Importance of the alternative oxidase (AOX) pathway in regulating cellular redox and ROS homeostasis to optimize photosynthesis during restriction of the cytochrome oxidase pathway in Arabidopsis thaliana.
ABSTRACT: The importance of the alternative oxidase (AOX) pathway, particularly AOX1A, in optimizing photosynthesis during de-etiolation, under elevated CO2, low temperature, high light or combined light and drought stress is well documented. In the present study, the role of AOX1A in optimizing photosynthesis was investigated when electron transport through the cytochrome c oxidase (COX) pathway was restricted at complex III.Leaf discs of wild-type (WT) and aox1a knock-out mutants of Arabidopsis thaliana were treated with antimycin A (AA) under growth-light conditions. To identify the impact of AOX1A deficiency in optimizing photosynthesis, respiratory O2 uptake and photosynthesis-related parameters were measured along with changes in redox couples, reactive oxygen species (ROS), lipid peroxidation and expression levels of genes related to respiration, the malate valve and the antioxidative system.In the absence of AA, aox1a knock-out mutants did not show any difference in physiological, biochemical or molecular parameters compared with WT. However, after AA treatment, aox1a plants showed a significant reduction in both respiratory O2 uptake and NaHCO3-dependent O2 evolution. Chlorophyll fluorescence and P700 studies revealed that in contrast to WT, aox1a knock-out plants were incapable of maintaining electron flow in the chloroplastic electron transport chain, and thereby inefficient heat dissipation (low non-photochemical quenching) was observed. Furthermore, aox1a mutants exhibited significant disturbances in cellular redox couples of NAD(P)H and ascorbate (Asc) and consequently accumulation of ROS and malondialdehyde (MDA) content. By contrast, WT plants showed a significant increase in transcript levels of CSD1, CAT1, sAPX, COX15 and AOX1A in contrast to aox1a mutants.These results suggest that AOX1A plays a significant role in sustaining the chloroplastic redox state and energization to optimize photosynthesis by regulating cellular redox homeostasis and ROS generation when electron transport through the COX pathway is disturbed at complex III.
Project description:Under high light, the rates of photosynthetic CO2 assimilation can be influenced by reductant consumed by both foliar nitrate assimilation and mitochondrial alternative electron transport (mAET). Additionally, nitrate assimilation is dependent on reductant and carbon skeletons generated from both the chloroplast and mitochondria. However, it remains unclear how nitrate assimilation and mAET coordinate and contribute to photosynthesis. Here, hydroponically grown Arabidopsis thaliana T-DNA insertional mutants for alternative oxidase (AOX1A) and uncoupling protein (UCP1) fed either NO3 (-) or NH4 (+) were used to determine (i) the response of NO3 (-) uptake and assimilation to the disruption of mAET, and (ii) the interaction of N source (NO3 (-) versus NH4 (+)) and mAET on photosynthetic CO2 assimilation and electron transport. The results showed that foliar NO3 (-) assimilation was enhanced in both aox1a and ucp1 compared with the wild-type, suggesting that foliar NO3 (-) assimilation is probably driven by a decreased capacity of mAET and an increase in reductant within the cytosol. Wild-type plants had also higher rates of net CO2 assimilation (A net) and quantum yield of PSII (?PSII) under NO3 (-) feeding compared with NH4 (+) feeding. Additionally, under NO3 (-) feeding, A net and ?PSII were decreased in aox1a and ucp1 compared with the wild type; however, under NH4 (+) they were not significantly different between genotypes. This indicates that NO3 (-) assimilation and mAET are both important to maintain optimal rates of photosynthesis, probably in regulating reductant accumulation and over-reduction of the chloroplastic electron transport chain. These results highlight the importance of mAET in partitioning energy between foliar nitrogen and carbon assimilation.
Project description:An improvement in photosynthetic rate promotes the growth of crop plants. The sink-regulation of photosynthesis is crucial in optimizing nitrogen fixation and integrating it with carbon balance. Studies on these processes are essential in understanding growth inhibition in plants with ammonium ( NH4+ ) syndrome. Hence, we sought to investigate the effects of using nitrogen sources with different states of reduction (during assimilation of NO3- versus NH4+ ) on the photosynthetic performance of Arabidopsis thaliana. Our results demonstrated that photosynthetic functioning during long-term NH4+ nutrition was not disturbed and that no indication of photoinhibition of PSII was detected, revealing the robustness of the photosynthetic apparatus during stressful conditions. Based on our findings, we propose multiple strategies to sustain photosynthetic activity during limited reductant utilization for NH4+ assimilation. One mechanism to prevent chloroplast electron transport chain overreduction during NH4+ nutrition is for cyclic electron flow together with plastid terminal oxidase activity. Moreover, redox state in chloroplasts was optimized by a dedicated type II NAD(P)H dehydrogenase. In order to reduce the amount of energy that reaches the photosynthetic reaction centers and to facilitate photosynthetic protection during NH4+ nutrition, non-photochemical quenching (NPQ) and ample xanthophyll cycle pigments efficiently dissipate excess excitation. Additionally, high redox load may be dissipated in other metabolic reactions outside of chloroplasts due to the direct export of nucleotides through the malate/oxaloacetate valve. Mitochondrial alternative pathways can downstream support the overreduction of chloroplasts. This mechanism correlated with the improved growth of A. thaliana with the overexpression of the alternative oxidase 1a (AOX1a) during NH4+ nutrition. Most remarkably, our findings demonstrated the capacity of chloroplasts to tolerate NH4+ syndrome instead of providing redox poise to the cells.
Project description:The present study reveals the importance of alternative oxidase (AOX) pathway in optimizing photosynthesis under osmotic and temperature stress conditions in the mesophyll protoplasts of Pisum sativum. The responses of photosynthesis and respiration were monitored at saturating light intensity of 1000 ?moles m(-2) s(-1) at 25°C under a range of sorbitol concentrations from 0.4 to 1.0 M to induce hyper-osmotic stress and by varying the temperature of the thermo-jacketed pre-incubation chamber from 25 to 10°C to impose sub-optimal temperature stress. Compared to controls (0.4 M sorbitol and 25°C), the mesophyll protoplasts showed remarkable decrease in NaHCO3-dependent O2 evolution (indicator of photosynthetic carbon assimilation), under both hyper-osmotic (1.0 M sorbitol) and sub-optimal temperature stress conditions (10°C), while the decrease in rates of respiratory O2 uptake were marginal. The capacity of AOX pathway increased significantly in parallel to increase in intracellular pyruvate and reactive oxygen species (ROS) levels under both hyper-osmotic stress and sub-optimal temperature stress under the background of saturating light. The ratio of redox couple (Malate/OAA) related to malate valve increased in contrast to the ratio of redox couple (GSH/GSSG) related to antioxidative system during hyper-osmotic stress. Further, the ratio of GSH/GSSG decreased in the presence of sub-optimal temperature, while the ratio of Malate/OAA showed no visible changes. Also, the redox ratios of pyridine nucleotides increased under hyper-osmotic (NADH/NAD) and sub-optimal temperature (NADPH/NADP) stresses, respectively. However, upon restriction of AOX pathway by using salicylhydroxamic acid (SHAM), the observed changes in NaHCO3-dependent O2 evolution, cellular ROS, redox ratios of Malate/OAA, NAD(P)H/NAD(P) and GSH/GSSG were further aggravated under stress conditions with concomitant modulations in NADP-MDH and antioxidant enzymes. Taken together, the results indicated the importance of AOX pathway in optimizing photosynthesis under both hyper-osmotic stress and sub-optimal temperatures. Regulation of ROS through redox couples related to malate valve and antioxidant system by AOX pathway to optimize photosynthesis under these stresses are discussed.
Project description:Electron transfer is the simplest chemical reaction and constitutes the basis of a large variety of biological processes, such as photosynthesis and cellular respiration. Nature has evolved specific proteins and cofactors for these functions. The mechanisms optimizing biological electron transfer have been matter of intense debate, such as the role of the protein milieu between donor and acceptor sites. Here we propose a mechanism regulating long-range electron transfer in proteins. Specifically, we report a spectroscopic, electrochemical, and theoretical study on WT and single-mutant Cu(A) redox centers from Thermus thermophilus, which shows that thermal fluctuations may populate two alternative ground-state electronic wave functions optimized for electron entry and exit, respectively, through two different and nearly perpendicular pathways. These findings suggest a unique role for alternative or "invisible" electronic ground states in directional electron transfer. Moreover, it is shown that this energy gap and, therefore, the equilibrium between ground states can be fine-tuned by minor perturbations, suggesting alternative ways through which protein-protein interactions and membrane potential may optimize and regulate electron-proton energy transduction.
Project description:The oxidation of water (H2O) to dioxygen (O2) is important in natural photosynthesis. One of nature's strategies for managing such multi-electron transfer reactions is to employ redox-active metal-organic cofactor arrays. One prototype example is the copper tyrosinate active site found in galactose oxidase. In this work, we have implemented such a strategy to develop a bio-inspired nickel phenolate complex capable of catalyzing the oxidation of H2O to O2 electrochemically at neutral pH with a modest overpotential. Employment of the redox-active ligand turned out to be a useful strategy to avoid the formation of high-valent nickel intermediates while a reasonable turnover rate (0.15 s-1) is retained.
Project description:The methylmenaquinol:fumarate reductase (Mfr) of Campylobacter jejuni is a periplasmic respiratory (redox) protein that contributes to the metabolism of fumarate and displays homology to succinate dehydrogenase (Sdh). Since chemically oxidized redox-enzymes, including fumarate reductase and Sdh, contribute to the generation of oxidative stress in Escherichia coli, we assessed the role of Mfr in C. jejuni after exposure to hydrogen peroxide (H2 O2 ). Our results show that a Mfr mutant (?mfrA) strain was less susceptible to H2 O2 as compared to the wildtype (WT). Furthermore, the H2 O2 concentration in the ?mfrA cultures was significantly higher than that of WT after exposure to the oxidant. In the presence of H2 O2 , catalase (KatA) activity and katA expression were significantly lower in the ?mfrA strain as compared to the WT. Exposure to H2 O2 resulted in a significant decrease in total intracellular iron in the ?mfrA strain as compared to WT, while the addition of iron to the growth medium mitigated H2 O2 susceptibility and accumulation in the mutant. The ?mfrA strain was significantly more persistent in RAW macrophages as compared to the WT. Scanning electron microscopy showed that infection with the ?mfrA strain caused prolonged changes to the macrophages' morphology, mainly resulting in spherical-shaped cells replete with budding structures and craters. Collectively, our results suggest a role for Mfr in maintaining iron homeostasis in H2 O2 stressed C. jejuni, probably via affecting the concentrations of intracellular iron.
Project description:When leaves receive excess light energy, excess reductants accumulate in chloroplasts. It is suggested that some of the reductants are oxidized by the mitochondrial respiratory chain. Alternative oxidase (AOX), a non-energy conserving terminal oxidase, was upregulated in the photosynthetic mutant of <i>Arabidopsis thaliana</i>, <i>pgr5</i>, which accumulated reductants in chloroplast stroma. AOX is suggested to have an important role in dissipating reductants under high light (HL) conditions, but its physiological importance and underlying mechanisms are not yet known. Here, we compared wild-type (WT), <i>pgr5</i>, and a double mutant of AOX1a-knockout plant (<i>aox1a</i>) and <i>pgr5</i> (<i>aox1a/pgr5</i>) grown under high- and low-light conditions, and conducted physiological analyses. The net assimilation rate (<i>NAR</i>) was lower in <i>aox1a/pgr5</i> than that in the other genotypes at the early growth stage, while the leaf area ratio was higher in <i>aox1a/pgr5</i>. We assessed detailed mechanisms in relation to <i>NAR</i>. In <i>aox1a/pgr5</i>, photosystem II parameters decreased under HL, whereas respiratory O<sub>2</sub> uptake rates increased. Some intermediates in the tricarboxylic acid (TCA) cycle and Calvin cycle decreased in <i>aox1a/pgr5</i>, whereas ?-aminobutyric acid (GABA) and N-rich amino acids increased in <i>aox1a/pgr5</i>. Under HL, AOX may have an important role in dissipating excess reductants to prevent the reduction of photosynthetic electron transport and imbalance in primary metabolite levels.
Project description:The redox state of the apoplast is largely determined by ascorbate oxidase (AO) activity. The influence of AO activity on leaf acclimation to changing irradiance was explored in wild-type (WT) and transgenic tobacco (Nicotiana tobaccum) lines containing either high [pumpkin AO (PAO)] or low [tobacco AO (TAO)] AO activity at low [low light (LL); 250 ?mol m-2 s-1 ] and high [high light (HL); 1600 ?mol m-2 s-1 ] irradiance and following the transition from HL to LL. AO activities changed over the photoperiod, particularly in the PAO plants. AO activity had little effect on leaf ascorbate, which was significantly higher under HL than under LL. Apoplastic ascorbate/dehydroascorbate (DHA) ratios and threonate levels were modified by AO activity. Despite decreased levels of transcripts encoding ascorbate synthesis enzymes, leaf ascorbate increased over the first photoperiod following the transition from HL to LL, to much higher levels than LL-grown plants. Photosynthesis rates were significantly higher in the TAO leaves than in WT or PAO plants grown under HL but not under LL. Sub-sets of amino acids and fatty acids were lower in TAO and WT leaves than in the PAO plants under HL, and following the transition to LL. Light acclimation processes are therefore influenced by the apoplastic as well as chloroplastic redox state.
Project description:The emergence of oxygen-producing (oxygenic) photosynthesis fundamentally transformed our planet; however, the processes that led to the evolution of biological water splitting have remained largely unknown. To illuminate this history, we examined the behavior of the ancient Mn cycle using newly obtained scientific drill cores through an early Paleoproterozoic succession (2.415 Ga) preserved in South Africa. These strata contain substantial Mn enrichments (up to ?17 wt %) well before those associated with the rise of oxygen such as the ?2.2 Ga Kalahari Mn deposit. Using microscale X-ray spectroscopic techniques coupled to optical and electron microscopy and carbon isotope ratios, we demonstrate that the Mn is hosted exclusively in carbonate mineral phases derived from reduction of Mn oxides during diagenesis of primary sediments. Additional observations of independent proxies for O2--multiple S isotopes (measured by isotope-ratio mass spectrometry and secondary ion mass spectrometry) and redox-sensitive detrital grains--reveal that the original Mn-oxide phases were not produced by reactions with O2, which points to a different high-potential oxidant. These results show that the oxidative branch of the Mn cycle predates the rise of oxygen, and provide strong support for the hypothesis that the water-oxidizing complex of photosystem II evolved from a former transitional photosystem capable of single-electron oxidation reactions of Mn.
Project description:Mitochondrial alternative oxidase (AOX) is involved in a large number of plant physiological processes, such as growth, development and stress responses; however, the exact role of AOX in response to drought remains unclear. In our study, we provide solid evidences that the activated AOX capacity positively involved in ethylene-induced drought tolerance, in tomato (Solanum lycopersicum), accompanied by the changing level of hydrogen peroxide (H2 O2 ) and autophagy. In AOX1a-RNAi plants, the ethylene-induced drought tolerance was aggravated and associated with decreasing level of autophagy. The H2 O2 level was relatively higher in AOX1a-RNAi plants, whereas it was lower in AOX1a-overexpressing (35S-AOX1a-OE) plants after 1-(aminocarbonyl)-1-cyclopropanecarboxylic acid (ACC) pretreatment in the 14th day under drought stress. Interestingly, the accumulation of autophagosome was accompanied by the changing level of reactive oxygen species (ROS) in AOX transgenic tomato under drought stress whether or not pretreated with ACC. Pharmacological scavenging of H2 O2 accumulation in AOX1a-RNAi (aox19) stimulated autophagy acceleration under drought stress, and it seems that AOX-dependent ROS signalling is critical in triggering autophagy. Lower levels of ROS signalling positively induce autophagy activity, whereas higher ROS level would lead to rapid programmed cell death (PCD), especially in ethylene-mediated drought tolerance. Moreover, ethylene-induced autophagy during drought stress also can be through ERF5 binding to the promoters of ATG8d and ATG18h. These results demonstrated that AOX plays an essential role in ethylene-induced drought tolerance and also played important roles in mediating autophagy generation via balancing ROS level.