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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
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
( 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.
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:
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:
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:
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.