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.

An L,L-diaminopimelate aminotransferase mutation leads to metabolic shifts and growth inhibition in Arabidopsis.

Lysine (Lys) connects the mitochondrial electron transport chain to amino acid catabolism and the tricarboxylic acid cycle. However, our understanding of how a deficiency in Lys biosynthesis impacts plant metabolism and growth remains limited. Here, we used a previously characterized Arabidopsis mutant (dapat) with reduced activity of the Lys biosynthesis enzyme L,L-diaminopimelate aminotransferase to investigate the physiological and metabolic impacts of impaired Lys biosynthesis. Despite displaying similar stomatal conductance and internal CO2 concentration, we observed reduced photosynthesis and growth in the dapat mutant. Surprisingly, whilst we did not find differences in dark respiration between genotypes, a lower storage and consumption of starch and sugars was observed in dapat plants. We found higher protein turnover but no differences in total amino acids during a diurnal cycle in dapat plants. Transcriptional and two-dimensional (isoelectric focalization/SDS-PAGE) proteome analyses revealed alterations in the abundance of several transcripts and proteins associated with photosynthesis and photorespiration coupled with a high glycine/serine ratio and increased levels of stress-responsive amino acids. Taken together, our findings demonstrate that biochemical alterations rather than stomatal limitations are responsible for the decreased photosynthesis and growth of the dapat mutant, which we hypothesize mimics stress conditions associated with impairments in the Lys biosynthesis pathway.

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.

Deciphering ploidal levels of Lippia alba by using proteomics.

Lippia alba (Mill.) N.E. Brown (Verbenaceae), popularly known as "lemon balm" or "bushy matgrass", is widely used in folk medicine due to its anti-inflammatory, antispasmodic, analgesic, and digestive properties. It was described as an autopolyploid complex with five cytotypes (2n = 30, 38, 45, 60 and 90). To enhance our understanding of the biological variation of the species, we investigated, comparatively, the proteomic profile of all ploidal levels (diploid, aneuploid, triploid, tetraploid, and hexaploid). Leaf proteins were extracted with subsequent separation by two-dimensional electrophoresis, spot analysis, and protein identification by mass spectrometry. By comparing the proteomic profile of diploid accession to the profile of the other ploidal levels we identified differential expression between the analysed spots. We identified 34 proteins with differential expression between the ploidal levels in comparison with the diploid. The identified proteins seem to play relevant roles in the primary metabolism of L. alba suggesting that a specific set of proteins was selected during the polyploidization process, being the triploid the most different one. Given that protein composition can substantially affect the desired therapeutic effect, we posit that further combination of proteomic and metabolomic studies may help to unravel genetic variations and phenotypic profiles in L. alba.

Evolution and functional implications of the tricarboxylic acid cycle as revealed by phylogenetic analysis.

The tricarboxylic acid (TCA) cycle, a crucial component of respiratory metabolism, is composed of a set of eight enzymes present in the mitochondrial matrix. However, most of the TCA cycle enzymes are encoded in the nucleus in higher eukaryotes. In addition, evidence has accumulated demonstrating that nuclear genes were acquired from the mitochondrial genome during the course of evolution. For this reason, we here analyzed the evolutionary history of all TCA cycle enzymes in attempt to better understand the origin of these nuclear-encoded proteins. Our results indicate that prior to endosymbiotic events the TCA cycle seemed to operate only as isolated steps in both the host (eubacterial cell) and mitochondria (alphaproteobacteria). The origin of isoforms present in different cell compartments might be associated either with gene-transfer events which did not result in proper targeting of the protein to mitochondrion or with duplication events. Further in silico analyses allow us to suggest new insights into the possible roles of TCA cycle enzymes in different tissues. Finally, we performed coexpression analysis using mitochondrial TCA cycle genes revealing close connections among these genes most likely related to the higher efficiency of oxidative phosphorylation in this specialized organelle. Moreover, these analyses allowed us to identify further candidate genes which might be used for metabolic engineering purposes given the importance of the TCA cycle during development and/or stress situations.