Foliar metabolite levels, including myoinositol, show correlation with grain yield in tropical maize field trials during drought, heat, and simultaneous drought

Protein synthesis and degradation represent substantial costs during plant growth. To obtain a quantitative measure of the rate of protein synthesis and degradation, we supplied ¹³CO₂ to intact Arabidopsis (Arabidopsis thaliana) Columbia-0 plants and analyzed enrichment in free amino acids and in amino acid residues in protein during a 24-h pulse and 4-d chase. While many free amino acids labeled slowly and incompletely, alanine showed a rapid rise in enrichment in the pulse and a decrease in the chase. Enrichment in free alanine was used to correct enrichment in alanine residues in protein and calculate the rate of protein synthesis. The latter was compared with the relative growth rate to estimate the rate of protein degradation. The relative growth rate was estimated from sequential determination of fresh weight, sequential images of rosette area, and labeling of glucose in the cell wall. In an 8-h photoperiod, protein synthesis and cell wall synthesis were 3-fold faster in the day than at night, protein degradation was slow (3%–4% d⁻¹), and flux to growth and degradation resulted in a protein half-life of 3.5 d. In the starchless phosphoglucomutase mutant at night, protein synthesis was further decreased and protein degradation increased, while cell wall synthesis was totally inhibited, quantitatively accounting for the inhibition of growth in this mutant. We also investigated the rates of protein synthesis and degradation during leaf development, during growth at high temperature, and compared synthesis rates of Rubisco large and small subunits of in the light and dark.

Complex I (NADH:ubiquinone oxidoreductase) is central to cellular NAD⁺ recycling and accounts for approximately 40% of mitochondrial ATP production. To understand how complex I function impacts respiration and plant development, we isolated Arabidopsis (Arabidopsis thaliana) lines that lack complex I activity due to the absence of the catalytic subunit NDUFV1 (for NADH:ubiquinone oxidoreductase flavoprotein1) and compared these plants with ndufs4 (for NADH:ubiquinone oxidoreductase Fe-S protein4) mutants possessing trace amounts of complex I. Unlike ndufs4 plants, ndufv1 lines were largely unable to establish seedlings in the absence of externally supplied sucrose. Measurements of mitochondrial respiration and ATP synthesis revealed that compared with ndufv1, the complex I amounts retained by ndufs4 did not increase mitochondrial respiration and oxidative phosphorylation capacities. No major differences were seen in the mitochondrial proteomes, cellular metabolomes, or transcriptomes between ndufv1 and ndufs4. The analysis of fluxes through the respiratory pathway revealed that in ndufv1, fluxes through glycolysis and the tricarboxylic acid cycle were dramatically increased compared with ndufs4, which showed near wild-type-like fluxes. This indicates that the strong growth defects seen for plants lacking complex I originate from a switch in the metabolic mode of mitochondria and an up-regulation of respiratory fluxes. Partial reversion of these phenotypes when traces of active complex I are present suggests that complex I is essential for plant development and likely acts as a negative regulator of respiratory fluxes.

Based on enzyme activity assays and metabolic responses to waterlogging of the legume Lotus japonicus, it was previously suggested that, during hypoxia, the tricarboxylic acid cycle switches to a noncyclic operation mode. Hypotheses were postulated to explain the alternative metabolic pathways involved, but as yet, a direct analysis of the relative redistribution of label through the corresponding pathways was not made. Here, we describe the use of stable isotope-labeling experiments for studying metabolism under hypoxia using wild-type roots of the crop legume soybean (Glycine max). [¹³C]Pyruvate labeling was performed to compare metabolism through the tricarboxylic acid cycle, fermentation, alanine metabolism, and the γ-aminobutyric acid shunt, while [¹³C]glutamate and [¹⁵N]ammonium labeling were performed to address the metabolism via glutamate to succinate. Following these labelings, the time course for the redistribution of the ¹³C/¹⁵N label throughout the metabolic network was evaluated with gas chromatography-time of flight-mass spectrometry. Our combined labeling data suggest the inhibition of the tricarboxylic acid cycle enzyme succinate dehydrogenase, also known as complex II of the mitochondrial electron transport chain, providing support for the bifurcation of the cycle and the down-regulation of the rate of respiration measured during hypoxic stress. Moreover, up-regulation of the γ-aminobutyric acid shunt and alanine metabolism explained the accumulation of succinate and alanine during hypoxia.

Abscisic acid (ABA) is a plant hormone that regulates a diverse range of cellular and molecular processes during development and in response to osmotic stress. In Arabidopsis (Arabidopsis thaliana), three Suc nonfermenting-1-related protein kinase2s (SnRK2s), SRK2D, SRK2E, and SRK2I, are key positive regulators involved in ABA signaling whose substrates have been well studied. Besides reduced drought-stress tolerance, the srk2d srk2e srk2i mutant shows abnormal growth phenotypes, such as an increased number of leaves, under nonstress conditions. However, it remains unclear whether, and if so how, SnRK2-mediated ABA signaling regulates growth and development. Here, we show that the primary metabolite profile of srk2d srk2e srk2i grown under nonstress conditions was considerably different from that of wild-type plants. The metabolic changes observed in the srk2d srk2e srk2i were similar to those in an ABA-biosynthesis mutant, aba2-1, and both mutants showed a higher leaf emergence rate than wild type. Consistent with the increased amounts of citrate, isotope-labeling experiments revealed that respiration through the tricarboxylic acid cycle was enhanced in srk2d srk2e srk2i. These results, together with transcriptome data, indicate that the SnRK2s involved in ABA signaling modulate metabolism and leaf growth under nonstress conditions by fine-tuning flux through the tricarboxylic acid cycle.

The plant tricarboxylic acid (TCA) cycle provides essential precursors for respiration, amino acid biosynthesis, and general nitrogen metabolism; moreover, it is closely involved in biotic stress responses and cellular redox homeostasis. To further understand the in vivo function of the TCA cycle enzymes, we combined affinity purification with proteomics to generate a comprehensive extra-pathway protein-protein interaction network of the plant TCA cycle. We identified 125 extra-pathway interactions in Arabidopsis (Arabidopsis thaliana) mostly related to the mitochondrial electron transport complex/ATP synthesis and amino acid metabolism but also to proteins associated with redox stress. We chose three high-scoring and two low-scoring interactions for complementary bimolecular fluorescence complementation and yeast two-hybrid assays, which highlighted the reliability of our approach, supported the intimate involvement of TCA cycle enzymes within many biological processes, and reflected metabolic changes reported previously for the corresponding mutant lines. To analyze the function of a subset of these interactions, we selected two mutants of mitochondrial glutaredoxin S15 and Amidase, which have not yet been analyzed with respect to their TCA cycle function, and performed metabolite profiling and flux analysis. Consistent with their interactions identified in this study, TCA cycle metabolites and the relative TCA flux of the two mutants were altered significantly.