Cytosolic NADP-thiol systems in oxidative signalling in plants – CYNTHIOL

Genetic analysis (production of double mutants in the Arabidopsis oxidative stress mutant, cat2). Functional analysis: phenotype, metabolomics, proteomics, transcriptomics. Quantitative redox proteomic analysis to identify post-translational modifications in the different lines. Redox-specific yeast-2-hybrid in vivo interaction assays and biochemical tests.

The program has just started, no significant results yet produced.

It is anticipated that that project will provide new insight into the components and pathways involved in plant stress signalling within the integrated in vivo context of the cellular network. As well as their significance to efforts to understand plant stress responses, results could be relevant to redox signaling in other organisms.

None to date

Oxidative stress signalling is an important part of plant responses to numerous environmental conditions that affect plant performance, vigour, and/or yield. Despite intense interest, it remains poorly understood how the effects of increased cellular oxidation caused by compounds such as H2O2 are mediated. A key candidate in transmitting oxidative signals is modified protein thiol/disulfide status, which is regulated by the NADPH-dependent glutathione and thioredoxin (TRX) systems. Recent data obtained by partners involved in this proposal have highlighted functions of the two systems, and interactions between them in stress response and plant development pathways. They also provided a first indication that perturbations in thiol-disulfide systems are important in transmitting H2O2 signals. However, little is known about the functional interplay between the two key thiol systems during oxidative stress. Although recent years have seen the identification of a growing number of target proteins whose thiol status is potentially affected by H2O2, glutathione and TRX, little or nothing is known about oxidative signalling through these pathways in vivo. The principal aims of the present project are to (1) establish the importance of the two thiol systems in oxidative stress signalling, and the interplay between them; (2) analyse the contribution of the enzymes that provide NADPH for the two systems during oxidative stress; and (3) identify candidate proteins that are involved in oxidative signalling though post-translational redox modifications. To achieve these objectives, we will use a combined genetic, transcriptomic, proteomic and biochemical approach. The genetic analysis will be based on the conditional catalase-deficient Arabidopsis mutant, cat2, in which controllable oxidation of NADPH-thiol systems can be triggered by easy modification of external conditions that affect the rate of intracellular H2O2 production. Using mutant lines available in the partner laboratories for glutathione reduction, glutathione synthesis, TRX reduction, and NADPH generation, secondary mutations will be introduced into the cat2 background. By comparison of double mutants with cat2 at the phenotypic, metabolomic, proteomic and transcriptomic levels, we will identify how modified status of specific thiol or NADPH-producing systems regulates the impact of oxidative stress on metabolic and signalling pathways. Quantitative redox proteomic analysis of proteins that are post-translationally modified in the different lines will allow us to identify novel candidates for transmitting oxidative stress signals. The interaction of candidate proteins with TRX or glutathione-dependent glutaredoxins will be analysed using redox-specific yeast-2-hybrid in vivo interaction assays, biochemical test and further genetic approaches. Together, the data will dissect the relative importance of different components of NADPH-thiol systems in oxidative stress and identify proteins with which the systems interact, thus providing incisive new insight into the redox signalling pathways through which stress conditions are perceived by plants.