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The role of antioxidant enzymes in photoprotection
Photosynthesis Research, 2006, vol. 88, no. 2, pp. 119-132.
Barry A. Logan (1), Dmytro Kornyeyev (2,3), Justin Hardison(1) and A. Scott Holaday(2)
(1) Department of Biology, Bowdoin College, 6500 College Station, Brunswick, ME 04011, USA
(2) Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409, USA
(3) Institute of Plant Physiology and Genetics, Vasylkivska St. 31/17, 03022 Kyiv, Ukraine
Abstract. The enzymatic component of the antioxidant system is discussed as one of the defensive mechanisms providing protection against excessive light absorption in plants. We present an analysis of attempts to improve stress tolerance by means of the creation of transgenic plants with elevated antioxidant enzyme activities and conclude that the effect of such transgenic manipulation strongly depends on the manner in which the stress is imposed. The following factors may diminish the differences in photosynthetic performance between transgenic plants and wild type under field conditions: effective functioning of the thermal dissipation mechanisms providing a primary line of defense against excessive light, long-term adjustments of the antioxidant system and other photoprotective mechanisms, the relatively low level of control over electron transport exerted by the Water�Water cycle, especially under warm conditions, and a decrease in the content of the transgenic product during leaf aging.
Keywords: antioxidants - photoinhibition - stress tolerance - transgenic plants - Water�Water cycle
Introduction
Photosynthetic electron transport can reduce molecular oxygen (O2) in the so-called �Mehler reaction� (Mehler 1951; Mehler and Brown 1952), yielding superoxide, a reactive oxygen species (ROS) capable of damaging or inactivating essential macromolecules (Halliwell and Gutteridge 1999). As atmospheric O2 concentrations increased over geologic time-scales, O2 photoreduction may have arisen as an unavoidable consequence of the mechanism of electron transport. Although superoxide itself may be damaging under some circumstances, it may cause greater harm by leading to the formation of the highly reactive hydroxyl radical from H2O2, the product of superoxide dismutation in the chloroplast (Bowler et al. 1992). In addition, singlet O2, another unstable ROS, can be formed via resonance energy transfer from triplet excited-state chlorophyll to ground-state O2 (Asada 1996; Niyogi 1999). ROS-mediated inactivation is thought to be one of the primary causes of the slowly reversible loss of photosynthetic activity commonly termed �photoinhibition,� which is sometimes observed in leaves exposed to light in combination with environmental stresses (Allen 1995; Niyogi 1999). In fact, the physiological effects of many environmental stresses, such as chilling temperatures, can be appreciated in terms of their ability to disrupt the balance between light energy absorption and light energy utilization via the Calvin-Benson cycle in a manner that favors O2 reduction and singlet O2 formation.
Plants possess multiple means of minimizing the deleterious effects of ROS. These include an integrated array of antioxidant enzymes and metabolites that detoxify those ROS that form, as well as a mechanism that safely dissipates excess absorbed light before it can lead to singlet O2 formation. This latter mechanism is referred to as thermal energy dissipation or feedback de-excitation and involves energy transfer to a chlorophyll-zeaxanthin dimer, which loses excitation energy as heat after charge separation followed by recombination (Holt et al. 2005). Collectively, thermal energy dissipation and antioxidants are thought to be �photoprotective� because they protect photosynthetic cells from the dangers of excess light absorption. Excellent reviews of the molecular biology and physiological ecology of thermal energy dissipation have been published (Demmig-Adams and Adams 1992, 1996; Gilmore 1997; Niyogi 1999) and we shall concentrate this review on the photoprotective role of antioxidant enzymes. However, it should be noted that thermal energy dissipation and antioxidation are closely connected. Xanthophylls can directly scavenge certain ROS (reviewed by Havaux and Niyogi 1999). Violaxanthin deepoxidase, the enzyme that transforms violaxanthin into zeaxanthin (the xanthophyll that can facilitate thermal energy dissipation), requires ascorbic acid, a linchpin in the chloroplast antioxidant system (Bratt 1995). In addition, electron flow to O2 photoreduction contributes to the low thylakoid lumen pH required for thermal energy dissipation (Li et al. 2002; Makino et al. 2002).
Unlike thermal energy dissipation, which proactively prevents ROS formation, antioxidants react with those ROS that have formed. In other words, they represent the second line of defense against photoinhibition. One may suggest that if thermal dissipation is capable of keeping excessive energy flow under control, the antioxidant system may not be especially important for photoprotection. However, a considerable body of literature suggests that this is not so. Plants adjust levels of antioxidants when acclimating to prevailing environmental conditions in concert with changes in the xanthophyll cycle. Adjustments in both processes are best understood as responsive to levels of excess light absorption. In addition, O2 photoreduction and subsequent ROS detoxification have been shown to influence chloroplast metabolism, as well as nuclear and chloroplastic gene expression in a manner that suggests that they are tightly interwoven into the regulatory regimes that preserve the balance between light use to power carbon assimilation and protection against the potentially harmful effects of absorbing too much light energy. In this review, we describe the current state of understanding of the antioxidant enzyme pathways for chloroplastic ROS detoxification, their role in photoprotection and their influence on the rate of photosynthetic electron transport, and assess attempts to enhance the stress tolerance of plants via transgenic overproduction of antioxidant enzymes.
The Water-Water Cycle
Chloroplastic superoxide is detoxified via a pathway referred to as the �Water-Water cycle� (Asada 1999). The name derives from the fact that water acts as both the source of electrons (at the oxygen-evolving complex associated with PSII) and the final product of the pathway. Thus, the Water-Water cycle produces nothing, but consumes considerable photogenerated reductant while ridding the chloroplast of potentially damaging ROS. Two enzymes functioning in series convert superoxide to water. Superoxide dismutase (SOD) first catalyzes the dismutation of superoxide to molecular oxygen and H2O2 (McCord and Fridovich 1969; Bowler et al. 1992; Alscher 2002). Ascorbate peroxidase, in turn, uses ascorbate to reduce H2O2 to water (Jablonski and Anderson 1982). It is critical that H2O2 be removed, because it can potentially inactivate Calvin-Benson cycle bisphosphatases via thiol oxidation (Charles and Halliwell 1981) and, more importantly, because it can readily interact with reduced transition metal cations via the Fenton reaction to form the highly reactive hydroxyl radical (Halliwell and Gutteridge 1999). PSI and its vicinity would seem acutely vulnerable to hydroxyl radical generation and subsequent attack, since PSI binds several Fe-S clusters and is also the principal site for generation of superoxide, which can reduce iron and other transition metal cations (Terashima et al. 1998). Immunogold labeling experiments suggest that, at least in some preparations, certain isoforms of SOD and APX are found in close association with PSI. This led Asada (1996) to propose the existence of a thylakoid super-enzyme complex, which could greatly minimize the potentially harmful effects of ROS generation by catalyzing superoxide detoxification in an assembly-line fashion, limiting ROS escape and the possibility that they could damage cellular constituents.
The one-electron oxidation product of ascorbate, monodehyrdoascorbate, formed by APX activity, can be recycled back to the reduced form via at least three mechanisms, two of which ultimately derive their reducing power from photosynthetic electron transport. Monodehydroascorbate can accept electrons directly from the electron transport chain, a reaction that is thought to occur at either the cytochrome b6/f complex or at PSI (Miyake and Asada 1992; Grace et al. 1995). It can also be reduced via the activity of monodehydroascorbate reductase, an enzyme that utilizes NADH, or to a lesser extent NADPH, as a reductant (Hossain et al. 1984). Lastly, monodehydroascorbate radicals can participate in a non-enzymatic dismutation yielding reduced ascorbate and the two-electron oxidation product of ascorbate, dehydroascorbate. Dehydroascorbate can, in turn, be reduced to ascorbate via the activity of dehydroascorbate reductase, which utilizes glutathione (GSH) as a reductant (Hossain and Asada 1984). Glutathione reductase completes this cadre of redox reactions by recycling GSH using NADPH as a reductant (Smith et al. 1989).
In addition to the largely enzyme-driven Water-Water cycle described above, ascorbate and GSH may also participate in superoxide scavenging via a reaction sequence that is non-enzymatic, with the exception of GSH recycling. Ascorbate can detoxify superoxide non-enzymatically (Halliwell and Gutteridge 1999) and is found at high concentrations in the chloroplast (>10 mM) (Foyer 1993). Furthermore, GSH can reduce dehydroascorbate to ascorbate under the alkaline conditions that are likely to prevail in the stroma during illumination (Foyer and Halliwell 1976; Winkler et al. 1994; Foyer and Noctor 2000, Noctor et al. 2000). The relative contributions of enzymatic versus non-enzymatic superoxide detoxification pathways are not known (see Asada 1999; Polle 2001) and may depend upon environmental conditions and the taxon.
The Response of the Water-Water Cycle to Environmental Stress
Plants adjust constituents of the Water-Water cycle in response to prevailing environmental conditions. Change in antioxidant activities/contents can be observed hours or days after a change in growth conditions (Logan et al. 1998b, 2003). The adjustment of levels of leaf antioxidants to a range of abiotic stresses, such as growth light intensity or chilling temperatures, can be best understood in terms of the effect that these stresses have on the absorption of light that exceeds the needs of photosynthetic electron transport, so-called excess light.
In a broad range of plant species, a strong correlation has been reported between the intensity of the light environment and foliar activities of a number of antioxidant enzymes and contents of ascorbate and GSH (Gillham and Dodge 1987; Mishra et al. 1993, 1995; Grace and Logan 1996; Logan et al. 1996, 1998a). However, it does not appear that the light intensity, per se, is as important as is the extent of excess light absorption. In a direct comparison of light acclimation in Vinca major and pumpkin, leaves of full-sun acclimated V. major possessed 31% greater SOD activities, 49% greater APX activities, and 61% greater reduced ascorbate contents than full-sun acclimated pumpkin leaves (when expressed per unit leaf fresh weight) (Logan et al. 1998a). Since V. major maintained 50% lower rates of photochemistry under ambient, full-sunlight exposed conditions in comparison to pumpkin (estimated from chlorophyll fluorescence emission) and absorbed greater levels of excess light as a consequence, these findings further support the hypothesis that antioxidant systems acclimate in response to the level of excess light, not simply to the intensity of the light environment. Conversely, leaves of hydroponic spinach grown in a growth chamber at 800 μmol photons m-2 s-1 possessed levels of SOD, APX, GR, and ascorbate that were statistically indistinguishable from levels found in leaves of spinach grown under similar conditions but only 400 μmol photons m-2 s-1 (B. Logan, T. Rosenstiel, B. Demmig-Adams, W. Adams, unpublished data). However, spinach grown at the higher light intensity maintained two-fold greater photosynthetic capacities, which presumably reduced the portion of the absorbed energy in excess of that needed for CO2 assimilation, thus explaining the absence of an acclimatory response of the Water-Water cycle components to a doubling in the growth light intensity.
Exposure to chilling temperatures has the potential to greatly increase the absorption of excess light. Chilling has a greater inhibitory effect on the enzyme-catalyzed reactions of the Calvin-Benson cycle than on the biophysical and redox reactions that make up light harvesting and electron transport. Thus, chilling can perturb the balance between the production and consumption of photogenerated reductant in a manner that favors O2 photoreduction. Many long-lived evergreens respond to the onset of wintertime cold temperatures by decreasing chlorophyll content (Adams and Demmig-Adams 1994; Verhoeven et al. 1996; Logan et al. 1998c; Burkle and Logan 2003) and reorganizing their light harvesting antennae into an energy dissipating state (Gilmore and Ball 2000; Matsubara et al. 2002; Adams et al. 2002, 2004). However, profound upregulation of levels of antioxidants is another hallmark of acclimation to cold temperatures (Schöner and Krause 1990; Anderson et al. 1992; Mishra et al. 1993; Logan et al. 1998c, 2003). Cold-induced increases in the levels of antioxidant enzymes compensate for the effect of lower temperatures on their activities and also presumably help cope with an enhanced rate of ROS formation (Lohrmann et al. 2004). In addition to up-regulation in overall activity, some species have been shown to respond to chilling with preferential expression of antioxidant enzyme isoforms with lower temperature optima and other biochemical features that would favor activity at colder temperatures (Guy and Carter 1984).
The Size of the O2 Photoreduction Electron �Sink�
The extent to which the Water-Water Cycle can serve a photoprotective role depends upon the magnitude of the electron flux to O2 photoreduction. However, measuring this flux has proven methodologically difficult. Some of the first attempts to do so used radiolabeled 18O2 to distinguish O2 derived from water splitting at the Oxygen-Evolving Complex from O2 consumed via photoreduction. Early measurements employing this method reported that electron flow to oxygen accounted for 10 to 30% of overall electron flow (Canvin et al. 1980; Furbank et al. 1982).
Electron transport that ultimately leads to O2 photoreduction is undetectable when measuring net O2 fluxes, but measurable via chlorophyll fluorescence-based estimates of electron transport. Therefore, one can compare electron transport rates derived from these two measures to estimate the rate of O2 photoreduction. In tropical trees measured at light saturation using this approach, 30% of the overall electron transport was dedicated to O2 photoreduction (Lovelock and Winter 1996).
The two approaches above used to estimate O2 photoreduction demand that measurements be made under non-physiological conditions that suppress photorespiration. More recently, Ruuska and colleagues (2000a) have applied transgenic technology to this problem by suppressing Rubisco activity in tobacco via the expression of an antisense construct to the Rubisco small subunit (reviewed in Badger et al. 2000). Antisense tobacco exhibit differing capacities for CO2 assimilation without parallel alterations in the capacities for photosynthetic electron transport (Ruuska et al. 2000a). Across a range of O2 and CO2 concentrations, calculated rates of electron transport closely matched the requirements for reductant, suggesting that O2 photoreduction is, at most, a minor flux, even under circumstances where the capacity to generate reductant greatly exceeds the capacity to consume it (Ruuska et al. 2000a).
An increase in the ratio of the quantum yields for electron transport (estimated from fluorescence) and CO2 fixation has been interpreted as a manifestation of greater fluxes to alternative electron sinks, in particular to the Water-Water cycle (Fryer et al. 1998). Adopting this approach, Miyake and Yokota (2000) consistently measured electron fluxes to alternative sinks in leaves of watermelon, ranging up to 20% of the overall electron fluxes. Hirotsu et al. (2004) determined that the acclimation of rice to low temperature led to an increase in the portion of photon energy utilized by alternative sinks from 10 to 15%.
Yet another method used to estimate O2 photoreduction via the Mehler reaction involves analyses of fluxes in the presence of glyceraldehyde, which prevents CO2 and O2 assimilation in the Calvin-Benson cycle by inhibiting the formation of ribulose bisphosphate via phosphoribulokinase (Wu et al. 1991). The data obtained by Wu et al. (1991) suggest that the Mehler reaction is not a substantial sink for electron flow.
None of the methods described above is free of complicating factors, including pleiotropic effects, the use of non-physiological gas concentrations, and the possibility that chlorophyll fluorescence analysis may over-emphasize the contribution of the uppermost layer of chlorophyllous cells. This, and the general lack of agreement among methods, leaves the size of the electron sink represented by O2 photoreduction unresolved. Multiple methods do agree, however, that significant electron flow to O2 occurs during photosynthetic induction (Neubauer and Yamamoto 1992; Ruuska et al. 2000b). Electron flow to O2 during induction may contribute to the low luminal pH required to induce thermal energy dissipation (Gilmore 1997; Makino et al. 2002). Even if the O2 photoreduction ultimately proves to be a relatively minor sink at steady state, the balance between photosynthetic ROS production and scavenging could still have profound effects on gene expression, as ROS and the redox status of certain chloroplastic constituents have recently been shown to regulate the transcription and post-transcriptional processing of nuclear and chloroplastic genes (reviewed in Barnes and Mayfield 2003; Link 2003; Baier and Deitz 2005; Fey et al. 2005; Pfannschmidt and Liere 2005). The dramatic responses of antioxidants to the imposition of environmental stresses indirectly, but strongly, suggests that O2 photoreduction is sensitive to environmental conditions and that adequate ROS scavenging capacity is essential for the proper functioning of the chloroplast.
Transgenic Manipulations of Photoprotection
Numerous studies imply that generation of ROS under illumination is one of the primary factors contributing to photoinactivation of the photosynthetic apparatus (Prasil et al. 1992; Andersson and Barber 1996; Melis 1999). Pigment-protein complexes of PSI and PSII, which are considered to be the main targets for photoinhibition, can be a source of ROS (Bradley et al. 1991; Foyer and Harbinson 1994; Osmond and Grace 1995). Moreover, ROS produced by PSI can augment photo-induced inactivation to PSII (Krieger et al. 2000; Tjus et al. 2001). Consequently, photoprotection of chloroplast constituents should depend on the efficiency of ROS removal by antioxidants. Indeed, the addition of antioxidants in vitro decreases the extent of photoinactivation (Barényi and Krause 1985; Richter et al. 1990; Tschiersch and Ohmann 1993; Tjus et al. 2001), and the lack of antioxidant enzymes is associated with a higher sensitivity to light treatment (Danna et al. 2003). These findings taken together with the observation that the response to many environmental stresses, particularly chilling, includes upregulation of antioxidant systems have led plant geneticists to attempt to improve the stress tolerance of some crop species by transforming plants with genes for chloroplast-targeted antioxidant enzymes (for reviews see Foyer et al. 1994; Allen 1995). Such attempts have met with mixed results, as indicated by the findings described in Table 1. Where enhanced environmental stress tolerance has been observed, its mechanism has rarely been probed in depth. In addition, artificially induced oxidative stress, for instance, the treatment with MV, is frequently imposed to study the effect of a transgene (see Table 1). Those experiments, however, do not provide information about the performance of transgenic plants under field conditions.
Increased tolerance to cold-induced photoinhibition was shown for transgenic poplar possessing between 100- and 1000-fold overexpression of chloroplast-targeted GR (Foyer et al. 1995). Enhanced cold tolerance was explained by more rapid recycling of ascorbate and GSH pools, the influence of higher levels of GSH on the stabilization of the enzymes requiring thiol groups for their activity and by the possible influence of altered GSH concentration on protein synthesis and gene expression. Tobacco plants (cv. Samsun) possessing increased GR activities (five- to eight-fold overexpression) have a lower rate constant of PSII photoinhibition at 20 oC (Tyystjärvi et al. 1999). However, this effect was not observed in another cultivar (cv. Bel W3), nor in transgenic poplar plants with a similar level of FeSOD overexpression. Transgenic tomato plants with a 60-fold increase in chloroplastic GR activity also did not show an improved resistance to photoinhibition (Bruggemann et al. 1999).
Research conducted on cotton plants indicates that the protective effect of transgenic overexpression of antioxidant enzymes may, in part, result from their ability to maintain the photosynthetic apparatus in a more oxidized state (Kornyeyev et al. 2001 2003a,b). PSII is more likely to experience photoinactivation when its final electron acceptor, QA, is reduced (Melis 1999). Similarly, light energy has a greater probability of leading to photoinhibition if is it absorbed by the antennae of �closed� PSII units (i.e. those already processing an electron) (Kornyeyev et al. 2004). Transgenic cotton with elevated activities of SOD, APX or GR (approximately three-, five-, and 35-fold overexpression, respectively) all maintained lower levels of QA reduction than the wild type when exposed to illumination under chilling conditions (Kornyeyev et al. 2001). No evidence for a direct effect of the transgenic modifications of ROS metabolism on PSII protection was found (Kornyeyev et al. 2001), although the enhanced PSI protection observed in GR and APX overproducing plants could be the direct result of ROS scavenging.
Consistent with the report that an effect of transformation is evident at only certain levels of a stress (Rubio et al. 1997), differences between transgenic and wild-type cotton were observed only at low temperatures (10 to 15�C) (Kornyeyev et al. 2003b). At warmer temperatures (i.e. 20 to 30 oC) antioxidant overproduction brings about no enhancement in resistance to photoinhibition. It may be that as the leaf temperatures rise, the contribution of CO2 fixation to the maintenance of electron flow overwhelms the effect of the transgenic manipulation. It may also be that native enzyme activity is adequate at warm, but not at chilling temperatures. These observations suggest the existence of a �thermal window� in which the effect of increased antioxidant activity may have a meaningful effect on PSII reduction state and thereby influence the level of PSII photoinhibition.
The effect of a single-enzyme transgenic modification may depend on the overall state of the antioxidant system. For example, a decrease in the activity of the key antioxidant enzymes caused by shading resulted in the disappearance of the beneficial effect of MnSOD overproduction on the methylviologen resistance of transgenic tobacco plants (Slooten et al. 1995). The overproduction of SOD itself can cause an increase in the activity of other antioxidant enzymes (Sen Gupta et al. 1993; Kingston-Smith and Foyer 2000); however, this effect is not always observed (Payton et al. 2001).
Most studies seeking to examine the stress tolerance of transgenic plants employ abruptly imposed, severe stresses. Typically, leaf tissues are detached from plants grown under near-optimal conditions and placed under stresses that exceed those that the species under study might encounter in the field. Experiments of this sort have yielded important insights into the mechanisms of chilling tolerance and the regulation of oxidative metabolism. However the enhanced stress tolerance that is occasionally observed under such conditions may not be predictive of enhanced stress tolerance under conditions typical of the field. For example, when leaf discs of warm-grown transgenic cotton that possessed ~four-fold higher chloroplastic APX activities were abruptly exposed to 10�C at 500 �mol photons m-2 s-1, they sustained lower levels of PSII and PSI photoinhibition in comparison to wild type (Fig. 1A) (Kornyeyev et al. 2001, 2003a). However, chilling tolerance was not enhanced when this same cotton genotype was grown in a growth chamber in which temperatures were lowered from 28 to 14�C over 9 days and held at for a subsequent 9-day period at 14�C (Fig. 1B) (J. Hardison, B. Logan, A.S. Holaday, unpublished data). The absence of an effect of APX overproduction under longer-term, gradually-imposed chilling may be explained, in part, by the fact that wild-type cotton acclimated to the latter chilling regime by upregulating native APX activity and abolishing significant genotypic differences in APX activity. Under similar experimental conditions, transgenic GR overproduction also failed to improve cotton chilling tolerance (Logan et al. 2003).
In field trials conducted in west Texas, where cotton is a primary crop, transgenic overproduction of GR (Kornyeyev et al. 2005) or APX (D. Kornyeyev, B. Logan, R. Allen, A. S. Holaday, unpublished data) failed to meaningfully improve photosynthetic performance. Over the course of the growing season from early June until mid-October, in Lubbock, Texas, the environmental conditions that enhance photoinactivation, namely, high light intensities in combination with chilling temperatures, were rarely observed because high light intensities quickly led to leaf warming. In addition, the wild-type and transgenic cotton plants had high capacities for non-photochemical, thermal dissipation of excitation energy that were rapidly engaged as light intensities climbed in the morning, most likely reducing the need for elevated levels of antioxidant enzymes (Kornyeyev et al. 2005). Thermal energy dissipation, which safely eliminates a large fraction of the photon energy absorbed by PSII antennae, may be the dominant photoprotective mechanism in cotton. The elevated activity of single antioxidant enzymes does not affect the levels of non-photochemical quenching of chlorophyll fluorescence nor the extent of de-epoxidation of xanthophyll cycle pigments (Kornyeyev et al. 2003a,b). In the case of APX overproduction, these data indicate that there is no noticeable competition between APX and violaxanthin de-epoxidase for reduced ascorbate, at least under chilling, moderate light conditions. The extent of transgenic enzyme overproduction also fell with leaf age, diminishing the difference in enzyme activity between transgenic and wild-type plants at the time of boll development (Kornyeyev et al. 2005).
Clearly, the nature and timing of the stress profoundly influences the response of transgenic plants with increased ROS scavenging capacity. This should be taken into account when assessing the utility of manipulating these systems as a strategy for developing more stress-tolerant crop varieties for agricultural use and underscores the need to design experiments that examine the performance of transgenic genotypes under realistic conditions of stress. We do not recommend basing the value of a transformation on its ability to enhance photosynthetic protection in the presence of MV or during a rapidly-imposed environmental stress. In addition, the effect of transgenic upregulation of antioxidant enzymes on plant defense deserves in-depth examination, as it may influence plant performance in the field.
Reactive Oxygen Species Metabolism and the Influence on Electron Transport and Redox State of the Chloroplast
As stated above, transgenic cotton plants overproducing chloroplast-targeted antioxidant enzymes maintained the leaf pool of QA in a more oxidized state (Kornyeyev et al. 2001). The reduction state of QA is determined by the balance between light energy inputs into PSII and downstream electron flow (Logan 2006). Any process that consumes reducing equivalents and thereby increases downstream electron flow will serve to lower the reduction state of PSII and lower its vulnerability to photoinhibition. Under conditions of stress, such as chilling, where Calvin-Benson cycle consumption of reducing equivalents is compromised, electron flow through the Water-Water cycle may serve in this capacity. This effect has been demonstrated in a series of experiments examining the performance of transgenic cotton with elevated activities of GR and APX in their chloroplasts (Kornyeyev et al. 2003a,b). In comparison to the wild-type plants, the transgenic plants maintain lower QA reduction states (Kornyeyev et al. 2001), higher rates of electron transport through PSII, and sustain less PSII photoinhibition (Kornyeyev et al. 2003a,b) during exposure to 10�C and 500 �mol photons m-2 s-1. The protective effect of APX or GR overproduction on PSII function can be abolished by inhibiting electron transfer from PSII with 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) (Kornyeyev et al. 2001), indicating that the effect of elevated antioxidant enzyme activity is exerted downstream of PSII. Thus, the PSII photoprotection conferred by antioxidant overexpression is not due to the direct effects of enhanced ROS scavenging, instead it is due to the effect this enhancement has upon the redox state of QA. Kornyeyev et al. (2003a,b) propose that the enhanced photochemistry maintaining QA in a more oxidized state than in wild-type leaves is largely due to an enhanced demand for reducing power in the chloroplasts of the transgenic plants during chilling. This hypothesis is supported by the following information:
(a) Although transgenic plants maintain greater PSI capacity (Kornyeyev et al. 2003a,b), as estimated by measuring the relative amount of oxidizable P700 present, the genotypic differences in PSI capacity are only observed after 2 h of chilling, while the greatest genotypic differences in electron transport are observed during photosynthetic induction, the very period when O2 photoreduction is likely to be substantial (Neubauer and Yamamoto 1992; Ruuska et al. 2000b);
(b) The enhanced photochemistry is not due to an increase in Calvin-Benson cycle activity, since rates of CO2 assimilation at 10oC do not differ between transgenic and wild-type plants despite differences in electron transport;
(c) Electron transport through PSII is greater for the transgenic plants than for the wild-type plants in the presence of glyceraldehyde that inhibits the Calvin-Benson cycle (Kornyeyev et al. 2003b);
(d) The content of GSH in leaves overproducing GR is higher than that of wild-type leaves during chilling (Kornyeyev et al. 2003b); and,
(e) in leaves overproducing APX, the illumination-dependent rise in H2O2 during chilling is dramatically reduced (Kornyeyev et al. 2003a). Thus, the in vivo activity of these enzymes is higher in transgenic than in wild-type leaves during chilling. Taken together, these data strongly suggest that the Water-Water cycle can serve as an alternative electron sink, at least under chilling conditions. Antioxidant enzyme overproduction is not likely to affect the rate of O2 photoreduction (i.e. the Mehler reaction). Rather, it increases the rate of ROS scavenging and, in doing so, increases the demand for reducing equivalents to recycle ascorbate and glutathione.
Concluding remarks
Although the rate of O2 photoreduction and subsequent ROS formation may not be substantial under optimum, steady-state conditions, data from a variety of experimental analyses indicate that the detoxification of ROS by antioxidants during exposure to stressful environments is essential for the protection of the photosynthetic apparatus. The capacity of the native antioxidant systems is highly responsive to the growth environment; nonetheless, native antioxidant systems can be overwhelmed by stresses such as chilling temperatures when they are abruptly imposed. However, transgenic overproduction of chloroplastic antioxidants has generally proven to be a poor strategy for protecting plants against stress in the field or experimental settings that simulate field conditions. Other strategies should be investigated if agriculturally meaningful gains in photosynthetic stress tolerance are to be realized. Identification of those sites which constrain electron transport and CO2 assimilation during and, perhaps more importantly, following the stress will be needed to develop these strategies.
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