Table Of ContentP. K. Stumpf and E. E. Conn
EDITORS-IN-CHIEF
Department of Biochemistry
and Biophysics
University of California
Davis, California
Volume 1 The Plant Cell N. E. Tolbert, Editor
Volume 2 Metabolism and Respiration David D. Davies, Editor
Volume 3 Carbohydrates: Structure and Function Jack Preiss, Editor
Volume 4 Lipids: Structure and Function P. K. Stumpf, Editor
Volume 5 Amino Acids and Derivatives B. J. Miflin, Editor
Volume 6 Proteins and Nucleic Acids Abraham Marcus, Editor
Volume 7 Secondary Plant Products E. E. Conn, Editor
Volume 8 Photosynthesis M. D. Hatch and N. K. Boardman, Editors
Volume 9 Lipids: Structure and Function P. K. Stumpf, Editor
Volume 10 Photosynthesis M. D. Hatch andN. K. Boardman, Editors
Volume 11 Biochemistry of Metabolism David D. Davies, Editor
Volume 12 Physiology of Metabolism David D. Davies, Editor
Volume 13 Methodology David D. Davies, Editor
Volume 14 Carbohydrates Jack Preiss, Editor
Volume 15 Molecular Biology Abraham Marcus, Editor
Volume 16 Intermediary Nitrogen Metabolism B. J. Miflin and Peter J. Lea, Editors
THE
BIOCHEMISTRY
OF PLANTS
A COMPREHENSIVE TREATISE
Volume 16
Intermediary Nitrogen
Metabolism
Editors
B. J. Miflin
Seeds Research and Development
CIBA-GEIGY
Basel, Switzerland
Peter J. Lea
Division of Biological Sciences
Institute of Environmental and Biological Sciences
Lancaster University
Lancaster, United Kingdom
ACADEMIC PRESS
Harcourt Brace Jovanovich, Publishers
San Diego New York Boston
London Sydney Tokyo Toronto
This book is printed on acid-free paper. @
Copyright © 1990 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.
San Diego, California 92101
United Kingdom Edition published by
Academic Press Limited
24-28 Oval Road, London NW1 7DX
Library of Congress Cataloging-in-Publication Data
(Revised for vol. 16)
The Biochemistry of plants.
Includes bibliographies and indexes
Contents: v. 1, The plant cell ~ v. 2, Metabolism
and respiration - [etc.] ~ v. 16, Intermediary
nitrogen metabolism.
1. Botanical chemistry, I. Stumpf, Paul K. (Paul
Karl), 1919- , II. Conn, Eric E.
QK861.B48 581.197 80-13168
ISBN 0-12-675416-0 (v. 16)
Printed in the United States of America
90 91 92 93 10 9 8 7 6 5 4 3 2 1
List of Contributors
Numbers in parentheses indicate the pages on which the authors' contributions begin.
J. W. Anderson (327), Botany Department, LaTrobe University, Bundoora,
Victoria 3083, Australia
Michael J. Boland (197), Center for Plant Science and Biotechnology, Biology
Department, Washington University, St. Louis, Missouri 63130
J. K. Bryan (161), Department of Biology, Syracuse University, Syracuse, New
York 13244
A. W. Galston (283), Biology Department, Yale University, New Haven, Con
necticut 06520
R. Kaur-Sawhney (283), Biology Department, Yale University, New Haven,
Connecticut 06520
Andris Kleinhofs (89), Department of Agronomy and Soils, and Program in
Genetics and Cell Biology, Washington State University, Pullman, Wash
ington 99164
David B. Layzell (1), Department of Biology, Queen's University, Kingston,
Ontario, Canada K7L 3N6
Peter J. Lea (121), Division of Biological Sciences, Institute of Environmental
and Biological Sciences, Lancaster University, Lancaster LAI 4YQ,
United Kingdom
John S. Pate (1), Department of Botany, The University of Western Australia,
Nedlands, Western Australia 6009, Australia
X List of Contributors
Sharon A. Robinson (121), Department of Biology, University College, London
WC1E 6BT, United Kingdom
Karel R. Schubert (197), Center for Plant Science and Biotechnology, Biology
Department, Washington University, St. Louis, Missouri 63130
George R. Stewart (121), Department of Biology, University College, London
WC1E 6BT, United Kingdom
A. F. Tiburcio (283), Biology Department, Yale University, New Haven, Con
necticut 06520
Carroll P. Vance (43), United States Department of Agriculture, Agricultural
Research Service, and The Department of Agronomy and Plant Genetics,
The University of Minnesota, St. Paul, Minnesota 55108
Robert L. Warner (89), Department of Agronomy and Soils, Washington State
University, Pullman, Washington 99164
General Preface
In 1950, a new book entitled "Plant Biochemistry" was authored by James
Bonner and published by Academic Press. It contained 490 pages, and much of
the information described therein referred to animal or bacterial systems. This
book had two subsequent editions, in 1965 and 1976.
In 1980, our eight-volume series entitled "The Biochemistry of Plants: A
Comprehensive Treatise" was published by Academic Press; this multivolume,
multiauthored treatise contained 4670 pages.
Since 1980, the subject of plant biochemistry has expanded into a vigorous
discipline that penetrates all aspects of agricultural research. Recently a large
number of research-oriented companies have been formed to explore and ex
ploit the discipline of plant biochemistry, and older established chemical com
panies have also become heavily involved in plant-oriented research. With this
in mind, Academic Press and the editors-in-chief of the treatise felt it impera
tive to update these volumes. Rather than have each chapter completely rewrit
ten, it was decided to employ the approach used so successfully by the editors of
Methods in Enzyrnology, in which contributors are invited to update those
areas of research that are most rapidly expanding. In this way, the 1980 treatise
constitutes a set of eight volumes with much background information, while
the new volumes both update subjects that are rapidly developing and discuss
some wholly new areas. The editors-in-chief have therefore invited the editors
of the 1980 volumes to proceed on the basis of this concept. As a result, new
volumes are forthcoming on lipids; general metabolism, including respiration;
carbohydrates; amino acids; molecular biology; and photosynthesis. Addi
tional volumes will be added as the need arises.
xii General Preface
Once again we thank our editorial colleagues for accepting the important task
of selecting authors to update chapters for their volumes and bringing their
volumes promptly to completion. And once again we thank Mrs. Billie Gabriel
and Academic Press for their assistance in this project.
P. K. Stumpf
E. E. Conn
It is with great sadness that we have learned of the untimely death of Dr. A. P.
Sims of the University ofEastAnglia on July 31st 1990. Tony Sims carried out a
series of elegant 15N labeling experiments, in collaboration with Brian Folkes on
the food yeast Candida utilis. The work provided the platform for all the later
investigations into nitrogen assimilation in plants. However, more than his
published work, Tony's enthusiasm, scientific integrity, wide knowledge, love of
his subject, and delight in open argument, stimulated the minds and activities of
his students and contemporaries and ensured that his influence on the subject
was extended widely. He is remembered with great respect by his colleagues in
the field of plant nitrogen metabolism, a number of whom have been fortunate
enough to receive their early training under his direction.
B. J. M, P. J. L.
Preface to Volume 16
Volume 5 of this series, "Amino Acid Derivatives," included chapters with
topics ranging from nitrogen fixation to amino acid accumulation in relation to
stress. Ten years later, Volume 16 now concentrates on the most rapid advances
in nitrogen metabolism: The genes for enzymes carrying out nitrogen fixation,
nitrate reduction, and ammonia assimilation in the early stages of nitrogen
metabolism have been cloned. Study of a range of mutants deficient in these
enzymes has increased our knowledge of the regulation of nitrogen metabo
lism. The dominating role of photorespiration in nitrogen metabolism of the
leaf and the continuous recycling of ammonia is well established. Our under
standing of pathways of biosynthesis of amino acids and polyamines has greatly
increased. Major advances in our knowledge of essential amino acid biosynthe
sis have been made as a result of interest generated by findings on the site of
action of new low-dose herbicides. New techniques using cloned genes of ani
mal or fungal enzymes as heterologous probes now provide shortcuts for the
isolation of genes that control amino acid biosynthesis in higher plants. For the
future, there is still considerable work waiting to be done on the purification and
characterization of these key enzymes.
We would like to take this opportunity to thank our numerous colleagues
with whom we have both worked over the last 10 years. These include Simon
Bright, Julie Cullimore, Leslie Fowden, Michael Kirkman, Alfred Keys, Ken
Joy, Mendel Mazelis, Sven Rognes, Peter Shewry, George Stewart, and Roger
Wallsgrove.
Finally, we would like to express our gratitude to Terry Bowden for consider
able help during the production of this volume, and to pay tribute to Janice
Turner's hard work in preparing the index.
Benjamin J. Miflin
Peter J. Lea
xiii
Energetics and Biological
/
Costs of Nitrogen
Assimilation
JOHN S. PATE
DAVID B. LAYZELL
I. Introduction
II. Calculating the Energy Costs of Chemical and Biochemical Processes
III. Direct Costs Associated with Nitrate Reduction
A. The Reduction Pathway and Sources of Reductant
B. Nitrate Reduction in Light and Darkness: Mechanics and Regulation
C. Assays for Nitrate-Reducing Ability of Plant Organs
D. The Site of Nitrate Reduction in Plant Species
IV. Direct Costs of N Fixation and Nitrogenase Functioning
2
A. N Fixation and H Evolution by Nitrogenase
2 2
B. Electron Allocation Coefficient (EAC) of Nitrogenase: Characteristics and Regulation
C. Uptake Hydrogenase Activity and Its Relevance to the Costs of N Fixation
2
V. Direct Costs of Ammonia Assimilation
VI. Ion Uptake and Transport and the Regulation of Ion Balance during Nitrogen Assimilation
VII. Nitrogen Assimilation and Its Relationships with Growth and Maintenance Respiration
VIII. Construction Costs for Organs Specializing in Nitrogen Assimilation
IX. Cost of Using Nitrogenous Compounds in Chemical Defense or Protection against Stress
X. Concluding Remarks
References
I. INTRODUCTION
With the notable exception of those species which feed heterotrophically by
parasitism, carnivorous habits, or through the agency of root mycorrhizae,
higher plants derive nitrogen from the environment principally, if not entirely,
as nitrate, ammonium, or atmospheric nitrogen. Utilization of the latter source
is restricted to those groups of plants capable of symbiosis with nitrogen-fixing
bacteria, cyanobacteria, or actinomycetes.
The generally accepted pathways of reduction of N0 ~ or N both lead to the
3 2
formation of ammonia, so that plants exploiting these more oxidized states of
The Biochemistry of Plants, Vol. 16 1
Copyright © 1990 by Academic Press, Inc.
All rights of reproduction in any form reserved.
2 John S. Pate and David B. Layzell
nitrogen must clearly require enzymatic machinery and energy inputs addi
tional to those involved when utilizing ammonium ions directly. Thereafter,
when the ammonia is incorporated into simple organic molecules such as
ureides, amides, or amino acids, the direct costs involved will be determined
principally by the metabolic routes being followed, and are therefore not likely
to differ significantly between species synthesizing identical sets of assimilatory
products. Finally, when these organic solutes are being consumed as sources of
nitrogen for protein synthesis, further metabolic rearrangement must take
place, involving partial or complete degradation of the primary products of
assimilation. Again, costs will depend on solutes concerned and metabolic
pathways implementing their utilization.
The philosophy of this chapter is that a full analysis of the energetics of
nitrogen assimilation requires consideration not only of the direct metabolic
costs stated above, but also of a number of diverse ancilliary processes imping
ing upon, or coming within, the general ambit of nitrogen assimilation. Promi
nent among these are energetic implications resulting from the localization of
assimilatory processes in photosynthetic or nonphotosynthetic parts of the
plant; uptake, transport, and storage of inorganic or organic solutes of nitrogen;
adjustments of ion imbalances caused by nitrogen assimilation; construction
and maintenance of structures specifically or indirectly associated with nitro
gen assimilation, and the deployment of nitrogen solutes as chemical defense
agents, or as osmotica under salt or water stress. As will be seen, costs relating to
each of these factors differ greatly between species, and within species with
nitrogen source being utilized. Several recent papers (Pate et al, 1981; Schu
bert, 1982; Mahon, 1983; Atkins, 1984; Saari and Ludden, 1985; Pate, 1986;
Layzell et al, 1988) have examined critically the methodologies used to mea
sure experimentally the costs of nitrogen assimilation and reviewed the large
body of data thereby obtained, particularly in relation to comparisons of legu
minous plants fixing N or assimilating N0 ~. This article will concentrate
2 3
more upon theoretically based discussions of the costs of nitrogen assimilation,
and thus provide a logical framework against which earlier published experi
mental data can be interpreted.
II. CALCULATING THE ENERGY COSTS OF CHEMICAL AND
BIOCHEMICAL PROCESSES
From a purely chemical standpoint, the energetics of the primary reactions of
any inert or biologically mediated process relate primarily to the bond energies
between atoms involved in the relevant chemical pathway(s), these characteris
tics in turn deriving from the theoretical standard free energy (AG°) of the
participating reactions. It is thus possible to predict whether a specific reaction
is thermodynamically favorable (negative AG°) or not (positive AG°), and, if
