development proceeds and reach their lowest level at Shumway's stage (Shumway, 1940) 22 when the thyroid anlage is formed. The physiological significance of thyroid hormone receptor mRNA and thyroid hormones of maternal origin remains to be clarified. B. Pituitary Control of Thyroid Function

The presence of thyroid-stimulating hormone in the amphibian pituitary has been confirmed indirectly by evidence of a difference in thyroid function between normal and hypophysectomized tadpoles ( R . pipiens) at TK stage I11 (Kaye, 1961) and by the stimulating effect of crude extracts prepared from the pituitaries of prometamorphic and climax Xenopus larvae on uptake of 13'1by the thyroid (Dodd and Dodd, 1976). Amphibian thyroid-stimulating hormone (TSH) has been considered to resemble mammalian TSHs, since metamorphic arrest by hypophysectomy is prevented by treatment with mammalian TSH (Dodd and Dodd, 1976). Eddy and

HORMONAL CONTROL OF AMPHIBIAN METAMORPHOSIS

113

Lipner (1976) used an antiserum against bovine TSH to immunoneutralize endogenous TSH circulating in the bullfrog tadpole. They observed that the antiserum blocked spontaneous metamorphosis, suggesting that amphibian TSH resembles mammalian TSH. A number of previous investigators have attempted to identify TSH cells in the pituitary of amphibians (see Holmes and Ball, 1974).Development in immunohistochemical techniques has permitted the identification of the hormone-producing cells in the amphibian pituitary with increasing accuracy (Doerr-Schott, 1980). Using antibodies against mammalian TSHP subunit, the ontogenic differentiation of pituitary TSH cells has been studied in several amphibian species such as X. faeuis (Moriceau-Hay et al., 1982), B . mefanostictus (Kar and Naik, 1986), B. cafamita, R . perezi (Garcia-Navarro et a f . , 1988b), H . nigrescens (Yamashita et a f . , 1991), and R . catesbeiana (Tanaka et a f . ,1991). In R . catesbeiana, cells immunoreactive for a subunit and for TSHP subunit appear simultaneously at Shumway’s stage 24, prior to the appearance of other pituitary glycoprotein hormones. Thyroid-stimulating hormone p-immunoreactive cells increase in number as metamorphosis progresses (Fig. 2). Among the pituitary hormones of the amphibia, TSH is the only remaining one for which details of physicochemical properties are still incomplete, since substantial amounts of highly purified TSH have not been obtained. This seems to be due to the presence of a relatively small amount of TSH in the pituitary, limited availability of pituitary glands for purification, and difficulty in eliminating gonadotropins, which are chemically related to TSH and abundantly present in the pituitary, from TSH preparations. Partial purification of TSH from bullfrog pituitary has been reported by MacKenzie et a f .(1978), but their preparation contained a considerable amount of follicle-stimulating hormone (FSH). Sakai et a f . (1991) succeeded in removing gonadotropins from a crude glycoprotein hormone preparation by immunoaffinity chromatography using monoclonal antibodies against bullfrog luteinizing hormone (LH)P and FSHP subunits (Park et a f . , 1987; Tanaka et af., 1990). This preparation was four times as potent as bovine TSH in stimulating the in uitro release of T, from the thyroid of prometamorphic bullfrog larvae. According to their report, the highly purified bullfrog LH retained 10-40% of the bioactivity of the TSH preparation, and bullfrog FSH had much weaker activity. Other pituitary hormones of bullfrog origin such as growth hormone, prolactin, and adrenocorticotropic hormone (ACTH) have no thyrotropic activity (Sakai et a f . , 1991). Moreover, the biological activity of a TSH preparation has been shown to be lost after incubation with an IgG fraction separated from antiserum against the a subunit of bullfrog LH/FSH (Sakai et al., 1990), suggesting that amphibian TSH has an a subunit similar or close to that of gonadotropins, as is the case for mammalian TSH. Very recently,

114

SAME KIKUYAMA E r AL.

FIG. 2 Distribution of immunoreactive a-subunit (A), TSHP (B), FSHP (C), and LHP (D) in the pituitaries glands of bullfrog tadpoles at TK stages V (left) and XXV (right). Bar = 50 pm. [Reproduced from Tanaka et al. (1991) with permission.]

HORMONAL CONTROL OF AMPHIBIAN METAMORPHOSIS

115

the same group have succeeded in purifying bullfrog TSH from their abovementioned preparation using high-pressure liquid chromatography. The product has 10 times the bioactivity of bovine TSH. It is evident that this is authentic amphibian TSH, since the N-terminal sequence of one of its subunits shows considerable sequence homology with mammalian TSHP subunits and the other subunit coincides with that of bullfrog LH/FSH. At present, no data on the circulating levels of TSH in metamorphosing tadpoles are available, because of the lack of a homologous RIA for amphibian TSH. C. Hypothalamic Control of TSH Secretion

In general, amphibian larvae in which the pituitary has been disconnected from the brain by removal of the primordium of the posterior hypothalamus (Hanaoka, 1967), implantation of the pituitary primordium into an ectopic site (Etkin and Lehrer, 1960), or dissection of the pituitary stalk (Etkin and Sussman, 1961) do not complete metamorphosis but are arrested in a state of prometamorphosis. Bufo juponicus larvae are exceptional in this respect. They finish metamorphosis even if their pituitary is dislocated as a result of removal of hypothalamic anlage at the embryonic stage (Kawamura and Kikuyama, 1987). However, climactic changes in these tadpoles are very slow in comparison with intact animals. All these findings indicate that the pituitary-thyroid axis is under the control of the hypothalamus. Thyroid-stimulating hormone is regarded as the major pituitary hormone that induces the release of thyroid hormone from the thyroid gland. However, studies on the identification of the hypothalamic hormone(s) that stimulates the release of TSH from the amphibian pituitary have not advanced far, mainly because of the lack of a suitable RIA for amphibian TSH. Instead, indirect criteria such as the acceleration of metamorphosis, increase in the plasma level of thyroid hormone after administration of a test substance, and in uirro release of thyroid hormone from a thyroid placed in medium in which a pituitary has been cultured in the presence of a test substance have been adopted. In mammals, pyro-Glu-His-Pro-NH, is known as thyrotropin-releasing hormone (TRH). Most experiments to demonstrate the acceleration of metamorphosis by injecting TRH into larvae of various species of amphibians have not shown a positive effect of the tripeptide (see Norris, 1983). However, Darras and Kuhn (1982) have demonstrated that injection of TRH into the abdominal vein of adult R. ridibundu causes elevation of plasma T4 levels. Gracia-Navarro er ul. (1990) presented morphological data indicating that injection of TRH into adult R. perezi enhances secre-

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tory function, i.e., a decrease of secretory granules, and development of endoplasmic reticulum and Golgi complex in the TSH cells. Denver (1988) has also demonstrated that the pituitary of adult R. pipiens incubated in the presence of synthetic TRH releases a substance that stimulates the release of T, from the thyroid in uitro. More recently, Denver and Licht (1989) have reported that TRH was not effective in a similar experimental model in which adult pituitary glands were substituted with larval ones. Moreover, they demonstrated that mammalian corticotropin-releasing factor (CRF) is a potent stimulator of the release of a thyroid-stimulating substance from the pituitary. Mammalian CRF is known to stimulate the release of ACTH from the distal lobe of R. catesbeiana (Tonon et a f . , 1986). Considering that ACTH has no thyroid-stimulating activity (Sakai et al., 1991), CRF may release a substance(s) that directly or indirectly (through the release of TSH) stimulates the release of thyroid hormone. Another neurohormone that may stimulate the release of thyroidstimulating substance from the pituitary is LH-releasing hormone (LHRH). Injection of mammalian LHRH into ranid frogs through the abdominal vein induces elevation of the plasma T4 level (Jacobs et a / . , 1988). The release of T, in uitro from the thyroid gland of R. pipiens is greatly enhanced when incubation is carried out in medium in which a pituitary gland has been cultured in the presence of LHRH (Denver, 1988). As mentioned above, a bullfrog LH preparation that is considered to be highly purified, judging from its electrophoretic and chromatographic patterns (Takahashi and Hanaoka, 1981; Hanaoka et al., 1984) and the results of amino acid sequence analysis (Hayashi et a f . , 1992a,b), has considerable thyroid-stimulating activity (Sakai et a/., 199 1). Therefore, it is assumed that LHRH causes the release of LH rather than TSH from the pituitary gland. Biochemical and histological studies have revealed that the tripeptide is abundantly present in the brain as well as in the skin of amphibians (Jackson and Reichlin, 1974; Taurog et al., 1974; Yasuhara and Nakajima, 1975; Giraud et al., 1979; Seki et a f . , 1983). Recently, localization of neurons containing mRNA encoding TRH has been demonstrated in the brain of Xenopus using an in situ hybridization technique (Zoeller and Conway, 1989). Increase of TRH in the tadpole brain during prometamorphosis and climax has been demonstrated biochemically (King and Millar, 1981) and histologically (Mimnagh et al., 1987; Taniguchi et a f . , 1990). Immunoassayable and immunostainable LHRH in the brain also increases as metamorphosis proceeds (King and Millar, 1981; Crim, 1984), but the increase is not as marked as that observed after metamorphosis (Millar et a / . , 1983). In summary, mammalian TRH seems to stimulate the release of TSH at least from the pituitary of adult amphibians, but the nature of TSH-

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releasing hormone in larvae is still obscure. Attempts have been made to identify TSH-releasing hormone in amphibians over the last 20 years, but little progress has been made, mainly because no specific and sensitive assay for amphibian TSH has been developed. Furthermore, unlike the case of mammalian hypothalamic hormones, no attempt has been made to test substances separated from the amphibian hypothalamus, for their TSH-releasing activity. The TSH preparation purified recently (II1,B) appears promising for the development of a RIA for amphibian TSH, and thus for the identification of TSH-releasing hormone. D. Thyroid Hormone Feedback

In amphibian larvae, a negative feedback effect of thyroid hormone on TSH secretion by the pituitary is functional from a relatively early stage. Kaye (1961) observed depression of iodine uptake by thyroids from R. pipiens tadpoles at TK stage I11 after exposure to thyroid hormone. The fact that administration of goitrogens to larvae induces hypertrophy of the thyroid gland also indicates the existence of a negative feedback mechanism (Dodd and Dodd, 1976). In this respect, it is of interest to note that although average plasma concentrations of thyroid hormone, especially T,, are high in climactic tadpoles, the values fluctuate widely among individuals (Fig. 3). A similar phenomenon has also been pointed out by Just (1972), who measured plasma levels of protein-bound iodine in R. pipiens larvae. This suggests that the release of thyroid hormone is pulsatile, possibly as a result of negative feedback of thyroid hormone. When the elevated levels of thyroid hormone act on the pituitary and/or hypothalamus, thyroid-stimulating activity will diminish. During this phase, circulating thyroid hormone will be cleared at least from the circulation. Thereafter, enhancement of thyrotropic stimulation, and thus another surge of thyroid hormone, may be induced upon release from thyroid hormone inhibition. It is also known that thyroid hormone induces the development of hypothalamic neurons, possibly containing releasing or inhibiting hormones, which send axons toward the median eminence. Through the median eminence, the hypothalamic hormones are conveyed to the pars distalis. According to Goos (1978), the number and size of these neurons increase gradually during the prometamorphic period. The median eminence starts to develop in early prometamorphosis. Thickening of the infundibular stalk (possibly as a result of an increase of axons extending from the hypothalamic neurons), penetration of capillaries, and formation of a capillary network take place as metamorphosis progresses further. Development of the median eminence can be induced experimentally by

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thyroid hormone. Interestingly, contact of epithelial pituitary tissue with the median eminence is indispensable for its development (Etkin et al., 1965).Thus, thyroid hormone acts to increase the capacity of the hypothalamus for stimulating the pituitary-thyroid axis. This so-called positive feedback of thyroid hormone is developmental or maturational in nature, and it enables the thyroid to secrete more of the hormone as metamorphosis proceeds. On the other hand, a negative feedback system is also operating throughout this period, and consequently, the thyroid hormone levels may fluctuate widely as metamorphosis progresses. Even if thyroid hormone levels are lowered temporarily by the negative feedback mechanism, metamorphosis will be completed in the end, because metamorphosis is an irreversible process of transformation. There are several reports suggesting the presence of positive feedback of thyroid hormone. Rana ornatiuenfris larvae from which the thyroid primordium had been removed at the embryonic stage were treated with a relatively low concentration of thyroid hormone after they had matured. They then received thyroid gland implants from hypophysectomized animals. I3'I uptake by the implants in the thyroidectomized tadpoles receiving T4was considerably enhanced in comparison with that in thyroidectomized tadpoles given no T4(Hanaoka, 1976).In this case, thyroid hormone

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seems to have induced elevation of the endogenous TSH level through its maturational effect on the hypothalamus, and thus thyroid function. Ambystoma mexicanum individuals metamorphosed responding to a single hypothalamic injection of T,, whereas the specimens that received the same amount of T, intraperitoneally did not metamorphose (Norris and Gem, 1976). Therefore, administered T, may act on the hypothalamus more effectively to bring about its maturation, thus making it capable of stimulating the pituitary and, in turn, the thyroid. Most of the RIA data on plasma thyroid hormone levels indicate that the levels decline at the final stage of climax. One possible explanation may be that the hypothalamic TSH-cell-stimulating neuronal system undergoes a certain irreversible (developmental) change due to the relatively high concentrations of endogenous thyroid hormone during climax, and that by the end of climax its function is considerably lowered. An instance of permanent decline of the hypothalamo-hypophyseal axis during the developmental period in terms of reproduction is well known in the rat. Male rats lose the ability for induction of the LH surge, whereas it is retained by female rats. This is due to secretion of testosterone by the testes of the male during the perinatal period, and this acts on the hypothalamic neurons that are involved in the LH' surge after the attainment of puberty to alter function irreversibly (Neil and Naftalin, 1981). Another possibility is that a regulatory system that stimulates less efficiently or inhibits TSH release is newly developed in the hypothalamus and becomes dominant around the end of metamorphosis.

111. Adrenocortical Hormones

A. Influence of Corticoids on Metamorphosis

It has long been known that adrenal corticoids administered together with thyroid hormone accelerate thyroid hormone-induced metamorphosis (Kaltenbach, 1968). This has led to studies of plasma corticoid levels during metamorphosis. According to Carstensen et al. (1961) and Macchi and Phillip (1966), corticosterone and aldosterone are the major corticoids secreted by the interrenal gland of amphibians. Radioimmunoassays for these steroids have been performed in larvae of several anuran species such as R. catesbeiana (Jaffe, 1981; Krug et al., 1983; Kikuyama et al., 1986), B . japonicus (Niinuma et al., 19891, X. laeuis (Jolivet-Jaudet and Leloup-Hatey, 1984), and A. tigrinum (Carr and Norris, 1990). Most of the data indicate that marked elevation of corticoid levels occurs in syn-

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chronization with the elevation of thyroid hormone levels (Fig. 4). The pattern of plasma corticoid levels in bullfrog tadpoles reported by Krug et ul. (1983) is somewhat different from that reported by others. Elevation of corticosterone levels starts rather early, and relatively high levels are maintained from stage XI11 onward. Aldosterone levels remain low. Instead, elevation of cortisol levels is synchronized with that of thryoid hormone levels. Cortisol was once thought not to be present in amphibians, but significant amounts seem to be secreted by the interrenals, at least during the larval period in bullfrogs. The existence of relatively high levels of circulating corticoids in metamorphosing tadpoles suggests that corticoids physiologically participate in metamorphic events. Kikuyama et al. (1982a) provided evidence that endogenous corticoids are involved in metamorphosis. They studied the effect of Amphenone B, an inhibitor of corticoid synthesis, on T,-induced metamorphosis in toad tadpoles, which were kept in thiourea throughout the experiment in order to avoid the effect of the inhibitor on endogenous thyroid hormone levels through the hypothalamo-hypophyseal thyroidal axis. Amphenone retarded T4induced tail resorption markedly. The effect of the inhibitor was nullified by corticoids added to the water in which tadpoles were kept. The effect of corticoids has also been reproduced in uitro (Kikuyama et al., 1983). The tail segments of B. juponicus larvae were cultured in a subthreshold concentration of T,. Addition of deoxycorticosterone to the medium induced shrinkage of the tail segments in accordance with hormone concentration. In the absence of thyroid hormone, corticoid had no effect. This suggests that corticoid potentiates the action of T,. Among the steroid hormones, including aldosterone, corticosterone, deoxycorti'2 1

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Developmental stages FIG. 4 Plasma aldosterone and corticosterone levels in bullfrog tadpoles at various stages of metamorphosis. [Modified from Jaffe (1981) and Kikuyama ef al. (1986).1

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costerone, cortisol, testosterone, estradiol, and progesterone, the firstnamed three were very effective in potentiating the action of T,. Cortisol was slightly effective, and the gonadal steroid hormones were not. It should be pointed out that corticosterone and aldosterone, which are regarded as the major corticoids in amphibians, have potent activity. It is notable that these corticoids potentiate not only the action of T, but also that of T, (Kikuyama et al., 1982b, 1983). Although this does not exclude the possibility that corticoids enhance the activity of T, by accelerating its conversion to more biologically active T,, it clearly indicates that another mechanism of corticoid-thyroid hormone interaction exists. Analysis of T, binding to the nuclear fraction of tail segment of toad tadpoles (Niki et al., 1981) or tail fin tissue from bullfrog tadpoles (Suzuki and Kikuyama, 1983) in the presence of aldosterone or corticosterone has revealed that these hormones increase nuclear T, binding in a dosedependent manner. Scatchard analysis revealed that the corticoids increase the maximum binding capacity for T, without affecting Kd values. This result suggests that the corticoids are involved, at least partly, in the increase of T,-binding sites in the tail nuclei around climax (11,A). Moreover, the steroid-induced increase of T, binding was blocked by an inhibitor of protein synthesis (cycloheximide) and an inhibitor of RNA synthesis (actinomycin D), indicating that the action of corticoids requires synthesis of a new protein(s) and a new RNA(s). Most of the work on thyroid hormone-corticoid interaction in metamorphosis has been carried out using tail shrinkage as a criterion. However, several reports have demonstrated that corticoids augment the action of thyroid hormone or act synergistically with it. In amphibians, there is a gradual shift of red blood cells from the larval to the adult type as metamorphosis progresses (Foxon, 1964). This transition is induced by thyroid hormone. Adult-type erythrocytes can be induced more precociously by combination of thyroid hormone and corticoid than by thyroid hormone alone (Asai and Tamanoi, 1978). Recently, it was shown that T, induces expression of the adult-type 63-kDa keratin gene and cornification of the larval epidermis (Mathisen and Miller, 1989). Cortisol acts synergistically with T, to increase keratin gene expression in isolated epidermal cells from Xenopus larvae. Moreover, the corticoid reduces the lag period required for induction of expression of the adult-type keratin gene by T, (Shimizu-Nishikawa and Miller, 1992). There have been few studies on the binding of corticoid hormones to the target tissue of thyroid hormone in amphibian larvae. It is known that the tail is one of the larval organs most sensitive to thyroid hormone. Among the tissues constituting the tail, epidermal tissue is considered to play an important role in the entire resorption of the tail. Tail blocks

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deprived of epidermis never undergo regression when cultured in medium containing T, (Niki et al., 1982). On the other hand, mesenchymal blocks of the tail respond to T, by regressing when placed in medium in which skin from the tail has been cultured (Niki et al., 1984). Corticoid-binding experiments have been conducted using the cytosol fraction of epidermal cells from the tail of bullfrog larvae (Yamamoto and Kikuyama, 1993). They observed that the number of binding sites for aldosterone decreased as metamorphosis progressed with no appreciable change in Kd. Analysis of steriod-binding specificity revealed significant displacement of aldosterone by corticosterone. It was pointed out that both aldosterone and corticosterone may have common binding sites in the epidermal cells of the tail. Presence of glucocorticoid receptor in the tail fin cytosol has been reported by Woody and Jaffe (1984). They observed that the binding capacity for dexamethasone was almost constant during pre and prometamorphosis and early climax (TK stage XXI). Tadpole intestine is also one of the targets of thyroid hormone. Binding of dexamethasone to the intestine cytosol has been studied by the same investigators. No change was noted in Kd during metamorphosis. Binding capacity was relatively high at premetamorphic stages and significantly reduced thereafter. The metabolic effects of corticoids during metamorphosis have been investigated in Xenopus larvae mostly by Hanke and coworkers. According to their results, corticoids are responsible for changes in water content and carbohydrate, lipid, and protein metabolism. These earlier studies have been reviewed by Dodd and Dodd (1976). There is an instance of corticoid hormone contributing to development of the central nervous system. In B. japonicus, catecholamine-containing neurons in the preoptic recess organ (PRO) become detectable around metamorphic climax by formaldehyde-induced fluorescence (Kikuyama er al., 1979). During premetamorphosis and early prometamorphosis, synthesis of catecholamines in the PRO seems to be very low, since immunoreactive tyrosine hydroxylase is not detectable. In thyroidectomized or hypophysectomized larvae, no monoaminecontaining neurons appear in the PRO. Thyroxine treatment induces development of PRO catecholamine neurons in the thyroidectomized tadpoles but not in the hypophysectomized tadpoles. Corticosterone induces the appearance of monoaminergic neurons in the PRO in the hypophysectomized larvae (Miyakawa et al., 1984). This suggests that corticosterone acts directly on the PRO neurons to accelerate catecholamine synthesis and that T, may act mainly on the hypothalamo-pituitary axis to enhance its adrenocorticotropic function. In mammals, it is known that T4 elicits the secretion of ACTH by the pituitary through its maturational effect on the neural structures in the brain (Lengrari er al., 1977). In amphibian larvae, T4is reported to elevate plasma corticosterone levels (Jaffe, 1981;

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Krug et al., 1983), although it is not definite whether this effect is mediated by ACTH. The role of PRO catecholamine neurons in amphibians has not been fully elucidated. In B. japonicus, catecholamine neurons seem to regulate the function of the pars intermedia. The larvae of this toad do not respond the background color and melanin granules in their dermal melanophores are in a state of constant dispersal until the catecholaminergic neurons develop in the PRO around climax. Moreover, the ability to change the body color in response to the color of the background never develops in specimens from which the PRO has been removed at the embryonic stage (Kato et a f . , 1991). B. Control of Adrenal Corticoid Secretion As stated above (III,A), there is evidence to suggest that the secretion of corticoids by the adrenal glands is enhanced during climax. If the production of adrenal corticoids in metamorphosing tadpoles is blocked by Amphenone B, hypertrophy of steroid-producing cells is observed (Kikuyama et a f . , 1983). This indicates the presence of a regulatory mechanism for corticoid secretion in amphibian larvae. Several substances are known to affect the secretion of corticoids by the adrenal glands in amphibians. However, it is not known to what extent they are involved in the control of corticoid secretion, and thus, metamorphosis, in amphibian larvae.

1. Hypophyseal Factors As in mammals, ACTH is generally believed to stimulate the synthesis and release of corticoids in amphibians (Buchmann et a f . , 1972; Vaudry et al., 1977; Delarue et a f . , 1979). Hypophysectomy in Xenopus larvae results in degenerative change of the interrenal tissue and decline of activity of A53p-hydroxysteroid dehydrogenase (3P-HSD), an enzyme that catalyzes the step from pregnenolone to progesterone in the biosynthesis of corticoids. Administration of ACTH to Xenopus larvae elicits the same changes in carbohydrate, lipid, and protein metabolism as those produced by treatment with adrenal corticoids (Dodd and Dodd, 1976). In late prometamorphic tadpoles of R. catesbeiana, aldosterone levels decline after hypophysectomy, and ACTH administration elevates the corticoid levels to normal. Interestingly, treatment of hypophysectomized tadpoles with T, increases the concentration of plasma aldosterone. Combined ACTH and T, treatment causes a marked increase in the plasma aldosterone level (Kikuyama e f a f . , 1986). The significant rise in plasma aldosterone triggered in hypophysectomized tadpoles by T, alone indicates that thyroid hormone induces development of an extrahypophyseal stimu-

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latory system for the steroidgenic cells and/or enhances the sensitivity of these cells to some stimulating factors other than those of pituitary origin. A similar conclusion has been obtained by a histochemical study of the effect of thyroid hormone on the activity of 3P-HSD in the steroidgenic cells in the adrenal glands of hypophysectomized bullfrog tadpoles (Hsu et al., 1984). It is known that 3P-HSD activity in the interrenal tissue of larval Xenopus (Leist et al., 1969) and R . catesbeiana (Hsii et al., 1980) rises as metamorphosis progresses, and then falls after the completion of metamorphosis. In bullfrog tadpoles, the enzyme activity is first detectable at Shumway stage 23, and thereafter it gradually rises (Hsu et al., 1980). The enzyme activity in hypophysectomized specimens remains as low as that in intact tadpoles at stages 24 and 25. Treatment with T, elevates the enzyme activity to the level at TK stage I, and with ACTH, to the level at stage X. Treatment with both T, and ACTH elevates the activity to the value for stage XXV. These results indicate that thyroid hormone enhances interrenal activity directly and/or indirectly. According to Dores et al. (1990), the processing of ACTH in A. tigrinum is developmentally regulated as in mammals. In larvae, neotene, postmetamorphic adults, and sexually mature adults, the molar ratios of ACTH to a-melanocyte-stimulating hormone (MSH) were 1.3 : 1 , 1 : 1.3, 1 : 1.3 and 8 : 1, respectively. Recently ovine CRF was found to be a potent stimulator of ACTH secretion by frog pituitary (Tonon et al., 1986). Immunoprecipitation analysis revealed that ACTH-immunoreactive substances in the pituitary and in the culture medium were increased by addition of ovine CRF (Fig. 5). Mammalian CRF-like immunoreactivity has been observed in hypothalamus of R. ridibunda (Tonon et al., 1985; Olivereau et al., 1987), Triturus

Gland Cont CRF

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FIG. 5 Immunoprecipitation analysis of synthesis and release of ACTH by the bullfrog anterior pituitary. Anterior lobes were incubated with [35S]methioninein the presence or absence of ovine CRF for 5 hr at 25°C. The pituitary extract and medium were subjected to immunoprecipitationwith normal rabbit serum (lane 1) anti-bullfrog ACTH (lane 2), or antiaMSH (lane 3) followed by SDS-PAGE and fluorography.

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cristatus (Fasolo et al., 1984), A . mexicanurn, Pleurodeles waltlii, Xenopus laeuis (Olivereau et al., 1987), and R . catesbeiana (Gonzalez and Lederis, 1988). In larval bullfrogs, CRF-like material in the median eminence is sparce or absent at premetamorphic stages, increases during prometamorphosis, and becomes most dense at climax (Carr and Norris, 1990). The results indicate that a CRF-like neuronal system develops just before the elevation of corticoidgenesis, which occurs around the onset of climax. Accordingly, the CRF-like substance may stimulate the secretion of ACTH, and thus bring about hypersecretion of corticoids in climactic tadpoles. Recently, amino acid sequence of Xenopus CRF has been reported by Stenzel-Poore et al. (1992). The neurointermediate lobes of amphibians seem to contain several peptides with corticotropic activity. In Xenopus, removal of the pars distalis results in a marked decline of plasma aldosterone levels, but they still remain slightly but significantly higher than the levels in totally hypophysectomized specimens (Iwamuro et al., 1989). Injection of a homogenate of the neurointermediate lobes from Xenopus or bullfrogs into hypophysectomized Xenopus causes marked elevation of plasma aldosterone (Iwamuro et al., 1989). A homogenate of intermediate lobes from R. ridibunda is also effective in enhancing the release of aldosterone from interrenal fragments in uitro (Delarue et al., 1979). Thus, the neurointermediate lobes appear to contain an aldosterone-releasing factor(s), although the activity might be that of ACTH, which is detectable in the pars intermedia by RIA (Vaudry et al., 1975). According to Leboulenger et al. (1986), the adrenal glands respond to a-MSH, desacetyl a-MSH, and y M S H to release aldosterone and corticosterone in uitro. Recently, Iwamuro et al. (1992) found that Nterminal peptide of proopiomelanocortin (POMC) also stimulates the release of aldosterone and corticosterone from the frog adrenal glands. This peptide is present in both the pars intermedia and the pars distalis. These results suggest the possibility that POMC peptides other than ACTH may also be involved in regulation of adrenal function. Another peptide family that has been found to stimulate the release of corticoids is arginine vasotocin (AVT) and its related peptides. Arginine vasotocin shows corticosteroid-releasingactivity in several anurans (Iwamuro et al., 1989, 1991a; Larcher et al., 1989, 1992; Kloas and Hanke, 1990). Mesotocin (MT), another posterior lobe hormone in amphibians, has much lower corticotropic potency than AVT (Iwamuro et al., 1989). In amphibians, AVT and MT were considered to be the final products present in the neural lobe resulting from the processing of their precursor molecules (Nojiri et al., 1987). Recently, however, it was found that the posterior lobes of several amphibian species contain a considerable amount of AVT-related peptides that are apparently derived from the provasotocin-neurophysin precursor (Acher, 1990), and that they exhibit

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hydroosmotic activity like AVT (RoullC et al., 1989). Nonamidated AVT C-terminally extended with glycine, lysine, and arginine (AVT-GlyLys-Arg: hydrin 1) and nonamidated AVT C-terminally extended with glycine (AVT-Gly; hydrin 2) are the peptides that have been isolated from the neurointermediate lobes of Xenopus and from those of several species of Ranidae and Bufonidae, respectively. These peptides are unique in that they are never found in urodeles (Chauvet et al., 1990) or other classes of vertebrates (Rouille et al., 1989; Michel et al., 1990), presumably because of their rapid molecular processing. The presence of these peptides in anuran neural lobes may indicate that formation of the AVT molecule is regulated at several steps of processing. Iwamuro et al. (1991a,b) isolated from the neurointermediate lobes of bullfrogs two active peptides that markedly stimulated the secretion of aldosterone by the interrenals in uitro and in uiuo. Sequence analysis revealed that they were AVT and hydrin 2. It was later confirmed that the latter also stimulated the release of corticosterone as well as aldosterone from interrenal slices of R. ridibunda (Larcher et al., 1992). Recently, another AVT-related peptide, nonamidated AVT C-terminally extended with glycine and lysine (hydrin 1 '), was found to be present in the Xenopus neural lobe. Both hydrin 1 and hydrin 1' have corticotropic activity, although their activity seems to be less potent than that of AVT when tested with Xenopus interrenal tissue in uitro (Iwamuro et al., 1992). Arginine vasotocin may also contribute to the elevation of corticoid levels by acting on the corticotrophs to elicit the release of ACTH (Jorgensen, 1976; Tonon et al., 1986). In bullfrog larvae, AVT-immunoreactive perikarya are not observed at premetamorphic stages, but at more advanced stages their number increases. AVT-irnmunoreactive materials in the posterior lobe increase markedly at TK stage XI1 and remains intense thereafter. In the median eminence region, AVT-immunoreactive staining is seen around the portal vessel at prometamorphic and climax stages but not at premetamorphic stages, suggesting the delivery of AVT to ACTH cells in metamorphosing tadpoles (Carr and Norris, 1990). It is worth mentioning that AVT is present in the adrenal chromaffin cells of R. ridibunda (Larcher et al., 1989). In amphibians, adrenal chromaffin cells are known to be intermingled with steroidgenic cells. This suggests that AVT is secreted in a paracrine manner and induces neighboring steroidgenic cells to release corticoids.

2. Nonhypophyseal Factors Several nonhypophyseal factors that may influence corticoid secretion by the amphibian interrenals are known, mainly from the work done by Vaudry and co-workers (Delarue et al., 1990). One candidate for the factor

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that exerts inhibitory control of corticoid secretion by the interrenals of amphibians is atrial natriuretic peptide (ANP), a substance having hypotensive, diuretic, natriuretic, and vasorelaxant activity. The ANP-like peptides are present in amphibian heart (Reineche et a f . , 1985; Netchitailo et a f . , 1987) and central nervous system (Netchitailo et al., 1987). The amino acid sequences of R. ridibunda (Lazure et al., 1988)and R . catesbeiana (Sakata et a f . , 1988) ANP have been reported. Lihrman et a f . (1988) studied the effect of synthetic ANP on perifused frog interrenal slices. They found that it does not inhibit the spontaneous secretion of aldosterone and corticosterone, but does inhibit the ACTHand angiotensin 11-stimulatedsecretion of the corticoids. Kloas and Hanke (1991b) also reported that human, rat, and frog ANPs and an extract of Xenopus heart inhibited the release of corticosterone and aldosterone from the adrenal glands of Xenopus. They also demonstrated the presence of a specific binding site for ANP in the adrenal tissue of Xenopus (Kloas and Hanke, 1991a). The finding that immunoreactive fibers are present in the vicinity of the interrenal cells in R. ridibunda (Lihrman et a f . , 1988) is of particular interest. On the basis of this finding, they proposed that in amphibians both “hormonal” ANP secreted by cardiocytes and “neurohormonal” ANP released from peptidergic nerve terminals distributed in the interrenal tissue may participate in the regulation of corticosteroid secretion. The ontogeny of immunoreactive ANP in cardiocytes of the toad B. japonicus has been studied by Hirohama et a f . (1989). Secretory granules in the atrial and ventricular cardiocytes first appear at the limb-bud stage, and the number of the granules increases rapidly in atrial but not in ventricular cardiocytes as metamorphosis proceeds. They also noted that in addition to the granules of ordinary size (1 10 nm in median diameter), larger granules (200-250 nm) appear in the metamorphosing tadpoles within the same cells. However, the physiological significance of these two granule types is not known. Angiotensin I1 is well known as a potent stimulator of steroidogenesis in the amphibian interrenals (see Nishimura, 1987). Angiotensin I1 is considered to act directly on the interrenals in ranid frogs. In Xenopus, however, it seems to act on the interrenals only indirectly. The interrenal tissue of Xenopus is devoid of binding sites for angiotensin I1 (Kloas et a f . , 1990). The catecholamine-producing cells, which are situated close to steroidproducing cells, contain immunoreactive vasoactive intestinal polypeptide (VIP) (Leboulenger et a f . , 1983; Kondo and Yui, 1984), Met-enkephalin (Leboulenger el a f . , 1983, 1984), and serotonin (Delarue et al., 1988a). Among them, VIP and serotonin exert dose-related stimulation of corticoid secretion by perifused interrenal fragments (Leboulenger et a f . , 1983,

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1984; Delarue et al., 1988b). Acetylcholine is also a stimulator of corticoid production by the adrenal glands (Benyamina et al., 1987). Conversely, dopamine exerts an inhibitory effect on steroid secretion by adrenocortical cells. It inhibits angiotensin II-induced but not ACTH- or seretonininduced corticoid secretion (Morra et al., 1990). The above-mentioned substances present in the nerve terminals or in the chromaffin cells situated closely to the steroid-producing cells may act as paracrine factors to regulate the secretion of corticoids.

IV. Prolactin A. Prolactin and Larval Growth and Metamorphosis Prolactin is known to be the most versatile among vertebrate hormones in its action. In amphibian larvae, prolactin has emerged as a hormone that regulates larval growth. Etkin and Lehrer (1960) studied the growth rate of R. pipiens tadpoles. The growth rate was reduced by hypophysectomy, but in hypophysectomized larvae with pituitary grafts, the growth rate was similar to that of normal tadpoles at an early stage of development and exceeded that of the normal animals at later stages. On the basis of these observations and by analogy with the mode of secretion of prolactin in mammals, they assumed that the pituitary grafts secrete a growthpromoting factor, possibly prolactin. In mammals, prolactin is the only adenohypophyseal hormone that is released autonomously when the pituitary is disconnected from the hypothalamus. The assumption that the growth-promoting factor was prolactin was later proved to be correct, although the secretory activity of growth-promoting hormone in pituitary grafts may have been overestimated. The excess growth observed by Etkin and Lehrer seems to have arisen partly from a hypothyroidal state due to hypophysectomy. They did not take into account the fact that thyroid hormone often suppresses growth in tadpoles. Stimulated by these findings, several investigators tested mammalian prolactin, which is the most readily available type, for growth-promoting activity in amphibian larvae. Berman et al. (1964) were the first to show that prolactin administration increases larval body weight and tail length. Almost all of the results reported by other investigators indicated that prolactin had potent growth-promoting activity. In parallel with tests of mammalian prolactin, growth hormone was also tested for growth-promoting activity. It was revealed that growth hormone is generally less active in larvae than prolactin, but more active than prolactin in adults (Dodd and Dodd, 1976). It is of interest to note that the organs that are very

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sensitive to prolactin are the so-called larval organs such as the tail and gills, which exist only in the larval period. As a result, tail length and tail height can be used to assess the response to prolactin. Growth-promoting activity of prolactin is also measured by monitoring the incorporation of labeled proline into the collagen fraction of the tail fin, since connective tissue growth in the tail fin is prominent following prolactin stimulation (Yoshizato and Yasumasu, 1970). Another important finding with regard to the action of prolactin in amphibian larvae is that prolactin antagonizes the action of thyroid hormone to suppress metamorphic changes. Administration of prolactin to metamorphosing tadpoles blocks tail resorption (Bern et al., 1967; Etkin and Gona, 1967). Tail segments cultured in medium containing T, or T, become gradually reduced in size. This effect of thyroid hormone is attenuated by adding prolactin to the medium (Derby and Etkin, 1968). This inhibitory effect of prolactin has been considered to be exerted on the “larval” organs (Dodd and Dodd, 1976). According to Wright et a / . (1979), however, long-term treatment with prolactin also antagonizes the action of thyroid hormone on the hind limbs, which develop during larval life. Recently this was confirmed by Tata et al. (1991), who showed that T,-induced development of the limb buds of Xenopus larvae in uitro is blocked by prolactin. At the initial stage of investigations into the effects of prolactin in amphibian larvae, mammalian prolactins were used exclusively because purified prolactin of amphibian origin was not available. From the early seventies, however, attention was directed to the endogenous prolactin. It was ascertained that injections of bullfrog anterior pituitary gland homogenate into tadpoles promoted overall growth and increased the amount of connective tissue in the tail, and that the morphological change in the tail was associated with elevation of collagen synthesis in the tail fin (Yoshizato et al., 1972). The presence of a prolactin-like hormone in the amphibian pituitary gland was reported by Nicoll and Nichols (1971). They obtained a protein band by applying a pituitary gland homogenate from several amphibian species to disc-gel electrophoresis and demonstrated that it had crop sac-stimulating activity. The preparations obtained similarly from adult and larval bullfrog pituitary glands exhibited potent activity in promoting collagen synthesis in the larval tail fin and in suppressing the T,-induced shrinkage of tail segments in uitro (Kikuyama et af., 1980). It was also revealed that administration of antiserum against ovine prolactin (Eddy and Lipner, 1975) or a bullfrog prolactin preparation separated electrophoretically (Clemons and Nicoll, 1977b) accelerated spontaneous metamorphosis, whereas injection of bullfrog growth hormone antiserum did not affect metamorphosis. These results indicated that the antimetamorphic factor present in the circulation was immunoneutralized by the antiserum and that the factor might be prolactin.

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Amphibian prolactin was first purified from bullfrog pituitaries and characterized by Yamamoto and Kikuyama (1981). Subsequently its amino acid sequence was determined by direct protein analysis (Yasuda et al., 1991) or deduced from its cDNA (Takahashi et al., 1990) (Fig. 6). The primary structure of bullfrog prolactin closely resembles that of prolactins of other tetrapods. Prolactins of other amphibian species such as B.japonicus (Yamamoto ef al., 1986b) and Cynops pyrrhogasfer (Matsuda et al., 1990a) have also been purified and characterized. All of these preparations exhibited considerable bioactivity (Yamamoto ef al., 1986b; Brown el al., 1991;Matsudaet al., 1991). Using these prolactins, antisera were produced and homologous RIA was developed (Yamamoto and Kikuyama, 1982b; Yamamoto e f al., 1989; Matsuda et al., 1990b). It is worth mentioning that powdered bullfrog anterior pituitary completely loses its activity in enhancing collagen synthesis in the tail fin of bullfrog larvae if it is preincubated with antiserum against bullfrog prolactin (Yamamoto and Kikuyama, 1982b). This indicates that the growth-promoting activity in the pituitary gland is due mostly to the prolactin it contains. It was also confirmed that T,-induced metamorphosis in bullfrog tadpoles is accelerated by injection of the antiserum (Yamamoto and Kikuyama, 1982a). Since prolactin is able to prevent thyroid hormone-induced changes in several organs such as the tail and hind limbs in uitro, it is considered that prolactin acts directly on these target organs of thyroid hormone. Recently, thyroid hormone was shown to up-regulate the thyroid hormone receptor mRNA in larval tissues (Yaoita and Brown, 1990; Kawahara e f al., 1991). According to Baker and Tata (1991), prolactin blocks this T,induced up-regulation of thyroid hormone receptor mRNA in organ cultures of tadpole tails. Accordingly, it is quite possible that prolactin lowers the activity of thyroid hormone at the level of its receptor. PRL: GH :

Q P I C P N G G T N C ~ PI T ~ A ~ D R ~ K L IS~ HS LY S S E M F N E F D E R F T ~ FPIMSLINWTNmIRAQHLBQMVADTYRDYERTYlB

L~HM~SEVQDlREAP~TltWKTVEVElQTKR~tEGMERlI~RIQPGDLENEl~ l ~ N R ~ F G N N O V F G N l ~ R V Y D R L R D L D ~ GRLE HL Dl ~D l P N V R N Y G V L T F T I SPWPGPASIPGD~NS~LFAFIN[%HULRRIS DKF DVNLRSEB G I A KNIGWSIFKKSM FIG.6 Amino acid sequences of bullfrog prolactin (PRL) (Takahashi ef al., 1990) and growth hormone (GH) (Takahashi et a!., 1992). The identical residues are shown by white letters. Gaps have been introduced to obtain maximal homology.

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The antimetamorphic action of prolactin may also be exerted in other ways. Gona (1967) pointed out that prolactin has a goitrogen-like action on the larval thyroid. He observed that prolactin blocked TSH-induced metamorphosis of bullfrog tadpoles and that thyroid glands in these animals appeared more stimulated than in those treated with TSH alone. Furthermore, he reported that thyroid I3'I uptake and serum PBI3'I are depressed by prolactin and that this effect is observed in prometamorphic tadpoles but not in climactic tadpoles (Gona, 1968). According to Anderson and Dent (1982a), prolactin accelerates the clearance of T, in the circulating blood of red-spotted newts, Notophthalmus viridescens. Kracht and Weber (1978) observed that prolactin blocks TSH-induced restoration of thyroidal epithelial activity in hypophysectomized adult redspotted newts. These results suggest that in the larval period, prolactin may act similarly to affect metamorphosis indirectly. However, a contradictory result has been reported. Thyroid glands from red-spotted newts were cultured in the presence of TSH and/or prolactin. Thyroidstimulating hormone but not prolactin stimulated the release of T, into the medium. Combination of TSH and prolactin enhanced the release of T, more than TSH alone (Anderson and Dent, 1982b). With regard to the mechanism of action of prolactin, Nicoll and coworkers (1990) have made an important finding. According to them, the liver of various vertebrate species secretes a prolactin synergist (synlactin), and prolactin but not growth hormone is effective in enhancing the secretion of synlactin. Synlactin itself, which is present in liver incubation medium, does not have prolactin-like activity as tested with pigeon crop sac, but it enhances the action of prolactin. Accordingly it does not mediate the effects of prolactin. It has been confirmed indirectly that synlactin does act synergistically with prolactin in amphibian larvae (Delidow et al., 1988). Bullfrog tadpoles in which endogenous prolactin was suppressed by CB154 received a prolactin pellet into the tail or spleen. The animals with a prolactin implant in the spleen showed a marked increase of tail height, indicative of prolactin action, whereas their plasma prolactin levels were not higher than those in specimens given a subcutaneous prolactin pellet. It was also confirmed that prolactin in combination with the incubation medium of liver from larvae with a pellet in the spleen stimulated the pigeon crop sac markedly in comparison with prolactin plus incubation medium of liver from larvae implanted with the pellet subcutaneously. It would be expected that the liver in animals with an intrasplenic prolactin pellet would be exposed to a higher concentration of hormone via the hepatic portal vein than the liver in animals with implants in other locations. It was concluded that the marked increase of tail height in the larvae with an intrasplenic pellet was due to enhanced secretion of synlactin. At the moment, it is not clear whether synlactin is effective when prolactin

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acts as a growth-promoting hormone, or as an antimetamorphic hormone, or both. Isolation and characterization of this substance remain to be done. It has also been noted that tadpoles also secrete an unknown factor(s) that alone stimulates the pigeon crop sac mucosa (Delidow et a / . , 1986). Responsiveness to prolactin should therefore be analyzed taking secondary factors like these liver factors, which are generated by prolactin, into consideration. Cellular distribution studies of prolactin cells in amphibian pituitary glands have been conducted by many investigators using antisera to mammalian prolactin. Prolactin-containing cells are generally distributed throughout the distal lobe (Doerr-Schott, 1980). Recently, prolactincontaining cells in larvae (Yamamoto et al., 1986a; Tanaka et a / . , 1991) as well as adults (Andersen et a / . , 1989; Tanaka et a / . , 1992) have been identified using an antiserum raised against bullfrog prolactin. Although the location of prolactin cells is very similar to that reported previously using antisera to mammalian prolactin, of particular interest is that prolactin cells contain both immunoreactive prolactin and a subunit of glycoprotein hormones within the same granules (Tanaka et a / . , 1992) (Fig. 7).

Electron micrographs of prometamorphic bullfrog larvae. a subunit and prolactin were immunolabeled with gold particles of 5- and IO-nm diameter, respectively. Note distribution of a subunit in peripheral zone of each secretory granule and distribution of prolactin in central zone. Bar = 0.5 jtm. FIG. 7

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B. Prolactin Levels during Metamorphosis Secretion of anterior pituitary hormone is generally regulated by hypothalamic hormones, which are transported to the pituitary via the median eminence. In amphibians, development of the median eminence (thickening of the infundibular stalk and enclosure of the capillaries) occurs around climax. For the formation of an adult-type median eminence, close contact of the epithelial hypophysis with the stalk and the presence of thyroid hormone above a certain circulating level are indispensable (see 11,D). Assuming that prolactin secretion in amphibians is under inhibitory hypothalamic control as in mammals, the inhibiting factor(s) will hardly reach the pituitary in premetamorphic and early prometamorphic tadpoles with a poorly developed median eminence. Thus, Etkin (1970) hypothesized that prolactin levels are high during premetamorphosis and early prometamorphosis, and decline at late prometamorphic stages as the median eminence starts to develop. High prolactin levels at earlier stages would favor larval growth, and low prolactin levels at later stages would allow thyroid hormone to act upon the tissue efficiently to induce metamorphic changes. This hypothesis was widely accepted, and determination of prolactin concentrations in the blood of tadpoles during development was awaited. Clemons and Nicoll (1977a) developed a homologous RIA for bullfrog prolactin using antibody against a prolactin preparation obtained by disc-gel electrophoresis. They measured prolactin concentrations in climactic tadpoles and found that they were higher at advanced than at early climax stages. Yamamoto and Kikuyama (1982b) also measured prolactin in the plasma of bullfrog tadpoles at various developmental stages by a homologous RIA employing chromatographically purified prolactin and its antiserum. The data obtained indicated that prolactin levels are relatively low during premetamorphosis and remain low during prometarnorphosis. A slight elevation occurs during early climax, followed by a marked elevation at late climax with a slight decline at the end of metamorphosis (Fig. 8). Prolactin concentrations in the pituitary gland are higher in climax tadpoles than in preclimax tadpoles (Yamamoto et al., 1986a). Recently, Takahashi et al. (1990) measured the pituitary content of prolactin mRNA in metamorphosing bullfrog tadpoles using bullfrog prolactin cDNA as a probe. They found that prolactin mRNA levels rose at mid climax (Fig. 8), suggesting that the increase in plasma and pituitary prolactin levels accompanied the increase in prolactin synthesis. These results indicated that the profile of prolactin levels in bullfrog tadpoles was different from what had been predicted. As well as the immunoassay result, there is evidence that the prolactin level may not be very high at premetamorphic stages. Collagen synthesis in the tail fin of premetamor-

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Developmental stages FIG.8 Changes in plasma levels of growth hormone (GH), prolactin (PRL), and PRL mRNA levels in pituitary glands during metamorphosis of bullfrog tadpoles. [Modified from Kobayashi and Kikuyama (1991), Takahashi et al. (1990), and Yamamoto and Kikuyama (1982b).]

phic bullfrog larvae is scarcely affected by injection of antiserum against bullfrog prolactin. Treatment with pimozide, a dopamine antagonist, markedly elevates collagen synthesis, and this elevation is completely nullified by the antiserum treatment. This indicates that the basal level of prolactin is so low that the PRL-neutralizing effect of the antiserum is undetectable (Yamamoto and Kikuyama, 1982a). Immunohistochemical observation of prolactin cells in the pituitary of metamorphosing tadpoles also suggests that during pre- and prometamorphosis, storage rather than release of prolactin is prevalent (Garcia-Navarro et al., 1988a). However, it was ascertained that even the relatively low levels of endogenous prolactin present during the preclimax period are effective in counteracting thyroid hormone. As mentioned in IV,A, relatively long treatment with antiserum against ovine prolactin (Eddy and Lipner, 1975) as well as bullfrog prolactin (Clemons and Nicoll, 1977b;Yamamoto and Kikuyama, 1982a) accelerated metamorphosis in bullfrog tadpoles to some degree. At early climax, the level of prolactin still remains relatively low, and its antimetamorphic action is overcome by thyroid hormone, since the plasma concentrations of both thyroid hormone and corticoids, which augment the action of thyroid hormone, rise markedly, as mentioned above (II,A, 111,A). Prolactin at late climax can hardly be considered an antimetamorphic hormone, since the tissue has already been subjected to high concentrations of thyroid hormone and has undergone considerable transformation.

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As seen from the results of the in uitro experiments cited above (IV,A), prolactin acts directly on the tail to antagonize thyroid hormone. Accordingly, tail tissue is expected to have specific receptors for prolactin. In fact, specific binding of prolactin to tail and kidney tissue has been reported by White and Nicoll(1979) and Carr and Jaffe (1980). Furthermore, Tarpey and Nicoll (1987) characterized the renal prolactin-binding site of adult R. catesbeiana and neotenic A . tigrinum. More recently, Dunand et al. (1988) isolated prolactin receptors from the kidney of B. marinus. According to Can- et al. (1981),prolactin binding capacity in the tail increases during development to a maximum at prometamorphosis, followed by a decrease at early climax, with no appreciable change in K d . Accordingly, extremely high concentrations of plasma prolactin at late climax may not be for the tail but for other organs. In this regard, it is of interest to note that prolactin binding in kidney is very low at premetamorphic stages and that it increases as metamorphosis progresses. This is consistent with the autoradiographic data of Gona (1982). The increase in prolactin binding to renal tissue seems to be induced by thyroid hormone (White and Nicoll, 1979; Carr et al., 1981). With regard to the effect of prolactin on induction of its own renal receptors, conflicting results, stimulatory (Carr et al., 1981) and inhibitory (White et al., 1981), have been reported. It has been reported that treatment of bullfrog tadpoles with prolactin for a relatively long period brings about suppression of thyroid hormoneinduced development of the active Na transport system in the skin (Takada, 1989). However, prolactin exhibits a “short-term” effect on skin from late climax tadpoles. When prolactin is applied to the isolated skin, a temporary elevation of active Na transport is observed (Eddy and Allen, 1979; Takada, 1986). This suggests that although prolactin may act as an antimetamorphic hormone on the development of the active Na transport system in the skin, it exerts a different effect on the system once the skin acquires it. It is well known that prolactin has a direct effect on osmoregulatory tissues and organs in amphibians (Brown and Brown, 1987). Tadpoles at the late climax stage attain an adult-like structure but still remain in an aquatic or semiaquatic environment. It is highly probable that prolactin at late climax stages is involved in osmoregulation. Toad (B. japonicus) tadpoles show preference for a terrestrial environment when they reach middle climax. Their prolactin levels are much lower than those in bullfrog larvae at comparable stages, although prolactin levels in the toad tadpoles rise moderately as metamorphosis progresses (Niinuma et al., 1991a). In this species, prolactin levels are high only when individuals stay temporarily in the water during the breeding season (Ishii et al., 1989; Yamamoto ef a[., 1989). This also suggests a requirement for prolactin in the process of adaptation to water.

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It should be mentioned that use of purified amphibian prolactin for the investigation of osmoregulatory actions of prolactin and a molecularbiological approach to the analysis of prolactin receptors are necessary for clarifying the roles of prolactin during climax. C. Control of Prolactin Secretion

With regard to the mechanisms involved in the prolactin surge at late climax, one report has indicated that hypothalamic stimulation is necessary for elevation of plasma prolactin; the stimulatory system develops in the hypothalamus around climax under the influence of thyroid hormone (11,B). When tadpoles are thyroidectomized at late prometamorphosis, they never show hypersecretion of prolactin. T, therapy induces elevation of prolactin levels. Stalk transection at mid climax (TK Stage XXII) does not block metamorphic processes, but it does block the prolactin hypersecretion seen at later stages. In these animals, T4 supplementation is not effective in inducing the hypersecretion (Kawamura et al., 1986). In mammals, it is well known that the isolated adenohypophysis autonomously secretes a large amount of prolactin, and that prolactin secretion is predominantly under the inhibitory influence of the hypothalamus. In bullfrog tadpoles, on the other hand, neither stalk transection nor ectopic transplantation of the pituitary gland induces hypersecretion of prolactin (Kawamura et al., 1986). There is also indirect evidence that autotransplantation of pituitaries does not result in hypersecretion of prolactin in tadpoles of other species (Enemar, 1978; Schultheiss, 1979). However, this does not mean that in the tadpoles the hypothalamic inhibitory mechanism is not operating. In amphibians, dopamine is a possible prolactininhibiting factor. When tadpoles are treated with a dopamine antagonist, acceleration of metamorphosis is retarded (Seki and Kikuyama, 1979; Kikuyama and Seki, 1980) and collagen synthesis in the tail fin is stimulated (Seki et al., 1982; Yamamoto and Kikuyama, 1982b), presumably as a result of release from the inhibitory control of prolactin secretion by dopamine (Seki and Kikuyama, 1982,1986). Accordingly, prolactin release in tadpoles appears to be under dual control, stimulatory and inhibitory, and enhancement of hypothalamic stimulation rather than depression of hypothalamic inhibition is necessary for the hypersecretion of prolactin, at least in bullfrog larvae. It is known that TRH stimulates the release of prolactin from the pituitary gland in vitra (Clemons et al., 1979; Kikuyama and Seki, 1983; Hall and Chadwick, 1984) and in viva Kuhn et al., 1985). At present, the tripeptide is regarded as the most potent prolactin-releasing substance contained in frog hypothalamic extract. An acid extract of bullfrog hypo-

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thalami stimulates the release of prolactin from the bullfrog pituitary gland in vitro. When the extract is fractionated chromatographically, significant prolactin-releasing activity is shown by the fraction presumed to contain TRH. This activity is markedly reduced by coincubation with the IgG fraction separated from antiserum against TRH (Seki et al., 1988). Isolation of the potent prolactin-releasing substance from bullfrog hypothalami has been performed recently. The substance was determined to be TRH on the basis of its amino acid composition, chromatographic behavior, and RIA data (Nakajima et al., 1991). During prometamorphosis, both monoaminergic (Aronsson, 1976) and TRH-immunoreactive (Taniguchi et al., 1990) fibers develop in the anterior lobe. These fibers all disappear when the animals reach climax. This is interesting, since dopamine and TRH are possibly involved in the control of prolactin secretion. It is probable that before the median eminence develops prolactin cells are provided with some releasing and inhibiting hormones directly from the nerve endings and that around the onset of climax prolactin cells start to receive these hypothalamic hormones in the circulating blood transported from the newly developed median eminence. In fact, both monoaminergic (Aronsson, 1976; Kikuyama et al., 1979) and TRH-immunoreactive (Mimnagh et al., 1987; Taniguchi et al., 1990) nerve terminals become abundant in the median eminence when tadpoles reach climax. Although TRH may be the major prolactin-releasing factor, it is quite possible that prolactin secretion is regulated by multiple factors. According to Koiwai et al. (1986), both VIP and peptide histidine isoleucine (PHI) stimulate the synthesis and release of prolactin by the bullfrog pituitary in uitro. Serotonin also moderately stimulates the release of prolactin from the pituitary in uitro (Seki and Kikuyama, 1982), although in mammals serotonin is known to exert its stimulatory effect on prolactin secretion via hypothalamic VIP (Kato et al., 1984). The fact that administration of parachlorophenylalanine, which depresses brain serotonin, to bullfrog tadpoles accelerates tail-height reduction (Kikuyama and Seki, 1983) also suggests the involvement of serotonin in the regulation of prolactin secretion in amphibians.

V. Growth Hormone

In earlier studies, growth hormone, along with prolactin, was often tested for its growth-promoting activity in amphibian larvae. In general, prolactin was a more potent stimulator of larval growth than growth hormone. In some species, however, growth hormone was as effective, or more so, than prolactin (Dodd and Dodd, 1976). This may indeed be true, but the

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effect may have been due to the use of mammalian hormones. There are some instances of lower vertebrates responding similarly to both prolactin and growth hormone of mammalian origin, whereas they discriminate between the two hormones of nonmammalian origin, and respond to them differently (Ishii and Kikuyama, 1984; de Luze et al., 1989). Recently, amphibian growth hormone was purified from bullfrog pituitary glands and characterized (Kobayashi et al., 1989b). The amino acid sequence of growth hormone has been deduced from cDNA (Pang and Chang, 1988; Takahashi et af., 1992) or determined by protein analysis (Kobayashi et al., 1991). In amphibian larvae, an apparent effect of amphibian growth hormone was observed in the hind limbs. When hypophysectomized B. japonicus tadpoles were injected with bullfrog growth hormone, growth of the hind limbs occurred, whereas bullfrog prolactin was not effective in this respect. Thyroid hormone also stimulated the hind limb growth. The effects of both hormones were additive (Fig. 9). Development of a homologous RIA made it possible to measure plasma growth hormone levels in amphibians. Clemons (1976) measured plasma samples from climactic bullfrog tadpoles and noticed an increase in circulating levels of growth hormone through TK stage XXIV. Subsequently Kobayashi and Kikuyama (1991) determined plasma growth hormone lev-

0

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Days alter treatment FIG. 9 Effect of thyroid hormone, bullfrog growth hormone (GH), and bullfrog prolactin (PRL) on the growth of hind limbs of hypophysectomized tadpoles of Bufo. GH (0.5 p g ) and PRL (1.5 pg) were administered intrapentoneally every other day. Bovine serum albumin (BSA) ( I .5 p g ) was administered as a control. T4 was given by immersion.

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els in both bullfrog larvae and adults. Growth hormone levels are low during premetamorphosis and gradually increase toward climax. The hormone levels rise sharply during late climax, reach maximum in juvenile frogs, and then gradually decline as they grow up (Fig. 7). Changes of growth hormone mRNA levels in the pituitary parallel those of the plasma growth hormone levels (Takahashi er al., 1992). These findings suggest that growth hormone contributes mainly to growth in postmetamorphic juvenile frogs. In fact, both mammalian growth hormone (Dodd and Dodd, 1976) and growth hormone preparations from amphibian pituitaries (Nicoll and Licht, 1971 ; Kikuyama et al., 1984) increase the body size of postrnetamorphic amphibians. Distribution of growth hormone-producing cells in the pituitary of amphibians has previously been studied employing antisera to mammalian growth hormone (Doerr-Schott, 1976). Recently, an antiserum raised against purified bullfrog growth hormone has become available. Investigation using this antiserum clearly demonstrated the location of growth hormone-containing cells in the pituitary of adults. They are localized in the dorsal part of the distal lobe (Kobayashi and Kikuyama, 1991; Yon er al., 1991). Ontogeny of growth hormone-immunoreactive cells has been investigated in bullfrog larvae (Kobayashi and Kikuyama, 1989). Growth hormone-containing cells first appeared in the central part of the pars distalis at Shumway stage 22, and increased in number as development proceeded. Around stage 25, growth hormone cells gradually take a position in the dorsal part of the distal lobe as seen in adult specimens. At early stages of development, a few growth hormone-positive cells were also prolactin-immunoreactive. The effect of growth hormone on growth can be measured by monitoring the in uitro uptake of [35S]sulfateby the xiphisternal cartilage from hypophysectomized juvenile Xenopus individuals treated with the hormone. Most of the labeled sulfate taken up by the cartilage is incorporated into chondroitin sulfates (Ishii and Kikuyama, 1984). In this assay system, bullfrog growth hormone was three times as potent as ovine growth hormone in stimulating the sulfate uptake by the cartilage, and bullfrog prolactin had much less activity. Addition of growth hormone to the incubation medium did not affect the uptake of labeled sulfate by the cartilage. Plasma from hypophysectomized Xenopus that had received injections of growth hormone stimulated the in uitro uptake of sulfate by the cartilage. The stimulating effect of the plasma was dependent on the dosage of growth hormone that had been given to the plasma donors (Kobayashi et al., 1989a). It is well known that in mammals, insulin-like growth factors (IGF-I and IGF11) mediate the mitogenic actions of growth hormone (Van Wyk, 1984). The presence of IGF-like immunoreactivity has also been reported in amphibians (Daughaday et al., 1985; Rothstein et al., 1980). Indirect evi-

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dence that the action of growth hormone in amphibians is mediated by an IGF-Hike substance has been obtained by Rothstein et al. (1980). According to them, DNA synthesis and mitosis in lens epithelium of bullfrog juveniles are abolished by hypophysectomy and restored by the treatment with IGF-I or growth hormone. Pancak-Roessler and Lee (1990) provided direct evidence that the liver of the toad B. woodhousei produces an IGF-Hike substance. However, it was only possible to demonstrate that the production of the substance was stimulated by mammalian prolactin and not by mammalian growth hormone. There is little information about the regulation of growth hormone secretion in amphibians. Somatostatin-containing nerve terminals (Olivereau et al., 1987) and growth hormone-releasing factor-immunoreactive terminals (Marivoet et al., 1988) are abundant in the median eminence of several amphibian species. According to Hall and Chadwick (1984), both TRH and hypothalamic extract stimulate the in uitro synthesis and release of growth hormone, as detected by disc-gel electrophoresis of the medium and pituitary homogenate, by the pituitary glands of R. catesbeiana, R. pipiens, and X . laeuis. Somatostatin has no effect alone, but it inhibits TRH- and hypothalamic extract-induced release of growth hormone. Gracia-Navarro et al. (1991) have studied the subcellular changes of growth hormone cells in the pituitary evoked by TRH and concluded that the tripeptide is effective in stimulating the release of growth hormone from the pituitary of R. perezi both in uiuo and in uitro. Isolation and characterization of growth hormone-releasing factor from the amphibian hypothalamus have not been done.

VI. Conclusion

Amphibian metamorphosis seems to be under multihormonal control, and the secretion and function of these hormones are regulated in various ways. In addition to the hormones mentioned above, several other hormones, such as melatonin (Wright et al., 1991) and mesotocin (Platt and LiCause, 1980), may be included among the hormones influencing metamorphosis. Much work remains to be done on the identification of hypothalamic factors that regulate the secretion of the pituitary hormones involved in metamorphosis. Although we have not covered the ground extensively here, the ontogeny of hypothalamic neurons that may control pituitary function is of particular interest. Recently, experimental manipulation of amphibian embryos has revealed that some cell masses that are closely apposed to the primordium of the epithelial pituitary at early embryonic

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stages migrate into the brain to become hypothalamic cells after the completion of neural tube formation (Kawamura and Kikuyama, 1992) or just before completion of brain morphogenesis (Murakami et al., 1992). These findings suggest that the hypophysis and hypothalamus constitute a single entity from the primary stage of histogenesis and that paracrine-type control of the hypophysis by the hypothalamus shifts to endocrine-type control as the portal system develops in the median eminence. The finding that thyroid hormone (Fujikura and Suzuki, 1991) and the mRNA of its receptor (Kawahara et al., 1991) exist in the amphibian egg may prompt study on the effect of thyroid hormone on some cells or tissues during embryogenesis. Recently, metamorphosis of some teleosts such as flounders was revealed to be hormonally controlled, as is the case in amphibians (Inui and Miwa, 1985; Miwa et al., 1988). In addition, involvement of various hormones, which are similar to those involved in amphibian metamorphosis, in parr-smolt transformation ofjuvenile salmonids has been postulated (see Dickhoff et al., 1990). Accordingly, information derived from the endocrinological studies of these phenomena in teleosts will become useful for the advancement of research into the hormonal control of metamorphosis in amphibians, and vice versa.

Acknowledgments The help of Noriyuki Takahashi and Keisuke Nakajima in the preparation of this manuscript is gratefully acknowledged. This work was supported in part by a grant-in-aid from the Ministry of Education, Culture and Science of Japan and a research grant from Waseda University to S.K.

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Control of Metabolism and Development in Higher Plant Plastids M. J. Emes and A. K. Tobin Plant Metabolism Research Group, Department of Cell and Structural Biology, School of Biological Sciences, University of Manchester, Manchester MI 3 9PL, United Kingdom

1. Introduction Eukaryotic cells are divided by a large number of independent membrane systems into separate interior compartments. Each separate membranebounded interior structure carries out particular cellular functions. Among the various compartments and membrane systems within the cell are the nucleus, mitochondria, Golgi, microbodies, peroxisomes, glyoxysomes, endoplasmic reticulum, and vacoules. The most fundamental difference between plants and other eukaryotic organisms is the presence of a unique additional compartment, the plastid. It would take several books to cover in depth the diversity in structure and function of these organelles (Kirk and Tilney-Bassett, 1978) and so it is the aim of this review to try to summarize only some of the more recent topical areas of higher plant plastid metabolism, and the evolutionary origin and development of plastids. The compartmentation of metabolism within an organelle provides a means of concentrating the enzymes of a pathway and its associated metabolites, enhancing the likelihood of an enzyme product encountering the subsequent enzyme in the sequence of reactions. Further, compartmentation of events often avoids futile cycling by opposing reactions, and may sequester together related pathways that interact through the supply and demand of substrate. The controlled interaction of metabolism that this preserves is dependent on the selective permeability of the surrounding membrane to particular substrates and on the accurate targeting of polypeptides, synthesized in the cytoplasm, to their final subcellular destination. Inrernarional Review of Cytology. Val. 145

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All of the above mechanisms are to be found in plastids, which may be either autotrophic or heterotrophic depending on their age and tissue localization. Plastids are the biosynthetic powerhouse of the plant cell, being responsible not only for the reductive biosynthesis of intermediary carbohydrates from CO, but also for the synthesis of starch, fatty acids, amino acids, and pigments and the generation of precursors for secondary metabolism. They also contain, to varying degrees, the glycolytic and oxidative pentose phosphate pathways, which may assume a more significant role in energy and reductant provision in nonphotosynthetic plastids. It is not our intention to give a comprehensive account of everything to do with higher plant plastids, and much of what is presented undoubtedly reflects our own bias. That is not to say that any of the many topics not covered, or that are dealt with cursorily, are any the less important. For example, we shall not deal with energy capture or photosynthetic electron transport, which produces the energy upon which all life ultimately depends. These have been amply reviewed elsewhere (Andreasson and Vanngard, 1988; Ghanotakis and Yocum, 1990; Glazer and Melis, 1987). At the metabolic end, more emphasis will be given to a comparison of different types of plastid and in particular, where a process occurs in both photosynthetic and nonphotosynthetic organelles, we will consider how pathways are integrated with the metabolism of the tissue in which they operate.

II. Physical and Molecular Structure of Plastids A. Types of Higher Plant Plastid

The “plastid” is a general term applied to subcellular organelles found exclusively in plants. As the name suggests these organelles are extremely plastic in both form and function and a variety of plastid types are found in different plant organs and at various stages of cellular development (Fig. 1). Despite this heterogeneity all plastids are developmentally interrelated and interconvertible (Fig. 2). Their nomenclature generally relates to the morphology, composition, or localization within the plant. For comprehensive reviews of plastid types the reader is referred to articles by Kirk and Tilney-Bassett (1978) and Thomson and Whatley (1980). In this review we shall largely divide the different plastids into two main groups-photosynthetic and nonphotosynthetic. Nonphotosynthetic plastids include the plastid precursors proplastids or “eoplasts” (Thomson and Whatley, 1980) and etioplasts, and specialized plastids that may be

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considered functionally mature. These specialized nonphotosynthetic plastids include chromoplasts, amyloplasts, and elaioplasts. The eoplast (eo = early) has been identified as the first stage in plastid development (Whatley, 1977). These organelles are roughly spherical in shape, have a dense stroma, and lack the inner membrane found in chloroplasts. They are found in meristematic cells, in the leaves of some species during dormancy (Gaff ef al., 1976; Hallam and Gaff, 1978), and at stages during the transition from egg to ripe seed (Heslopp-Harrison, 1972). They originate maternally during zygote formation and consequently contribute toward inheritance of maternal characteristics (Kuroiwa, 1991). The eoplast contains DNA, RNA, ribosomes, and proteins and it divides within the meristematic region to maintain, or increase, its population within the cell (Whatley, 1980). The etioplast may be regarded as a nonphysiological precursor of the chloroplast that occurs in dark-grown plants. The major characteristic of these plastids is the presence of a fine, crystalline prolamellar body. Despite the increasing number of reports of major deviations from the normal development of chloroplasts in light-grown leaves, the study of light-dependent etioplast development still forms the basis of our “understanding” of plastid biogenesis. Some of these discrepancies will be referred to in later sections of this review and it is hoped that future work will take account of the likelihood that etioplast development cannot be used as a universal model. The specialized nonphotosynthetic plastids may be considered to be functionally mature in that they fulfill a metabolic role within the cell. This implies that they do not function as precursors, as is the case for the eoplasts and etioplasts discussed above. Although it is true that they do not act as universal progenitors, it would be misleading to suggest that they are rigidly fixed to serve a particular function, as many of these plastids are readily interconvertible. Different types of nonphotosynthetic plastid are usually localized in different types of tissue. Amyloplasts, for example, are mainly found in storage tissues, such as roots, tubers, endosperm, and cotyledons. They are also found in the root cap, where they may be involved in the geotropic response. The main function of the amyloplast appears to be to synthesize and store starch as a food reserve. Starch fills most of the internal space of these plastids and in some cases this leads to a distortion of their normal, spherical shape. Amyloplasts may be formed directly from eoplasts (Whatley, 1977) or by dedifferentiation of chloroplasts (Cran and Possingham, 1972; Senser ef af., 1975). Elaioplasts are oil-rich plastids, usually restricted to oil glands that are found in the stems of a number of cacti and in epidermal cells of some members of the Liliaceae and Orchidaceae (Kirk and Tilney-Bassett, 1978). These contain predominantly terpenoid compounds, which may serve allelopathic roles.

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Chromoplasts, as their name suggests, are pigment-containing plastids present in fruit, petals, and sepals. They accumulate hydrophobic carotenoid pigments that are localized in the chromoplast membranes. The chloroplast is the photosynthetic plastid of higher plant cells. It contains chlorophyll and is found in all photosynthetic parts of a plant, mainly in the leaves but also in stems, tendrils, cotyledons, and runners. Chloroplasts may also be found in a particular organ at a certain stage of its development, for example, in unripe (green) fruit where there may be quite significant rates of photosynthesis (Blanke and Lenz, 1989). There is considerable variation in chloroplast structure and metabolism and this will be discussed in detail later. Apart from the structural changes that occur during chloroplast biogenesis, one major example of chloroplast heterogeneity occurs in C4 plants where mesophyll cell chloroplasts are deficient in Calvin cycle enzymes, whereas bundle sheath chloroplasts are somewhat deficient in PSI1 proteins (Bassi et al., 1985; Broglie et al., 1984; Schuster et al., 1985). Furthermore, chloroplast composition varies in response to environmental influences such as light intensity (Malkin and Fork, 1981).

6 . The Chloroplast Genome

In many, but not all, plants certain plastid characteristics are inherited maternally in a non-Mendelian manner. This “cytoplasmic inheritance” was recognized in higher plants in the early 1900s and has recently been reviewed in this series by Kuroiwa (1991). Some mitochondria1 mutations are also transmitted uniparentally (Sager, 1972). These early observations clearly indicated that both chloroplasts and mitochondria possess their own distinct genomes. A sign of the interest and activity within this field, as a result mainly of the progress in molecular techniques, is that within 30 years of the discovery of chloroplast DNA (Ris and Plaut, 1962) the entire nucleotide sequence of the chloroplast genome has now been elucidated for two species of higher plants-rice (Oryza satiua, Hiratsuka et a / . , 1989) and tobacco (Nicotiana tabacum, Shinozaki et al., 1986)-and for the liverwort Marchanria polymorpha (Ohyama et al., 1986). The chloroplast genome has been the subject of a number of recent reviews (Sugiura, 1986, 1989a,

FIG. 1 (a) Pea root plastid. S, starch; P, plastoglobuli; L, lamellae. (Courtesy of Dr. C. G . Bowsher.) (b) Spinach leaf chloroplast. S, starch; M, mitochondrion; T, thylakoids. Bar = 1 pm. (Courtesy of Dr. E. Sheffield.)

M. J. EMES AND A. K. TOBlN

154 Senescent Chloroplast

Etioplast

\

FIG.2

Mature

Plastid types and their interconversion. [Redrawn from Thomson and Whatley (1988).]

b; Jukes and Osawa, 1990) and the aim here is to produce an overview and to highlight current areas of interest that merit further attention. The chloroplast genome is a covalently closed circle of double-stranded DNA of between 120 and 160 kb. The DNA is associated with the inner envelope or with thylakoid membranes where aggregates of 10-20 DNA molecules occur (Herrmann and Possingham, 1980). Plastids contain between 22 and 900 genome copies, although it is still unclear why this should be the case (Bendich, 1987). The chloroplast genome is highly conserved in both size and gene arrangement compared to the nuclear and mitochondria1 genomes of plants. Restriction maps of chloroplast DNA from a large number of species (Lim et al., 1990; Birky, 1988; Tippetts et al., 1991; Fluhr and Edelman, 1981; Bovenberg et al., 1981; Bowman et al., 1981; Chu et al., 1981; Link et al., 1981; Ma and Smith, 1985) show a remarkable similarity in gene order. The general structure, which occurs in all families of the angiosperms and in all of the families of gymnosperms (one family), pteridophytes (three families), and byrophytes (two families) studied to date, is divisible into

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three distinct regions: a large single-copy sequence (LSC) of 78-100 kb, a smaller single copy sequence (SSC) of 20-24 kb, and a duplicated region of 12-30 kb, which contains the rRNA genes. This duplicated region is usually inverted (referred to as the “inverted repeat” region (IR) and is found in all of the families of angiosperms studied to date, with one exception. In one group, consisting of several tribes in the subfamily Papinonideae of the legume family (Fabaceae), which includes Pisum safiuum and Viciafaba, the entire segment of the inverted repeat is absent (Koller and Delius, 1980; Palmer and Thompson, 1982; Crouse et al., 1986; Lavin et al., 1990). In these species, and in those where the inverted repeat region is very much larger than normal, for example in Geranium, there has been extensive rearrangement of the genome (Palmer, 1985). This rearrangement of the genome is a rare event and has yet to be understood. Apart from these rare exceptions, the chloroplast genome of all angiosperm species is extremely similar in size, gene content, gene order and arrangement, confirmation, and repeat structure. It is the most slowly evolving organelle genome (Palmer, 1985). The highly conserved nature of chloroplast DNA and the relatively slow rate of evolution (approxisubstitutions per site per year, Zurawski and Clegg, mately 1.5 X 1987) are features that are of considerable use in phylogenetic studies of plant evolution. There has been considerable interest in the use of comparative restriction maps of cpDNA for phylogeny reconstruction (Palmer, 1987; Palmer et a f . , 1988). A detailed discussion of this type of analysis is outside the remit of this review, but the reader is referred to excellent reviews on this subject (Zurawski and Clegg, 1987; Palmer, 1985, 1987, 1990; Palmer et al., 1988). The complete sequencing of the chloroplast genome of rice (Hiratsuka et a f . , 1989) and tobacco (Shinozaki et a f . , 1986) and of the liverwort M. polymorpha (Ohyama et al., 1986) has enabled a detailed comparison to be made of the sequences and the size and positioning of the open reading frames (orfs) and spacer regions, and this has revealed some general structural features of the chloroplast genome (Shimada and Sugiura, 1991). The genome contains all of the chloroplast rRNA genes (3-5 genes) and over 62 other genes for proteins sythesized within the chloroplast. These include RNA polymerase subunits, photosystem components, and polypeptides homologous to the mitochondria1 NADH dehydrogenase subunits (Shimada and Sugiura, 1991). These genes can be divided into two groups-those coding for photosynthetic components and those required for transcription, translation, or replication of the genome (Table I). There are four kinds of rRNAs coded for in the chloroplast. The 23S, 5S, and 4.5s rRNA are associated with the 50s ribosomal subunit, and the 16s is associated with the 30s subunit. The 4.5s rRNA is unique to the chloroplasts of higher plants. Because the rRNA genes are present in the inverted

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EMES AND A. K. TOBIN

TABLE I Plasld Genes and Their Products

Protein complex

Polypeptide

Gene

Reference"

I. Genes involved in chloroplast gene expression a subunit I2 1-kDap subunit 78-kDap' subunit Ribosomal RNAs 4 3 3 , 5S, 16S, 23s Transfer RNAs 30 tRNAs Ribosomal proteins Large subunit proteins (9 genes) Small subunit proteins (12 genes)

RNA polymerase

Rubisco ATP synthase

PSI PSI1

Cytochrome b,,f

11. Genes involved in photosynthesis Large subunit CF, a subunit CF, p subunit CF, E CFo subunit I CFo subunit I11 2 7 - k D a F ~protein (subunit IV) A1 (CP1) subunit of WOO apoprotein A2 (CP2) subunit of WOO apoprotein 9-kDa apoprotein of FeS centers A and B DI reaction center protein (32-kDa herbicidebinding protein, or Qe) 47-kDa polypeptide 43-kDa polypeptide D2 reaction center protein 9-kDa subunit of cytochrome b,,, 4kDa subunit of cytochrome b,,, 24-kDa G protein 10-kDa phosphoprotein 3.2-kDa hydrophobic polypeptide Cytochrome f Cytochrome b6 Subunit IV 111. Other genes NADH dehydrogenase (NDl-5)

rpoA rpoB rpoC rrn trn rPl rPs

2 3-6 7-10 3,5,6,11 3,5,6,12

rbcL atpA atpB atbE atpF atpH atpI psaA psaB psaC psbA

13 14 15,16 15,16 17-20 17-20 17-20 21 21 22,23 16

psbB psbC psbD psbE psbF psbG psbH psbL petA petB petC

24 24 25,26 27 27 28 3,29 30 31 32,33

1

32,33

ndhA-F

a I . Sijben-Muller, G., Hallick, R., Alt, J., Westhoff, P., and Herrmann, R. G. (1986). Nucleic Acids Res. 14, 1029-1044; 2. Ohme, M., Tanaka, M.,Chunwongse, J., Shinozaki, K., and Sugiura, M.(1986). FEBS Lett. 200,87-90; 3. Shinozaki, K., Ohme, M.,Tanaka, M., Wakasugi, T., Hayashida, N., Matsubayashi, T., Zaita, N., Chunwongse, J., Obokata, J., Yamaguchi-Shinozaki, K., Ohto, C., Torazawa, K., Meng, B. Y ., Sugita, M., Deno, H., Kamogashira, T., Yamada, K., Kusuda, J., Taikawa, F., Kato, A., Tohdoh, N., Shimada, H., and Sugiura, M. (1986). EMBO J. 5, 2043-2049; 4. Cozens, A. L., and Walker, J. E. (1986). Biochem. J. 236,453-460; 5 . Ohyama, K., Fukuzawa, H., Kohchi, T., Shirai, H., Sano, T., Sano, S., Umesono, K., Shiki, Y., Takeuchi, M., Chang, Z., Aota, S., Inokuchi, H., and Ozeki, H. (1986). Nature (London) 322, 572-574;,6. Hiratsuka, J., Shimada, H.,

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TABLE I (Continued) Whittier, R., Ishabashi, T.. Sakamoto, N., Mori, M., Kondo, C., Honji, Y., Sun, C.-R., Meng, B. Y., Li, Y. Q.. Kanno, A., Nishizawa, Y.,Hirai, A., Shinozaki, K., and Sugiura, M. (1989). Mol. Gen. Genet. 217, 185-194; 7. Bedbrook, J. R., Kolodner, R., and Bogorad, L. (1977). Cell (Cambridge. Mass.) 11, 739-749; 8. Schwarz, Z., and Kossel, H. (1980). Nature (London) 283, 739-742; 9. Edwards, K., and Kossel, H. (1981). Nucleic Acids Res. 9, 2853-2869; 10. Takaiwa, F., and Sugiura, M. (1980). Mol. Gen. Genet. 180, 1-4; 1 1 . Wakasugi, T., Ohme, M.,Shinozaki, K., and Sugiura, M. (1986). Plant Mol. Biol. 7, 385-392; 12. Sugita, M., and Sugiura, M. (1983). Nucleic Acids Res. 11, 1913-1918; 13. Mclntosh, L., Poulsen, C., and Bogorad, L. (1980). Nature (London) 288, 556-560; 14. Westhoff, P., Nelson, N., Bunemann, H., and Herrmann, R. G. (1981). Curr. Genet. 4, 109-120; 15. Krebbers, E. T., Larrinua, I. M., Mclntosh, L., and Bogorad, L. (1982). Nucleic Acids Res. 10, 4985-5002; 16. Zurawski, G., Bottomly, W., and Whitfield, P. R. (1982). Proc. Natl. Acad. Sci. U.S.A. 79,6260-6264; 17. Howe, C. J . , Auffret, A. D., Doherty, A., Bowman, C. M., Dyer, T. A., and Gray, J. C. (1982). Proc. Natl. Acad. Sci. U . S . A . 79, 6903-6907; 18. Cozens, A. L., Walker, J. E., Phillips, A. L., Huttly, A. K., and Gray, J. C. (1986). EMBO J . 5, 217-222; 19. Bird, C. R., Koller, B., Auffret, A. D., Huttly, A. K., Howe, C. J., Dyer, T. A., and Gray, J. C. (1985). EMBO J . 4, 1381-1388; 20. Hennig, J . , and Herrmann, R. G. (1986). Mol. Gen. Genet. 203, 117-128; 21. Westhoff, P., Alt, J., Nelson, N., Bottomley, W., Bunemann, H., and Herrmann, R. G. (1983). Plant Mol. Biol. 2,95-107; 22. Hayashida, N., Matsubayashi, T., Shinozaki, K., Sugiura, M., Inoue, K., and Hiyama. T. (1987). Curr. Genet. 12,247-250; 23. Hoj, P. B., Svendsen, I., Scheller, H. V., and Moller, B. L. (1987). J . B i d . Chem. 262, 12676-12684; 24. Westhoff, P., Alt, J., and Herrmann. R. G. (1983). EMBO J . 2, 2229-2237; 25. Alt, J., Moms. J., Westoff, P., and Herrmann, R. G. (1984). Curr. Genet. 8, 597-606; 26. Holschuh, K., Bottomley, W., and Whitfield, P. R. (1984). Nucleic Acids Res. l2, 8819-8834; 27. Herrmann, R. G., Ah, J., Schiller, B., Widger, W. R., and Cramer, W. A. (1984). FEBS Lett. 176, 239-244; 28. Steinmetz, A. A., Castroviegjo, M., Sayre, R. T., and Bogorad, L. (1986). J . Biol. Chem. 261, 2485-2488; 29. Farchaus, J . , and Dilley, R. A. (1986). Arch. Biochern. Biophys. 244, 94-101; 30. Webber, A. N., Hird, S. M., Packmann, L. C . , Dyer, T. A., and Gray, J. C. (1989). Plant Mol. Biol. 12, 141-151; 31. Willey, D. L., Auffret, A. D., and Gray, J. C. (1984). Cell (Cambridge, M u s s . ) 36, 555-562; 32. Phillips, A. L., and Gray, J. C. (1984). Mol. Gen. Genet. 194,477-484; 33. Heinemeyer, W., Alt, J., and Herrmann, R. G. (1984). Curr. Genet. 8, 543-549.

repeat region there are two copies of each of these genes per genome. All of the tRNAs used in chloroplast protein synthesis are believed to be coded for in the chloroplast genome. These are thought to number 30 tRNAs in all (Shinozaki et af., 1986; Hiratsuka et af.,1989). The tRNA genes appear to be highly conserved, with over 80% sequence homology among the rice, tobacco, and liverwort genes (Shimadaand Sugiura, 1991). In higher plants the tRNA genes are scattered throughout the cpDNA, whereas in Euglena most of them are clustered together (Hallick et al., 1984). Chloroplast ribosomes contain around 60 ribosomal proteins, of which 20 may be coded for in the chloroplast (Eneas-Filho el af., 1981). Chloroplast-encoded photosynthetic proteins, and their respective genes, are shown in Table 1. Other putative polypeptides coded for in the chloro-

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plast that do not fall into either of these two groups include sequences homologous to the mitochondria1 NADH dehydrogenase complex (Ohyama et al., 1986; Shinozaki er al., 1986). There are also a number of orfs that remain to be identified (Shimada and Sugiura, 1991). The chloroplast genes and products identified to date are summarized in Table I. An interesting deviation from the “normal” genome of higher plant chloroplasts occurs in the parasitic angiosperm Epifagus uirginiana. This plant is entirely nonphotosynthetic and grows parasitically on beech trees. The plastids of E. uirginiana lack thylakoids (Walsh et al., 1980) and, significantly, have a much smaller genome than that of photosynthetic plants due to the deletion of all of the photosynthetic and chlororespiratory (ndh) genes (depamphilis and Palmer, 1990; Morden et al., 1991; Wolfe et al., 1992). The SSC region of the Epifagus plastid genome is only 0.26 the size of tobacco plastid DNA (Wolfe et al., 1992) due not only to the lack of photosynthetic and chlororespiratory genes but also to the absence of two ribosomal protein genes (rpsl5 and 4 3 2 ) (Wolfe et al., 1992). Another ribosomal protein gene, rpslb, has also been lost from the LSC region of this genome (Morden et al., 1991). It remains to be established whether these “missing” ribosomal genes have been transferred to the nucleus (see Section D). Although the sequencing of the chloroplast genome has been a considerable milestone in this field-indeed, it might even have closed the subject for many researchers-the recent reports of RNA editing in chloroplasts (Hoch et al., 1991; Kudla et al., 1992; Cattaneo, 1992) suggest that this, perhaps, is not the end of the story. The editing of mRNA was first discovered in trypanosomes (Benne et al., 1986) and is particularly common in mitochondria, including those from plants, where extensive editing occurs (Covello and Gray, 1989; Gualberto et al., 1989; Hiesel et al., 1989). Different types of editing take place in different systems, for example, single base substitutions, small deletions, or insertions (Benne, 1990; Schuster et al., 1991), and these yield functional mRNA. In plant mitochondria, the most common form is C+ U editing, although occasionally a U-to-C substitution is found (Cattaneo, 1991). It was generally believed that editing did not occur in chloroplasts, since the amino acid sequences predicted from the gene sequence of mRNAs encoded by chloroplast DNA were extremely reliable and unambiguous (Shinozaki et al., 1986; Ohyama et al., 1986; Hiratsuka et al., 1989). Kossels group, however, have noted two examples of variant amino acids in the deduced sequences of protein products of chloroplast genes. The first report (Hoch et al., 1991) was of the rp12 gene (which codes for a subunit of a ribosomal protein; Shinozaki et al., 1986; Hiratsuka et al., 1989; Shimada and Sugiura, 1991) of maize chloroplasts where a comparison between the genomic sequence and the cDNA sequence of the corresponding mRNA showed that a C+U conver-

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sion had taken place. Thus, although the genomic sequence has an ACG codon, this is edited to produce an AUG initiation codon in the mRNA transcript. Similar C+U editing was found in another chloroplast gene, psbL (which codes for a 3.2-kDa polypeptide of PSII; Webber et al., 1989) from tobacco (Kudla et a f . , 1992). Again, this creates an initiation codon, AUG, in the mRNA. This observation, from a dicot species, together with the initial report using a monocot, maize, suggests that RNA editing in chloroplasts may be a relatively widespread phenomenon, although it is not, yet, possible to estimate how frequently this may occur. This is currently a subject of considerable research activity since, if more examples are found, it may be necessary to revise values of the lengths of open reading frames or of identified genes if it is found that RNA editing has created previously unidentified initiation or termination codons. There is, thus, a case for caution in deducing amino acid sequences and open reading frames directly from chloroplast DNA sequences. Another recent and exciting advance in this field has been the successful transformation of the tobacco chloroplast genome (Svab et a f . ,1990; Staub and Maliga, 1992), which now paves the way for future investigations into chloroplast gene expression (see next section). Although stable transformation of the chloroplast genome had been carried out with the unicellular alga, Chfamydomonas (Boynton et a f . , 1988), higher plant chloroplasts pose a number of additional problems. Most notable of these is the large number of genome copies-higher plant cells may contain 100 or more plastids, each of which has several copies of the genome. In order, then, to replace all of these copies with the transformed genome it is necessary to exert an extremely strong selection pressure in favor of the “foreign” gene. Techniques for the transformation of plastids have been described recently by Chasan (1992) and will not be discussed in detail. The successful approach used particle bombardment-where DNA-coated tungsten particles are shot into cells-to introduce DNA into tobacco plastid genomes (Svab et a f . , 1990; Staub and Maliga, 1992). In the first study (Svab et a f . , 1990) it was found that one of the introduced markers, streptomycin resistance, which was not selected for, became segregated out in the next generation. At the time it was thought that this might preclude the successful introduction of long, homologous sections of DNA into the tobacco plastid genome. Staub and Maliga (1992) later concluded that this loss of streptomycin resistance was not due to frequent recombination but was, rather, the result of the selective advantage that the loss of this characteristic conferred on the recombinant. In the later study, a larger DNA fragment, including the same 16s rDNA (which encodes 16s rRNA) as in the previous study (Svab et a f . , 1990), together with six unselected markers [five restriction fragment length polymorphism (RFLP) markers and a streptomycin-resistance marker] and the selected streptinomycin-

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resistance marker, was introduced into different functional domains of the genome (Staub and Maliga, 1992). The results confirmed that there was stable integration and that the introduced sequences were not only correctly copied into the second copy of the inverted repeat region but were also passed on to subsequent generations. Progeny from selfed transformants, or from crosses where the transformant was the maternal parent, were resistant to both antibiotics. Plastid transformation, then, offers a number of exciting possibilities for future investigation of the chloroplast genome. One of these, as suggested by Chasan (1992), is the introduction of insertional inactivation mutations into the unidentified orfs of the chloroplast genome. This might, then, enable the assignment of functions to these, as yet, unknown chloroplast genes.

C. Interactions between Nuclear and Chloroplast Genomes during Chloroplast Development and Differentiation

The diversity of plastid types and the flexibility with which these organelles interconvert and differentiate have led to many studies into how these processes may be regulated. Although the plastid genome codes for a number of its own proteins, as discussed above, many more are coded for within the nucleus. Photosynthesis, for example, requires the expression of several hundred genes, of which only approximately 120 are present within the chloroplast genome. This “division of labor” between the two genomes is exemplified by Rubisco, whose large subunit (LSU) is coded for and synthesized within the chloroplast, and whose small subunit (SSU) is nuclear encoded, cytosolically synthesized, and imported into the chloroplast, where the holoenzyme is constructed (Ellis, 1977, 1984). The way in which this “dispersal” of genes has arisen will be discussed in the next section. In this section we shall highlight conflicting reports into aspects of chloroplast gene expression and nuclear-chloroplast genome interactions that remain to be fully resolved. For previous reviews on these subjects refer to Gruissem (1989), Mullet (1988), Ellis (1984), Taylor (1989), and Kuroiwa (1991). Although the division of plastids is an important aspect of their biogenesis, this has recently been reviewed in some detail in this series (Kuroiwa, 1991) and will not, therefore, be discussed here. Plastid type is largely a function of the cell type within which it is located. The majority of proplastids within a leaf meristem, for example, will differentiate into chloroplasts. Not all leaf cells, however, contain fully developed chloroplasts. The epidermal and vascular cells contain either immature or undifferentiated proplastids (Kirk and Tilney-Bassett, 1978; Whatley, 1979). Even within the mesophyll cells of C, plants there

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is a clear progression in structural and biochemical development of the chloroplast as the cells mature (Tobin and Rogers, 1992). Significant differences may also be seen between the fully differentiated chloroplasts of different cell types. In NADP-malic enzyme-type C4 plants, for example, the chloroplasts are dimorphic, being agranal in the bundle sheath cells (Hatch, 1987). Fully differentiated plastids are also capable of “dedifferentiation” where, for example, mature chloroplasts of flowers and fruits may be transformed into pigmented chromoplasts (Grierson, 1986). Similarly, under particular conditions, the yellow or orange rind of Citrus fruits may regreen as the chromoplasts redifferentiate into chloroplasts (Mayfield and Huff, 1986; Thomson et al., 1967). Plastid differentiation therefore requires both spatial and temporal information, i.e., organ-specific, cell-specific, and developmental stage-specific signals that control and regulate gene expression. This type of information, i.e., positional and developmental, is eukaryotic in nature and is, perhaps, unlikely to be interpreted directly by the prokaryotic-type plastid genome. Thus, it has been suggested that the genetic information within the nucleus ultimately controls plastid genome expression (Taylor, 1989). This is not a unidirectional transfer of information, however, as there is evidence that plastid factors control the expression of some nuclear genes (Borner, 1986; Taylor, 1989). Comparisons of restriction maps of DNA from different types of plastid, for example, daffodil chromoplasts and chloroplasts (Hansmann, 1987; Thompson, 1980), tomato fruit chromoplasts and chloroplasts (Hunt et al., 1986; Iwatsuki et al., 1985; Marano and Carillo, 1991), and chromoplasts from Capsicum annuum (Gounaris et al., 1986), indicate that plastid diversity is unlikely to be due to differences in plastid DNA (ptDNA) sequence. One exception occurs in the parastic angiosperms, such as E. uirginiana, whose leaf plastids lack thylakoids and are nonphotosynthetic due, in part, to the complete absence of photosynthetic genes from the plastid genome (see earlier). In all other cases, then, it seems that differential gene expression is the major cause of plastid diversity. This could arise from selective expression of either nuclear or plastid genes, or both. It is still not entirely clear how selective expression is achieved and there are conflicting ideas. Also, the extent to which plastid genome expression is regulated at the transcriptional level is still unclear. It was generally thought that different mechanisms operated in the regulation of nuclear and plastid gene expression-the expression of nuclear genes encoding plastid proteins being regulated primarily at the transcriptional level, whereas plastid gene expression was regulated primarily at the posttranscriptional and translational level (Taylor, 1989; Gruissem, 1989).The regulation of expression of nuclear genes will not be discussed further, except where this is likely to influence, or be influenced by, chloroplast

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gene expression (Taylor, 1989; Kuhlemeier et af., 1987; Mullet, 1988; Borner, 1986). A number of reports have indicated that transcriptional control exerts only a limited influence upon chloroplast gene expression. Ngernprasirtsiri et al., (1988a) suggested that the amyloplast genome was virtually inactive in sycamore cell suspension cultures, as no transcripts could be detected, with the exception of the gene for 16s rRNA and psbA, the latter two presumably being transcriptionally regulated. The inactive genes, furthermore, were methylated in the amyloplasts but not in the chloroplasts (Ngernprasirtsiri et af., 1988~).Transcriptional activity was, however, found in spinach root amyloplasts when assayed using run-on experiments. The fact that transcription of genes for photosynthetic proteins could be detected in these nonphotosynthetic plastids suggested that most, if not all, plastid genes may be constitutively transcribed (Gruissem et af., 1987; Deng and Gruissem, 1988). The overall rate was much lower than that in the chloroplasts but the relative rate of transcription was the same for each gene (Deng and Gruissem, 1987), thus indicating no selective transcriptional control. Constitutive transcription of the plastid genome has also been detected in developing chromoplasts of sunflower (Heliunthus annuus) and pepper (C. annuurn) (Kuntz et ul., 1989). Work by Gruissems’ group has added support to the view that the major changes that occur in RNA levels in plastids during chloroplast development (Deng and Gruissem, 1987, 1988;Gruissem, 1989)and chloroplast-to-chromoplast transformation during tomato fruit ripening (Piechulla et al., 1985, 1986) are a consequence of changes in RNA stability (Gruissem, 1989; Mullet, 1988), i.e., are post-transcriptional. This is supported by work of Klein and Mullet (1986), who used an in vitro transcription assay to find that, during etioplast to chloroplast development in barley, there was no coordination between RNA levels and transcription rates, due, perhaps, to differences in stability of RNA. Most of the studies into transcriptional regulation of genome expression during chloroplast development have been carried out using etiolated tissue. There is considerable evidence from run-on transcription experiments that there is only a limited role for transcriptional regulation during etioplast-to-chloroplast transformation in spinach (Deng et al., 1987; Westhoff et al., 1988) and barley (Klein and Mullet, 1986; Klein ef al., 1988; Krupinska and Apel, 1989). This does not necessarily mean that the same is true during the natural differentiation of chloroplasts from proplastids in light-grown leaves. Indeed, a recent report now suggests that transcription plays a significant role in regulating plastid gene expression during normal leaf development in barley (Krupinska, 1992). Chloroplasts isolated from the tips of 5- and 7-day-old light-grown barley leaves and a protoplast-enriched fraction prepared from the leaf base were analyzed in

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run-on transcription experiments. Clear differences were observed in both the rates and the products of transcription in relation to chloroplast maturity. General transcriptional activity was 5- .to 10-fold lower in 7-day-old than in 5-day-old leaf tip chloroplasts. Some genes, the two ndh genes that code for subunits of the NADH-ubiquinone reductase (Hiratsuka et al., 1989), appeared not to be transcribed at any stage of barley chloroplast development. Others, which include some of the tRNA genes such as tmV, were transcribed at a relatively constant rate throughout chloroplast development. A third group of genes, notably the rrn operon, were transcribed fastest in proplastids and then declined with chloroplast age. Thus, whereas there is an increase in transcriptional activity during etioplast to chloroplast development in barley (Krupinska and Apel, 1989) there is a 5-fold decrease in the rate of transcription during chloroplast development in light-grown barley leaves (Krupinska, 1992). A similar decrease was found during chloroplast development in spinach grown in a greenhouse (Deng and Gruissem, 198’). It therefore appears that there are different mechanisms involved in the regulation of plastid gene expression during etioplast and proplastid differentiation. Hence extrapolation from the artificial experimental system of “de-etiolation” to the situation that occurs in light-grown plants must be made with extreme caution. There are a number of mechanisms that could cause developmentspecific changes in gene transcription. One of these, DNA methylation, has already been mentioned briefly. DNA methylation has been implicated in the regulation of plastid differentiation during conversion of chloroplasts to chromoplasts in ripening tomatoes, and in amyloplasts of sycamore cell cultures (Ngernprasirtsiri et al., 1988a,b; Kobayashi et al., 1990),whereby methylation is correlated with plastid gene inactivation. Krupinska (1992), however, found no evidence to suggest that the variation in plastid gene expression in light-grown barley leaves was caused by changes in methylation of plastid DNA. This adds further support to a similar observation on chromoplast formation during tomato fruit ripening. Comparison of restriction digests of ptDNA from green leaf chloroplasts and from mature red fruit chromoplasts, using restriction enzymes that distinguished between methylated and nonmethylated bases, showed no differences in methylation of the DNA from either plastid type (Marano and Carrillo, 1991). There is, as yet, no explanation for these conflicting results and we await further developments in this field. Another factor that has been implicated in transcriptional regulation is that of gene copy number. Several authors have found the photogenes psbA, psbB, and rbcL to be active in green tissue, such as leaves and stems, but inactive in nonphotosynthetic organs, such as roots (Sasaki and Kuroiwa, 1989; Sasaki et al., 1990). The organ-specific variation in transcript level was similar to the difference in gene dosage and Sasaki er

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al. (1990) suggested that gene dosage affects the organ-specific expression of the photogenes. In contrast, Baumgartner et al. (1989) found that there was no direct relationship between gene dosage and transcriptional activity. Indeed, transcriptional activity per DNA template varied up to fivefold during barley leaf development (Baumgartner et al., (1989). The presence of transcripts of the photogenes psbA and rbcL at all stages of embryo development in Arabidopsis, including the nongreen proembryo stage, suggests that, in contrast to previous reports (Sasaki and Kuroiwa, 1989), chlorophyll is not necessary for the expression of these genes at the RNA level (Degenhardt et al., 1991). The presence of chlorophyll is, however, thought to be necessary for the translation of transcripts of several chloroplast genes encoding chlorophyll-binding proteins (Mullet, 1988; Gray, 1992). One interesting mechanism that could serve to control plastid gene expression at the transcriptional level is via changes in the activity of RNA polymerase in the plastids, as suggested by Krupinska (1992). Although plastid RNA polymerases have yet to be fully characterized, there is evidence of a number of isoenzymes (Bottomley et al., 1971; Lerbs et al., 1983, 1985; Smith and Bogorad, 1974; Tewari and Goel, 1983; Greenberg et al., 1984; Gruissem, 1989), at least some of the subunits of which are nuclear encoded and some chloroplast encoded (Gruissem, 1989). Thus, some nuclear control could be exerted over plastid transcriptional activity (Gruissem, 1989). Other mechanisms involved in regulating plastid gene expression, such as RNA splicing, RNA stability (Gruissem, 1989), and DNA-binding proteins (Kuriowa, 1991), have been reviewed elsewhere. D. Evolutionary Origin of Plastids

There have been two main theories on the evolutionary origin of organelles: the endosymbiotic theory, first proposed by Schimper (1883), and the episomal theory of Raff and Mahler (1972). The endosymbiotic theory proposes that the chloroplast has evolved from free-living prokaryotes that were taken up by a host cell in which they continued to live as endosymbionts. This theory has received increasing support in recent years and is now becoming established as the more likely explanation of the origin of the chloroplast (Schiff, 1980; Margulis, 1981 ;Cavalier-Smith, 1982; Lewin and Cheng, 1989; Stackebrandt, 1989. Recent developments in this field will be discussed below. The episomal theory was first proposed to explain the origin of the mitochondria (Raff and Mahler, 1972). In this scheme, an aerobic prokaryotic cell formed intracellular organelles initially by invagination of the inner cell membrane. The organellar genome then arose by incorporation of a stable plasmid that contained the appro-

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priate genes. This theory has certain attractions in that it provides a means of explaining how genes may have become dispersed between organelles. Plasmids may become integrated within chromosomal DNA and, following excision, may then incorporate foreign DNA sequences to form a novel plasmid. In this way, genes could be transferred from one organelle to another. A strong argument against this theory is that there is no similarity between the base composition of the chloroplast DNA and that of the nucleus. It is, however, possible that a distinct and unrepresentative part of the nuclear DNA became detached to form the chloroplast DNA molecule or, alternatively, that the chloroplast DNA has diverged through subsequent evolution (Schiff, 1980). A closely related hypothesis, the “cluster clone theory” proposed by Bogorad (Bogorad et al., 1973; Bogorad, 1975) suggests that the modem eukaryotic cell arose as a result of the segregation of clusters of genes into separate subcellular compartments where they subsequently evolved into mitochondria, chloroplasts, and nuclei. In other words, whereas the endosymbiotic theory proposes that two or more cells combined to make one, in the cluster clone and episomal theories a single cell became partitioned to form subcellular components. The cluster clone theory is concerned more with seeking to explain how the partitioning and segregation of the genomes of organelles arose rather than finding the initial origin (Bogorad, 1975). There is considerable evidence to show that gene dispersal is an ongoing event and that genes have been transferred during the comparatively recent period of speciation (Brennicke, 1991; Gantt et al., 1991; Nugent and Palmer, 1991). For example, the caxll gene, which codes for cytochrome oxidase subunit 11, is found in the mitochondria1 genome of mammals, fungi, and most higher plants. In some species of legume this gene has become transferred to the nucleus (Nugent and Palmer, 1991). This transfer has been suggested to have occurred after the evolutionary divergence of monocotyledons and dicotyledons, some 60-200 million years ago (Nugent and Palmer, 1991). Thus, gene dispersal mechanisms operated well after the period when eukaryotic cells first arose. The endosymbiotic theory cannot, therefore, be dismissed simply on the grounds that it does not explain how the genes for multienzyme complexes became distributed among different genomes. Schuster and Brennicke (1987) suggest that one mechanism for gene dispersal may be the transport of RNA from one organelle to another. Such a process would ensure that only transcribed sequences would be transferred, hence avoiding nonfunctional transfers (Brennicke, 1991). The endosymbiotic theory for the origin of chloroplasts was first proposed over a century ago by Schimper (1883) and later by Mereschkowsky (1905). The suggestion was that chloroplasts arose from blue-green algae, which invaded eukaryotic cells and became established as endosymbionts (Margulis, 1970). There was good reason to support such a hypothesis:

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chloroplasts have a predominately prokaryotic nature; most notably, they have a circular DNA molecule and 70s ribosomes, they are capable of dividing within the eukaryotic cell, and they have their own genetic and protein synthesizing systems, as discussed above. Furthermore, bluegreen algae are known to form endosymbioses with eukaryotes (Tiffany, 1951). To substantiate the endosymbiotic theory it is necessary to identify the chloroplast progenitor. For some years the lack of a suitable candidate was a flaw in the hypothesis. Although the cyanobacteria had been implicated, there were a number of differences between these organisms and the chloroplasts of higher plants, which suggested that the two were not closely related. The photosynthetic apparatus, for example, differs in both structure and composition. Cyanobacteria lack chlorophyll b, a major pigment in higher plant chloroplasts. Phycobilins, phycocyanin and phycoerthryin, serve as accessory pigments in cyanobacteria but are absent from higher plant chloroplasts. The thylakoids of cyanobacteria are single and unstacked, whereas those of higher plant chloroplasts are organized into stacked (granal) and unstacked (stromal) regions. The discovery of a single-celled alga, Prochloron, which contained chlorophylls a and b, lacked physobilins, and had stacked thylakoids, caused considerable excitement and reinvigorated this field (Lewin, 1975, 1976, 1977; Lewin and Withers, 1975; Newcornb and Pugh, 1975). There has since been considerable debate over whether Prochloron is the primeval ancestor (Whitton and Carr, 1982; Van Valen and Maiorana, 1980; Lewin, 1981; Margulis, 1981; Lewin and Cheng, 1989; Cavalier-Smith, 1982). Prochloron is one of three genera of the Prochlorales (Lewin, 1977), the others being Prochlorothrix (Burger-Wiersma et al., 1986) and Prochlorococcus (Chisholm el al., 1988). Ironically, it has taken years of technological advances, for example, in DNA sequence analysis, to return with increasing support for the original proposal of Schimper (1883) that the most likely chloroplast ancestor was a cyanobacterium and not a prochlorophyte. Giovannoni et al. (1988) compared sequences of 16s rRNAs from 29 cyanobacteria, from the cyanelle of Cyanophora paradoxa, and from chloroplasts of M. polymorpha (liverwort). Phylogenetic trees were created and interpreted as showing that the chloroplasts and the cyanobacteria form a coherent phylogenetic group and that the chloroplasts should be viewed as a subline of the cyanobacteria. The cyanelle, which is the photosynthetic organelle of C. parudoxa, was also considered to be within the cyanobacterial radiation (Turner et al., 1989; Giovannoni et al., 1988). Two papers support this view and dismiss the Prochloron ancestory of the chloroplast. Palenik and Haselkorn (1992) compared partial sequences of the rpoCl gene (which codes for a subunit of DNA-dependent RNA polymerase) from a number of prochlorophytes and cyanobacteria; the

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cyanelle of C. paradoxa; and chloroplasts of Euglena, M. polymorpha, spinach, and maize. The analysis showed that the prochlorophytes were a highly divergent group and were not monophyletic. The phylogeny of the chloroplasts and the cyanelle of C. paradoxa was the same as that inferred from 16s rRNA analysis (Giovannoni et al., 1988). In no case did any of the prochlorophytes group with the chloroplasts to the exclusion of the cyanelle. The cyanelle, however, branched with the chloroplasts in 80% of the replicates (Palenik and Haselkorn, 1992). Urbach et al. (1992) analyzed sequence data of 16s rRNA from prochlorophytes and also reached the conclusion that this was a polyphyletic group. These authors concluded that none of the known species of prochlorophytes is specifically related to chloroplasts. They further suggested that chlorophyll b was acquired by the prochlorophytes and the green chloroplast ancestor in convergent evolutionary events. In this study, also, the chloroplasts branched with the C. paradoxa cyanelle, thus supporting the view that these descended from a common ancestor (Giovannoni et a f . , 1988; Douglas and Turner, 1991).

111. Metabolism of Higher Plant Plastids A. Translocation of Metabolites across the Plastid Envelope

The selective permeability of organelles to metabolites, through the operation of transporter proteins within membranes, offers the means by which metabolism within an organelle may be controlled, and also a medium through which the metabolic/bioenergetic status of that organelle may be communicated to the rest of the cell. Studies of purified chloroplasts have provided a rich vein of insight into the exchange of metabolites between the cytoplasm and plastids, though as different types of plastids are investigated, it is becoming increasingly apparent that a single model will not serve for all. C , chloroplasts are capable of counterexchanging inorganic orthophosphate and triose-phosphates, dicarboxylates and amino acids, and amides. These transporters serve as a means of exporting photosynthate to the cytoplasm, controlling partitioning between sucrose and starch, exporting products of nitrogen assimilation, and indirectly moving reducing power and energy between the two compartments. Much of our understanding of these processes has originated from studies by Heldt and co-workers (Heldt and Fliigge, 1987, 1992). Our intention here is to summarize what is currently known and to highlight future directions.

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1. The Phosphate Translocator The best studied of these transporters is the phosphate translocator of the inner chloroplast envelope. It is the largest protein fraction of the chloroplast envelope and may account for up to 15% of envelope protein. It catalyzes the counterexchange of inorganic phosphate (Pi), triosephosphate, and 3-phosphoglyceric acid (PGA), which are competitive with each other at a common binding site. The spinach chloroplast Pi translocator is probably the best characterized, consisting of a dimer of two identical polypeptide chains each 29 kDa as determined by SDS-PAGE. A fulllength cDNA clone has been obtained for the spinach protein (Flugge et af., 1989) encoding the entire 404 amino acids of the precursor protein with a molecular weight of 44,000. This protein can be imported into spinach chloroplasts where it is processed to a polypeptide of the same size as the native polypeptide in the inner envelope (now estimated to be M,36,000).It is a very hydrophobic protein with several a-helical regimes that probably traverse the inner membrane of the chloroplast envelope. Recently a cDNA clone for the pea chloroplast Pi translocator has been sequenced showing considerable homology (87%) with the mature spinach protein (Willey et al., 1991). The transit peptides from the peaand spinach Pi translocators are very long (72 and 80 amino acids, respectively), show less homology (45%), and are very different from those for stromal and thylakoid proteins. Import of the phosphate translocator is energy dependent, requiring ATP hydrolysis for its insertion into the inner membrane. This ATP could be generated in the cytoplasm or internally in the chloroplast, but a component of the proton motive force does not appear to be involved in protein import. There has recently been a suggestion that the clone obtained from pea might in fact not code for the Pi translocator, but for a chloroplast import receptor protein involved in the import of Rubisco SSU (Schnell et al., 1990). The cloned cDNA sequences reported were identical to those of Flugge et al. (1989), and it is unlikely that the same protein could function as both a Pi translocator and an import receptor protein. Both groups used 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) to inhibit the function of the mature proteins in chloroplasts, though it may be that this is not a specific inhibitor of the Pi translocator and the kinetics for the inhibition of the two processes may be different. Flugge et al. (1991) have recently translated in uitvo the cDNA clone to the 29-kDa protein and demonstrated that it shares properties with those of the phosphate translocator determined separately, and suggest that there may be common epitopes between the import receptor protein and the Pi translocator, which may account for the results of Schnell and co-workers. The physiological properties and substrate specificity of the phosphate translocator vary among species and plastid types. The phosphate translocator of C4chloroplasts is able to counterexchange phosphoenolpyruvate

METABOLISM AND DEVELOPMENT IN PLASTIDS

169

(PEP) and 2-phosphoglycerate in addition to those substrates mentioned earlier, distinguishing it from that in C, mesophyll chloroplasts (Huber and Edwards, 1977; Day and Hatch, 1981). Further, the phosphate translocators of chloroplasts from bundle sheath and mesophyll cells of Panicum miliaceum were found to have different kinetic properties, implying that there may be differential expression of different phosphate translocators within a species (Heldt et al., 1990a). This possibility has been further extended with the consideration of phosphate translocators in plastids of nonphotosynthetic cells. The first demonstration that such organelles possess the ability to counterexchange phosphate with other metabolites was achieved with preparations of pea root plastids (Emes and Traska, 1987). Subsequently it was shown that glucose 6-phosphate (G6P) could enter these root plastids and was able to sustain nitrite reduction (Bowsher et al., 1989). A thorough reappraisal of the pea root plastid phosphate translocator showed that in addition to the counterexchange that could be carried out by chloroplasts, this translocator could also import glucose 6-phosphate (but not glucose 1-phosphate) in exchange for triose-phosphate/ Pi (Borchert et al., 1989). This distinguishes the root plastid phosphate translocator from its leaf counterpart, which is virtually impermeable to glucose 6-phosphate. Using antibodies raised against the Pi translocator from C3chloroplasts, other groups have shown, in Western blot analysis, that envelope membranes from cauliflower buds (Alban et a f . , 1988) and sycamore cells (Alban et a / . , 1988; Ngernprasirtsiri et al., 1988b) possess a protein with antigenic similarity to the leaf protein. The observation that wheat endosperm amyloplasts preferentially incorporate glucose 1-phosphate into starch raises the possibility that there may be yet another type of Pi translocator in some species (Tyson and ap Rees, 1988). Clearly we now need more subtle probes to begin to delineate and identify the cell-specific expression of different phosphate translocators in a wide range of species and tissues if we are to understand their role more completely. A further important question is whether the nature ofthe phosphate translocator changes during development, particularly in leaves (Hoinghaus and Feierabend, 1985). For example, in what form does carbon move across the plastid envelope in young leaf cells, which are not photosynthetically active, and how does this differ from mature leaf cells and senescing chloroplasts, where presumably, in the latter case, photosynthate is no longer being exported? A summary of the specificities of phosphate translocators studied so far is shown in Table I1 (Heldt et al., 1991). 2. Organic Acid Transport

The movement of organic acids across the envelope of chloroplasts, leucoplasts, and amyloplasts is necessary to maintain amino acid biosynthe-

170

M. J. EMES AND A. K. TOBlN TABLE II Specificity of Phosphate Translocators ~~

Chloroplasts

Pi (Km) DHAP (Ki) PGA (Ki) PEP (Ki) G6P (K,,,)

Spinach leaf"

Maize mesophyllb

Leucoplasts Pea rootC

0.2 0.13 0.15 4.7

0.045 0.084 0.053 0.086 -

0.18 0.11 0.31 0.2 0.33

40

Note. All values are rnillimolar (mM). a Fliegge et af. (1978). Gross et al. (1990). Borchert et al. (1989).

sis, photosynthesis (C, and CAM), photorespiration, and transfer of reducing equivalents between the cytoplasm and the plastid. Chloroplasts are able to transport dicarboxylates including L-malate, 2-oxoglutarate, Laspartate, L-glutamate, and L-glutamine. Although undirectional transport is possible, it is much slower than counterexchange processes. The counterexchange of 2-oxoglutarate and glutamatelglutamine probably involves two translocators with different but overlapping substrate specificities (Lehner and Heldt, 1978; Woo et al., 1987). This is supported by observations with Arubidopsis mutants, in which one mutant line transported dicarboxylates but not glutamine, and the other transported glutamine in preference to dicarboxylates (Somerville and Somerville, 1985). Although oxaloacetate can be transported by the dicarboxylate transporters referred to in the preceding paragraph, the in uiuo concentration of oxaloacetate is about two orders of magnitude lower than malate (Heldt et al., 1990b), suggesting its transport via this route would be unlikely. However, an oxaloacetate translocator has been demonstrated in maize mesophyll chloroplasts, where it is involved in carbon movement in C4 photosynthesis. In malic enzyme-type C4 plants, oxaloacetate produced by PEP carboxylase in the cytoplasm is reduced in mesophyll chloroplasts to malate, prior to transfer to the bundle sheath cells. The K,,, of this translocator for oxaloacetate is very low (9 p M ) and is relatively insensitive to malate (Ki1.4 mM) (Hatch et al., 1984). Such properties permit effective counterexchange of oxaloacetate and malate across the chloroplast (Heldt et al., 1990a). A similar translocator has also been found in spinach chloroplasts (Ebbighausen et al., 1987) and the ability to transfer reductant from one side of the chloroplastenvelopeto the other is probably a function of the NAD(P)H/NAD(P)+ ratio in the chloroplast and cytosol,

METABOLISM AND DEVELOPMENT IN PLASTIDS

171

and the stromal NADP' malate dehydrogenase, which is controlled by the NADPH/NADP+ ratio (Scheibe, 1987). In C4plants the malate produced in the mesophyll stroma is decarboxylated in the bundle sheath cells yielding CO, (for the Calvin cycle) and pyruvate. Pyruvate passes back to the mesophyll cells where it is converted to PEP by the pyruvate phosphate dikinase located in the stroma of mesophyll chloroplasts. This necessitates entry of pyruvate into the organelle and is driven by a light-dependent cation gradient ( H + , Na+ depending on species) across the envelope, against a concentration gradient (Flugge et al., 1985; Ohnishi et al., 1990). Pyruvate transport may also take place in nonphotosynthetic plastids, where it is needed by the pyruvate dehydrogenase complex during fatty acid biosynthesis. This is likely to be the case where glycolysis within these organelles is incomplete and pyruvate cannot be generated internally by oxidation of hexose phosphates (Frehner et al., 1990).

3. Envelope Permeability during Chloroplast Development To date, there has been little study of the changes in chloroplast envelope permeability that may take place during leaf development. Chloroplasts from young pea leaves are permeable to adenylates (Robinson and Wiskich, 1977a,b) but this is not true for chloroplasts from mature leaf material (Walker, 1976), suggesting that such changes occur. Hampp and Schmidt (1976) examined changes in chloroplast envelope permeability during greening of Auena plastids. From osmotic swelling studies etioplasts appeared to be more permeable to organic acids and amino acids. After returning etiolated material to the light for 72 hr, chloroplasts were no longer permeable to sucrose, glycine, glutamate, or succinate. Malate uptake into rye etioplasts was low compared with that in chloroplasts (Hoinghaus and Feierabend, 1985), whereas phosphate translocator activity was hardly detectable in chloroplasts from old leaves and in ribosomedeficient plastids. Clearly there is considerable scope for further investigation into this area as more specific probes (e.g., antibodies, cDNA clones) to translocators become available. B. C 0 2 Fixation

The fundamental metabolic pathway of fixation of CO, in the light was determined by Calvin and co-workers in the mid 1950s(Calvin, 1956).This well-known cycle involves a complex sequence of interactions in which the generatiodregeneration of the acceptor molecule of CO,, ribulose bisphosphate (RuBP), is achieved at the expense of NADPH and ATP

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M. J. EMES AND A. K. TOBIN

derived from the photochemical reactions of the thylakoid. The enzymes of the reductive pentose phosphate cycle, as it is often termed, the regulation of the pathway through, e.g., light activation of enzymes, and its coordination with partitioning into sucrose and starch have been well documented and nonspecialist readers requiring an informative summary are referred to MacDonald and Buchanan (1990). This discussion will be confined to some of the more interesting recent discoveries about the primary carboxylating enzyme of photosynthesis, ribulose bisphosphate carboxylase (Rubisco), which catalyzes the reaction RuBP

+ COZ

M2 + -+

2 PGA

during photosynthesis and M’+

RuBP

+ 02-+PGA + phosphoglycollate

when photorespiration occurs (see Section III,E, 1,b). Rubisco, which is located in the chloroplast stroma, constitutes up to 50% of the total soluble protein in a leaf, earning it the deserved reputation as the most abundant single protein in the world (Ellis, 1979). This and the proposal that the physiological measurement of photosynthesis, determined a s net C 0 2 fixation, can be closely modeled by understanding the biochemistry of Rubisco (Farquhar and von Caemmerer, 1982) give it a significance above any other enzyme.

1. Rubisco Activation Rubisco is a large enzyme (molecular weight, 550,000) made up of two types of subunits: 8 LSU, each approximately 54,000, and 8 SSU, each approximately 15,000. The LSU is chloroplast encoded and SSU is nuclear encoded (see Section 11,C). The active site of the enzyme is found on the LSU and a clear biochemical function for SSU has not been ascribed thus far. Recently it has been found that there may be as many as five isoforms of SSU in maize and two in spinach, differing in isoelectric points, though the significance of this is unclear (Ren et al., 1991). With all forms of Rubisco the enzyme can exist in either an inactive or an active form. Activation is achieved when the enzyme is complexed with CO, and Mg2+. The CO, molecule involved in activation is distinct from the substrate molecule of CO, and binds to the epsilon amino group of a lysyl residue to form a carbamate that is stabilized by the binding of a divalent metal cation, which in uiuo is magnesium (Andrews and Lorimer, 1987)

173

METABOLISM AND DEVELOPMENT IN PLASTIDS slow

E(lnact,"e)+

A

co,

* E.

fast A COZ<,"act,"e) +

Mg2+

* E.

A

COz.MgZ+(actl"e)

The activation state of Rubisco changes substantially in response to irradiance, increasing with light intensity (Machler and Nosberger, 1980; Perchorowicz et al., 1982; V u et al., 1983, Fig. 3), and is closely correlated with photosynthetic rate (Salvucci et al., 1986). This activation state can occur spontaneously in vitro by incubation of inactive enzyme with CO, and Mg2+.However, whereas carbamylation is the most likely mechanism regulating the Rubisco activation state, the conditions in the chloroplast of an illuminated leaf mitigate against a high activation state by this spontaneous mechanism (Andrews and Lorimer, 1987). Further, binding of RuBP to sites on the decarbamylated (inactive) form of the enzyme in vitro severely hinders spontaneous activation, and in vivo would probably prevent activation by carbamylation in the light (Jordan and Chollet, 1983). In other words, spontaneous activation by CO, and magnesium alone cannot properly account for the observed in vivo activation. At least part of the solution to this enigma has come with the discovery of Rubisco activase. This was discovered as a result of a nuclear gene mutation of Arabidopsis thaliana, named rca, that possessed a perfectly normal, wild-type Rubisco enzyme, but was unable to activate Rubisco in the light (Somerville et al., 1982). Analysis of the soluble polypeptides of chloroplasts from the rca mutant indicated that two polypeptides were missing that were present in the wild-type (Salvucci et al., 1985). These

100

. 1600 E'

1200

.a00

2

32 JJJJ

11)

I

d

. 400

20

0 6

12

18

Time of Day (h) FIG.3 Light activation of Rubisco in soybean leaves. Total activity when fully carbamylated was measured at different times of day when growth was under natural illumination. (0) Rubisco activity; (0)PAR [Redrawn from Vu et al. (1983).]

174

M. J. EMES AND A. K. TOBIN

subunits constitute Rubisco activase. Rubisco activase has a molecular weight of about 200,000 and is a heterotetramer with subunits of mass 43 and 47 kDa (Salvucci et al., 1987). Antibodies raised against Rubisco activase were used to confirm its presence in a number of plants including C, species. Streusand and Portis (1987) demonstrated that activation of Rubisco enzyme by Rubisco activase was ATP dependent. Both the activation activity and the ATPase activity show a sigmoidal response to ATP concentration and are inhibited by ADP (Robinson and Portis, 1989). The dependence of Rubisco activation on ATP and its regulation by ADP could, in principle, coordinate COz fixation with the production of ATP by the thylakoids, though there is some doubt whether this has a significant role in uiuo (Portis, 1990). The mechanism by which Rubisco activase brings about a change in Rubisco enzyme activity is not fully established. However, the more or less coincidental discovery of another in uiuo mechanism for the regulation of Rubisco enzyme activity may provide an important clue. It had generally been thought that Rubisco activity in plant extracts could be fully activated by carbamylation with CO, and Mg2+.However, in the early eighties (Ku et al., 1982; Vu et al., 1983; Servaites et al., 1984) it was demonstrated that in some species, the maximal activatable Rubisco activity in leaves was considerably reduced in leaves at the end of a night period (Fig. 3). Subsequently an inhibitor was isolated (Gutteridge et al., 1986) from Solanum tuberosum and shown to be carboxyarabinitol 1phosphate (CAI P). This naturally occurring inhibitor binds tightly to Rubisco active sites, preventing carboxylation, and its concentration in the leaf varies inversely with light intensity. Carboxyarabinitol 1-phosphate closely resembles keto-carboxyarabinitol bisphosphate (CABP), which is the six-carbon transition-state intermediate of the carboxylation step (Schloss and Lorimer, 1982); CABP is also a powerful inhibitor of the carboxylation reaction. Seeman et al. (1990)have compiled a list of species apparently showing dark inhibition of Rubisco (the criterion often used to determine whether the species synthesizes CAlP) and it would seem not to be universally present in all species. Of 58 species examined, 37 exhibited dark inhibition of Rubisco and 21 did not. Recently Holbrook et al. (1992) examined 75 species from 21 tribes of the Papilionodeae for dark inhibition of Rubisco. A wide range in the extent of dark inhibition was found, and it was concluded that regulation by CAlP is not of recent origin, nor is it necessarily less pronounced among evolutionarily advanced species. However, direct measurements of CAlP are fraught with difficulties and the failure to demonstrate dark inhibition of Rubisco or direct measurement of CAlP may be due to rapid turnover of CAlP or lack of an available pool of precursors for its synthesis, or because Rubisco from different species may have different affinities for CA 1P (Gutteridge

METABOLISM AND DEVELOPMENT IN PLASTIDS

175

et al., 1986; Seeman et al., 1990). For example, Rubisco from spinach leaves does not appear to exhibit dark inhibition, yet Salvucci et al. (1988) have detected the first enzyme in the pathway of CAlP breakdown in spinach, implying that it may be present after all, but has not so far been detected by the assay methods used. It is likely that all CAlP is bound in uiuo to Rubisco, implying that a free pool of the inhibitor would be negligible. The concentration of CAlP and its effectiveness in inhibiting Rubisco therefore lies in its relationship to the number of Rubisco active sites. Phaseolus uulgaris accumulates CAlP in excess of the number of catalytic sites available (Kobza and Seeman, 1988), more than any other species examined so far, making it a prime target for the study of CAlP metabolism. At present, we know little about the synthesis and degradation of CAlP other than that it can be broken down by a chloroplastic phosphatase (Holbrook et a f . , 1989). The CAlPase breaks down CAlP phosphohydrolytically after release from Rubisco. It is stimulated by fructose bisphosphate and RuBP, favoring its operation as the Calvin cycle functions, thereby further enhancing Rubisco activation (Holbrook et al., 1991).The relationship between the foregoing discussion and Rubisco activase comes through the important observation of Robinson and Portis (1988) that the activase can catalyze the release of CAlP from Rubisco, but does not metabolize it. The authors suggest that light-dependent reversal of CAlP inhibition in uiuo may require Rubisco activase. In the dark, the activase would be inhibited by a low ATPIADP ratio, effectively coordinating Rubisco activity with the light reactions of photosynthesis. It seems that there is good evidence that Rubisco activase is ubiquitous in higher plants. This does not appear to be the case with CAlP and therefore the universal applicability of this model is at present open to question.

2. Decreased Rubisco in Transgenic Plants A number of papers have recently investigated the impact of reducing the amount of Rubisco protein by antisense RNA technology on the rate of photosynthesis. Most recently, Hudson ef al. (1992) have used RNA to SSU cloned from tobacco to make antisense transformants in the same species. This resulted in a reduction in the total amount of Rubisco, with the transformed plants possessing about 18% of the activity of the wild type. The consequence of this was that photosynthesis was reduced by 63%. Given the size of the reduction in Rubisco, perhaps this is not too surprising in light of the earlier discussion. The earlier work of Stitt’s group using an identical approach, again with tobacco, has been more illuminating. They studied photosynthesis in a number of transgenic plants covering a range of Rubisco activities obtained with antisense to SSU and generated by Bogorad’s group (Rodermel ef al.,

176

M. J. EMES AND A. K. TOBIN

1988). The impact of decreased Rubisco on growth, carbon and nitrogen content, and shootIroot ratio was marked when Rubisco content fell below 57% of the wild type (Quick etal., 1991a).Of more interest in this particular discussion is the study of photosynthesis in plants with 57- 100% Rubisco (Quick et al., 1991b; Stitt et al., 1991), in which biomass and partitioning are much less effected. Stitt and colleagues have used these mutants in a flux control analysis (Kacser, 1987) of the importance of Rubisco in controlling CO, assimilation. Flux control analysis aims at obtaining a quantitative estimate of the importance of an enzyme to a metabolic pathway by assessing the impact that small fractional changes in an enzyme's activity (dEIE) have on flux, also measured as a fractional change (dJIJ). The ratio of these two values (dJIJldEIE) extrapolated to the wild type gives the control coefficient for that step, Ci.In a given, defined pathway, the sum of the control coefficients for all reactions of that pathway equals 1. Under the ambient conditions used of 340 pmol quanta rn-, * s-' and ambient CO,, a 43% decrease in Rubisco had only a marginal effect on CO, fixation, with a control coefficient for Rubisco of less than 0.1. However, further reduction in the amount of Rubisco had a dramatic effect on photosynthesis, the latter being reduced parallel with the enzyme (Quick et al., 1991b). These figures suggest that under the ambient conditions employed, in the wild-type Rubisco is in approximately 40% excess of what is actually needed to maintain photosynthesis. One of the compensating mechanisms that the transgenic tobacco plants seem to adopt to maintain photosynthesis is to increase the activation state of Rubisco. A 43% decrease in amounts of Rubisco was at least partially compensated for by a 31% increase in activation, resulting from an increased ATP/ADP ratio, favoring Rubisco activase. More detailed study of photosynthesis in these transgenic plants at varied light intensity, CO,, and air humidity (Stitt et al., 1991) indicates that the control coefficient of Rubisco is much higher at high light intensities, lower CO,, or increasing air humidity. The value obtained for CL varied with the combinations of conditions applied, but the real value of this approach is that it shows how control of metabolism by an enzyme shifts under different environmental conditions. An important step now will be to obtain decreased activity mutants/transgenic plants for other steps of the Calvin cycle to determine their relative importance to photosynthesis.

C. Starch Synthesis Starch is made in both photosynthetic and nonphotosynthetic plastids. In leaves it rapidly turns over, being synthesized during the day and broken

METABOLISM AND DEVELOPMENT IN PLASTIDS

177

down at night. By contrast, in nonphotosynthetic cells, such as developing endosperm, it is synthesized in amyloplasts during grain-filling and broken down extracellularly during germination (Duffus, 1984). In roots it can be synthesized and degraded, but turnover is minimal (Hargreaves and ap Rees, 1988). There is a fundamental difference between starch synthesis in chloroplasts and amyloplasts. In the former all the prerequisites for synthesis-fixed carbon and ATP-are generated within the organelle, requiring no import from the cytoplasm: in the latter, all substrates have to be imported into the amyloplast to make starch. This means that the degree of analogy that can be drawn between the two types of plastid is inevitably limited. The pathway of starch synthesis common to both types of plastid can be considered the transfer of glucosyl units via ADP-glucose to starch catalyzed by starch synthase (SS) in combination with branching enzyme (BE) (Fig. 4). Studies of the biochemical regulation of this pathway in chloroplasts have centered on ADP-glucose pyrophosphorylase and have been well reviewed (Beck and Ziegler, 1989; Preiss, 1988). This enzyme is activated by physiological concentrations of phosphoglyceric acid and inactivated by inorganic orthophosphate (Preiss, 1988) and simple, elegant models have been presented for the role of these modulators in controlling partitioning of photosynthate between starch in the chloroplast and sucrose in the cytoplasm. Although the nonphotosynthetic enzyme has been less extensively studied, its regulatory properties are similar to those of the leaf enzyme if careful extraction procedures are observed (Plaxton and Preiss, 1987). Starch synthase exists in both soluble and starch-granulebound forms (MacDonald and Preiss, 1985). The precise roles of these two forms are not known and the proportions of the soluble to starch-granulebound form may vary (Smith, 1990). The soluble starch synthase activity itself may be resolved into isoforms in some species (Denyer and Smith, 1992). The granule-bound starch synthase, sometimes referred to as the “waxy protein,” appears to be essential for amylose synthesis in potato tubers (van der Leij et al., 1991). When the expression of this protein is inhibited by antisense constructs in potato, amylose-free starch is produced in the tubers (Visser et d.,1991). Branching enzyme, which brings about the formation of amylopectin, also probably exists as a number of isoenzymes (Boyer and Preiss, 1979). The importance of the role of these isoenzymes can be deduced from studies of the r-locus on starch synthesis in developing pea cotyledons (Smith, 1988). Here it has been shown that the loss of one form of BE leads to a reduction in starch synthesis in the developing embryos. Starch branching enzyme may influence the overall rate of starch synthesis since it has been shown that the activity of purified starch synthase is considerably stimulated by starch branching enzyme (Pollock and Preiss, 1980).

FBP

G6P

tf f f

G1P

ADP-Glucose

ADP-Glucose ?

ADP

FIG. 4 Summary diagram of starch synthesis in photosynthetic and nonphotosynthetic plastids. Key features are the involvement of fructose bisphosphatase (a) in the chloroplast pathway; the presence of a phosphate translocator capable of transporting hexose phosphate (b) in nonphotosynthetic plastids; a putative adenylate translocator (c) in amyloplasts; and the regulation of ADP-glucose pyrophosphorylase (d) by PGA and Pi. The synthesis of starch from ADP-glucose is taken to include starch synthase and starch branching enzyme activity. Hatched area indicates amyloplast envelope; open area represents chloroplast envelope.

METABOLISM AND DEVELOPMENT IN PLASTIDS

179

It has previously been assumed that starch in the amyloplasts of storage tissues is derived from triosephosphate imported from the cytoplasm. This was largely based on the demonstration of an active phosphate/ triosephosphate translocator in the chloroplast envelope (Heldt and Rapley, 1970) and the fact that chloroplasts contain all the necessary enzymes to convert triose phosphate to starch. This view was enhanced by the demonstration that nonphotosynthetic plastids also contain a Pi translocator (Emes and Traska, 1987; Ngernprasirtsiri el al., 1988a) and reports that nongreen plastids contain fructose bisphosphatase (MacDonald and ap Rees, 1983; Journet and Douce, 1985; Echeverria et al., 1988). There is now good evidence that the number of enzymatic steps from precursor carbohydrate to starch is greater in the chloroplast than in most amyloplasts. In the chloroplast, triose phosphate generated in the light is converted to glucose 1-phosphate, dependent upon chloroplastic fructose bisphosphatase. It has recently been shown that nonphotosynthetic tissues that store starch lack this enzyme (Entwistle and ap Rees, 1990a)and that any apparent fructose bisphosphatase activity is in fact due to the contaminating presence of the reversible pyrophosphate-dependent phosphofructokinase (ap Rees et al., 1991; Entwistle and ap Rees, 1990b). Since the latter is exclusively cytosolic there appears to be no obvious route from triosephosphate to hexose phosphate within the amyloplast. The exception to this is the developing pea embryo, whose plastids probably contain fructose bisphophatase, reflecting their origin in cotyledons. It has been suggested that in this tissue, the enzyme may be confined to plastids that contain little starch (Smith et af., 1990a). The above observations imply that the substrate entering the amyloplast for starch synthesis is not a triose but a hexose, bypassing the need for fructose bisphosphatase. There is strong support for this from experiments in which potato tuber (Viola et al., 1991) and developing wheat grain (Keeling et af., 1988) were supplied with [1-’3C]-and [6-’)C] glucose. If hexose supplied to whole cells enters the cytoplasm and is converted to starch via triosephosphate metabolism in the amyloplast, then there should be complete randomization of label between carbon atoms 1 and 6 as a consequence of the triosephosphate isomerase activity located in both cell compartments. Consequently the hexose units found in newly synthesized starch should have equal proportions of [1-l3C1and [6-I3C],irrespective of the form of labeled glucose supplied. In fact, redistribution of label between carbon atoms 1 and 6 in starch glucosyl units is only 15-20% and this can be accounted for by substrate cycling in the cytoplasm (Keeling el a / . , 1988; Viola et al., 1991; Hatzfeld and Stitt, 1990). This line of evidence gives strong support to the view that carbon destined for starch synthesis enters the amyloplast not as triosephosphate but as hexose/ hexose phosphate. Studies of nitrite reduction with pea root leucoplasts

180

M. J. EMES AND A. K. TOBIN

indicate that these plastids, which also contain starch, are able to import glucose 6-phosphate (Bowsher e f a f . , 1989) and the presence of a phosphate translocator able to transport glucose 6-phosphate but not glucose 1-phosphate has been reported for the same root plastids (Borchert et al., 1989). Determination of the preferred form of carbon entering amyloplasts has proved difficult (by comparison with chloroplasts) because of the problems of purifying intact organelles in sufficient quantities. Tyson and ap Rees (1988) fed radiolabeled precursors to amyloplasts isolated from wheat endosperm. Only glucose 1-phosphate supported starch synthesis when account was taken of rates of label incorporation into preparations that had been deliberately lysed. However, the authors did not report whether addition of ATP had any effect on rates of incorporation of precursors into starch. By contrast, preparations of maize endosperm amyloplasts were better able to synthesize starch from triose phosphate, glucose 1-phosphate, and glucose 6-phosphate being ineffective as substrates (Echeverria er al., 1988). Fructose bisphosphate supplied to the same amyloplasts supported starch synthesis at approximately half the rate of triose phosphates. This is difficult to reconcile with the previous discussion, which implies that maize endosperm amyloplasts lack fructose bisphosphatase (Entwistle and ap Rees, 1990a). More recently, there is evidence that in purified amyloplasts from developing pea embryos (which may possess fructose bisphosphatase) the only substrate to support starch synthesis in purified organelles at rates approximate to in uiuo values was glucose 6-phosphate (Hill and Smith, 1991) and such rates of starch synthesis were ATP-dependent (Table 111). Glucose 1-phosphate and triose phosphates were ineffective as substrates for starch synthesis in the presence or absence of ATP. Although fructose bisphosphate was a relatively good substrate for starch synthesis, this did not depend on the intactness of the preparation. The data also imply that such nonphotosynthetic plastids may possess an adenylate translocator. Evidence in support of this comes from an immunochemical detection of an adenylate translocator in amyloplasts of sycamore suspension cultures (Ngernprasirtsiri et al., 1989). Taken together, all the above lines of evidence point to hexose phosphate entering the amyloplast for starch synthesis, not triose phosphate. The source of ATP for starch synthesis remains open. Clearly there is a prima facie case for an adenylate translocator (Table 111).The significance of this in relation to the operation of a triose phosphate shuttle that could indirectly transport ATP across the amyloplast envelope remains an open question. Recently Akazawa and co-workers have suggested that ADP-glucose may enter directly from the cytoplasm to support starch synthesis (Pozueta-Romero et al., 1991a,b). In support of this they suggest that

METABOLISM AND DEVELOPMENT IN PLASTIDS

181

TABLE 111 Incorporationof I4C-Labeled Substrates into Starch by Plastids Isolated from Developing Pea Embryos

Substrate (all 2 mM in presence of 1 mMATP)

Incorporation into starch (%)

Glucose 6-phosphate Glucose 1-phosphate

100

0.2

5.7

Fructose 6-phosphate Fructose bisphosphate Dihydroxyacetone phosphate

0

0 4.5

Glucose Fructose

11.9 ~

~

~

~~

Note. Activity is expressed as a percentage of that obtained when supplying glucose 6-phosphate and ATP, and after taking account of label incorporation into starch in lysed preparations of plastids. [Adapted from Hill and Smith (1991) with permission.]

ADP-glucose may be formed directly in the cytoplasm by sucrose synthase (Pozueta-Romero et al., 1991~).This is an interesting suggestion and is still consistent with the 13Cdata referred to earlier. However, it raises the question why plants have ADP-glucose pyrophosphorylase, as the need for this would be obviated by such a route of biosynthesis. Further, there are clearly identified mutants of plastidial phosphoglucomutase (Kiss and Sack, 1989) and ADP-glucose pyrophosphorylase (Lin et al., 1988) in which starch synthesis is negligible in all organs. This is difficult to reconcile with the proposal that ADP-glucose enters the plastid directly from the cytoplasm and is the main route for starch synthesis. It may be that such a pathway is possible, but the isolation of the above mutants suggests that if it functions at all in uiuo it may be of only minor importance. Nonetheless, it is an interesting proposal and warrants further verification. Since it is apparent that the pathway of starch synthesis in chloroplasts (postformation of triosephosphate via the Calvin cycle) is longer than in amyloplasts, it follows that the potential for control of starch synthesis is not confined to ADP-glucose pyrophosphorylase, starch synthase, and branching enzyme. Stitt and collaborators have recently investigated the control of starch synthesis in leaves using decreased activity mutants of Clarkia xantiana (Kruckeberg et al., 1989), A . rhaliana (Neuhaus and Stitt, 1990), and P . satiuum (Smith et al., 1990b) to determine the flux

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control coefficients (CL) of a number of enzymes. Interestingly Stitt and co-workers have found that in reduced activity mutants of C. xantiana under conditions of high light intensity, phosphoglucose isomerase has a significant control coefficient for starch synthesis. What is surprising about this result is that this is an enzyme that catalyzes an equilibrium reaction and whose maximal in uitro activity is usually regarded as being in excess of that required to support starch synthesis in the chloroplast. The flux control coefficient appears to indicate that there is no large excess of plastidial phosphoglucose isomerase. In Arabidopsis leaves growing in high light most of the control of starch synthesis is invested in ADPglucose pyrophosphorylase, but in low light control is moved away from the committed enzymes of starch synthesis. An outline of our current understanding of the pathway of starch synthesis in green and nongreen plastids is given in Fig. 4. D. Fatty Acid Synthesis

The plastid is the major site of de nouo fatty acid synthesis in higher plants (Harwood, 1991).Two enzyme complexes are involved: acetyl CoA carboxylase (ACC) and fatty acid synthetase (FAS). These two complexes together form long-chain fatty acids from acetyl CoA. The reactions of FAS are catalyzed in association with acyl carrier protein (ACP). In this section we discuss the role of the plastid in these processes and the differences in fatty acid biosynthesis in photosynthetic and nonphotosynthetic tissue. For recent reviews on wider aspects of this subject, see Ohlrogge and Kuo (1984), Stumpf (1987), Harwood (1988, 1991), Andrews and Ohlrogge (1990), and Slabas et af. (1992). For a recent review on the involvement of carnitine in fatty acid metabolism, see Wood et al. (1992). 1. Acetyl CoA Carboxylase Acetyl CoA carboxylase catalyzes carboxylation of acetyl CoA to form malonyl CoA, in a two-step reaction: (a) Acetyl CoA

+ HCOj + BCCP + ATP+malonyl CoA + ADP + Pi (b) BCCP-C02 + acetyl CoA+BCCP + malonyl CoA

+ BCCP-C02

In plants, ACC is a multifunctional polypeptide with three functional domains (Harwood, 1988). Biotin is attached covalently to the biotin carboxyl carrier domain. C 0 2is activated and attached to biotin by the biotin carboxylase domain-reaction (a)-to form carbox yl-biotin (BCCP-C02). The CO, is then transferred from this to acetyl CoA by the acetyl CoA : malonyl CoA transcarboxylase domain-reaction (b)-to form malonyl

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CoA. Acetyl CoA carboxylase has been localized in the chloroplasts of leaves (Kannangara and Jensen, 1975;Mohan and Kekwick, 1980;Nikolau et al., 1981;Thompson and Zalik, 1981).In nonphotosynthetic tissue there is some evidence of plastidial and cytosolic isoenzymes. Plastids isolated from the endosperm of developing castor bean seeds (Finlayson and Dennis, 1983) and from avocado mesocarp (Mohan and Kekwick, 1980) have high activities of ACC but, because of the fragility of isolated plastids, the possibility of cytosolic ACC cannot be entirely ruled out. Indirect evidence from the developing jojoba bean does suggest that ACC is present in both the plastid and the cytosol (Ohlrogge et al., 1978). 2. Fatty Acid Synthetase

All of the subsequent steps in fatty acid biosynthesis in plants require the participation of ACP, whose properties and isoenzymes will be discussed later. Acyl carrier protein serves as a cofactor rather than an enzyme in these reactions. Acyl groups, including the substrates, intermediates, and products of fatty acid biosynthesis, are attached to ACP via a thioester link to the 4’-phosphopantetheine prosthetic group of the protein. Fatty acid synthetase is a dissociable type I1 enzyme consisting of at least seven distinct enzymes (Table IV; Caughey and Kekwick, 1982; Hoj and Mikkelsen, 1982; Schuz ef af., 1982; Shimakata and Stumpf, 1982a,b). For more detailed information on the properties of each enzyme, see Stumpf (1987), Harwood (1988), and Slabas er al. (1992). The precise stoichiometry of FAS components has yet to be elucidated. Nor is it clear TABLE IV Enzymes of Fatty Acid Synthetase

I . Acetyl CoA : ACP Transacetylase Acetyl CoA + ACPeAcetyl ACP + CoA 2. Malonyl CoA : ACP Transacylase Malonyl-CoA + ACPSMalonyl ACP f CoA 3. P-Ketoacyl ACP Synthetase I (KAS 1) + ACP Acyl-ACPfC2.,,,, + Malonyl ACPeP-Ketoacyl ACPfC4.C16) 4. 0-Ketoacyl-ACP Synthetase I1 (KAS 11) Palmitoyl ACP + malonyl ACPeP-Ketostearoyl ACP + C 0 2 + ACP 5. 0-Ketoacyl ACP Reductase P-Ketoacyl-ACP + NADPH + H+=D-P-Hydroxyacyl ACP + NADP+ 6. D-P-Hydroxyacyl ACP Dehydrase D-0-Hydroxyacyl ACPZtrans-2-Enoyl ACP + H 2 0 7. Enoyl ACP Reductase 2-Enoyl ACP + NADH + H + e A c y l ACP + NAD+

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whether the enzymes exist together as a complex in uiuo, although this seems likely. The first reaction, catalyzed by acetyl CoA : ACP transacetylase, involves the transfer of the acetyl group from acetyl CoA to ACP. This enzyme has a low specific activity in extracts from developing seeds of Cuphea lutea, safflower, and rapeseed and from leaves of pea and spinach (Stumpf, 1987), and was often classed as the “rate-limiting step” in fatty acid synthesis. There is now some doubt whether this is, indeed, the first step in the pathway. A thiolactomycin-sensitive acetoacetyl ACP synthetase activity (KAS 111) was discovered by Jackowski and Rock (1987) in Escherichia coli and later found in plants ( Jaworski et al., 1989; Walsh et al., 1990). This enzyme catalyzes a condensation between acetyl CoA and malonyl ACP to form acetoacetyl CoA. This, then, may replace acetyl CoA :ACP transacetylase as the first step in fatty acid synthesis. Malonyl ACP is formed by the activity of malonyl CoA :ACP transacetylase. The enzyme has been purified from a number of tissues, including barley chloroplasts (Hoj and Mikkelsen, 1982), avocado fruit (Caughey and Kekwick, 1982), soybean (Guerra and Ohlrogge, 1986), and leek (Lessire and Stumpf, 1983). Although in some species there appears only to be one isoform, in others there is evidence of tissue-specific isoforms. For example, in soybean two isoenzymes were found in the leaves but only one in the seeds (Guerra and Ohlrogge, 1986). In leek leaf tissue a comparison was made of the enzyme from epidermal and parenchyma cells (Lessire and Stumpf, 1983). Differences were found in the molecular weights of the enzymes from the two different cell types and it remains to be seen whether these are true isoenzymes. The next step in fatty acid synthesis, leaving aside the possibility that KAS I11 may be involved, is the condensation of acetyl and malonyl groups to yield acetoacetyl CoA. This is catalyzed by P-ketoacyl ACP-synthetase (KAS). The enzyme transfers acyl groups from a range of substrates. Two forms have been identified from higher plants; these differ in their substrate specificity. Synthetase I (KAS I) uses acyl ACPs in the range C2-C,4 (Shimakata and Stumpf, 1983); KAS I has recently been purified to homogeneity from oilseed rape (Mackintosh et al., 1989). Synthetase 2 (KAS 11) is only active with palmitoyl ACP or myristoyl ACP and is therefore necessary for stearate formation (Shimakata and Stumpf, 1982b,c). Two forms of P-ketoacyl ACP reductase that differ in their reductant requirement have been reported. In avocado mesocarp extracts (nonphotosynthetic) there are two distinct isoenzymes (Caughey and Kekwick, 1982). One has a requirement for NADPH as reductant and the other has a greater affinity for NADH. In safflower (Shimakata and Stumpf, 1982a), spinach (Shimakata and Stumpf, 1982d), and barley (Hoj and Mikkelsen, 1982) NADPH is the preferred reductant.

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The sixth enzyme in fatty acid synthesis, D-P-hydroxyacyl ACP dehydrase, has been purified to homogeneity from spinach (Shimakata and Stumpf, 1982d) and partially purified from safflower (Shimakata and Stumpf, 1982a). Only one form has, so far, been detected and this utilizes a range of 2-enoyl ACPs from C4 to CI6. Two forms of enoyl ACP reductase have been detected. Type I is specific for NADH (NADPH is ineffective) and uses crotonyl ACP as substrate (Shimakata and Stumpf, 1982d). Type I1 has a higher affinity for NADPH than NADH and uses 2-decenyl ACP as substrate. Both isoenzymes are present in castor bean, safflower, and rape seeds (Slabas et al., 1984), although only type I appears to be present in spinach leaves (Shimakata and Stumpf, 1982d) and avocado (Caughey and Kekwick, 1982). The enzyme has been localized, by immunogold labeling, in the chloroplasts of rape leaves (Slabas et al., 1992). It has been suggested that the source of NADH for the type I isoform in spinach chloroplasts is from electrons flowing through NADPH :ferredoxin reductase, which may also reduce NAD (Stumpf, 1987). The process of fatty acid synthesis may be regarded as a limited cycle of condensation, keto reduction, dehydration, and enoyl reduction, which may then be repeated to increase the chain length of the fatty acid until it is 16 carbons long. The palmitoyl ACP, thus formed, has three alternative fates. It may be elongated further to yield a C,, fatty acid, it may remain within the plastid to be used in glycerolipid biosynthesis, or it may be hydrolyzed to a free fatty acid, by a soluble acyl ACP thioesterase, and exported from the plastid.

3. Acyl Carrier Protein Acyl carrier protein is a small-11 kDa (Simoni et al., 1967)-acidic protein that serves as a cofactor in fatty acid biosynthesis. Evidence for its existence in higher plants came from the work of Overath and Stumpf (1964), who found that extracts of avocado mesocarp could be separated into two components. Each of the extracts was inactive alone, but when combined they were able to synthesize palmitic and stearic acids. One component was heat- and acid-stable and could substitute for E. coli ACP in a reconstituted system. Since this early characterization of ACP, the protein has been purified from a number of different plant sources (Ohlrogge, 1987). Complete amino acid sequences have been obtained, and cDNAs isolated from spinach leaves (Scherer and Knauf, 1987) and roots (Schmid and Ohlrogge, 1990), barley leaves (Hansen, 1987), and maize seedlings (Souciet and Weil, 1992), and from developing seeds of Brassica campestris (Scherer and Knauf, 1987) and B. napus (Safford et al., 1988). Genomic clones have been obtained from Arubidopsis (Lamppa and Jacks,

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1991 ; Post-Beittenmiller et al., 1989), barley (Hansen and von WettsteinKnowles, 1991), and B. napus (de Silva et al., 1990). There are a number of different types of ACP. Two forms, ACP I and ACP 11, have been found in leaves of barley (Hoj and Svendsen, 1984), spinach (Kuo and Ohlrogge, 1984), and castor bean (Ohlrogge and Kuo, 1985). Both isoenzymes are present in etiolated leaves of barley, although ACP I increases relative to ACP I1 during greening (Hoj and Svendsen, 1984). Both leaf isoforms are present within the chloroplast (Ohlrogge et al., 1979). Although ACP I is the major form in leaves, it is barely detectable in seeds, where there appears to be only one isoenzyme, ACP I1 (Ohlrogge and Kuo, 1985). There is also evidence of a third isoform, ACP 111, in barley leaves (Hoj and Svendsen, 1984). There appear to be both constitutive and tissuespecific forms of ACP. In spinach, a root ACP I1 isoform is also expressed in the leaves and seeds (Schmid and Ohlrogge, 1990). Recent studies of Arabidopsis suggest that there are several isoforms. From immunoblots of SDS-PAGE, three putative ACP isoforms were detected in Arabidopsis leaves and one isoform in roots (Batty and Ohlrogge, 1990). Analysis by urea-PAGE, however, detected four leaf isoforms, four root isoforms (identical to those in the leaf), and two seed isoforms. Thus, there would appear to be at least three constitutively expressed isoforms present in all tissue, as well as at least two seed-specific and one tissue-specific isoform each in roots and leaves (Hlosek-RadojciC et al., 1992). These authors propose that there is a multigene family of at least four, and possibly six, ACP genes in Arabidopsis. At present, it remains unclear why there are different ACP isoforms, particularly since they may all be located within the plastid. It is possible that they may be distributed between plastids in different types of cell, or that there are different sites within the plastid (Ohlrogge, 1987). Alternatively, the isoforms may relate to the different reactions in which ACP participates. Apart from the reactions of FAS, ACP is also involved in the desaturation of stearate, the release of oleate by acyl ACP hydrolase, and acyl group transfer to glycerol 3-phosphate and monoacylglycerol 3phosphate. Guerra et al. (1986) tested the hypothesis that ACP I and ACP I1 may be differentially involved in these reactions so that these pathways could be regulated by altering the relative expression or activity of one isoform relative to the other. The K , value of oleoyl ACP thioesterase was 10-fold higher for ACP I1 than for ACP I, whereas the acyl ACP glycerol-3-phosphate acyltransferase had a lower affinity for oleoyl ACP I than for oleoyl ACP 11. Both reactions involve the same substrate, oleoyl ACP, which is present in the plastid. Thus, differential expression of the ACP isoenzymes in the plastid could alter the proportion of oleate that is retained in the plastid for glycerolipid synthesis, or exported into the cytosol (Ohlrogge, 1987). Finally, multiple forms of ACP-and of other

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enzymes of FAS-may be required in order to serve the different roles of fatty acid synthesis in plants, such as oil accumulation in seeds, membrane formation, and the production of the cuticle by epidermal cells. 4. Sources of Acetyl CoA for Fatty Acid Biosynthesis The precursor for fatty acid biosynthesis in plastids is acetyl CoA and this does not readily cross the plastid membranes (Brooks and Stumpf, 1965). The pyruvate dehydrogenase complex (PDC), which forms acetyl CoA from pyruvate, has been detected in chloroplasts (Camp and Randall, 1985) and in nonphotosynthetic plastids (Rapp et af., 1987) as well as in the mitochondria (Miernyk et af., 1985, 1987). The activity of PDC in chloroplasts is much lower than that in the mitochondria (Williams and Randall, 1979) and an indirect pathway was proposed by Stumpf and co-workers, whereby acetyl CoA is generated within the mitochondria (Murphy and Stumpf, 1981; Liedvogel and Stumpf, 1982; Murphy and Walker, 1982). The acetyl CoA was purported to be hydrolyzed to acetate by a mitochondria1acetyl CoA hydrolase and the free acetate then moves, by diffusion (Jacobsen and Stumpf, 1972), into the chloroplasts. Acetyl CoA synthetase in the chloroplast stroma (Kuhn et af., 1981)then converts the acetate to acetyl CoA. Free acetate is an effective substrate for fatty acid biosynthesis in isolated spinach chloroplasts (Stumpf, 1987) and in nonphotosynthetic plastids isolated from pea roots (Sparace et al., 1988). Although acetyl CoA hydrolase has been found in isolated spinach leaf mitochondria (Liedvogel and Stumpf, 1982) the enzyme could not be detected in pea leaf mitochondria (Givan and Hodgson, 1983). The availability of free acetate to the chloroplast is also unclear. Whereas Kuhn et af. (1981) estimated acetate concentrations to be as high as 1 mM, Liedvogel (1985) detected only 0.065 mM free acetate in young spinach leaves. Steady-state concentrations do not in any way relate to the rate at which acetate is generated for utilization by the chloroplast and this still remains unkown. The “acetate pathway” has been criticized by Wood et af. (1992) on the grounds that it is inefficient, in requiring the hydrolysis and resynthesis of acetyl CoA. These authors propose that acyl groups are transferred as acylcarnitine derivatives (reviewed in Wood et af., 1992). Carnitine acyltransferases catalyze the conjugation of acyl CoAs to L-carnitine to form L-acylcarnitine: acyl CoA

+ L-carnitine

L-acylcarnitine + CoASH

The reaction is freely reversible. Whereas acyl CoAs do not readily cross membranes, acylcarnitines are transported via translocases, which act as carnitine/acylcarnitine antiporters. These have been studied in detail in

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mammalian systems (Pande, 1975; Pande and Parvin, 1980; Ramsay and Tubbs, 1975,1976)but have yet to be characterized in plant cells. Carnitine long-chain and short-chain (CAT) acyltransferases have been found in barley etioplasts (Thomas et al., 1982, 1983) and in pea chloroplasts (McLaren et al., 1985) as well as in plant mitochondria (Wood et al., 1983, 1984; Burgess and Thomas, 1986; Thomas and Wood, 1986; Gerbling and Gerhardt, 1988). The most convincing demonstration of the effect of the carnitine/acylcarnitine system on fatty acid biosynthesis has come from the work of Masterson et al. (199Oa) using isolated pea chloroplasts. These authors measured the rate of incorporation of I4C-labeled substrates into fatty acids. Acetyl CoA incorporation was negligible (25 pmol mg-’ chlorophyll min-’) in the absence of carnitine but in the presence of 1 mM L-carnitine the rate increased ninefold. The conclusion was that an external CAT was converting acetyl CoA to acetylcarnitine, which was then transported across the chloroplast envelope. L-Acetylcarnitine was incorporated into fatty acids at a far greater rate than was acetate, pyruvate, or citrate (Masterson et al., 1990a; Fig 5 ) , indicating that the “carnitine pathway” was the preferred route for fatty acid synthesis in pea chloroplasts. Inhibitor titrations, using D-carnitine and deoxycarnitine, showed that whereas these compounds decreased the rate of L-acetylcarnitine-dependent fatty acid synthesis, they were without effect on acetate incorporation into fatty acids (Masterson et al., 199Ob). Masterson et al. (1990a) proposed that 200

i

I

0

0.2

1

I

I

1

I

0.4

0.6

0.8

1.o

Substrate Concentration (mM) FIG. 5 Rate of incorporation of I4C-labeled substrates into fatty acids by isolated pea chloroL-Acetylcarnitine; (A) acetate; (0)pyruvate. [Redrawn from Masterson et al. plasts. (0)

(1990a,b), with permission.]

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acetyl CoA could be transferred from the mitochondria to support fatty acid synthesis within the chloroplasts. Acetyl CoA could be converted to acetylcarnitine via CAT present in the mitochondria1 matrix (Burgess and Thomas, 1986). Acetylcarnitine could then be transported out of the mitochondria on a carnitine :acylcarnitine translocator in the inner mitochondrial membrane and into the chloroplast on a similar translocator. There it could be converted to acetyl CoA by the chloroplastic CAT (McLaren et al., 1985). The translocators involved in this process have yet to be characterized but the scheme has a number of attractions. It is energetically more conservative than the “acetate pathway” and it is also more controllable. Whereas acetate is freely diffusable, the transport of acylcarnitines via specific translocators provides a means of controlling the rate of acyl group transfer between organelles. As an alternative to importing acyl groups, the plastid may generate its own acetyl CoA via plastidial PDC. The source of pyruvate for this reaction is glycolysis and there are some differences in the capacity of plastids to carry out this process. Mature chloroplasts may not have a complete glycolytic pathway, as phosphoglyceromutase may be absent (Stitt and ap Rees, 1979; Dennis and Miernyk, 1982). This would explain the very low rates of incorporation of 14C02into fatty acids in isolated chloroplasts (Givan, 1983). Triosephosphates could, however, be transported from the chloroplast, via the phosphate translocator, and converted to pyruvate by glycolytic enzymes in the cytosol. Pyruvate could then be moved back into the chloroplast either via a translocator, or by diffusion (Proudlove and Thurman, 1981). Nonphotosynthetic plastids from a range of species and organs contain the full glycolytic sequence of enzymes. Evidence comes from work on leucoplasts of developing castor oilseed (Simcox el al., 1977; Miernyk and Dennis, 1982, 1983) and of cauliflower (Journet and Douce, 1985), chromoplasts of daffodil flowers (Liedvogel and Kleinig, 1980), and plastids from safflower and linseed cotyledons (Browse and Slack, 1985). Plastids isolated from castor bean endosperm will incorporate hexose, phosphoglycerate, and pyruvate into fatty acids (Miernyk and Dennis, 1983), although it is not clear which substrate is actually imported from the cytosol in vivo. Further characterization of the translocators in nonphotosynthetic plastids is required.

5. Sources of ATP and Reductant for Fatty Acid Biosynthesis In chloroplasts, photosynthesis is the likely source of ATP and NADPH required for fatty acid biosynthesis. In nonphotosynthetic plastids NADPH could be generated by the oxidative pentose phosphate pathway, although there have been few studies directly related to this process. In

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nonphotosynthetic plastids with a complete glycolytic pathway some of the reducing power could come from the glyceraldehyde-3-phosphatedehydrogenase reaction. Fatty acid synthesis, from hexose or 3-phosphoglyceric acid, by isolated castor bean leukoplasts requires externally added ATP (Miernyk and Dennis, 1983). This requirement can be replaced by the addition of PEP, which generates ATP within the plastid via pyruvate kinase (Ireland et al., 1980; Boyle et al., 1990). The rate of fatty acid biosynthesis is much higher with internally generated ATP than when it is supplied externally (Boyle et al., 1990),indicating that adenylate transport may be limiting the flux under these conditions. 2-Phosphoglycerate will also generate ATP but at a slower rate than with PEP (Boyle et al., 1990). Similar results have been obtained with chromoplasts from daffodil flowers (Kleinig and Liedvogel, 1980). The incorporation of [14C]acetateinto fatty acids in isolated pea root plastids was found to be dependent on exogenously supplied ATP (Kleppinger-Sparace et al., 1992). This could be replaced, to some extent, by the addition of ADP alone, which is thought to generate ATP via a plastidial adenylate kinase. However, ADP was less effective than ATP, as the rate of fatty acid synthesis was only 24% of the ATP-stimulated rate. Components of the DHAP shuttle-DHAP, oxaloacetate, and Pi-gave approximately 40% of the control ATP-dependent rate and this was stimulated to 100% of the control by the addition of ADP. The authors concluded that ATP could be generated inside the root plastids by substrate level phosphorylation at phosphoglycerate kinase (KleppingerSparace et al., 1992). Thus, DHAP enters on the phosphate translocator and is converted to 3-phosphoglycerate, which may then be exported on the same translocator. Although, in this study, acetate was used as the substrate for fatty acid synthesis, the authors do not discount the possibility that pea root plastids may be capable of synthesizing fatty acids from glycolytic intermediates. E. Amino Acid Synthesis

Amino acids are the basic building blocks of proteins and are also a means of transporting nitrogen between tissues such as roots, leaves, and fruits. They are precursors in the synthesis of cofactors such as biotin, thiamine pyrophosphate, and coenzyme A and in the synthesis of chlorophyll and many other nitrogen containing compounds. Despite their obvious importance, our understanding of amino acid biosynthesis in plants is still far from complete, though in nearly all cases, their synthesis is carried out in plastids. It is not the intention here to go into the complexities of the biosynthesis of all the different families of protein and nonprotein amino acids. The subjects of plant amino transferases (Givan, 1980; Ireland and

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Joy, 1985), the biosynthesis of branched-chain amino acids, aromatic amino acids, histidine, arginine, and proline (Bryan, 1990), and the sulfur amino acids (Anderson, 1990) have been covered elsewhere, and in a recent review in this series (Sechley et al., 1992). Our aim here is to deal with recent developments in our understanding of those aspects of the assimilation of inorganic nitrogen into amino acids that take place in plastids.

1. Metabolic Sources of Ammonia for Biosynthesis a. Nitrate Assimilation The major turnover of inorganic nitrogen is through the routes of primary assimilation and photorespiratory reassimilation of ammonia in leaves. The principle source of inorganic nitrogen available to higher plants is nitrate, which is taken up by the roots and assimilated there or transported to leaves for assimilation. The topic of nitrogen assimilation has recently been covered (Sechley et al., 1992) and we will deal with it primarily from the point of view of the involvement of plastids and the interaction of plastidial nitrogen assimilation with other metabolic pathways.

The first step in the intracellular pathway of nitrate assimilation is catalysis by nitrate reductase. This substrate-inducible enzyme is a molybdoflavohaem-containing protein about which much is known at the biochemical level of understanding and about which we are discovering more at the molecular level of control (Kleinhofs and Warner, 1990). This reaction is confined to the cytoplasm or at the very least is located outside plastids. The product of the reaction, nitrite, then enters the plastid. The mechanism by which it does so has not been precisely determined. Osmotic swelling studies carried out with isolated chloroplasts suggest that the anion enters passively by diffusion (Heber and Purczeld, 1978). However, the concentrations of nitrite used in such experiments are unphysiologically high at 20 mM and may tell us little about the in uiuo situation, where concentrations of nitrite are more likely to be less than 1 mM. A suggestion has been made that there is a nitrite transporter in the envelope of pea chloroplasts (Brunswick and Cresswell, 1988) from studies using photosynthetic electron transport regulators. However, the uptake of nitrite was followed through its disappearance from the chloroplast suspension medium and it is not apparent how this could be distinguished from

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the process of nitrite reduction. Direct import of nitrite into the organelle was not determined. On entering the plastid, nitrite is reduced to ammonia by nitrite reductase. This enzyme exists as a monomer (molecular weight, 6O,OOO-64,OOO)and has been purified from a number of sources including leaves (Small and Gray, 1984; Ida and Mikami, 1986; lp et al., 1990) and roots (Ida et al., 1974; Bowsher et al., 1988). Nitrite reductase contains a sirohaem prosthetic group and a (4 Fe-4s) cluster at its active center. There are also reports that the native nitrite reductase may have a larger molecular weight of 85,000 and that a 24,000 M , component that binds ferredoxin is lost on purification (Hirasawa and Knaff, 1985). This matter remains unresolved, although the protein of M , 61,000 from a number of sources (see above) will also use ferredoxin as an electron donor, and the sequence derived from full-length clones suggests the same value (Back er al., 1988). A variety of environmental and developmental stimuli regulate expression of the nitrite reductase genes. Like nitrate reductase, the enzyme is induced in plants by the application of nitrate. Nitrite reductase cDNA has been cloned from maize (Lahners et al., 1988) and spinach (Back et al., 1988) and used to study the expression of the enzyme. Rothstein and co-workers have recently examined maize root, leaf, and cell suspensions and found that nitrite reductase mRNA is induced transiently after application of nitrate, with a half-life as short as 30 min (Kramer et al., 1989), although enzyme activity continues to increase long after this period. Induction of the enzyme in leaves is also apparently dependent on light as well as nitrate (Gupta and Beevers, 1983). In maize, although there was little diurnal fluctuation in the amount of nitrite reductase protein following de nuuu synthesis, when plants were grown under a 16-hr light/&hr dark regime, there was considerable cycling of the mRNA. Nitrite reductase mRNA increased considerably in the dark period, declining in the light period in both shoots and roots (Bowsher et al., 1991). This implies that the synthesis of message follows an endogenous diurnal rhythm, which can be partly disrupted by growing the plants in continuous darkness (Bowsher et al., 1991). By contrast to the synthesis of mRNA, nitrite reductase protein does not seem to show oscillations in activity, suggesting that there may be considerable post-transcriptional control of this enzyme and that it is relatively stable once made. The electron donor for nitrite is ferredoxin, which in leaves is reduced by the activity of photosystem I. This means that, potentially, nitrite reductase (and glutamate synthase, see later) could compete with the process of CO, reduction (Calvin cycle) for photochemically generated electrons. In C4species, this will not occur, as the Calvin cycle is confined to the bundle sheath cells, whereas nitrite reductase is probably confined to the mesophyll (Yamaya and Oaks, 1988; Hare1 et al., 1977). In C3species

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the potential for competition has been examined in spinach and soybean leaf chloroplasts (Robinson, 1986). Surprisingly,it appears that even in conditions of low light intensity where the supply of reductant for either NO2- or CO, is limiting, there is no inhibition of CO, fixation by nitrite, or of nitrite reduction by CO,. The reduction of nitrite in nonphotosynthetic plastids clearly depends upon the oxidation of photosynthate previously generated in the light and transported to the site of nitrogen assimilation. Reductant for nitrate assimilation in roots is generated through the oxidation of carbohydrate (Aslam and Huffaker, 1982). Since nitrite reductase is plastidial (Emes and Fowler, 1979a) questions arise concerning the form in which the reductant is channeled into the root plastid. The entire sequence of oxidative pentose phosphate pathway enzymes has been shown to be located in root plastids as well as the cytosol (Emes and Fowler, 1979b) and there is convincing evidence that it is the oxidation of glucose 6-phosphate via this pathway that provides reductant for the assimilation of nitrite (Emes and Fowler, 1983; Bowsher er al., 1989). The evidence for this arises from studies of purified pea root plastids and is twofold. First, glucose 6-phosphate and ribose 5-phosphate are able to support nitrite reduction in intact but not broken root plastids. Second, by following the release of CO, from specifically labeled carbon atoms of glucose 6-phosphate supplied to intact root plastids, there is a marked stimulation of CO, evolution from carbon atom one when nitrite is supplied at the same time. The latter is indicative of increased flux through the oxidative pentose phosphate pathway and is not brought about by the application of anions other than nitrite. That this stimulation of carbohydrate oxidation is due to a demand for reductant by nitrite reductase is shown by the observation that it is not observed if the latter enzyme has not been induced (Bowsher et al., 1989). In relation to this, glucose 6-phosphate has been shown to enter pea root plastids via a phosphate translocator (Borchert er al., 1989), supporting the view that this is a likely source of reductant for assimilatory processes in root plastids. However, the oxidative pentose phosphate pathway generates NADPH, which is not an electron donor for root or leaf nitrite reductase (Bowsher er al., 1988), raising the question of the nature of the immediate electron donor. A number of groups have shown that roots contain a protein capable of supporting either nitrite reduction or ferredoxin-dependent glutamate synthase (Suzuki et al., 1985; Ninomiya and Sato, 1984) and recently published amino acid composition data suggest that it is a different protein from leaf ferredoxin (Wada et al., 1986, 1989; Morigasaki et al., 1990). Using a pre-ferredoxin promoter from A. thuliuna to direct p-Glucuronidase (GUS) expression in transgenic tobacco, Weisbeek and co-workers (Vorst et al., 1990) have shown that there is a small amount of ferredoxin mRNA

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in roots. However, in Arabidopsis there appears to be only one gene copy for ferredoxin, whereas other studies have suggested that there are at least two ferredoxins in other species (Sakihama and Shin, 1987). Whatever the type of ferredoxin in roots, there is a need for an oxidoreductase capable of transferring electrons from NADPH to ferredoxin. Such a protein has been indicated in maize roots (Suzuki et al., 1985) and purified from bean hypocotyls (Hirasawa et al., 1990) and spinach roots (Morigasaki et al., 1990). The latter was shown to have an amino acid composition slightly different than that of its leaf counterpart. More significantly, N-terminal amino acid sequencing indicated that the root enzyme is possibly 12 amino acids shorter than its leaf counterpart, and of the 23 amino acids sequenced in the root enzyme, only 8 were invariant and conserved in the leaf protein. There seems to be a strong case for suggesting that the root protein is significantly different from leaf ferredoxin : NADPH oxidoreductase. Further characterization of this system is essential. An important consideration is the bioenergetics of this process since, in the leaf, transfer of reductant from NADPH to ferredoxin is considered inefficient, although this in itself may not be important since reduction of nitrite through glucose Qphosphate oxidation is functional in purified plastids (Emes and Bowsher, 1991). A final consideration here is the coordination of expression of such an electron transport system with the onset of nitrate assimilation. We have recently shown that a root ferredoxin and ferredoxin reductase activity are induced and present in pea root plastids during the induction of nitrite reductase (Hucklesby et al., 1990; C. G. Bowsher and M. J. Emes, unpublished results). In the absence of nitrate assimilation, root plastid ferredoxin could be detected in small amounts using a pea leaf ferredoxin antibody on Western blots. Some ferredoxin is required constitutively to support ferredoxin-dependent glutamate synthase, but considerably more is detectable in root plastids after the onset of nitrate assimilation, presumably to meet the demands of nitrite reductase. Molecular characterization of this induction is in progress in the author’s laboratory.

b. Photorespiration Photorespiration (Fig. 6 ) is the major source of ammonia in green leaves in the light. Rates of ammonia production from photorespiratory glycine metabolism have been estimated to exceed, by 10-fold, the rate of ammonia production from nitrite reduction (Keys et al., 1978). There is now considerable evidence, primarily from work with mutants, that reassimilation is via the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle in the chloroplast (see below; Sechley et al., 1992; Wallsgrove et a f . , 1992). Although photorespiration cannot be considered a unique property of the chloroplast, it is apparent from Fig. 6 that many of the reactions of the

195

METABOLISM AND DEVELOPMENT IN PLASTIDS

3. PGA

P-glycolate glycerate

4

glycerate

4

NH3

(2)

glycolate I

4

glycolate

%

02

NH3

FIG. 6 The photorespiratory nitrogen cycle. Numbers refer to the following enzymes: ( I ) Rubisco; (2) phosphoglycolate phosphatase; (3) glycolate oxidase; (4) catalase; ( 5 ) serine : glyoxylate aminotransferase; (6) glutamate :glyoxylate aminotransferase; (7) glycine decarboxylase; (8) serine hydroxymethyltransferase; (9) hydroxypyruvate reductase; (10) glycerate kinase; ( I 1) glutamine synthetase; (12) glutamate synthase. The exact stoichiometry

is not shown, for reasons of clarity.

196

M. J. EMES AND A. K. TOBIN

pathway occur within this organelle. The whole process involves reactions within the chloroplast, mitochondria, peroxisomes, and cytosol and, as will become apparent, this means that the development of the pathway requires coordinated biogenesis of organelles. The photorespiratory pathway has been discussed recently in this series (Sechley et al., 1992) and only those reactions that involve or interact with the chloroplast will be mentioned here. Photorespiration, which is a light-dependent uptake of 0, and release of CO,, arises from the reactions of Rubisco in the chloroplast. Rubisco has two distinct catalytic activities (see Section III,B,l), the carboxylase reaction, which is responsible for fixing C 0 2into two molecules of PGA, and the oxygenase reaction, which is a potentially wasteful reaction, as it produces only one molecule of PGA and also generates 2-phosphoglycolate, a compound that cannot be directly incorporated into the Calvin cycle. The relative rates of carboxylase and oxygenase activities depend on the relative concentrations of oxygen and CO, at the active site of Rubisco (Keys, 1986). In C, and, to some extent, in CAM plants decarboxylation reactions lead to elevated C 0 2concentrations, which consequently reduce photorespiratory flux. In C, plants, temperature may alter the relative rates of the two Rubisco reactions by changing the relative solubilities of CO, and 0, (Keys, 1986; Fuhrer and Erismann, 1984). The subject of CO, diffusion and transport into the chloroplasts has been reviewed recently (Machler el al., 1990). The generation of 2-phosphoglycolate, via Rubisco oxygenase, thus presents a potential drain of carbon from the Calvin cycle and photorespiration may be viewed as a wasteful process. Attempts to reduce this activity, by site-directed mutagenesis of Rubisco, have so far been unsuccessful, as the carboxylase and oxygenase activities share the same active site and indeed react with the same enediol intermediate (Keys, 1986; Ogren, 1984). There are, however, differences in the CO, : 0, “specificity factor” of Rubisco enzymes from different species ( Jordan and Ogren, 1981; Parry et al., 1989) and this offers hope for future improvement. The reactions of the photorespiratory pathway perform a “scavenging” function in returning the carbon lost at the Rubisco oxygenase step to a form that can be utilized by the Calvin cycle (Fig. 5 ) . Three of the four carbons that leave the chloroplast, as two molecules of glycolate (2 x 2C), are returned to the chloroplast as glycerate (3C). Carbon is lost at the mitochondria1 stage, during the oxidative decarboxylation and deamination of glycine via the glycine decarboxylase complex (GDC). This releases CO, and NH,, both of which may be reassimilated in the chloroplast (see below). The significance of this scavenging process becomes apparent when the pathway is blocked, either by chemical inhibition or in mutants that lack one of the enzymes. Chemical inhibition of photorespiratory

METABOLISM AND DEVELOPMENT IN PLASTIDS

197

enzymes leads to a decrease in the rate of photosynthesis (Lawyer and Zelitch, 1979; Jenkins et al., 1982; Ikeda et al., 1984) and many of these inhibitors are effective herbicides. In many, but not all, photorespiratory mutants C 0 2 fixation rates decrease on transfer of plants from 1% O2 to air (Blackwell et al., 1988). It is generally agreed that the main cause of photosynthetic inhibition, when photorespiration is blocked, is the depletion of Calvin cycle intermediates (Blackwell et al., 1988). Another potential cause is the accumulation of photorespiratory intermediates, such as ammonia, which may uncouple the chloroplasts. There is evidence to suggest that this does not occur in v i m . In barley mutants deficient in either GS or GOGAT there is no direct correlation between ammonia accumulation and photosynthetic inhibition (Blackwell et al., 1988). In wheat, the addition of ammonium chloride through the transpiration stream results in a much greater accumulation of ammonium than occurs in similar leaves treated with the GS inhibitor L-methionine sulfoximine, and yet there is no effect on photosynthetic activity (Walker et al., 1984). It seems likely that ammonia is sequestered in the vacuole under these conditions. The inhibition of photosynthesis that results from inhibition of photorespiratory ammonia assimilation is attributable to the lack of carbon recycling through the peroxisomal aminotransferase reactions (Fig. 5 ; Blackwell er al., 1988). Following the formation of 2-phosphoglycollate by Rubisco, any disruption to the photorespiratory cycle clearly leads to deleterious effects on plant metabolism. It is therefore essential that the enzymes of the pathway are always sufficiently active to ensure efficient recycling of carbon and nitrogen. Studies on the development of photorespiration during leaf growth show that there is a high degree of coordination of enzyme activities (Fig. 7; Tobin et al., 1988, 1989; Tobin and Rogers, 1992). In light-grown grasses, such as wheat, growth from a basal meristem results in a natural developmental gradient of increasing cell and chloroplast development toward the tip. At the leaf base the plastids are relatively immature, with unstacked thylakoids, and no detectable rates of photosynthesis (Tobin et al., 1988). The rate of photosynthesis and photorespiration increases with the maturity of the leaf cells (Tobin et al., 1988)and there is a concomitant increase in enzyme activity. Rubisco protein per mesophyll cell, for example, increases 20-fold during primary leaf development in wheat (Dean and Leech, 1982). Synthesis of the large and small subunits of Rubisco is highly coordinated (Dean and Leech, 1982) and there appears to be no accumulation of free subunits (Nivison and Stocking, 1983). The 46- and 41-kDa Rubisco activase polypeptides develop parallel with the Rubisco subunits in barley leaves (Zielinski et al., 1989). Rubisco levels continue to increase in the cell after the chloroplasts have ceased to divide; hence, there is an increase in the concentration of Rubisco in the chloroplasts

198

M. J. EMES AND A. K. TOBIN

V)

a

b

r;

40

20 0

2

4

6

8

10

-

Distance from Leaf Base (cm) FIG. 7

Changes in photorespiratory enzyme activities during development of a light-grown primary wheat leaf. (U) Chloroplast glutamine synthetase activity (GS,); (m) Glycollate oxidase; (0) glycine decarboxylase (actual values X 10 to aid comparison). Data from Tobin e t a / . (1988, 1989).

during leaf development (Dean and Leech, 1982). The increase in activity of photorespiratory enzymes in the chloroplast (GS,), peroxisome (glycolate oxidase), and mitochondrion (GDC) closely parallels the change in Rubisco levels (Fig. 7; Tobin et af., 1985, 1988; Rogers et af., 1991). GS, activity per cell increases more than 50-fold and, as with Rubisco, its concentration within the chloroplast increases with development (Tobin et al., 1985). Thus, although present in different subcellular compartments, there appears to be a coordinated development of photorespiratory enzymes. The mechanism involved in this coordination has yet to be elucidated, although the expression of all of these enzymes is considerably influenced by light (Canovas et af., 1986; Evstigneeva et af., 1981; Guiz et al., 1979; Mann et af., 1979; Nishimura et af., 1982; Hondred et af., 1987; Betsche and Eking, 1989; Day et al., 1985; Walker and Oliver, 1986). Phytochrome may also be involved (Morohashi, 1987; Hecht et af., 1988; Kansara et al., 1989). 2. Ammonia Assimilation

a . Ammonia Assimilation in Chloroplasts The assimilation of ammonia, from whatever source, in photosynthetic cells is via the GWGOGAT cycle (Miflin and Lea, 1980; for recent reviews, see Lea et af., 1990; Sechley et

METABOLISM AND DEVELOPMENT IN PLASTIDS

199

al., 1992). There are two isoenzymes of GS in leaves: GS, in the chloroplast and GS, in the cytosol (Mann et al., 1979). The relative proportion of GS isoenzymes in leaves differs between species (McNally et al., 1983) and is influenced by light (Hire1 et a f . , 1982) and by plant development (Tobin et al., 1985). In pea leaves, growth at high concentrations of CO, was reported to reduce the expression of GS, mRNA (Edwards and Coruzzi, 1989). No measurement was made of either GS activity or protein in this study and there appears to be no change in the level of either of these in barley grown at high concentrations of CO, (R. M. Wallsgrove, unpublished; Wallsgrove et a/., 1992). It is important to note that, as there are strong developmental influences on the levels of photorespiratory enzymes, including GS2,in leaves (see previous section) any effect of high C 0 2 on expression may be an indirect effect resulting from a change in plant growth. Two GOGAT isoenzymes are also present in leaves. One is Fd-dependent and the other is NADH-dependent. Both are localized in the chloroplast (Wallsgrove et al., 1979). The Fd-dependent GOGAT is by far the most active isoform in light-grown leaves (Matoh et a/., 1979; Wallsgrove et al., 1982; Matoh and Takahashi, 1982). In young, light-grown pea seedlings, NADH-GOGAT is the predominant form but the relative activity of Fd-GOGAT increases rapidly within the first few days of seedling growth and, after 17 days, represents 97% of total GOGAT activity (Matoh and Takahashi, 1982). Mutants lacking either GS2 and/or Fd-GOGAT have been isolated in Arabidopsis (Somerville and Ogren, 1980) and barley (Kendall et al., 1986, 1987; Blackwell et al., 1987,1988). All of the evidence from labeling studies and analysis of these mutants indicates that most, if not all, ammonia assimilation in leaves occurs within the chloroplast (Lea et al., 1990). There are exceptions. Some plants lack GS, (McNally et al., 1983) and would therefore assimilate ammonia in the cytosol, via GS,. In these species the glutamine formed in the cytosol would then have to be transported into the chloroplast for further reaction with GOGAT. Yu and Woo (1988) have identified a specific glutamine translocator on the inner chloroplast envelope and have proposed a three-translocator model to account for the participation of GS, in the photorespiratory ammonia cycle.

b. Ammonia Assimilation in Nonphotosynthetic Plastids In nonphotosynthetic plant material, such as roots, root-nodules, and plant cell cultures, the main source of ammonium is not photorespiration but processes of nitrate assimilation, nitrogen fixation, deamidation, and secondary metabolism (e.g., the activity of phenylalanine ammonia lyase). All the evidence for ammonia assimilation in such systems is in favor of the operation

200

M. J. EMES AND A.

K. TOBIN

of the glutamine synthetaselglutamate synthase pathway. Isoforms of both enzymes have been shown to exist but their intracellular distribution between the compartments of the cytosol and plastid are apparently in general contrast to the situation in leaves. Earlier studies had shown that a small proportion of cellular glutamine synthetase was associated with plastids of pea roots (Miflin, 1974; Emes and Fowler, 1979a). However, by far the greater proportion (90%) was found to be cytosolic, and other cell-fractionation studies with a wide range of species suggested that GS was absent from root plastids (Suzuki et al., 1981). Purification of glutamine synthetase by DEAE anion exchange chromatography revealed only one form in root extracts, corresponding to the cytosolic form in leaves (Mann et al., 1979). However, a number of recent observations suggest that there may be a genuine GS isozyme within nonphotosynthetic plastids bearing some relationship to the process of nitrate assimilation. Emes and Fowler (1983)demonstrated that GS activity, measured in pea root plastids by either the transferase or semibiosynthetic assay, increased during the induction of nitrate assimilation. This has recently been confirmed by Vezina and Langlois (1989), who have demonstrated the presence of a 44-kDa subunit of GS in pea root plastids in addition to a cytoplasmic 38-kDa polypeptide. The abundance of the 44-kDa protein in plastids increased markedly after pretreatment of roots with nitrate. In nongreen rice cell cultures plastidial GS, identified as a 44-kDa polypeptide, increased in response to nitrate, whereas the cytosolic 41-kDa GS protein declined over a time period of 72 hr (Hayakawa et al., 1990). Vezina et al. (1987) have demonstrated that there are two isoenzymes of GS in pea and alfalfa roots that are separable by DEAE chromatography and that the ratio of synthetase : transferase activity is different for the two enzymes. In etiolated seedlings of tomato only GS, could be detected initially. After exposure of above-ground parts to 40 hr of continuous light, this form apparently declined and was replaced by GS, in roots, although whether this was actually located in plastids was not determined (Galvez et al., 1990). A minor plastidic band of GS has also been detected in 2-day-old seedlings of P. vulgaris (Bennett and Cullimore, 1989). If roots lack plastidial GS then ammonia, produced by nitrite reduction, would have to leave the organelle and reenter as glutamine in order for the glutamate synthase reaction to occur. This presumes that glutamate synthase in roots is entirely plastid located as in the leaf. Glutamate synthase exists in two forms in roots-NADH and ferredoxin dependent. In nonnodulated root enzymes there appears to be a small constitutive level of NADH-glutamate synthase, whereas the ferredoxin-dependent form increases substantially during root development (Matoh and Takahashi, 1982). There seems to be general agreement that the ferredoxin- and NADH-dependent enzyme

METABOLISM AND DEVELOPMENT IN PLASTIDS

201

is located in nonphotosynthetic plastids (Emes and Fowler, 1979a; Suzuki et al., 1981). By contrast the NADH form in lupin nodules is cytosolic (Boland and Benny, 1977), whereas in nodules of P. vulgaris there are two NADH forms, both of which are plastid located and one of which increases during nodulation (Chen and Cullimore, 1988; Chen ef al., 1990). Until we have a more detailed picture of precisely which cells in a particular tissue express these enzymes, it will prove difficult to rationalize the significance of their intracellular localization. For example, in developing maize kernels, GS is found in the maternal pedicel region, whereas NADH- and ferredoxin-dependent GOGAT were located in the endosperm (Muhitch, 1991). F. Pathways of Carbohydrate Oxidation

In other organisms, the ubiquitous pathways of glycolysis and the oxidative pentose phosphate pathway (OPPP) are not associated with any membrane-bound organelle and it was assumed that plants would follow this pattern. However, it has become increasingly apparent that plant cells are unique in having part or all of these pathways duplicated in plastids as well as cytosol (Dennis and Miernyk, 1982). A number of different approaches have been used to ascertain whether all or part of glycolysis and the OPPP exist within nonphotosynthetic plastids and chloroplasts. Answers to this question are by no means clear-cut and vary depending on a number of factors, which include plant species and the type of tissue from which the plastids are isolated. In addition, the developmental stage at which the tissue is examined will affect the levels of glycolytic and OPPP enzymes detected in the plastids. Table V summarizes the results of some of the studies that have examined the intracellular location of the enzymes of glycolysis. It is clear that some plastids, e.g., chloroplasts of pea, lack a complete glycolytic sequence, whereas others, e.g., developing castor bean endosperm, have been shown to possess all the intermediate steps from glucose to oxidation of pyruvate. Likewise, some plastids have a complete OPPP, e.g., in pea roots (Emes and Fowler, 1979b), whereas other do not (Frehner et al., 1990) and lack glucose-6-phosphate dehydrogenase. All plastids examined seem to possess the upper half of glycolysis, at least from glucose 6-phosphate metabolism as far as the formation of triose phosphate. However, the specific activities and proportions of enzymes in the lower half of glycolysis are generally much lower, and in particular the demonstration of the presence of phosphoglyceromutase within an organelle has been problematical (Stitt and ap Rees, 1979). The difficulty with a negative result is that there is always room for doubt, as artifacts sometimes arise from the way the experiments are performed.

TABLE V Location of Glycolytic Enzymes in Plastids

Species/tissue Wheat endosperm Soybean suspension cultures Developing castor endosperm Germinating castor endosperm Sycamore suspension cultures Cauliflower buds Pea leaf Spinach leaf

Developing pea cotyledons Pea roots

H / F HPI PGM PFK Aid TPI N D + ND +

+

+ + +

+

+

+ ND

GAPDH

+ (NAD) + (NAD)

PGK PGaM Enol PK PDC

+

+

ND

ND

+

F

+

+

+

+

+ +

+

-

+

+

+

+

+

+

H

+

+

+

+

4

H

+

+

+

+

-

+

+

+

+

F

+

+

-

+

Miernyk and Dennis (1982, 1983); Simcox er al. (1977) ND Nishimura and Beevers (1979)

+

+

+

ND Frehner et a / . (1990)

+

+

+

ND

+

+

+ +

+

+

+

-

+ (NADP")

+

+

+

+

N D +

+

+

+

ND

+ (NAD)

+

+

+

+

+ (NAD)

+

-

+

+

+ + ND Entwistle and ap Rees (1988) ND ND ND MacDonald and ap Rees (1983) +

+

+ (NADP)

Reference

ND

+

Journet and Douce (1985) Stitt and ap Rees (1979); Stitt er a1 (1978); Elias and Givan (1979) ND ND ND Miihlbach and Schnarrenberger (1978); Schnarrenberger and Oeser (1974); Kelly and Latzko (1977); Liedvogel (1985) + + + Denyer and Smith (1988, and personal communication) + + + Bowsher et al. (1989); Trimming and Emes (1992)

Note. + , present; -, not found; ND, not determined. Abbreviations: H/F, hexokinase/fructokinase; PGM, phosphoglucomutase; PGaM, phosphoglyceromutase; HPI, hexosephosphate isomerase; PFK, phosphofructokinase; Ald, aldolase; TPI, triosephosphate isomerase; GAPDH, glyceraldehyde phosphate dehydrogenase (NAD and NADP dependent); PGK, phosphoglycerate kinase; Enol, enolase; PK, pyruvate kinase; PDC, pyruvate dehydrogenase complex. llAssurned present as part of functioning Calvin cycle.

METABOLISM AND DEVELOPMENT IN PLASTIDS

203

Early reports (Simcox et al., 1977) suggested that castor bean plastids lacked glucose-6-phosphate dehydrogenase. However, subsequent studies (Satoh et al., 1983) showed that the enzyme was indeed present in these organelles and that the reason it had not been detected originally was because of the sensitivity of the enzyme to low temperature storage and detergents. There are also clear differences in tissue complements of plastidic enzymes. Dennis et al. (1991) reported that the levels of plastid enolase in the castor oil plant varied, depending on the tissue and its age. Plastidic enolase from castor oil seed increased during development to reach a maximum of 30% of the total cellular activity at the time of greatest oil synthesis. In the germinating seed, only 10% of activity was found in plastids; none was found in mature leaf chloroplasts and 7% in chloroplasts of immature leaves and root plastids. The regulation of these pathways in plastids is not well understood with the possible exception of carbohydrate oxidation in chloroplasts. Here it is apparent that in order to avoid futile cycling of substrates in the light, both glycolysis and the OPPP are restricted through mechanisms of light inactivation involving changes in pH, stromal Mg2+,NADPH/NADP+, and oxidationheduction of disulfide bridges (Heuer et al., 1982; Srivastava and Anderson, 1983), although transitory starch degradation can occur in the light, implying that such controls can be overridden (Stitt and Heldt, 1981). The functions of plastidic glycolysis and the OPPP are to provide NAD(P)H and ATP for biosynthetic reactions such as fatty acid synthesis (Dennis and Miernyk, 1982; see Section 111,DS). There is also good evidence that the OPPP in root and other nonphotosynthetic plastids functions to supply reductant for nitrogen metabolism (see Section III,E,l) and probably for fatty acid synthesis (Simcox et al., 1977) and is likely to fulfil similar functions in chloroplasts of darkened leaves. The presence of plastidic pyruvate dehydrogenase complex suggests that glycolysis may also lead to the generation of acetyl CoA for fatty acid synthesis in these organelles (Denyer and Smith, 1988; see Section III,D,5). Glycolysis and the OPPP in chloroplasts are involved in the breakdown of starch, which is hydrolyzed in the dark (Stitt and Heldt, 1981). Glycolysis is also present in its entirety in amyloplasts of developing wheat endosperm, which are organelles committed to synthesis of starch, not breakdown (Entwistle and ap Rees, 1988), though the function of glycolysis in these organelles is not readily apparent. There is clearly a need for much greater effort in establishing the inter- and intracellular distribution and operation of both these major pathways of carbohydrate oxidation in a wider range of species and in determining their significance and control.

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M. J. EMES AND A. K. TOBIN

IV. Summary In conclusion, plastids represent a unique organelle capable of great diversification in terms of physical and genetic structure and metabolism both within and between species. Inside a single plant many types of plastid may be found arising from a common progenitor. The basis of this differentiation remains poorly understood but it is clear that even in adjacent cells plastids may differ in their enzyme/protein content. Within individual tissues and organs a great deal more study is required on this aspect using techniques such as immunogold localization of proteins and in situ DNA hybridization to determine which plastid proteins are being expressed. Without this our biochemical appreciation of plastid metabolism in uiuo will be hampered. The study of the metabolism of nonphotosynthetic plastids is receiving increasing attention, particularly as the major primary products of biosynthesis and yield for human consumption-lipids, amino acids, and starch-are all made within these organelles in storage organs and seeds. It is hoped that the present review, while summarizing our current understanding in these areas, will also have provided suggestions for future exploration.

Acknowledgments We thank the Royal Society (University Research Fellowship to A. K. Tobin), the Agricultural and Food Research Council, and the Science and Engineering Research Council for financial support and Ms M. A. Cooper for drawing the figures.

References Alban, C., Joyard, J., and Douce, R. (1988). Plant Physiol. 88, 709-717. Anderson, J. W. (1990). I n “The Biochemistry of Plants” (B. J. Miflin and P. J. Lea, eds.), Vol. 16, pp 327-381. Academic Press, London. Andreasson, L.-E., and Vanngard, T. (1988). Annu. Rev. Plant Physiol. Plant Mol. Biol. 39, 379-41 1. Andrews, J., and Ohlrogge, J . (1990). I n “Plant Physiology, Biochemistry and Molecular Biology” (D. T. Dennis and D. H. Turpin, eds.), pp. 339-352. Longman, Harlow, England. Andrews, T. J., and Lorimer, G. H . (1987). I n “The Biochemistry of Plants” (M. D. Hatch and N. K. Boardman, eds.), Vol. 10, pp. 131-218. Academic Press, Orlando, FL. a p Rees, T., Entwistle, T. G., and Dancer, J. E. (1991). I n “Compartmentation of Plant Metabolism in Non-Photosynthetic Tissues” (M. J. Emes, ed.), pp. 95-1 10. Cambridge Univ. Press, Cambridge. Aslam, A., and Huffaker, R. C. (1982). Plant Physiol. 70, 1009-1013. Back, E., Burkhart, W., Mayer, M., Privalle, L., and Rothstein, S. (1988). Mol. Gen. Genet. 212,20-26.

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Thomas, D. R., Noh Hj Jalil, M., Cooke, R. J., Yong, B. C. S., Ariffin, A., McNeil, P. H., and Wood, C. (1982). Planta 154, 60-65. Thomas, D. R., Noh Hj Jalil, M., Ariffin, A., Cooke, R. J., McLaren, I., Yong, B. C. S., and Wood, C. (1983): Planta 158, 259-263. Thompson, J. A. (1980). Z. Naturforsch. B: Anorg. Chem., Org. Chem. 35B, 1101-1103. Thompson, L . W., and Zalik, S. (1981). Plant Physiol. 67, 655-661. Thornson, W. W., and Whatley, J. M. (1980). Annu. Rev. Plant Physiol. 31, 375-394. Thornson, W. W., Lewis, L. N., and Coggins, C. W. (1967). Cytologia 32, 117-124. Tiffany, L. H. (1951). In “Manual of Phycology” (G. M. Smith, ed.), pp. 307-308. Ronald Press, New York. Tippetts, M. T., Robertson, D. L., and Smith, M. A. (1991). Mol. Cell. Biochem. 100,61-70. Tobin, A. K., and Rogers, W. J. (1992). In “Plant Organelles: Compartmentation of Metabolism in Photosynthetic Cells” (A. K. Tobin, ed.), pp. 293-323. Cambridge Univ. Press, Cambridge. Tobin, A. K., Ridley, S. M., and Stewart, G. R. (1985). Planta 163, 544-548. Tobin, A. K., Sumar, N., Patel, M., Moore, A. L., and Stewart, G. R. (1988). J. Exp. Bot. 39, 833-843. Tobin, A. K., Thorpe, J. R., Hylton, C. M., and Rawsthorne, S. (1989). Plant Physiol. 91, 1219-1225. Trimming, B. A., and Emes, M. J. (1992). Submitted. Turner, S., Burger-Wiersma, T., Giovannoni, S. J., Mur, L. R., and Pace, N. R. (1989). Nature (London) 337, 380-382. Tyson, R. H., and ap Rees, T. (1988). Planta 175, 33-38. Urbach, E., Robertson, D. L., and Chisholm, S. W. (1992). Nature (London) 355,267-270. van der L e i , F. R., Visser, R. G. F., Poustein, A. S., Jacobsen, E., and Veenstra, W. J. (1991). Mol. Gen. Gene?. 228, 240-248. Van Valen, L. M., and Maiorana, V. C. (1980). Nature (London) 287, 248-250. Vezina, L. P., Hope, W. J., and Joy, K. W. (1987). Plant Physiol. 83, 58-62. Vezina, L. P . , and Langlois, J. R. (1989). Plant Physiol. 90, 1129-1 133. Viola, R., Davies, H. V., and Chudeck, A. R. ( 1 9 9 1 ) . Plunru 183, 202-208. Visser, R. G. F., Somhorst, I., Kuipersd, G. J., Rouys, N. J., Feenstra, W. J., and Jacobsen, E. (1991). Mol. Gen. Genet. 225, 289-296. Vorst, 0.. van Dam, F., Oosterhoff-Teertstra, R., Smeekens, S., and Weisbeek, P. (1990). Plant Mol. Biol. 14, 991-999. Vu, J. C. V., Allen, L. H., and Bowes, G. (1983). Plant Physiol. 73, 729-734. Wada, K., Ouda, M., and Matsubara, M. (1986). Plant Cell Physiol. 27, 407-415. Wada, K., Ouda, M., and Matsubara, H. (1989). J. Biochem. (Tokyo) 105, 619-625. Walker, D. A. (1976). In “Encyclopedia of Plant Physiology, New Series’’ (C. R. Stocking and U. Heber, eds.), Vol. 3, pp. 85-136. Springer-Verlag, Berlin. Walker, J . L., and Oliver, D. J. (1986). Arch. Biochem. Biophys. 248, 626-638. Walker, K. A., Keys, A. J., and Givan, C. V. (1984). Plant Physiol. 75, 60-66. Wallsgrove, R. M., Lea, P. J., and Miflin, B. J. (1979). Plant Physiol. 63, 232-236. Wallsgrove, R. M., Lea, P. J., and Miflin, B. J. (1982). Planta 154,473-476. Wallsgrove, R. M., Baron, A. C., and Tobin, A. K. (1992). In “Plant Organelles, Compartmentation of Metabolism in Photosynthetic Cells” (A. K. Tobin, ed.), pp. 79-96. Cambridge Univ. Press, Cambridge. Walsh, M. A., Rechel, E. A., and Popovich, T. M. (1980). Am. J. Bor. 67, 833-837. Walsh, M. C. Klopfenstein, W. E., and Harwood, J. L. (1990). Phytochemistry 29, 3797-3799. Webber, A. N., Hird, S. M., Packmann, L . C., Dyer, T. A., and Gray, J. C. (1989). Plant Mol. Biol. 12, 141-151.

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Biomineralization and Eggshells: Cell-Mediated Acellular Compartments of Mineralized Extracellular Matrix Jose L. Arias: David J. Fink: Si-Qun Xiao: Arthur H. Heuer! and Arnold I. Caplad *Department of Animal Biological Sciences, Faculty of Veterinary Sciences, University of Chile, Casilla 2, Santiago, Chile TCollaTek, Inc., Columbus, Ohio 43201 *Department of Materials Science and Engineering, Case Western Reserve University, Cleveland, Ohio 44106 §Skeletal Research Center, Department of Biology, Case Western Reserve University, Cleveland, Ohio 44106

Introduction

The eggshell is a microenvironmental compartment for housing developing embryos of a number of species and, in this respect, constitutes a widespread evolutionary strategy across phylogeny. This unique microenvironment not only provides physical protection to the embryo, but also regulates gas, water, and ionic exchange (Romanoff and Romanoff, 1949; Wangensteen and Rahn, 1971; Wangensteen et al., 1971), and in some calcareous eggshells, such as those of aves and some reptiles, provides a source of calcium for the formation of the embryonic skeleton (Johnston and Comar, 1955; Tyler and Simkiss, 1959; Romanoff, 1967; Simkiss, 1967; Coleman and Terepka, 1972; Crooks and Simkiss, 1974; Dieckert et al., 1989). In this regard, it has not been possible to obtain high degrees of viability or complete embryonic development in shell-less culture of chicken embryos (Vollmar, 1936; Romanoff, 1943; Boone, 1963; Ramsey, 1970; Elliott and Bennett, 1971; Comer and Richter, 1973; Dunn, 1974; Auerbach et al., 1974; Dunn and Boone, 1975, 1976, 1977, 1978; Tuan, 1980; Dunn et al., 1981; Rowlett and Simkiss, 1987; Dugan et al., 1991; Miura et al., 1991). Not all eggshells are calcified, and calcification is not an exclusive privilege of the upper phyla. The eggshells of approximately half of the Internotionnl Reuiew of Cytology, Vol. 145

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land snail species are calcified (Meenakshi et al., 1974a; Tompa, 1976, 1977;Meenakshi and Watabe, 1977;Watabe and Dunkelberger, 1979)and, among the amniotes, it is possible to describe three groups of eggshells as related to the extent of mineralization (Hirsch and Packard, 1987): 1. Soft-, parchment-, or membrane-like eggshells. With minute amounts of granular calcite, these eggshells are characteristic of snakes, most lizards, and monotremes. Because of their low quantities of mineral, only a few fossil specimens have been identified as belonging to this category (Romer and Price, 1939; Kitching, 1979;Sexton et af., 1979;Heulin, 1990). 2. Eggshells with a pliable calcareous layer. Eggshells of most contemporary turtles, of the tuatara, and of some fossil chelonians belong to this group (Packard er al., 1982; Hirsch, 1983). There is a clear demarcation, in these eggshells, between the thick organic membrane of the inside and the thinner, external, crystalline layer formed by loosely opposed columns of mineral (Hirsch and Packard, 1987). 3. Eggshells with a rigid calcareous layer. Eggshells of all crocodiles, all birds, some turtles and geckos, and all identified dinosaur eggshells belong to this group (Erben, 1970; Ferguson, 1982; Ewert et af., 1984; Hirsch and Packard, 1987). The crystalline layer is formed by tightly abutted columns of mineral that are normal to the eggshell surface. Although other reviews have focused on the formation, structure, organization, or chemical composition of eggshells (Frobose, 1928; Simkiss, 1961, 1967, 1968, 1975; Schmidt, 1962; Wilbur and Simkiss, 1968; Tyler, 1969; Simkiss and Taylor, 1971; Krampitz and Witt, 1979; Board, 1982; Leach, 1982; Parsons, 1982; Schleich and Kaestle, 1988;Arias and Fernandez, 1989; Simkiss and Wilbur, 1989), the present review attempts to provide an integrated summary of the cell biology, morphological organization, crystallography, chemical composition, process of mineralization, and biological function of avian eggshells. In addition, this review discusses emerging concepts of biomineralization and speculates about the mechanism of eggshell assembly and its implications for fabrication of polymer-ceramic composites.

II. Structural Organization and Composition of the Avian Eggshell

The first structural characterization of the avian eggshell was done in the middle of the nineteenth century. The pioneering studies by Wilhelm von Nathusius showed that the eggshell is an intricate, highly ordered structure

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composed of multiple layers of membranes and calcified matrix (Tyler, 1964). During this era, Dickie (1848) inaccurately suggested that the cuticle which covers the egg was an epithelium over a basement membrane. Perhaps the most visionary study of the eggshell was that of Purkyne (1855), who was able to draw with extraordinary preciseness that which can be now observed only by scanning electron microscopy. From information derived from microscopic studies of the developing eggshell and of partially demineralized shells, and by analysis of chemical composition, the eggshell can be characterized as a multilayered, polymer-ceramic composite (Fig. 1). Superficial observation following a simplified dissection allows the recognition of only three main layers: an outer mucous layer, an intermediate calcified zone, and an inner fibrous membrane layer. A more detailed and complex analysis of the eggshell reveals a fourth discrete component as illustrated in Fig. 1. This (mammillary) layer is difficult to define clearly because it overlaps both the inner portion of the calcified layer and the outer region of the membranous layer. The Nomina Anatomica Avium (Baumel et al., 1979)recognizes only three layers: cuticula, testa, and membrana testae. However, for those authors, testa comprises the stratum mamillarium and spongiosum. Crystallographic analysis of the calcified layer differentiates three sublayers in the calcified layer (see below). In this review, the four classical layers will be considered: ( a ) shell membranes; ( b ) the mammillary layer; (c) palisades; and ( d )cuticle. When pertinent, subdivisions and synonymy of the layers will be indicated.

palisade (spongy)

mammillary or

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A. Shell Membrane Layer

The shell membranes (synonym: membranae testae) are the most internal layer of the eggshell and are formed by two nonmineralized fibrillar sublayers, the outer, -48-pm-thick membrana testae externa, and the inner, -22-pm-thick membrana testae interna or putaminis. These two sublayers strongly adhere to each other around almost the entire inner surface of the shell, but are separated at the obtuse side of the egg defining the boundaries of the air chamber. The inner sublayer has thinner fibers than the outer layer (Fig. 2a). Fibers in both layers have a central core with a smooth and homogeneous outer sheath due to the presence of an amorphous material that has been referred to as mantle (Masshoff and Stolpmann, 1961; Simons and Wiertz, 1963; Bellairs and Boyde, 1969; Hoffer, 1971; Draper et al., 1972). Some authors were able to dissect more than two sublayers of the shell membranes (Moran and Hale, 1936; Simons and Wiertz, 1963), but these dissections may be artifactual. In preparations of full-thickness sections of eggshell for transmission electron microscope (TEM) analysis, it appears that the upper section of the outer membrane is integral with the base of the mammillary layer (Fig. 2c). Fibers in this upper zone appear to be calcified only in the mantle. The predominant components of the shell membranes are highly insoluble proteins that are resistant to typical protein extraction techniques (Krampitz et al., 1972, 1974). Early studies suggested that proteins of the shell membranes were similar to keratin because of their similar amino acid composition (Abderhalden and Ebstein, 1906; Calvery, 1933; Jones and Mecham, 1944; Munks et al., 1945; Wolken, 1951; Baker and Balch, 1962; Britton and Hale, 1977). Later studies based on the structure (Terepka, 1963a; Hoffer, 1971), amino acid analyses (Wedral et al., 1974), solubilities (Vadehra et al., 1971), and the absence of epitopes recognized by antikeratin antibodies (Arias et al., 1991b)demonstrated the inaccuracy of this suggestion. In fact, X-ray diffraction studies (Rudall, 1950) and the detection of hydroxyproline (Balch and Cooke, 1970), hydroxylysine (Candlish and Scougall, 1969), and lysine-derived cross-links (Crombie et al., 1981) were the first indication of the collagenous nature of the proteins of the shell membranes. It was also believed that elastin could be a component of the shell membranes, due to the occurrence of desmosine and isodesmosine (Baumgartner et al., 1978; Harris et al., 1980; Starcher and King, 1980; Youtz et a f . , 1984). However, the protein of the shell membrane is resistant to elastase (Leach et al., 1981). Amino acid analysis of avian and reptilian eggs shows that the eggshell membranes have a high content of cysteine and proline (Krampitz et a f . , 1972; Crombie et al., 1981).

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The desmosine and isodesmosine cross-links are formed extracellularly by oxidative deamination of a-amino groups in a reaction dependent on lysyl oxidase and a later condensation of three allysine residues with one lysine (Davidson and Giro, 1986; Kagan, 1986). A high activity of lysyl oxidase has been observed in the oviduct isthmus (Harris et al., 1980). Although these cross-links are characteristic of elastin molecules, they are also found in mature collagen (Kagan, 1986). Some authors have suggested that the avian and reptilian eggshell membranes have proteins rich in desmosine and isodesmosine but that these proteins are different from keratin, collagen, and elastin (Leach et al., 1981; Cox et al., 1982; Youtz et al., 1984). However, the collagenous nature of the shell membrane proteins can be inferred from the effect of copper and ascorbate deficiency and the use of lathyrogenic diets on shell matrix and membrane formation (Barnett et al., 1957; Thornton and Moreng, 1958, 1959; Bannister et al., 1971; Baumgartner et a f . , 1978). Immunohistochemical studies confirm the presence of types I, V, and X collagens in the shell membranes, being mainly type I in the outer sublayer and type V in the inner one (Wong er al., 1984), with type X observed throughout the entire shell membranes (Arias et al., 1991~). Pepsin treatment of type X collagen extracted from shell membranes removes, at least, the amino-terminal domain of the molecule, which shows electrophoretic mobility comparable to that of standard type X collagen obtained from hypertrophic chondrocytes (Arias et al., 1992b). These collagens are embedded in a proteoglycan-like material (Wong et al., 1984), which could be related to the reported sulfated glycoproteins (Picard et al., 1973; Paul-Gardais et al., 1974). However, only in the most external fibers of the outer shell membrane can the occurrence of keratan sulfate be demonstrated (Arias et al., 1992b). Interestingly, the familiar banding of collagen-rich fibers has not been observed in the TEM studies (Fig. 2) of eggshell membranes (S.-Q. Xiao, S. Baden, and A. H. Heuer, unpublished data, 1992). Although the insolubility of the eggshell membrane proteins has been attributed to the generation of disulfide bonds due to the high content of cysteine, the fact that the shell membranes are not solubilized by reducing agents such as mercaptoethanol or dithiothreithol does not support this disulfide hypothesis (Arias et a f . , 1992b). However, the detailed composition and localization of the eggshell membrane proteins remain unknown. The precise function of the shell membranes is not well understood, but it is known that the shell membranes are essential for normal calcification of the shell. Indirect evidence demonstrates that the structure of the shell membranes guides the topographic pattern of crystal deposition. Thus, the pattern of mineral deposited by the snail Otala, after replacement of

FIG.2 TEM micrographs of the eggshell membrane. Samples were prepared from physically separated eggshell membranes (a) or full thickness sections of mineralized, unfertilized eggshells (b, c) that were embedded in Spurr medium, stained with osmium and lead citrate (a, b) or osmium only (c), and sectioned by ultramicrotomy. (a) Fibers of the inner (IM) and outer (OM) eggshell membranes. (Photo by J. E. Dennis.) (b) Enlargement of a fiber cluster showing mantle (M)and fiber cores (FC) of the fibers shown in (a). (c) Fibers in the outer zone of the eggshell membrane are integrated into the rnarnmillary layer and display calcified mantles (CM).(Photo by S . - Q . Xiao.)

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FIG. 2

Continued

a piece of its shell with hen eggshell membrane, corresponds closely to that of the inner surface of the hen eggshell and not to that of the normal regenerated shell of the snail (Meenakshi et af., 1974b). Most of the deformities and embryonic mortality observed in eggs from mature hens can be correlated with deficiencies in the amino acid composition and thickness of the shell membranes, with aspartic acid and proline being the most important amino acids (Britton, 1977; Britton and Hale, 1977; Blake er al., 1985; Peebles and Brake, 1985). In addition, the amino acid composition of eggshell membranes may play an important role in the formation of soft-shelled and shell-less eggs (Klingensmith er al., 1988). In fact, this layer, together with the mammillary layer, is responsible for the initiation of the calcification of the shell (Creger er al., 1976; Stemberger et al., 1977; Dieckert et al., 1989). This appears to be a well-regulated process in which small amounts of intrusive materials deposited on the outer shell membrane during their fabrication can produce anomalous mineralization of the eggshell (Costello er af., 1985). The palisade layer and the shell membranes increase their permeabilities to gases during incubation (Kutchai and Steen, 1971). This enhancement in permeability is due mainly to the solubilization of calcium carbonate and mobilization of calcium ions to the embryo. A key element in these

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processes is the close association that becomes established during early development between the shell membranes and the chorioallantoic membrane. Such solubilization is due to the transformation of carbonate to bicarbonate and not by the action of HC1, as was suggested (Narbaitz, 1974). Carbonic anhydrase has been identified in the chorioallantoic membrane (Anderson et al., 1981). The processes of solubilization and mobilization are visualized as a resorption in the inner shell membrane and erosion of the cores with continued development (Doskocil et al., 1985; Bond et al., 1988). This decalcification in incubating eggs is manifested in eggs of the domestic hen between the morning and the evening of the fifteenth day after fertilization by the physical detachment of the membrane from the palisades (see Fig. 5) (J. P. Rodriguez, J. L. Arias, T.-M. Wu, M. Agarwal, D. J. Fink, A. I. Caplan, and A. H. Heuer, unpublished data, 1992). Prior to this time, attempts to physically detach the membrane cause tearing with the outer portion of the membrane firmly attached to the shell. (In in uitro experiments, partial decalcification with EDTA is necessary to physically detach the membrane from the shell.) B. Mammillary Layer

-

The 100-pm-thick mammillary layer (synonyms: cone layer, stratum mamillarium, granular shell membrane) is characterized by the presence of discrete aggregations of organic matter that intermix with the fibrillar material of the outer sublayer of the shell membranes (see below) (Sajner, 1955; Fuji and Tamura, 1970). The knobs, cores, or mammillae, as they are variously described, are suggested to be the sites where the initiation of crystal formation takes place (Robinson and King, 1963; Stemberger et al., 1977; Dieckert et al., 1989). In fact, the organic matrix cores of the mammillary layer are surrounded by spicular to prismatic spherulite crystallites of aragonite in dinosaur eggshells (Erben, 1970; Erben and Newesely, 1972) and of calcite in the ostrich (Sauer et al., 1975), hen (Kelly, 1901; Heyn, 1963c), and quail (Quintana and Sandoz, 1978), which appear to nucleate around the core regions and grow radially from their periphery (Schmidt, 1960, 1964). Aragonite crystals on the inner portion of avian eggs, reported by Erben (1970), are not present in domestic American eggs (Wu et al., 1992); however, some eggs have been found with vaterite microcrystals in their stead (Wu et al., 1992). The mammillary layer is difficult to visualize by scanning electron microscopy (SEM) of intact shells but organic matrix remnants of these structures are evident in decalcified material (Fig. 3). Analysis by TEM reveals regions of mixed organic matrix and calcite crystals (Fig. 4).

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FIG. 3 SEM micrographs of fully and partially demineralized eggshells. (a) The outer portion of a demineralized membrane after immersion of an intact eggshell in 15% EDTA for 1 hr. (Photo by T.-M. Wu.) The raised features, which have been denoted as “knobs” previously (Wu et a!., 1992), are now known to be collapsed deposits of matrix material. (b) These structures can be seen clearly in a partially demineralized fractured surface that has been immersed in 15% EDTA for 14 hr. The region adjacent to the membrane preferentially demineralized, leaving some freestanding matrix (M), which has collapsed in one region to give the feature marked “K” which is identical to the knob features in (a). Note also a remnant of the palisade calcite (C). (Photo by S.-Q. Xiao and M. Agarwal.)

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FIG. 4 TEM micrographs of the mammillary layer of mineralized eggshell, prepared as described in the legend to Fig. 2. (Photos by S.-Q. Xiao.) (a) Unstained section of mammillary layer indicating mixed calcitic (C) and matrix-rich (M)regions, and outer shell membrane fibers (F). Crystalline regions display high contrast in unstained sections. (b) Osmium-stained section illustrating matrix-rich region (M), fibers (F), calcified mantles (CM), and calcite (C) in the mammillary zone. Crystalline regions display low contrast in stained sections, whereas fibers and matrix-rich regions of the mantle show high contrast.

The mammillary layer is rich in neutral mucopolysaccharides (Simkiss, 1958; Robinson and King, 1968; Robinson, 1970), hexosamines, hexoses, and sialic acid (Cooke and Balch, 1970a). Protein-acid polysaccharide complexes have also been histochemically detected (Simkiss and Tyler, 1957),together with reducing groups (sulfydryls and/or phenols) (Sirnkiss, 1958). Keratan sulfate has been immunohistochemically detected (Arias et af., 1992b). The inability to detect uronic acid suggested to Cook and Balch (1970a) that proteoglycans of the chondroitin sulfate type did not play a role in the initiation of shell calcification. Interestingly, carbonic anhydrase has been found in the mamillae (Robinson and King, 1963), although its origin has been questioned (Diamantstein et al., 1964; Krampitz et al., 1974). Independent of its origin, inhibition of carbonic anhydrase by DDT, DDE, sulfanilamide, or acetazolamide causes thinning of the eggshells (Bitman et af., 1970; Bird et af., 1983; Lundholm, 1990a),

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FIG. 4 Continued

which suggests some relationship to the mineralization of the eggshell. Although the detailed chemical composition of the mammillary components remains unresolved, the close relationship between the density of the mammillary layer and the thickness and quality of the shell suggests that these structures are intimately involved in mineral deposition (Robinson and King, 1970; Simons, 1971; Fujii, 1974; Bunk and Balloun, 1978). The experimental decalcification of eggshells with EDTA or acetic acid (J. L. Arias, T.-M. Wu, V. J. Laraia, M. S. FernBndez, A. H. Heuer, and A. I. Caplan, unpublished data, 1992), or the natural removal of calcium from the shell by the embryo during development (Terepka, 1963b),occurs around the mammillae and in the interface between the “spherulitic” crystals and the calcitic columns proper (Fig. 5). In this regard, subregions of the mammillary layer consisting of a baseplate closely associated with the outer shell membrane and a calcium reserve body extended outward from the baseplate have been observed (Dieckert et a / . , 1989). These subregions, referred to as calcium reserve assembly (CRA) units, have been proposed to be the primary source of calcium for the developing embryo (see below).

FIG. 5 SEM micrographs of eggshell from 17-day incubated, fertilized eggs. (Photos by T.-M. Wu.) (a) Base of the mammillary layer showing natural decalcification of the calcium reserve bodies. (b) Surface of the outer shell membrane physically separated from the eggshell in (a). Residual calcitic crystals surround a polymeric surface, presumed to be of similar composition to the knob remnants in Fig. 3a. (c) Higher magnification image of a single calcitic ring in (b). Note the similarity to the crystalline fragment in Fig. 3b.

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FIG. 5

Continued

C. Palisade This calcified layer (synonyms: testa, calcified layer, column layer, crystalline layer, shell proper, spongila, spongy layer or stratum spongiosum) is not easily defined because the exact line of demarcation between the inner portion of this layer and the outer portion of the mammillary layer is arbitrary. However, it can be operationally defined as the region beginning above the mammillary layer, where the crystal front is confluent, and ending below the cuticle. The palisade corresponds to the thickest layer of the eggshell (200-350 pm) and is composed of integrated inorganic and organic components. The inorganic material is most often calcite in avian eggs and aragonite in reptilian eggs (Erben, 1970); rarely occurring vaterite is found in ampullarid snail shells (Hall and Taylor, 1971; Meenakshi et al., 1974a) and in some bird eggs (Gould, 1972; Tullet et al., 1976; Board and Perrott, 1979). The organic component (shell matrix or spongiosa), which constitutes about 2-5% (dry weight) of the whole layer and is 2% protein (N X 6.25) (Tyler and Geake, 1953, 1958), is obtained after decalcification with 20% EDTA (Baker and Balch, 1962)or 5% acetic acid followed by extensive dialysis (Arias et al., 1992b). Crystallographic and morphologic studies of the palisade reveal three subzones (Stewart, 1935; Terepka, 1963a; Perrott er al., 1981; Sharp and Silyn-Roberts, 1984):

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1. Cone layer. This inner zone of the palisade is formed by crystals that show no preferred orientation in X-ray diffraction (XRD) patterns (Wu et al., 1992) and that are interspersed in the mammillary layer (Fig. 6a) (Schmidt, 1962; Heyn, 1963a,b; Cain and Heyn, 1964). The crystals of this zone are the first crystals to be deposited during shell formation (Heyn, 1963a,b,c; Robinson and King, 1963; Creger et al., 1976; Stemberger et al., 1977; Dieckert et al., 1989) and adopt a “brick and mortar” structure (S.-Q. Xiao, S. Baden, and A. H. Heuer, unpublished data, 1992) (Figure 6a); the long axes of the bricks are perpendicular to the columnar axes of the palisades. 2. Central layer. In this zone, the crystals gradually develop a modest crystallographic texture, as revealed by XRD, the brick and mortar morphology becomes better developed (Fig. 6b), and there are many gas vesicles between the crystals. 3. Surface vertical crystal layer or external layer. This zone is 3 to 8 pm thick and corresponds to a region with an XRD texture and some vertical reorientation of the crystals (Fig. 6c) (Cain and Heyn, 1964; Favejee et al., 1965; Wu et al., 1992; S.-Q. Xiao, S. Baden, and A. H. Heuer, unpublished data, 1992) (see below). This region contains a relatively small number of gas vesicles between the crystals compared to the other zones (Parsons, 1982; Sharp and Silyn-Roberts, 1984). It has been suggested that this zone is shaped by a continued accretion of calcium carbonate even after shell matrix deposition ceases (Fujii, 1974). In each of these subzones, the dominant crystal morphological crosssection (the brick) is an approximately 1-pm-wide x 0.3-pm-high platelike crystal deposited within an organic network (Figs. 6a and 6b). In some cases, pseudo-single crystals, -100 pm in maximum dimensions, are present (Fig. 6d) in the cone layer; they are strong evidence for matrix control of the initial stages of crystallization (S.-Q. Xiao, S. Baden, and A. H. Heuer, unpublished data, 1992). The inorganic portion of the palisade is structured as columns between which the shell pores are defined. The pores of the hen’s egg (see Board, 1982) consist of broad, funnel-like openings that penetrate the palisade as single unbranched channels of irregular internal surface and terminate in clefts formed between adjacent mammillary knobs (Parsons, 1982). A hen’s egg has 7000 to 17,000 pores (Tyler and Geake, 1953), which are 10-20 pm in diameter at their inner side and 20-60 pm across their upper openings. These pores are more concentrated at the obtuse side of the egg (Romanoff and Romanoff, 1949),and their density is inversely related with embryonic mortality (Peebles and Brake, 1985). The palisade is the shell layer most resistant to gas exchange, with all of the gas exchange taking place through the pores (Paganelli, 1980). This permeability to gases increases with incubation (Kutchai and Steen, 1971).

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Infrared analysis of the material at the intersection of the palisade and the mammillary layer shows the presence of small amounts of phosphate (Lorenz et a / . , 1943). However, microchemical analysis of the shell section of Fig. 6c using energy dispersive spectroscopy reveals that most of the phosphorous is localized in the cuticle, and electron diffraction reveals the presence of three diffraction rings (S.-Q. Xiao, S. Baden, and A. H. Heuer, unpublished data, 1992), suggesting an inorganic phosphate. On the other hand, the shell matrix, that is, the remaining organic material obtained after decalcification of the palisade, is closely related to the calcite crystals and forms a continuous band around the egg. This band is formed by layers of organic matrix distributed not only parallel but normal to the eggshell surface ( J . L. Arias, T.-M. Wu, V. J. Laraia, M. S. Fernandez, A. H. Heuer, and A. I. Caplan, unpublished data, 1992) in such a way that the inorganic and organic moieties interpenetrate one another (Pooley, 1979). Studies indicate that components of the shell matrix, especially carbohydrates, are not uniformly distributed around the band of shell matrix (Tyler and Simkiss, 1959; Cooke and Balch, 1970b). There is also an inverse relationship between shell thickness and the percentage of total eggshell mass comprised by the matrix (Mather et al., 1962; Tyler, 1969). Shell-less eggs have no matrix (Mather et a / . , 1962). The main portion of the shell matrix is composed of 11% polysaccharide and 70% proteins (Baker and Balch, 1962).Older studies demonstrated the presence in the shell matrix of glycoproteins and polysaccharide-proteins (Simkiss and Tyler, 19571, presumably proteoglycans. The presence of such proteoglycans can be inferred from the reported determination of glycocyamine, galactosamine, galactose, and uronic acid, whereas the detection of mannose, fucose, and sialic acid indicates the presence of glycoproteins (Baker and Balch, 1962; Frank et a / . , 1965; Krampitz and Engels, 1975; Abatangelo et al., 1978; Salevsky and Leach, 1980). Also indicative of the presence of proteoglycans is the reported similarity of the amino acid composition of the proteins of the shell matrix with those of the core protein of the chondroitin sulfate proteoglycan of cartilage, and the indirect evidence of the presence of chondroitin-4-sulfate and dermatan sulfate glycosaminoglycans constituting 35% of the total polysaccharides of the shell matrix (Baker and Balch, 1962; Leach, 1982).Also in this regard, the occurrence in the isthmus of the oviduct of large amounts of enzymes that form 3’-phosphoadenosine 5’-phosphosulfate (PAPS) suggests that this enzyme is involved in the sulfation of glycosaminoglycans (Suzuki and Strominger, 1958, 1960a,b,c). A chelating role for calcium during shell formation has been attributed to these proteoglycans (mucopolysaccharides) (Simkiss and Tyler, 1958; Simkiss, 1961; Diamantstein, 1966). However, the exact nature, composition, distribution, and function of these putative proteoglycans has not been completed (see below).

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FIG. 6 TEM micrographs of the mineralized zones of unstained eggshell, prepared as described in the legend to Fig. 2. (Photos by S.-Q. Xiao.) (a) Mammillary zone indicating mixed regions of calcitic (C) and matrix-rich (M) regions, and outer shell membrane fibers (F). (b) Midsection of the palisade illustrating “brick-like” calcitic structures. (c) Outer zone of

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eggshell illustrating transition from surface vertical (SV) to cuticle (CU) region. (d) Pseudosingle calcitic crystal observed in the eggshell above the mammillary layer, extending to the center of the palisade. The inset is a selected-area electron diffraction pattern from this region demonstrating the pseudo-single crystal nature.

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When the palisade is decalcified with EDTA, a soluble (40%) and an insoluble fraction (60%) are obtained. Hyaluronic acid and some calciumbinding proteins have been detected in the soluble fraction (Heaney and Robinson, 1976; Krampitz and Engels, 1974, 1975; Krampitz et af., 1977, 1980; Abatangelo et al., 1978; Cortivo et al., 1982). The proteins have abundant acidic amino acid residues (aspartic and glutamic acids); also, y-carboxyglutamic acid has been detected in some of these proteins. The y-carboxyglutamic acid forms from a post-translational modification of glutamic acid, which is regulated by vitamin K. The shell matrix proteins containing y-carboxyglutamic acid have been named ovocalcin (Krampitz et al., 1980). Other mineralized biological systems such as bone, teeth, and molluscan shells also have y-carboxyglutamic acid-rich proteins (King, 1978). Such observations strongly suggest that the binding of calcium is a central role of these ionized carboxylate groups. Some of the calcium-binding proteins of the shell matrix have been described as containing carbohydrate and ester sulfate moieties (Krampitz et al., 1977). Keratan and dermatan sulfate have also been described in the shell matrix (Arias et al., 1992b)as well as carbonic anhydrase (Krampitz et al., 1974). It is interesting to note that many of the shell proteins are synthesized from precursors in the liver and not in the shell gland of the oviduct (Eckert et al., 1986; Schade, 1987). Although there was a suggestion about the presence of a collagen-like substance in the shell matrix (Almquist, 1934), present data do not support this suggestion. The shell matrix is not evenly distributed throughout the palisade but is particularly concentrated about one-third of the way into the shell (Cooke and Balch, 1970b; Dieckert et al., 1989).

D. Cuticle The most external layer (synonyms: cuticular layer, cuticula, cover, or tegmentum) of the eggshell is referred to as the cuticle (Fig. 6c). This proteinaceous layer covers the entire calcified portion of the shell to a depth of about 10 pm (Parsons, 1982),but its surface is irregular and varies in thickness from 0.5 to 12.8 pm. This variation in thickness reflects the presence of star-shaped cracks and flake-like layers, some of which delineate and cover the opening of the shell pores. In some avian species like the quail, the cuticle is mineralized-not in the form of regular crystals but as a fine powder or a chalky cover (Schmidt, 1958; Quintana and Sandoz, 1978;Board, 1982). As already noted, the cuticle in avian eggshells appears to contain fine particles of an inorganic phosphate (S.-Q. Xiao, S. Baden, and A. H. Heuer, unpublished data, 1992).

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The cuticle is easily extracted (or demineralized in those species in which it is mineralized) with 5% EDTA for 90 min (Baker and Balch, 1962) or with 0.25% sodium hypochlorite for 5 min at 40°C (Peebles and Brake, 1986). Although the cuticle has been considered to have a composition similar to that of the shell membranes (Romanoff and Romanoff, 1949; Cooke and Balch, 1970a), it is known to be composed mainly of mucin (Moran and Hale, 1936; Haines and Moran, 1940) and phosphorus (Tullett, 1987), in the form of glycoproteins having high molecular weights and containing hundreds of carbohydrate chains (see Roussel et al., 1988). However, the precise composition of the cuticle mucin has not been determined. It contains many disulfide linkages and free sulfhydryl groups and differs from the proteins of the other layers in having a higher content of lysine, glycine and tyrosine (Baker and Balch, 1962). Analysis for sugars shows the presence of galactose, mannose, fucose, and hexosamine, but not uronic acid (Baker and Balch, 1962). The cuticle in brown-shelled eggs also contains protoporphyrin (Poole, 1965; Kennedy and Vevers, 1976; Schwartz et al., 1975) and, in blue- and green-shelled eggs, biliverdin (Kennedy and Vevers, 1976), although pigmentation can be formed in other layers of the shell as well (Tyler, 1969). The cuticle appears to protect the egg from microbial invasion, possibly due to mechanisms of recognition and binding of bacteria to carbohydrate residues, as has been described for many glycoproteins (see Roussel et al., 1988). However, its main role appears to be to protect against excessive water loss by a mechanism that depends on the environmental humidity (Peebles and Brake, 1986; Peebles et al., 1987). The cuticle has a high conductance for gases compared to that of the rest of the shell (Paganelli, 1980). However, the permeabilities of both water and gases are dependent on the environmental humidity in such a way that it is difficult to study the two phenomena independently. It has been suggested that the removal of the cuticle may provide the means to improve gas exchange if appropriate adjustments in humidity can serve to prevent excessive water loss (Peebles et al., 1987).

111. Fabrication of Eggshells

The different eggshell layers are sequentially fabricated during passage of the egg through the oviduct (Fig. 7) (in most species of birds, only the reproductive organs of the left side are functional). The oviduct is a highly convoluted, muscular duct that functions to transport the ovum away from the ovary, promote fertilization of the ovum, and control the deposition of albumen and formation of the eggshell. On the basis of differences in

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7

OVARY INFUNDIBULUM \ Ovum fertilized 15-30 min

MAGNUM Albumen 2-3 h n

SHELL GLAND (uterus) Shell calcification 20-26 hn

FIG 7 Schematic drawing of the avian oviduct. [Reprinted from Fink et a / . (1992) with permission.]

morphologic features and biologic functions, the oviduct is organized into five regions (Coste, 1847; Hodges, 1974; Solomon, 1983). These regions are, from proximal to distal: the infundibulum, which receives the ovum and in which fertilization occurs; the magnum, which secretes albumen; the isthmus, which secretes precursors of the shell membranes; the uterus or shell gland, in which deposition of calcium carbonate takes place; and the vagina, from which the egg is expelled. The time of oviposition is crucial for shell calcification. Prostaglandindependent shortening of the oviposition time results in the laying of softshelled (partially calcified) or shell-less (uncalcified) eggs (Hertelendy et af., 1974, 1975; Hester et al., 1991), whereas prostaglandin inhibition produces hard-shelled eggs (Hertelendy et al., 1974; Hammond and Ringer, 1978; Shimada and Asai, 1978). Calcium transfer across shell gland cells is calbindin-dependent (Corradino et al., 1968; Bar and Hurwitz, 1973), and the process is unaffected by vitamin D (Bar et af., 1990). Calmodulin involvement has also been suggested in calcium transport regulation in the shell gland (Lundholm, 1990b), which is mediated by the columnar epithelium of the shell gland (Gay and Schraer, 1971). Although the microscopic structure and ultrastructure of the isthmus have been studied quite thoroughly (Surface, 1912; Tehver, 1930; Richard-

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son, 1935; Turchini and Broussy, 1938; Hoffer, 1971; Wyburn et al., 1973; Solomon, 1983), little is known about the nature of the process by which the amorphous secretory product of the isthmus glands is transformed into the double-layered, fibrous shell membranes. This lack of progress is related to unsuccessful assays for keratin precursors, because keratin was thought to be a shell membrane component. It is now known that keratin does not occur in shell membranes (Arias et al., 1991b). In addition, the biochemical analysis of the isthmus (Taylor and Hertelendy, 1960; Suzuki and Strominger, 1960a,b,c; Suzuki, 1962; Schraer and Schraer, 1965; Wood and Anastassiadis, 1965; Moo-Young et al., 1968)is not sufficiently sensitive to provide chemical characterization of the shell membrane precursors. In the secretion granules of the isthmus principal cells, an electrondense core and a less dense cortex resembling the ultrastructure of the shell membrane fibers have been described (Hoffer, 1971). With monoclonal antibodies, it has been possible to detect specific components of the shell membranes and follow their assembly throughout the oviduct. Type X collagen has been detected as a main component of the shell membranes (Arias er al., 1991a), and this collagen also is observed in the glandular cells of the isthmus (Arias et al., 1991d).Type I collagen was immunohistologically colocalized with type X collagen in the shell membranes of mineralized eggs only after pepsin treatment of the tissue (Arias et al., 1991~). However, shell membranes obtained before they reach the uterus stain intensely for types X and I collagen, which suggests that some rearrangement between the two collagen types occurs during the passage of the shell membranes and results in the masking of the type I collagen epitope. How the intense activity of lysyl oxidase occurring in the isthmus (Harris et al., 1980) affects cross-linking between these two collagen types is not known, but there is a progressive increase in the diameter of the shell membrane fibers deposited from the beginning to the end of the isthmus (Fujii et al., 1970). In addition, in virro studies have shown that type V collagen is able to regulate the diameter of type I collagen fibrils by limiting the growth of type I collagen into thick fibrils (Adachi and Hayashi, 1986). If this effect is applicable to shell membranes, the difference in diameter of the thinner fibers of the inner layer, where type V collagen is present (Wong et al., 1984), compared to the thicker fibers of the outer layer, might be explained. The enzymes necessary to form PAPS are present in the isthmus region of the oviduct (Suzuki and Strominger, 1958, 1960a); PAPS is involved in the sulfation of the glycosaminoglycan components of proteoglycans and is found in the shell matrix, but not in the membranes. Additionally, it has been shown that the amino acid composition of the organic matrix has a crucial influence on the type of calcium carbonate crystal to be formed,

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favoring the more acidic matrices involved in calcite formation in shellfish (Watabe, 1974); similar suggestions have been proposed for eggshells (Board and Perrott, 1979). Experiments with crude shell matrix extracts may explain how keratan sulfate per se influences crystal growth or whether other molecules are also involved. Mineralization of the eggshell is initiated in the final region of the isthmus (Creger et al., 1976; Stemberger et al., 1977).This process is first observed (Stemberger et al., 1977) when tiny calcium-rich, carbonate-deficient grains, 1-10 pm in diameter, reach a maximum density of 2700/mm2and increase to a size range of 28-90 pm. These particles are enmeshed in the fibers of the outer shell membrane and serve as the bases of the mammillae. In the shell gland proper, the particles grow laterally to form a confluent layer of 60- to 150-pm-diameter palisades, which then grow to a thickness of about 200 pm. The region at the base of the palisade layer, within the mammillary layer, has been identified as the calcium reservoir for the developing embryo (Dieckert et al., 1989). Based on histological evaluation of developing unfertilized and fertilized/hatched eggs, four distinct regions of this CRA have been observed: the base plate, by which the mammillae are inserted into the outer shell membrane; the calcium reserve body, forming the primary reservoir for embryonic calcium; the calcium reserve body cap, which terminates the zone of resorbable calcium; and the crown, which separates the calcium reserve from the base of the palisade proper. Investigations of eggshells from hatched chicks demonstrate that the base plate and calcium reserve bodies become progressively decalcified as the embryo develops (Terepka, 1963a,b). Residual organic matrix films remain on the outer membrane surface and within the base of the palisades, and highly textured, crystalline remnants of the base of the mammillae adhere to the outer membrane. Given the similarity of crystallite dimensions and surface densities, the first two steps described above (Stemberger et al., 1977) appear to represent the development of base plate structures, which then are progressively mineralized to form the calcium reserve bodies. From comparison of the dimensions of the first structures to form and the eventual palisade dimensions, it appears that each palisade must be formed by the aggregation of three to eight calcium reserve body structures. This aggregation is also apparent from the stained section of ground eggshell (cf. Fig. 3c of Dieckert et al., 1989). Except for the outermost region in association with the mammillae, where the mantle of the fibers entrapped in the mammillae contain calcite crystals, (Figs. 2c and 4) the eggshell membranes never mineralize naturally. The inability of crystals to grow into the shell membranes has been

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explained by a progressive lack of availability of ions as the crystals grow outward, because the ions are being supplied from the oviduct cells on the outside of the egg (Schmidt, 1957, 1962; Simkiss and Wilbur, 1989). Crystals nucleate within the mammillary layer and initially grow upward as discrete groups. The morphology is produced by crystal surfaces growing at approximately the same rate in what must be a constant ionic environment. Although it has been suggested that some crystals arising within the mammillae form the columns of the palisade layer (Schmidt, 1960, 1962), it was also suggested that the crystals forming the palisade were not continuous with the first-formed crystals and must, therefore, have a separate origin and sequence of formation (Young, 1950; Dughi and Sirugue, 1962). It has been suggested that only a few of the initially formed crystals continue to grow; therefore, as the shell increases in thickness, it consists of individual crystals packed tightly together and growing vertically to the surface (Tyler, 1969). However, Masshoff and Stolpmann (1961) argued that the shell structure consists of minute and separate calcite crystals embedded in a net of organic matrix. It is apparent from the older literature, and demonstrated with great clarity in Figs. 4 and 6, that there is concomitant deposition of shell matrix with small crystals within the palisade. How the matrix regulates crystal growth, and the effect of the matrix phase on the mechanical properties of the eggshell, particularly its mucopolysaccharide and nitrogen (protein) content (Bronsch and Diamantstein, 1965; Simons et al., 1966), remain areas that require further work. In spontaneous calcite growth, which is a process driven by the properties of supersaturated solutions without interference of an organic substrate, crystal growth may be controlled by factors such as interfacial energy or attachment kinetics, so that certain surfaces grow faster than others. X-ray diffraction and SEM and TEM of avian eggshells have demonstrated some crystallographic texture, but only toward the outside of the shell-shell interiors are essentially randomly oriented. The modest and somewhat variable texture that develops (Wu et al., 1992) is different in avian shell than in shells from other animals, such as reptiles, and involves the intensity ratios (1014) : (1018) and (1074) : (0006), which are greater at the exterior of the shell than at the interior ((1014) is the most intense X-ray reflection in calcite). The texture that does arise probably develops from the preferential deposition of certain crystal habits and possibly suggests that the shell matrix contributes to the control of crystal orientation during shell formation (Sharp and Silyn-Roberts, 1984; SilynRoberts and Sharp, 1986; J. P. Rodriguez, J. L. Arias, T.-M. Wu, M. Agarwal, D. J. Fink, A. I. Caplan, and A. H. Heuer, unpublished data, 1992; J. L. Arias, T.-M. Wu, V. J. Laraia, M. S. Fernandez, A. H. Heuer,

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and A. I. Caplan, unpublished data, 1992). Whether development of modest texture in the eggshell involves fast-growing surfaces or preferentially slow-growing ones is not yet clear. Scanning electron microscope investigations of fracture surfaces reveal a different morphology in the portion of the palisades adjacent the membrane compared to that adjacent the cuticle. This has led to interpretations relating the morphology of fracture surfaces to the mechanism(s) of crystal deposition (Silyn-Roberts and Sharp, 1986). However, the results of our combined SEM and TEM studies (S.-Q. Xiao, S. Baden, and A. H. Heuer, unpublished data, 1992) suggest that the reported fracture structures probably result from the familiar mirror, mist, and hackle feature observed on fracture surfaces of glass and other brittle materials (Lawn and Wilshaw, 1975), which are related primarily to details of crack propagation and not to ultrastructural features. The fracture surfaces do suggest, however, that eggshell fractures are initiated within the mammillary layer. In fact, Fig. 1 was taken from a shell in which the outer surface was in tension and the previously reported smooth fracture surface of the mammillae was absent. Termination of shell formation has been a matter of controversy. It has not been established whether the end of shell formation is a consequence of (a) an arrest of calcium secretion, (b) changes in uterine fluid composition, or (c) inhibition of calcite growth (Klingensmith and Hester, 1983). There is a premature oviposition of eggs induced by intrauterine injection of orthophosphate or pyrophosphate (Ogasawara et al., 1975; Klingensmith and Hester, 1983). In this regard, a concomitant appearance of inorganic phosphorus in the uterine fluid (Ogasawara et al., 1974) and mucosa (Klingensmith and Hester, 1985) and of insoluble organic phosphorus associated with cuticle deposition (Nys et al., 1991) has been described. Phosphate has been shown to poison calcite formation (Simkiss, 1964). These data support the hypothesis of a role for phosphorus in the termination mechanism of shell formation. A similar phosphoproteindependent mechanism has recently been proposed in the regulation of carbonate biomineralization of molluscan shells (Borbas et al., 1991). In summary, the end of shell formation occurs with a decrease in the amount of collectable uterine fluid, a decline in bicarbonate and calcium concentrations, and the appearance of insoluble organic phosphorus, despite a supersaturated milieu that still surrounds the shell (Nys et al., 1991).

IV. Eggshells as Models of Biomineralization

It is useful to look at eggshell formation in another context. The avian eggshell is one of the most rapidly mineralizing biological systems known:

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5 g of calcium carbonate are crystallized in the fewer than 20 hr required to fabricate the shell in situ (Simkiss, 1961). Like other hard tissues, eggshells are fabricated by living cells by combining an organic matrix with a crystalline inorganic filler (Arias et al., 1991a)to produce a multilayered, mineral-organic composite (Calvert and Mann, 1988). This natural or biological composite ceramic is fabricated at the relatively low temperature of 40°C, without the need for the high temperatures required to fabricate man-made ceramics. As in most mineralized tissues, the deposition of the inorganic filler and the deposition of organic matter occur simultaneously. In contrast to other hard tissues, including bone, tooth, and mollusc shell, the cells involved in eggshell formation are not intrinsic components of the shell; mineral deposition is effected by specialized oviduct cell populations in an “assembly-line’ ’ sequence as the egg is transported down the oviduct (Fig. 7). Therefore, distinct regions of the oviduct or distinctive oviduct cells must have more specialized control of distinct parts of eggshell fabrication-the cells in the isthmus secrete membrane components, and perhaps the initial calcified structures upon which subsequent crystal growth proceeds, whereas the bulk of the mineral phase is deposited in the uterus (shell gland). Because the secretory cells do not become an intrinsic part of the eggshell, and because the secretory events occur in a relatively short time frame, there is only a limited portfolio of regulatory mechanisms available to the cells of the oviduct. For example, in order to regulate mineralization, specific populations of cells might secrete (or stop secreting) a variety of chemical species into the fluid of the oviduct:

1. Calcium and/or carbonate ions (or other species containing these ions) into the medium surrounding the developing eggshell, perhaps by some facilitated diffusion mechanism 2. Biopolymers involved in one or more aspects of eggshell fabrication 3. Activating factors such as hydrolytic enzymes that might remove or produce structural components or active regulatory constituents 4. Ionic materials that might locally adjust the pH or ionic strength, for example, in the oviduct fluid Based on comparison of the time spent by the developing egg in each region, it appears that specialized cells in the upper, tube-like structures of the oviduct may perform a single, spatially controlled secretory function as the egg is transported through each region. On the other hand, the “ceramic processing” that occurs in the shorter, pouch-like shell gland may require the same tissue to perform multiple, temporally regulated secretions.

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A working hypothesis (Fink et al., 1992) for the steps involved in eggshell mineralization is illustrated in Fig. 8; the known components of this process, and unanswered assembly issues, are summarized in Table I. The ordering of the development of each structural feature may be subject to debate inasmuch as sequential steps may tend to occur simultaneously at different regions of the developing shell. The eggshell is a natural example of heterogeneous nucleation, that is, crystal nucleation onto a substrate that is chemically different from the crystal that is being nucleated. As the effectiveness of the crystal growth and inhibition processes depends upon the structure and chemistry of the

FIG. 8 Schematic model of a working hypothesis of the sequence and approximate zones of

mineralization of the avian eggshell. The numbered features are identified in Table I. [Reprinted from Fink et al. (1992) with permission.]

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BlOMlNERALlZATlON AND EGGSHELLS TABLE I Steps in the Formation of the Avian Eggshell: A Working Hypothesis

Region

Description

Organic constituents

1

Inner membrane formed.

Collagens (types I, V, and X). Proteoglycan(s) in mantle?

2

Outer membrane formed.

Collagens (types I and X). Proteoglycan(s) in mantle?

3

“Base plate” insertion into outer membrane.

Proteoglycan(s) (keratan sulfate?).

4

“Calcium reserve body” mineralized.

Keratan sulfate. Separate cap matrix?

5

Nucleation initiated at periphery of knobs. Membrane and calcium reserve bodies encapsulated in calcite crystals. “Crown” formation.

Matrix regulated.

6

7 8

Matrix regulated? Proteoglycan(s).

Palisade growth.

Dermatan sulfate-concentration highest at base of palisade. Mechanism of crystal size regulation?

“Pore” constructed.

Spontaneous or matrix regulated?

10

Surface vertical layer terminates palisade.

Depleted in dermatan sulfate.

11

Cuticle deposition.

Mucin (glycoproteins). Color bodies.

9

Note. Table reprinted with permission (Fink et al., 1992).

interfaces between organic substrate, mineral, and medium (Heuer er al., 1992), the acellular eggshell, which experiences only limited turnover, is a useful model for studying matrix-regulated biomineralization processes. It is important that the different compartments of the eggshell can be easily separated to allow the differential study of their organization and structure, and also to permit the use of new combinations of substrates to study the molecular control of biomineralization. Shell membranes, isolated together with remnants of the mammillary layer and matrix, and incubated with calcium chloride in an atmosphere of ammonium carbonate, act as a substrate to promote calcium carbonate nucleation and growth. Importantly, the deposition of these crystals is restricted to only certain regions of the mammillae (Wu el al., 1992). As in the calcification of the eggshell (Tyler, 1969; Fujii, 1974), the deposition of calcium carbonate crystals starts on the periphery of the organic matrix cores of the mammillae and gradually progresses from them in an outward and lateral direction, while crystal growth is inhibited from progressing inward. Only a few crystals grow around and enclose those fibers that are in close association with the mammillary material.

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During in uitro mineralization of eggshell membranes, with either calcium carbonate or calcium phosphate, it has been shown that the shell membranes have the ability to inhibit mineral deposition, probably due to an inhibitory effect of type X collagen on mineral deposition onto type I collagen (Arias et al., 1992a; Wu et al., 1992). Selective deposition of calcium phosphate crystals on shell membrane fibers occurs after, at least, nonhelical domains of type X collagen are chemically or enzymatically removed. Crude preparations of shell matrix influence the crystallization of both calcium carbonate and calcium phosphate (Arias et al., 1991a, 1992b; Wu et al., 1992), such that it enhances precipitation of calcium phosphate from a supersaturated solution, and modifies the morphology and crystal size and morphology of calcite on demineralized membranes (Wu et al., 1992).

V. Concluding Remarks The calcification of the eggshell occurs in three main steps: (a) fabrication of an organic matrix (shell membranes and mammillae) by the cells of the isthmus; (b) nucleation of calcium carbonate crystals within the mammillary layer; and (c) space-filling growth of calcite from these first-formed crystals, probably regulated by concomitant shell matrix deposition in the shell gland. The constancy of eggshell formation and shaping is an admirable example of the precise interaction of organic and inorganic moieties in a relatively simple and rapid formative system, and can be contrasted to the formation of other hard tissues such as bone and teeth. The complementary use of biological, chemical, and crystallographic approaches demonstrates that the eggshell is a very promising model for the study of biomineralization. The interaction between organic matrices and inorganic crystals can be studied in this system without the interference of cells that normally populate other biomineralizing systems. Understanding the principles governing the natural synthesis and fabrication of the eggshell and other bioceramic composites is an essential aspect of designing new materials that mimic biological systems (Heuer el al., 1992). Control of crystal nucleation, growth, shape, orientation, and size is of interest to material scientists who want to mimic the structure, properties, and performance relationships of these natural bioceramics. Additionally, if biologists could understand the regulation of these relatively simple processes occurring in eggshell formation, they may gain insight into more complex processes controlling other hard tissue development, maintenance, and repair. Lastly, poultry scientists could use this

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basic knowledge to improve and optimize the mechanical properties for these natural food packages, the eggshells.

Acknowledgments Some of the studies reported here were supported by the National Institutes of Health and the Department of Energy through Battelle Pacific Northwest Laboratories and the University of Chile, Santiago. Fruitful collaborations with M. Agarwal, S. Baden, D. A. Carrino, J. E. Dennis, M. S. Fernandez, V. J. Laraia, Jr., 0. Nakamura, J. P. Rodriguez, and T.-M. Wu are also gratefully acknowledged.

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Regulation of lntracellular Movements in Plant Cells by Environmental Stimuli Reiko Nagai Department of Biology, College of General Education, Osaka University, Toyonaka, Osaka 560, Japan

1. Introduction

Living things are constantly exposed to fluctuations in various environmental factors such as light, temperature, and humidity, and to mechanical and chemical stresses. They sometimes suffer from invasion by other organisms and from physical injury. Unlike animals, most plants are unable to change their location to escape from adverse environmental conditions. Instead, plant cells possess elegant stimulus-response systems by which they detect and respond to such fluctuations or potentially damaging situations in a rapid and appropriate manner. In plant cells, various forms of intracellular movement, such as the orientation movement of chloroplasts, the traumatotactic or premitotic migration of the nucleus, and streaming of the cytoplasm, are observed. These movements are thought to be essential for accomplishment of efficient photosynthesis, cell division at a suitable site, and appropriate delivery of substances that are required for growth and differentiation of plant cells. It has become evident that a spatially well-organized motile apparatus drives these movements as in animal cells. Changes in the pattern of movement or induction of a new pattern of movement are two of the most sensitive responses of plant cells to a given environmental stimulus. The reaction to a stimulus is assumed to be composed of a chain of, at least, three elementary processes: (a) perception of the stimulus, which includes direct physical and chemical interactions between the stimulus and a receptor molecule(s), resulting in some changes in the state of the receptor system; (b)transduction of changes produced in the receptor system, with sequential modification of the changes, to a specific effector system; and (c) the final response, completed by the effector system. International Review of Cytology, Vol. I45

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In this chapter, an attempt is made to describe examples of such responses in terms of movement of the cytoplasm, chloroplasts, and nucleus, all of which are known to be induced by environmental stimuli, such as light, low temperature, wounding, and chemicals. An attempt is also made to review our present understanding of the following subjects: ( a )possible receptor systems specific for each type of environmental stimulus; (6) a motile apparatus that functions as a specific effector; and (c) a possible signal-transduction chain that conveys information between a receptor system and an effector system, in other words, the processes involved in regulation of the motile apparatus.

II. Light-Induced lntracellular Movement

A. Photodinesis

The streaming of the cytoplasm observed in plant cells can be of two types. In one case, streaming persists under natural conditions. In the other case, streaming is induced or the rate of streaming increases as a result of a physical or chemical stimulus. Hauptfleisch (1892) termed the former type “primary streaming” and the latter “secondary streaming.” In cases where streaming is induced by light irradiation, the streaming is referred to as “photodinesis” (Fitting, 1925).The distribution of organelles in the cytoplasm does not change after the perception of light and the response is independent of the direction of irradiation. Photodinesis was investigated by several early researchers after Moore (1888a,b) first observed it in leaf cells of Elodea and Vallisneria. More detailed investigations began around 1960,with an analysis of responses from a more modern perspective. The classical studies have been well reviewed by Haupt (1959b) and Kamiya (1959, 1960, 1962). 1. Phenomena In dark-adapted cells of Elodea canadensis, streaming involves only small colorless organelles. The chloroplasts apparently remain motionless and are randomly distributed in the cytoplasmic layers along the walls that are normal (anticlinal) and parallel (periclinal) to the leaf surface. Light irradiation increases the rate of cytoplasmic streaming within 1 to 5 min, depending on the fluence of actinic light. When the rate of streaming reaches a maximum, the chloroplasts start to participate in the streaming of the cytoplasm. Under irradiation at sufficiently high fluence, the cytoplasm preferentially streams along the anticlinal walls, a phenomenon known as

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rotational streaming or cyclosis. Even when the chloroplasts are located along the periclinal wall prior to irradiation, they eventually participate in cyclosis after they have been translocated from the periclinal to the anticlinal walls (Seitz, 1964). Similar phenomena have been observed in the epidermal cells of Vallisneria spiralis (Seitz, 1967). In the mesophyll cells of V . gigantea, the cytoplasm apparently remains motionless in darkness after careful pretreatment (Takagi and Nagai, 1983). Upon irradiation with actinic light, small organelles sporadically initiate agitation (Kamiya, 1962). In time the agitation becomes the saltatory movement (Rebhun, 1964) that is responsible for local streamlets. Within several minutes, these streamlets are organized as a closed circuit that rotates unidirectionally along the side walls. Once the cytoplasm streams at a constant rate, the nucleus, chloroplasts, and cytoplasmic particles move together at a rate of 10 to 20 ,um/sec (Takagi and Nagai, 1985). In root hairs of Hordeum uulgare, rotational streaming of the cytoplasm is accelerated by irradiation at high fluence, even after a short light pulse. The accelerated streaming continues for 1 h or longer in darkness (Keul, 1976).

2. Perception of Light The action spectrum for photodinesis in the epidermal cells of V. spiralis has major peaks at around 370 and 450 nm and a very small peak at 680 nm (Seitz, 1967). Peaks at 450 and 370 nm disappeared in the presence of potassium iodide, which is assumed to act as a quencher of the triplet-excited state of flavins. However, even after treatment with this drug, regions of the action spectrum around 430, 480, and 680 nm remain relatively uneffected. Treatment with 343' ,4'-dichloropheny1)-1, l-dimethylurea (DCMU), an inhibitor of photosynthesis, specifically reduces the effect of light at these wavelengths. Thus, two different photoreceptors, flavins and photosynthetic pigments, have been proposed to function in the perception of light. Using linearly polarized light, Seitz (1967) identified action dichroism in the blue region of the action spectrum. The reaction was induced more effectively by irradiation with polarized blue light (B) that was vibrating parallel to the long axis of the cell than that vibrating perpendicularly. This observation suggests that the photoreceptor molecules reside in the cortical layer of the cytoplasm and that they are oriented parallel to the cell surface. In the red region of the spectrum, no such preferential effect of linearly polarized light could be found. Similar action dichroism was found with E. canadensis (Seitz, 1964). Cooperation of phytochrome and photosynthetic pigments in photodinesis was proposed for the first time in experiments with the mesophyllic

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cells of V. gigantea (Takagi et al., 1990). In such cells that have been briefly preirradiated with red light (R), the dependence on wavelength of the induction of streaming has two distinct peaks, a major one at 650 nm and a minor one at 450 nm (Fig. la). When a brief irradiation with far-red light (FR) is applied immediately after irradiation with R, the minor peak ceases to be significant, and the major peak remaines unchanged (Fig. lb). The photoreversible effect of R and FR, in which light of 450 nm becomes effective or noneffective, can be observed repeatedly after alternating brief cycles of irradiation (Fig. 2). Irradiation with FR itself has a definite inhibitory effect on streaming (Fig. 3). Blue or red light seems to be effective in inducing streaming only in the presence of the FR-absorbing form of phytochrome (Pfr). The induction of streaming by continuous irradiation with B after a brief preirradiation with R is suppressed in the presence of DCMU or atrazine, two inhibitors of photosynthesis (Fig. 4). The effect of photosynthetic pigments is separable from that of phytochrome. Although continuous irradiation with B does not induce streaming

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Wavelength (nm) FIG. 1 Dependence on wavelength of the induction of streaming in mesophyll cells of V. gigantea. The specimen was continuously irradiated with monochromatic light either after irradiation with R (650.0 nm) for 1 min (a) or after alternating irradiation with R and FR (729.4 nm) for 1 min each (b). The fluence rate of each monochromatic light was 10.3 pmoll m2 . sec. The ratio of N , (the number of streaming cells) to Nlold(the total number of cells

observed) obtained after 1000 sec of irradiation with monochromatic light is expressed as a relative value, the ratio for light at 650 nm being taken as 1.O. [Reprinted from Takagi et a / . (1990) by permission of The American Society of Plant Physiologists.]

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FIG. 2 Photoreversible effects of R and FR on the induction of streaming in mesophyll cells gigantea. Specimens were continuously irradiated with B (447.8 nm, 10.3 pmol/ mz . sec) either immediately after the last dark treatment of the pretreatment procedures or after alternating irradiation with R and FR for 1 min each. N , was counted at 5-min intervals, and the ratio of N , to NtOd was plotted as a percentage against the duration of irradiation with B. [Reprinted from Takagi et al. (1990) by permission of The American Society of Plant Physiologists.]

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FIG.3 Dependency on wavelength of the cessation of streaming in mesophyll cells of V. gigantea. Streaming was induced by irradiation with continuous R (650.0 nm, 10.3 pm/ m2 . sec), and then the specimens were continuously irradiated with monochromatic light at 7.5 pmol/rn2 . sec. The ratio of the number of cells in which streaming stopped during the 10-min period after irradiation with monochromatic light to N,,,, is expressed as a relative value, the ratio for light at 729.4 nm being taken as 1 .O. [Reprinted from Nagai and Takagi (1991) by permission of Shujunsha Co., Ltd, Tokyo.]

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%

Time FIG. 4 Inhibitory effects of DCMU on the induction of streaming in mesophyll cells of V. gigantea. Specimens were treated with DCMU at lo-’ M in the dark for 0, 30, and 60 min.

Then the specimens were continuously irradiatedwith B (447.8 nm, 10.3 pmollm2 . sec) after a 1-min irradiation with R (650 nm, 10.3 pmol/m2 . sec). The ratio of N , to N,,,,, was plotted against the duration of irradiation. [Reprinted from Takagi e? a / . (1990) by permission of The American Society of Plant Physiologists.]

by itself, streaming can be rapidly induced if a brief irradiation with R is superimposed on background irradiation with B. The duration of the apparent latent period, between the start of irradiation with R and the initiation of streaming, depends upon the timing of the exposure to R. The shorter the time during which cells are exposed to background irradiation with B, the longer is the latent period. In other words, in cells in which photosynthesis has taken place for longer than 6 min, streaming can be induced upon the formation of Pfr with an apparent latent period of as little as 65 to 75 sec. Thus, the induction of streaming in the mesophyllic cells of V. gigantea appears to require both the presence of Pfr and some factor(s) related to photosynthesis. The presence of phytochrome in V. gigantea was confirmed spectrophotometrically in crude extracts of leaves (Takagi et al., 1990). Flavins and cytochrome have been proposed as the photoreceptors responsible for photodinesis in the root hairs of Hordeum, because the action spectrum has major peaks at about 366 and 450 nm and a minor peak at 550 nm (Keul, 1976). Since a limited effect of R could be reversed by FR, the involvement of phytochrome has also been proposed in this system (Augsten and Finke, 1978). Recently, photoreceptors functioning in the blue to ultraviolet (UV) region of the action spectra, but not above 520 nm, were collectively designated B photoreceptors (Senger, 1984; Senger and Schmidt, 1986).

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3. Changes in the Mechanical Properties of the Cytoplasmic Matrix It is generally accepted that, for cytoplasmic streaming to occur, at least two conditions must be fulfilled: a motive force must be generated and the cytoplasmic matrix must have appropriate mechanical properties (Kamiya, 1959; Seitz, 1987; Tazawa, 1968). The latter component is often referred to as the cytoplasmic viscosity. Streaming of the cytoplasm can be accelerated through an increase in the motive force and/or a decrease in the cytoplasmic viscosity. Experiments involving centrifugation methods have demonstrated that light can affect cytoplasmic viscosity. The passive mobility of chloroplasts, namely the extent of displacement of chloroplasts by centrifugal force, has been used as an index of cytoplasmic viscosity. Cytoplasm with lower viscosity can be displaced more easily by centrifugation. In leaves of €€elodea densa, Virgin (1951, 1952, 1954) found that light produced an increase in the passive mobility of chloroplasts at very low fluence, a decrease above the dark control level at moderate fluence and, finally, an increase again at high fluence. Blue light was most effective in inducing these responses. Similar results were obtained with the epidermal cells of V. spiralis (Seitz, 1967, 1987). Furthermore, Seitz found that an increase in the passive mobility of chloroplasts induced at high fluence precedes the initiation of rotational streaming of the cytoplasm by several minutes, and that the action spectra for the two responses are identical. These findings indicate not only that the light-induced increase in the passive mobility of chloroplasts and the photodinesis observed in these cells share common photoreceptor systems, but also that the two responses are connected in terms of the process of signal transduction. In these experiments, cells were centrifuged at relatively high centrifugal forces, and the results obtained before and after centrifugation were simply compared. In these systems, the initial process of the reaction could not be analyzed. Using a centrifuge microscope with stroboscopic illumination (CMS; Kamitsubo et al., 1988), in which continuous observation of real-time changes in the behavior of chloroplasts at high optical resolution is possible during centrifugation, Takagi et al. (1992) examined the effects of light irradiation on the passive mobility of chloroplasts in mesophyll cells of V. gigantea. When the dark-adapted cells were centrifuged at 5 to 20 x g, the chloroplasts migrated in a direction parallel to that of the centrifugal force. The rate of this passive gliding did not change as long as the cell was observed under a constant centrifugal force (Fig. 5A). A brief irradiation with R increased the rate of the passive gliding of chloroplasts after an apparent latent period of 15 to 30 sec (Fig. 5B). This acceleration of

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Time (rnin) FIG. 5 Effects of light irradiation on the passive gliding of chloroplasts produced by centrifugal force in mesophyll cells of V. gigantea. Each specimen was observed under the minimal centrifugal force required for induction of passive gliding of chloroplasts: (A) 5.7 x g ; (B) 4.7 x g ; (C) 6.3 x g ; (D) 5.5 X g . The average rate of gliding of all chloroplasts in a cell was recorded at intervals of 1 to 3 rnin and plotted in terms of relative values. (A) Cells were observed under stroboscopic illumination with green light (500.5 nm). (B) Cells were irradiated with R (650.0 nm, 14.7 pmol/m2 . sec) for 2 sec at time 0 and then irradiated with FR (746.0 nm, 13.1 pmol/m2 . sec) for 10 sec, 5 rnin after the end of the R irradiation. (C) Brief irradiation with R (2.2 sec) and then with FR (13.3 sec) at time 0 (R . FR). (D) Cells were briefly irradiated with R (2.2 sec) in the presence of DCMU. After irradiation with FR (B, C), the specimens were observed under light with a wavelength of more than 690.0 nm. [Reprinted from Takagi et al. (1992) by permission of Springer-Verlag, Vienna.]

passive gliding was not observed if a brief irradiation with FR was applied immediately after the end of the preceding irradiation with R (Fig. 5C). Once the rate of the passive gliding of chloroplasts had been increased by a brief irradiation with R, there was a delay after the subsequent irradiation with FR of 5 min or more before the rate of gliding returned to the control value (Fig. 5B). Irradiation with B did not affect the passive gliding of chloroplasts. These results suggest that the passive mobility of chloroplasts may increase when the phytochrome in the cell is converted to the Pfr form, and that the increase may be negated when Pfr is converted back to the R-absorbing form of phytochrome (Pr). Stroboscopic illumination centrifuge microscopy enabled Takagi el al. (1992) to demonstrate that the phytochrome-mediated acceleration of the passive gliding of chloroplasts is separable from the processes in which

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photosynthetic pigments are involved. A prolonged irradiation with R induced not only the acceleration of passive gliding but also an apparently active movement of chloroplasts, which was observed in a direction that differed from the direction of the centrifugal force. Irradiation with B was found to induce the apparently active movement of chloroplasts if it was applied after a brief irradiation with R, which, by itself, did not induce the apparently active movement of chloroplasts. Furthermore, in the presence of DCMU, the passive gliding of chloroplasts still could be accelerated by a brief irradiation with R (Fig. 5D), whereas the apparently active movement of chloroplasts was never'observed. Induction of apparently active movement seems to occur depending on photosynthesis only in the presence of Pfr. By exploring factors that function during the apparent latent period, between the perception of R and the increase in the passive mobility of chloroplasts, we may be able to obtain important information about the primary action of phytochrome in this system. In dark-grown leaves of Triticum,Virgin (1987) demonstrated that irradiation with R increased the time necessary for displacement of the contents of cells by centrifugal force, and this effect was negated by irradiation with FR. He postulated that phytochrome controls the viscosity of the cytoplasm, with phototransformation to the Pfr form resulting in a more viscous cytoplasm. 4. Motile Systems

Much informatiofl about the mechanics of cytoplasmic streaming has been obtained from studies with internodal cells of Characeae (Kamiya, 1960, 1962, 1981, 1986; Kuroda, 1990; Tazawa and Shimmen, 1987). The most reasonable mechanism responsible for the rotational cytoplasmic streaming in characean cells is understood to operate as follows. Streaming is caused by the unidirectional sliding of endoplasmic organelles, which are equipped with a myosin-like protein, along bundles of microfilaments that are anchored on the stationary files of chloroplasts. The direction of streaming is thought to be determined by the polarity of fibrous actin (Factin), which makes up the microfilaments. Filamentous structures of 4nm diameter, which appear to be frayed out from the surface of the endoplasmic organelles, are thought to be effective in driving the rest of the endoplasm (Yoneda and Nagai, 1988). The presence of adenosine triphosphate (ATP) and Mg2+ions is indispensable for generation of the motive force (Williamson, 1975; Tazawa e l al., 1976; Shimmen and Tazawa, 1983a,b). a. Micro#lamentSystems Several studies have been carried out to determine whether the above-mentioned mechanism is applicable to the motile

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systems in cells that exhibit photodinesis. Experiments with cytochalasin B (CB), a specific inhibitor of many cellular responses that are mediated by microfilament systems (Wessels etal., 1971; Hepler and Palevitz, 1974), have provided some evidence for the involvement of microfilaments in the generation of the motive force for streaming. Cytochalasin B inhibits the streaming of chloroplasts in E. canadensis (Forde and Steer, 1976) and mesophyllic cells of V. asiatica (Ishigami and Nagai, 1980). Using this drug, Ishigami and Nagai (1980) examined the structural stability of the microfilaments. To do this, they compared the number of cells (N,) in which the direction of the streaming was reversed before and after the treatment with CB to the total number of cells treated (N,,,,,). The ratio increased with increases in the duration of treatment and of N , to Ntotal reached 50% after 24 hr at 10 to 100 pg/ml of CB. However, lead acetate, which reversibly inhibits the cytoplasmic streaming in characean cells (Kamitsubo, 1976), produced no reversal in the direction of the streaming. The results suggest the following conclusions: (a) The motile system in the mesophyll cells of Vallisneria is similar to that in characean cells. (b)The microfilaments do not seem to be as rigidly constructed as those of the characean cells in which the structural integrity of the microfilament bundles is perserved after treatment with CB (Bradley, 1973; Williamson, 1972). (c) The structural integrity of the microfilaments is not so labile that the simple cessation of streaming (by lead acetate) leads to the reversal of the polarity of the microfilaments. In the cytoplasm from lysed protoplasts of V. gigantea, Yamaguchi and Nagai (1981) were the first to observe bundled structures of microfilaments in a paracrystalline array similar to that found in characean cells (Kamiya and Nagai, 1982; Nagai and Hayama, 1979a). The optical diffraction pattern from the bundles was identical to that obtained from paracrystals of skeletal muscle F-actin (Moore et al., 1970). The microfilaments in the bundles could be decorated with skeletal muscle heavy meromyosin to form arrowheaded complexes. Yamaguchi and Nagai (1981) further confirmed, with epidermal cells of V. gigantea in which the cytoplasm rotated along the anticlinal walls, that the bundles of microfilaments resided in the vicinity of the cell membrane along the anticlinal walls and that the longitudinal axis of these bundles was parallel to that of the streaming cytoplasm. The direction of rotational streaming in epidermal cells of V. gigantea did not change after centrifugation of the cells at 8000 X g for 15 min (Yamaguchi and Nagai, 1981). In Elodea, a reversal of the direction of streaming occurred in only 7% of cells after centrifugation at 350,000 x g for 30 min (Beams, 1949). The bundles of microfilament are assumed to be anchored somehow in the cortical layer of the cytoplasm. Some idea of how the bundles of microfilaments maintain their structure was obtained

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from experiments with the mesophyllic cells of V. gigantea, in which the bundles also serve as tracks for streaming cytoplasm. The configuration, localization, and distribution of the microfilament bundles do not seem to change and are independent of occurrence of streaming (Takagi and Nagai, 1983). The three-dimensional organization of the bundles was clearly demonstrated in single mesophyllic cells isolated by enzymatic digestion (Masuda et ul., 1991). The circular arrangement of the bundles, visualized by staining with fluorescein isothiocyanate (F1TC)-conjugated phalloidin, which is known to bind specifically to F-actin, corresponded exactly to the pattern of streaming cytoplasm observed in situ (Fig. 6a). Masuda et a f . (1991) further noted that the streaming exhibited unusual patterns when single cells underwent plasmolysis in hypertonic solutions. They confirmed that the array of microfilament bundles changed dramatically upon plasmolysis (Figs. 6b-6d). Both plasmolysis and the disturbance of tracks were suppressed when inhibitors of proteases were added to the solution of enzymes used for isolation of single cells. In addition, an exogenously applied protease promoted the disturbance of tracks in an isotonic solution. These findings strongly suggest that the bundles of microfilament are stabilized through the adhesion of the cell membrane to the cell wall, and some protease-sensitive factor(s) is implicated in the stabilization. Other reports also demonstrate the crucial involvement of the cell wall in the arrangement and organization of the cytoskeleton in plant cells (Kropf et af., 1988; Akashi et a / . , 1990; Akashi and Shibaoka, 1991). Similar plasmolysis-induced disturbances of tracks for streaming cytoplasm were observed in Elodea (Kuster, 1910) and in Chara (Hayashi and Kamitsubo, 1959).

b. Myosin Some lines of evidence about myosin in plants are presented. A protein isolated from Nitelfa (Kato and Tonomura, 1977) exhibits properties in vitro similar to those of skeletal muscle myosin (“conventional myosin” or “myosin 11”) in terms of molecular weight; sensitivity of ATPase activity to ethylenediaminetetraacetic acid (EDTA), Ca2+ ions, and Mg2+ions; superprecipitation with actin; actin-activated ATPase activity; and the ability to form bipolar aggregates. Similarly, the occurrence of proteins that resemble myosin I1 and myosin I (Pollard and Korn, 1973; Warrick and Spudich, 1987) has been reported in Pisum (Ma and Yen, 1989), and in Lycopersicon (Vahey et al., 1982) and Heraefeum (Sokolov et al., 1986), respectively. Kohno et af. (1991) demonstrated that muscle F-actin can move on the surface of a coverslip coated with a crude extract from pollen tubes of Liliurn. Such movement depends on the presence of Mg2+ ions and ATP, suggesting that the extract contains a myosin-like protein. Immunoblotting revealed two polypeptides of 200 and 110 kDa, respectively, from Chara corallina that cross-reacted with a monoclonal

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FIG. 6 Arrangements of microfilaments in isolated mesophyll cells of V . gigantea. Isolated cells were stained with FITC-phalloidin either in an isotonic solution (a) or in a hypertonic solution (b-d). [Reprinted from Masuda et al. (1991) by permission of Springer-Verlag,

Vienna.]

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antibody against the myosin heavy chain from mouse 3T3 cells (Grolig et al., 1988; Qiao et al., 1989). Two polypeptides of 220-230 and 85 kDa, respectively, from the green alga Ernodesmis were found to cross-react with an affinity-purified antibody against the myosin heavy chain from slime mold (La Claire, 1991). Both types (myosin I and myosin 11) of myosin-like proteins seem to be present in these cells. Results of the various attempts of localizing myosin in the internodal cells of Characeae (Williamson, 1975; Nagai and Hayama, 1979a,b; Grolig et al., 1988; Qiao et al., 1989) suggested that myosin is associated with endoplasmic organelles and/or endoplasmic reticulum. Indirect immunofluorescence microscopy revealed the localization of myosin in other plants. Immunofluorescence staining with monoclonal antibodies against the subfragment 1 (S I) of skeletal muscle myosin or against light meromyosin from mouse 3T3 cells generated numerous fluorescent spots, which were thought to represent endoplasmic vesicles and/or organelles in the pollen tubes of Nicotiana (Tang et al., 1989). Similar patterns of staining were observed in pollen tubes of Alopecurus and Secale with antibodies against bovine skeletal and smooth muscle myosin (Heslop-Harrison and Heslop-Harrison, 1989). In Nicotiana, it was further noted that the nuclear envelope of both generative and vegetative cells was stained with the S1 fragment-specific antibody (Tang et al., 1989). Similar results were also reported in pollen tubes of Hyacinthus and Helleborus (Heslop-Harrison and Heslop-Harrison, 1989). In the green alga Emodesmis, immunofluorescence due to myosin was found on the surface of chloroplasts, in the nuclei, and in the cytoplasmic strands connecting the plastids (La Claire, 1991). There is one report of the possible involvement of myosin in photodinesis. Ohsuka and Inoue (1979) partially purified a myosin-like protein from Egeria (Elodea). The molecular mass was estimated by gel filtration to be 400 to 500 kDa. The presence of a heavy chain of 180 kDa was confirmed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The protein exhibited ATPase activity that could be stimulated to a greater extent by addition of Ca” ions than of Mg2+ions. The EDTA(Kf )-ATPase activity of the protein at higher ionic strengths was rather low. The protein aggregated to form bipolar filaments at lower ionic strengths. Finally, the protein bound to F-actin from skeletal muscle. These characteristics indicated that the protein resembled myosin 11. However, no information about the localization of the protein in situ is available to date.

5. Factors That Function in Signal Transduction

a. ATP It was suggested that, in V . spiralis, ATP mediates between the perception of light and the increase in the passive mobility of chloroplasts

264 RElKO NAGAI that ultimately leads to streaming that is induced by high-fluence irradiation. Seitz (1971, 1972, 1978, 1979a,b, 1980, 1987) proposed a hypothesis, referred to as the “ATP hypothesis” by Haupt and Wagner (1984), for the way in which light affects the intracellular availability of ATP. The hypothesis is based on the following observations: (a) The energy source for the actomyosin-based motility is ATP; (b)a release from the rigor state of muscle actomyosin requires ATP, and ATP may weaken the anchoring of chloroplasts to the cell cortex; and ( c ) ATP can completely substitute for the light stimulus in the dark. Under high-fluence irradiation, as a result of the saturation of the photosynthetic machinery, a surplus of ATP, produced via cyclic photophosphorylation, is added to the ATP produced via oxidative phosphorylation, which is enhanced as a result of activation of B photoreceptors. The consequent high level of ATP increases the passive mobility of chloroplasts and then induces cytoplasmic streaming. This hypothesis has also been adopted as a possible explanation for the light-dependent orientation movement of chloroplasts (see Section 11,B). Adenosine triphosphate has been shown to be virtually the only source of energy for cytoplasmic streaming with tonoplast-free cell models (Williamson, 1975; Tazawa e? al., 1976) and plasmalemma-permeabilized cell models (Shimmen and Tazawa, 1983a,b)of Characeae. The rate of streaming changes depending on the concentration of ATP. The apparent Michaelis constant is 60 to 80 p M , and the rate of streaming reaches a maximum at 200 p M ATP (Shimmen, 1978). The concentration of ATP in the cytoplasm, 0.5 to 4 mM (Hatano and Nakajima, 1963; Keifer and Spanswick, 1979), is much higher than this level. In intact cells of Chum, Reid and Walker (1983) found an almost linear relationship between the level of ATP and the rate of streaming. To modify the level of intracellular ATP, they used metabolic inhibitors, such as carbonyl cyanide rn-chlorophenylhydrazone (CCCP) and dicyclohexylcarbodimide (DCCD). They suggested that these inhibitors produced changes in several factors that are known to affect cytoplasmic streaming, such as the level of adenine nucleotides, the level of free Ca2+ions, and the pH of the cytoplasm. Before the ATP hypothesis can be accepted, it seems necessary to examine the level of intracellular ATP in V. spiralis kept in the darkness. If the level turns out to be higher than the concentration at which the rate of streaming reaches its maximum, the hypothesis would lose its validity. It may also be necessary to demonstrate that the level of free Caz+ions and the pH of the cytoplasm do not change prior to the induction of cytoplasmic streaming. Several further contradictions have been pointed out that render the details of the ATP hypothesis open to still further investigation (Haupt, 1982; Haupt and Wagner, 1984).

b. Cd+ Ions It has been suggested that, in the mesophyllic cells of V. gigantea, Ca2’ ions are involved in signal transduction between the

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perception of light and the induction of cytoplasmic streaming. The hypothesis comes from the assertion that Caz+ions regulate the actomyosinbased motile systems in both muscle and nonmuscle cells, and that Ca2+ ions are known to act as the “second messenger” in many signaltransduction processes, not only in animal cells but also in plant cells (Hepler and Wayne, 1985; Takagi and Nagai, 1992). A great deal of information about the role of Ca2+ions in streaming of the cytoplasm has accumulated for characean cells (Kamiya, 1981, 1986; Kuroda, 1990; Tazawa and Shimmen, 1987). Williamson (1975) first noted that the concentration of Ca2+ ions in the cytoplasm ([Ca2+],) has to be kept at 0.1 p M or lower for streaming to be maintained, even in the presence of ATP. The transient cessation of streaming upon excitation of the plasma membrane, which has been proven to be the result of a temporary loss of motive force (Tazawa and Kishimoto, 1968), was suggested to be due to a transient increase in [Caz+l,(Hayama et al., 1979). Rotation of chloroplasts observed in an isolated endoplasmic drop was inhibited by injection of Ca2+ ions, but not by that of Mg2+or K+ ions (Hayama and Tazawa, 1980). Streaming in intact cells was also inhibited by injection of Ca2+ ions (Kikuyama and Tazawa, 1982). By microinjection of the photoprotein aequorin, Williamson and Ashley (1982) directly demonstrated that the action potential produces an increase in [Ca2+],,concomitantly with inhibition of streaming. Tominaga and Tazawa (1981) and Tominaga et al. (1983) showed that streaming was inhibited by about 40% at 0.5 p M Ca2+ ion concentration and almost completely at 1 p M in a plasmalemma-permeabilized cell model. However, in tonoplast-free cell models, Ca2+ions at 1 mM were needed to bring about a complete cessation of streaming. Since most of the endoplasm in tonoplast-free cell models is lost after removal of the tonoplast, these findings suggest that a CaZ+-sensitizingcomponent(s) is present in the intact endoplasm. Several further studies using reconstituted systems (Tazawa and Shimmen, 1987; Shimmen, 1988; Kuroda, 1990; Higashi-Fujime, 1991) revealed that in characean cells the sensitivity to Ca2+ions is associated with myosin. In this connection, the proposal of Tominaga et al. (1987) should be mentioned, namely that CaZ+-dependent,calmodulin-independent phosphorylation of myosin is involved in the cessation of streaming, whereas calmodulin-dependent dephosphorylation of myosin is involved in the recovery of streaming. The presence of calmodulin in Cham has been demonstrated by radioimmunoassay (Tominaga et al., 1985). The motile apparatus in Vallisneria cells seem to be similar to that in characean cells. It may be reasonable to expect that the mode of regulation of the cytoplasmic streaming by Ca2+ions in Vallisneria is similar to that in characean cells. In the mesophyll cells of V . gigantea, even in the dark, the streaming can be induced by treatment with a solution that contains

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Ca2+ions at less than M and the divalent cationophore A23187. When this solution is replaced by others with various concentrations of Ca2+ ions, the induced streaming is found to be maintained at lops and M Ca2+ion concentrations. In contrast, streaming is gradually inhibited by lo-' and M Ca2+ ion concentrations. After streaming has stopped completely at M concentration and the solution is replaced again, the streaming can be induced by to 10-6M Ca2+ion concentration. However, only transient saltatory movements are observed at 1O-j and 10-4M concentrations. Unless the duration of treatment exceeds 3 hr, the induction and cessation of streaming can be alternately brought about by application of lop7and lop4M concentrations, respectively (Takagi and Nagai, 1986). Thus, in the mesophyllic cells of V. gigantea, as well as in characean cells, lower concentrations of Ca2+ ions are favorable for streaming and higher concentrations of Ca2+ion have an inhibitory effect. Changes in the level of Ca2+ion concentration in the cytoplasm after light irradiation were examined by electron microscope cytochemistry (Takagi and Nagai, 1985). In the mobile cytoplasm after irradiation with R, barely any precipitate was formed in the calcium-specific reaction with antimony. The amount of precipitate markedly increased in cytoplasm that had been rendered immobile by irradiation with FR. Red light may induce streaming by decreasing [Ca2'lc and FR may inhibit streaming by increasing [Ca2+],. To confirm that fluxes of Ca2+ions across the cell membrane are involved in the light-dependent changes in [Ca2+],, mesophyll protoplasts were isolated by enzymatic digestion (Takagi and Nagai, 1988). Irradiation with R induced an increase in the concentration of Ca2+ ions in a test solution that bathed the protoplasts, indicative of an efflux of Ca2+ions from the protoplasts. Subsequent irradiation with FR produced a rapid decrease in the concentration of Ca2+ ions to the dark control level. Vanadate suppressed both the acceleration of the efflux of Ca2+ions and the induction of streaming caused by irradiation with R. The efflux of Ca2+ ions may be an energy-dependent process. In the presence of nifedipine or La3+ions, commonly used blockers of Ca2+channel, both the influx of Ca2+ions and the cessation of streaming caused by irradiation with FR were inhibited. Ca2+channels in the cell membrane may be involved in the cessation of streaming. As mentioned above, streaming is controlled via cooperation of phytochrome and photosynthetic pigments. A similar requirement for Pfr and photosynthesis in the acceleration of the efflux of Ca2+ ions has been demonstrated (Takagi et al., 1990). Figure 7 demonstrates typical examples of such an assay. In Fig. 7a, the concentration of Ca2+ions in the test solution increased after a 10-min irradiation with B subsequent to a 1-min irradiation with R. The increased concentration of Ca2+ ions fell to the original level within 10 min of further continuous irradiation with FR.

267

REGULATION OF INTRACELLULAR MOVEMENTS Efflux

Influx

L -uL& R:l min FR:10 min

E 0

f

&

v)

3

Dark:@) min

R 1 min

I

B:10 min

+10-’u DCMU B:IO min

Efflux

b

I

xul

Influx

fl U

1 B:10 min

1

AAO.OO1

U U R:l min 8:s min

FIG. 7 Effects of light irradiation on fluxes of Ca2+ions in protoplasts isolated from mesophyll cells of V. gigantea. Changes in the concentration of Ca2+ ions in the test solutions that bathed the mesophyllic protoplasts were measured spectrophotometrically using the Ca2’sensitive dye murexide. The ordinate is the difference between the absorbance at 544 nm (the measuring wavelength) and that at 500 nm (the reference wavelength) of murexide. Efflux indicates an increase in the concentration of Ca2+ions and influx indicates a decrease. (a) About lo4 protoplasts were irradiated with B (447.8 nm, 1.7 pmol/m2 . sec) for 10 min after a I-min irradiation with R (650.0 nm, 2.0 pmol/m2 . sec). After spectrophotometric measurements, the same protoplasts were irradiated with FR (729.4 nm, 1.9 pmol/m2 . sec) for 10 min and then kept in darkness for 60 min in the presence of lo-’ M DCMU. Then the second series of irradiations with R and B was applied. (b) Another lo4 protoplasts were irradiated with B for 10 min, then with R for 1 min, and finally with B for 5 min. The concentration of Ca2+ ions was measured after each irradiation. The spectrophotometric measurements were stopped during each round of irradiation with actinic light. [Reprinted from Takagi et al. (1990) by permission of The American Society of Plant Physiologists.]

After treatment of the protoplasts with lO-’M DCMU in the dark, a brief irradiation with R and a subsequent continuous irradiation with B no longer accelerated the efflux of Ca2+ions. In Fig. 7b, no Ca2+ions were released by a 10-min irradiation with B alone, whereas R irradiation for 1 min between a prior 10-min and a subsequent 5-min irradiation with B accelerated the efflux of Ca2+ions. It appears that [Ca”], may be regulated

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by modulation of movements of Ca2+ions across the cell membrane, under the cooperative control of phytochrome and photosynthetic pigments, and that the consequent changes in [Ca2+],,in turn, bring about the induction and cessation of streaming. The various results from studies of photodinesis in the mesophyllic cells of V . gigantea now pose a new intriguing question: how does light control the transport of Ca2+ions across the cell membrane? B. Orientation Movement of Chloroplast

Light is also known to induce the movement of chloroplasts from a defined pattern to a newly defined pattern. This type of movement has been called orientation movement. In contrast to photodinetic responses, which are rather rapid, chloroplasts in the orientation response move slowly until a new quasi-stationary pattern is generated and usually remain in this pattern until a change in conditions calls for a transient movement to the new pattern. Orientation movement of chloroplasts has been studied in a wide variety of plant cells since Senn (1908) first made a comprehensive examination of this phenomenon. Several reviews have been published covering whole field (Haupt, 1959a, 1973,1982; Haupt and Wagner, 1984; Zurzycki, 1962; Britz, 1979) and on limited aspects of it (Haupt, 1965, 1987; Haupt and Weisenseel, 1976; Schonbohm, 1972, 1980, 1987; Seitz, 1972; Zurzycki, 1972, 1980; Wagner and Grolig, 1985). 1. Phenomena When exposed to unidirectional light, the chloroplasts in epidermal cells of V . gigantea (Izutani et al., 1990) and of V. spiralis (Seitz, l967,1979a,b) migrate to the periclinal walls of the cells (Fig. 8a) if the intensity of the light is low or moderate (low-fluence arrangement; Haupt, 1965, 1973) and they move to the anticlinal walls, then participate in cyclosis of the cytoplasm (Fig. 8b) in high-intensity light (high-fluence arrangement; Haupt, 1965, 1973). In darkness, the chloroplasts are distributed randomly all around the cytoplasm (dark arrangement). The responses are similar in the leaves of the moss Funaria (Senn, 1908; Zurzycki, 1980), and in the fronds of the duckweed Lemna (Senn, 1908; Zurzycki, 1980; Zurzycki et al., 1983). The chloroplasts in the siphonaceous alga Vaucheria gather above and below the central axis of its tubular body in low-intensity light (diastrophe; Senn, 1908) and gather along the flanks in high-intensity light (parastrophe; Senn, 1908). In the unicellular green alga Eremosphaera, organelles aggregate around the nucleus in the center of the cell under irradiation with high fluence (systrophe; Senn, 1908; Weidinger and Rup-

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FIG. 8 Orientation movements of chloroplasts in epidermal cells of V. gigantea. (a) The

specimen was continuously irradiated with R (650.0 nm) at low fluence (2.2 pmol/mZ. sec). Photographs of the P side of cells were taken at 0, 1, and 4 hr, respectively, after the onset of irradiation. (b) The specimen was irradiated with B (451 .O nm) at high fluence (1 1.1 pmol/ m2 . sec) and photographed 0, 5 , and 40 rnin after the onset of irradiation. [Reprinted from Izutani et al. (1990) by permission of Pergamon Press Ltd., Oxford.]

pel, 1985). In protonemata of the fern Adiantum (Yatsuhashi et al., 1985), the chloroplasts respond in a manner similar to that of the chloroplasts in Vaucheria. In the filamentous green alga Mougeotia and in the unicellular green alga Mesotaenium, only one large ribbon-shaped chloroplast, which divides the central vacuole in half, rotates to expose its face or profile to low- or high-intensity white light, respectively. Unlike the chloroplasts in other plants, only the edges of this chloroplast glide along the peripheral cytoplasm. The response to low-fluence light can be completed in Mougeoria upon exposure to light for only seconds or even fractions of microseconds during a subsequent dark period of 15 to 45 min (Haupt, 1959~; Weisenseel, 1968a,b; Haupt and Polacco, 1979). In Mesotaenium, however, orientation requires repeated pulses of light during the entire process of movement, with dark intervals not exceeding a few minutes (Haupt and Reif, 1979). The one large chloroplast in each epidermal cell of Selaginella martensii is situated near the wall, facing the mesophyll cells, that is

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opposite side to the light source, in low-fluence light and in darkness, and it moves to one of the anticlinal walls under high-fluence light (Senn, 1908; Mayer, 1964). The chloroplast in the alga Hormidiumflaccidum orients to light as does that of Selaginellu. However, the low-fluence movement, mainly toward the rear wall, is observed only if the cells are surrounded by air; if the cells are immersed in paraffin oil, each chloroplast goes preferentially to the front wall (Senn, 1908; Scholz, 1976a,b). In general, under low- or moderate-fluence light, the chloroplasts seem to be situated in regions where absorption of light is highest. By contrast, they occupy regions where light absorption is lowest under high-fluence light. These phenomena may be tentatively interpreted as being related to the optimal use of the light energy for photosynthesis at low fluence and protection against damage by strong light. Rearrangements of chloroplasts that are not related to the direction of light have also been reported. In the developing thallus of Caulerpa (Dawes and Barilotti, 1969) or Acetabularia (Koop et al., 1978), the tip becomes pale in the dark as a result of the basipetal movement of the chloroplasts together with the cytoplasm. During the daylight hours, movement to the apical region is accelerated, and hence, the tip region becomes greenish in color. The daily movements persist in continuous darkness or light, demonstrating that the movements are under the control of a circadian rhythm (Koop et al., 1978). In the green alga Ulua lactuca L., the position of chloroplasts also changes according to a circadian rhythm. Each cell contains one large irregularly cup-shaped chloroplast that migrates between the outer (periclinal) cell wall in the daytime and the anticlinal cell wall at night. The rhythm persists with a period close to 24 hrSunder conditions of constant temperature and continuous light or darkness (Britz and Briggs, 1976, 1983; Britz et al., 1976). Light is considered to act indirectly on the movements by synchronizing the circadian rhythm. In the tubulous coenocyte Dichotomosiphon tuberosus, chloroplasts together with other cell organelles move in a manner similar to that of the chloroplasts in Caulerpa and Acetabularia but the movements are not circadian. When a part of a cell is illuminated with a white or blue microbeam, the chloroplasts and the cell organelles accumulate in the illuminated region (Maekawa et al., 1986). Accumulation of the chloroplasts in the region illuminated by a blue microbeam has also been reported in Vaucheria (Fischer-Arnold, 1963; Blatt and Briggs, 1980; Blatt et al., 1980,1981)and in the marine coenocytic green alga Bryopsis plumosa (Mizukami and Wada, 1981). 2. Perception of Light A number of investigations have been performed to determine the nature, the intracellular location, and the molecular orientation of photoreceptor

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pigments. In many kinds of plant, such as Vaucheria (Fischer-Arnold, 1963; Blatt, 1983), Selaginella (Mayer, 1964), Lemna, and Funaria (Zurzycki, 1962; Lechowski, 1972), the orientation movement of the chloroplast in both low- and high-intensity light has been shown to be controlled by near-UV and B, and to be independent of light of wavelengths longer than 500 nm. Systrophe in Eremosphaera is caused most effectively by B (Weidinger and Ruppel, 1985). In epidermal cells of V. spiralis, the action spectrum for high-fluence light has a large peak and a small peak in the blue and near-UV region, respectively, and a very small peak at 680 nm (Seitz, 1967). Combining the action spectrum with results from the experiments with various inhibitors, Seitz proposed that a flavin and photosynthetic pigments act as photoreceptors (cf. Section 11,A). Seitz (1979a) further investigated the photoreceptor pigment that is active in the response to low-intensity light by examining the centrifugability (passive mobility) of the chloroplasts at the periclinal wall. The light produced a decrease in the centrifugability, with peaks of activity at 430 and 480 nm. Combining these result with those from the experiments with metabolic inhibitors, he suggested that the movement of chloroplasts from the anticlinal to the periclinal wall is under control of chlorophylls a and b. Thus, a different action spectrum in low- and high-intensity light was obtained, indicating that two different photoreceptor systems are involved. In epidermal cells of V . gigantea (Izutani et al., 1990), movement of the chloroplasts to the periclinal wall is most effectively induced by red light (Fig. 9a), even at a fluence of only 0.1 pmol/m2.sec, but it was inhibited at a fluence higher than 9.0 pmol/ m*.seL. Light of 750 nm completely reversed the effect of green light (550 nm), which was somewhat effective when it was used for photography

Wavelength

FIG. 9 Dependence of wavelength of the orientation movement of chloroplasts in epidermal cells of V . gigantea. (a) Under low-intensity light. (b) Under high-intensity light. The asterisk in each figure represents the effect of the irradiation with green light (552.0 nm, 2.5 pmol/ m2 . sec, 3 min) that was used for photography. [Reprinted from Izutani et al. (1990) by permission of Pergamon Press Ltd., Oxford.]

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(asterisk in Fig. 9a). These observations suggest that phytochrome acts in the response to low-intensity light because Pfr is assumed to be generated by exposure to green light. The action spectrum in the high-intensity range, above 5.6 pmol/m2-sec(Fig. 9b), suggests that the B photoreceptors act in the perception of light. The low-fluence responses (profile to face) in Mougeoria and Mesotaenium are most effectively induced by R (Senn, 1908). Action spectra and a typical R/FR antagonism have been used to demonstrate that phytochrome is the photoreceptor pigment (Haupt, 1959c; Haupt and Thiele, 1961). In linearly polarized light, only those cells respond for which the electrical vector of the light (the vibration plane) is perpendicular to the long axis (action dichroism; Haupt, 1970). The action dichroism points to a defined orientation of the photoreceptor molecules, and hence, to an association with or very close proximity to the cell membrane in a somehow stable manner. Furthermore, by a combination of polarization of light and the microbeam technique, it was demonstrated that Pr is oriented with its transition moments preferentially parallel to the cell surface and aligned helically to the long axis of the cell; however, Pfr is oriented perpendicular to the surface (Haupt, 1960, 1968; Haupt and Bock, 1962; Haupt er a f . , 1969). The dichroic orientation of phytochrome has been used to explain the perception of the direction of light and the orientation movement of the chloroplast (see also Kraml et af., 1984). At the front and rear ( i e . , proximal and distal to the light source) of the cell the surface-parallel Pr can absorb light that is vibrating in every direction. At the flanks, however, Pr can absorb only those components of the light that vibrate parallel to the cell axis. Thus, under polarized R and low-fluence white light, Pr at the front and rear of the cell is transformed effectively to Pfr, and at the flanks it remains predominantly in the Pr form. Consequently, a tetrapolar gradient of Pfr is formed in the cylindrical cell even under saturating irradiation. The edges of the chloroplast orient within the Pfr gradient as though repelled by the highest concentration of Pfr. Reports have been presented suggesting that there exists, in addition to the phytochrome system, further sensor systems, each of which is active under B or green light and is active only when there is little Pfr within the cell. That is, the low-fluence response is induced by continuous, weak B (Haupt, 1971)in the presence of a strong background of diffuse FR (GabryS et al., 1984; Walczak et a f . , 1984; GabryS, 1985). The response is also induced by linearly polarized green light (550 nm, 0.9 pmol/m2.sec) if the electric vector of the green light is vibrating perpendicularly to the cell axis, but if it is parallel no response is observed, even with a fluence as high as 13.9 pmol/m2-sec. Unpolarized FR (727 nm, 12.2 pmol/m2.sec), given alone, has no effect on the reorientation of chloroplast. Simultaneous and continuous irradiation with polarized green light, together with unpolarized FR, does lead to the response (Lechowski and Biaczyk, 1987).

REGULATION OF INTRACELLULAR MOVEMENTS

273

The movement from face to profile (high-fluence response), can be brought about by strong B. A portion of B is considered to be absorbed by phytochrome, resulting in gradient of Pfr and the rest is absorbed by B photoreceptors to induce the response. Movement can be induced by a combination of R and B, but it cannot be induced by R alone irrespective of the fluence. It is R that determines the direction of movement when R is applied together with low-intensity B. The effect of B absorbed by B photoreceptors is referred to as a tonic effect. An interaction between phytochrome and an auxiliary, B-sensitive, tonic system is assumed to be indispensable for the movement from the face to profile position (Schonbohm, 1963, 1971, 1980). GabryS et al. (1985) postulated that a photoproduct formed by the B-mediated reaction, having a lifetime of 2 to 3 min, interacts with phytochrome during its transformation or with its final Pfr form. Reorientation of chloroplast to the face position in Mesotaenium can be induced under the effect of phytochrome. The phytochrome effect is strongly potentiated by B (Kraml et al., 1988). A 10-min irradiation with R is hardly effective, but it induces a strong response if given together with 10 min of B, which by itself is nearly ineffective. Even 1 min of R can be highly effective, if it is followed by 10 min of B. Red light is alone responsible for the directionality . For potentiation of the phytochrome effect, B has been postulated to have a dual and sequential effect. In the first phase, it interacts with Pfr for at least 1 min to produce an internal signal that stores the directional information. In the second phase, movement, as controlled by the internal signal, depends on B for its realization. Thus, movement stops after B is switched off. Phytochrome was recently purified from Mesotaenium caldariorum and its molecular weight and biochemical properties were determined (Kidd and Lagarias, 1990). An action spectrum for the low-fluence response of chloroplast movement (diastrophe) in protonemata of the fern Adiantum capillus-ueneris L. was determined using polarized light that was vibrating perpendicularly to the axis of the protonema (Yatsuhashi et al., 1985). The spectrum had several peaks in the blue region around 450 nm and one in the red region at 680 nm (Fig. 10). The action of R was suppressed by nonpolarized FR given simultaneously or alternately, whereas the action of B was not. Blue light at high fluence (87.4 pmollm2-sec)caused the accumulation of the chloroplasts at the flanks of cells (parastrophe). However, high-fluence R (2.8 mmol/m2.sec) caused diastrophe (Yatsuhashi and Wada, 1990). Chloroplast movement was also induced by local irradiation with a narrow beam of monochromatic light (Yatsuhashi et al., 1985). Under a low-fluence beam of B (0.03 to 3.8 pmollm2.sec), chloroplasts moved toward the beam and aggregated inside the beam (positive response), whereas at high fluences (37.9 pmol/m2.sec and higher) they moved to the outside of the area of the beam (negative response). A red beam caused a

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300

400

500

600

700

800

Wavelength (nm)

FIG. 10 Action spectrum for the response of Adianrum chloroplasts to low-fluence light. [Reprinted from Yatsuhashi ef al. (1985) by permission of Springer-Verlag, Heiderberg.]

positive response at fluences up to 0.6 mmol/m2.sec, but a negative response at high fluences (1.3 and 2.6 mmol/m2.sec),a result that is assumed to be due to the R destroying some fraction of the phytochrome in the beam area, whereas scattered light from the beam produces a relatively high level of Pfr in the neighborhood of the beam. The chloroplasts move toward the region of higher levels of Pfr. Under simultaneous background irradiation with R or B at fluences that cause a low-fluence response in whole cells, the chloroplasts move out of the beam area only when R is used as background irradiation. From these results, Yatsuhashi and Wada (19%) concluded that the light-oriented movement of Adiantum chloroplasts is caused by R and B, mediated by phytochrome and another unidentified photoreceptor(s), respectively, and that this movement depends on a local gradient of Pfr or of photoexcited B photoreceptors. It was proposed that the absorption axes of the Pr and of the B photoreceptors are parallel to the cell surface and, on average, in helices of opposite handedness, the angle between the absorption axis of the Pr and the cell axis is larger than that in the case of the B photoreceptors, whereas the absorption axis of the Pfr is perpendicular to the cell surface (Yatsuhashi et al., 1987). In contrast to the situation in Adiantum, the phytochrome system is not involved in the movement of chloroplasts in the homosporous fern Pteris uittata L. The orientation movement of chloroplasts is apparent only after irradiation with polarized B. With a microbeam of B, both the low- and the high-fluence responses of chloroplasts can be induced (Kadota et al., 1989). In Pteris, the phytochrome system has been shown to be active in

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the germination response (Sugai and Furuya, 1967,1985), in the regulation of cell elongation (Furuya et al., 1967; Kadota and Furuya, 1977; Kadota et al., 1979), and in the regulation of cell division (Furuya et al., 1967; Wada and Furuya, 1972, 1974). Taking the above results with their own results that phytochrome is not involved in either the regulation of the tropic response or the photoorientation of chloroplasts, Kadota et al. (1989) suggested that there must exist, in a fern gametophyte cell, two independent populations of phytochrome, namely dichroic phytochrome located at the cell periphery and nondichroic phytochrome located elsewhere in the cell, and that Pteris lacks the dichroic form.

3. Mechanics and the Transduction Chain a . Epidermal Cells of Vallisneria The behavior of the chloroplasts in the cytoplasmic layer that faces the periclinal wall (P side) was examined (Izutani et al., 1990) with a time-lapse video system. In light of 725 nm, the chloroplasts maintain agitational movements (Kamiya, 1962), with no positional changes. The movements can be somewhat accelerated, becoming saltatory (Rebhun, 1964), upon irradiation with low-intensity R. Some of the chloroplasts migrate from the cytoplasmic layer that faces the anticlinal walls (A sides) to the P side and others move in the opposite direction. The chloroplasts that migrate into the center of the P side lose their mobility and stay there, which brings about the gradual accumulation of chloroplasts from the center to the periphery of the P side. Chloroplasts often pass underneath the accumulated chloroplasts. The nucleus exhibits saltatory movements throughout the orientation movements of the chloroplasts. The results suggest that R irradiation not only activates the movement of chloroplasts but also renders the chloroplasts immobile on the P side, supposedly via some anchoring mechanism. Such putative anchoring is further suggested by the following observations. When dark-adapted cells were irradiated with B at high intensity, almost all the chloroplasts moved to the A sides within 40 min (highfluence response). However, the chloroplasts in cells preirradiated with R remained in the center of the P side upon irradiation with B, even though the chloroplasts at the periphery of the mass of chloroplasts changed their location continually. When cells in which the agitation and the orientation movement had been inhibited by the presence of CB during R irradiation were subsequently irradiated with B, rapid movement of the chloroplasts to the A sides occurred after removal of the drug, but a considerable number of chloroplasts remained at the center of the P side. Without preirradiation with R, B fully induced the normal orientation movement after removal of the drug. The anchoring of the chloroplasts was also indicated by a study using CMS (Takagi et al., 1991). Whereas the minimal

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centrifugal force required to induce passive gliding of the chloroplasts in the dark-adapted specimens was usually 25 to 40 x g, the force increased to over 50 x g in the case of specimens that had been preirradiated with low-fluence R for 3 to 4 hr. Red light seems to affect the mechanical properties of the cytoplasmic matrix that surrounds each chloroplast and to cause them to associate with the ectoplasm andor the plasma membrane in the P side. The mechanism whereby this effect occurs remains to be determined. The high-fluence response of the chloroplasts in the epidermal cells of V. gigantea to B is completed in a few tens of minutes. Prior to the orientation movement, the chloroplasts in the P side are induced to move with an apparent latent period of about 120 sec upon the onset of the light. The induction of the chloroplast movement may result from activation of the microfilament system because CB completely inhibits the response (Izutani et al., 1990). Blue light, on the other hand, renders the cytoplasm more fluid. Under a constant centrifugal force, the rate of passive gliding of the chloroplasts increases three- to fivefold after an apparent latent period of 40 to 80 sec (Takagi et al., 1991). Changes in the mechanical properties of the cytoplasmic matrix seem to precede the activation of chloroplast movement. Similar results with respect to the action of B have been obtained in leaf cells of Helodea (Virgin, 1952, 1954)and in epidermal cells of V. spiralis (Seitz, 1967). acid Ethyleneglycol-bis (2-aminoethylether) - N , N, N ' , "tetraacetic (EGTA) can induce cyclosis (Yamaguchi and Nagai, 1981), and mimics B in terms of its effect on the mechanical properties of the cytoplasmic matrix. When EGTA was applied to cells instead of irradiation with B, in the presence of CB, acceleration of the passive gliding was observed under a constant centrifugal force (Takagi et al., 1991). It appears, therefore, that Ca2+ions are involved in both the changes in the mechanical properties of the cytoplasmic matrix and the activation of the microfilament-based motile apparatus, which together result in cyclosis accompanied by the movement of chloroplasts along the A sides of cells. With respect to the orientation movement of the chloroplasts in epidermal cells of V. spiralis, Seitz (1972) proposed that the chloroplast movements are directed by differences in the availability of ATP, which is formed via oxidative and photosynthetic phosphorylation, between the P side and the A sides (the ATP hypothesis; see Section 11,A).As mentioned above, in epidermal cells of V. gigantea, Rat low intensity induces anchoring of the chloroplasts which spreads from the center to the periphery of the P side. Moreover, chloroplasts can pass underneath the accumulated chloroplasts, which is difficult to conceptualize given the homogeneous concentration of ATP available in the P side. Therefore, it seems that the complex behavior of the chloroplasts cannot be explained only by a

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difference in the availability of ATP between the P side and the A sides. It is necessary to postulate production of some functional substances and their spatial organization on the P side under the control of R.

b. Cells of Mougeotia and Mesotaenium i. Motile Apparatus. Several reports on the motile system in Mougeoria cells have accumulated. Schonbohm (1972, 1973, 1975, 1987) was first to notice specific fibrillar structures and to characterize them. In dark- as well as FR-sensitized cells, a few, relatively long, thick fibrils can be seen, which emerge from the cytoplasm that encircles the single chloroplast, run through vacuoles, oriented more or less perpendicularly to the long axis of the cell, and seem to stabilize the position of the chloroplast. Upon irradiation with light of high or low fluence, the vesicles situated in the cytoplasm around the chloroplast become motile, and then another set of fine cytoplasmic filaments, oriented more or less normally to the cell’s axis, are newly generated at several loci along the margin of the chloroplast. The cytoplasmic filaments are crowded with vesicles that are often seen as chains of vesicles. The number and the length of these filaments or of the vesicle chains increase with time. Simultaneously, the chloroplast starts to rotate. After the orientation movement of the chloroplast, profile to face or face to profile, is completed, the filaments disappeared and the movement of vesicles slows down and then ceases completely. These observations suggest that the filaments are active contractile elements involved in the movement of the chloroplast. The orientation movement of the chloroplast is reversibly inhibited by CB (Wagner er al., 1972; Schonbohm, 1973, 1975). The Pfr-controlled increase in the number of the filaments is also inhibited by this drug (Schonbohm, 1975). Electron microscopy revealed a structure, composed of microfilaments, 5 to 10 nm in diameter, running between the edge of the chloroplast and the cytoplasm along the cell wall (Wagner and Klein, 1978). The microfilaments could be decorated with heavy meromyosin from skeletal muscle in a cell homogenate (Marchant, 1976) and in spread protoplasts (Klein er a f . , 1980). These early observations suggested that actin is involved in the orientation movement of the chloroplast in Mougeoria cells. Using rhodamine (Rh)-phalloidin, Grolig et a f . (1990) confirmed the localization of F-actin in “interphase” cells (in which the chloroplast exhibits no movement). Staining revealed a low level of fluorescence due to Rh-phalloidin throughout the cytoplasm. An enhanced level of such fluorescence was found around the nucleus and in two parallel fringes along each longitudinal edge of the chloroplast. Electron microscopy revealed a meshwork of 7-nm filaments which was sparse in the cytoplasm that covered the face of the chloroplast, dense in the thin layer of cortical cytoplasm, and densest in the region where the chloroplast meets with the

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cortical cytoplasm. These findings indicate that organization of the actin cytoskeleton in Mougeotia cells seems not to be involved in bundling of the filaments in interphase cells. However, the ability of the actin filaments to bundle was revealed in protoplasts derived from cells at interphase. In these protoplasts, fluorescent fibrillar structures, with no apparent specific orientation with respect to the chloroplast, were abundant. Thus the actin filaments appear able to respond to changes in the physiological state of the cells by generating bundles. Mineyuki and Nagai (1991) observed the dynamic behavior of the cytoplasmic filaments in connection with the movement of the chloroplasts from the face to the profile position. Figure I l a shows a number of filaments that appear to emanate from the moving edge of the chloroplast. Figure I l b shows the fluorescent image of such filaments, which was obtained by staining with FITC-phalloidin after pretreatment of specimens with rn-maleimidobenzoyl N-hydroxysuccinimide ester (Sonobe and Shibaoka, 1989). Thus, it appears that the filaments are composed of F-actin. Mineyuki and Nagai further observed that fibrils, which appeared relatively thick and free from the vesicles in “interphase” cells, were not stained with FITC-phalloidin but were stained with fluoro-

FIG. 11 Cytoplasmic filaments emanating from the edge of a chloroplast in Mougeotia sp. (a) Photograph taken using differential interference microscopy 10 min after the chloroplast had started to move from the face to the profile position. (b) Fluorescence image, stained at almost the same time as in (a) with FITC-phalloidin after pretreatment with m-maleimide benzoyl N-hydroxy-succinimide ester (MBS). Bar = 10 pm. ( Y . Mineyuki and R . Nagai.)

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chrome 3,3-dihexyloxacarbocyanineiodide (Y. Mineyuki and R. Nagai, unpublished). This observation suggests that the fibrils may correspond to the endoplasmic reticulum (Terasaki et al., 1984; Quader and Schnepf, 1986; Lichtscheidl and Url, 1990) or the mitochondria (Hatano and Ueda, 1988; Hayashi and Ueda, 1989). Thus, each orientation movement of the chloroplast seems to require the steady assembly of F-actin at the moving front of the chloroplast and the disassembly of F-actin when the movement of the chloroplast is completed. One of the crucial issues in the movement of chloroplasts is the mechanism of the assembly and disassembly of actin filaments, which may also contain processes of polymerization and depolymerization of actin. There must be an involvement of various actin-binding proteins, for example, gelsolin, which functions under the control of Ca2+ ions (Yin and Stossel, 1979; Stossel et al., 1985). Possible involvement of profilin (Carlsson et al., 1976; Lassing and Lindberg, 1985; Hartwig et al., 1989) also should be investigated. True myosin has not been identified in plants but N-ethylmaleimide (Klein, 1981) has been reported to inhibit the movement of chloroplasts. In experiments that involved centrifugation of cells in the longitudinal direction, it was found that anchoring of the chloroplast to the cortical cytoplasm can be increased by R irradiation with kinetics similar to the kinetics of the orientation movement and the kinetics of the increase in number of cytoplasmic filaments. Anchorage is loosened when the movement of chloroplasts is completed, in accordance with the decrease in the number of filaments (Schonbohm, 1972, 1987). Cytochalasin B inhibits the Pfr-mediated increase in the anchorage of chloroplasts (Schonbohm and Meyer-Wegener, 1989). There are many reports of microtubules in Mougeotia cells (Foos, 1970, 1971; Marchant and Fowke, 1977; Galway and Hardham, 1986, 1991; Kakimoto and Shibaoka, 1986; White et al., 1990). It was previously thought that they were not involved in the orientation movement of the chloroplast (Foos, 1971 ; Schonbohm, 1975). However, Serlin and Ferrell (1989) found that exposure to microtubule inhibitors such as colchicine, nocodazole, and vinblastine resulted in normal light-induced movement of the chloroplast but also in a reduction in the length of time required for reorientation of the chloroplast. The effect was specific because lumicolchicine had no effect. By contrast, taxol, a microtubule-stabilizing agent, increased the time required for reorientation. Serlin and Ferrell suggested that microtubules do influence the rotation of chloroplasts, probably through effects on cytoplasmic fluidity or on interactions with the microfilaments. ii. Signal Transduction. The biochemical steps in the photosensory transduction chain between the putative gradient of Pfr and the activity of the motor apparatus are, as yet, unclear. However, two hypotheses have

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been proposed. According to one hypothesis, the influx of Ca2+ ions through the plasmalemma, which is directly coupled to the tetrapolar gradient of phytochrome in its Pfr form, results in a tetrapolar gradient of Caz+ ions in the cytoplasm. The gradient in Ca2+ions spatially controls the activity of the actin-myosin system, causing the orientation movement of the chloroplast (Haupt and Weisenseel, 1976; Weisenseel, 1986). This hypothesis is supported by the observation that the rate of uptake of 4sCa2+ ions from the medium increases much more in R-irradiated cells than in unirradiated cells, but this increase is not observed when R is followed by FR (Dreyer and Weisenseel, 1979). Further support comes from experiments with probes coated with the ionophore A23187, which can cause the rotation of chloroplasts in the dark if the probes bilaterally abut on the cell wall closest to the edge of the chloroplast (Serlin and Roux, 1984). No rotation occurs if Ca2+ions are omitted from the external medium. When the ionophore is given unilaterally, there is no response by chloroplasts. However, results have been reported that contradict the hypothesis mentioned above. Roux (1984) observed a R-induced efflux of Ca2+ions from dark-adapted cells. He explained the result by suggesting that R induces a very small transient influx of Ca2+ions across the plasma membrane, which cannot be detected for technical reasons, and a coincident release of Ca2+ions from intracellular stores, which together increase the intracellular level of Ca2+ions. The net efflux of Ca2+ions would result from a stimulation of the Ca2+-ATPasepump on the plasma membrane. Uptake of 45Ca2+ions was shown to be restricted to the pectic layer of the cell wall (Grolig, 1986). Schonbohm et al. (1990a,b) showed that the organic blockers of entry of Ca2+ ions, such as diltiazem, nifedipine and ruthenium red, and inorganic blockers, namely La3+ and Co2+ ions, did not affect the low- or high-fluence movement of the chloroplast. Only at toxic concentrations or after long-term incubations, for example, several days, was the movement inhibited. Even under Ca2+-deficientconditions, the R-induced movement was not inhibited. The second hypothesis postulates that the reorientation results from a gradient of sites of anchorage to actin in the plasmalemma. It is proposed that phytochrome as Pfr and Pr controls the anchorage sites. Actin-myosin interactions are triggered by cytoplasmic Ca2+ions which, it is suggested, are released from internal stores or taken up through the plasmalemma; in this case any possible gradient of Caz+ions is not taken into consideration (Wagner and Klein, 1981; Wagner and Grolig, 1985; Grolig and Wagner, 1988). Calcium-sequestering vesicles, as internal stores of Ca2+ions, have been presented. Their abilities in calcium binding were indicated in uiuo by the pattern of fluorescence from chlorotetracycline (CTC), a probe whose fluorescence has been shown to be a function of lipid-associated calcium (Caswell, 1979), and by X-ray microanalysis (Wagner and Ross-

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bacher, 1980; Wagner and Klein, 1981; Rossbacher et al., 1984). It has been suggested that the phenolic component of the vesicular matrix allows the exchange of Ca2+ ions and guarantees calcium homeostasis in the cytoplasm (Grolig and Wagner, 1987, 1989). Increases in the vesicular load of neutral red, which is specifically adsorbed by phenolics in uiuo, causes inhibition of the movement of chloroplasts (Russ et al., 1988; Grolig and Wagner, 1989). Red lightlfar-red light-dependent changes in the calciumbuffer capacity of the vesicles in uiuo were demonstrated by monitoring the fluorescence of CTC (Jacobshagen et al., 1986). Thus, it is stressed that a decrease in levels of Ca2+ ions should inhibit the movement of chloroplasts under low-intensity irradiation. The role of calmodulin in mediating movement of the chloroplast was suggested by the inhibitory effect of a calmodulin-antagonist drug, N-(6-aminohexyl)-5-chloro-lnaphthalenesulfonamide (W-7) (Serlin and Roux, 1984). Calmodulin, isolated from Mougeotia and Mesotaenium (Wagner et al., 1984,1987;Jacobshagen et al., 1986) is suggested to mediate the transfer of the Ca2+signal to some cytoskeletal regulatory protein, via the activation of a protein kinase, postulated to be myosin light chain kinase (MLCK) (Altmiiller et al., 1984). Interactions between myosin molecules, phosphorylated on their light chains, with actin anchored at Pr zones are then established to reorient the chloroplast. Figure 12 is a schematic representation of the hypothetical network of events from absorption of light by phytochrome to the photoorientational response of the chloroplast. Various results that fail to support the second hypothesis mentioned above have been presented. Tests of the effects of two calmodulin antagonists, namely trifluroperazine and W-7, on the movement of chloroplasts and on the Hr-mediated decrease in centrifugability under continuous R showed that neither phenomenon was inhibited by either drug at “physiological” concentrations. Neutral red did not inhibit the low-fluence response of the chloroplast that is induced by pulses or continuous irradiation with R (Schonbohm et al., 1990a,b). These results clearly contradict the earlier results published by Wagner et al. (1984,1987) and Jacobshagen et al. (1986). Schonbohm and Schonbohm (1984) postulated an essential role for biophenolic compounds within the signal-transduction chain of the low-fluence as well as the high-fluence motile response of the chloroplast. A protein kinase was detected and partially purified (Roberts, 1989) from Mougeotia by use of a synthetic peptide, KM-14, which contains the sequence of the regulatory light chain of smooth muscle myosin that is phosphorylated by the calcium-calmodulin-dependent MLCK. Unlike MLCK, however, the KM- 14 kinase exhibits a dependence on micromolar levels of Ca2+ions in the presence of physiological concentrations of Mg2+ ions, and in the absence of two known calcium effectors, calmodulin and phosphatidylserine/diacylglycerol.The possibility has been pointed out

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cw

vac

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vac

cyt pl

FIG. 12 Schematic representation of the upper half of a Mougeotia cell in cross section. Phytochrome is indicated as Pfr (T) and Pr (I), respectively, and is proposed to control anchorage sites for actin (dotted lines) at the plasmalemma (pi) and, thereby, to control the reorientation movement of the chloroplast (chi) after activation of the interaction between actin and myosin (caged bars), which is under the control of Ca*+ions, Ca2+-calmodulin (CaCaM), and myosin light chain kinase (MLCK). The chloroplast is shown turning away from high concentrations of Pfr to a high concentration of Pr. cw, cell wall; vac, vacuole; cyt, cytoplasm. [From Wagner and Grolig (1985) by permission of Plenum Press.]

that calcium-dependent, calmodulin-independent protein kinase is associated with the actomyosin cytoskeleton, and phosphorylation of the sequence in myosin may be associated with the control of the contractile activity of actomyosin. Russ et al. (1991)recently measured cytoplasmic levels of free Ca2+ ions to be 0.92 2 0.29 pmol. 1- I in M. scalaris by loading filamentous cells and protoplasts with the fluorescent calcium-sensitive dye 1-[2-amino-5(6-carboxyindol-2-yl)-phenoxy]-2-(2’-amino-5’-methylphenoxy)-ethaneN,N,N’,N’-tetraacetic acid (indo-1). Pulses of light (365 nm, 1.7 sec) caused an increase in [Ca2+],that was nearly independent of the external concentration of Ca2+ ions, suggesting that the increase resulted from a release of Ca2+ ions from intracellular stores. Increased [Ca2+],, higher under UV light than under B or R, was suggested to affect the velocity of rotation of chloroplasts since the movement mediated by UV was faster than that in cells irradiated with B or R. Using membrane fractions prepared by osmotic lysis of protoplasts from M. cufduriorum, Berkelman and Lagarias (1990)investigated its calcium transport system. The preparation exhibited a high rate of ATP-dependent uptake of Ca2+. Thus, it is reasonable to suppose that [Ca”], can be

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lowered, in the presence of ATP, to a level similar to the reported [Ca2+], in characean cells (Williamson and Ashley, 1982).Sucrose gradient centrifugation and analysis of marker enzyme allowed separation of two pools of activity, one being similar to the activity of a vanadate-insensitive ATPase and the other being similar to that of a pyrophosphatase, which is considered to be associated with the tonoplast. Precise examination of the effects of light irradiation on a reconstituted Ca2+ transport system composed of the membrane fraction and phytochrome is expected to help in elucidation of the early steps of the signal-transduction pathway.

c. Protonemal Cells of the Fern Adiantum In the dark, chloroplasts in the fern Adiantum capillus-ueneris L. move in random directions in the cytoplasm. Movement of the chloroplasts is inhibited by CB but not by colchicine (Kadota and Wada, 1992). Under polarized light, they exhibit orientation movement and gather at the site at which the absorption of light by the oriented dichroic photoreceptors seems to be maximal (Yatsuhashi et al., 1985), and they stay there until the light is turned off. Thus, it appears that some anchoring mechanism keeps the chloroplasts in place. Rhodamine-phalloidin staining revealed two types of actin filaments in the dark, that is, thick filaments running roughly parallel to the cell axis and a meshwork of fine filaments. However, after treatment for 4 hr with polarized light, when the photoorientation of chloroplasts had been completed, thick actin filaments encircled the margin of each chloroplast and the fine meshwork of actin appeared to be less abundant (Kadota and Wada, 1989). Kadota and Wada speculate that the photoinduced changes in the organization of actin may block the movement of chloroplasts and anchor them in the place. The mechanism of the event is, as yet, unclear. In this connection it should be mentioned that in Vaucheria sessilis, a microbeam of B causes concomitant reticulation of actin bundles with aggregation of chloroplasts (Blatt and Briggs, 1980; Blatt et al., 1980). Prior to the aggregation of chloroplasts, an outward current due to an efflux of protons is observed. It is suggested that this efflux of proton via hyperpolarization of the membrane brings about a redistribution of other ions and, in particular, a change in local concentrations of Ca2+ ions, which in turn give rise to the reticulation of the actin bundles (Blatt et al., 1981). Breakage of actin bundles and resultant cessation of cytoplasmic streaming has been reported in pollen tubes, when the intracellular concentration of Ca2+ion was increased in the presence of A23187 (Kohno and Shimmen, 1988). d . The Green Alga Dichotomosiphon Maekawa et al. (1986) described the details of the manner of orientation movement of the cytoplasm in the coencytic green alga D . tuberosus. The cytoplasm, including a number of

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chloroplasts, nuclei, and other small organelles move along multistriated cytoplasmic strands, which are aligned mostly parallel to the longitudinal axis of the tubular body of the cell. Streaming is bidirectional, being both acropetal and basipetal. When the algal body is irradiated, the rate of acropetal migration of the organelles increases, whereas basipetal migration continues, and the organelles gradually accumulate at the cell apex. Upon cessation of irradiation, the organelles migrate basipetally and accumulate at the basal region of the algal body. Local illumination with white light or B causes local reversible accumulation of the organelles, which migrate from both the apical and the basal ends of the algal body. More organelles migrate from the basal end than from the apical end. The light-dependent translocation of the organelles can be inhibited by colchicine but not by CB (Maekawa et al., 1986). Two types of array of microtubules were revealed by electron microscopy and by staining with monoclonal antibodies against tubulin (Maekawa and Nagai, 1988). One type of microtubule, seen mostly individually, lies along the cortical cytoplasm (cortical microtubules; Fig. 13a) and the other type, found singly and in bundles, is seen in the inner portion of the cytoplasm (endoplasmic microtubules; Figs. 13b and Fig. 14a). The longitudinal axis of each microtubule parallels that of the cell. Figure 14a shows a cross-sectional image of bundles of microtubules in the endoplasm, and some of them are seen to be connected via three-forked cross-bridges (Fig. 14b, arrowheads). Changes in the arrangement of endoplasmic microtubules are directly related to the light-induced translocation of the organelles. In the dark, small (such as that indicated by thin arrow in Fig. 14a) and medium-sized (medium arrow) bundles are evenly distributed along the whole length of

FIG. 13 Immunofluorescentstaining of microtubules. (a) Ectoplasmic microtubules. (b) Endoplasmic microtubules. Bar = 50 pm. [Reprinted from Maekawa and Nagai (1988) by permission of Springer-Verlag, Vienna.]

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FIG. 14 Cross-sectional image of bundles of microtubules in the endoplasm of Dichotornosiphon tuberosis. (a) The large, medium, and small arrows represent large, medium, and small bundles, respectively. Bar = I p m . (b) Three-forked cross-bridges (arrowheads) linking neighboring microtubules in a bundle. Bar = 0.1 p m . [Reprinted from Maekawa and Nagai (1988) by permission of Springer-Verlag, Vienna.]

the cells (dark type). In the light, a few small bundles are found in the apical region and large bundles (thick arrow) become conspicuous in the basal region (light type). The number of microtubules seems to decrease in the apical region and increase in the basal region in the light. The local accumulation of the organelles is accompanied by decomposition of bundles at the irradiated region and formation of large bundles in the unirradiated basal region (Maekawa and Nagai, 1988). Conversion of arrays of microtubules from the dark to the light type and vice versa is assumed to be the result of changes in the polymerization-depolymerization equilibrium of microtubules and of changes in the microtubule-bundling activity in the cell. As one possible candidate for a regulatory factor, a 90-kDa microtubule-associated protein (MAP), was partially purified and characterized by Maekawa er al. (1990). Western blotting demonstrated that the protein cross-reacted with an affinitypurified antibody against a bovine adrenal 190-kDa MAP (Murofushi et al., 1986; now termed MAP4; Aizawa et al., 1991) and with antibodies raised against a synthetic peptide that is identical to the tubulin-binding domain of the 190-kDa MAP and of a T protein (Aizawa et al., 1989). The partially purified 90-kDa protein had the ability to bundle microtubules when mixed with a tubulin fraction from D . tuberosus in the presence of taxol. Double-immunofluorescence microscopy revealed that the fiber-like

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patterns of staining obtained with the affinity-purified antibody against 190-kDa MAP and with the tubulin-specific antibody coincided with one another (Maekawa et al., 1990). Examination by immunoelectron microscopy showed that antibodies specific for the 190-kDa MAP were localized at sites that may correspond to the location of the 90-kDa MAP (Fig. 15). Further examination is needed to demonstrate that modulations of bundling activity occur in light- and space-dependent ways. In this connection, the report by Mori et af. (1991) is suggestive in that the assemblypromoting activity of MAP4 was inhibited by site-specific phosphorylation by protein kinase C. A protein with a molecular weight different from that of the 90-kDa MAP has been isolated from carrot cells and its ability to bundle microtubules has been demonstrated (Cyr and Palevitz, 1989). It is known that tip-growing organisms generate an endogenous electrical current such that positive charge flows into the tip and exits from the trunk (Nuccitelli, 1983). Such a current exists in the case of Dichotomosiphon (N. Kami-ike and R. Nagai, unpublished), which exhibits a typical tip growth. A tip-to-base gradient of Ca2+ ions, which results from an influx of Ca2+ ions at the tip, has been reported (Jaffe et al., 1975; Reiss and Herth, 1979; Saunders and Hepler, 1981; Brownlee and Wood, 1986). If it is true in Dichotomosiphon that the electrical current carries more Ca2+ions under irradiation with light, Ca2+ ions would be more concentrated at the tip than in the trunk. Ca2+ion alone, at high enough concentrations (above 10-6M), can be expected to depolymerize microtubules or to act together with calmodulin and MAPSfor depolymerization of microtubules (Hawser et al., 1984;Cyr, 1991). Ca2+ions at lower concentrations

FIG. 15 Immunoelectron micrograph of Dichotomosiphon tuberosus obtained after reaction with an antibody against the 190-kDa MAP from the bovine adrenal gland. Deposition of colloidal gold particles is obvious at the sites at which the MAP is expected to be localized. Arrowheads indicate microtubules in cross section. Bar = 0.1 pm. (Y. Maekawa and R. Nagai.)

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in the trunk would activate polymerization of tubulin into microtubules which, in turn, would form bundles with the 90-kDa MAP. Identification of protein translocators and their possible relevant interactions with microtubules remain to be investigated. C. Orientation Movement of Nucleus

Migration of the nucleus to the site of its division is a prerequisite for appropriate development of plants. Light-dependent formation of side branches in the mosses Phycomitrium turbinatum (Jensen, 1981) and Physcomitrella patens (Doonan er al., 1985, 1986) have been reported to occur in accordance with the precisely controlled migration of nuclei. The apical cell of the moss, at the filamentous stage, extends by tip growth and divides by means of an oblique cross-wall. After the division, one of the daughter nuclei in the newly formed subapical cell comes to reside in the resting position, which is 40 to 50 pm behind the new crosswall. After two to three apical cell cycles in the dark, subapical cells of P . patens become competent to divide again upon brief exposure to light and they produce side branches (Doonan et a f . , 1986). This response to light is saturated by a 2-sec irradiation of 25.1 pmol/m*.sec at 665 nm. Approximately 4 to 6 hr after irradiation, a bulge is observed on the cell flank close to the leading edge of the subapical cell. This bulge increases in size as the nucleus migrates toward it from its resting position, with a concomitant migration of chloroplasts, which finally enter the bulge. The movement of the nucleus is associated with thick strands of cytoplasm that run from the nucleus into the bulge. Division occurs at the base of the bulge. One daughter nucleus enters the branch initial, while the other returns to the position of the parent nucleus about midway along the flank of the cell. This process takes from 10 to 12 hr. Using indirect immunofluorescence with monoclonal tubulin-specific antibodies, Doonan et al. (1986) were the first to show the coherent, nonfragmented nature of microtubules that connect the nucleus to the apical cross wall. Disruption of microtubules by the herbicide cremart reversibly abolishes nuclear migration and cell division. Stabilization of microtubules with taxol largely overcomes the effects of cremart, an indication that microtubules are a herbicide-sensitive target during nuclear migration. Involvement of microtubules in the directed movement of the nucleus has also been demonstrated in other plants (Schmiedel and Schnepf, 1979a,b; Vogelmann et al., 1981;Meindl, 1983;Hepler and Palevitz, 1974; Britz, 1979; Gunning and Hardham, 1982). Both light-induced branching of subapical caulonemal cells of P . patens and induction of buds by exogenous cytokinin in caulonemal cells of

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Funaria are initiated by a localized swelling in target cells at the presumptive sites of asymmetric divisions, with subsequent nuclear migration to those regions. These observations suggest that in an earlier step, linking the perception of the stimulus to initiation of the events, similar mechanisms may be operative. Cytokinin-induced division of target caulonemal cells of F. hygrometrica is preceded by localized changes in levels of membrane-associated calcium and an increase in inward current that predicts the site of the asymmetric division within the cell (Saunders and Hepler, 1981; Saunders, 1986). The current is blocked by gadolinium nitrate, an inhibitor of Ca2+uptake, a result that indicates that Ca2+ions are a component of the current (Saunders, 1986). A23187 plus exogenous Ca” ions can induce the initial division of target cells in the absence of cytokinin (Saunders and Hepler, 1982). Both nuclear migration and subsequent division are blocked if uptake of Ca2+ions from the external medium is inhibited (Saunders and Hepler, 1983). Saunders (1986) suggested that cytokinin-induced concentration of ion channels over presumptive bud sites, as indicated by an increase in the inward current, may be envisioned to exert spatial control of the cytoplasmic ion concentration and to stimulate formation of buds by establishing a new growth zone and directing nuclear migration and cell division. The formation of branch in P . patens is known to be most effectively induced by R (Doonan et al., 1985). Red light, in an effect that can be reversed by FR, stimulates the branching of F. hygrornetrica (LarpentGourgaud et al., 1974). The cytokinin-induced initiation of buds is also enhanced by a pulse of R and this effect can be partially reversed by FR (Simon and Naef, 1981). The results suggest that the initiation of buds is under the control of phytochrome.

111. Responses of Streaming Cytoplasm t o Low Temperatures

Low temperature is known to cause physiological malfunctions and structural disorders in plants that have high sensitivity to cold, whereas plants that tolerate chilling temperatures have developed characteristic mechanisms for withstanding such unfavorable conditions (Lyons, 1973; Lyons et al., 1979; Ilker et al., 1979; Graham and Patterson, 1982; Steponkus, 1982). Streaming cytoplasm responds rapidly to low temperature. Streaming in chill-sensitive plants ceases or is perceptible after 1 or 2 min at 10°C and it invariably ceases promptly at 5 or 0°C. By contrast, streaming in chill-resistant plants shows only a steady reduction in rate, equivalent to

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a constant Q,, between 25 and 0°C. The cytoplasm is still seen clearly to be streaming even at 0°C. (von Sachs, 1864; Lewis, 1956; Das et al., 1966; Patterson and Graham, 1977; Patterson et al., 1979; Woods er al., 1984a). Cessation of streaming is often accompanied by dramatic changes in subcellular structure. In sensitive plants, for example, watermelon and tomato, at chilling temperatures the complex network of transvacuolar strands, along which active streaming of organelles is normally observed, disappears and the cytoplasm becomes vesiculated. During rewarming of the chilled cells, the vesicles fuse into pleiomorphic blebs, which gradually stretch out into fully functional strands (Patterson et al., 1979; Woods et al., 1984a). Similar events have been reported by Das et al. (1966) in single tobacco cells in microculture. The time taken to recover a normal structure and streaming activity is related to the duration of the exposure to chilling temperatures, and to plant species (Woods et al., 1984a). These events are not seen during the chilling and rewarming of cells of chill-resistant plants Digitalis purpurea and Veronica persica (Woods et al., 1984a). In the epidermal cells of onions, which are commonly referred to as coldtolerant plants, cold treatment was observed by Quader er al. (1987, 1989) to induce breakage of transvacuolar strands and to alter dramatically the organization, shape, and distribution of at least two of the three domains of the endoplasmic reticulum (ER). The cisternae and long tubular strands disintegrated into short ER tubules which showed rapid agitational motion. Long-distance movement was inhibited. The ER was partly reorganized during recovery from cold treatment. Bundles of actin filaments, visualized by staining with Rh-phalloidin, could withstand cold stress for at least several hours. Quader et al. suggested that low temperatures most likely influence either the interactions of the force-generating system, probably myosin, with actin filaments, or the force-generating mechanism of the actomyosin-driven intracellular movement, but that low temperatures do not affect the integrity of actin filaments. It has been proposed that cellular membranes in sensitive plants undergo a physical phase transition from a normal, flexible liquid-crystalline state to a solid-gel structure at the temperature critical for chilling injury, and that this is the primary event in response to chilling (Lyons, 1973; Lyons et al., 1979). Such a change in state would be expected to bring about changes in permeability, which in turn would lead to a disturbance in the ion balance in cells. Woods et al. (1984b) suggested that the rapid effects of low temperature upon cyclosis and the structure of the cytoplasm might be due to a breakdown in compartmentalization of intracellular Ca2+ions, with a resultant increase in [Ca2'],. Using CTC, Woods er al. (1984b) observed a consistent decrease in the signal as cells of chill-sensitive tomato plants (Lycopersicon esculentum) were cooled below their threshold temperature for chilling sensitivity. On rewarming, as the temperature

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rose above the chilling threshold, there was an increase in the fluorescent signal. By contrast, trichomes of D . purpurea showed no such changes. The uncoupling agent CCCP and the Ca2+chelator EGTA induced marked decreases in the fluorescent signal in cells from both species. Direct manipulation of [Ca2+],by use of a series of EGTA-Ca2+ buffers combined with the Ca2+ionophore A23187 caused changes in the structure and motility of the cytoplasm that closely resembled the changes caused by chilling temperatures. Minorsky (1985) also suggested that chilling induces an increase in the cytosolic level of free Ca2' ions, which could then be responsible for the cessation of cytoplasmic streaming. Yoshida et al. (1989a,b)found that the reversible decrease in the activity of the tonoplast H+-ATPase and in its proton-translocating activity was the earliest response to chilling in hypocotyls of chill-sensitive mung bean (Vigna radiata L. wilczek). The reversible changes occurred when the tissues suffered no permanent injury. Taking into account the function of the proton-translocating ATPase of the tonoplast in the regulation of cellular pH and the proton antiports for Ca2+ions (Ohsumi and Anraku, 1983; Blumwald and Poole, 1986), Yoshida et al. (1989a) suggest that the chillinduced decline in the H+-ATPase activity and the impairment of the proton-translocating ability, whether reversible or irreversible, might result in a perturbation of the cellular compartmentalization of solutes and ions, and of protons and Ca2+ions in particular. The fluorescence from fluorescein diacetate, a pH-sensitive fluorophore (Slavik, 1983), was shown to be markedly reduced after suspension-cultured cells from mung bean seedlings were chilled for 24 hr, suggesting acidification of the cytoplasm (Yoshida et al., 1989b).

IV. Wound-Induced Movement of the Nucleus and the Cytoplasm A. Higher Plant Cells

Wound-induced intracellular movements are closely related to the woundinduced cell division of cells. In tissues of most higher plants, after either natural or experimental wounding, cells adjacent to the wound respond by reentry into the mitotic cycle and one or more divisions. Healing of experimentally induced wounds has provided valuable information in attempts to analyze the mechanism of cell division and plant morphogenesis, since such analysis can be conducted under controlled conditions. Sinnott and Bloch (1941a,b) were the first to show that new cell plates are always deposited parallel to the surface of the wound in cells adjacent to

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slit wounds. The phenomenon has been reported in various tissues of different plants. Even after puncture wounds, the new planes of division are induced in such a way that the wound is encircled. The new walls meet exactly and in the diagrams of Sinnott and Block, they give the appearance of having been drawn by a single stroke of the pen (Comer, 1978;Robbertse and McCully, 1979; Gunning, 1982; Hardham and McCully, 1982a,b; Schulz, 1987, 1988; Venverloo, 1990; Venverloo and Libbenga, 1987; Goodbody and Lloyd, 1990; Hush et al., 1990). There are excellent reviews that cover a whole event in wound healing (Bloch, 1941, 1952; Lipetz, 1970). Within a short time after wounding, various characteristic events occur in the cytoplasm (Biinning, 1959). Ziegler (1954) described precisely the early events after wounding in the epidermal cells of Allium sepa. The rate of streaming of the cytoplasm usually increases considerably within minutes. The temperature of the reacting cells rises rapidly during the first 20 min. The nuclei in the cells adjacent to the wound decrease slightly in size after about 1 hr but then they increase in size again until the sixteenth hour after wounding (Birkholz, 193 1). The cytoplasm becomes displaced toward the face of the cell nearest the wound and then disperses again around 10 hr. Next, the nuclei migrate toward the wound, a phenomenon that was first discovered by Tang1 (1884) and is referred to as traumatotactic nuclear migration. Using time-lapse video-microscopy and epidermal cells of Tradescantea leaves, Goodbody and Lloyd (1990) revealed that each nucleus migrates independently of nuclei in neighboring cells and that the migration occurs at variable rates, via transvacuolar strands as well as around the cortical cytoplasm, such that each nucleus ends up at the face of the wound. The traumatotactic migration is inhibited by ethionine, puromycin, and actinomycin D. Thus, it appears that this process depends on the synthesis of new mRNA and a new protein(s) after wounding. The synthesis of these molecules begins after a lag phase of about 1.5 hr. Inhibition by actinomycin D is reversible (Schnepf and Klump, 1975). The movement of nuclei is inhibited by CB or CD but not by colchicine, results that suggest that involvement of actin filaments (Schnepf and von Traitteur, 1973; Goodbody and Lloyd, 1990). The presence of actin filaments was confirmed by Rh-phalloidin staining (Goodbody and Lloyd, 1990). A belt of fine, cortical-actin filaments, parallel to the wound, was formed within 15 min after wounding. Migration of nuclei to the cell walls adjacent to the wound involved pronounced association of actin filaments with the nucleus. Later still, actin strands were seen to line up from cell to cell, parallel to the wound, anticipating the future plane of division. In protoplasts derived from BY-2 tobacco suspension-cultured cells, nuclei that are originally situated at the periphery migrate to the center of the

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protoplasts and are supported there by transvacuolar strands. This positioning of nuclei is maintained in the presence of propyzamide, a microtubule inhibitor, but not in the presence of CB. Thus, it appears that actin filaments alone can tether the nucleus, as appears to be the case for the nuclei during traumatotactic migration (Katsuta and Shibaoka, 1988). At the next stage, the nucleus migrates into the center of the cell from the wound site to which the nucleus had migrated before metaphase, for example, after 10 to 20 hr in leaf explants of Nautifocalyx fynchii(Venverloo et a f . , 1980), passing along transvacuolar strands (premitotic migration). Only the few ranks of cells nearest to the wound are supposed to undergo subsequent division that involves premitotic migration of the nucleus to the center of the cell. This premitotic migration is a normal feature of division in unwounded, vacuolated cells. Movement of organelles also occurs along all strands of the cytoplasm. While traumatotactic migration involves actin but not microtubules, premitotic migration of nuclei appears to involve microtubules. Bakhuizen et al. (1985) proposed that microtubules that radiate from the nucleus into the transvacuolar strands have a role first in mobilizing the nucleus, and then in stabilizing it in the plane of division. Using leaf explants of N . fynchii, Venverloo and Libbenga (1987) showed that migration of the nucleus to the center of the cell was inhibited by colchicine but not by CB. In cultured BY-2 cells, it was observed that premitotic migration of nuclei, accompanied by increasing numbers of transvacuolar strands composed of both microtubules and actin filaments, was not prevented by treatment with CD (Katsuta et a f . , 1990). However, Katsuta et a f . supposed that microtubules do not play an exclusive role in the migration of the premitotic nucleus since CD cannot completely destroy actin filaments and since simultaneous treatment with CD and propyzamid reduced the number of cells with a central nucleus to below 1%. Sinnott and Bloch (1940) observed that transvacuolar strands gradually aggregated, anastomosing into a diaphragm, the phragmosome. Formation of the phragmosome starts some hours after nuclear migration and persists during the successive stages of mitosis. The cell plate follows the course of the phragmosome exactly until the lateral wall is reached. This method of division is not limited to wounded tissue but is also observed in meristems of many types of plant, in which comparatively large vacuolated cells divide (Bloch, 1941; Sinnott and Bloch, 1945). In the phragmosome, both continuous bundles of microtubules that radiate from the surface of the nucleus and actin filaments associated with microtubules bridge the nucleus to the cell cortex. Other cytoplasmic strands, which are perpendicular to the plane of division, correspond to the polar strands (Sinnott and Bloch, 1940), also contain microtubules and actin filaments

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(Flanders et al., 1990; Goodbody and Lloyd, 1990; Katsuta et al., 1990; Lloyd, 1991). Next, or simultaneously with formation of the phragmosome, a band of cortical microtubules (preprophase band) is deposited at the site at which the phragmosome makes contact with the cell cortex. The preprophase band (PPB) is thought to be involved in the determination of the site of division (Pickett-Heaps and Northcote, 1966a,b; Palevitz and Hepler, 1974a,b; Gunning, 1982; Wick, 1991). The PPB has been shown not to regulate the premitotic migration of nucleus in tobacco BY-2 cells since migration of the nuclei occurs in the presence of aphidicolin, an inhibitor of DNA polymerase a that suppresses the formation of the PPB (Katsuta et al., 1990). Microtubules begin to disappear at late prophase from the phragmosome and the PPB, leaving actin filaments as the components of the transvacuolar cytoplasmic strands. Katsuta et al. (1990) suggested that the premitotic array of microtubules is set up as a scaffold for construction of the array of actin filaments. The array of actin filaments is, thereafter, responsible for maintaining the position of the mitotic apparatus. Venverloo and Libbenga (1987) demonstrated a requirement for actin filaments in maintenance of the mitotic apparatus at the equatorial plane, providing evidence that CB disrupted this positioning. In carrot suspension cells, it has been demonstrated that the nucleus is supported throughout cell division by actin filaments (Traas et al., 1987) and that actin filaments are present between the equatorial plane of the mitotic spindle and the cell cortex (Lloyd and Traas, 1988). The direction of expansion of the phragmoplast has also been suggested to be controlled by actin filaments since they connect the margin of the phragmoplast to the cell cortex (Kakimoto and Shibaoka, 1987). Using injured pea roots, Hush et al. (1990) examined arrays of cortical microtubules in cells at the wound tip. At interphase, arrays of microtubules underwent a reorientation from a transverse alignment to orientation in a plane parallel to the surface of the wound. Reorientation, which preceded the radial expansion of cells, occurred between 2 and 5 hr after wounding. A PPB then developed before cell division occurred, the new cell wall being positioned in exactly the same plane as the reoriented interphase array of microtubules. A new axis of alignment of cortical microtubules was established some 15 to 20 hr before formation of the phragmosome. Tang1 (1884) observed that the wound effect could be communicated through several layers of cells, a phenomenon that led him to the discovery of plasmodesmata. Goodbody and Lloyd (1990) showed that in Tradescantia leaves, the wound signal appears to be effective over approximately four cell diameters. If two individual punctured cells are separated by

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more than this distance, each wound is separately encircled by specifically dividing cells. When the wounds are closer together, the circles fuse to form one common dumbbell-shaped pattern without dividing cells around the two axes between the punctures. In slit wounds, also, mitosis is activated in cells that are three or four ranks away from the wound. Signals that emanate from a wound have been discussed, but remain to be characterized. Schnepf and Volkmann (1974) reported that thorough washing of wounded tissue inhibits traumatotaxis of the nuclei, suggesting that the signal(s) is of a chemical nature. A localized stimulation of cell divisipn and differentiation is now suggested to result from the diffusion of a wound-related hormone, such as auxin or ethylene, away from the site of the wound. However, the question remains how the diffusion of some chemical species, which presumably results in a concentration gradient through the tissue around the wound site, can result in the precise, new orientation of walls as cells divide within an array of cells that is highly ordered. As a more reasonable explanation for the proliferative aspect of wound reactions, Lintilhac and Vesecky (1981) proposed a response to physical stress. They argued that living tissue is in a condition of mechanical equilibrium, with internal regions of mutually opposed tension and compression, and that any external force causes a rearrangement of these equilibrium stresses such that a new family of division planes becomes possible. Ionic currents have recently been suggested to be involved in the process of wound healing and regeneration in higher plants (Gender, 1979; Davies and Schuster, 1981). The wound stimulus appears to induce a large inward current, which may involve an influx of Ca” ions and which is focused directly over the wound site. Hush et al. (1990), working with wounded pea roots, suggested that if cytosolic levels of Ca2+ ions are increased, then an intracellular gradient of Ca2+ions may serve to determine the new polar axis, possibly via effects on the cortical microtubules. B. Siphonous Green Algae

When we consider the large size and cellular nature of siphonous green algae, it seems clear that wounding must have serious consequences in these plants. The wound-healing process has been studied in representatives of all three orders of coenocytic Chlorophyceae. Some Codiales, such as Caulerpa (Lohr and Dawes, 1974; Dreher et al., 1978), Bryopsis (Burr and West, 1971; Burr and Evert, 1972), and Udotea (Mariani-Colombo and Postai, 1978; Mariani-Colombo and De Carli, 1980; Mariani-Colombo e f al., 1980), and the Dasycladales AcetabuZaria, (Menzel, 1981), have been reported to have the ability to seal a

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wound quickly with one or more insoluble plugs composed of proteins, polysaccharides, or both. The healed cells are able to continue to grow or to regenerate new plants or parts of plants. Dreher et al. (1978) described precisely the cytological changes that accompany wounding and recovery from wounding in Caulerpa simpliciuscufa. The following processes can be successively detected after wounding: (a) the immediate expulsion of a globule of viscous, colorless liquid that gels rapidly in seawater and adheres to the cut end of the tissue; ( 6 ) deposition of an internal wound plug during which process the cytoplasm migrates along the cell wall and adheres closely to the inside edge of the wound plug; ( c ) contraction of the cytoplasm away from the wound site and the formation of a large number of vesicles between the cytoplasm and the surface of the internal wound plug; ( d )gradual extension of the retracted cytoplasm to the inner surface of the internal wound plug with a corresponding resorbtion of some of the vesicular material; and ( e ) the synthesis of a new cell wall that begins within 11 hr and is complete after 6 days. The same pattern was described in Bryopsis (Burr and West, 1971), suggesting that similar mechanisms of wound healing may be operative. In a few genera of Siphonocladales, an entirely different response to wounding has been reported. For example, in Boergesenia, spherical tiny protoplasts (coenocysts or aplanospores), derived from the protoplasm, are induced by a puncture wound (Enomoto and Hirose, 1972; Ishizawa et al., 1979). Investigations by La Claire (1982a) showed that no wound plug is formed during healing in Ernodesmis, Boergesenia, Cladophoropis, and Siphonocfadus. The response to wound in these genera involves substantial protoplasmic motility, which includes retraction of the cell contents from the wound site. The protoplasm then either closes around the central vacuole or breaks up into numerous spherical protoplasts. Healing in Boodfea resembles the process described for Bryopsis. Motility related to wound healing in E. verticiffatais quite distinct from cytoplasmic streaming or the saltatory movement of organelles. Saltatory movements of chloroplasts can be detected only with time-lapse microcinematography, which ceases when the concentration of free Ca2+ions in the cytoplasm is higher than a threshold value, as is the case in characean cells. Wound-related motility is induced only when micromolar levels of free Ca2+ions and ATP are present, indicating that the motility resembles muscle motility and the majority of types of nonmuscle motility. La3+ions at 1 mM completely and reversibly inhibits the motility. Therefore, it has been postulated that an influx of Ca2+ions upon wounding, through calcium channels and from the external medium, is able to trigger motility (La Claire, 1982b, 1983, 1984a). The dynamic behavior of actin upon wounding, as revealed by immunofluorescence microscopy with actin-specific antibodies has been observed

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as follows: (a) Either fine punctate fluorescence or none at all is detected in intact cells, even though the presence of actin can be confirmed by other methods (La Claire, 1984b). (b) A reticulate pattern of fluorescence appears throughout the cortical cytoplasm shortly after wounding. (c) Slender, longitudinal bundles of actin filaments become evident in contracting regions of the cells. (d) Thicker bundles of actin filaments are observed in the contracted regions, suggesting contraction of the bundles. ( e ) Bundles of actin filaments are no longer evident after healing. (f)In cells wounded in Ca2+-freemedium, no actin bundles are ever observed, even though the actin is visible as a reticulate pattern of fluorescence (La Claire, 1989). It is noteworthy that wounding seems to induce very rapid (with a 2-min lag) and extensive synthesis of actin or the transformation of actin from an unpolymerized form to fibers, which further assemble to form bundles. Localized longitudinal arrays of actin bundles, the formation of which may be controlled by Ca2' ions, appear to be associated with longitudinal contraction of the cytoplasm and centripetal closure of the wound itself. Disappearance of the bundles after healing is a further interesting event. Ernodesmis seems to provide an excellent model system for studies of the regulatory mechanism of cytoskeletal organization and its functions. The localization of myosin also changes upon wounding (La Claire, 1991). Within 5 min of wounding, a faint reticulate pattern of immunofluorescence due to myosin becomes detectable, at the level of the plasma membrane. Doubly labeled preparations demonstrate that the reticulum also contains actin. Myosin becomes colocalized with actin in the slender linear arrays mentioned above. These results indicate that actin and myosin together form the motile apparatus that functions in contraction of cells during healing of the wound. Calmodulin is colocalized with actin-containing microfilaments in extensive, longitudinal bundles and in the reticulum (Goddard and La Claire, 1991a,b). Cytoplasmic motility can be inhibited in a dose-dependent manner by calmodulin antagonists. In cells treated with such inhibitors, bundles of microfilaments did not assemble or were very sparse. Immunogold labeling by anti-calmodulin IgG was associated with amorphous material studding the microfilament bundles. Goddard and La Claire proposed that calmodulin binds indirectly to actin by activating an actin-binding regulatory protein that functions in early stages of the transduction sequence leading to functional microfilament bundles. In contrast to the postulated role for actin, microtubules are not considered to be directly involved in wound-induced motility in these plants because amiprophos-methyl and cold treatment depolymerize most cortical microtubules without inhibiting wound-related motility (La Claire, 1987). A review has recently come out about contractile movements in the Siphonocladales (La Claire, 1992).

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V. Responses of the Cytoplasm t o Various Chemicals

As mentioned above (Section IV,A), wounding induces cytoplasmic streaming in cells near the wound site. Early workers suggested that some substances derived from injured cells might exert a stimulatory effect on the cytoplasm and referred to these substances as wound hormones (Haberlandt, 1902; see Bloch, 1941). In order to determine whether the rate of cytoplasmic streaming could be used as a measure of the immediate physiological changes brought about by auxin within cells, many investigators have examined the effects of auxin on streaming of the cytoplasm. In general, there is a parallelism between the effects of auxin on growth and its effect on streaming, although the effective concentrations differ between the two processes and among plants. Thimann and Sweeney (1937) suggested that the effects of auxin on streaming are closely connected with one of the first stages of the effect of auxin on the growth process. The effects of auxin on cytoplasmic streaming in various plant tissues have been reviewed by Kamiya (1959). The mechanism of acceleration of streaming by auxin remains to be determined. A growth substance that is acidic in nature and probably identical to indolacetic acid or one of its homologs was found in cut discs and slices of potato tubers (Hemberg, 1943). Hemberg supposed that products of the decomposition of injured cells served as activators for the formation of auxin or of a wound hormone of the type proposed by Haberlandt (1902). Fitting (1925) emphasized the probability of a hormonal effect on streaming and respiration in wound cells. He also found that an extract from leaves of V . spiralis, even at 106-fold dilution, could induce streaming in cells of Vallisneria. In his search for the effective chemical agents, Fitting found that amino acids, in particular L-histidine (L-His) and methylL-histidine (M-L-His), were effective at a concentration as low as M. L-Histidine was so effective that Fitting assumed that, in the leaf extract, the factor that induced the cytoplasmic streaming was L-His or an analog of this amino acid. He proposed the term “chemodinesis” for the induction of cytoplasmic streaming by chemical agents (Fitting, 1936). Recently, Tazawa el al. (1991) examined the effects of L-His and its derivatives (1-M-L-His, 3-M-~-His),as well as those of light, on the induction of rotational cytoplasmic streaming in leaf cells of Egeria densa, in an attempt to clarify difference between the mechanisms of induction by chemical and physical stimuli. All three amino acids penetrated the cells. L-Histidine induced streaming along the anticlinal walls via displacement of cytoplasm and chloroplasts from the periclinal wall, a pattern that resembled the pattern induced by light. The effective concentration of LHis was about 0.01 mM and the effect was almost saturated at 0.1 mM.

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A derivative of L-His, 3-M-~-His,was slightly less effective than L-His; 1-M-L-His was, however, almost ineffective in inducing streaming, not only in Egeria but also in Vullisneriu. This latter result contradicts Fitting’s report (1936) that l-M-L-His was more effective than L-His. The level of ATP in leaves kept in the dark (620 nmol/g fresh weight) was almost the same as that in leaves treated with 0.1 mM L-His for 20 min (640 nm/g fresh wt). Thus, it is evident that L-His does not act by increasing the intracellular level of ATP. The L-His-induced rotational streaming was inhibited with CB, as is the streaming induced by light irradiation. There is a difference in the time needed for mobilization of chloroplast between chemodinesis and photodinesis, the former requiring a much longer time than the latter. Furthermore, the number of chloroplast mobilized from the periclinal wall is larger in photodinesis. Light failed to increase the level of L-His in leaves. The results suggest that both light and L-His generate a common signal that leads to the induction of cytoplasmic streaming. Adenosine triphosphate may not be the signal in question. To gain some information about the signal, Tominaga et al. (1991) monitored changes in cytoplasmic pH (pH,) and vacuolar pH (pH,) during treatment of leaves of E. densa both with L-His and with light. For this purpose, they used in uiuo 31P-nuclearmagnetic resonance, which allows measurement of physiological parameters, such as pH, or pH,, under noninvasive conditions. Both irradiation and treatment with L-His increased the pH, by 0.3 units compared with the dark control value (average pH at 6.96). In contrast, pH, changed only slightly with both treatments. Tominaga et al. suggested that alkalization of the cytoplasm was brought about by these two different stimuli, perhaps via activation of the proton pump in the plasmalemma, and that alkalization may be involved in the mechanism of activation of cytoplasmic streaming. However, it is not clear at present whether the increase in pH, is linked, in the signal-transduction sequence, to the decrease in [Ca2+],that must be associated with the activation of motility (see section 11,A). Felle (1988a,b) reported that the increase in pH, and the decrease in [Ca”], are interrelated in Riccia and Zea. It is necessary now to examine the temporal changes in pH, and [Ca”], in Egeriu cells at early stages after receipt of the stimuli. Results of such an examination may provide information about which change occurs first in the transduction sequence and about the way in which the two factors are interrelated.

VI. Concluding Remarks

In the foregoing pages, I have reviewed various types of intracellular movement that are induced in response to environmental stimuli. From

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studies on the perception of stimuli, the signal-transduction pathways and the motile apparatus, several unified views have been developed. In some steps, however, certain problems still remain to be solved. In addition, several new findings, derived from recent precise observations, require us to consider novel explanations. In the following paragraphs, I will mention various issues that remain to be resolved. Photoreceptor: The action spectra of light-induced movements in most plant species have peaks in the blue and near UV regions. Events regulated by UV-B in plants, in addition to motility, are widely known. However, the nature of the photoreceptor(s) and the signal-transduction mechanism(s) that mediate the effects of UV-B are, as yet, unknown. Heterotrimeric guanosine triphosphate-binding (GTP-binding) regulatory proteins (G proteins) have been identified in a wide variety of animal cells as part of signal-transduction systems. The presence of a G protein was recently reported in association with the plasma membrane of the apical buds of etiolated peas seedling (Warpeha et al., 1991). A 40-kDa polypeptide was recognized by polyclonal antisera raised against the a subunit of the G protein transducin, from bovine retina. It has been suggested that the protein may function as the a subunit of a G protein that is active, at the low-fluence range, in light-mediated developmental processes in higher plants. It will be interesting to investigate whether such a protein is also involved in various types of intracellular movement induced by UV-B. Phytochrome has been identified as a rather exceptional photoreceptor for the orientation movement of chloroplasts in Mougeotia and Mesotaenium. Involvement of phytochrome has been newly confirmed in photodinesis in mesophyll cells of V. gigantea, where it acts in cooperation with photosynthetic pigments (Takagi et al., 1990). The primary action of phytochrome is, as yet, unknown. Observation that phytochrome in the Pfrform in mesophyll cells of Vullisneria (Takagi et al., 1992) exerts its effects on the mechanical properties of the cytoplasm prior to induction of intracellular movements is expected to open a way to solve the problem. Phytochrome has been reported to exist in multiple forms, namely as photolabile and photostable pools at the physiological level and as genetically distinct molecular species at the protein level (Furuya, 1989). It will be of interest, in terms of the physiology of phytochrome, to determine which pool and which molecular species are functioned in these cells. Plant hormone: If we assume that wound-induced streaming of cytoplasm serves as a means of transport of materials for cell division and differentiation after wounding, the role of auxin in the induction of streaming by wounding cannot be ignored. In this connection, it should be mentioned that auxin receptors, namely a protein with a 22-kDa subunit and a 42-kDa dimer, have been identified in maize endoplasmic membranes

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(Jones and Venis, 1989; Jones et al., 1989) and in plasma membranes of zucchini and tomato (Hicks et al., 1989a,b),respectively. A 65-kDaprotein has also been shown to bind auxin specifically. The protein has been found in a wide range of plants, localized in the nucleus and, probably, also in the cytosol (Prasad and Jones, 1991). It should be also mentioned that externally applied indole-3-acetic acid at 1 p M triggers oscillations in the membrane potential, pH,, and [Ca2'], with a period of 20 to 30 min and that a reduction in pH, (acidification) increases [Ca2+],(Felle, 1988a). 2,4-Dichlorophenoxyaceticacid, an analog of auxin, has also been confirmed to induce, over a period of 4 min, a decrease in pH, of 0.1 to 0.2 pH units and an increase in [Ca2+],from 280 to 380 nM (Gehring et al., 1990). The interrelationship between [Ca2+], and pH, in signal-transduction pathways clearly merits further investigation. The induction of streaming by ethylene has not been reported to date. Ca2+ions: There can be no doubt about the crucial role of Ca2+ions in the mediation between each stimulus and the final response. However, the mode of involvement of Ca2+ions differs from cell to cell. In skeletal muscle, contraction is evoked by Ca2+ions at concentrations above low6 M , whereas streaming of cytoplasm in characean cells (Williamson and Ashley, 1982; Tominaga et al., 1983) and in mesophyllic cells of V. gigantea (Takagi and Nagai, 1986) is inhibited by Ca2+ions at concentrations M. In Mougeotia cells, an increase in the concentration higher than of Ca2+ions is suggested to be essential for accomplishment of reorientation of the chloroplasts. Information has accumulated about various calcium-binding proteins and chains of reactions that mediate the action of Ca2+ions in animal cells. The presence and mode of regulation of such proteins have not yet been fully investigated in plant cells. Motile systems: Actin filaments, in bundled form, play the role of tracks along which organelles move and they are anchored in the ectoplasmic layer of cells. Studies on the mechanism of anchoring of actin filaments (Kropf et al., 1988; Masuda et al., 1991) and also of microtubules (Akashi and Shibaoka, 1991) have provided evidence that cytoskeletal elements have a close relationship to the extracellular matrix, namely the cell wall in the case of plant cells, as has been generally accepted to be the case in animal cells. It is an important task for the future to identify what kinds of molecule are involved and the ways in which they participate in the organization and stabilization of the cytoskeleton in plant cells. Bundles of actin filaments in some cases, for example, in Mougeotia (Schonbohm; 1973;Mineyuki and Nagai, 1991)and Ernodesmis (La Claire, 1989), are formed and disintegrate at each initiation and termination of the movement of organelles. Formation of a belt of actin filaments parallel to the wound surface (Goodbody and Lloyd, 1990)may be one of the earliest responses in higher plants, occurring prior to the traumatotactic migration

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of nuclei to the wall adjacent to the wound. Investigations of mechanisms that regulate these dynamic rearrangements of actin filaments, in terms of molecular species of proteins and their mode of involvement, seem to be crucial if we are to understand the transduction chains involved in stimulus-induced movements in these cells. Encirclement of chloroplasts by bundles of actin filament, which may function to anchor the chloroplasts at their destination, after reorientation movement has been achieved in Adianturn represents another type of rearrangement of actin filaments (Kadota and Wada, 1989). It will be of interest to determine whether this process resembles the orientation movement of chloroplasts to the periclinal wall, which is induced under light of low fluence in epidermal cells of Vullisneria (Seitz, 1967, 1979a,b; Izutani et al., 1990). Myosin has been isolated and characterized from only a limited number of plants. Its intracellular localization and mode of regulation by [Ca”], ions and regulatory protein(s) are crucial issues to be clarified. There are many reports of microtubule-based movements. The involvement of microtubules in the migration of nucleus to the appropriate sites prior to division of cells seems to have sound experimental evidences. However, the mechanism of the cell cycle-dependent construction of arrays of microtubules that associate with the nucleus must be clarified. Reports on the green alga Dichotornosiphon (Maekawa and Nagai, 1988) present another aspect of the dynamic rearrangement of arrays of microtubules that occurs in concert with the light-dependent orientation of the cytoplasm. A novel MAP of 90 kDa was identified in the course of studies on the regulation of the rearrangement of microtubules in this alga (Maekawa et al., 1990). Purification of the protein and examination of its interaction with microtubules in uitro may open a way toward resolution of the problem. Nothing is known in plant cells about the motor proteins kinesin and cytoplasmic dynein, which interact with microtubules, even though detailed investigations have been carried out in animal cells (Vale, 1987; McIntosh and Porter, 1989; Warner and McIntosh, 1989; Warner et al., 1989). Such proteins in plant cells need prompt attention. It seems that we are now at the beginning of a long and difficult series of investigations at the molecular level. Current problems associated with such investigations may, in general, be due to the cell wall, the small amount of cytoplasm, and the large vacuoles that contain many very active proteases. Reliable methods for overcoming these difficulties must be developed. Acknowledgments My sincere thanks go to Professor Emeritus Noburo Kamiya of Cell Physiology at Osaka University for critical reading of the manuscript. I also thank Dr. Shingo Takagi for criticism and help with preparation of figures and photographs.

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A

Accessory genes, urease in plant cells and, 86, 88, 92, 98-99 Acetabularia, environmental stimuli in plant cells and, 270, 294 Acetate, higher plant plastids and, 187-189 Acetohydroxamate, urease in plant cells and, 75 N-Acetylaspartate (NAA), nuclear magnetic resonance and, 43, 45-46 Acetylcholine, hormonal control of amphibian metamorphosis and, 128 Acetyl CoA, higher plant plastids and, 187-189, 203 Acetyl CoA carboxylase (ACC), higher plant plastids and, 182-183 Acidification environmental stimuli in plant cells and, 290, 300 nuclear magnetic resonance and, 42 Acidity, urease in plant cells and, 83 Actin, environmental stimuli in plant cells and, 300-301 F-actin, 260-261, 277-279 light, 278-280, 283 low temperature, 289 wounding, 291-292, 295-296 Activation, higher plant plastids and, 172- 176 Actomyosin, environmental stimuli in plant cells and, 264-265, 282, 289 Acyl carrier protein (ACP), higher plant plastids and, 182-185 Adenylate, higher plant plastids and, 180, 190 Adiantum, environmental stimuli in plant cells and, 269, 273-274, 283, 301 ADP, higher plant plastids and, 174-177, 180-182, 190

31 1

Adrenocortical hormones, control of amphibian metamorphosis and, 105, 119-123 secretion, 123-128 Adrenocorticotropic hormone (ACTH), control of amphibian metamorphosis and, 113, 116, 122-126, 128 Aldosterone, control of amphibian metamorphosis and, 119-120, 122-123, 125-127 Allantoate, urease in plant cells and, 75-77 Allantoic acid, urease in plant cells and, 73-74, 76, 78, 95 Allantoin, urease in plant cells and, 73, 75-79, 95-96 AIIium sepa, environmental stimuli in, 291 Allopurinol, urease in plant cells and, 77-79, 96-97 Ambystoma gracile, hormonal control of metamorphosis and, 107 Ambystoma mexicanum, hormonal control of metamorphosis and, 119, 125 Ambystoma tigrinum, hormonal control of metamorphosis and, 107, 119, 124, 135 Amides higher plant plastids and, 167 urease in plant cells and, 74, 77 Amidinohydrolases, urease in plant cells and, 74-75,7940 Amidohydrolases, urease in plant cells and, 74-76 Amino acids eggshells and biomineralization and, 220, 223, 231, 234, 237 environmental stimuli in plant cells and, 297 higher plant plastids and, 204 fatty acid synthesis, 185 structure, 158-159

312 synthesis, 190-201 translocation, 167-169, 171 hormonal control of amphibian metamorphosis and, 116, 127, 137-138, 139 nuclear magnetic resonance and, 37, 45 urease in plant cells and, 98 biochemical genetics, 86, 89, 91 elimination, 81, 83 metabolic origins, 67-68, 74, 77 Ammonia higher plant plastids and, 191-201 urease in plant cells and, 66,74,83-84,89 Ammonium higher plant plastids and, 197, 199 nuclear magnetic resonance and, 3 Amniotic fluid, nuclear magnetic resonance and, 37 Amphenone, contror of amphibian metamorphosis and, 120 Amphibian metamorphosis, hormonal control of, see Hormonal control of amphibian metamorphosis Amyloplasts, higher plant metabolism, 169, 171, 203 starch synthesis, 177, 179-180 structure, 151, 161, 163 Anchorage-dependent cells, nuclear magnetic resonance and, 14-15, 37 Angiosperms, higher plant plastids and, 154, 158, 161 Angiotensin 11, control of amphibian metamorphosis and, 127, 129 Antibiotics, higher plant plastids and, 160 Antibodies eggshells and biomineralization and, 220 environmental stimuli in plant cells and, 263, 285-286, 295 higher plant plastids and, 169, 174 hormonal control of amphibian metamorphosis and, 133 monoclonal, see Monoclonal antibodies urease in plant cells and, 93 Antigens, see also Epitopes higher plant plastids and, 169 urease in plant cells and, 92 Aphids, urease in plant cells and, 77 Apparent diffusion coefficient (ADC), nuclear magnetic resonance and, 27 Arabidopsis plastids and amino acid synthesis, 193-194, 199

INDEX

fatty acid synthesis, 185-186 metabolism, 170, 173 starch synthesis, 181-182 structure, 164 urease in plant cells and, 98-99 Arabidopsis thuliuna, urease in plant cells and, 82, 90 Ardisia, urease in plant cells and, 97 Arginase, urease in plant cells and, 66-68, 71-72, 82-83 Arginine, urease in plant cells and, 98 elimination, 80-83 metabolic origins, 66-72, 78-79 Arginine vasotocin (AVT), control of amphibian metamorphosis and, 125-126 ATP environmental stimuli in plant cells and, 295, 298 chloroplasts, 276-277, 282-283 photodinesis, 259, 263-265 higher plant plastids and CO, fixation, 171, 174-176 fatty acid synthesis, 189-190 metabolism, 168, 203 starch synthesis, 177, 180-181 nuclear magnetic resonance and, 2, 7, 19, 42-43 urease in plant cells and, 80 ATPase environmental stimuli in plant cells and, 261, 263, 280, 283, 290 higher plant plastids and, 174 Atrazine, environmental stimuli in plant cells and, 254 Atrial natriuretic peptide (ANP), control of amphibian metamorphosis and, 127 Autoradiography, hormonal control of amphibian metamorphosis and, 135 Auxin, environmental stimuli in plant cells and, 294, 297, 299-300 Auena, plastids and, 171 Avocado, plastids and, 184-185 Azaserine, urease in plant cells and, 78

B

Background urease in plant cells, 92, 95 Bacteria, see also specific bacteriu eggshells and biomineralization and, 235 urease in plant cells and, 65, 98-99 biochemical genetics, 88-90, 92

313

INDEX

elimination, 80, 83 metabolic origins, 66, 74, 76, 79 nickel metabolism, 92-97 Barley plastids and metabolism, 184-186, 188, 197, 199 structure, 162- 163 urease in plant cells and, 81 Beans environmental stimuli in, 290 plastids and, 183, 185-186, 189, 194, 203 urease in plant cells and, 68,76-77,89-91 Biomineralization and eggshells, see Eggshells, biomineralization and Biotin, higher plant plastids and, 182, 190 Blood, nuclear magnetic resonance and, 29 Blood flow, cerebral, nuclear magnetic resonance and, 32 Boergesenia, environmental stimuli in, 295 Brain hormonal control of metamorphosis and, 107, 137, 141 nuclear magnetic resonance and, 19, 24, 29, 32, 35, 43-45 urease in plant cells and, 83 Branching enzyme, higher plant plastids and, 177 Brussica, plastids and, 185-186 Breast cancer cells, nuclear magnetic resonance and, 37, 39 Bryopsis, environmental stimuli in plant cells and, 270, 294-295 Bufo cularnita, hormonal control of metamorphosis and, 113 Bufo juponicus, control of metamorphosis and adrenocortical hormones, 119-120, 122-123, 127 growth hormone, 138 prolactin, 130, 135 thyroid hormone, 107, 1 I5 Bufo marinus, hormonal control of metamorphosis and, 135 Bufo melanostictus, hormonal control of metamorphosis and, I13 Bufo woodhousei, hormonal control of metamorphosis and, 140

C

Calbindin; eggshells and biomineralization and, 235

Calcification, eggshells and biomineralization and, 217-218, 236, 244 models, 24 I , 243 structure, 223, 226, 229, 234 Calcite, eggshells and biomineralization and, 238-240 Calcium eggshells and biomineralization and, 217, 24 1 fabrication, 236, 238, 240 structure, 223, 227, 231, 234 environmental stimuli in plant cells and, 298, 300-301 chloroplasts, 279-283, 286 low temperature, 289-290 nucleus, 288 photodinesis, 261, 263-268 wounding, 294, 296 nuclear magnetic resonance and, 3, 20 Calcium-binding proteins, eggshells and biomineralization and, 234 Calcium carbonate, eggshells and biomineralization and, 244 fabrication, 236-237 models, 241, 243-244 structure, 221, 230 Calcium chloride, eggshells and biomineralization and, 243 Calcium phosphate, eggshells and biomineralization and, 244 Calcium reserve assembly (CRA), eggshells and biomineralization and, 227, 238 Calibration, nuclear magnetic resonance and, 24 Calmodulin, environmental stimuli in plant cells and, 265, 281-282, 286, 2% Cancer cells, nuclear magnetic resonance and, 37, 39, 42 Cancer therapy, nuclear magnetic resonance and, 50 Cupsecum annuum, plastids and, 161 Carbohydrates eggshells and biomineralization and, 230, 234-235 higher plant plastids and, 150, 193, 201-203 hormonal control of amphibian metamorphosis and, 122-123 Carbon higher plant plastids and amino acid synthesis, 192-193, 196-197

314 C 0 2 fixation, 174, 176 fatty acid synthesis, 184-185, 188 starch synthesis, 177, 179-181 structure, 160 translocation, 167-171 urease in plant cells and, 66, 74, 76 Carbonate, eggshells and biomineralization and, 244 fabrication, 236-238, 240 models, 241, 243-244 structure, 221, 230 Carbon dioxide higher plant plastids and, 150 amino acid synthesis, 192-193, 196-197, 199 fatty acid synthesis, 182, 189 fixation, 171-176 urease in plant cells and, 69, 71, 74-76 Carbonic anhydrase, eggshells and biomineralization and, 224, 226, 234 Carboxyarabinitol bisphosphate, higher plant plastids and, 174 Carboxyarabinitol I-phosphate, higher plant plastids and, 174-175 Carboxylase, urease in plant cells and, 80 Cardiovascular system images, nuclear magnetic resonance and, 31 Carnitine, higher plant plastids and, 182, 187- 189 Carotenoids, higher plant plastids and, 153 Catecholamines, hormonal control of amphibian metamorphosis and, 122-123 Caulerpa, environmental stimuli in plant cells and, 270, 294-295 Cellular suspension, nuclear magnetic resonance and, 14, 19, 35 Central nervous system, hormonal control of metamorphosis and, 112, 122, 127 Centrifugal force, environmental stimuli in plant cells and chloroplasts, 271, 276, 279, 281 photodinesis, 257, 259-360 Centrifuge microscopy, environmental stimuli in plant cells and, 258 Cerebrospinal fluid, nuclear magnetic resonance and, 37 Chara, environmental stimuli in, 261, 264-165 Chemicals, environmental stimuli in plant cells and, 251-252 Chemical shift, nuclear magnetic resonance and, 7-8, 43-44

INDEX

Chemical shift imaging, nuclear magnetic resonance and, 23, 43 Chlamydomonas. plastids and, 159 Chlorophyll, higher plant plastids and, 164, 166-167, 190 Chloroplasts environmental stimuli in plant cells and, 252, 300-301 chemicals, 297-298 light, 268-275 nucleus, 287 photodinesis, 252-253, 257-259, 263 transduction chain, 275-287 wounding, 295 higher plant amino acid synthesis, 191, 194, 196-199 carbohydrate oxidation, 201, 203 CO, fixation, 172-173 evolution, 164-167 fatty acid synthesis, 183-189 genome, 153-164 starch synthesis, 177, 179-182 structure, 151 translocation, 167-171 Chlorotetracycline, environmental stimuli in plant cells and, 280-281, 289 Choline, nuclear magnetic resonance and, 43, 45-46 Chondroitin sulfate, eggshells and biomineralization and, 226, 231 Chromoplasts, higher plant metabolism, 175, 190, 197 structure, 151, 153, 161-163 Chromosomes, higher plant plastids and, 165 Cirrus, plastids and, 161 Cladophoropsis, environmental stimuli in, 295 Clarkia xantiana, higher plant plastids and, 181-182 Clones higher plant plastids and, 165, 168, 185, 192 urease in plant cells and, 86, 89, 91, 98 Cluster clone theory, higher plant plastids and, 165 Colchicine, environmental stimuli in plant cells and, 283-284, 291-292 Collagen eggshells and biomineralization and, 221, 234, 237, 244 hormonal control of amphibian metamorphosis and, 129-130, 133-134, 136

315

INDEX

Complementary DNA higher plant plastids and, 158, 168, 185, 192 hormonal control of metamorphosis and, 111, 130, 133, 138 urease in plant cells and, 89 Connective tissue, hormonal control of amphibian metamorphosis and, 129 Contrast agents, nuclear magnetic resonance and, 30-31 Corticoids, control of amphibian metamorphosis and, 119-123, 125-128 Corticosteroids, control of amphibian metamorphosis and, 125, 127 Corticosterone, control of amphibian metamorphosis and, 119-122, 125-127 Corticotropin-releasing factor (CRF), hormonal control of amphibian metamorphosis and, 116, 124-125 Cortisol, control of amphibian metamorphosis and, 120-121 Cotyledons higher plant plastids and, 151, 153, 177, 179, 189 urease in plant cells and, 95 elimination, 82-84 metabolic origins, 68-69, 71-72, 76-78 Cowpea, urease in plant cells and, 77-78 Creatine, nuclear magnetic resonance and, 45-46 Creatine phosphate, nuclear magnetic resonance and, 2, 19 Crystallization, eggshells and biomineralization and, 230, 238-239, 241, 243-244 Cuphea lurea, plastids and, 184 Cuticle, eggshells and biomineralization and, 219, 234-235, 240 Cyanelles, higher plant plastids and, 166-167 Cyanobacteria, higher plant plastids and, 166 Cyanophora paradoxa, plastids and, 166- 167 C ycloheximide hormonal control of amphibian metamorphosis and, 121 urease in plant cells and, 91 Cyclosis, environmental stimuli in plant cells and, 253, 268, 276, 289 Cyclotriphosphazatriene, urease in plant cells and, 82

Cytochalasin B, environmental stimuli in plant cells and chemicals, 298 light, 260, 275-277, 284 wounding, 291-293 Cytokinin, environmental stimuli in plant cells and, 287-288 Cytoplasm environmental stimuli in plant cells and, 251-252, 301 chemicals, 297-298 chloroplasts, 268-270, 276-282, 284 low temperature, 288-290 nucleus, 287-288 photodinesis, 252-253, 257-261, 266 wounding, 290-296 higher plant plastids and, 149 amino acid synthesis, 191, 200 starch synthesis, 177, 179-181 translocation, 167- 168, 170 urease in plant cells and, 69, 84, 90-91 Cytoplasmic inheritance, higher plant plastids and, 153 Cytoplasmic streaming, environmental stimuli in plant cells and chemicals, 297 chloroplasts, 283 low temperature, 288-290 photodinesis, 252, 257, 259-261, 265 wounding, 295 Cytoskeleton, environmental stimuli in plant cells and, 261, 278, 281-282, 296, 300

D Dark adaptation, environmental stimuli in plant cells and, 257, 276, 280 Decalcification, eggshells and biomineralization and, 227, 229, 231 Decarboxylation, urease in plant cells and, 67 Decoupling, nuclear magnetic resonance and, 9, 44 5-Deiodinase, hormonal control of metamorphosis and, 107 Deiodination, hormonal control of metamorphosis and, 110 Deoxycorticosterone, control of amphibian metamorphosis and, 120-121 Depolymerization, environmental stimuli in plant cells and, 279, 285-286, 296

316

INDEX

Depth resolved surface spectroscopy (DRESS), pathology and, 21 Desmosine, eggshells and biomineralization and, 220-221 Diamagnetic substances, nuclear magnetic resonance and, 30 Dicarboxylates, higher plant plastids and,

167,170 3-(3',4'-Dichlorophenyl)I, 1-dimethylurea (DCMU), environmental stimuli in plant cells and, 253-254,259,267 Dichotomosiphon tuberosus, environmental stimuli in plant cells and, 270,283-287,

301 Diffusion, nuclear magnetic resonance and,

Eggshells, biomineralization and, 217-218,

244-245 fabrication, 235-240 models, 240-244 structure, 218-219 cuticle, 234-235 mamillary layer, 224-229 palisade, 229-234 shell membrane layer, 220-224 EGTA, environmental stimuli in plant cells and, 276,290 Elaioplasts, higher plant, 151 Elastase, eggshells and biomineralization and, 220 Elastin, eggshells and biomineralization and,

220-221

26-29 Digitalis purpurea, environmental stimuli in,

289-290 DNA complementary, see Complementary DNA higher plant plastids and, 151,204 evolution, 165-166 genome, 153-155,158-159,161,

163-164 hormonal control of amphibian metamorphosis and, 140 ribosomal, see Ribosomal DNA Dopamine, hormonal control of amphibian metamorphosis and, 128,134,136-137 Drug resistance, nuclear magnetic resonance and, 37 Drugs, nuclear magnetic resonance and, 20,

31, 37,41

Electrocardiogram, nuclear magnetic resonance and, 31 Electrolytes, nuclear magnetic resonance and, 19 Electron microscopy, environmental stimuli in plant cells and, 266,277,284 Electron transport, higher plant plastids and, 150, 191,193

Electrophoresis eggshells and biomineralization and, 221 environmental stimuli in plant cells and,

263 hormonal control of amphibian metamorphosis and, 116,129, 133,

140 urease in plant cells and, 85, 88-89,89 SDS-PAGE, see SDS-PAGE Elodea, environmental stimuli in, 252,

260-261,263 E

Echo planar imaging (EPI), nuclear magnetic resonance and, 29,32 Echo time, nuclear magnetic resonance and,

11-12,23 Ectoplasm, environmental stimuli in plant cells and, 276,300 Edema, nuclear magnetic resonance and, 46 EDTA eggshells and biomineralization and, 227,

229,234 environmental stimuli in plant cells and,

261,263 Egeria, environmental stimuli in, 263,

297-298

Elodea canadensis, environmental stimuli in, 252-253,260 Embryos eggshells and biomineralization and, 217,

223,227,238 higher plant plastids and, 164,180-181 hormonal control of amphibian metamorphosis and, 106,115,118,

123,140-141 urease in plant cells and, 98 biochemical genetics, 84-86 elimination, 80-84 metabolic origins, 67,69,71-72,77 nickel metabolism, 92-93,95 Endocrine mechanisms, hormonal control of amphibian metamorphosis and, 105,141 Endodesmis, environmental stimuli in, 263,

296,300

317

INDEX

Endoplasm, environmental stimuli in plant cells and, 259, 263, 265, 284, 299 Endoplasmic reticulum environmental stimuli in plant cells and, 263, 289 hormonal control of amphibian metamorphosis and, I16 Endosperm higher plant plastids and carbohydrate oxidation, 201, 203 fatty acid synthesis, 183, 189 metabolism, 169, 180 structure, 151 urease in plant cells and, 68 Endosymbiotic theory, higher plant plastids and, 164-166 Environment, higher plant plastids and, 176 Environmental stimuli in plant cells, 251-252, 298-301 chemicals, 297-298 light chloroplasts, 268-275 nucleus, 287-288 transduction chain, 275-287 low temperature, 288-290 photodinesis, 252-259 motile systems, 259-263 signal transduction, 263-268 wounding higher plants, 290-294 siphonous green algae, 294-296 Enzymes, see also specific enzymes eggshells and biomineralization and, 231, 241, 244 environmental stimuli in plant cells and, 261, 266 higher plant plastids and, 2-4, 149 amino acid synthesis, 191-194, 196-198, 200-201 carbohydrate oxidation, 201-203 C 0 2fixation, 172-176 fatty acid synthesis, 182-185, 187, 189 starch synthesis, 177, 179, 182 structure, 153, 161 translocation, 170 hormonal control of amphibian metamorphosis and, 123-124 nuclear magnetic resonance and, 33 urease in plant cells and, 65, 84, 98-99 biochemical genetics, 86, 89 metabolic origins, 66, 72, 76 Eoplasts, higher plant, 150-151

Epidermal cells, environmental stimuli in plant cells and, 275-277 Epifagus uirginiana, plastids and, 158, 161 Episomal theory, higher plant plastids and, 164-165 Epithelium eggshells and biomineralization and, 219, 236 hormonal control of amphibian metamorphosis and, 118, 131, 133, 140 urease in plant cells and, 83 Epitopes, see also Antigens eggshells and biomineralization and, 220, 237 higher plant plastids and, 168 Eremosphaera, environmental stimuli in plant cells and, 268, 271 Erythrocytes hormonal control of amphibian metamorphosis and, 110, 121 nuclear magnetic resonance and, 19 Escherichia coli higher plant plastids and, 184-185 urease in plant cells and, 92 Estradiol, control of amphibian metamorphosis and, 121 Ethylenediaminetetraacetic acid, see EDTA Eth yleneglycol-bis(2-amhoethylether)N,N,N’,N’-tetraacetic acid, see EGTA Etioplasts, higher plant, 150-151, 162-163, 171, 188 Euglena, plastids and, 157, 167 Eukaryotes higher plant plastids and, 149, 165-166 urease in plant cells and, 80 Eurycea bislineata, hormonal control of metamorphosis and, 107 Evolution eggshells and biomineralization and, 217 higher plant plastids and, 149, 155, 164-167, 174 Excitation, nuclear magnetic resonance and, 5-6, 45

F

F-actin, environmental stimuli in plant cells and, 260-261, 277-279

318

INDEX

Far-red (FR) light, environmental stimuli in plant cells and chloroplasts, 272, 277, 280-281 nucleus, 288 photodinesis, 254, 258-259, 266 Fast low-angle shot (FLASH) sequences, nuclear magnetic resonance and, 29 Fatty acid synthesis, higher plant plastids and, 150, 171, 182-190, 203 Fatty acid synthetase (FAS), higher plant plastids and, 182-187 Feedback, hormonal control of amphibian metamorphosis and, 117-1 19 Ferredoxin, higher plant plastids and, 192-194, 200-201 Ferromagnetic substances, nuclear magnetic resonance and, 30 Flavin, environmental stimuli in plant cells and, 253, 271 Flow imaging, nuclear magnetic resonance and, 25-26 Fluorescein isothiocyanate (FITC), environmental stimuli in plant cells and, 261, 278 Fluorescence environmental stimuli in plant cells and light, 263, 277-278, 280-282 low temperature, 290 wounding, 296 hormonal control of amphibian metamorphosis and, 122 Fluorine, nuclear magnetic resonance and, 20, 32, 41 Follicle-stimulating hormone, control of amphibian metamorphosis and, 113, 115 Fourier acquired steady state (FAST), nuclear magnetic resonance and, 29 Fourier transform (FT), nuclear magnetic resonance and, 4, 6 Free induction decay (FID), nuclear magnetic resonance and, 4 Fructose bisphosphatase, higher plant plastids and, 177, 180 Funaria, environmental stimuli in, 268, 271, 288

G Gadolinium inflow, nuclear magnetic resonance and, 29

Gas exchange, eggshells and biomineralization and, 230, 235 Gastric glands, urease in plant cells and, 83 Gene expression higher plant plastids and, 159-162, 164 hormonal control of amphibian metamorphosis and, 105, 121 Gene products, urease in plant cells and, 88-92 Genes accessory, see Accessory genes higher plant plastids and, 204 evolution, 165-166 metabolism, 173, 186, 194 structure, 153-164 structural, see Structural genes Genetics, urease in plant cells and, 65, 79, 98-99 accessory genes, 86, 88 gene products, 88-92 metabolic origins, 75, 78 structural genes, 84-87 Genotype, urease in plant cells and, 93, 97 Geranium, plastids and, 155 Germination higher plant plastids and, 177 urease in plant cells and elimination, 80-83 metabolic origins, 71-72 nickel metabolism, 92, 94-95 Gliosis, nuclear magnetic resonance and, 50 Globulin, urease in plant cells and, 68 Glucorticoids, control of amphibian metamorphosis and, 122 Glucose higher plant plastids and, 177, 179-181, 20 1 nuclear magnetic resonance and, 37-38 Glucose-1-phosphate, higher plant plastids and, 169, 179-180 Glucose-6-phosphate, higher plant plastids and, 169, 180, 193-194, 201, 203 Glucose pyrophosphorylase, higher plant plastids and, 181-182 Glutamate higher plant plastids and, 169, 171 urease in plant cells and, 69 Glutamate decarboxylase, urease in plant cells and, 69

319

INDEX

Glutamine higher plant plastids and, 169, 199-200 urease in plant cells and, 74, 78 Glutamine synthetase, higher plant plastids and, 197-201 Glycine decarboxylase complex (GDC), higher plant plastids and, 196, 198 Glycol ysis higher plant plastids and, 150, 171, 189-190, 201-203 nuclear magnetic resonance and, 37, 45 Glycoproteins eggshells and biomineralization and, 221, 231, 235 hormonal control of amphibian metamorphosis and, 113, 132 urease in plant cells and, 91 Glycosaminoglycans, eggshells and biomineralization and, 231, 237 Glycosylation, urease in plant cells and, 91 Glyoxylate, urease in plant cells and, 75-76 Glyoxysomes, urease in plant cells and, 76 Golgi complex higher plant plastids and, 149 hormonal control of amphibian metamorphosis and, 116 Gonadotropins, control of amphibian metamorphosis and, 113 G protein, environmental stimuli in plant cells and, 299 Growth factors, nuclear magnetic resonance and, 17 Growth hormone, control of amphibian metamorphosis and, 113, 129, 131, 137- I40 Growth-promoting activity, hormonal control of amphibian metamorphosis and, 128-130, 132, 137 GTP, environmental stimuli in plant cells and, 299 P-Guanidinopropionate, urease in plant cells and, 72, 78 Gymnosperms, higher plant plastids and, 154

H Heart hormonal control of amphibian metamorphosis and, 107, 127 nuclear magnetic resonance and, 43-44

HeLa cells, nuclear magnetic resonance and, 35, 31 Helicobacter pylori, urease in plant cells and, 83 Helodea, environmental stimuli in, 257, 276 Heraeleum, environmental stimuli in, 261 Hexose, higher plant plastids and, 179-180, 189 Higher plant plastids, see Plastids, higher plant High-pressure liquid chromatography (HPLC), hormonal control of amphibian metamorphosis and, 115 Histidine, environmental stimuli in plant cells and, 297-298 Homology higher plant plastids and, 155, 157, 168 hormonal control of amphibian metamorphosis and, 115, 130, 133, 138 urease in plant cells and, 92 Hordeum uulgare, environmental stimuli in, 253 Hormidium jlaccidum, environmental stimuli in, 270 Hormonal control of amphibian metamorphosis, 105-106, 108-109, 140- 141 adrenocortical hormones, 119-123 secretion, 123-128 growth hormone, 137-140 prolactin larval growth, 128-132 levels, 133-137 thyroid hormone, 105-107, 110-1 12 feedback, 117-1 19 hypothalamic control, 115-1 17 pituitary control, 112-1 15 Hormones environmental stimuli in plant cells and, 294, 297, 299-300 nuclear magnetic resonance and, 37 Humidity eggshells and biomineralization and, 235 environmental stimuli in plant cells and, 25 1 higher plant plastids and, 176 Hyacinfhus, environmental stimuli in, 263 Hybridization higher plant plastids and, 204

320

INDEX

hormonal control of amphibian metamorphosis and, 112, 116 Hydrolysis eggshells and biomineralization and, 24 1 higher plant plastids and, 168, 187 urease in plant cells and biochemical genetics, 89-90 elimination, 80, 82 metabolic origins, 71, 75, 77-78 Hydrophobicity, higher plant plastids and, 153, 168 3P-Hydroxysteroid dehydrogenase, control of amphibian metamorphosis and, 123- 124 Hynobius nigrescens, hormonal control of metamorphosis and, 107 Hyperammonemia, urease in plant cells and, 83 Hypersecretion, hormonal control of amphibian metamorphosis and, 136 Hypophyseal factors, hormonal control of amphibian metamorphosis and, 123-126 Hypophysectomy, control of amphibian metamorphosis and adrenocortical hormones, 122-125 growth hormone, 138-140 prolactin, 128, 131 thyroid hormone, 112, 118 Hypothalamus control of amphibian metamorphosis and, 140- 141 adrenocortical hormones, 122, 124 growth hormone, 140 prolactin, 128, 133, 136-137 thyroid hormone, 115-1 19 hormonal control of amphibian metamorphosis and, 140-141 Hypoxia, nuclear magnetic resonance and, 25, 42, 50

I Imaging, nuclear magnetic resonance and, 13, 25, 32 cardiovascular system, 31 diffusion, 26-29 magnetic angiography, 25-26 magnetization transfer, 29-30

metabolites, 30 microscopy, 31-32 perfusion, 29 receptor imaging, 30-31 Immunofluorescence environmental stimuli in plant cells and, 263, 285, 295-296 hormonal control of amphibian metamorphosis and, 110 Immunoglobulin G , hormonal control of amphibian metamorphosis and, 113, 137 Immunoprecipitation, hormonal control of amphibian metamorphosis and, 124 Immunoreactivity, control of amphibian metamorphosis and adrenocortical hormones, 122, 124, 126- 127 growth hormone, 139-140 prolactin, 132, 137 thyroid hormone, 113 Indirect immunofluorescence microscopy, environmental stimuli in plant cells and, 263, 287 Inhibitors control of amphibian metamorphosis and adrenocortical hormones, 120, 127-128 growth hormone, 140 prolactin, 129, 133, 135-137 thyroid hormone, 110, I17 eggshells and biomineralization and, 226, 236, 240, 242, 244 environmental stimuli in plant cells and, 300 chloroplasts, 271, 275-277, 279-281, 283-284, 286 photodinesis, 253-254, 260-261, 265-266 wounding, 291-296 higher plant plastids and amino acid synthesis, 193, 196-197 metabolism, 168, 174-175, 188 urease in plant cells and, 95, 98 biochemical genetics, 90-92 elimination, 80, 82, 84 metabolic origins, 69, 71-72, 75, 77-79 Inorganic phosphate eggshells and biomineralization and, 23 1, 234 higher plant plastids and, 168-169, 179, 190 nuclear magnetic resonance and, 2, 7, 19, 42-43

321

INDEX

Insulin-like growth factors, control of amphibian metamorphosis and, 139-140 Intravoxel incoherent motion (IVIM) imaging, pathology and, 27, 29 Inverted repeat (IR) region, higher plant plastids and, 155, 157, 160 Iodine, hormonal control of amphibian metamorphosis and, 117, 131 Iopanoic acid, hormonal control of amphibian metamorphosis and, I10 Ischemia, nuclear magnetic resonance and, 25, 29, 35, 42, 44, 50 Isodesmosine, eggshells and biomineralization and, 220-221

K Keratan sulfate, eggshells and biomineralization and, 234, 238 Keratin eggshells and biomineralization and, 220-221, 237 hormonal control of amphibian metamorphosis and, 121 225-Ketoacyl ACP-synthetase (KAS), higher plant plastids and, 184 Kinetics eggshells and biomineralization and, 239 environmental stimuli in plant cells and, 279 higher plant plastids and, 168-169 nuclear magnetic resonance and, 33-34 Klebsiella aerogenes, urease in plant cells and, 89, 92

L

Lactate, nuclear magnetic resonance and, 30, 43,45-46 Large-single copy (LSC) sequence, higher plant plastids and, 155, 158 Larmor frequency, nuclear magnetic resonance and, 3-5 Larval growth, hormonal control of amphibian metamorphosis and, 128-132, 134, 137-139

Larhyrus satiua, urease in plant cells and, 67 Lectins, urease in plant cells and, 84, 98 Legumes higher plant plastids and, 163 urease in plant cells and, 74, 98 Lemna, environmental stimuli in, 268, 271 Leukoplasts, higher plant plastids and, 169-170, 179, 190 Light, environmental stimuli in plant cells and, 251, 299, 301 chemicals, 297-298 chloroplasts, 268-275 motile systems, 259-263 nucleus, 287-288 photodinesis, 252-259 signal transduction, 263-268 transduction chain, 275-287 Lilium, environmental stimuli in, 261 Lipids higher plant plastids and, 204 hormonal control of amphibian metamorphosis and, 122-123 nuclear magnetic resonance and, 45 Liver eggshells and biomineralization and, 234 hormonal control of amphibian metamorphosis and, 107, 131-132, I40 nuclear magnetic resonance and, 31, 35, 43-44 urease in plant cells and, 83 Liverwort, plastids and, 153, 155, 157, 166-167 Localization environmental stimuli in plant cells and, 263 hormonal control of amphibian metamorphosis and, 112 Lupinus texensis, urease in plant cells and, 82 Luteinizing hormone, control of amphibian metamorphosis and, 113, 115-1 16, I19 Luteinizing hormone-releasing hormone, control of amphibian metamorphosis and, 116 Lycopersicon, environmental stimuli in, 261, 289 Lysine, eggshells and biomineralization and, 220-221 Lysyl oxidase, eggshells and biomineralization and, 221, 237

322

INDEX

M Magnesium environmental stimuli in plant cells and, 259, 261, 263, 265 higher plant plastids and, 172-174, 203 nuclear magnetic resonance and, 3, 19-20 Magnetic resonance imaging (MRI), pathology and, 7, 10, 46, 50-51 applications, 41, 43-44 techniques, 23 Magnetic resonance imaging (MRI) microscopy, pathology and, 31-32 Magnetic resonance spectroscopy (MRS), pathology and, 7, 23, 41, 43 Magnetization transfer contrast (MTC), pathology and, 30 Maize environmental stimuli in, 299 plastids and amino acid synthesis, 192, 194, 201 metabolism, 170, 172, 180, 185 structure, 159, 167 urease in plant cells and, 94 Malate, higher plant plastids and, 170-171 Malignancy, nuclear magnetic resonance and, SO Mammillary layer, eggshells and biomineralization and, 243-244 fabrication, 238-240 structure, 219-220, 224-231 Maps, higher plant plastids and, 161 Marchanria polymorpha, plastids and, 153, 155, 166-167 Melanoma cells, nuclear magnetic resonance and, 39 Melanophores, hormonal control of amphibian metamorphosis and, 123 Meromysoin, environmental stimuli in plant cells and, 260, 263, 277 Mesotaeniurn, environmental stimuli in, 269, 272-273, 281, 299 Messenger RNA higher plant plastids and, 158-159, 192-193, 199 hormonal control of amphibian metamorphosis and, 11 1-1 12, 130, 133, 139, 141 Metabolites, nuclear magnetic resonance and, 30, 43-49 Metamorphosis, amphibian, hormonal control of, see Hormonal control of amphibian metamorphosis

Methylation, higher plant plastids and, 162-163 Methylobacterium, urease in plant cells and, 93 Microfilaments, environmental stimuli in plant cells and, 259-262, 276-277, 279, 296 Microtubule-associated protein (MAP), environmental stimuli in plant cells and, 285-287, 301 Microtubules, environmental stimuli in plant cells and light, 279, 284-287 wounding, 292-294, 296 Mineralization and eggshells, see Eggshells, biomineralization and Mitochondria environmental stimuli in plant cells and, 279 higher plant plastids and metabolism, 187-189, 196, 198 structure, 153-155, 158, 164-165 urease in plant cells and, 68-69, 71, 83 Mitosis environmental stimuli in plant cells and, 292-294 hormonal control of amphibian metamorphosis and, 140 Monoclonal antibodies eggshells and biomineralization and, 237 environmental stimuli in plant cells and, 261, 263, 284 Mougeofia, environmental stimuli in, 269, 272, 277-279, 281-282, 299-300 Mucin, eggshells and biomineralization and, 235 Mucopolysaccharides, eggshells and biomineralization and, 239 Multidrug resistance, nuclear magnetic resonance and, 50 Muscles, environmental stimuli in plant cells and, 260-261, 263, 277 Mutagenesis, higher plant plastids and, 196 Mutation higher plant plastids and amino acid synthesis, 196-197, 199 metabolism, 170, 173, 176, 181-182 structure, 153, 160 urease in plant cells and, 98-99 biochemical genetics, 85-86, 88, 90, 92 elimination, 80-81, 84

323

INDEX

metabolic origins, 69, 71, 75, 78 nickel metabolism, 92-94 Myocardial infarction, nuclear magnetic resonance and, 43-44 Myosin, environmental stimuli in plant cells and, 289, 296, 301 chloroplasts, 279-282 photodinesis, 259, 261, 263, 265 Myosin light chain kinase (MLCK), environmental stimuli in plant cells and, 28 1 Myrothecium uerrucaria, urease in plant cells and. 80

N NADH, higher plant plastids and, 200-201 NADH hydrogenase, higher plant plastids and, 158 NADPH, higher plant plastids and fatty acid synthesis, 184-185, 189 metabolism, 193-194, 203 translocation, 170-171 Neoplasia, nuclear magnetic resonance and, 35, 42 Neurotransmitters, nuclear magnetic resonance and, 45 Nickel, urease in plant cells and, 65, 99 biochemical genetics, 88, 90-92 elimination, 80-81 metabolism, 92-97 Nicotiana, environmental stimuli in, 263 Nicotiana tabacum, plastids and, 153 Nitella, environmental stimuli in, 261 Nitrate, higher plant plastids and, 191-194, 200 Nitrite, higher plant plastids and, 179, 191, 193 Nitrite reductase, higher plant plastids and, 192- 194 Nitrogen eggshells and biomineralization and, 239 higher plant plastids and aminoacid synthesis, 190-191,193,197, 199 metabolism, 167, 176, 203 nuclear magnetic resonance and, 3 urease in plant cells and, 97-99 biochemical genetics, 90 elimination, 80-83

metabolic origins, 66-68, 72-77, 79 nickel metabolism, 95-96 Nonhypophyseal factors, hormonal control of amphibian metamorphosis and, 126-128 Nonphotosynthetic plastids, higher plant, 150, 204 amino acid synthesis, 199-201 carbohydrate oxidation, 201, 203 fatty acid synthesis, 182-183, 189-190 starch synthesis, 177, 180 structure, 150, 161, 163 translocation, 169 Notophthalmus viridescens, hormonal control of amphibian metamorphosis and, 131 Nuclear magnetic resonance, 1-7,46,50-51 applications ex vivo NMR spectroscopy, 34-40 imaging of metabolites, 43-49 in vivo NMR spectroscopy, 35, 39, 41-43, 50 imaging, 25, 32 cardiovascular system, 31 diffusion, 26-29 magnetic resonance angiography, 25-26 magnetization transfer, 29-30 metabolites, 30 microscopy, 31-32 perfusion, 29 receptor imaging, 30-31 magnetization transfer, 29-30, 33-34 observed parameters chemical shift, 7-8 peak intensity, 11 peak shape, 12 relaxation times, 9-1 1 spin coupling, 8-9 spin echo, 11-12 spectroscopy, 13 cell studies, 13-18 nuclear characteristics, 17, 19-20 in vivo tissue studies, 20-24 urease in plant cells and, 76 Nuclear Overhauser effect (NOE), nuclear magnetic resonance and, 9, 44 Nucleation, eggshells and biomineralization and, 242, 244 Nucleotides higher plant plastids and, 153 urease in plant cells and, 78, 86, 91 Nucleus, environmental stimuli in plant cells and, 252, 300-301

324

INDEX

light, 287-288 wounding, 290-296 Nutrition, urease in plant cells and, 66, 75, 83

0 Oats, urease in plant cells and, 67 Open reading frames higher plant plastids and, 158 urease in plant cells and, 91 Organelles environmental stimuli in plant cells and, 289, 295, 300 chloroplasts, 268, 270, 284-285 photodinesis, 252-253, 263 higher plant plastids and, 149-150, 204 metabolism, 189, 191, I%, 201, 203 starch synthesis, 177, 180 structure, 150-151, 160, 164-165 translocation, 167, 169, 171 urease in plant cells and, 90 Organic acids, higher plant plastids and, 169- 171 Ornithine, urease in plant cells and, 68-69, 72 Ornithine aminotransferase (OAT), urease in plant cells and, 69 Ornithine transcarbarnylase (OTC), urease in plant cells and, 69 Orthophosphate, eggshells and biomineralization and, 240 Oryza sativa, plastids and, 153 Otala, eggshells and biomineralization and, 22 1 Oviposition, eggshells and biomineralization and, 236, 240 Ovocalcin, eggshells and biomineralization and, 234 Oxaloacetate, higher plant plastids and, 170 Oxidation environmental stimuli in plant cells and, 264, 276 higher plant plastids and, 193-194, 201-203 Oxygen higher plant plastids and, ]%-I97 nuclear magnetic resonance and, 3, 14,29, 32, 39, 41

P

Palisades, eggshells and biomineralization and, 219, 229-234, 238-240 Panicum miliaceum, plastids and, 169 Paramagnetic substances, nuclear magnetic resonance and, 30-31 Pathogenesis, urease in plant cells and, 83-84, 98 Pathology, nuclear magnetic resonance and, see Nuclear magnetic resonance Pea environmental stimuli in, 293-294 plastids and amino acid synthesis, 191, 193-194, 199-200 carbohydrate oxidation, 201 fatty acid synthesis, 184, 187-188, 190 starch synthesis, 177, 179-181 translocation, 168-171 urease in plant cells and, 67-69, 74, 82, 90, 99 Peak intensity, nuclear magnetic resonance and, 11 Peak shape, nuclear magnetic resonance and, 12 Peanut, urease in plant cells and, 67 Pectin, urease in plant cells and, 94 Pentose phosphate pathways, oxidative, higher plant plastids and, 150, 189, 193, 201, 203 Pepsin, eggshells and biomineralization and, 237 Peptide histidine isoleucine (PHI), hormonal control of amphibian metamorphosis and, 137 Peroxisomes, higher plant plastids and, 196, 198 PH eggshells and biomineralization and, 241 environmental stimuli in plant cells and, 290, 298, 300 nuclear magnetic resonance and, 19-20, 41, 45 urease in plant cells and, 66, 92-93 Phase-based angiography, nuclear magnetic resonance and, 26 Phaseolus uuigaris plastids and, 175, 200-201 urease in plant cells and, 76 Phenotype, urease in plant cells and, 81,85, 88, 94

325

INDEX

Phenylphosphorodiamidate (PPD), urease in plant cells and, 75-77, 82, 91, 95-96 Phosphate eggshells and biomineralization and, 231, 240, 244 inorganic, see Inorganic phosphate nuclear magnetic resonance and, 36 Phosphate translocator, higher plant plastids and, 168-170, 179-180, 189, 193 3’-Phosphoadenosine 5’-phosphosulfate (PAPS), eggshells and biomineralization and, 23 I , 237 Phosphocreatine (PCr), nuclear magnetic resonance and, 42-45 Phosphoglycerate, higher plant plastids and, 169, 189-190, 196 3-Phosphoglyceric acid (PGA), higher plant plastids and, 168, 177, 190, 196 Phospholipids, nuclear magnetic resonance and, 2, 19, 37, 39, 42, 45 Phosphomonoesters, nuclear magnetic resonance and, 42 Phosphorus eggshells and biomineralization and, 231, 235, 240 nuclear magnetic resonance and, 2 imaging, 34-35, 37, 41-42, 44-45 techniques, 19, 23-24, 33 Phosphorylation environmental stimuli in plant cells and, 264, 276, 282, 286 higher plant plastids and, 190 Photodinesis, environmental stimuli in plant cells and, 252-259, 298 motile systems, 259-263 signal transduction, 263-268 Photoreceptors, environmental stimuli in plant cells and, 256, 264, 270-274, 299 Photorespiration, higher plant plastids and, 194- 199 Photosynthesis environmental stimuli in plant cells and, 25 1 chloroplasts, 270-271, 276 photodinesis, 253-254, 256, 259, 264, 266, 268 higher plant plastids and, 150 amino acid synthesis, 191, 197-198 C 0 2 fixation, 172-173, 175-176 fatty acid synthesis, 182, 189 genome, 160-162

metabolism, 170 structure, 150, 153, 155, 157-158, 166 Phragmosomes, environmental stimuli in plant cells and, 292-293 Phycomitrium turbinatum, environmental stimuli in, 287 Physcornitrella patens, environmental stimuli in, 287-288 Phytochrome, environmental stimuli in plant cells and, 288, 299 photodinesis, 254, 256, 258-259, 266, 268 Pimozide, hormonal control of amphibian metamorphosis and, 134 Pink-pigmented, facultative methylotroph (PPFM), urease in plant cells and, 93, 96-99 Pisum, environmental stimuli in plant cells and, 261 Pisum satiuum, plastids and, 155, 181 Pituitary, control of amphibian metamorphosis and, 105, 140 adrenocortical hormones, 122, 124 prolactin, 128-130, 132-134, 136-137 thyroid hormone, 112-1 15, 117, 119 Plant cells environmental stimuli in, see Environmental stimuli in plant cells urease in, see Urease in plant cells Plant hormone, environmental stimuli in plant cells and, 299-300 Plant plastids, see Plastids, higher plant Plasma, control of amphibian metamorphosis and adrenocortical hormones, 119, 122-123, 125

growth hormone, 138-139 prolactin, 131, 133-136 thyroid hormone, 111, 115-117, 119 Plasma membrane environmental stimuli in plant cells and, 265, 276, 280, 296, 299-300 hormonal control of amphibian metamorphosis and, 110 Plasmids higher plant, 164-165 urease in plant cells and, 92 Plasmolysis, environmental stimuli in plant cells and, 261 Plastids, environmental stimuli in plant cells and, 263

326 Plastids, higher plant, 149-150, 204 metabolism amino acid synthesis, 190-201 carbohydrate oxidation, 201-203 C 0 2 fixation, 171-176 fatty acid synthesis, 182-190 starch synthesis, 176-182 translocation, 167- I7 1 structure chloroplast genome, 153-160 evolutionary origns, 164-167 genome interactions, 160-164 types of plastid, 150-154 Point resolved spectroscopy (PRESS), nuclear magnetic resonance and, 21 Polymerization, environmental stimuli in plant cells and, 279, 285, 287 Polypeptides environmental stimuli in plant cells and, 261, 263, 299 higher plant plastids and, 149 metabolism, 168, 173, 182, 197, 200 structure, 155- 157 Polysaccharide, eggshells and biomineralization and, 23 1 Positron emission tomography (PET), nuclear magnetic resonance and, 41 Potassium, nuclear magnetic resonance and, 20 Potato environmental stimuli in, 297 plastids and, 177, 179 urease in plant cells and, 90 Pregnenolone, control of amphibian metamorphosis and, 123 Preoptic recess organ (PRO), hormonal control of amphibian metamorphosis and, 122-123 Preprophase band, environmental stimuli in plant cells and, 293 Prochloron, higher plant plastids and, 166 Prochlorophyte, higher plant plastids and, 166- 167 Progesterone, control of amphibian metamorphosis and, 121, 123 Prokaryotes, higher plant plastids and, 161, 164, 166 Prolactin, control of amphibian metamorphosis and, 113, 138-139

INDEX larval growth, 128-132 levels, 133-137 Proline, urease in plant cells and, 68-69 Proopiomelanocortin (POMC), control of amphibian metamorphosis and, 125 Proplastids, higher plant, 160, 162-163 Protease environmental stimuli in plant cells and, 261, 301 urease in plant cells and, 84, 98 Proteins eggshells and biomineralization and, 220-221, 226, 231, 234-235, 239 environmental stimuli in plant cells and, 299-301 chloroplasts, 281, 285-287 photodinesis, 259, 261, 263 wounding, 291, 295 higher plant plastids and, 204 amino acid synthesis, 190-194, 199 fatty acid synthesis, 182-187 genomes, 153, 155-158, 160-162, 164 metabolism, 167-169, 172, 175, 177 structure, 151, 166 hormonal control of amphibian metamorphosis and, 117, 122-123, 129-130, 138 nuclear magnetic resonance and, 7, 31 urease in plant cells and, 65-66, 98-99 biochemical genetics, 84-86, 89, 91-92 elimination, 80-82 metabolic origins, 67-68, 71, 77 nickel metabolism, 95 Protein kinase, environmental stimuli in plant cells and, 281-282, 286 Proteoglycans, eggshells and biomineralization and, 221, 226, 23 1, 237 Proton density images, nuclear magnetic resonance and, 25-26 Protons environmental stimuli in plant cells and, 290 nuclear magnetic resonance and, 31 imaging. 41-42 magnetization transfer, 44-45 spectroscopy, 35, 37-38 urease in plant cells and, 83 Proton spectroscopy, pathology and, 2, 19, 43

327

INDEX

Protoplasts, environmental stimuli in plant cells and light, 260, 266-267, 278, 282 wounding, 292, 295 psbA, higher plant plastids and, 162-163 psbB, higher plant plastids and, 163-164 Pteris, environmental stimuli in, 274-275 Pulse sequences, nuclear magnetic resonance and, 20, 24, 45 Pumpkin, urease in plant cells and, 67-69, 72 Purines, urease in plant cells and, 73-79 Puromycin, environmental stimuli in plant cells and, 291 Pyruvate, higher plant plastids and, 171, 187-190, 203 Pyruvate dehydrogenase complex (PDC), higher plant plastids and, 187, 189

R Radiofrequency, nuclear magnetic resonance and, 4-5, 9-11 applications, 38, 44 techniques, 21, 24, 31 Radioimmunoassay (RIA) control of amphibian metamorphosis and adrenocortical hormones, 119, 125 growth hormone, 138 prolactin, 130, 133, 137 thyroid hormone, 107, 115, 117, 119 environmental stimuli in plant cells and, 265 Rana calmitans, hormonal control of metamorphosis and, 107 Rana catesbeiana, control of metamorphosis and adrenocortical hormones, 119, 123-125, 127 growth hormone, 140 prolactin, 135 thyroid hormone, 107, 113, 116 Rana ornariuenrris, hormonal control of metamorphosis and, 118 Rana perezi, hormonal control of metamorphosis and, 113, 115, 140 Ranupipiens, control of metamorphosis and, 106, 108-109 growth hormone, 140 prolactin, 128 thyroid hormone, 112, 116-1 17

Rana ridibundu, hormonal control of metamorphosis and, 115, 124-127 r b d , higher plant plastids and, 163-164 Receptor imaging, nuclear magnetic resonance and, 30-31 Region of interest (ROI), nuclear magnetic resonance and, 29, 42-43, 45 Relaxation times, nuclear magnetic resonance and, 9-11, 31-32, SO Repetition time, nuclear magnetic resonance and, 11-12, 31 Restriction fragment length polymorphism higher plant plastids and, 159 urease in plant cells and, 86 Retinal epithelial cells, nuclear magnetic resonance and, 38 Rhodamine, environmental stimuli in plant cells and, 277, 283, 289, 291 Ribosomal DNA, higher plant plastids and, 159 Ribosomal RNA, higher plant plastids and, 155-156, 159, 162, 166-167 Ribosomes higher plant plastids and, 151, 155, 157-158, 171 urease in plant cells and, 84 Ribulose bisphosphate, higher plant plastids and, 171, 173 Rice plastids and, 153, 155, 157, 200 urease in plant cells and, 67, 80 RNA, see also Messenger RNA; Ribosomal RNA; Transfer RNA higher plant plastids and, 151, 158-159, 162, 164-165, 175 hormonal control of amphibian metamorphosis and, 121 RNA polymerase, higher plant plastids and, 155-156, 164, 166 Rubisco, higher plant plastids and amino acid synthesis, 196-198 CO, fixation, 172-176 large subunit (LSU), 160, 172 small subunit (SSU), 160, 168, 172, 175 structure, 160 translocation. 168

S

Saturation, nuclear magnetic resonance and, 26, 29-30

328 Saturation transfer, nuclear magnetic resonance and, 33 Scanning electron microscopy (SEM), eggshells and biomineralization and, 219, 224, 239-240 SDS-PAGE higher plant plastids and, 168 urease in plant cells and, 89 Secretion eggshells and biomineralization and, 24 1 hormonal control of amphibian metamorphosis and, 116, 123-128, 136- 137 Sedimentation, nuclear magnetic resonance and, 14 Seeds higher plant plastids and, 151, 184, 186-187, 204 urease in plant cells and, 65-66, 98 biochemical genetics, 84, 89, 91 elimination, 80-83 metabolic origins, 67-73, 75, 77-78 nickel metabolism, 93, 97 Seluginella, environmental stimuli in, 269-271 Selection, urease in plant cells and, 81 Sequences eggshells and biomineralization and, 239, 24 1 higher plant plastids and metabolism, 168, 171, 185, 193-194, 20 1 structure, 155, 157-158, 160, 165- 167 hormonal control of amphibian metamorphosis and, 115-1 16, 126-127, 130, 138 nuclear magnetic resonance and applications, 35, 44 techniques, 20, 24-25, 27, 29 urease in plant cells and, 86, 89, 91, 98 Serotonin, control of amphibian metamorphosis and, 127-128, 137 Shell membranes, eggshells and biomineralization and, 2 19-224 Signal-to-noise ratio, nuclear magnetic resonance and, 6, 9 applications, 32, 44-45 techniques, 15, 23

INDEX

Signal transduction, environmental stimuli in plant cells and, 252, 298-299, 301 chloroplasts, 279-283 photodinesis, 257, 263-268 Siphonocludus, environmental stimuli in, 295 Siphonous green algae, environmental stimuli in, 294-2% Skeletal muscles, environmental stimuli in plant cells and, 260-261, 263, 277 Small single copy (SSC) sequences, plastids and, 155, 158 Sodium hormonal control of amphibian metamorphosis and, 135 nuclear magnetic resonance and, 20 Solanurn tuberosum, plastids and, 174 Somatostatin, control of amphibian metamorphosis and, 140 Soybean plastids and, 184, 192 urease in plant cells and, 65-66, 98-99 biochemical genetics, 84-92 elimination, 81-84 metabolic origins, 67-69, 72-73, 75-79 nickel metabolism, 92-97 Spectroscopy, nuclear magnetic resonance and, 2-3, 5 , 7-9, 46, 50 applications, 34-43 cell studies, 13-18 imaging of metabolites, 43-44 nuclear characteristics, 17, 19-20 techniques, 26, 33 in uivo tissue studies, 20-24 Spinach, plastids and amino acid synthesis, 192, 194 C 0 2 fixation, 172, 175 fatty acid synthesis, 184-187 structure, 162-163, 167 translocation, 168, 170 Spin coupling, nuclear magnetic resonance and, 8-9 Spin decoupling, nuclear magnetic resonance and, 8-9 Spin density, nuclear magnetic resonance and, 26 Spin echo, nuclear magnetic resonance and, 11-12, 25, 27 Spleen hormonal control of amphibian metamorphosis and, 131 nuclear magnetic resonance and, 31

329

INDEX

Starch, higher plant plastids and, 167, 172, 176-182, 203-204 Starch synthase, higher plant plastids and, 177, 181 Steady-state free precession (SSFP) sequences, nuclear magnetic resonance and, 29 Steroids, control of amphibian metamorphosis and, 119-121, 124, 126- 128 Stimulated echo acquisition mode (STEAM), nuclear magnetic resonance and, 21,45 Streaming, environmental stimuli in plant cells and chemicals, 297 chloroplasts, 283-284 low temperature, 288-290 photodinesis, 252-254,256-257,260-261, 264-266 primary streaming, 252 secondary streaming, 252 wounding, 295 Streptomycin, higher plant plastids and, 159 Structural genes, urease in plant cells and, 84-90,98 Succinate, urease in plant cells and, 71 Sucrose, higher plant plastids and, 167, 171-172 Sugar, eggshells and biomineralization and, 235 Suppression, urease in plant cells and, 80 Synlactin, control of amphibian metamorphosis and, 131

T

Taxol, environmental stimuli in plant cells and, 285, 287 Temperature, low, environmental stimuli in plant cells and, 251-252, 288-290, 291 Testosterone, control of amphibian metamorphosis and, 119, 121 Thiolactomycin, higher plant plastids and, 184 Thylakoids, higher plant plastids and metabolism, 168, 172, 174, 197 structure, 153, 161, 166

Thyroid hormones, control of amphibian metamorphosis and, 105-107, 110-1 12, 141 adrenocortical hormones, 124 feedback, 117-1 19 growth hormone, 138 hypothalamus, 115-117 pituitary, 112-1 15 prolactin, 130, 133-136 Thyroid-stimulating hormone (TSH), control of amphibian metamorphosis and, 112-113, 115-117, 119, 131 Thyrotropin-releasing hormone (TRH), control of amphibian metamorphosis and, 115-116, 136-137, 140 Thyroxine (T4), control of amphibian metamorphosis and, 107, 110, 112-113, 115-116, 118-119 adrenocortical hormones, 120-124 prolactin, 129-131, 136 Tissue analysis, urease in plant cells and, 92 Tissue specificity higher plant plastids and, 184, 186 nuclear magnetic resonance and, 31 Tissue studies, nuclear magnetic resonance and, 45, 50 imaging, 25-26, 30-32 magnetization transfer, 33-34 spectroscopy, 20-24 Tobacco environmental stimuli in plant cells and, 291, 293 plastids and metabolism, 175-176, 193 structure, 153, 155, 157-159 urease in plant cells and, 80, 90 Tomato plastids and, 161-163 urease in plant cells and, 81 Tonoplasts, environmental stimuli in plant cells and, 283, 290 Tradescanria, environmental stimuli in plant cells and, 291, 293 Transcription higher plant plastids and, 161-164 hormonal control of amphibian metamorphosis and, 112 Transfer RNA, higher plant plastids and, 156-157, 163 Transgenic plants, plastids and, 175-175 Translation, higher plant plastids and, 161

330

INDEX

Translocation environmental stimuli in plant cells and, 284,287, 290 higher plant plastids and amino acid synthesis, 193, 199 fatty acid synthesis, 189-190 metabolism, 167-171 starch synthesis, 179-180 urease in plant cells and, 77, 94 Transmission electron microscopy (TEM), eggshells and biomineralization and fabrication, 239-240 structure, 220-221, 224, 232-233 Transplants, nuclear magnetic resonance and, 41 Traumatotactic migration, environmental stimuli in plant cells and, 292, 294, 300 Trifluoromethane, nuclear magnetic resonance and, 32 Triiodothyronine (T3), control of amphibian metamorphosis and, 107, 110, 121-122, 129-130 Triose phosphate, higher plant plastids and, 168-169, 179-180, 201 Triticum, environmental stimuli in plant cells and, 259 Triturus cristafus, hormonal control of metamorphosis and, 124-125 Tubulin, environmental stimuli in plant cells and, 284, 286-287 Tumors, see also specific tumors nuclear magnetic resonance and, 1, 3, 50 applications, 35, 39, 41-44 techniques, 17, 19-21, 31 Turnover eggshells and biomineralization and, 243 higher plant plastids and, 177 urease in plant cells and, 66-68, 98

U Ubiquitous urease in plant cells, 84-86, 92 Udotea, environmental stimuli in, 294 Ultraviolet light, environmental stimuli in plant cells and, 256, 282, 299 Urea in plant cells biochemical genetics, 88, 90 elimination, 81, 83-84

metabolic origins, 71-72, 74-76, 79 nickel metabolism, 82-97 Urease in plant cells, 65-66, 97-99 biochemical genetics accessory genes, 86-88 gene products, 88-92 structural genes, 84-87 elimination, 79-80 germination, 81-82 loss of protection, 82-84 protein deposition, 80-81 metabolic origins, 66 arginine, 66-72 purines, 73-79 nickel metabolism, 92-97 Ureides, urease in plant cells and, 66, 98 elimination, 81, 83 metabolic origins, 73-79 nickel metabolism. 95-97

V

Vacubles, environmental stimuli in plant cells and, 269, 277, 292, 295, 298 Vallisneria, environmental stimuli in, 299-301 chemicals, 297-298 chloroplasts, 268, 271, 275-277 photodinesis, 252-254,256-257,260-261, 263-265, 268 Vanadate, environmental stimuli in plant cells and, 266, 283 Vasoactive intestinal polypeptide (VIP), control of amphibian metamorphosis and, 127, 138 Vaucheria, environmental stimuli in, 268-271, 283 Vesicles eggshells and biomineralization and, 230 environmental stimuli in plant cells and light, 263, 277-278, 281 low temperature, 289 wounding, 295 Vicia faba plastids and, 155 urease in plant cells and, 67 Viscosity, environmental stimuli in plant cells and, 257, 259

331

INDEX

Vitamin D, eggshells and biomineralization and, 236 Vitamin K, eggshells and biomineralization and, 234 Vittellogenesis, hormonal control of amphibian metamorphosis and, 112

W

Water suppression, nuclear magnetic resonance and, 45 Wheat plastids and, 179, 197, 203 urease in plant cells and, 67 Wounding, environmental stimuli in plant cells and, 252, 290-297, 299

Xenopus, control of metamorphosis and adrenocortical hormones, 121, 123-127 growth hormone, 139 prolactin, 129 thyroid hormone, 107, 110, 112 Xenopus laeuis, control of metamorphosis and, 106, 108-109 adrenocortical hormones, 119, 125 growth hormone, 140 thyroid hormone, 107, 1 1 1 , 113 X-ray diffraction, eggshells and biomineralization and, 230, 239 Xylem, urease in plant cells and, 77, 79

Y Yeast, urease in plant cells and, 74-75

x Z Xanthine dehydrogenase, urease in plant cells and, 77, 95

Zinc, urease in plant cells and, 94

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