Keshav Dahal

Mitochondrial Alternative Oxidase Maintains Respiration and Preserves Photosynthetic Capacity during Moderate Drought in Nicotiana tabacum

Alternative oxidase reduces feedback on the photosynthetic apparatus that can otherwise limit photosynthetic capacity during moderate drought. The mitochondrial electron transport chain includes an alternative oxidase (AOX) that is hypothesized to aid photosynthetic metabolism, perhaps by acting as an additional electron sink for photogenerated reductant or by dampening the generation of reactive oxygen species. Gas exchange, chlorophyll fluorescence, photosystem I (PSI) absorbance, and biochemical and protein analyses were used to compare respiration and photosynthesis of Nicotiana tabacum ‘Petit Havana SR1’ wild-type plants with that of transgenic AOX knockdown (RNA interference) and overexpression lines, under both well-watered and moderate drought-stressed conditions. During drought, AOX knockdown lines displayed a lower rate of respiration in the light than the wild type, as confirmed by two independent methods. Furthermore, CO2 and light response curves indicated a nonstomatal limitation of photosynthesis in the knockdowns during drought, relative to the wild type. Also relative to the wild type, the knockdowns under drought maintained PSI and PSII in a more reduced redox state, showed greater regulated nonphotochemical energy quenching by PSII, and displayed a higher relative rate of cyclic electron transport around PSI. The origin of these differences may lie in the chloroplast ATP synthase amount, which declined dramatically in the knockdowns in response to drought. None of these effects were seen in plants overexpressing AOX. The results show that AOX is necessary to maintain mitochondrial respiration during moderate drought. In its absence, respiration rate slows and the lack of this electron sink feeds back on the photosynthetic apparatus, resulting in a loss of chloroplast ATP synthase that then limits photosynthetic capacity.

Knockdown of mitochondrial alternative oxidase induces the ‘stress state’ of signaling molecule pools in Nicotiana tabacum, with implications for stomatal function

The mitochondrial electron transport chain (ETC) includes an alternative oxidase (AOX) that may control the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS and RNS act as signaling intermediates in numerous plant processes, including stomatal movement. The role of AOX in controlling ROS and RNS concentrations under both steady-state and different stress conditions was evaluated using Nicotiana tabacum plants lacking AOX as a result of RNA interference. A potential functional implication of changes in ROS and RNS homeostasis was also evaluated by examining stomatal function. The leaves of nonstressed AOX knockdowns maintained concentrations of H2O2 and nitric oxide (NO) normally seen in wildtype plants only under stress conditions. Further, guard cell NO amounts were much higher in knockdowns. These guard cells were altered in size and were less responsive to NO as a signal for stomatal closure. This, in turn, compromised the stomatal response to changing irradiance. The results reveal a role for AOX in stomata. A working model is that guard cell AOX respiration maintains NO homeostasis by preventing over-reduction of the ETC, particularly during periods when high concentrations of NO acting as a signal for stomatal closure may also be inhibiting cyt oxidase respiration.

Alternative oxidase: a respiratory electron transport chain pathway essential for maintaining photosynthetic performance during drought stress

Photosynthesis and respiration are the hubs of energy metabolism in plants. Drought strongly perturbs photosynthesis as a result of both diffusive limitations resulting from stomatal closure, and in some cases biochemical limitations that are associated with a reduced abundance of key photosynthetic components. The effects of drought on respiration, particularly respiration in the light (RL ), are less understood. The plant mitochondrial electron transport chain includes a non-energy conserving terminal oxidase called alternative oxidase (AOX). Several studies have shown that drought increases AOX transcript, protein and maximum capacity. Here we review recent studies comparing wild-type (WT) tobacco to transgenic lines with altered AOX protein amount. Specifically during drought, RL was compromised in AOX knockdown plants and enhanced in AOX overexpression plants, compared with WT. Significantly, these differences in RL were accompanied by dramatic differences in photosynthetic performance. Knockdown of AOX increased the susceptibility of photosynthesis to drought-induced biochemical limitations, while overexpression of AOX delayed the development of such biochemical limitations, compared with WT. Overall, the results indicate that AOX is essential to maintaining RL during drought, and that this non-energy conserving respiration maintains photosynthesis during drought by promoting energy balance in the chloroplast. This review also outlines several areas for future research, including the possibility that enhancement of non-energy conserving respiratory electron sinks may be a useful biotechnological approach to increase plant performance during stress.

Alternative oxidase respiration maintains both mitochondrial and chloroplast function during drought.

The mitochondrial electron transport chain (ETC) terminates at cytochrome (cyt) oxidase or alternative oxidase (AOX). In Nicotiana tabacum leaves, mitochondrial respiration in the light (RL ) declined with increasing drought severity but then increased under extreme drought, despite a steep decline in maximal cyt oxidase activity. This increased RL was absent in AOX knockdown lines, while AOX overexpression lines showed enhanced RL relative to the wild-type (WT). Cyt oxidase activity under extreme drought was higher in overexpressors and lower in knockdowns, compared with the WT, providing evidence that AOX acted to maintain cyt pathway function. The rate of RL was a strong determinant of the reduction state of the photosynthetic ETC during drought. As such, the maximal quantum yield of photosystem II was compromised in knockdowns, compared with the WT, during extreme drought. By contrast, overexpressors maintained their instantaneous leaf water-use efficiency equally as high during extreme drought as when they were well watered. In both mitochondria and chloroplasts, protein carbonyl accumulation during extreme drought was strongly increased in knockdowns, and decreased in overexpressors, relative to WT. Hence the ability of AOX to maintain critical mitochondrial and chloroplast functions during extreme drought is likely due, at least in part, to its ability to reduce oxidative damage.

Potential for increased photosynthetic performance and crop productivity in response to climate change: role of CBFs and gibberellic acid

We propose that targeting the enhanced photosynthetic performance associated with the cold acclimation of winter cultivars of rye (Secale cereale L.), wheat (Triticum aestivum L.), and Brassica napus L. may provide a novel approach to improve crop productivity under abiotic as well as biotic stress conditions. In support of this hypothesis, we provide the physiological, biochemical, and molecular evidence that the dwarf phenotype induced by cold acclimation is coupled to significant enhancement in photosynthetic performance, resistance to photoinhibition, and a decreased dependence on photoprotection through non-photochemical quenching which result in enhanced biomass production and ultimately increased seed yield. These system-wide changes at the levels of phenotype, physiology, and biochemistry appear to be governed by the family of C-repeat/dehydration-responsive family of transcription factors (CBF/DREB1). We relate this phenomenon to the semi-dwarf, gibberellic acid insensitive (GAI), cereal varieties developed during the “green revolution” of the early 1960s and 1970s. We suggest that genetic manipulation of the family of C-repeat/dehydration-responsive element binding transcription factors (CBF/DREB1) may provide a novel approach for the maintenance and perhaps even the enhancement of plant productivity under conditions of sub-optimal growth conditions predicted for our future climate.

Improved photosynthetic performance during severe drought in Nicotiana tabacum overexpressing a nonenergy conserving respiratory electron sink.

Chloroplasts have means to manage excess reducing power but these mechanisms may become restricted by rates of ATP turnover. Alternative oxidase (AOX) is a mitochondrial terminal oxidase that uncouples the consumption of reducing power from ATP synthesis. Physiological and biochemical analyses were used to compare respiration and photosynthesis of Nicotiana tabacum wild-type (WT) plants with that of transgenic lines overexpressing AOX, under both well-watered and drought stress conditions. With increasing drought severity, AOX overexpression acted to increase respiration in the light (RL ) relative to WT. CO2 and light response curves indicated that overexpression also improved photosynthetic performance relative to WT, as drought severity increased. This was not due to an effect of AOX amount on leaf water status or the development of the diffusive limitations that occur due to drought. Rather, AOX overexpression dampened photosystem stoichiometry adjustments and losses of key photosynthetic components that occurred in WT. The results indicate that AOX amount influences RL , particularly during severe drought, when cytochrome pathway respiration may become increasingly restricted. This impacts the chloroplast redox state, influencing how the photosynthetic apparatus responds to increasing drought severity. In particular, the development of biochemical limitations to photosynthesis are dampened in plants with increased nonenergy conserving RL .

Long-Term Growth Under Elevated CO2 Suppresses Biotic Stress Genes in Non-Acclimated, But Not Cold-Acclimated Winter Wheat

This study compared the photosynthetic performance and the global gene expression of the winter hardy wheat Triticum aestivum cv Norstar grown under non-acclimated (NA) or cold-acclimated (CA) conditions at either ambient CO2 or elevated CO2. CA Norstar maintained comparable light-saturated and CO2-saturated rates of photosynthesis but lower quantum requirements for PSII and non-photochemical quenching relative to NA plants even at elevated CO2. Neither NA nor CA plants were sensitive to feedback inhibition of photosynthesis at elevated CO2. Global gene expression using microarray combined with bioinformatics analysis revealed that genes affected by elevated CO2 were three times higher in NA (1,022 genes) compared with CA (372 genes) Norstar. The most striking effect was the down-regulation of genes involved in the plant defense responses in NA Norstar. In contrast, cold acclimation reversed this down-regulation due to the cold induction of genes involved in plant pathogenesis resistance; and cellular and chloroplast protection. These results suggest that elevated CO2 has less impact on plant performance and productivity in cold-adapted winter hardy plants in the northern climates compared with warmer environments. Selection for cereal cultivars with constitutively higher expression of biotic stress defense genes may be necessary under elevated CO2 during the warm growth period and in warmer climates.

Coordinated regulation of photosynthetic and respiratory components is necessary to maintain chloroplast energy balance in varied growth conditions

Highlight Plants use the alternative oxidase to modulate the rate of mitochondrial respiration in the light, maintaining chloroplast energy balance and hence optimizing photosynthesis under varied growth conditions.

Improved chloroplast energy balance during water deficit enhances plant growth: more crop per drop

Mitochondrial alternative oxidase respiration aids in balancing the thylakoid membrane proton circuit, thus improving both photosynthesis and growth during a prolonged water deficit.

Growth at Elevated CO2 Requires Acclimation of the Respiratory Chain to Support Photosynthesis

Mitochondrial alternative oxidase respiration supports source leaf carbon and energy balance during growth at elevated CO2. Plants will experience an elevated atmospheric concentration of CO2 (ECO2) in the future. Growth of tobacco (Nicotiana tabacum) at ECO2 more than doubled the leaf protein amount of alternative oxidase (AOX), a non-energy-conserving component of mitochondrial respiration. To test the functional significance of this AOX increase, wild-type tobacco was compared with AOX knockdown and overexpression lines, following growth at ambient CO2 or ECO2. The ECO2-grown AOX knockdowns had a reduced capacity for triose phosphate use (TPU) during photosynthesis compared with the other plant lines. This TPU limitation of CO2 assimilation was associated with an increased accumulation of glucose-6-phosphate, sucrose, and starch in the leaves of the knockdowns. Under TPU-limiting conditions, the size of the proton gradient and proton motive force across the thylakoid membrane was enhanced in the knockdowns relative to the other plant lines, suggesting a restriction of chloroplast ATP synthase activity. This restriction was not due to a decline in ATP synthase (AtpB) protein amount. The knockdowns also displayed a photosystem stoichiometry adjustment at ECO2, which was absent in the other plant lines. Additional experiments showed that the way in which AOX supports photosynthesis at ECO2 is distinct from its previously described role in supporting photosynthesis during water deficit. The results are discussed in terms of how AOX contributes to TPU capacity and the maintenance of chloroplast ATP synthase activity at ECO2. Overall, the evidence suggests that AOX respiration is needed to maintain both the carbon and energy balance in photosynthetic tissues during growth at ECO2.