SummaryThe 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 H 2 O 2 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.
HighlightPlants 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.
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, Q -site inhibitor of Complex III) increased NO amount more than treatment with myxothiazol (Myxo, Q -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.
Chloroplasts use light energy and a linear electron transport (LET) pathway for the coupled generation of NADPH and ATP. It is widely accepted that the production ratio of ATP to NADPH is usually less than required to fulfill the energetic needs of the chloroplast. Left uncorrected, this would quickly result in an over-reduction of the stromal pyridine nucleotide pool (i.e., high NADPH/NADP+ ratio) and under-energization of the stromal adenine nucleotide pool (i.e., low ATP/ADP ratio). These imbalances could cause metabolic bottlenecks, as well as increased generation of damaging reactive oxygen species. Chloroplast cyclic electron transport (CET) and the chloroplast malate valve could each act to prevent stromal over-reduction, albeit in distinct ways. CET avoids the NADPH production associated with LET, while the malate valve consumes the NADPH associated with LET. CET could operate by one of two different pathways, depending upon the chloroplast ATP demand. The NADH dehydrogenase-like pathway yields a higher ATP return per electron flux than the pathway involving PROTON GRADIENT REGULATION5 (PGR5) and PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE1 (PGRL1). Similarly, the malate valve could couple with one of two different mitochondrial electron transport pathways, depending upon the cytosolic ATP demand. The cytochrome pathway yields a higher ATP return per electron flux than the alternative oxidase (AOX) pathway. In both Arabidopsis thaliana and Chlamydomonas reinhardtii, PGR5/PGRL1 pathway mutants have increased amounts of AOX, suggesting complementary roles for these two lesser-ATP yielding mechanisms of preventing stromal over-reduction. These two pathways may become most relevant under environmental stress conditions that lower the ATP demands for carbon fixation and carbohydrate export.
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 AAsensitive 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.
Plant mitochondria are proposed to act as signaling organelles in the orchestration of defense responses to biotic stress and acclimation responses to abiotic stress. However, the primary signal(s) being generated by mitochondria and then interpreted by the cell are largely unknown. Recently, we showed that mitochondria generate a sustained burst of superoxide (O2-) during particular plant-pathogen interactions. This O2- burst appears to be controlled by mitochondrial components that influence rates of O2- generation and scavenging within the organelle. The O2- burst appears to influence downstream processes such as the hypersensitive response, indicating that it could represent an important mitochondrial signal in support of plant stress responses. The findings generate many interesting questions regarding the upstream factors required to generate the O2- burst, the mitochondrial events that occur in support of and in parallel with this burst and the downstream events that respond to this burst.
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.
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