Table Of ContentAntioxidants
in
Higher Plants
Edited by
Ruth G. Alscher
Associate Professor
Department of Plant Pathology, Physiology, and Weed Science
Virginia Polytechnic Institute and State University
Blacksburg, Virginia
John L. Hess
Associate Professor
Department of Biochemistry and Nutrition
Virginia Polytechnic Institute and State University
Blacksburg, Virginia
First published 1993 by CRC Press
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© 1993 by Taylor & Francis
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INTRODUCTION
Oxidative stress in biological organisms is a consequence of life as it is currently
defined on this planet. Our purpose is to consider key metabolites in higher plants
that have capacity to react as antioxidants at various levels of absorbed energy and
oxidation states of oxygen. We have intentionally not considered key enzymes,
superoxide dismutase (SOD), catalase, and general peroxidases. Each of these
protein classes has been extensively studied and, with the tools of molecular biology,
remarkable progress is being made in our understanding of their responses to ox
idative stress. Another topic of related interest, photorespiration, is also not included
in this volume. It must be recognized, however, that the cycling of carbon through
assimilation of C02 to key intermediates and its reoxidation to C02 provides a
means of dissipating energy while minimizing losses of carbon from the plant. It
is understood that constitutive levels of enzymes of this pathway in plants are
required as a consequence of the current low C02/02 ratio. This metabolism defines
a functional antioxidant pathway requiring chloroplasts, peroxisomes, and mito
chondria to: catalyze the consumption of 02, generate H202, dismute the peroxide
with the peroxisomal catalase, and cycle carbon. However, the extent to which this
pathway is modulated in response to other environmental stressors has received
little attention.
The substrates that function as electron donors in more general antioxidant
metabolism and molecules that absorb energy directly are the focus of our review
and comment. These compounds must serve as key structures upon which to develop
comparative chemical analogs that may be used to study antioxidant metabolism
in order to understand the function and compartmentalization of the natural anti
oxidants.
A premise for our interest in these compounds is that the oxidative burden on
organisms has increased due to anthropogenic factors among which air pollution is
a major component. Both light intensity and ambient energies also change over
time and most recently include increases in the ambient UV. Fundamental mech
anisms for both oxidative and photochemical damage are understood in terms of
free radical chemistry or direct energy dissipation through thermal processes. Re
sponses by plants to extremes in temperature and drought stress correlate with
responses typically observed from increased oxidative stress. Hence, a continued
emphasis on exploring susceptibility of plants to oxidative and photochemical stress
is needed in order to understand competitive relationships in ecosystems and effects
on overall productivity.
Oxidative stress may be initiated by the generation of organic free radicals that
results in self-propagating autooxidation reactions. These radicals form in response
to either direct excitation from incident radiation or secondarily from reactions with
oxygen radicals or metastable forms of reduced oxygen, i.e., OH’, H202, singlet
02, and 02_. The damage to biological systems resides in the formation of other
organic peroxides propagated through typical radical chain reactions.
Protection to organisms may be expressed through various strategies. We have
learned much from model systems using synthetic antioxidants and inhibitors of
oxidation chemistry. Successful protection may result from: direct quenching of the
reactive oxygen species, disruption of the free radical chain reaction, or direct
absorption of ambient radiation. In each of these reactions it is essential that the
final product of the chemistry or the excitation result in a product which no longer
carries the properties of the free radical or a reactive excited state. Conceptually,
it is when oxidative stress exceeds the normal capacity of the plant cell to dissipate
absorbed energy and/or free radical formation that irreversible damage is initiated.
The extent to which specific enzymes have evolved to accomplish this protection
directly depends on the properties and abundance of specific substrates.
In the investigation of these systems in higher plants much progress has been
made in describing relationships between oxygen, ascorbate, and glutathione. The
following chapters summarize current research efforts that provide insight into how
plants with differential sensitivities to oxidative stress use these metabolites and
associated enzymes. This antioxidant capacity is relegated to the aqueous phase of
the plant cell more directly than the membrane and likely occurs within organelles
as well as the cytosol. However, it needs to be recognized that reactive, water
soluble organic peroxides may be released from membrane oxidations and thus
react with thiols or other scavengers in the aqueous phase.
Glutathione is a ubiquitous tripeptide that mediates redox cycling between
ascorbate and NADPH. It is essential for maintaining the cellular redox status.
Much is known about glutathione function, and the rapid changes observed in
glutathione contents under oxidative conditions indicate its importance in antioxidant
defense. While our knowledge about the regulation of glutathione biosynthesis and
degradation in plants is still limited, much recent research has focused on the effects
of glutathione on gene expression and on the molecular biology of the associated
enzyme, glutathione reductase.
Ascorbate plays a pivotal and clearly defined role in the scavenging of hydrogen
peroxide. In the chloroplast, the ascorbate system is important as a detoxification
system and as a regulator of electron flow in vivo. The reduction of molecular
oxygen to hydrogen peroxide, and, eventually, to water in photosynthesis aids
membrane energization, also affording protection through triggering processes in
the thylakoid that lower intrinsic quantum yield of Photosystem II, helping to prevent
photoinhibitory damage. Precise mechanisms involving ascorbate remain unclear
for other biochemical and physiological functions of plants. In addition to its role
as a primary antioxidant, it is also an important secondary antioxidant. It maintains
the a-tocopherol pool that scavenges radicals in the inner regions of membranes.
Ascorbate and a-tocopherol share the unique property of a high capacity for free
radical scavenging combined with low reactivities with oxygen.
The carotenoids may function to mediate potential damage from excited states
of pigment molecules and singlet oxygen. It is expected that these conversions
result in dissipation of energy through loss of thermal energy. Protection from
photooxidative damage involves the carotenoids in preventing the formation of
reactive singlet oxygen. The carotenoids also function in maintaining the structural
integrity of the photosynthetic apparatus.
The conversion of violaxanthin to zeaxanthin is mediated by oxygen uptake
and cycling back to violaxanthin requires ascorbate. Substantial evidence now exists
to correlate levels of zeaxanthin with the capacity of a plant to minimize damaging
effects caused by incident radiation in excess of that required to saturate reductive
metabolism. It is zeaxanthin that is involved in a thermal energy dissipation process.
The positive correlation of the components of the xanthophyll cycle with growth
of plants in high light intensity is consistent with a greater capacity for thermal
energy dissipation than occurs in shade-grown plants.
Vitamin E, a-tocopherol, is an exquisite, lipophilic, free radical scavenger
which, like the carotenoids, exists within biological membranes. As a free radical
trap it directly interrupts free radical chain reactions. As with glutathione, mech
anisms exist that allow cycling of the quinone (oxidized) and quinols (reduced)
using reduced ascorbate or NADFH directly. For these substrates, a-tocopherol
should function to trap alkylperoxyl free radicals at the expense of normal reducing
equivalents available in the cell.
Finally, various classes of phenylpropanoid derivatives, for example, the fla-
vonoids, lignans, tannins, lignins, also function as antioxidants and many of these
compounds contribute exclusively to the browning reactions observed in injured or
pathogen-invaded plant tissues. Some classes of these compounds are now used
commercially as antioxidants. Like the carotenoids, many of these aromatic com
pounds serve to minimize the effects of direct photobleaching, particularly that
initiated by UV irradiation, and irreversible oxidative damage. Much additional
information is required about phenylpropanoids, their turnover, and the enzymology
associated with their metabolism. As with the antioxidants discussed in the other
chapters, the response of phenylpropanoids to environmental factors will lead to a
greater understanding of their contribution to protecting plants against oxidative
damage.
It is our intention that the reader become more aware of the varied, yet con
sistent, antioxidant metabolism available to the higher plant cell. Much remains to
be learned about the expression and regulation of this metabolism.
Ruth Grene Alscher
John L. Hess
THE EDITORS
Ruth Grene Alscher’s central focus of research is on the ways in which green
plant cells respond and adapt to environmental cues. She received her undergraduate
degree in Biochemistry from Trinity College, Dublin, Ireland in 1965. Subsequently,
she studied effects of light on chloroplast composition at Washington University,
St. Louis where she received an MA in Botany in 1968. In Dr. Paul Castelfranco’s
laboratory at the University of California at Davis, her thesis research (Ph.D. in
Plant Physiology, 1972) involved chlorophyll biosynthesis in greening tissue. From
1975 to 1977, she was an NIH postdoctoral fellow in the laboratory of Dr. André
Jagendorf at Cornell University where she studied the effect of anoxia on the
translational capacity of thylakoid-bound polyribosomes. She was a member of the
Environmental Biology Program of Boyce Thompson Institute at Cornell University
from 1979 to 1988, and an Adjunct Assistant Professor of Plant Biology at Cornell
from 1985 to 1988. Her work during the Ithaca years was centered on the responses
of antioxidants to the oxidative stress imposed by air pollutants. She has continued
to pursue these questions as a faculty member at Virginia Tech. Together with Drs.
J. Hess, C. Cramer, and E. Grabau, she is currently investigating the molecular
and metabolic roles of the plastid antioxidant proteins glutathione reductase and
superoxide dismutase in the resistance of green plant cells to oxidative stress.
John L. Hess has been interested in factors that impact photosynthesis in higher
plants throughout his career. His research has focused on oxidative metabolism in
both plants and animals and continues to explore how cells accommodate oxidative
stress. Following the completion of the A.B. degree in Chemistry at Franklin and
Marshall College, Lancaster, Pennsylvania, he pursued a M.S. in Chemistry at the
University of Delaware while studying the use of pectinases to release cells from
leaf tissue in order to quantify distributions of plastoquinone and ubiquinone. He
studied aspects of glycolate metabolism with Dr. N. E. Tolbert and earned a Ph.D.
degree in Biochemistry in 1966. He completed postdoctoral work with Dr. A. A.
Benson at Scripp’s Institution of Oceanography (1966) and with Drs. J. Berry and
O. Bjorkman at the Carnegie Institution of Washington Plant Biology Laboratory
(1978), where he was able to pursue work on the association of pigments with
membranes and the response of plant metabolism to temperature stress. He has
been a faculty member in the Department of Biochemistry and Nutrition, Virginia
Polytechnic Institute and State University since 1967. He teaches students at all
levels and pursues two research interests. How leaf metabolism responds to the
combined effects of temperature stress and air pollutant exposure is studied with
his colleagues, Drs. R. G. Alscher and B. I. Chevone. The metabolic response of
the rat lens to systemic delivery of oxidants and potential sources of free radicals
is studied in collaboration with Dr. G. E. Bunce.
THE CONTRIBUTORS
William W. Adams III Alfred Hausladen
Assistant Professor Research Associate
Department of Environmental, Department of Biochemistry
Population, and Organismic Biology Duke University School of Medicine
University of Colorado Durham, North Carolina
Boulder, Colorado
Ruth G. Alscher John L. Hess
Associate Professor of Plant Associate Professor
Physiology Department of Biochemistry and
Department of Plant Pathology, Nutrition
Physiology, and Weed Science Virginia Polytechnic Institute and State
Virginia Polytechnic Institute and State University
University Blacksburg, Virginia
Blacksburg, Virginia
Barbara Demmig-Adams Norman G. Lewis
Assistant Professor Director and Professor
Department of Environmental, Institute of Biological Chemistry
Population, and Organismic Biology Washington State University
University of Colorado Pullman, Washington
Boulder, Colorado
Kenneth E. Pallett
Christine H. Foyer Head
Research Director Molecular Biochemistry
Laboratory of Metabolism and Cellular Biology Department
INRA Rhône-Poulenc Secteur Agro
Versailles, France Lyon, France
Andrew J. Young
Senior Lecturer
School of Biological and Earth
Sciences
Liverpool John Moores University
Liverpool, England
TABLE OF CONTENTS
Chapter 1
Glutathione............................................................................................................... 1
Alfred Hausladen and Ruth G. Alscher
Chapter 2
Ascorbic Acid..........................................................................................................31
Christine H. Foyer
Chapter 3
Carotenoids..............................................................................................................59
Kenneth E. Pallett and Andrew J. Young
Chapter 4
The Xanthophyll Cycle...........................................................................................91
Barbara Demmig-Adams and William W. Adams, III
Chapter 5
Vitamin E, a-Tocopherol......................................................................................Ill
John L. Hess
Chapter 6
Plant Phenolics......................................................................................................135
Norman G. Lewis
Index 171
Chapter 1
GLUTATHIONE
Alfred Hausladen and Ruth G. Alscher
TABLE OF CONTENTS
I. Introduction....................................................................................................2
IL Properties of Glutathione and Glutathione-Dependent Enzymes
in the Antioxidative System..........................................................................3
A. Glutathione and Homoglutathione...................................................3
1. Properties...............................................................................3
2. Subcellular and Intraorgan Distributiono f
Glutathione...........................................................................3
3. Assay.....................................................................................4
B. Dehydroascorbate Reductase...........................................................4
1. Properties...............................................................................4
2. Enzymatic vs. Nonenzymatic Reduction of
Dehydroascorbate.................................................................4
3. Assay.....................................................................................5
C. Glutathione Reductase......................................................................5
1. Overview..............................................................................5
2. Subunit Composition.............................................................5
3. Isozymes................................................................................6
4. Light Activation...................................................................6
5. Subcellular Localization......................................................6
6. Assay.....................................................................................6
D. Other Glutathione-Dependent Enzymes...........................................7
III. Biosynthesis and Metabolism of Glutathione...............................................7
A. The y-Glutamyl Cycle......................................................................7
B. Properties and Localization of Enzymes of the y-
Glutamyl Cycle.................................................................................9
C. Modification of GSH Concentration................................................9
D. Regulation of GSH Biosynthesis...................................................10
E. Effect of Light on GSH Biosynthesis.............................................11
IV. Active Oxygen in Plants.............................................................................12
A. Formation of Active Oxygen...........................................................12
1. The Role of Oxygen in the Formation of Free
Radicals................................................................................12
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