The role of the electron-transfer flavoprotein: ubiquinone oxidoreductase following carbohydrate starvation in Arabidopsis cell cultures.

KEY MESSAGE: The functional absence of the electron-transfer flavoprotein: ubiquinone oxidoreductase (ETFQO) directly impacts electrons donation to the mitochondrial electron transport chain under carbohydrate-limiting conditions without major impacts on the respiration of cell cultures. Alternative substrates (e.g., amino acids) can directly feed electrons into the mitochondrial electron transport chain (mETC) via the electron transfer flavoprotein/electron-transfer flavoprotein: ubiquinone oxidoreductase (ETF/ETFQO) complex, which supports plant respiration during stress situations. By using a cell culture system, here we investigated the responses of Arabidopsis thaliana mutants deficient in the expression of ETFQO (etfqo-1) following carbon limitation and supplied with amino acids. Our results demonstrate that isovaleryl-CoA dehydrogenase (IVDH) activity was induced during carbon limitation only in wild-type and that these changes occurred concomit with enhanced protein content. By contrast, neither the activity nor the total amount of IVDH was altered in etfqo-1 mutants. We also demonstrate that the activities of mitochondrial complexes in etfqo-1 mutants, display a similar pattern as in wild-type cells. Our findings suggest that the defect of ETFQO protein culminates with an impaired functioning of the IVDH, since no induction of IVDH activity was observed. However, the functional absence of the ETFQO seems not to cause major impacts on plant respiration under carbon limiting conditions, most likely due to other alternative electron entry pathways.

Heat stress-mediated effects on the morphophysiological, biochemical, and ultrastructural parameters of germinating Melanoxylon brauna Schott. seeds.

KEY MESSAGE: The present study showed that the heat stress (40 °C) caused changes in morphophysiological, biochemical, and ultrastructural parameters to the seeds Melanoxylon brauna, ultimately leading to loss of germination capacity. Temperature is an abiotic factor that influences seed germination. In the present study, we investigated morphophysiological, biochemical, and ultrastructural changes during the germination of Melanoxylon brauna seeds under heat stress. Seed germination was evaluated at constant temperatures of 25 and 40 °C. The samples consisted of seeds soaked in distilled and ionized water for 48 and 96 h at both temperatures. For the evaluation of internal morphology, the seeds were radiographed. Ultrastructural parameters were assessed using transmission electron microscopy (TEM). The production of reactive oxygen species (ROS), content of malondialdehyde (MDA) and glucose, carbonylated proteins, and activity of the enzymes (superoxide dismutase-SOD, ascorbate peroxidase-APX, catalase-CAT, peroxidase-POX, glucose-6-phosphate dehydrogenase-G6PDH, lipase, α- and ß-amylase, and protease) were measured by spectrophotometric analysis. An 82% reduction in the germination of M. brauna seeds was observed at 25 °C, and 0% at 40 °C. TEM showed that seeds submitted to heat stress (40 °C) had poorly developed mitochondria and significantly reduced respiration rates. The content of ROS and protein carbonylation in seeds subjected to 40 °C increased compared to that at 25 °C. The activity of antioxidant enzymes, namely SOD, APX, CAT, and POX, was significantly reduced in seeds subjected to heat stress. Glucose content, G6PDH, and lipase activity also decreased when the seeds were exposed to heat stress. Conversely, α- and ß-amylase enzymes and the protease increased due to the increase in temperature. Our data showed that the increase in temperature caused an accumulation of ROS, increasing the oxidative damage to the seeds, which led to mitochondrial dysfunction, ultimately leading to loss of germination.

Biochemical and functional characterization of a mitochondrial citrate carrier in Arabidopsis thaliana.

A homolog of the mitochondrial succinate/fumarate carrier from yeast (Sfc1p) has been found in the Arabidopsis genome, named AtSFC1. The AtSFC1 gene was expressed in Escherichia coli, and the gene product was purified and reconstituted in liposomes. Its transport properties and kinetic parameters demonstrated that AtSFC1 transports citrate, isocitrate and aconitate and, to a lesser extent, succinate and fumarate. This carrier catalyzes a fast counter-exchange transport as well as a low uniport of substrates, exhibits a higher transport affinity for tricarboxylates than dicarboxylates, and is inhibited by pyridoxal 5'-phosphate and other inhibitors of mitochondrial carriers to various degrees. Gene expression analysis indicated that the AtSFC1 transcript is mainly present in heterotrophic tissues, and fusion with a green-fluorescent protein localized AtSFC1 to the mitochondria. Furthermore, 35S-AtSFC1 antisense lines were generated and characterized at metabolic and physiological levels in different organs and at various developmental stages. Lower expression of AtSFC1 reduced seed germination and impaired radicle growth, a phenotype that was related to reduced respiration rate. These findings demonstrate that AtSFC1 might be involved in storage oil mobilization at the early stages of seedling growth and in nitrogen assimilation in root tissue by catalyzing citrate/isocitrate or citrate/succinate exchanges.

Data-Mining Bioinformatics: Connecting Adenylate Transport and Metabolic Responses to Stress.

Adenine nucleotides are essential in countless processes within the cellular metabolism. In plants, ATP is mainly produced in chloroplasts and mitochondria through photophosphorylation and oxidative phosphorylation, respectively. Thus, efficient adenylate transport systems are required for intracellular energy partitioning between the cell organelles. Adenylate carriers present in different subcellular compartments have been previously identified and biochemically characterized in plants. Here, by using data-mining bioinformatics tools, we propose how, and to what extent, these carriers integrate energy metabolism within a plant cell under different environmental conditions. We demonstrate that the expression pattern of the corresponding genes is variable under different environmental conditions, suggesting that specific adenylate carriers have distinct and nonredundant functions in plants.

Differential impact of amino acids on OXPHOS system activity following carbohydrate starvation in Arabidopsis cell suspensions.

Plant respiration mostly depends on the activity of glycolysis and the oxidation of organic acids in the tricarboxylic acid cycle to synthesize ATP. However, during stress situations plant cells also use amino acids as alternative substrates to donate electrons through the electron-transfer flavoprotein (ETF)/ETF:ubiquinone oxidoreductase (ETF/ETFQO) complex to the mitochondrial electron transport chain (mETC). Given this, we investigated changes of the oxidative phosphorylation (OXPHOS) system in Arabidopsis thaliana cell culture under carbohydrate starvation supplied with a range of amino acids. Induction of isovaleryl-CoA dehydrogenase (IVDH) activity was observed under carbohydrate starvation which was associated with increased amounts of IVDH protein detected by immunoblotting. Furthermore, activities of the protein complexes of the mETC were reduced under carbohydrate starvation. We also observed that OXPHOS system activity behavior is differently affected by different amino acids and that proteins associated with amino acids catabolism are upregulated in cells following carbohydrate starvation. Collectively, our results support the contention that ETF/ETFQO is an essential pathway to donate electrons to the mETC and that amino acids are alternative substrates to maintain respiration under carbohydrate starvation.