Download Understanding oxidative stress and antioxidant functions in order to

Plant Physiology Preview. Published on November 2, 2010, as DOI:10.1104/pp.110.166181
Running Title: Protection from Oxidative Stress.
Corresponding author: Christine H. Foyer
Centre of Plant Science
Research Institute of Integrative and Comparative Biology
Faculty of Biological Science University of Leeds
Leeds
LS2 9JT
UK
Tel +44 (0)113 343 1421; Fax +44(0)113 323 3144
Email: [email protected]
Research area: Update for Special Issue “Enhancing photosynthesis”
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Copyright 2010 by the American Society of Plant Biologists
Update: Understanding oxidative stress and antioxidant functions in order to enhance
photosynthesis
Christine H. Foyer1 and Shigeru Shigeoka2
1
Centre for Plant Sciences, Faculty of Biology, University of Leeds, Leeds, LS2 9JT, UK.
2
Department of Advanced Bioscience, Kinki University, Nakamachi, Nara 631-8505, Japan
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Footnotes: Part of this work was supported by EU funded project on Chloroplast Signals:
COSI (ITN-GA-2008-215174), and part by a Grant-in-aid for Scientific Research (A) (S.S.
:22248042) from the MEXT, JAPAN.
Author to whom correspondence should be addressed: Email: [email protected]
Tel +44 (0)113 343 1421; Fax +44(0)113 323 3144
The author(s) responsible for distribution of materials integral to the findings presented in
this article in accordance with policy described in the instructions for Authors
(www.plantphysiol.org) is: Christine H. Foyer ([email protected])
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Photosynthesis is a well established source of reactive oxygen species (ROS) in plants. The
photosynthetic transport chain operates in an aerobic environment and thus regulatory
systems are required to minimize ROS production. An efficient antioxidant network is also
essential in order to process ROS effectively and to maintain intracellular ROS pools at low
levels. The importance of the antioxidant network in maintaining high rates of
photosynthesis has been demonstrated in many studies using molecular genetics approaches
to either disable antioxidant enzymes or boost their activities or the abundance of low
molecular weight molecular antioxidants such as ascorbate (AsA) and reduced glutathione
(GSH). However, such studies have often failed to take into account the photosynthetic
production of reductants and oxidants within the context of cellular redox homeostasis and
redox signaling. Our understanding of the interactions of redox signaling pathways with
photosynthesis has greatly increased in recent years and this provides new opportunities for
enhancing photosynthesis particularly under conditions that favor cellular oxidation. Here
we consider current concepts of oxidative stress and oxidative signaling in relation to
photosynthesis and whether ROS scavenging systems have evolved to be “leaky” in order
that ROS can function as a useful signaling system.
Oxidative Stress and the role of reactive oxygen species
The field of redox biology has recently witnessed a dramatic reappraisal of the importance
of ROS. Due to the reactivity of ROS and because they are unavoidable by-products of
oxygenic photosynthesis, only the more negative aspects of ROS generation are often
considered in relation to observations. Thus, light-driven ROS production is generally
described as harmful because it can cause irreversible damage to photosynthetic
components. It is thus generally often suggested that ROS production should be minimized
at all costs. However, despite their potential for causing harmful oxidations, it is now well
established that ROS are also powerful signaling molecules, which are involved in the
control of plant growth and development as well as priming acclimatory responses to stress
stimuli (Jones, 2006; Foyer and Noctor, 2005 a, b; 2009). Nevertheless, in many studies
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involving photosynthetic ROS generation, the signaling function of these powerful
metabolites is largely ignored and interpretations are all too frequently based solely on the
notion that ROS exert their principal effects through chemical toxicity and their abilities to
cause damage. Within this context the term “oxidative stress” has become largely
synonymous with “oxidative damage”, to cellular components, particularly in situations
where oxidative inactivation exceeds that of repair or replacement. Furthermore, it is often
suggested that the accumulation of damaged cellular components and associated loss of
function leads to cell death but the mechanisms that cause cell death in this case are
generally vague or undefined. Relatively few studies to date have considered
photosynthetic ROS generation in the context of the light-driven production of powerful
signaling molecules, whose abundance provides essential information to the cell concerning
imbalances between energy-generating and energy-utilizing processes in the photosynthetic
electron transport (PET) system. Within this context, increased ROS production leading to
enhanced oxidation in high light may be considered to be a powerful signal that not only
decreases photosystem II (PSII) activity but also stimulates gene expression, particularly
with regard to acclimation and defense genes. While concepts of ROS function in signaling
or damage in photosynthesis are largely irrelevant to the description of the basic
biochemical mechanisms by which ROS oxidize cellular components, the philosophical
choice between ‘damage’ and ‘signaling’ in data analysis, remains crucial to the evaluation
of the physiological significance of these mechanisms.
Oxidant production and the regulation of photosynthesis
Oxygenic photosynthesis is a dynamic and flexible process that powers life on earth, in
which water oxidation on the lumen side of PSII is an indispensable step. The light driven
PET system drives electrons from water through to NADP, generating the proton gradient
that facilitates ATP synthesis. Intriguingly, a key feature of PSII is its vulnerability to
light-induced damage, which is considered to be a consequence of the production of singlet
oxygen (Vass and Cser, 2009) in the PSII reaction centre leading to irreversible oxidation
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of the D1 protein (Krieger-Liszkay et al., 2008). This sensitivity means that the PSII
reaction centre has to be re-built about once every 30 min even under relatively low
irradiances. The damage and repair process thus occurs under all light intensities. A
limitation of the PET system only occurs when the rate of damage exceeds that of repair
and this makes a major contribution to the processes called photoinhibition (Vass and Aro,
2008). In many circumstances where the rate of damage is fast such as at high light
intensities, the rate repair is also rapid (Ohad et al., 1984) and so a high level of PSII
activity can be maintained.
It is widely accepted that efficient regulation of PET serves to minimize the production of
singlet oxygen at PSII, as well as the generation of superoxide and H2O2, which occurs
predominantly on the reducing side of photosystem I (PSI) (Genty and Harbinson, 1996).
Within this context, reversible decreases in the efficiencies of both photosystems are
intrinsic to the regulation of light use by photosynthesis (Genty and Harbinson, 1996).
Inherent limitations on the capacity for electron transport through the cytochrome b6/f
(cytb6f) complex favor over-reduction of PSII, which exacerbates PSII turnover (Vass and
Cser, 2009; Genty and Harbinson, 1996). Of the strategies that can be employed to protect
PSII under conditions of limiting PET capacity, the most important is non-photochemical
quenching (NPQ), which dissipates the excitation energy of chlorophyll a molecules safely
as heat (Niyogi et al., 1998).
The majority of electron flow follows a linear, non-cyclic route from water through PSII,
the cytb6f complex and PSI to NADP, leading to the generation of both NADPH and ATP.
However, the operation of a cyclic electron flow pathway around PSI provides a
mechanism whereby ATP production can be increased relative to NADPH. Moreover, the
operation of cyclic electron flow is also considered to prevent over-reduction of the
acceptor side of PSI (Munekage et al., 2008). This is important because superoxide and
H2O2 are generated by PET components on acceptor side of PSI. Cyclic electron flow may
also help to limit singlet oxygen production at PSII because it enhances protonation in the
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lumen, which triggers protective NPQ mechanisms (Rumeau et al., 2007). The exact
contribution of the linear and cyclic pathways to overall electron flow depends on cell type
and environmental conditions. However, in C3 leaves under optimal growth conditions, the
major function of cyclic electron flow around PSI is considered to be the augmentation of
ATP production relative to NADPH in order to balance the energy budget of the
chloroplast.
While many fundamental questions remain concerning the components involved in cyclic
electron flow and the extent to which this pathway operates in C3 plants, there is a general
consensus of opinion that cyclic electron flow is an essential component of the repertoire of
chloroplast mechanisms that serve to co-ordinate energy metabolism and balance redox
status. A further mechanism of regulation for photosynthesis that prevents over-reduction
of the PSI acceptor side and the chloroplast stroma is the “malate valve” system, which
transfers reducing equivalents to the cytosol (Scheibe et al., 2005). This pathway, which is
activated by the thioredoxin (TRX)-regulated activation of chloroplastic NADP-dependent
malate dehydrogenase, is an essential component for the stromal redox homeostasis
network because it allows the export of excess reducing power and thus relieves electron
pressure in the chloroplast. In this way, the regulation of metabolite distribution can be
used to balance cellular redox status in order to achieve metabolic and photosynthetic
control. However, to date little attention has been paid to how the regulated distribution of
other metabolites, particularly antioxidants, may alter the stromal redox homeostasis
network and affect the regulation of photosynthesis.
The antioxidant network of chloroplasts
Photosynthesis is an important source of cellular oxidants (Foyer and Noctor, 2003). Even
under optimal conditions, the calculated rate of H2O2 formation by the PET chain in the
chloroplasts during photosynthesis in C3 leaves is nearly as high (4 μmol.m–2.s–1) as the
amount produced in the peroxisomes as a result of the glycolate oxidation in the
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photorespiratory pathway (10 μmol. m–2.s–1 ; Foyer and Noctor, 2003). While it is often
stated that the amount of H2O2 in chloroplasts can increase by several orders of magnitude
during stress, such notions must be viewed with caution because of the inherent
complexities of obtaining accurate measurements of H2O2 contents in intact tissues and
isolated organelles (Queval et al., 2008). Current methods do not allow accurate
estimations of the H2O2 concentration of the stroma under either optimal or stress
conditions. Regardless of any uncertainties about absolute values for H2O2 levels in
photosynthetic tissues, it has long been recognized that H2O2 is a potent inhibitor of
photosynthesis because even at low concentrations (10 μM) it can inhibit CO2 fixation by
50% because of the oxidation of the thiol-modulated enzymes of the Calvin cycle.
Therefore, the balance between ROS-production and ROS-scavenging in chloroplasts is
delicate and must be strictly controlled. AsA and GSH are the most abundant and best
characterized water-soluble antioxidants in plants, and they accumulate to millimolar
concentrations in chloroplasts. Although these metabolites scavenge ROS separately, they
have long been considered to function together in the AsA/GSH cycle (Foyer and Halliwell,
1976) and water-water cycle (Asada, 1999) to metabolize H2O2 (Figure 1) and to dissipate
excess excitation energy in chloroplasts.
The reduction of ground state molecular oxygen to superoxide on the acceptor side of PSI
in the Mehler reaction is the first step of a series of reactions that together has been called
“the water-water cycle” (Asada, 2000). This incorporates superoxide dismutase (SOD),
AsA peroxidase (APX) and the AsA-GSH cycle (Figure 1) in a mechanism that builds up
ΔμH+ and facilitates ATP formation at the expense of NADPH and reductant (Asada, 1999).
APX utilizes AsA as a specific electron donor to reduce H2O2 to water with the
concomitant generation of monodehydroascorbate (MDA), a univalent oxidant of AsA.
While MDA is spontaneously converted to AsA and dehydroascorbate (DHA), it is also
rapidly reduced to AsA by the action of a NAD(P)H-dependent MDA reductase (MDAR).
DHA reductase (DHAR) utilizes GSH to reduce DHA and thereby regenerate AsA. GSH is
then regenerated from glutathione disulfide (GSSG) by the action of glutathione reductase
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(GR) using NAD(P)H. While different APX and SOD isoforms are located in the stroma
and thylakoid membrane, the chloroplastic GR and DHAR enzymes are localized in the
stroma. A characteristic property of APX, particularly the chloroplastic APX forms, is their
susceptibility to oxidative inactivation in the absence of AsA (Ishikawa and Shigeoka
2008). Under low AsA concentrations (less that 20 µM) the activity chloroplastic APXs is
rapidly lost in the presence of H2O2, with a half-inactivation time of less than 30 sec. In
contrast, the cytosolic and peroxisomal APXs only lose activity after more than an hour.
The chloroplastic APXs are the primary targets for inactivation if chloroplast ascorbate
accumulation is impaired. Depletion of chloroplast AsA and inactivation of chloroplast
APXs have therefore been considered as limitations of photosynthetic efficiency in stress
conditions (Ishikawa and Shigeoka, 2008) and therefore potential targets for improvement.
In addition to the AsA-GSH cycle, other proteins that are important in chloroplast ROS
detoxification are peroxiredoxin (PRX, particularly 2-CysPrx and PrxQ), glutathione
peroxidase (GPX), sulfiredoxin and cyclophilin, which function together with Trx and
Trx-like proteins in the chloroplasts (Dietz 2011). The thiol-based catalytic mechanism
used by PRX to reduce H2O2, consists of a peroxidative reduction, followed by
regeneration that can involve a variety of electron donors such as TRX, glutaredoxin
(GRX), cyclophilins, GSH and AsA. GPXs can use both GSH and TRX as reducing
substrates and they can detoxify lipid peroxides as well as H2O2 (Herbette et al., 2002).
Because TRX is a more efficient substrate than GSH and their high rates of
TRX-dependent peroxidase activity, plant GPXs have been assigned as to the PRX protein
family (Dietz et al., 2006). In the GPX system, the regeneration of reduced TRX is linked
to the PET chain through either ferredoxin (Fd)-TRX reductases (FTR) or
NADPH-dependent TRX reductases (NTR). While the catalytic rates of plant GPXs and
their affinities for H2O2 are rather low compared to APX, the PRX and GPX pathways
provide an alternative pathway to the water-water cycle in the light (Figure 1), particularly
if the AsA-GSH cycle is impaired. However, lipid peroxides are also efficient substrates for
the chloroplast GPXs. The detoxification of lipid peroxides, which exacerbate the lipid
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peroxidation cascade reactions, may be as equally important as H2O2 detoxification in
terms of maintaining optimal photosynthetic functions in the light. AsA-GSH cycle and the
PRX-dependent detoxification pathways may be equally important in vivo and serve
interfacing functions. Our view is that the two detoxification pathways are tailored to suit
specific niches in defense metabolism and their relative importance probably varies
according to the prevailing environmental conditions.
The observation that chloroplastic mDHARs and APXs are enhanced in antisense
Arabidopsis plants with suppressed chloroplastic-located PRX (Baier et al., 2000), suggests
that regulatory compensation mechanisms exist between the pathways, so that one pathway
may be enhanced to compensate for losses in the other pathway. Such observations of
interactions between the AsA-GSH pathway and the PRXs demonstrate cross-talk between
the individual ROS-metabolizing pathways of the chloroplasts. The AsA-GSH pathway has
a higher specificity for H2O2 and the chloroplast APX has higher activities than PRXs but
the PRXs have a broad specificity of towards lipid peroxides and/or reactive nitrogen
species, such as nitric oxide (NO). For example, values for the catalytic efficiencies
(Kcat/KM ; L.mol-1.s-1) for plant 2-CysPrx with H2O2 range between 2.5.104 to 1.8.103 with
a value of 7.3.103 with tBOOH (Dietz, 2011). In comparison, the value for AsA-dependent
H2O2 reduction is 0.9.106 L. mol-1.s-1 (Asada, 1999).
Like ROS, NO is an important plant signaling molecule. NO reacts rapidly with the
superoxide anion to produce peroxynitrite. It is likely that peroxynitrite is produced in
chloroplasts, where it may fulfill signaling functions. Moreover, in marked contrast to the
situation in animals, peroxynitrite does not appear to be toxic to plants, which appear to
function without problem even in the presence of high levels of this metabolite. A key
feature of the regulation of NO metabolism is its reaction with GSH to form
nitrosoglutathione (GSNO), which can then react with other thiols to form nitrosothiols
(Lindermayr et al., 2005). GSNO serves as a storage pool of NO and it probably also fulfils
as yet unknown signaling functions. Like glutathionylation, protein S-nitrosylation can
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modify both protein function and activity (Noctor et al., 2011)
While the AsA/GSH cycle operates largely within the stroma, it is also linked to the
functions of a hydrophobic antioxidant, α-tocopherol (Toc), which is maintained in its
reduced form by AsA. Carotenoids and tocopherols are the most abundant groups of
lipid-soluble antioxidants in chloroplasts (DellaPenna and Pogson, 2005). Toc is
accumulated to high concentrations in chloroplasts, where it serves to prevent lipid
peroxidation by removal of singlet oxygen and lipid peroxyl radicals (Krieger-Liszkay et
al., 2008). The resultant tocopheroxyl radicals are reduced back to Toc by AsA and the
action of the AsA/GSH cycle. The Arabidopsis vte1 mutants that are deficient in Toc show
a significant accumulation of AsA and GSH. In contrast, VTE1-overexpressing plants,
which accumulate Toc, have lower AsA and GSH levels (Kanwischer et al., 2005). Such
observations provide further evidence of the close relationships that exist between
chloroplast antioxidants, which compensate for each other. Moreover, these data not only
demonstrate the high degree of interaction between the chloroplast antioxidant pathways
but they also suggest that there are multiple sites of reciprocal control. However, while the
biosynthetic pathways for AsA (Smirnoff et al., 2001), GSH (Mullineaux and Rausch,
2005) and Toc (DellaPenna and Pogson, 2005) are now well established, much remains to
be understood regarding how the synthesis and accumulation of these essential antioxidants
is controlled and regulated in a coordinated manner. The factors that control the
intracellular partitioning of metabolites between the different compartments of the cell and
the antioxidant transport systems are of particular importance to the overall regulation of
photosynthesis and its effective operation over a wide range of environmental conditions.
While recent years have witnessed an increase in our understanding of the roles of ROS in
the signaling systems that coordinate antioxidant gene expression, very little information is
available to date on how the low molecular weight antioxidants participate in this control.
Genetic manipulation of antioxidant defenses of chloroplasts
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A wide range of strategies, including molecular genetics and genetic engineering
approaches, have been used to enhance the tolerance of plants to abiotic stress (Mittler and
Blumwald, 2010). Many attempts have been made over the last two decades to protect
photosynthesis against stress-induced inhibition by manipulation of component antioxidant
enzymes and extensive literature exists showing that enhancing the capacity of the
water-water cycle through genetic engineering, including the overexpression of SOD, GR,
and DHAR, can improve the tolerance of plants to abiotic stress (Logan et al., 2006).
Overall, the enhancement of chloroplast antioxidant defenses has proved to be one of the
most effective ways of protecting plant cells from abiotic stress (for example, Ishikawa and
Shigeoka, 2008; Chang et al., 2009).
Taken together, studies that demonstrate that an enhancement of antioxidant capacity gives
added protection to photosynthesis illustrate the point that there is not an excess capacity
for ROS destruction in the chloroplasts. Rather, such findings suggest that the capacity for
ROS detoxification in the chloroplasts is balanced with regard to ROS production. This is
perhaps surprising given the susceptibility of the photosynthetic processes to oxidative
inactivation. However, if one considers that ROS generated in photosynthesis participate in
a redox signaling cascade then the importance of regulation of both ROS abundance and
the life-time of the ROS signal becomes apparent. The available data suggest that ROS
detoxification pathways are not present in amounts that would totally remove all the ROS
from the cellular environment. Rather, there appears to be a level of co-ordination between
ROS producing- and ROS-metabolizing processes that allows a pool of ROS to persist in
the cellular environment. This co-ordinate regulation allows increases in the available ROS
pool to occur during stress, presumably for signaling purposes.
The size of the ROS signaling pool will depend on the relative rates of ROS production and
destruction, the latter of which can be finely tuned with regard to enzyme activation and
gene expression. In this context, the balance between oxidant production and removal by
the antioxidant system would essentially determine the strength and lifetime of the ROS
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signal, as illustrated in Figure 2. In addition, the oxidation of some proteins could trigger
altered pathways of gene expression while oxidation of other proteins such as those in PSII
reaction center might further provide a mechanism that would enable more rapid
adjustment of PSI and PSII stoichiometries to changing environmental conditions.
Oxidative modifications to specific amino acids is a widespread and generally accepted
mechanism of marking proteins for proteolysis,
Within the context of the “oxidative stress-induced damage”, hypothesis, the added
protection of photosynthesis during stress that has been obtained by enhancing chloroplast
antioxidant network provides evidence that it is possible to mitigate potentially harmful
reactions caused by enhanced oxidative load (Logan et al., 2006). For example, transgenic
tobacco and Arabidopsis plants overexpressing tAPX in the chloroplasts exhibited
enhanced tolerance to high light and paraquat treatment, a combination that is designed to
maximize photooxidative stress (Ishikawa and Shigeoka, 2008). Similarly, transgenic
tobacco overexpressing KatE, a gene encoding an E. coli catalase (CAT), in the stroma,
resulted in a greatly enhanced tolerance to photo-oxidative stress (Miyagawa et al., 2000).
In this case the enhanced tolerance was explained in terms of compensation for inactivation
of chloroplastic APXs by KatE.
The physiological significance of the high susceptibility
of chloroplastic APXs to inactivation by ROS remains a mystery if one considers only the
potential for increased ROS-induced damage. However, from a signaling perspective, the
inactivation of chloroplastic APXs may have a regulatory role in facilitating redox
signaling pathways under conditions of high ROS production or oxidative stress.
Ectopic expression of useful genes from cyanobacteria into the chloroplasts of higher plants
has also proved to be effective at enhancing tolerance to abiotic stress. For example, the
GPX-like proteins of Synechocystis PCC 6803 are able to reduce unsaturated fatty acid
hydroperoxides using NADPH as an electron donor, but not GSH and TRX. The expression
of Synechocystis GPX in Arabidopsis chloroplasts resulted in a suppression of
photoinhibition and an increased tolerance to photooxidative stress (Gaber et al., 2004).
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Moreover, the expression of a cyanobacterial flavodoxin, which is iso-functional with Fd,
in chloroplasts enhanced tolerance to a range of abiotic stresses (Zurbriggen et al., 2008).
Flavodoxins are considered to prevent electron misrouting and thus contribute to the stress
tolerance by reducing the probability of ROS formation (Zurbriggen et al., 2008).
The evidence described above confirms the importance of the antioxidative enzymes in
chloroplasts, and the value of genetically-enhanced antioxidants and other defense
compounds as a mechanism for stabilizing photosynthesis in stress situations. Other
metabolites can also serve protective functions in chloroplasts. For example,
oligosaccharides such as galactinol and raffinose, which often accumulate to high
concentrations, can also act as antioxidants and function as osmoprotectants in plant cells.
However, they differ from AsA, GSH and tocopherol as such molecules are generally
destroyed as a result of oxidation and they cannot be recycled. Transgenic Arabidopsis
plants overexpressing galactinol synthases (GolS1 and GolS2), key enzymes in the
synthesis of galactinol and raffinose, had increased levels of these oligosaccharides, and
enhanced tolerance to enhanced oxidation caused by exposure to paraquat, chilling and
osmotic stress (Nishizawa et al., 2008). Thus, these oligosaccharides can function to protect
plant cells from oxidative damage (Nishizawa et al., 2008). Similarly, it is well established
that the enhanced biosynthesis of glycinebetaine, an osmoprotectant, increases the tolerance
of plants to various types of abiotic stress (Chen and Murata, 2008).
While glycinebetaine
does not scavenge ROS directly, glycinebetaine synthesis produces H2O2 that activates
ROS-scavenging enzymes and thus mitigates against oxidation. The accumulation of
glycinebetaine appears to be more effective in the chloroplasts than in the cytosol,
suggesting its ability to protect the photosynthetic machinery (Chen and Murata, 2008).
Antioxidants and redox signaling in chloroplasts
Extensive evidence now points to important roles for oxidative and reductive signaling in
the regulation of photosynthesis, with antioxidants such as AsA and GSH having multiple
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signaling functions (Foyer and Noctor, 2009). While manipulation of the antioxidant
network has repeatedly shown that depletion of individual antioxidant components has a
negative effect on photosynthesis, a number of studies have demonstrated that our original
hypotheses concerning ROS and antioxidants are simplistic and in some cases naïve. For
example, while depletion of either CAT or cytosolic APX has a general negative impact,
tobacco plants that are deficient in both CAT and cytosolic APX show a less marked
phenotype than the parent lines (Riszhsky et al., 2002). Thus, whether the functions and
action of ROS and antioxidants associated with photosynthesis are often most usefully
described within the framework of ‘damage’ or ‘signaling’ these metabolites are part of the
much more complex cellular system that governs cell fate in terms of triggering
programmed cell death or enhancing defenses to ensure survival. Thanks to the pioneering
work of Klaus Apel and his colleagues (for example Laloi et al., 2007), it is now well
established that singlet oxygen generated by photosynthesis triggers a genetically
programmed suicide pathway that leads to cell death (Figure 2). However, it has only more
recently been shown that metabolically generated H2O2 also causes cell death through a
genetically programmed pathway that requires salicylic acid (Mhamdi et al., 2010) and is
modulated by myo-inositol (Chaouch and Noctor, 2010). Manipulation of these signaling
pathways thus provides a novel mechanism to protect photosynthesis and leaf cells from the
cell death triggered by metabolically generated ROS.
While superoxide production in the Mehler reaction is undoubtedly of crucial importance to
the chloroplast redox system, the production of H2O2 during the glycolate oxidase reaction
of photorespiration is often the most significant in terms of overall oxidant production in C3
plants (Foyer and Noctor, 2003). Despite the high rates of H2O2 generation in the
peroxisomes of C3 plants in air, cat2 mutants that are deficient in the major leaf CAT
isoform do not accumulate H2O2. Rather, the total pool size is greatly increased and there is
a low GSH/GSSG ratio (Queval et al., 2007; 2009). This has lead to the hypothesis that
many of the responses of plant cells to enhanced oxidation including changes in gene
expression caused by metabolically-generated H2O2 are transmitted through changes in the
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GSH pool rather than by H2O2 per se (Queval et al., 2010). Moreover, unlike the double
mutants lacking both CAT and cytosolic APX, the double mutants lacking both CAT and
cytosolic GR have a more severe phenotype than the cat2 single mutants (Mhamdi et al.,
2010). This finding may support the view that GR and GSH participate in AsA-independent
H2O2 metabolism as described above, as well as in the AsA-GSH pathway.
While AsA and GSH act alongside CAT in high capacity redox-homeostatic
H2O2-processing pathways, these antioxidants have discrete and specific functions in
photosynthesis and associated redox signaling (Foyer and Noctor, 2009). In addition to the
AsA-GSH cycle, AsA has several other important roles in photosynthesis. It is a cofactor
for violaxanthin de-epoxidase, an enzyme required for NPQ formation, and it participates
in the abscisic acid-mediated regulation of stomatal closure. Finally, it can strongly
influence the expression of both nuclear and chloroplast genes encoding photosynthetic
components (Kiddle et al., 2003). Given the many roles of AsA in the regulation of
photosynthesis it is hardly surprising that AsA-deficient plants show zeaxanthin depletion
and an inhibition of photosynthesis when exposed to high light (Muller-Moule´ et al.,
2004).
Involvement in chloroplast to nucleus retrograde signaling pathways
The chloroplast ROS, AsA and GSH pools have direct interactions with the photosynthetic
machinery and they also participate in signaling pathways that enable photosynthesis to
acclimate to environmental change. Long-term acclimation to high light is known to
involve increases in antioxidants and decreases in light-harvesting antenna size that is
likely due to changes in gene expression (Foyer and Noctor, 2009). Moreover, ROS and
GSH are considered to act as second messengers in the pathways responsible for high light
stress perception and long distance signal transduction (Szechyn´ ska-Hebda, et al., 2010).
The high light stress signaling pathways also interact with hormone signaling pathways.
For example, abscisic acid (ABA) pathways participate in systemic light signaling, a
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mechanism that appears to involve H2O2 generation in bundle sheath cells (Mullineaux, et
al., 2006). Accumulating evidence links AsA, ROS, NO and ABA signaling pathways in
the regulation of stomatal closure and at the level of information flow from the chloroplast
to the nucleus in order to co-ordinate plastid and nuclear gene expression, in the process
called “retrograde”, signaling (Pfannschmidt et al., 2008). In particular, ABA-insensitive-4
(ABI4) has been shown to be important in the regulation of nuclear genes in response to
environmental and developmental cues (Koussevitzky et al., 2007). While much remains to
be discovered regarding the nature of the crosstalk between the different signaling
pathways, it is probable that some degree of control is exerted through the abundance of
redox metabolites, as illustrated in Figure 2. Metabolite crosstalk between different
pathways involving oxidants and antioxidants not only serves as a mechanism of reciprocal
control but it also provides a hub of redox information that readily interfaces with different
types of retrograde signals, such as Mg-protophorphyrin IX, that are generated by
chloroplasts and that are responsible for the fine-tuning of genes encoding photosynthetic
proteins in the nucleus (Pfannschmidt et al., 2008).
Intermediates of chlorophyll biosynthesis that produce ROS by photodynamic action are
considered to be important in chloroplast-to-nucleus retrograde signaling (Rintamäki et al.,
2009). Much of our current understanding of the role of singlet oxygen signaling in
chloroplast to nucleus retrograde signaling pathways has been provided by the analysis of
Arabidopsis mutants that are deficient in the control of tetrapyrrole biosynthesis, which can
be distinguished from the wild-type by the strong protochlorophyllide fluorescence and
were hence named fluorescent or flu (Meskauskiene et al., 2001). Changes in the gene
expression patterns of the flu mutants relative to the wild type showed that the pathway of
singlet oxygen-dependent control of nuclear gene expression was different from that of
H2O2 (Laloi et al., 2007). Moreover, singlet oxygen generation in the flu mutant activated a
broad range of signaling pathways involved in biotic and abiotic stress responses but this
process was found to be essentially antagonistic to the action of H2O2 on certain gene
expression patterns (Laloi et al., 2007). Further analysis of the flu mutants revealed that the
17
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inactivation of a nuclear-encoded plastid protein called EXECUTER1 was sufficient to
abolish singlet oxygen–dependent stress responses and attenuate the extent of
superoxide-induced up-regulation of nuclear gene expression (Wagner et al., 2004). A
second related nuclear-encoded protein, called EXECUTER2, has also implicated in singlet
oxygen-dependent control of nuclear gene expression (Lee et al., 2007). Both EXECUTER
proteins function in retrograde signaling pathways in the chloroplasts and control singlet
oxygen-responsive genes in order to regulate the accumulation of chlorophyll and
carotenoids, particularly in high light stress (Lee et al., 2007).
In summary, enhancing the activities of antioxidant enzymes and/or the accumulation of
low molecular weight antioxidants by genetic manipulation increases tolerance to a variety
of stresses, through more efficient removal of ROS. Thus, by making ROS removal more
efficient the photosynthetic processes are desensitized to environmental change.
Antioxidant systems have evolved not to completely remove ROS but to allow these signals
to persist within the cellular environment. The persistence of ROS in stressful
environmental conditions serves to limit the capacity of photosynthesis in situations where
energy-producing and energy-utilizing processes are not well balanced. Hence, high or
enhanced antioxidant capacity can be considered to be generally beneficial because it
desensitizes photosynthesis to over-reduction in the PET chain and in some cases such as
enhanced water-water cycle activity it can even help alleviate over-reduction. While, there
is little information to date concerning how increased antioxidant capacity influences
retrograde signaling pathways from the chloroplasts to the nucleus, plants with enhanced
antioxidants often show altered local and systemic stress responses. To date, manipulations
of plant antioxidant defenses have been largely limited to a small number of enzymes. The
elucidation of redox signaling pathways related to both ROS and antioxidants could provide
much more useful molecular tools for up-regulation of whole suites of genes with protective
functions that could make the photosynthetic system much less susceptible to light and
ROS-mediated modifications.
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Legends to figures
Figure 1.
Ascorbate (AsA-GSH cycle left) and thioredoxin-dependent (TRX/GPX
right) pathways of H2O2 removal in chloroplasts.
AsA, ascorbate; DHA, dehydroascrobate; DHAR, DHA reductase; Fd, ferredoxin; FNR,
ferredoxin NADPH reductase; FTR, ferredoxin thioredoxin reductase; GPX, glutathione
peroxidase; GR, glutathione reductase; GRX, glutaredoxin; GSH, reduced glutathione;
GSSG, oxidized glutathione; MDA, monodehydroascorbate; MDAR, MDA reductase;
NTR, NADPH thioredoxin reductase; PRX, peroxiredoxin; Trx, thioredoxin ; SOD,
superoxide dismutase.
Figure 2.
A simple scheme of the interactions between cellular redox state and
photosynthesis within the context of plant stress responses and interactions with
hormone signaling pathways. Enhanced oxidation can either lead to either programmed
cell death (A) or to enhanced sustainability (B) depending on the intensity of the oxidative
signal and the nature of the signaling pathway that is activated.
25
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Copyright © 2010 American Society of Plant Biologists. All rights reserved.
AsA
GSSG
NAD(P)H
AsA-dependent pathway
DHAR
GR
Trx-dependent pathway
GSH
DHA
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Fdred
Trxox
NADPH
H2O
MDA
APX
APX
MDAR
GPX
NTR
NADP+
NAD(P)H
PRX
FTR
Fdox
NAD(P)+
Trxred
H2O2
NAD(P)+
AsA
SOD
O2-
O2
NADP+
Fdred
ee-
Photosynthetic
Electron Transport
FNR
High Light
Environmental Stress
Perception
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Photosynthesis
Antioxidants
Enhanced Oxidation
Hormones
Hormones
A
Cell
Death
Defense
responses
B
Enhanced
Sustainability
ROS
Cellular Redox Homeostasis