Structural and Biophysical Characterization of Purified Recombinant Arabidopsis thaliana's Alternative Oxidase 1A (rAtAOX1A): Interaction With Inhibitor(s) and Activator.

In higher plants, alternative oxidase (AOX) participates in a cyanide resistant and non-proton motive electron transport pathway of mitochondria, diverging from the ubiquinone pool. The physiological significance of AOX in biotic/abiotic stress tolerance is well-documented. However, its structural and biophysical properties are poorly understood as its crystal structure is not yet revealed in plants. Also, most of the AOX purification processes resulted in a low yield/inactive/unstable form of native AOX protein. The present study aims to characterize the purified rAtAOX1A protein and its interaction with inhibitors, such as salicylhydroxamic acid (SHAM) and n-propyl gallate (n-PG), as well as pyruvate (activator), using biophysical/in silico studies. The rAtAOX1A expressed in E. coli BL21(DE3) cells was functionally characterized by monitoring the respiratory and growth sensitivity of E. coli/pAtAOX1A and E. coli/pET28a to classical mitochondrial electron transport chain (mETC) inhibitors. The rAtAOX1A, which is purified through affinity chromatography and confirmed by western blotting and MALDI-TOF-TOF studies, showed an oxygen uptake activity of 3.86 µmol min-1 mg-1 protein, which is acceptable in non-thermogenic plants. Circular dichroism (CD) studies of purified rAtAOX1A revealed that >50% of the protein content was α-helical and retained its helical absorbance signal (ellipticity) at a wide range of temperature and pH conditions. Further, interaction with SHAM, n-PG, or pyruvate caused significant changes in its secondary structural elements while retaining its ellipticity. Surface plasmon resonance (SPR) studies revealed that both SHAM and n-PG bind reversibly to rAtAOX1A, while docking studies revealed that they bind to the same hydrophobic groove (Met191, Val192, Met195, Leu196, Phe251, and Phe255), to which Duroquinone (DQ) bind in the AtAOX1A. In contrast, pyruvate binds to a pocket consisting of Cys II (Arg174, Tyr175, Gly176, Cys177, Val232, Ala233, Asn294, and Leu313). Further, the mutational docking studies suggest that (i) the Met195 and Phe255 of AtAOX1A are the potential candidates to bind the inhibitor. Hence, this binding pocket could be a 'potential gateway' for the oxidation-reduction process in AtAOX1A, and (ii) Arg174, Gly176, and Cys177 play an important role in binding to the organic acids like pyruvate.

CRISPR/Cas9 mutants of tomato MICRORNA164 genes uncover their functional specialization in development.

Plant MICRORNA164 (miR164) plays diverse regulatory functions by post-transcriptional repression of certain NAM/ATAF/CUC-domain transcription factors. However, the involvement of miR164 in fleshy fruit development and ripening remains poorly understood. Here, de novo prediction of tomato (Solanum lycopersicum) MIR164 genes identified four genes (SlMIR164a-d), of which SlMIR164d has an atypically long pre-miRNA. The roles of the fruit expressed SlMIR164a, b, and d were studied by analysis of their Clustered Regularly Interspaced Short Palindromic Repeats mutants. The slmir164bCR mutant plants exhibited shoot and flower abnormalities characteristic of ectopic boundary specification, whereas the shoot and flower development of slmir164aCR and slmir164dCR mutants were indistinguishable from wild-type. Strikingly, the knockout of SlMIR164a practically eliminated sly-miR164 from the developing and ripening fruit pericarp. The sly-miR164-deficient slmir164aCR fruits were smaller than the wild-type, due to reduced pericarp cell division and expansion, and displayed intense red color and matte, instead of glossy appearance, upon ripening. We found that the fruit skin phenotypes were associated with morphologically abnormal outer epidermis and thicker cuticle. Quantitation of sly-miR164 target transcripts in slmir164aCR ripening fruits demonstrated the upregulation of SlNAM3 and SlNAM2. Specific expression of their miR164-resistant versions in the pericarp resulted in the formation of extremely small fruits with abnormal epidermis, highlighting the importance of their negative regulation by sly-miR164a. Taken together, our results demonstrate that SlMIR164a and SlMIR164b play specialized roles in development: SlMIR164b is required for shoot and flower boundary specification, and SlMIR164a is required for fruit growth including the expansion of its outer epidermis, which determines the properties of the fruit skin.

Current approaches to measure nitric oxide in plants.

Nitric oxide (NO) is now established as an important signalling molecule in plants where it influences growth, development, and responses to stress. Despite extensive research, the most appropriate methods to measure and localize these signalling radicals are debated and still need investigation. Many confounding factors such as the presence of other reactive intermediates, scavenging enzymes, and compartmentation influence how accurately each can be measured. Further, these signalling radicals have short half-lives ranging from seconds to minutes based on the cellular redox condition. Hence, it is necessary to use sensitive and specific methods in order to understand the contribution of each signalling molecule to various biological processes. In this review, we summarize the current knowledge on NO measurement in plant samples, via various methods. We also discuss advantages, limitations, and wider applications of each method.

A discrete role for alternative oxidase under hypoxia to increase nitric oxide and drive energy production.

Alternative oxidase (AOX) is an integral part of the mitochondrial electron transport and can prevent reactive oxygen species (ROS) and nitric oxide (NO) production under non-stressed, normoxic conditions. Here we assessed the roles of AOX by imposing stress under normoxia in comparison to hypoxic conditions using AOX over expressing (AOX OE) and anti-sense (AOX AS) transgenic Arabidopsis seedlings and roots. Under normoxic conditions stress was induced with the defence elicitor flagellin (flg22). AOX OE reduced NO production whilst this was increased in AOX AS. Moreover AOX AS also exhibited an increase in superoxide and therefore peroxynitrite, tyrosine nitration suggesting that scavenging of NO by AOX can prevent toxic peroxynitrite formation under normoxia. In contrast, during hypoxia interestingly we found that AOX is a generator of NO. Thus, the NO produced during hypoxia, was enhanced in AOX OE and suppressed in AOX AS. Additionally, treatment of WT or AOX OE with the AOX inhibitor SHAM inhibited hypoxic NO production. The enhanced levels of NO correlated with expression of non-symbiotic haemoglobin, increased NR activity and ATP production. The ATP generation was suppressed in nia1,2 mutant and non symbiotic haemoglobin antisense line treated with SHAM. Taken together these results suggest that hypoxic NO generation mediated by AOX has a discrete role by feeding into the haemoglobin-NO cycle to drive energy efficiency under conditions of low oxygen tension.

Isolation and Structural Studies of Mitochondria from Pea Roots.

For structural and respiratory studies, isolation of intact and active mitochondria is essential. Here, we describe an isolation method which gave good yield and intact mitochondria from 2-week-old pea (Pisum sativum) roots grown hydroponically under standard growth conditions. We used Percoll gradient centrifugation for this isolation procedure. The yield of purified mitochondria was 50 µg/g FW. Isolated mitochondria maintained their structure which was observed by using MitoTracker green in confocal microscope and scanning electron microscopy (SEM). Intact mitochondria are clearly visible in SCM images. Taken together this isolation method can be used for physiological and microscopic studies on mitochondria.

Genetic engineering of AtAOX1a in Saccharomyces cerevisiae prevents oxidative damage and maintains redox homeostasis.

This study aimed to validate the physiological importance of Arabidopsis thaliana alternative oxidase 1a (AtAOX1a) in alleviating oxidative stress using Saccharomyces cerevisiae as a model organism. The AOX1a transformant (pYES2AtAOX1a) showed cyanide resistant and salicylhydroxamic acid (SHAM)-sensitive respiration, indicating functional expression of AtAOX1a in S. cerevisiae. After exposure to oxidative stress, pYES2AtAOX1a showed better survival and a decrease in reactive oxygen species (ROS) when compared to S. cerevisiae with empty vector (pYES2). Furthermore, pYES2AtAOX1a sustained growth by regulating GPX2 and/or TSA2, and cellular NAD (+)/NADH ratio. Thus, the expression of AtAOX1a in S. cerevisiae enhances its respiratory tolerance which, in turn, maintains cellular redox homeostasis and protects from oxidative damage.

Alternative Oxidase Pathway Optimizes Photosynthesis During Osmotic and Temperature Stress by Regulating Cellular ROS, Malate Valve and Antioxidative Systems.

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 µmoles 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.

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.

BACKGROUND AND AIMS: 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. METHODS: 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. KEY RESULTS: 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. CONCLUSIONS: 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.

Physiological role of AOX1a in photosynthesis and maintenance of cellular redox homeostasis under high light in Arabidopsis thaliana.

As plants are sessile, they often face high light (HL) stress that causes damage of the photosynthetic machinery leading to decreased photosynthesis. The importance of alternative oxidase (AOX) in optimizing photosynthesis is well documented. In the present study, the role of AOX in sustaining photosynthesis under HL was studied using AOX1a knockout mutants (aox1a) of Arabidopsis thaliana. Under growth light (GL; 50 µmol photons m(-2) s(-1)) conditions, aox1a plants did not show any changes in photosynthetic parameters, NAD(P)/H redox ratios, or respiratory O2 uptake when compared to wild-type (WT). Upon exposure to HL (700 µmol photons m(-2) s(-1)), respiratory rates did not vary between WT and aox1a. But, photosynthetic parameters related to photosystem II (PSII) and NaHCO3 dependent O2 evolution decreased, while the P700 reduction state increased in aox1a compared to WT. Further, under HL, the redox state of cellular NAD(P)/H pools increased with concomitant rise in reactive oxygen species (ROS) and malondialdehyde (MDA) content in aox1a compared to WT. In presence of HL, the transcript levels of several genes related to antioxidant, malate-oxaloacetate (malate-OAA) shuttle, photorespiratory and respiratory enzymes was higher in aox1a compared to WT. Taken together, these results demonstrate that under HL, in spite of significant increase in transcript levels of several genes mentioned above to maintain cellular redox homeostasis and minimize ROS production, Arabidopsis plants deficient in AOX1a were unable to sustain photosynthesis as is the case in WT plants.

Attenuation of hydrogen peroxide-mediated oxidative stress by Brassica juncea annexin-3 counteracts thiol-specific antioxidant (TSA1) deficiency in Saccharomyces cerevisiae.

Brassica juncea annexin-3 (BjAnn3) was functionally characterized for its ability to modulate H2O2-mediated oxidative stress in Saccharomyces cerevisiae. BjAnn3 showed a significant protective role in cellular-defense against oxidative stress and partially alleviated inhibition of mitochondrial respiration in presence of exogenously applied H2O2. Heterologous expression of BjAnn3 protected membranes from oxidative stress-mediated damage and positively regulated antioxidant gene expression for ROS detoxification. We conclude that, BjAnn3 partially counteracts the effects of thioredoxin peroxidase 1 (TSA1) deficiency and aids in cellular-protection across kingdoms. Despite partial compensation of TSA1 by BjAnn3 in cell-viability tests, the over-complementation in ROS-related features suggests the existence of both redundant (e.g. ROS detoxification) and distinct features (e.g. membrane protection versus proximity-based redox regulator) of both proteins.