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CELLULAR REDOX HOMEOSTASIS, GLUTATHIONE REDUCTION AND PATHOGEN DEFENCE IN ARABIDOPSIS THALIANA1 Laurent Marty, Markus Wirtz, Andreas J. Meyer and Rüdiger Hell Heidelberg Institute of Plant Sciences, University of Heidelberg, Im Neuenheimer Feld 360, D-69120 Heidelberg, Germany Abstract Cellular redox signals are assumed to contribute to fundamental processes such as cell differentiation, cell death and stress responses. In contrast to established signal transduction pathways including Ca2+ signalling and phosphorylation cascades the mechanisms of redox signalling are largely unclear. Glutathione is a major constituent of redox homeostasis in several compartments of the plant cell, including the organelles, cytosol and ER. In addition glutathione has been discussed with respect to redox control in photosynthesis, pathogen defence and many other processes (May et al. 1998). In most cases reactive oxygen species (ROS) are likely to trigger imbalances in the ratio of reduced glutathione (GSH) to glutathione disulfide (GSSG). ROS will give rise to oxidative stress, unless the arsenal of antioxidants such as ascorbic acid and tocopherol in conjunction with ROS detoxifying enzymes successfully degrade ROS. Different specificities towards ROS and subcellular locations need to be considered in this process: catalase is exclusively found in peroxisomes, while superoxide dismutases and the ascorbate-glutathione cycle are present in cytosol, plastids and mitochondria. Ascorbate peroxidase, on the other hand, is not only present in the latter compartments but also attached to the thylakoid membrane and the microsomal fraction (Noctor 2006). Glutathione is involved in various reaction types in tolerance against cold treatment, heat shock, heavy metals, drought, xenobiotics and pathogens (Rennenberg and Brunold 1994; May et al. 1998). It may directly react via its sulfhydryl group and is thus being consumed. This refers to chemical or glutathione peroxidase-mediated electron transfer to radicals or formation of conjugates with electrophile reactants catalyzed by glutathione-S-transferases that are subsequently transported to vacuoles for degradation Sulfur Metabolism in Higher Plants, pp. xx-xx Edited by A. Sirko et al. 2009 Backhuys Publishers, Leiden, The Netherlands initiated by γ-glutamyltranspeptidases (Grzam et al. 2007). In contrast, in the ascorbateglutathione cycle GSSG is recycled by reduction back to GSH under consumption of NADPH by glutathione reductase (GR; Noctor 2006). Among the above listed roles of glutathione in stress responses the tolerance against pathogens is one of the least investigated ones. Vanacker et al. (2000) reported large inoculation-dependent changes in both the total amount of glutathione and in the redox state of the glutathione pool in resistant barley cultivars after inoculation with powdery mildew, but not in the susceptible isolate. A putative role for GSH in the defence against pathogens is to change the redox status of the NPR1 (non-expressor of PR genes) protein (Mou et al. 2003). Reduction of NPR1 oligomers leads to translocation from cytosol to the nucleus to mediate transcription of salicylic acid-induced genes that encode pathogenesis-related (PR) proteins (Eulgem 2005). A causal link between defence against pathogens and impaired GSH synthesis is also supported by the observation that the Arabidopsis thaliana pad2 mutant, which has lowered GSH levels and decreased concentrations of camalexin, shows enhanced susceptibility towards the oomycete Phytophthora brassicae (Parisy et al. 2007). The contents of glutathione were also observed to increase in wild type Arabidopsis plants upon infection with bacterial and oomycete pathogens, pointing to the activation of primary sulfur metabolism upon infection (Kruse et al. 2007). Fig. 1. Gene expression pattern of glutathione reductase genes GR1 and GR2 from Arabidopsis thaliana in response to various biotic and abiotic stress factors. Modified from Genevestigator database version 2 (Zimmermann et al. 2004). The existing data suggest that cellular glutathione contents or the GSH/GSSG ratio or both potentially contribute to pathogen defence. Since GRs are responsible to keep the GSH/GSSG ratio in balance, the roles of GRs in Arabidopsis were investigated with respect to pathogen resistance. Arabidopsis contains two GR genes encoding GR2 (At3g54660) that is dual targeted to plastids and mitochondria and GR1 (At3g24170) that constitutes the cytosolic isoform. Gene names were adopted from TAIR8 release (www.arabidopsis.org). An overview of expression responses to biotic and abiotic stress factors using the microarray database Genevestigator (Zimmermann et al. 2004) showed that mostly cytosolic GR1 but not organellar GR2 was up-regulated (Fig. 1). The GR1 gene responded with 2 to 3-fold increase in mRNA levels when leaves were infected with the fungus Botrytis cinerea, the oomycete Phytophthora infestans or the bacterium Pseudomonas syringae. In order to further elucidate the specific role of GR1 in plant defence, a T-DNA insertion mutant of GR1 was isolated from the SALK collection (line 105794) and challenged with the pathogen P. brassicae HH. Homozygous knock-outs were identified by PCR (Fig. 2A), and it was confirmed that these mutants had no GR1 expression (data not shown). Compared to wild type the GSSG contents were increased five times in the gr1-1 leaf tissue, shifting glutathione to a more oxidized state (Fig. 2B). Fig. 2. Isolation and characterization of an Arabidopsis gr1-1 T-DNA insertion mutant. (A) PCR genotyping of gr1-1 mutant. Upper panel: PCR performed with gene-specific primers. Lower panel: PCR performed with a T-DNA- and one gene-specific primer. Templates used for PCRs were the following: lane 1: Col-0, lanes 2 and 3: homozygous insertion mutant, lane 4: heterozygous insertion mutant. (B) Contents of reduced glutathione (GSH) and glutathione disulfide (GSSG) in leaves of eight week old Col-0 and homozygous gr1-1 Arabidopsis plants. Six-week old plants were plug-infected with P. brassicae HH. The disease progress was evaluated after 4 days by bright-field microscopy of lactophenol-trypan blue-stained tissue (Fig. 3). To distinguish potential effects caused by altered redox state and total content of glutathione, the pad2 mutant that contains only about 30 % of wild type glutathione (Parisy et al. 2007) at an unchanged GSH/GSSG ratio was treated the same way. The pad2 mutant was found to be unable to induce defence reaction like hypersensitive response (HR) and thus is hyper-susceptible to P. brassicae HH (Roetschi et al. 2001). In contrast to the pad2 mutant gr1-1 showed resistance and defence mechanism induction comparable to wild type. Thus, the redox state of cytosolic glutathione pool had apparently no effect on resistance, at least in this pathosystem. However, it cannot be excluded that other pathogen interactions indeed depend on the redox state of the cytosolic glutathione pool. Also, short-term alterations could contribute to signal transduction but would not be detectable using this approach. Fig. 3. Resistance of Arabidopsis Col-0 wild type, gr1-1 and pad2 mutants to Phytophthora brassicae HH. Five days after inoculation, the tissue was stained with lactophenol-trypan blue and analyzed by bright field microscopy. Lactophenol-trypan blue stains dead cells and fungal structures. (A) Col-0. Dark stained cells (arrow) have undergone HR defence reaction. (B) The pad2 mutant did not show any HR upon infection with P. brassicae and pathogen mycelium was visible in the infected mesophyll tissue (mycelium is indicated by arrows). (C) gr1-1 tissue challenged with P. brassicae showed HR reaction and no pathogen growth within the tissue was observed. Scale bar: 25µm. References Eulgem, T. 2005. Regulation of the Arabidopsis defense transcriptome. Trends Plant Sci. 10: 7178. Grzam, A., Martin, M.N., Hell, R. and Meyer, A.J. 2007. -Glutamyl transpeptidase GGT4 initiates vacuolar degradation of glutathione S-conjugates in Arabidopsis. FEBS Lett. 581: 3131-3138. Kruse, C., Jost, R., Lipschis, M., Kopp, B., Hartmann, M. and Hell, R. 2007. Sulfur-enhanced defence: effects of sulfur metabolism, nitrogen supply, and pathogen lifestyle. Plant Biol. 9: 608-619. May, M., Vernoux, T., Leaver, C., Van Montagu, M. and Inze, D. 1998. Glutathione homeostasis in plants: implications for environmental sensing and plant development. J. Exp. Bot. 49: 649-667. Mou, Z., Fan, W. and Dong, X. 2003. Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes. Cell. 113: 935-944. Noctor, G. 2006. Metabolic signalling in defence and stress: the central roles of soluble redox couples. Plant Cell Environ. 29: 409-425. Parisy, V., Poinssot, B., Owsianowski, L., Buchala, A., Glazebrook, J. and Mauch, F. 2007. Identification of PAD2 as a gamma-glutamylcysteine synthetase highlights the importance of glutathione in disease resistance of Arabidopsis. Plant J. 49: 159-172. Rennenberg, H. and Brunold, C. 1994. Significance of glutathione metabolism in plants under stress. Progr. Bot. 55: 143-156. Roetschi, A., Si-Ammour, A., Belbahri, L., Mauch, F., and Mauch-Mani, B. 2001. Characterization of an Arabidopsis-Phytophthora pathosystem: resistance requires a functional PAD2 gene and is independent of salicylic acid, ethylene and jasmonic acid signalling. Plant J. 28: 293-305. Vanacker, H., Carver, T.L. and Foyer, C.H. 2000. Early H 2O2 accumulation in mesophyll cells leads to induction of glutathione during the hyper-sensitive response in the barley-powdery mildew interaction. Plant Physiol. 123: 1289-1300. Zimmermann, P., Hirsch-Hoffmann, M., Hennig, L. and Gruissem, W. 2004. GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiol. 136: 2621-2632.