AOX1a Expression in Arabidopsis thaliana Affects the State of Chloroplast Photoprotective Systems under Moderately High Light Conditions.

Alternative oxidase (AOX) in the mitochondrial electron transport chain is considered important for sustaining photosynthesis under high light conditions. Here, we examined the effects of the AOX pathway on the state of chloroplast photoprotective systems. Arabidopsis thaliana plants (4 weeks old), comprising three genotypes (wild type [WT], overexpressing [XX-2] and antisense [AS-12] lines for AOX1a), were exposed to moderately high light conditions (MHL, 400 µmol m-2 s-1) in a short-term experiment (8 h). After 8 h of MHL, the WT and XX-2 plants showed stable non-photochemical quenching (qN) and violaxanthin cycle activity. Antisense plants displayed the lowest level of qN and a lower de-epoxidation state (DEPS) relative to plants of the same line after 4-6 h MHL, as well as compared to WT and XX-2 plants after 8 h MHL. The decline in DEPS in AS-12 plants was attributed to an insufficient violaxanthin de-epoxidase activity, which in turn was associated with a decrease in reduced ascorbate levels in the chloroplasts and leaves. Simultaneously, gene expression and the activity of ascorbate peroxidase in the antisense line increased after 8 h of MHL, supporting the compensatory effect of the antioxidant system when AOX1a expression is suppressed. This study emphasizes the role played by AOX in modulating the photoprotection processes and in the maintenance of relationships between mitochondria and chloroplasts by influencing ascorbate content.

Responses of genes of DNA repair, alternative oxidase, and pro-/antioxidant state in Arabidopsis thaliana with altered expression of AOX1a to gamma irradiation.

PURPOSE: High doses of gamma (γ) irradiation cause oxidative stress and DNA damage. Alternative oxidase (AOX) catalyzes the energy-dissipating cyanide-resistant alternative pathway in plant mitochondria and is an important part of the cellular defense network under stress conditions. In this study, Arabidopsis thaliana plants with an altered expression of the AOX1a gene were exposed by high dose-rate ionizing radiation to assess the expression of genes of DNA repair and pro-/antioxidant states to elucidate the functional significance of AOX in plant stress response. MATERIALS AND METHODS: Five-week-old A. thaliana plants, either with basal AOX1a gene expression (wild-type Colombia-0 (Col-0)), antisense silencing of AOX1a (AS-12), and overexpression of the gene (XX-2), were γ-irradiated at a dose of 200 Gy. Gene expression and biochemical analyses were performed 12 h after irradiation. RESULTS: Acute γ-irradiation caused different responses between the genotypes. XX-2 plants, either control or irradiated, showed the highest expression of AOX1a gene and AOX protein, and the lowest expression of DNA repair genes. Wild type and AS-12 plants exposed to γ-irradiation upregulated another stress-induced gene, AOX1d, and DNA repair genes. Furthermore, a higher activity of Mn-dependent superoxide dismutase (Mn-SOD) was observed in the irradiated AS-12 plants than in the untreated plants of this line. However, AS-12 plants were less effective than Col-0 plants in controlling the accumulation of the superoxide anion. XX-2 plants had the lowest reactive oxygen species (ROS) levels among the genotypes. CONCLUSIONS: AS-12 plants display a compensatory mechanism by increasing the expression of AOX1d and the synthesis of the AOX protein, as well as by Mn-SOD activation. However, these were insufficient to maintain the background level of embryonic lethal mutations, and thereby the reproductive capacity. These results highlight the importance of AOX in the successful adaptation of plants to acute γ-irradiation, and indicate that AOX1a plays a key role in the regulation of the stress response.

The gene expression profiles of mitochondrial respiratory components in Arabidopsis plants with differing amounts of ALTERNATIVE OXIDASE1a under high intensity light.

We compared the expression of mitochondrial alternative oxidase (AOX) and other non-phosphorylating respiratory components (NPhPs) in wild type and AOX1a transgenic Arabidopsis thaliana following short-term transfer of plants to higher irradiance conditions to gain more insight into the mechanisms of AOX functioning under light. The AOX1a overexpressing line (XX-2) showed the highest amount of AOX1a transcripts and AOX1A synthesis during the entire experiment, and many NPhPs genes were down-regulated after 6-8 h under the higher light conditions. Antisense AS-12 plants displayed a compensatory effect, typically after 8 h of exposure to higher irradiance, by up-regulating their expression of the majority of genes encoding AOX and other respiratory components. In addition, AS-12 plants displayed 'overcompensation effects' prior to their transfer to high light conditions, i.e., they showed a higher expression level of certain genes. As a result, the ROS content in AS-12, as in XX-2, was consistently lower than in the wild type. All NPhPs genes share, in common with AOX1a, light- and stress-related cis-acting regulatory elements (CAREs) in their promoters. However, the expression of respiratory genes does not always depend on the level of AOX1a expression. This suggests the presence of multiple combinations of signaling pathways in gene induction. Based on our results, we outline possible directions for future research.

Altered levels of AOX1a expression result in changes in metabolic pathways in Arabidopsis thaliana plants acclimated to low dose rates of ultraviolet B radiation.

UV-B is a damaging component of solar radiation that inevitably reaches the Earth's surface. Plants have developed response mechanisms to adapt to UVB exposure. The alternative oxidase (AOX) catalyzes the ATP-uncoupling cyanide-resistant alternative pathway (AP) in plant mitochondria and is thought to be an important part of the cellular defense network under stress conditions. This study aimed to unravel the poorly understood functional significance of AOX1a induction in Arabidopsis thaliana leaves exposed to ecologically relevant doses of UVB radiation, by comparing wild-type (WT) plants with plants with modified expression of the AOX1a gene, either downregulated by antisense (AS-12) or overexpressed (XX-2). UVB exposure resulted in a phenotypic difference between lines. AOX1a overexpression resulted in the highest induction of AOX1A synthesis and MnSOD activity, and the lowest ROS level without pronounced changes in the phenotype relative to other genotypes. In AS-12 plants, expression of the majority of the genes encoding AOX was detected, other non-phosphorylating pathway components and antioxidant enzymes increased along with anthocyanin accumulation in leaves, and the ROS content was lower than in the WT. In addition to the expected AOX1 protein size (34 kDa), an AOX1 30 kDa band appeared under UVB exposure in all genotypes. However, in AS-12, the alterations in the transcript level and in the abundance of AOX1 protein isoforms induced by UVB could not fully functionally compensate for the lack of AOX1A. This was confirmed by the observed low AP capacity and increased levels of the oxidized form of ascorbate. These results highlight the importance of AOX in plant response to UVB for the control of a balanced metabolism, and indicate that AOX1a plays a key role in the regulation of the stress response.

Expression profiles of genes for mitochondrial respiratory energy-dissipating systems and antioxidant enzymes in wheat leaves during de-etiolation.

Mitochondrial respiratory components participate in the maintenance of chloroplast functional activity. This study investigates the effects 48h de-etiolation of spring wheat seedlings (Triticum aestivum L., var. Irgina) on the expression of genes that encode energy-dissipating respiratory components and antioxidant enzymes under continuous light conditions. The expression of AOX1a following the prolonged darkness exhibited a pattern indicating a prominent dependence on light. The expression of other respiratory genes, including NDA2, NDB2, and UCP1b, increased during de-etiolation and dark-to-light transition; however, changes in the expression of these genes occurred later than those in AOX1a expression. A high expression of NDA1 was detected after 12h of de-etiolation. The suppression of AOX1a, NDA2, NDB2, and UCP1b was observed 24h after de-etiolation when the photosynthetic apparatus and its defence systems against excess light were completely developed. The expression patterns of the respiratory genes and several genes encoding antioxidant enzymes (MnSOD, Cu-ZnSOD, t-APX, GR, and GRX) were quite similar. Our data indicate that the induction of nuclear genes encoding respiratory and antioxidant enzymes allow the plants to control reactive oxygen species (ROS) production and avoid oxidative stress during de-etiolation.

Light regulation of mitochondrial alternative oxidase pathway during greening of etiolated wheat seedlings.

This study deals with effects of de-etiolation (48h) of spring wheat (Triticum aestivum L., var. Irgina) seedlings on differential expression of AOX1 genes, levels of AOX protein and the alternative respiratory pathway (AP) capacity. As a result of exposure to continuous irradiation of dark-grown wheat seedlings, the respiratory activity and AP capacity in leaves significantly increased during the first 6h of studies. Expression of AOX1a was up-regulated by light and proved consistent with changes in the AP capacity. Effects on expression of AOX1c were less pronounced. Immunoblot analysis showed three distinct bands of AOX with molecular weights of 34, 36 and 38kDa, with no significant changes in the relative levels during de-etiolation. The lack of a clear correlation between AOX protein amount, AOX1a expression, and AP capacity suggests post-translational control of the enzyme activation. The AOX1a suppression and a decrease in the AP capacity correlated with the sugar pool depletion after 24h of the de-etiolation, which may mean a possible substrate dependence of the AOX activity in the green cells. More efficient malate oxidation by mitochondria as well as the higher AOX capacity during the first 6h of de-etiolation was detected, whereas respiration and AOX capacity with exogenous NADH and glycine increased after 6 and 24h, respectively. We conclude that AOX plays an important role during development of an actively photosynthesizing cell, and can rapidly adapt to changes in metabolism and photosynthesis.