Metabolite modification in oxidative stress responses: A case study of two defense hormones.

Studies of the Arabidopsis cat2 mutant lacking the major leaf isoform of catalase have allowed the potential impact of intracellular H2O2 on plant function to be studied. Here, we report a robust analysis of modified gene expression associated with key families involved in metabolite modification in cat2. Through a combined transcriptomic and metabolomic analysis focused on the salicylic acid (SA) and jasmonic acid (JA) pathways, we report key features of the metabolic signatures linked to oxidative stress-induced signaling via these defence hormones and discuss the enzymes that are likely to be involved in determining these features. We provide evidence that specific UDP-glycosyl transferases contribute to the glucosylation of SA that accumulates as a result of oxidative stress in cat2. Glycosides of dihydroxybenzoic acids that accumulate alongside SA in cat2 are identified and, based on the expression of candidate genes, likely routes for their production are discussed. We also report that enhanced intracellular H2O2 triggers induction of genes encoding different enzymes that can metabolize JA. Integrated analysis of metabolite and transcript profiles suggests that a gene network involving specific hydrolases, hydroxylases, and sulfotransferases functions to limit accumulation of the most active jasmonates during oxidative stress.

Measuring Stress-Induced Changes in Defense Phytohormones and Related Compounds.

Measuring quantitative changes in plant hormones and derivatives is crucial to understand how reactive oxygen species trigger signaling cascades to regulate stress responses. In this chapter, we describe the liquid chromatography-mass spectrometry procedure that we use to extract and quantify salicylic acid (SA), jasmonic acid (JA), and related compounds in common extracts of Arabidopsis tissue. The method can provide quantitative data on SA, SA glucosides, and JA, as well as information on oxidized and conjugated forms of these compounds and related derivatives of benzoic acid.

Carbon-use strategies in stem radial growth of two oak species, one Temperate deciduous and one Mediterranean evergreen: what can be inferred from seasonal variations in the δ13C of the current year ring?

Tree ring synthesis is a key process in wood production; however, little is known of the origin and fate of the carbon involved. We used natural 13C abundance to investigate the carbon-use process for the ring development in a temperate deciduous (Quercus petraea (Matt.) Liebl.) and a Mediterranean evergreen (Quercus ilex L.) oak. The sapwood carbon reserves, phloem sucrose contents, stem respired CO2 efflux and their respective carbon isotope compositions (δ13C) were recorded over 1 year, in the native area of each species. The seasonal δ13C variation of the current year ring was determined in the total ring throughout the seasons, as well as in slices from the fully mature ring after the growth season (intra-ring pattern). Although the budburst dates of the two oaks were similar, the growth of Quercus ilex began 50 days later. Both species exhibited growth cessation during the hot and dry summer but only Q. ilex resumed in the autumn. In the deciduous oak, xylem starch storage showed clear variations during the radial growth. The intra-ring δ13C variations of the two species exhibited similar ranges, but contrasting patterns, with an early increase for Q. petraea. Comparison between δ13C of starch and total ring suggested that Q. petraea (but not Q. ilex) builds its rings using reserves during the first month of growth. Shifts in ring and soluble sugars δ13C suggested an interspecific difference in either the phloem unloading or the use of fresh assimilate inside the ring. A decrease in ring δ13C for both oaks between the end of the radial growth and the winter is attributed to a lignification of ring cell walls after stem increment. This study highlighted the differences in carbon-use during ring growth for evergreen and deciduous oaks, as well as the benefits of exploring the process using natural 13C abundance.

The metabolomics of oxidative stress.

Oxidative stress resulting from increased availability of reactive oxygen species (ROS) is a key component of many responses of plants to challenging environmental conditions. The consequences for plant metabolism are complex and manifold. We review data on small compounds involved in oxidative stress, including ROS themselves and antioxidants and redox buffers in the membrane and soluble phases, and we discuss the wider consequences for plant primary and secondary metabolism. While metabolomics has been exploited in many studies on stress, there have been relatively few non-targeted studies focused on how metabolite signatures respond specifically to oxidative stress. As part of the discussion, we present results and reanalyze published datasets on metabolite profiles in catalase-deficient plants, which can be considered to be model oxidative stress systems. We emphasize the roles of ROS-triggered changes in metabolites as potential oxidative signals, and discuss responses that might be useful as markers for oxidative stress. Particular attention is paid to lipid-derived compounds, the status of antioxidants and antioxidant breakdown products, altered metabolism of amino acids, and the roles of phytohormone pathways.

Comparison of seasonal variations in water-use efficiency calculated from the carbon isotope composition of tree rings and flux data in a temperate forest.

Tree-ring δ(13) C is often interpreted in terms of intrinsic water-use efficiency (WUE) using a carbon isotope discrimination model established at the leaf level. We examined whether intra-ring δ(13) C could be used to assess variations in intrinsic WUE (W(g), the ratio of carbon assimilation and stomatal conductance to water) and variations in ecosystem WUE (W(t) , the ratio of C assimilation and transpiration) at a seasonal scale. Intra-ring δ(13) C was measured in 30- to 60-µm-thick slices in eight oak trees (Quercus petraea). Canopy W(g) was simulated using a physiologically process-based model. High between-tree variability was observed in the seasonal variations of intra-ring δ(13) C. Six trees showed significant positive correlations between W(g) calculated from intra-ring δ(13) C and canopy W(g) averaged over several days during latewood formation. These results suggest that latewood is a seasonal recorder of W(g) trends, with a temporal lag corresponding to the mixing time of sugars in the phloem. These six trees also showed significant negative correlations between photosynthetic discrimination Δ calculated from intra-ring δ(13) C, and ecosystem W(t), during latewood formation. Despite the observed between-tree variability, these results indicate that intra-ring δ(13) C can be used to access seasonal variations in past W(t).

Acclimation to high CO2 in maize is related to water status and dependent on leaf rank.

The responses of C(3) plants to rising atmospheric CO(2) levels are considered to be largely dependent on effects exerted through altered photosynthesis. In contrast, the nature of the responses of C(4) plants to high CO(2) remains controversial because of the absence of CO(2) -dependent effects on photosynthesis. In this study, the effects of atmospheric CO(2) availability on the transcriptome, proteome and metabolome profiles of two ranks of source leaves in maize (Zea mays L.) were studied in plants grown under ambient CO(2) conditions (350 +/- 20 µL L(-1) CO(2) ) or with CO(2) enrichment (700 +/- 20 µL L(-1) CO(2) ). Growth at high CO(2) had no effect on photosynthesis, photorespiration, leaf C/N ratios or anthocyanin contents. However, leaf transpiration rates, carbohydrate metabolism and protein carbonyl accumulation were altered at high CO(2) in a leaf-rank specific manner. Although no significant CO(2) -dependent changes in the leaf transcriptome were observed, qPCR analysis revealed that the abundance of transcripts encoding a Bowman-Birk protease inhibitor and a serpin were changed by the growth CO(2) level in a leaf rank specific manner. Moreover, CO(2) -dependent changes in the leaf proteome were most evident in the oldest source leaves. Small changes in water status may be responsible for the observed responses to high CO(2,) particularly in the older leaf ranks.

Impact of carbohydrate supply on stem growth, wood and respired CO2 delta13C: assessment by experimental girdling.

The present study examines the impact of the C source (reserves vs current assimilates) on tree C isotope signals and stem growth, using experimental girdling to stop the supply of C from leaves to stem. Two-year-old sessile oaks (Quercus petraea) were girdled at three different phenological periods during the leafy period: during early wood growth (Girdling Period 1), during late wood growth (Girdling Period 2) and just after growth cessation (Girdling Period 3). The measured variables included stem respiration rates, stem radial increment, delta(13)C of respired CO(2) and contents of starch and water-soluble fraction in stems (below the girdle) and leaves. Girdling stopped growth, even early in the growing season, leading to a decrease in stem CO(2) efflux (CO(2R)). Shift in substrate use from recently fixed carbohydrate to reserves (i.e., starch) induced (13)C enrichment of CO(2) respired by stem. However, change in substrate type was insufficient to explain alone all the observed CO(2R) delta(13)C variations, especially at the period corresponding to large growth rate of control trees. The below-girdle mass balance suggested that, during girdling periods, stem C was invested in metabolic pathways other than respiration and stem growth. After Girdling Period 1, the girdle healed and the effects of girdling on stem respiration were reversed. Stem growth restarted and total radial increment was similar to the control one, indicating that growth can be delayed when a stress event occurs early in the growth period. Concerning tree ring, seasonal shift in substrate use from reserves (i.e., starch) to recently fixed carbohydrate is sufficient to explain the observed (13)C depletion of tree ring during the early wood growth. However, the inter-tree intra-ring delta(13)C variability needs to be resolved in order to improve the interpretation of intra-seasonal ring signals in terms of climatic or ecophysiological information. This study highlighted, via carbohydrate availability effects, the importance of the characterization of stem metabolic pathways for a complete understanding of the delta(13)C signals.

Conditional modulation of NAD levels and metabolite profiles in Nicotiana sylvestris by mitochondrial electron transport and carbon/nitrogen supply.

Environmental controls on leaf NAD status remain poorly understood. Here, we analyzed the effects of two key environmental variables, CO(2) and nitrogen, on leaf metabolite profiles, NAD status and the abundance of key transcripts involved in de novo NAD synthesis in wild-type (WT) Nicotiana sylvestris and the CMSII mutant that lacks respiratory complex I. High CO(2) and increased N supply both significantly enhanced NAD(+) and NADH pools in WT leaves. In nitrogen-sufficient conditions, CMSII leaves were enriched in NAD(+) and NADH compared to the WT, but the differences in NADH were smaller at high CO(2) than in air because high CO(2) increased WT NADH/NAD(+). The CMSII-linked increases in NAD(+) and NADH status were abolished by growth with limited nitrogen, which also depleted the nicotine and nicotinic acid pools in the CMSII leaves. Few statistically significant genotype and N-dependent differences were detected in NAD synthesis transcripts, with effects only on aspartate oxidase and NAD synthetase mRNAs. Non-targeted metabolite profiling as well as quantitative amine analysis showed that NAD(+) and NADH contents correlated tightly with leaf amino acid contents across all samples. The results reveal considerable genotype- and condition-dependent plasticity in leaf NAD(+) and NADH contents that is not linked to modified expression of NAD synthesis genes at the transcript level and show that NAD(+) and NADH contents are tightly integrated with nitrogen metabolism. A regulatory two-way feedback circuit between nitrogen and NAD in the regulation of N assimilation is proposed that potentially links the nutritional status to NAD-dependent signaling pathways.

Cotranslational proteolysis dominates glutathione homeostasis to support proper growth and development.

The earliest proteolytic event affecting most proteins is the excision of the initiating Met (NME). This is an essential and ubiquitous cotranslational process tightly regulated in all eukaryotes. Currently, the effects of NME on unknown complex cellular networks and the ways in which its inhibition leads to developmental defects and cell growth arrest remain poorly understood. Here, we provide insight into the earliest molecular mechanisms associated with the inhibition of the NME process in Arabidopsis thaliana. We demonstrate that the developmental defects induced by NME inhibition are caused by an increase in cellular proteolytic activity, primarily induced by an increase in the number of proteins targeted for rapid degradation. This deregulation drives, through the increase of the free amino acids pool, a perturbation of the glutathione homeostasis, which corresponds to the earliest limiting, reversible step promoting the phenotype. We demonstrate that these effects are universally conserved and that the reestablishment of the appropriate glutathione status restores growth and proper development in various organisms. Finally, we describe a novel integrated model in which NME, protein N-alpha-acylation, proteolysis, and glutathione homeostasis operate in a sequentially regulated mechanism that directs both growth and development.

Crosstalks between myo-inositol metabolism, programmed cell death and basal immunity in Arabidopsis.

BACKGROUND: Although it is a crucial cellular process required for both normal development and to face stress conditions, the control of programmed cell death in plants is not fully understood. We previously reported the isolation of ATXR5 and ATXR6, two PCNA-binding proteins that could be involved in the regulation of cell cycle or cell death. A yeast two-hybrid screen using ATXR5 as bait captured AtIPS1, an enzyme which catalyses the committed step of myo-inositol (MI) biosynthesis. atips1 mutants form spontaneous lesions on leaves, raising the possibility that MI metabolism may play a role in the control of PCD in plants. In this work, we have characterised atips1 mutants to gain insight regarding the role of MI in PCD regulation. METHODOLOGY/PRINCIPAL FINDINGS: - lesion formation in atips1 mutants depends of light intensity, is due to PCD as evidenced by TUNEL labelling of nuclei, and is regulated by phytohormones such as salicylic acid - MI and galactinol are the only metabolites whose accumulation is significantly reduced in the mutant, and supplementation of the mutant with these compounds is sufficient to prevent PCD - the transcriptome profile of the mutant is extremely similar to that of lesion mimic mutants such as cpr5, or wild-type plants infected with pathogens. CONCLUSION/SIGNIFICANCE: Taken together, our results provide strong evidence for the role of MI or MI derivatives in the regulation of PCD. Interestingly, there are three isoforms of IPS in Arabidopsis, but AtIPS1 is the only one harbouring a nuclear localisation sequence, suggesting that nuclear pools of MI may play a specific role in PCD regulation and opening new research prospects regarding the role of MI in the prevention of tumorigenesis. Nevertheless, the significance of the interaction between AtIPS1 and ATXR5 remains to be established.

Kinetic 12C/13C isotope fractionation by invertase: evidence for a small in vitro isotope effect and comparison of two techniques for the isotopic analysis of carbohydrates.

The natural (13)C/(12)C isotope composition (delta(13)C) of plants and organic compounds within plant organs is a powerful tool to understand carbon allocation patterns and the regulation of photosynthetic or respiratory metabolism. However, many enzymatic fractionations are currently unknown, thus impeding our understanding of carbon trafficking pathways within plant cells. One of them is the (12)C/(13)C isotope effect associated with invertases (EC 3.2.1.26) that are cornerstone enzymes for Suc metabolism and translocation in plants. Another conundrum of isotopic plant biology is the need to measure accurately the specific delta(13)C of individual carbohydrates. Here, we examined two complementary methods for measuring the delta(13)C value of sucrose, glucose and fructose, that is, off-line high-performance liquid chromatography (HPLC) purification followed by elemental analysis and isotope ratio mass spectrometry (EA-IRMS) analysis, and gas chromatography-combustion (GC-C)-IRMS. We also used these methods to determine the in vitro (12)C/(13)C isotope effect associated with the yeast invertase. Our results show that, although providing more variable values than HPLC approximately EA-IRMS, and being sensitive to derivatization conditions, the GC-C-IRMS method gives reliable results. When applied to the invertase reaction, both methods indicate that the (12)C/(13)C isotope effect is rather small and it is not affected by the use of heavy water (D(2)O).

Leaf and twig delta13C during growth in relation to biochemical composition and respired CO2.

In deciduous trees, the delta(13)C values of leaves are known to diverge during growth from those of woody organs. The main purpose of this study is to determine whether the divergence in delta(13)C between leaves and current-year twigs of Fagus sylvatica (L.) is influenced by changes (i) in the relative contents of organic matter fractions and (ii) in the delta(13)C of respired CO(2). The delta(13)C values of bulk matter, extractive-free matter, lignin, holocellulose, starch, soluble sugars, water-soluble fraction and respired CO(2), as well as their relative contents in bulk matter were determined. The delta(13)C values of biochemical fractions and respired CO(2) showed very similar temporal variations for both leaves and twigs. Variations in bulk matter delta(13)C during growth were, therefore, poorly explained by changes in biochemical composition or in respiratory fractionation and were attributed to the transition from (13)C-enriched reserves (mainly starch) to (13)C-depleted new photoassimilates. The divergence between leaves and twigs was related to higher values of soluble sugar delta(13)C in twigs. However, the difference between lignin and holocellulose delta(13)C varied during growth. This phenomenon was attributed to the delay between holocellulose and lignin deposition. These results may have implications for analysis of organic matter delta(13)C in trees and forest ecosystems.