A. Scott Holaday

The role of antioxidant enzymes in photoprotection

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.

Over-expression of chloroplast-targeted Mn superoxide dismutase in cotton (Gossypium hirsutum L., cv. Coker 312) does not alter the reduction of photosynthesis after short exposures to low temperature and high light intensity

Transgenic cotton plants from several independently-transformed lines expressing a chimeric gene encoding a chloroplast-targeted Mn superoxide dismutase (SOD) from tobacco exhibit a three-fold increase in the total leaf SOD activity, strong Mn SOD activity associated with isolated chloroplasts, and a 30% and 20% increase in ascorbate peroxidase and glutathione reductase activities, respectively. The Mn SOD plants did exhibit a slightly enhanced protection against light-mediated, paraquat-induced cellular damage but only at 0.3 µM paraquat. In addition, photosynthetic rates at 10°C and 15°C were similar to those of controls, and the immediate recovery of photosynthesis after a 35-min exposure to 5°C and full sun was only slightly better than that for wild-type plants. The recovery for longer exposure times was comparable for both genotypes as was the deactivation of the H2O2-sensitive, Calvin-cycle enzyme, stromal fructose 1,6-bisphosphatase (FBPase). Compared to the controls, Mn SOD plant leaves in full sun prior to chilling stress had a lower activation of FBPase, a higher ratio of oxidized to reduced forms of ascorbate, and a higher total glutathione content. After 35 min at 5°C in full sunlight, total glutathione had risen in control leaves to 88% of the Mn SOD plant values, and oxidized to reduced ascorbate ratios were higher for both genotypes. However, an 80% increase in the ratio of oxidized to reduced glutathione occurred for Mn SOD plant leaves with no change for controls. This increased demand on the ascorbate-glutathione cycle is circumstantial evidence that high Mn SOD activity in the chloroplast leads to increased H2O2 pools that could, in some manner, affect photosynthetic recovery after a stress period. We postulate that the pool sizes of reduced ascorbate and glutathione may restrict the ability of the ascorbate-glutathione cycle to compensate for the increased activity of SOD in cotton over-producing mitochondrial Mn SOD in chloroplasts during short-term chilling/high light stress.

Elevated chloroplastic glutathione reductase activities decrease chilling-induced photoinhibition by increasing rates of photochemistry, but not thermal energy dissipation, in transgenic cotton

The effect of the overproduction of glutathione reductase (GR+) in cotton (Gossypium hirsutum L. cv.Coker 312) chloroplasts on the response of photosynthetic parameters to chilling in the light was examined. After 180 min at 10°C and 500 μmol photons m-2 s-1 in the chamber of an oxygen electrode, leaf discs of GR+ plants exhibited lower levels of sustained PSII photoinhibition than leaf discs of wild-type plants. No genotypic differences in thermal energy dissipation, leaf pigment composition, or the dynamics of xanthophyll cycle de-epoxidation were observed. The rate of induction and steady-state levels of photochemistry were greater for GR+ in comparison to wild-type plants. Enhanced photochemistry in GR+ plants could not be attributed to higher rates of CO2 assimilation at 10°C. Although GR overproduction afforded some increased protection against PSI photoinactivation, suggesting improved scavenging of reactive oxygen species, higher PSI activities could not completely explain the greater rates of photochemistry. Pools of glutathione and ascorbate were significantly more reduced in GR+ plants. Increased demand for reducing power to maintain these constituents in the reduced state may contribute to the higher rates of photochemistry observed in GR+ plants.

Photosynthetic/photorespiratory characteristics of C3−C4 intermediate species

The extent of photorespiration, the inhibition of apparent photosynthesis (APS) by 21% O2, and the leaf anatomical and ultrastructural features of the naturally occurring C3−C4 intermediate species in the diverse Panicum, Moricandia, and Flaveria genera are between those features of representative C3 and C4 plants. The greatest differences between the photosynthetic/photorespiratory CO2 exchange characteristics of the C3−C4 intermediates and C3 plants occur for the parameters which are measured at low pCO2 (i.e., the CO2 compensation concentration and rates of CO2 evolution into CO2-free air in the light). The rates of APS by the intermediate species at atmospheric pCO2 are similar to those of C3 plants.The mechanisms which are responsible for reducing photorespiration in the C3−C4 intermediate species are poorly understood, but two proposals have been advanced. One emphasizes the importance of limited C4 photosynthesis which reduces O2 fixation by ribulose 1,5-bisphosphate carboxylase/oxygenase, and, thus, reduces photorespiration by a CO2-concentrating mechanism, while the other emphasizes the importance of the internal recycling of photorespiratory CO2 evolved from the chloroplast/mitochondrion-containing bundle-sheath cells. There is no evidence from recent studies that limited C4 photosynthesis is responsible for reducing photorespiration in the intermediate Panicum and Moricandia species. However, preliminary results suggest that some, but not all, of the intermediate Flaveria species may possess a limited C4 cycle. The importance of a chlorophyllous bundle-sheath layer in the leaves of intermediate Panicum and Moricandia species in a mechanism based on the recycling of photorespiratory CO2 is uncertain.Therefore, although they have yet to be clearly delineated, different strategies appear to exist in the C3−C4 intermediate group to reduce photorespiration. Of major importance is the finding that some mechanism(s) other than Crassulacean acid metabolism or C4 photosynthesis has (have) evolved in at least the majority of these terrestrial intermediate species to reduce the seemingly wasteful metabolic process of photorespiration.

Transgenic cotton over-producing spinach sucrose phosphate synthase showed enhanced leaf sucrose synthesis and improved fiber quality under controlled environmental conditions

Prior data indicated that enhanced availability of sucrose, a major product of photosynthesis in source leaves and the carbon source for secondary wall cellulose synthesis in fiber sinks, might improve fiber quality under abiotic stress conditions. To test this hypothesis, a family of transgenic cotton plants (Gossypiumhirsutum cv. Coker 312 elite) was produced that over-expressed spinach sucrose-phosphate synthase (SPS) because of its role in regulation of sucrose synthesis in photosynthetic and heterotrophic tissues. A family of 12 independent transgenic lines was characterized in terms of foreign gene insertion, expression of spinach SPS, production of spinach SPS protein, and development of enhanced extractable Vmax SPS activity in leaf and fiber. Lines with the highest Vmax SPS activity were further characterized in terms of carbon partitioning and fiber quality compared to wild-type and transgenic null controls. Leaves of transgenic SPS over-expressing lines showed higher sucrose:starch ratio and partitioning of 14C to sucrose in preference to starch. In two growth chamber experiments with cool nights, ambient CO2 concentration, and limited light below the canopy, the transgenic line with the highest SPS activity in leaf and fiber had higher fiber micronaire and maturity ratio associated with greater thickness of the cellulosic secondary wall.

Photosynthetic/photorespiratory CO2 exchange characteristics of the C3-C4 intermediate species, Moricandia arvensis☆

Abstract The results of photosynthetic/photorespiratory CO2 exchange measurements with the crucifer, Moricandia arvensis, were similar to those found for the C3-C4 intermediate species, Panicum milioides. The CO2 compensation point (Γ) values for both species were significantly lower than those of the C3 plant, Glycine max, at O2 concentrations greater than 2%. At 21% O2, the Γ-values for M. arvensis and P. milioides approached those exhibited by C3 species as the light intensity decreased from 700 to 50 μE·m−2·s−1. Both the Γ and net photosynthesis of M. arvensis and P. milloides were less sensitive to changes in O2 concentration than were these parameters for G. max. The inhibition of M. arvensis photosynthesis by 21% O2 was 25.5 ± 1.4% as compared to 29.3 ± 1.4% for G. max and 23.1% for P. milioides. Furthermore, since the ratio of CO2 evolution in the light to net photosynthesis for M. arvensis was only 45% of that for G. max, we concluded that, as in the case of P. milioides, photorespiration and O2 inhibition of photosynthesis in this crucifier were less than found in representative C3 plants.

Excitation pressure as a measure of the sensitivity of photosystem II to photoinactivation

The appearance of a new hypothesis implicating the oxygen-evolving complex as the dominant target of PSII photoinactivation (the ‘manganese cluster’ mechanism) suggests that the inactivation of PSII can be predicted on the basis of the total amount of incident photons, and challenges the role that electron transport and thermal dissipation of excitation energy play in mitigating PSII photoinactivation. This viewpoint article discusses evidence showing that minimising of the amount of energy reaching closed PSII reaction centres (i.e. the excitation pressure) is important for photoprotection. Examples are described where the parameters derived from excitation pressure correlate with the level of PSII photoinactivation, whereas the counting of incident photons does not. These examples confirm the role of electron transport and thermal energy dissipation as factors modulating PSII photoinactivation, and validate strategies that are aimed at understanding and improving PSII resistance to photoinactivation by analysis and manipulation of photoprotective processes. The authors conclude that an integrated model that incorporates various mechanisms of PSII photoinactivation and analysis of their contribution is needed. In addition, the role of UV light in naturally occurring PSII photoinactivation is evaluated. It is suggested that, when compared with visible light, the damaging effect of UV light may be limited under field conditions.

The differential response of photosynthesis to high temperature for a boreal and temperate Populus species relates to differences in Rubisco activation and Rubisco activase properties

Significant inhibition of photosynthesis occurs at temperatures only a few degrees (<or= 10 degrees C) above the optimum, resulting in a considerable loss of potential productivity. Most studies of heat stress have focused on crop or weedy annual plants, whereas similar studies with trees have been limited in number. As temperature is a major factor limiting the geographic ranges of most plants, the aim of this study was to use two Populus species adapted to contrasting thermal environments for determining the factors that constrain photosynthetic assimilation (A) under moderate heat stress in tree species. Consistent with its native range in temperate regions, Populus deltoides Bartr. ex Marsh. exhibited a significantly higher temperature optimum for A than did Populus balsamifera L., a boreal species. The higher A exhibited by P. deltoides at 33-40 degrees C compared to that for P. balsamifera was associated with a higher activation state of Rubisco and correlated with a higher ATPase activity of Rubisco activase. The temperature response of minimal chlorophyll a fluorescence for darkened leaves was similar for both species and was not consistent with a thylakoid lipid phase change contributing to the decline in A in the range of 30-40 degrees C. Taken together, these data support the idea that the differences in the temperature response of A for the two Populus species could be attributed to the differences in the response of Rubisco activation and ultimately to the thermal properties of Rubisco activase. That the primary sequence of Rubisco activase differed between the species, especially in regions associated with ATPase activity and Rubisco recognition, indicates that the genotypic differences in Rubisco activase might underlie the differences in the heat sensitivity of Rubisco activase and photosynthesis at moderately high temperatures.

Transgenic overproduction of glutathione reductase does not protect cotton, Gossypium hirsutum (Malvaceae), from photoinhibition during growth under chilling conditions

In some studies, tissues from plants that have been genetically transformed to overproduce antioxidant enzymes sustain less damage when abruptly exposed to short-term chilling in the laboratory. However, few studies have examined the performance of transgenic plants during longer-term growth under chilling conditions. We compared growth of transgenic cotton that overproduces glutathione reductase (GR+; ∼40-fold overproduction) to growth of the wild type in a controlled environment chamber as leaf temperature was lowered from 28° to 14°C over 9 d and for a subsequent 9-d period at 14°C. In wild-type and GR+ cotton, chilling temperatures resulted in decreased dark-adapted F(v)/F(m) (the ratio of variable to maximal fluorescence; a measure of maximum photosystem II quantum yield) and mid-light period photosystem II quantum yield, coupled with increased 1 - q(P) (a nonlinear estimate of the reduction state of the primary quinone acceptor of photosystem II). The capacity for photosynthetic oxygen evolution decreased during the first portion of the chilling exposure, but recovered slightly during the second half. At no point during the chilling exposure did the performance of GR+ plants differ significantly from that of wild-type plants in any of the above parameters. The absence of an effect of GR overproduction under longer-term chilling may be explained, in part, by the fact that wild-type cotton acclimated to chilling by upregulating native GR activity.

Effects of water deficit on gas-exchange parameters and ribulose 1,5-bisphosphate carboxylase activation in wheat

Abstract Two spring wheat (Triticum aestivum L.) genotypes, Anza, drought resistant, and Chenab 70, drought susceptible, were used to study the effects of a slowly developing water stress on gas-exchange parameters and ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPCase; EC 4.1.1.39) activities. By day 7 of the experiment, the two genotypes exhibited similar declines in stomatal conductance (g) (by 77%) and photosynthetic CO2 assimilation rate (A) (by 33%). However, their leaf relative water content (RWC) was still 90% and total (CO2/Mg2+ activated) RuBPCase activities had declined only by 12%. By day 12, RWC decreased to 67% for Chenab 70 and 58% for Anza plants, while g was reduced by more than 95%, and A was reduced by 71% for both genotypes. Thus, Anza plants maintained the same A as Chenab 70 plants even though their RWC was less. Total soluble protein decreased by 30% for Chenab 70 and 43% for Anza plants, while a 12% average reduction in chlorophyll occurred. Over the 12-day experiment, an average 31% reduction in RuBPCase activity occurred for both genotypes. However, the proportion of RuBPCase in the activated state did not change (for Anza) or declined by only 9% (for Chenab 70) over the course of the experiment. These data suggest that changes in in vivo RuBPCase activities are not a major consequence of moderate to severe water deficits in spring wheat.