Cross-Platform Comparison of Amino Acid Metabolic Profiling in Three Model Organisms Used in Environmental Metabolomics.

Environmental metabolomics is a promising approach to study pollutant impacts to target organisms in both terrestrial and aquatic environments. To this end, both nuclear magnetic resonance (NMR)- and mass spectrometry (MS)-based methods are used to profile amino acids in different environmental metabolomic studies. However, these two methods have not been compared directly which is an important consideration for broader comparisons in the environmental metabolomics field. We compared the quantification of 18 amino acids in the tissue extracts of Daphnia magna, a common model organism used in both ecotoxicology and ecology, using both 1H NMR spectroscopy and liquid chromatography with tandem MS (LC-MS/MS). 1H NMR quantification of amino acids agreed with the LC-MS/MS quantification for 17 of 18 amino acids measured. We also tested both quantitative methods in a D. magna sub-lethal exposure study to copper and lithium. Again, both NMR and LC-MS/MS measurements showed agreement. We extended our analyses with extracts from the earthworm Eisenia fetida and the plant model Nicotiana tabacum. The concentrations of amino acids by both 1H NMR and LC-MS/MS, agreed and demonstrated the robustness of both techniques for quantitative metabolomics. These findings demonstrate the compatibility of these two analytical platforms for amino acid profiling in environmentally relevant model organisms and emphasizes that data from either method is robust for comparisons across studies to further build the knowledge base related to pollutant exposure impacts and toxic responses of diverse environmental organisms.

Evidence that mitochondrial alternative oxidase respiration supports carbon balance in source leaves of Nicotiana tabacum.

Alternative oxidase (AOX) represents a non-energy conserving pathway within the mitochondrial electron transport chain. One potential physiological role of AOX could be to manage leaf carbohydrate amounts by supporting respiratory carbon oxidation reactions. In this study, several approaches tested the hypothesis that AOX1a gene expression in Nicotiana tabacum leaf is enhanced in conditions expected to promote an increased leaf carbohydrate status. These approaches included supplying leaves with exogenous carbohydrates, comparing plants grown at different atmospheric CO2 concentrations, comparing sink leaves with source leaves, comparing plants with different ratios of source to sink activity, and examining gene expression over the diel cycle. In each case, the pattern of AOX1a gene expression was compared with that of other genes known to respond to carbohydrates and/or other factors related to source:sink activity. These included GPT1 and GPT3 (that encode chloroplast glucose 6-phosphate/phosphate translocators), SPS (that encodes sucrose phosphate synthase), SUT1 (that encodes a sucrose/H+ symporter involved in phloem loading) and UCP1 (that encodes a mitochondrial uncoupling protein). The AOX1a transcript amount was higher following the leaf sink-to-source transition, and in plants with higher source relative to sink activity due to increasing plant age. Further, these effects were amplified in plants grown at elevated CO2 to stimulate source activity, particularly at end-of-day time periods. The AOX1a transcript amount was also higher following treatment of leaves with carbohydrate, in particular sucrose. Overall, the results provide evidence that, while source leaf sucrose accumulation may signal for a down-regulation of sucrose synthesis and transport, it also signals for means to manage the excess cytosolic carbohydrate pools. This includes increased AOX respiration to support carbon oxidation pathways even if energy charge is high, in combination perhaps with some return flux of carbohydrate from cytosol to stroma through the GPT3 translocator. As discussed, these activities could contribute to maintaining plant source:sink balance, as well as photosynthetic and phloem loading capacity.

The Role of Alternative Oxidase in the Interplay between Nitric Oxide, Reactive Oxygen Species, and Ethylene in Tobacco (Nicotiana tabacum L.) Plants Incubated under Normoxic and Hypoxic Conditions.

The transgenic tobacco (Nicotiana tabacum L.) plants with the modified levels of alternative oxidase (AOX) were used to evaluate the physiological roles of AOX in regulating nitro-oxidative stress and metabolic changes after exposing plants to hypoxia for 6 h. Under normoxia, AOX expression resulted in the decrease of nitric oxide (NO) levels and of the rate of protein S-nitrosylation, while under hypoxia, AOX overexpressors exhibited higher NO and S-nitrosylation levels than knockdowns. AOX expression was essential in avoiding hypoxia-induced superoxide and H2O2 levels, and this was achieved via higher activities of catalase and glutathione reductase and the reduced expression of respiratory burst oxidase homolog (Rboh) in overexpressors as compared to knockdowns. The AOX overexpressing lines accumulated less pyruvate and exhibited the increased transcript and activity levels of pyruvate decarboxylase and alcohol dehydrogenase under hypoxia. This suggests that AOX contributes to the energy state of hypoxic tissues by stimulating the increase of pyruvate flow into fermentation pathways. Ethylene biosynthesis genes encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC oxidase, and ethylene-responsive factors (ERFs) were induced during hypoxia and correlated with AOX and NO levels. We conclude that AOX controls the interaction of NO, reactive oxygen species, and ethylene, triggering a coordinated downstream defensive response against hypoxia.

The flexibility of metabolic interactions between chloroplasts and mitochondria in Nicotiana tabacum leaf.

To examine the effect of mitochondrial function on photosynthesis, wild-type and transgenic Nicotiana tabacum with varying amounts of alternative oxidase (AOX) were treated with different respiratory inhibitors. Initially, each inhibitor increased the reduction state of the chloroplast electron transport chain, most severely in AOX knockdowns and least severely in AOX overexpressors. This indicated that the mitochondrion was a necessary sink for photo-generated reductant, contributing to the 'P700 oxidation capacity' of photosystem I. Initially, the Complex III inhibitor myxothiazol and the mitochondrial ATP synthase inhibitor oligomycin caused an increase in photosystem II regulated non-photochemical quenching not evident with the Complex III inhibitor antimycin A (AA). This indicated that the increased quenching depended upon AA-sensitive cyclic electron transport (CET). Following 12 h with oligomycin, the reduction state of the chloroplast electron transport chain recovered in all plant lines. Recovery was associated with large increases in the protein amount of chloroplast ATP synthase and mitochondrial uncoupling protein. This increased the capacity for photophosphorylation in the absence of oxidative phosphorylation and enabled the mitochondrion to act again as a sink for photo-generated reductant. Comparing the AA and myxothiazol treatments at 12 h showed that CET optimized photosystem I quantum yield, depending upon the P700 oxidation capacity. When this capacity was too high, CET drew electrons away from other sinks, moderating the P700+ amount. When P700 oxidation capacity was too low, CET acted as an electron overflow, moderating the amount of reduced P700. This study reveals flexible chloroplast-mitochondrion interactions able to overcome lesions in energy metabolism.

Nitric Oxide Turnover Under Hypoxia Results in the Rapid Increased Expression of the Plastid-Localized Phosphorylated Pathway of Serine Biosynthesis.

The plant mitochondrial electron transport chain influences carbon and nitrogen metabolism under near anoxic conditions through its involvement in the phytoglobin-nitric oxide cycle, where the respiratory chain reduces nitrite to nitric oxide (NO), followed by NO conversion to nitrate by class 1 phytoglobin. Wild type (WT) and transgenic tobacco (Nicotiana tabacum L.) with differing amounts of alternative oxidase (AOX) were used to manipulate NO generation under hypoxia, and to examine whether this in turn influenced the gene expression of two stress-related amino acid biosynthetic pathways, the plastid-localized phosphorylated pathway of serine biosynthesis (PPSB), and the γ-aminobutyric acid (GABA) shunt. Under hypoxia, leaf NO emission rate was highest in AOX overexpressors and lowest in AOX knockdowns, with WT showing an intermediate rate. In turn, the rate of NO emission correlated with the degree to which amino acids accumulated. This amino acid accumulation was associated with the increased expression of the enzymes of the stress-related amino acid biosynthetic pathways. However, induction of the PPSB occurred much earlier than the GABA shunt. This work shows that high rates of NO turnover associate with rapid gene induction of the PPSB, establishing a clear link between this pathway and the maintenance of carbon, nitrogen and energy metabolism under hypoxia.

Distinctive mitochondrial and chloroplast components contributing to the maintenance of carbon balance during plant growth at elevated CO2.

Plant carbon balance depends upon the difference between photosynthetic carbon gain and respiratory carbon loss. In C3 plants, growth at an elevated atmospheric concentration of CO2 (ECO2) stimulates photosynthesis and raises the leaf carbohydrate status, but how respiration responds is less understood. In this study, growth of Nicotiana tabacum at ECO2 increased the protein amount of the non-energy conserving mitochondrial alternative oxidase (AOX). Growth at ECO2 increased AOX1a transcript amount, and the transcript amount of a putative sugar-responsive gene encoding a chloroplast glucose-6-phosphate/phosphate translocator (GPT3). We suggest that the elevated amounts of AOX and GPT3 represent distinctive mitochondrial and chloroplast mechanisms to manage an excessive cytosolic pool of sugar phosphates. AOX respiration could consume cytosolic sugar phosphates, without this activity being restricted by rates of ATP turnover. GPT3 could allow accumulating cytosolic glucose-6-phosphate to return to the chloroplast. This could feed starch synthesis or a glucose-6-phosphate shunt in the Calvin cycle. AOX and GPT3 activities could buffer against Pi depletions that might otherwise disrupt mitochondrial and chloroplast electron transport chain activities. AOX and GPT3 activities could also buffer against a down-regulation of photosynthetic capacity by preventing a persistent imbalance between photosynthetic carbon gain and the activity of carbon utilizing sinks.

Roles for Plant Mitochondrial Alternative Oxidase Under Normoxia, Hypoxia, and Reoxygenation Conditions.

Alternative oxidase (AOX) is a non-energy conserving terminal oxidase in the plant mitochondrial electron transport chain (ETC) that has a lower affinity for oxygen than does cytochrome (cyt) oxidase. To investigate the role(s) of AOX under different oxygen conditions, wild-type (WT) Nicotiana tabacum plants were compared with AOX knockdown and overexpression plants under normoxia, hypoxia (near-anoxia), and during a reoxygenation period following hypoxia. Paradoxically, under all the conditions tested, the AOX amount across plant lines correlated positively with leaf energy status (ATP/ADP ratio). Under normoxia, AOX was important to maintain respiratory carbon flow, to prevent the mitochondrial generation of superoxide and nitric oxide (NO), to control lipid peroxidation and protein S-nitrosylation, and possibly to reduce the inhibition of cyt oxidase by NO. Under hypoxia, AOX was again important in preventing superoxide generation and lipid peroxidation, but now contributed positively to NO amount. This may indicate an ability of AOX to generate NO under hypoxia, similar to the nitrite reductase activity of cyt oxidase under hypoxia. Alternatively, it may indicate that AOX activity simply reduces the amount of superoxide scavenging of NO, by reducing the availability of superoxide. The amount of inactivation of mitochondrial aconitase during hypoxia was also dependent upon AOX amount, perhaps through its effects on NO amount, and this influenced carbon flow under hypoxia. Finally, AOX was particularly important in preventing nitro-oxidative stress during the reoxygenation period, thereby contributing positively to the recovery of energy status following hypoxia. Overall, the results suggest that AOX plays a beneficial role in low oxygen metabolism, despite its lower affinity for oxygen than cytochrome oxidase.

Does the stromal concentration of Pi control chloroplast ATP synthase protein amount in contrasting growth environments?

Changes in the growth environment can generate imbalances in chloroplast photosynthetic metabolism. Under water deficit, stomatal closure limits CO2 availability such that the production of ATP and NADPH by the thylakoid membrane-localized electron transport chain may not match the consumption of these energy intermediates by the stroma-localized Calvin-Benson cycle, thus challenging energy balance. Alternatively, in an elevated CO2 atmosphere, carbon fixation by the Calvin-Benson cycle may outpace the activity of downstream carbohydrate-utilizing processes, thus challenging carbon balance. Our previous studies have shown that, in both of the above scenarios, a mitochondrial alternative oxidase contributes to maintaining energy or carbon balance, highlighting the importance of photosynthesis-respiration interactions in optimizing photosynthesis in different growth environments. In these previous studies, we observed aberrant amounts of chloroplast ATP synthase protein across the different transgenic plant lines and growth conditions, compared to wild-type. Based on these observations, we develop here the hypothesis that an important determinant of chloroplast ATP synthase protein amount is the stromal concentration of inorganic phosphate. ATP synthase is a master regulator of photosynthesis. Coarse control of ATP synthase protein amount by the stromal inorganic phosphate status could provide a means to coordinate the electron transport and carbon fixation reactions of photosynthesis.

Signaling interactions between mitochondria and chloroplasts in Nicotiana tabacum leaf.

Research has begun to elucidate the signal transduction pathway(s) that control cellular responses to changes in mitochondrial status. Important tools in such studies are chemical inhibitors used to initiate mitochondrial dysfunction. This study compares the effect of different inhibitors and treatment conditions on the transcript amount of nuclear genes specifically responsive to mitochondrial dysfunction in leaf of Nicotiana tabacum L. cv. Petit Havana. The Complex III inhibitors antimycin A (AA) and myxothiazol (MYXO), and the Complex V inhibitor oligomycin (OLIGO), each increased the transcript amount of the mitochondrial dysfunction genes. Transcript responses to OLIGO were greater during treatment in the dark than in the light, and the dark treatment resulted in cell death. In the dark, transcript responses to AA and MYXO were similar to one another, despite MYXO leading to cell death. In the light, transcript responses to AA and MYXO diverged, despite cell viability remaining high with either inhibitor. This divergent response may be due to differential signaling from the chloroplast because only AA also inhibited cyclic electron transport, resulting in a strong acceptor-side limitation in photosystem I. In the light, chemical inhibition of chloroplast electron transport reduced transcript responses to AA, while having no effect on the response to MYXO, and increasing the response to OLIGO. Hence, when studying mitochondrial dysfunction signaling, different inhibitor and treatment combinations differentially affect linked processes (e.g. chloroplast function and cell fate) that then contribute to measured responses. Therefore, inhibitor and treatment conditions should be chosen to align with specific study goals.

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

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.

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

The non-energy-conserving alternative oxidase (AOX) respiration of plant mitochondria is known to interact with chloroplast photosynthesis. This may have consequences for growth, particularly under sub-optimal conditions when energy imbalances can impede photosynthesis. This hypothesis was tested by comparing the metabolism and growth of wild-type Nicotiana tabacum with that of AOX knockdown and overexpression lines during a prolonged steady-state mild to moderate water deficit. Under moderate water deficit, the AOX amount was an important determinant of the rate of both mitochondrial respiration in the light and net photosynthetic CO2 assimilation (A) at the growth irradiance. In particular, AOX respiration was necessary to maintain optimal proton and electron fluxes at the chloroplast thylakoid membrane, which in turn prevented a water-deficit-induced biochemical limitation of photosynthesis. As a result of differences in A, AOX overexpressors gained more biomass and knockdowns gained less biomass than wild-type during moderate water deficit. Biomass partitioning also differed, with the overexpressors having a higher percentage, and the knockdowns having a lower percentage, of total above-ground biomass in reproductive tissue than wild-type. The results establish that improving chloroplast energy balance by using a non-energy-conserving respiratory electron sink can increase photosynthesis and growth during prolonged water deficit.

The occurrence and control of nitric oxide generation by the plant mitochondrial electron transport chain.

The plant mitochondrial electron transport chain (ETC) is bifurcated such that electrons from ubiquinol are passed to oxygen via the usual cytochrome path or through alternative oxidase (AOX). We previously showed that knockdown of AOX in transgenic tobacco increased leaf concentrations of nitric oxide (NO), implying that an activity capable of generating NO had been effected. Here, we identify the potential source of this NO. Treatment of leaves with antimycin A (AA, Qi -site inhibitor of Complex III) increased NO amount more than treatment with myxothiazol (Myxo, Qo -site inhibitor) despite both being equally effective at inhibiting respiration. Comparison of nitrate-grown wild-type with AOX knockdown and overexpression plants showed a negative correlation between AOX amount and NO amount following AA. Further, Myxo fully negated the ability of AA to increase NO amount. With ammonium-grown plants, neither AA nor Myxo strongly increased NO amount in any plant line. When these leaves were supplied with nitrite alongside the AA or Myxo, then the inhibitor effects across lines mirrored that of nitrate-grown plants. Hence the ETC, likely the Q-cycle of Complex III generates NO from nitrite, and AOX reduces this activity by acting as a non-energy-conserving electron sink upstream of Complex III.

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

Mitochondria have a non-energy-conserving alternative oxidase (AOX) proposed to support photosynthesis, perhaps by promoting energy balance under varying growth conditions. To investigate this, wild-type (WT) Nicotiana tabacum were compared with AOX knockdown and overexpression lines. In addition, the amount of AOX protein in WT plants was compared with that of chloroplast light-harvesting complex II (LHCB2), whose amount is known to respond to chloroplast energy status. With increased growth irradiance, WT leaves maintained higher rates of respiration in the light (RL), but no differences in RL or photosynthesis were seen between the WT and transgenic lines, suggesting that, under non-stress conditions, AOX was not critical for leaf metabolism, regardless of growth irradiance. However, under drought, the AOX amount became an important determinant of RL, which in turn was an important determinant of chloroplast energy balance (measured as photosystem II excitation pressure, EP), and photosynthetic performance. In the WT, the AOX amount increased and the LHCB2 amount decreased with increased growth irradiance or drought severity. These changes in protein amounts correlated strongly, in opposing ways, with growth EP. This suggests that a signal deriving from the photosynthetic electron transport chain status coordinately controls the amounts of AOX and LHCB2, which then both contribute to maintaining chloroplast energy balance, particularly under stress conditions.

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.

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.

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 .

In planta analysis of leaf mitochondrial superoxide and nitric oxide.

Superoxide (O2(-)) and nitric oxide (NO) are produced within plant mitochondria and may have signaling functions within the cell. Here we describe semiquantitative fluorescence imaging-based approaches to estimate the mitochondrial amount of these reactive and short-lived species within intact leaf tissue. We also outline a biochemical method using oxyhemoglobin to measure NO within a whole leaf tissue extract. This quantitative method, while not specifically evaluating mitochondrial localized NO, does nonetheless provide another independent measure of NO that can be useful.

Mitochondrial alternative oxidase maintains respiration and preserves photosynthetic capacity during moderate drought in Nicotiana tabacum.

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.

A lack of mitochondrial alternative oxidase compromises capacity to recover from severe drought stress.

Alternative oxidase (AOX) constitutes a nonenergy conserving branch of the mitochondrial electron transport chain. AOX activity may be important to avoid reactive oxygen species (ROS) generation by the chain, particularly during abiotic stress. We compared leaf AOX1a transcript and AOX protein amounts in wild-type (WT) Nicotiana tabacum plants experiencing mild to severe drought. Mild to moderate drought resulted in a progressive and modest increase in AOX amount, accompanied by a progressive increased expression of different ROS-scavenging components. Under these conditions, transgenic plants with suppressed AOX amount, due to an RNA interference construct, were not compromised in their ability to manage ROS load and prevent cellular damage. Severe drought stress, particularly when combined with increased irradiance, strongly increased AOX amount in WT tobacco and this coincided with an increase in total respiration and, despite further induction of ROS-scavenging systems, some evidence of cellular damage. Under these severe conditions, plants lacking AOX suffered more cellular damage than WT and, at the most severe stage, were found to downregulate rather than upregulate the transcript level of several important ROS-scavenging components. At this stage, WT plants could still recover rapidly after rewatering, but the recoverability of AOX knockdown plants was strongly compromised.