Download Drought-induced responses of photosynthesis and antioxidant

ARTICLE IN PRESS
Journal of Plant Physiology 161 (2004) 1189–1202
www.elsevier.de/jplph
REVIEW
Drought-induced responses of photosynthesis and
antioxidant metabolism in higher plants
Attipalli Ramachandra Reddya,*, Kolluru Viswanatha Chaitanyaa,
Munusamy Vivekanandanb
a
School of Life Sciences, Pondicherry University, Pondicherry 605 014, India
Department of Biotechnology, School of Life Sciences, Bharatidasan University, Tiruchirapalli 620 024, India
b
Received 16 October 2003; accepted 7 January 2004
KEYWORDS
Abscisic acid;
Antioxidants;
Antioxidative enzymes;
Drought;
Higher plants;
Photosynthesis
Summary
Environmental stresses trigger a wide variety of plant responses, ranging from altered
gene expression and cellular metabolism to changes in growth rates and crop yields. A
plethora of plant reactions exist to circumvent the potentially harmful effects caused
by a wide range of both abiotic and biotic stresses, including light, drought, salinity,
high temperatures, and pathogen infections. Among the environmental stresses,
drought stress is one of the most adverse factors of plant growth and productivity.
Understanding the biochemical and molecular responses to drought is essential for a
holistic perception of plant resistance mechanisms to water-limited conditions.
Drought stress progressively decreases CO2 assimilation rates due to reduced stomatal
conductance. Drought stress also induces reduction in the contents and activities of
photosynthetic carbon reduction cycle enzymes, including the key enzyme, ribulose1,5-bisphosphate carboxylase/oxygenase. The critical roles of proline and glycinebetaine, as well as the role of abscisic acid (ABA), under drought stress conditions
have been actively researched to understand the tolerance of plants to dehydration.
In addition, drought stress-induced generation of active oxygen species is well
recognized at the cellular level and is tightly controlled at both the production and
consumption levels in vivo, through increased antioxidative systems. Knowledge of
sensing and signaling pathways, including ABA-mediated changes in response to
drought stress, is essential to improve crop management. This review focuses on the
ability and strategies of higher plants to respond and adapt to drought stress.
& 2004 Elsevier GmbH. All rights reserved.
Abbreviations: A, foliar photosynthetic rate; AA, ascorbic acid; ABA, abscisic acid; APX, ascorbate peroxidase; CAT, catalase; COD,
choline oxidase; CMO, choline monoxygenase; CDH, choline dehydrogenase; DHA, dehydroascorbate; FBPase, fructose-1,6bisphosphatase; GlyBet, glycine betaine; GR, glutathione reductase; LEA, late embryogenesis abundant; MDA, monodehydroascorbate;
MDAR, monodehydroascorbate reductase; POD, peroxidase; ROS, reactive oxygen species; RWC, relative water content; RuBP, ribulose1,5-bisphosphate; SPS, sucrose phosphate synthase; SOD, superoxide dismutase
*Corresponding author. Tel.: þ91-0413-2655991; fax: þ91-0413-2655211.
E-mail address: [email protected] (A.R. Reddy).
0176-1617/$ - see front matter & 2004 Elsevier GmbH. All rights reserved.
doi:10.1016/j.jplph.2004.01.013
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Introduction
Plants are subjected to several harsh environmental stresses that adversely affect growth, metabolism, and yield. Drought, salinity, low and high
temperatures, flood, pollutants, and radiation are
the important stress factors limiting the productivity of crops (Lawlor, 2002). Several biotic (insects,
bacteria, fungi, and viruses) and abiotic (light,
temperature, water availability, nutrients, and soil
structure) factors affect the growth in higher plants
(as reviewed by Lichtenthaler, 1996, 1998). Among
these, drought is a major abiotic factor that limits
agricultural crop production. Plants experience
drought stress either when the water supply to
roots becomes difficult or when the transpiration
rate becomes very high. These two conditions often
coincide under arid and semi-arid climates. Water
stress tolerance is seen in almost all plant species
but its extent varies from species to species.
Although the general effects of drought on plant
growth are fairly well known, the primary effects of
water deficit at the biochemical and the molecular
levels are not well understood (Zhu, 2002; Chaitanya et al., 2003; Chaves et al., 2003). In order to
improve the agricultural productivity within the
limited land resources, it is imperative to ensure
higher crop yields against unfavorable environmental stresses. Understanding plant responses to the
external environment is of greater importance and
also a fundamental part for making the crops stress
tolerant. In this article we provide an overview of
the current understanding of photosynthetic carbon metabolism under drought. In addition, we will
describe the cellular antioxidative defense strategies used to circumvent the deleterious effects of
drought in the leaves of higher plants.
Photosynthesis under drought
The foliar photosynthetic rate, A, of higher plants
is known to decrease as the relative water content
(RWC) and leaf water potential decrease (Lawlor
and Cornic, 2002). However, the debate continues
as to whether drought mainly limits photosynthesis
through stomatal closure or through metabolic
impairment (Tezara et al., 1999; Lawson et al.,
2003). Stomatal limitation was generally accepted
to be the main determinant of reduced photosynthesis under drought stress (Cornic, 2000). This
has been attributed to decreases in both A and
internal CO2 concentration, which finally inhibits
total photosynthetic metabolism. Several nonstomatal effects are also attributed for stomatal
A.R. Reddy et al.
closure during drought. These include photophosphorylation (Meyer and Genty, 1999), ribulose-1,5bisphosphate (RuBP) regeneration (Lawlor, 2002),
rubisco activity (Medrano et al., 1997) and ATP
synthesis (Tezara et al., 1999). Considering the past
literature as well as the current information on
drought-induced photosynthetic responses, it is
evident that stomata close progressively with
increased drought stress, followed by reduced net
photosynthetic rates. It is well known that leaf
water status always interacts with stomatal conductance and a good correlation between leaf
water potential and stomatal conductance always
exists, even under drought stress. It is now clear
that there is a drought-induced root-to-leaf signaling, which is promoted by soil drying through the
transpiration stream, resulting in stomatal closure.
This chemical signal is now known to be abscisic
acid (ABA) and a direct correlation between the
xylem ABA content and stomatal conductance has
been demonstrated (Socias et al., 1997). The
closure of stomata under drought has also been
implicated due to changes in plant nutritional
status, xylem sap pH, farnesyl tranferase activity,
xylem hydrolic conductivity, and leaf-to-air vapor
pressure deficit (Oren et al., 1999). However,
differences in the complex regulation of stomatal
conductance among several species and genotypes
in response to leaf water potential and ABA makes
it difficult to see a clear pattern in photosynthetic
responses to drought. Decrease in RWC has been
known to induce stomatal closure and thus a
parallel decrease A (Cornic, 2000). Nevertheless,
it has been emphasized that a high degree of coregulation exists between stomatal opening and
photosynthesis (Farquhar et al., 2001; Hubbard
et al., 2001). Thus, stomatal movements are very
dynamic, involving complex regulation by several
environmental factors, and stomatal conductance
should be taken as an integrative parameter to
assess photosynthetic responses under drought.
The limitation of photosynthesis under drought
through metabolic impairment is a more complex
phenomenon than stomatal limitation. Changes in
the cellular carbon metabolism are likely to occur
early in the dehydration processes. Drought generally reduces the biochemical capacity for carbon
assimilation and utilization. The rate of photosynthesis in higher plants depends on the activity of
ribulose-1, 5-bisphosphate carboxylase/oxygenase
(rubisco) as well as synthesis of RuBP (Ramachandra
Reddy, 1996; Tezara et al., 1999; Chaitanya et al.,
2002a; Parry et al., 2002). Despite several studies
on photosynthetic carbon assimilation under
drought, a definitive conclusion regarding the most
sensitive changes in rubisco metabolism remains
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Photosynthetic and antioxidant responses under drought stress
elusive. Rubisco quantity limits photosynthesis in
most cases, although in some cases the regeneration of RuBP has been shown to limit the photosynthetic capacity (Vu et al., 1999). Studies on
transgenic plants suggest that rubisco may not be
the main limitation in chloroplast metabolism
(Tezara et al., 1999). However, most of the
evidence on drought-induced changes indicates
that the amount and activity of rubisco really
control photosynthetic carbon assimilation. The
quantity of rubisco in leaves is controlled by the
rate of synthesis and degradation of the enzyme,
even in stressful environments. Decreased synthesis
of rubisco under drought was evidenced by a rapid
decrease in the abundance of rubisco small subunit
(rbcS) in tomato (Vu et al., 1999). Parry et al.
(2002) suggested that rubisco activity is regulated
to match the capacity of the leaf to regenerate
RuBP. Loss of rubisco activity has been reported in
several plants under drought (Parry et al., 2002;
Chaitanya et al., 2003). In tobacco, initial and total
activities of rubisco were decreased under drought
and this decrease was due to the reduction in the
apparent Kcat ; rather than the changes in the
activation state of the enzyme (Parry et al.,
2002). These studies indicate that irreversible
damage to rubisco should be taking place in the
leaves, or there is a reduction in the total soluble
protein. Inhibition of rubisco activity was due to
binding of inhibitors like CA1P with rubisco. Total
activity of rubisco has been used as an indicator to
estimate the percentage of catalytic sites that are
blocked by inhibitors. The decrease in the initial
and total activities of rubisco with an increase in
drought stress was related to an increase in CA1P
(Parry et al., 2002). However, the interactions of
rubisco with tight binding inhibitors have been
known to be advantageous in vivo, as they could
prevent rubisco from degradation by proteases. It is
thus believed that although drought stress reduces
rubisco activity, the carboxylating enzyme is
otherwise protected from protease degradation. It
is quite interesting to know that the release of tight
binding receptors requires the participation of
rubisco activase and ATP hydrolysis. Rubisco activase is an abundant protein, which regulates the
active site conformation of rubisco, removes the
tight-bound inhibitors, and then allows rubisco to
undergo rapid carboxylation. This suggests that
there is now evidence that rubisco activase activity
also decreases with increasing drought stress
(Chaves et al., 2002). Under these conditions, the
removal of inhibitors from rubisco binding sites by
rubisco activase is now known to be impaired due
to reduced ATP concentrations under drought stress
conditions (Tezara et al., 1999).
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The rate of photosynthesis also depends on the
synthesis of RuBP. The response of photosynthesis
to RuBP content was linear, indicating that the
supply of RuBP determines the net photosynthetic
activity. However, limited data are available on the
actual role of RuBP regeneration in relation to the
rubisco activity. Tezara et al. (1999) reported that
limitations in RuBP regeneration could result from
an inadequate supply of ATP and/or NADPH to the
PCR cycle, or from a decreased rate of turn over
caused by low enzyme activity either in stressed or
non-stressed plants. The reactions of RuBP to 3-PGA
always decreased with reducing RWC, which suggest that regeneration of RuBP was inhibited under
drought. RuBP contents and synthesis are presumed
to be under the control of the PCR cycle or the
supply of ATP and NADPH to the PCR cycle. ATP
synthesis was known to limit A at low RWC because
of the inhibition of photophosphorylation in waterstressed sunflower leaves (Lawlor, 2002). Under
conditions of drought, reduction in chloroplast
volume might also lead to desiccation within the
chloroplast, which in turn leads to conformational
changes in rubisco. Drought stress conditions are
also known to acidify the chloroplast stroma,
resulting in inhibited rubisco activity (Meyer and
Genty, 1999). However, C4 plants are known to use
water more efficiently than C3 plants, but they also
need rubisco to achieve a given rate of photosynthesis. The primary carboxylating enzyme in C4
plants, phosphoenol pyruvate carboxylase (PEPcase), is also inhibited under drought (Boyer et al.,
1997).
It is also known that the limitation of PCR cycles
per se under drought stress conditions is due to
partial reduction in the activities of the other
enzymes involved in the cycle. The activities of
phosphoribulo kinase and fructose-1,6-bisphosphatase (FBPase) were also reduced with decreases in
RWC (Haupt-Herting and Fock, 2002). Drought also
results in changes in the ratio of the end products
of photosynthesis, starch, and sucrose. With reduced water potentials, the activities of FBPase
and sucrose phosphate synthase (SPS) declined,
indicating that the rate of sucrose synthesis is
strongly influenced by drought stress (Haupt-Herting and Fock, 2002). The inhibited activities of
FBPase and SPS might regulate the synthesis of
sucrose and starch and as well as their partitioning
under drought stress. The altered starch/sucrose
ratios under drought stress should certainly cause
changes in the Pi flux across the chloroplast
membrane. Reduced levels of Pi in the chloroplasts
inhibit ATP synthesis, which in turn has a greater
impact on photophosphorylation and PCR cycle
(Tezara et al., 1999). Sometimes the combined
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effects of heat and light stress on photosynthesis
superimposed with drought will be more complex
(Wingler et al., 1999). Under these unfavorable
conditions, plants are known to lose chlorophyll
(Havaux and Tardy, 1999) or divert the absorbed
light to other processes, like thermal dissipation to
protect the photosynthetic apparatus (DemingAdams and Adams, 1996). Photochemical efficiency
under drought can also be monitored by measuring
chlorophyll fluorescence to assess the photosynthetic functioning (Osmond et al., 1999), while
multispectral fluorescence images are also currently being used to study plant responses to
drought stress (Lichtenthaler, 1997).
Drought-induced oxidative stress in
plants
Acclimation of plants to changing environmental
conditions such as drought stress is essential for
survival and growth. Plant responses to drought
stress, especially in reference to chloroplast
metabolism, are complex. Drought stress is known
to inhibit photosynthetic activity in tissues due to
an imbalance between light capture and its
utilization (Foyer and Noctor, 2000). Down regulation of photosystem II (PSII) activity results in an
imbalance between the generation and utilization
of electrons, apparently resulting in changes in
quantum yield. These changes in the photochemistry of chloroplasts in the leaves of droughtstressed plants result in the dissipation of excess
light energy in the PSII core and antenna, thus
1
generating active oxygen species (O
2 , O2, H2O2,
OH), which are potentially dangerous under
drought stress conditions (Peltzer et al., 2002).
Also, changes in the photosynthetic electron transport under drought inevitably lead to the formation
of superoxide radicals (O
2 ), since the molecular
oxygen competes with NADP for reduction at the
acceptor side of photosystem I (PSI). Although
photosynthetic electron transport is considerably
tolerant to drought stress, variations of 20–30%
(reduction in the leaf chloroplast photosynthetic
electron transport) among plant species are not
uncommon. Drought not only causes dramatic loss
of pigments, but also leads to disorganization of
thylakoid membranes (Ladjal et al., 2000). Photochemical reactions associated with PSII have been
shown to be more susceptible to drought. Loss or
decline in D1 and D2 proteins of PSII have been
reported, which were associated with the loss of
PSII chemistry (Lu and Zhang, 1999). Inhibition of
CO2 assimilation, coupled with the changes in
A.R. Reddy et al.
photosystem activities and photosynthetic electron
transport capacity, results in accelerated production of active oxygen via the chloroplast Mehler
reaction (Asada, 1999). Thus, during drought, there
is considerable potential for increased accumulation of superoxide and hydrogen peroxide resulting
from the increased rate of O2 photoreduction in
chloroplasts (Robinson and Bunce, 2000). PSII
down-regulation and thermodynamic constraints
exert a restraining control on the rate of electron
flow in the drought-stressed leaves, and the oxygen
free radicals are responsible for most of the
oxidative damage in biological systems. Deleterious
effects of free radicals on biological structures
include DNA nicking, amino acid and protein
oxidation, and lipid peroxidation (Asada, 1999;
Johnson et al., 2003). Reactive oxygen species
(ROS) attack the most sensitive biological macromolecules in cells to impair their function. The
damaged targets are recovered by repair or by
replacement via de novo biosynthesis. However,
under intense stressful conditions and due to
severely damaged target molecules, a catastrophic
cascade of events set in, resulting in cell death.
The destiny of cells under stressful environments is
determined by the duration of stress as well as the
protective capacity of the plant. ROS plays a crucial
role in causing cellular damage under drought
stress. The sequence of events in the plant tissue
subjected to drought stress are: (1) increased
production of ROS and of oxidized target molecules; (2) increases in the expression of genes for
antioxidant functions; (3) increases in the levels of
antioxidative systems and antioxidants; and (4)
increased scavenging capacity for ROS, resulting in
tolerance against the drought stress (Mano, 2002).
Secondary products of ROS in plant cells during
stress include lipid peroxides and thiol radicals.
Although a series of regulatory mechanisms have
evolved within the plant cell to limit the production of these toxic molecules, oxidative damage
remains a potential problem, since it causes
perturbations in metabolism, such as a loss of coordination between energy production (source) and
energy utilization (sink) processes during photosynthesis in green leaves in stressful environments.
Mechanisms of ROS detoxification exist in all
plants and can be categorized as enzymatic [superoxide dismutase (SOD), catalase (CAT), ascorbate
peroxidase (APX), peroxidase (POD), glutathione
reductase (GR) and monodehydroascorbate reductase (MDAR)] and non-enzymatic (flavanones, anthocyanins, carotenoids and ascorbic acid (AA)).
The degree to which the activities of antioxidant
enzymes and the amount of antioxidants increase
under drought stress will be extremely variable
ARTICLE IN PRESS
Photosynthetic and antioxidant responses under drought stress
among several plant species and even between two
cultivars of the same species. The level of response
depends on the species, the development, and the
metabolic state of the plant, as well as the duration
and intensity of the stress. Many stress situations
cause an increase in the total foliar antioxidant
activity (Pastori et al., 2000), but little is known
about the coordinative control of activity and
expression of the different antioxidant enzymes in
plant cells that are subjected to drought stress.
Several studies have reported enhanced stress
tolerance related to overproduction of chloroplastic SOD (Arisi et al., 1998; Foyer, 2002). Induction
of oxidative stress in drought-stressed plants has
also been well known (Ramachandra Reddy et al.,
2000; Chaitanya et al., 2002b; Mano, 2002).
Antioxidant components are also known to be
distributed among all photosynthetic cells in higher
plants. The distribution of antioxidative enzymes
between mesophyll and bundle sheath cells of C4
plants has been described (Foyer, 2002). In maize
leaves, GR and DHAR were exclusively localized in
mesophyll cells whereas most of the SOD and APX
were localized in mesophyll and bundle sheath cells.
CAT and MDHAR were approximately equally distributed between mesophyll and bundle sheath
cells. H2O2 was found to accumulate only in
mesophyll cells (Doulis et al., 1997; Foyer, 2001).
These localization studies are very interesting,
because enzymes of the PCR cycle, which are very
sensitive to H2O2, are found only in bundle sheath
chloroplasts. Such studies on C4 plants indicate that
oxidative damage was not uniformly distributed
between mesophyll and bundle sheath cells of C4
plants. Kingston-Smith and Foyer (2000) suggested
that oxidative damage under stressful conditions in
C4 plants is restricted to bundle sheath tissue
because of inadequate antioxidant protection in this
tissue. However, very little mechanistic information
is available on drought-induced antioxidative metabolism between the two cell types in C4 plants. In
contrast, few experiments on woody plants have
shown differing antioxidant system responses to
drought stress, ranging from no effects to decreases
in the activity in certain antioxidative enzymes
(Kronfub et al., 1998). The reason for this contrasting behavior among plant species might be due to
the plants’ gradual adjustment to the changing
climate, thus allowing for an acclimation process.
Plant responses to drought stress
The responses of plants to drought stress are highly
complex, involving deleterious and/or adaptive
1193
changes. In particular, if the drought stress is under
field conditions, the plant responses can be
modified synergistically or antagonistically. Early
responses of plants to drought stress usually help
the plant to survive for some time, while the
acclimation of the plant subjected to drought is
indicated by the accumulation of certain new
metabolites associated with the structural capabilities to improve plant functioning under drought
stress (Pinhero et al., 2001). Acclimation-related
changes in the root/shoot ratio or the temporary
changes in accumulation of reserves in the stem
under drought stress are accompanied by the
changes in carbon and nitrogen metabolism (Noctor
et al., 2002). A continuous oxidative assault on
plants during drought stress has led to the presence
of an arsenal enzymatic (discussed above) and nonenzymatic antioxidant defenses to counter the
phenomenon of oxidative stress in plants. The
non-enzymatic plant antioxidants can be classified
into two major types: (1) AA-like scavengers, and
(2) pigments such as carotenoids (Conklin, 2001).
AA is an important antioxidant, which reacts not
only with H2O2 but also with O
2 , OH and lipid
hydroperoxidases. Protective functions of AA are
also assayed in a wide variety of common human
ailments and diseases. In recent years, clear
evidence has emerged that elevated dietary intake
of AA lowers the incidence of cancer, cardiovascular disease, and other oxidative stress disorders
(Noctor and Foyer, 1998; Grassmann et al., 2002;
Munne-Bosch and Algere, 2003). On the other hand,
AA has been implicated in several types of
biological activities in plants: (1) as an enzyme
co-factor, (2) as an antioxidant, and (3) as a donor/
acceptor in electron transport at the plasma
membrane or in the chloroplasts, all of which are
related to oxidative stress resistance (Conklin,
2001). In chloroplasts, the so-called ‘‘HalliwellAsada’’ pathway indicates that APX uses AA and
oxidizes it to monodehydroascorbate (MDA). MDA
may give rise to dehydroascorbate (DHA). Both MDA
and DHA will then be reduced to regenerate the
ascorbate pool. This type of scavenging is thought
to occur near PSI, thereby minimizing the risk of
escape and reaction of ROS with each other (Foyer
and Noctor, 2000). AA is water-soluble and also has
an additional role in protecting or regenerating
oxidized carotenoids or tocopherols (Imai et al.,
1999). AA is a major metabolite in chloroplasts of
higher plants and represents about 10% of the
soluble carbohydrate pool in leaves (Noctor and
Foyer, 1998). AA is also known to function as the
‘‘terminal antioxidant’’ because the redox potential of AA/MDA pair (þ280 mv) is lower than that of
most of the bioradicals (Scandalios et al., 1997).
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1194
The levels of glutahione reductase and DHA
reductase increased upon water stress in Sporobolus stapfianus (Conklin, 2001). An increase in MVdependent photoproduction of MDHA was also
observed in lettuce leaves due to water stress
(Conklin, 2001). However, very little is known about
the regulation of AA biosynthesis in higher plants.
Nevertheless, AA concentrations are reported to
increase in response to wounding (Robinson and
Bunce, 2000). It is conceivable that adaptations of
intracellular AA to drought stress might clearly
depend on the balance between the rates and
capacity of AA biosynthesis and turnover related to
antioxidant demand (Chaves et al., 2002). The
biosynthesis of AA from hexose phosphate and its
involvement in protection against photooxidative
stress suggest that there may be links between
photosynthesis and the AA pool size.
Although the antioxidant defense system is
impaired under stressful conditions, plants are able
to get rid of excessive energy by thermal dissipation associated with an increase in the concentration of xanthophyll pigments, zeaxanthin, and
antheraxanthin, at the expense of violoxanthin in
water stressed plants (Alonso et al., 2001). The
high proportion of xanthophylls under stress conditions may serve as a protection mechanism in
leaves. The activation of the xanthophyll cycle has
also been detected in several other plants subjected to drought stress (Foyer, 2001). Mechanisms
are not known of a precise regulation to prevent
potentially destructive side reactions involving AOS
that damage photosynthetic pigments and proteins.
In addition, the xanthophyll cycle may be involved
in lowering the yield of triplet chlorophyll formation by pre-emptive quenching of the excited
singlet state of chlorophyll (Rontein et al., 2002).
It is thus presumed that the role of antioxidants and
carotenoid pigments in regulating photosynthetic
electron transport is crucial. Thus, a complex
relationship between xanthophyll cycle-dependent
energy quenching and formation of AOS exists in
photosynthetic systems of plants under drought
stress. a-tocopherol, found in green parts of plants
also scavenges lipid peroxy radicals through the
concerted action of other antioxidants (MunneBosch and Alegre, 2002). Further, tocopherols were
also known to protect lipids and other membrane
components by physically quenching and chemically reacting with 1O2 in chloroplasts, thus
protecting the structure and function of PSII (Trebst
et al., 2002).
Many plants and other organisms cope with
osmotic stress by synthesizing and accumulating
some compatible solutes, which are termed as
osmoprotectants or osmolytes. These compounds
A.R. Reddy et al.
are small, electrically neutral molecules, which are
non-toxic even at molar concentrations (Alonso
et al., 2001). During osmotic stress, plant cells
accumulate solutes to prevent water loss and to reestablish cell turgor. The solutes that accumulate
during the osmotic adjustment include ions such
as Kþ, Naþ, and Cl or organic solutes that include
nitrogen-containing compounds, such as proline
and other amino acids, polyamines and quaternary ammonium compounds like glycine betaine
(GlyBet) (Tamura et al., 2003). Other osmolytes,
that are produced in response to stress, include
sucrose, polyols, sugar alchohols (pinitol), and
oligosacharides. The organic solutes are compatible
with cellular processes and accumulate to high
levels in the cytosol with increasing drought.
Production of osmolytes is a general way to
stabilize membranes and maintain protein conformation at low leaf water potentials. The synthesis
and accumulation of osmolytes varies among plant
species as well as among different cultivars of the
same species. Osmolytes play a major role in
osmotic adjustment and also protect the cells by
scavenging ROS (Pinhero et al., 2001). Proline is
also known to be involved in reducing the photodamage in the thylakoid membranes by scavenging
and/or reducing the production of 1O2. Proline
accumulation in plants is caused, not only by the
activation of proline biosynthesis, but also by the
inactivation of proline degradation, thereby resulting in a decrease in the level of accumulated
proline in rehydrated plants. Proline degradation to
glutamic acid via D1 pyrroline-5-carboxylate in
higher plants is catalyzed by proline dehydrogenase
and D1 pyrroline-5-carboxylate dehydrogenase in
mitochondria (Bohnert and Jenson, 1996). It can
also be inferred that proline acts as a free radical
scavenger and may be more important in overcoming stress than in acting as a simple osmolyte.
Such studies open a new avenue of research for
metabolic engineering in several agriculturally
important crop plants for drought resistance.
Accumulation of other amino acids like glycine,
serine, and glutamate are known to regulate and
integrate the metabolism in stressed photosynthetic tissues (Lawlor and Cornic, 2002). Proline
concentrations increase many fold with reduced
leaf water potentials, and at this stage photosynthesis is known to be quite reduced (Morot-Guadry
et al., 2001). A more common explanation for the
accumulation of proline is that it confers advantages by protecting membranes and proteins when
RWC decreases. In plant cells, osmolytes are
typically confined to the chloroplasts and cytoplasmic compartments that together occupy 20% or less
of the volume of mature cells (Ain-Lhout et al.,
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Photosynthetic and antioxidant responses under drought stress
2001). Natural osmolyte concentrations in plant
cells can reach 200 mM or more, and such concentrations are osmotically significant and have pivotal
roles in maintaining cell turgor and driving the
gradient for water uptake under stress (Rhodes and
Samaras, 1994).
H3C
H3C
N
CH2
H2C
CH2
H2C
C
-
COO
H3C
Glycine Betaine
COON
H2
L-proline
Accumulation of GlyBet occurs in some, but not
in all, higher plants. Most of the GlyBet is
synthesized in chloroplasts as two enzymes, namely
choline monoxygenase (CMO) and betaine aldehyde
dehydrogenase, are responsible for GlyBet synthesis chloroplastically. GlyBet synthesis can be
induced by both drought and salt stress (NaCl,
KCl, MgCl2, Na2SO4) by over-expression of CMO, and
betaine aldehyde dehydrogenase (Nakamura et al.,
2001). Progressive drought and salinity were also
known to induce late embryogenesis abundant
(LEA) proteins in vegetative organs, which can
stabilize enzyme complexes and the structure of
cell membranes (Chourey et al., 2003; Liang et al.,
2003). The increased concentrations of GlyBet
under drought stress situations clearly suggest that
this osmolyte has an important role in protecting
plant cell mechanisms under conditions of drought.
A physiological role of GlyBet in alleviating osmotic
stress was proposed based on accumulation of
GlyBet in plants subjected to drought (Jun et al.,
2000). GlyBet has been shown to protect enzymes
and membranes and also to stabilize PSII protein
pigment complexes under stressful conditions
(Papageorgiou and Morata, 1995). A moderate
stress tolerance, as shown by dry weight production
in transgenic plants, was noticed based on relative
shoot growth studies under stress conditions, like
drought (Jun et al., 2000).
Abscisic acid (ABA) responsiveness to
water deficit in plants
The plant hormone ABA is produced de novo under
water deficit conditions and plays a major role in
response and tolerance to dehydration (Shinozaki
1195
and Yamaguchi-Shinozaki, 1999). ABA is synthesized
from xanthophylls via violaxanthin, xanthoxin and
ABA-aldehyde (C-40 pathway). The conversion of
violaxanthin to xanthoxin is the rate-limiting step
in ABA biosynthesis under drought stress. The
involvement of drought-induced ABA and ethylene
in shoot and root growth is still a controversial
subject (Robert and LeNoble, 2002). Under
drought-stressed conditions, stomata close in response to either a decline in leaf turgor and/or
water potential, indicating that stomatal responses
are closely linked to soil moisture content and leaf
water status. Much is known about the role of ABA
in closing the stomata as well as ABA production in
dehydrating roots and ABA circulation in the plant
(Wilkinson and Davies, 2002). However, little is
understood about the exact relationship between
water deficit and ABA long-distance signaling and
the nature of interactions between ABA and other
chemical signals, like ethylene and cytokinins
(Sauter and Hartung, 2000). ABA-induced stomatal
closure causes depression in net CO2 uptake, which
involves mechanisms at both the stomatal and
chloroplast levels. The mid-day decline in stomatal
conductance in several plant species under drought
conditions may be due to increased sensitivity to
xylem-carried ABA, which is induced by low leaf
water potentials (Wilkinson and Davies, 2002).
Also, the decline in intercellular CO2 following
stomatal closure apparently induces a down regulation of photosynthetic machinery to match the
available carbon substrate. The amount of ABA in
xylem sap can increase substantially as a function
of reduced water availability in the soil and this
might result in an increased ABA concentration in
different compartments of the leaf. Zhang and
Outlaw (2001) reported that stressing Vicia faba
roots could change ABA concentrations at the guard
cell apoplast and that the apoplastic guard cell ABA
concentration correlated with changes in stomatal
aperture more effectively than did the guard cell
symplastic fraction. These studies indicate that
apoplastically facing guard cell ABA receptors seem
to be important in the responses to stress signals
experienced by plants. Increases in the xylem sap
ABA and leaf ABA were correlated with reduced
stomatal conductance under partial root drying
conditions in grape vines (Stoll et al., 2000).
Deciphering the molecular events in
plants under drought
Plants respond to water deficit and adapt to semiarid drought conditions by various physiological,
ARTICLE IN PRESS
1196
biochemical, anatomical, and morphological
changes, including transitions in gene expression.
Plants also adapt different types of life strategies
to cope and resist drought stress. Two such
strategies are drought avoidance and drought
tolerance. Drought avoidance is the ability of the
plant to maintain high tissue water potential under
drought conditions, while drought tolerance is a
plant’s ability to maintain its normal functions even
at low tissue water potentials. Drought avoidance is
usually achieved through morphological changes in
the plant, such as reduced stomatal conductance,
decreased leaf area, development of extensive root
systems and increased root/shoot ratios (Levitt,
1980). On the other hand, drought tolerance is
achieved by cell- and tissue-specific physiological,
biochemical, and molecular mechanisms, which
include specific gene expression and accumulation
of specific proteins under drought stress. A variety
of genes are induced by drought stress, and
functions of such gene products have been predicted from sequence homology with known proteins (Bohnert and Jenson, 1996; Shinozaki et al.,
1999). Drought stress-induced genes function, not
only in protecting plant cells from dehydration, but
also in regulating certain genes for signal transduction in response to drought.
During the past two decades, several specific
proteins have been characterized in droughtstressed plants that can be classified as LEA,
dissication stress protein, response to ABA, dehydrins (dehydration-induced proteins), cold regulation proteins, proteases (for protein turnover),
enzymes required for the biosynthesis of various
osmoprotectants (sugars, proteins and GlyBet), the
detoxification enzymes (SOD, CAT, APX, POD, GR),
and protein factors involved in the regulation of
signal transduction and gene expression, such as
protein kinases and transcription factors (Tzvi
et al., 2000). The mRNAs corresponding to the
genes of antioxidant enzymes are induced by
drought stress (Xu et al., 1996). Transgenic plants
expressing LEA proteins exhibited more tolerance
to water deficit conditions (Bray, 2002). Some of
the stress-inducible genes that encode proteins,
such as D1 pyrroline-5-carboxylate synthase (P5CS,
a key enzyme for proline biosynthesis), was overexpressed in transgenic plants to produce a stress
tolerant plant (Kishore et al., 1995). Dehydrins are
another group of drought-induced proteins with a
consensus 15-amino acid sequence (EKKGIMDKIKEKLPG), that is involved in hydrophobic interactions leading to macromolecule stabilization and
that may act synergistically with compatible
solutes (Close, 1996). Plastid lipid-associated proteins, termed fibrillin/chloroplastic drought stress
A.R. Reddy et al.
protein-34 proteins, substantially increased in
several dicot and monocot plants in response to
water deficit (Langenkaneper et al., 2001). Plant
fibrillins have been reported to stabilize hydrophobic carotenoids as well as to participate in
the structural stabilization and protection of
thylakoids during dehydration (Schwartz et al.,
2001). The accumulation of free proline in response
to osmotic stress is known to be regulated by a
rate-limiting enzyme P5CS in higher plants and
antisense transgenics of Arabidopsis with P5CS
cDNA showed morphological alterations in leaves
that were hypersensitive to osmotic stress (Nanjo
et al., 1999). In these plants, proline deficiency
specifically affected structural proteins of cell
walls, suggesting that proline is not only an
osmoreuglator in stressed plants but it is also
involved in osmotolerance and morphogenesis in
plants.
The characterization of biosynthetic pathways
leading to different compatible solutes (polyols,
sugars, aminoacids, GlyBet, and related compounds) and the exploitation of different genes
through transgenic plant research suggest the
importance of the accumulation of these compounds in the acclimatization of plants to drought
stress (Sakamoto and Murata, 2002). Transgenic
production of these compatible solutes can protect
plants to a greater extent from stress, even when
they are present at low and osmotically insignificant levels (Sakamoto and Murata, 2002). There
has been considerable progress recently in the
molecular characterization of GlyBet biosynthesis,
which encouraged the introduction of GlyBet
biosynthetic enzymes into GlyBet-deficient plants.
The first demonstration in plants of engineered
synthesis of GlyBet showed over-expression of the
gene for choline oxidase (COD) from Arthrobacter
globiformis in the chloroplasts of Arabidopsis and
rice (Hayashi et al., 1997; Sakomoto et al., 1998).
GlyBet accumulated at a concentration of
50–100 mM in the chloroplasts. Tobacco has
been engineered to produce bacterial choline
dehydrogenase (CDH) and COD as well as plant
CMO (Ain-Lhout et al., 2001). Metabolic engineering of choline biosynthesis in plants could enhance
plants’ resistance to osmotic stress. The key
enzyme of the plant choline synthesis pathway is
phosphoethanolamine-N-methyl transferase and
over-expression of this enzyme in transgenic
tobacco increased the phosphocholine by 5-fold
and the free choline by 50-fold (Mc Neil et al.,
2001). The capacity of such transgenic plants to
accumulate GlyBet seems to depend on the plant
species and on the subcellular localization of the
engineered enzyme.
ARTICLE IN PRESS
Photosynthetic and antioxidant responses under drought stress
Complex mechanisms seem to be involved in
gene expression and signal transduction in
response to drought stress. However, the precise
molecular mechanisms of regulation of plant
genes to water deficit conditions remain elusive.
The expression patterns of genes induced by
drought stress indicate broad variations in the
timing of induction of different genes. Many
drought-inducible genes are known to respond to
ABA levels in leaves (Shinozaki and YamaguchiShinozaki, 1999; Zhu, 2002). Mutants with ABA
deficiency were used to analyze drought-inducible
genes and the results indicate that several genes
are induced by exogenous ABA treatment. There
are ABA-independent and ABA-dependent regulatory systems of gene expression under drought
stress. Several mutants in ABA signaling have
been identified and their genes encode protein
phosphatase and farnesyl transferase (Qin and
Zeevart, 2002). These studies clearly indicate
that protein dephosphorylation and protein
farnesylation are involved in ABA signaling. The
role of ABA in drought-stress signal transduction has
been genetically analyzed with ABA insensitive
mutants in several plant species. Maize vp1 and
Arabidpsis abi1, abi2, abi3 and abi4 have been
extensively characterized and their genes have
been cloned (Bonetta and Mc Court, 1998). Two
steps in the conversion of xanthoxin to ABA
aldehyde and oxidation of ABA-aldehyde to ABA
are determined by Arabidopsis aba2 and aba3
mutants, respectively (Marin et al., 1996). Recently, Iuchi et al. (2000) showed that the cowpea
drought inducible VuNCED1 gene encodes the 9-cisepoxy-carotenoid dioxygenase, a key enzyme in
ABA biosynthesis and its product is localized in
plastids. Analysis of transgenic plants in which the
VuNCED1 gene is over-expressed or suppressed by
antisense RNA should give us more information on
its function in ABA biosynthesis in drought-stressed
plants. However, the molecular mechanism of
regulation of ABA biosynthesis under stress conditions is yet to be established. Further, nitrate
reductase activity decreased under drought stress
in a number of plant species and was strongly downregulated at the mRNA level in Arabidopsis (Foyer
et al., 1998). A number of genes induced by water
deficit may also be predicted to play a role in
altering cell wall characteristics, including a polygalactouronase-like gene product (Bray, 2002).
Microarray assays using full-length cDNAs obtained
from Arabidopsis plants subjected to different
stresses have recently been reported (Seki et al.,
2001). The physiological, biochemical and molecular responses to drought stress in higher plants are
summarized in Fig. 1.
1197
Perspectives of future research
Plant productivity is greatly affected by environmental stresses, and drought stress is among the
worst scourges of agriculture. Water deficit occurs,
not only during drought, but also during cold
conditions and causes turgor stress at the cellular
level. Thus, a change in the osmotic potential
across the plasma membrane should be one of the
triggers of the stress response at the molecular
level, thus resulting in an oxidative burst under
drought stress conditions. An understanding of the
mechanistic basis for changes in plant gene
expression in response to environmental cues is
beginning to emerge. The foremost challenge in
improving the crop productivity in the near future
is to identify molecular events for better understanding of how to improve drought tolerance.
During the last 10 years, many drought-induced
genes have been cloned in a variety of plants. The
transcriptional regulatory regions of drought-induced genes should be completely analyzed to
understand the expression of these genes under
water-deficit. The Arabidopsis genome project is to
assign functions to all the 20,000 genes of
Arabidopsis by 2010, which should give us complete
metabolic maps of related genes. Assessing the
relative contribution of each gene to dehydration
tolerance and eliminating those that do not
measurably effect stress tolerance is a major
challenge. All recent transgenic approaches used
single genes that led to marginal stress tolerance.
Hence, future work should introduce sets of genes
in order to express quantitative traits, determined
by multiple genes, under drought stress. Metabolic
engineering of osmoprotectant pathways was successful for certain model plants subjected to stress.
But there is a long way to raise the accumulation
levels of these osmoprotectants to overcome side
effects related to accumulation and to prove the
value of engineered osmoprotectants in major
crops under field stress conditions. We are now
moving towards a complete understanding of the
whole plant signaling process and the intensity of
chemical signaling under drought. These signals
may vary between environments and plant species.
The actual role of the ascorbate-glutathione
cycle should provide a valuable intellectual framework, which will contribute to an exciting area of
stress relations in higher plants. The identification
of factors that sense alterations in glutathione pool
will be a valuable addition to the tools that are
available for manipulation of plant stress responses. We can also expect rapid progress in a
better understanding of AA metabolism, eventually
leading to controlled manipulation of AA content
ARTICLE IN PRESS
1198
A.R. Reddy et al.
Fig. 1. Physiological, Biochemical and Molecular responses to drought stress in higher plants.
for improved nutritional quality and drought stress
resistance. The role of AA in a wide number of
cellular processes influences growth and development of plants. The goal of increasing AA biosynthesis in transgenic plants should be aimed at
improving the stress tolerance in plants. To date,
no AA transporters across the cell membrane have
been identified. Future work on AA biosynthesis and
its regulation should greatly benefit all fields of
plant biology. Despite recent progress in characterization of the protective action of proline and
GlyBet in transgenic plants, there remains a critical
gap, in terms of protective effects of these
osmoprotectants between phenomena in vivo and
in vitro. Our understanding of ABA action at the
molecular level during drought stress is incomplete. It is important to consider that ABA signaling
is part of a complex web of stress pathways. It
would be also interesting to know whether inhibitors of translational processing have effects on
ABA-inducible gene expression in plants under
drought stress. Signaling pathways concerning the
initiation by different stressors, such as drought,
salt, UV radiation, and ozone, might change gene
expression or the amount and activity of different
proteins, but these are not yet established. Therefore, the drought and salt signal transduction
pathways and the fine tuning of plant sensing and
signaling systems to unfavorable environments are
still in need of further study. Molecular analysis of
these factors should provide a better understanding
of the signal transduction cascades under drought
stress. The interactive effects of elevated CO2
concentrations and drought/salt stress on physiological, biochemical, and molecular responses of
plants in the changing global climate scenario are
still far from yielding a clear picture. Also, our
knowledge of the metabolic changes that contribute to dehydration tolerance is limited. Recently,
the role of ROS-scavenging systems in plant stress
tolerance has gained attention through the use of
transgenic approaches by manipulating the levels
of antioxidant enzymes. Therefore, it is conceivable that decreasing oxidative stress offers an
avenue to counter the effects of environmental
stresses. Such studies are to be extended to the
whole plant level and the impact of gene expression under drought on several plant metabolic
ARTICLE IN PRESS
Photosynthetic and antioxidant responses under drought stress
processes is to be assessed. The eventual practical
application of all of these studies should be
directed toward the engineering of several worldwide food crops with increased drought stress
tolerance.
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