Toshihiro Obata

The use of metabolomics to dissect plant responses to abiotic stresses

Plant metabolism is perturbed by various abiotic stresses. As such the metabolic network of plants must be reconfigured under stress conditions in order to allow both the maintenance of metabolic homeostasis and the production of compounds that ameliorate the stress. The recent development and adoption of metabolomics and systems biology approaches enable us not only to gain a comprehensive overview, but also a detailed analysis of crucial components of the plant metabolic response to abiotic stresses. In this review we introduce the analytical methods used for plant metabolomics and describe their use in studies related to the metabolic response to water, temperature, light, nutrient limitation, ion and oxidative stresses. Both similarity and specificity of the metabolic responses against diverse abiotic stress are evaluated using data available in the literature. Classically discussed stress compounds such as proline, γ-amino butyrate and polyamines are reviewed, and the widespread importance of branched chain amino acid metabolism under stress condition is discussed. Finally, where possible, mechanistic insights into metabolic regulatory processes are discussed.

Identification of the 2-Hydroxyglutarate and Isovaleryl-CoA Dehydrogenases as Alternative Electron Donors Linking Lysine Catabolism to the Electron Transport Chain of Arabidopsis Mitochondria

This study indicates that Lys catabolism can directly channel electrons to the mitochondrial electron transport chain in addition to sustaining the operation of the tricarboxylic acid cycle under carbon-limiting conditions. The process of dark-induced senescence in plants is relatively poorly understood, but a functional electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) complex, which supports respiration during carbon starvation, has recently been identified. Here, we studied the responses of Arabidopsis thaliana mutants deficient in the expression of isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase to extended darkness and other environmental stresses. Evaluations of the mutant phenotypes following carbon starvation induced by extended darkness identify similarities to those exhibited by mutants of the ETF/ETFQO complex. Metabolic profiling and isotope tracer experimentation revealed that isovaleryl-CoA dehydrogenase is involved in degradation of the branched-chain amino acids, phytol, and Lys, while 2-hydroxyglutarate dehydrogenase is involved exclusively in Lys degradation. These results suggest that isovaleryl-CoA dehydrogenase is the more critical for alternative respiration and that a series of enzymes, including 2-hydroxyglutarate dehydrogenase, plays a role in Lys degradation. Both physiological and metabolic phenotypes of the isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase mutants were not as severe as those observed for mutants of the ETF/ETFQO complex, indicating some functional redundancy of the enzymes within the process. Our results aid in the elucidation of the pathway of plant Lys catabolism and demonstrate that both isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase act as electron donors to the ubiquinol pool via an ETF/ETFQO-mediated route.

Developmental Stage Specificity and the Role of Mitochondrial Metabolism in the Response of Arabidopsis Leaves to Prolonged Mild Osmotic Stress

When subjected to stress, plants reprogram their growth by largely unknown mechanisms. To provide insights into this process, the growth of Arabidopsis (Arabidopsis thaliana) leaves that develop under mild osmotic stress was studied. Early during leaf development, cell number and size were reduced by stress, but growth was remarkably adaptable, as division and expansion rates were identical to controls within a few days of leaf initiation. To investigate the molecular basis of the observed adaptability, leaves with only proliferating, exclusively expanding, and mature cells were analyzed by transcriptomics and targeted metabolomics. The stress response measured in growing and mature leaves was largely distinct; several hundred transcripts and multiple metabolites responded exclusively in the proliferating and/or expanding leaves. Only a few genes were differentially expressed across the three stages. Data analysis showed that proliferation and expansion were regulated by common regulatory circuits, involving ethylene and gibberellins but not abscisic acid. The role of ethylene was supported by the analysis of ethylene-insensitive mutants. Exclusively in proliferating cells, stress induced genes of the so-called “mitochondrial dysfunction regulon,” comprising alternative oxidase. Up-regulation for eight of these genes was confirmed with promoter:β-glucuronidase reporter lines. Furthermore, mitochondria of stress-treated dividing cells were morphologically distinct from control ones, and growth of plants overexpressing the alternative oxidase gene was more tolerant to osmotic and drought stresses. Taken together, our data underline the value of analyzing stress responses in development and demonstrate the importance of mitochondrial respiration for sustaining cell proliferation under osmotic stress conditions.

The Metabolic Response of Arabidopsis Roots to Oxidative Stress is Distinct from that of Heterotrophic Cells in Culture and Highlights a Complex Relationship between the Levels of Transcripts, Metabolites, and Flux

Metabolic adjustments are a significant, but poorly understood, part of the response of plants to oxidative stress. In a previous study (Baxter et al., 2007), the metabolic response of Arabidopsis cells in culture to induction of oxidative stress by menadione was characterized. An emergency survival strategy was uncovered in which anabolic primary metabolism was largely down-regulated in favour of catabolic and antioxidant metabolism. The response in whole plant tissues may be different and we have therefore investigated the response of Arabidopsis roots to menadione treatment, analyzing the transcriptome, metabolome and key metabolic fluxes with focus on primary as well as secondary metabolism. Using a redox-sensitive GFP, it was also shown that menadione causes redox perturbation, not just in the mitochondrion, but also in the cytosol and plastids of roots. In the first 30 min of treatment, the response was similar to the cell culture: there was a decrease in metabolites of the TCA cycle and amino acid biosynthesis and the transcriptomic response was dominated by up-regulation of DNA regulatory proteins. After 2 and 6 h of treatment, the response of the roots was different to the cell culture. Metabolite levels did not remain depressed, but instead recovered and, in the case of pyruvate, some amino acids and aliphatic glucosinolates showed a steady increase above control levels. However, no major changes in fluxes of central carbon metabolism were observed and metabolic transcripts changed largely independently of the corresponding metabolites. Together, the results suggest that root tissues can recover metabolic activity after oxidative inhibition and highlight potentially important roles for glycolysis and the oxidative pentose phosphate pathway.

SALT-RESPONSIVE ERF1 Regulates Reactive Oxygen Species–Dependent Signaling during the Initial Response to Salt Stress in Rice

Salinity is a common environmental constraint that is rapidly recognized by plants. This work demonstrates that early sensing of salt stress in rice involves a ROS-mediated response, in which the transcription factor SERF1 and the mitogen-activated protein kinase MAPK5 play a central role. Early detection of salt stress is vital for plant survival and growth. Still, the molecular processes controlling early salt stress perception and signaling are not fully understood. Here, we identified SALT-RESPONSIVE ERF1 (SERF1), a rice (Oryza sativa) transcription factor (TF) gene that shows a root-specific induction upon salt and hydrogen peroxide (H2O2) treatment. Loss of SERF1 impairs the salt-inducible expression of genes encoding members of a mitogen-activated protein kinase (MAPK) cascade and salt tolerance–mediating TFs. Furthermore, we show that SERF1-dependent genes are H2O2 responsive and demonstrate that SERF1 binds to the promoters of MAPK KINASE KINASE6 (MAP3K6), MAPK5, DEHYDRATION-RESPONSIVE ELEMENT BINDING2A (DREB2A), and ZINC FINGER PROTEIN179 (ZFP179) in vitro and in vivo. SERF1 also directly induces its own gene expression. In addition, SERF1 is a phosphorylation target of MAPK5, resulting in enhanced transcriptional activity of SERF1 toward its direct target genes. In agreement, plants deficient for SERF1 are more sensitive to salt stress compared with the wild type, while constitutive overexpression of SERF1 improves salinity tolerance. We propose that SERF1 amplifies the reactive oxygen species–activated MAPK cascade signal during the initial phase of salt stress and translates the salt-induced signal into an appropriate expressional response resulting in salt tolerance.

PLGG1, a plastidic glycolate glycerate transporter, is required for photorespiration and defines a unique class of metabolite transporters

Photorespiratory carbon flux reaches up to a third of photosynthetic flux, thus contributes massively to the global carbon cycle. The pathway recycles glycolate-2-phosphate, the most abundant byproduct of RubisCO reactions. This oxygenation reaction of RubisCO and subsequent photorespiration significantly limit the biomass gains of many crop plants. Although photorespiration is a compartmentalized process with enzymatic reactions in the chloroplast, the peroxisomes, the mitochondria, and the cytosol, no transporter required for the core photorespiratory cycle has been identified at the molecular level to date. Using transcript coexpression analyses, we identified Plastidal glycolate glycerate translocator 1 (PLGG1) as a candidate core photorespiratory transporter. Related genes are encoded in the genomes of archaea, bacteria, fungi, and all Archaeplastida and have previously been associated with a function in programmed cell-death. A mutant deficient in PLGG1 shows WT-like growth only in an elevated carbon dioxide atmosphere. The mutant accumulates glycolate and glycerate, leading to the hypothesis that PLGG1 is a glycolate/glycerate transporter. This hypothesis was tested and supported by in vivo and in vitro transport assays and 18O2-metabolic flux profiling. Our results indicate that PLGG1 is the chloroplastidic glycolate/glycerate transporter, which is required for the function of the photorespiratory cycle. Identification of the PLGG1 transport function will facilitate unraveling the role of similar proteins in bacteria, archaea, and fungi in the future.

Molecular mechanisms of desiccation tolerance in the resurrection glacial relic Haberlea rhodopensis

Haberlea rhodopensis is a resurrection plant with remarkable tolerance to desiccation. Haberlea exposed to drought stress, desiccation, and subsequent rehydration showed no signs of damage or severe oxidative stress compared to untreated control plants. Transcriptome analysis by next-generation sequencing revealed a drought-induced reprogramming, which redirected resources from growth towards cell protection. Repression of photosynthetic and growth-related genes during water deficiency was concomitant with induction of transcription factors (members of the NAC, NF-YA, MADS box, HSF, GRAS, and WRKY families) presumably acting as master switches of the genetic reprogramming, as well as with an upregulation of genes related to sugar metabolism, signaling, and genes encoding early light-inducible (ELIP), late embryogenesis abundant (LEA), and heat shock (HSP) proteins. At the same time, genes encoding other LEA, HSP, and stress protective proteins were constitutively expressed at high levels even in unstressed controls. Genes normally involved in tolerance to salinity, chilling, and pathogens were also highly induced, suggesting a possible cross-tolerance against a number of abiotic and biotic stress factors. A notable percentage of the genes highly regulated in dehydration and subsequent rehydration were novel, with no sequence homology to genes from other plant genomes. Additionally, an extensive antioxidant gene network was identified with several gene families possessing a greater number of antioxidant genes than most other species with sequenced genomes. Two of the transcripts most abundant during all conditions encoded catalases and five more catalases were induced in water-deficient samples. Using the pharmacological inhibitor 3-aminotriazole (AT) to compromise catalase activity resulted in increased sensitivity to desiccation. Metabolome analysis by GC or LC–MS revealed accumulation of sucrose, verbascose, spermidine, and γ-aminobutyric acid during drought, as well as particular secondary metabolites accumulating during rehydration. This observation, together with the complex antioxidant system and the constitutive expression of stress protective genes suggests that both constitutive and inducible mechanisms contribute to the extreme desiccation tolerance of H. rhodopensis.

Diurnal changes of polysome loading track sucrose content in the rosette of wildtype Arabidopsis and the starchless pgm mutant

Ribosome loading is closely coordinated with the sucrose supply during diurnal cycles in Arabidopsis. Growth is driven by newly fixed carbon in the light, but at night it depends on reserves, like starch, that are laid down in the light. Unless plants coordinate their growth with diurnal changes in the carbon supply, they will experience acute carbon starvation during the night. Protein synthesis represents a major component of cellular growth. Polysome loading was investigated during the diurnal cycle, an extended night, and low CO2 in Arabidopsis (Arabidopsis thaliana) Columbia (Col-0) and in the starchless phosphoglucomutase (pgm) mutant. In Col-0, polysome loading was 60% to 70% in the light, 40% to 45% for much of the night, and less than 20% in an extended night, while in pgm, it fell to less than 25% early in the night. Quantification of ribosomal RNA species using quantitative reverse transcription-polymerase chain reaction revealed that polysome loading remained high for much of the night in the cytosol, was strongly light dependent in the plastid, and was always high in mitochondria. The rosette sucrose content correlated with overall and with cytosolic polysome loading. Ribosome abundance did not show significant diurnal changes. However, compared with Col-0, pgm had decreased and increased abundance of plastidic and mitochondrial ribosomes, respectively. Incorporation of label from 13CO2 into protein confirmed that protein synthesis continues at a diminished rate in the dark. Modeling revealed that a decrease in polysome loading at night is required to balance protein synthesis with the availability of carbon from starch breakdown. Costs are also reduced by using amino acids that accumulated in the previous light period. These results uncover a tight coordination of protein synthesis with the momentary supply of carbon.

Rice shaker potassium channel OsKAT1 confers tolerance to salinity stress on yeast and rice cells.

We screened a rice (Oryza sativa L. ‘Nipponbare’) full-length cDNA expression library through functional complementation in yeast (Saccharomyces cerevisiae) to find novel cation transporters involved in salt tolerance. We found that expression of a cDNA clone, encoding the rice homolog of Shaker family K+ channel KAT1 (OsKAT1), suppressed the salt-sensitive phenotype of yeast strain G19 (Δena1–4), which lacks a major component of Na+ efflux. It also suppressed a K+-transport-defective phenotype of yeast strain CY162 (Δtrk1Δtrk2), suggesting the enhancement of K+ uptake by OsKAT1. By the expression of OsKAT1, the K+ contents of salt-stressed G19 cells increased during the exponential growth phase. At the linear phase, however, OsKAT1-expressing G19 cells accumulated less Na+ than nonexpressing cells, but almost the same K+. The cellular Na+ to K+ ratio of OsKAT1-expressing G19 cells remained lower than nonexpressing cells under saline conditions. Rice cells overexpressing OsKAT1 also showed enhanced salt tolerance and increased cellular K+ content. These functions of OsKAT1 are likely to be common among Shaker K+ channels because OsAKT1 and Arabidopsis (Arabidopsis thaliana) KAT1 were able to complement the salt-sensitive phenotype of G19 as well as OsKAT1. The expression of OsKAT1 was restricted to internodes and rachides of wild-type rice, whereas other Shaker family genes were expressed in various organs. These results suggest that OsKAT1 is involved in salt tolerance of rice in cooperation with other K+ channels by participating in maintenance of cytosolic cation homeostasis during salt stress and thus protects cells from Na+.

Thioredoxin, a master regulator of the tricarboxylic acid cycle in plant mitochondria

Significance The present work extends redox-based change in enzyme activity to the TCA cycle of plant mitochondria. Thioredoxin (TRX) was found to regulate the activity of enzymes of the mitochondrial cycle (succinate dehydrogenase and fumarase) and of an enzyme associated with it (ATP-citrate lyase) by modulating thiol redox status. A combination of experiments based on mutant and carbon isotope labeling analyses provides evidence that flux through this pathway is coordinately modulated by TRX at the enzyme level of both mitochondria and cytosol. The results provide in vivo confirmation of earlier in vitro results and further show that mitochondria resemble plastids in using TRX and redox status to regulate the main carbon flux pathway of the organelle. Plant mitochondria have a fully operational tricarboxylic acid (TCA) cycle that plays a central role in generating ATP and providing carbon skeletons for a range of biosynthetic processes in both heterotrophic and photosynthetic tissues. The cycle enzyme-encoding genes have been well characterized in terms of transcriptional and effector-mediated regulation and have also been subjected to reverse genetic analysis. However, despite this wealth of attention, a central question remains unanswered: “What regulates flux through this pathway in vivo?” Previous proteomic experiments with Arabidopsis discussed below have revealed that a number of mitochondrial enzymes, including members of the TCA cycle and affiliated pathways, harbor thioredoxin (TRX)-binding sites and are potentially redox-regulated. We have followed up on this possibility and found TRX to be a redox-sensitive mediator of TCA cycle flux. In this investigation, we first characterized, at the enzyme and metabolite levels, mutants of the mitochondrial TRX pathway in Arabidopsis: the NADP-TRX reductase a and b double mutant (ntra ntrb) and the mitochondrially located thioredoxin o1 (trxo1) mutant. These studies were followed by a comparative evaluation of the redistribution of isotopes when 13C-glucose, 13C-malate, or 13C-pyruvate was provided as a substrate to leaves of mutant or WT plants. In a complementary approach, we evaluated the in vitro activities of a range of TCA cycle and associated enzymes under varying redox states. The combined dataset suggests that TRX may deactivate both mitochondrial succinate dehydrogenase and fumarase and activate the cytosolic ATP-citrate lyase in vivo, acting as a direct regulator of carbon flow through the TCA cycle and providing a mechanism for the coordination of cellular function.