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From Master 2 Recherche Biosciences Végétales

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Study of ROS production systems during early steps of Arabidopsis germination

Laboratoire d'accueil : UMR5546
Equipe d'accueil : Evolution et Expression de Peroxydases
Encadrant(e)(s) : Christophe Dunand, dunand@scsv.ups-tlse.fr

Context:
Germination is controlled by external factors such as temperature, water and light and by hormone balance. Recently, reactive oxygen species (ROS) have been shown to act as messengers during plant development, stress responses and programmed cell death. We analyzed the role of ROS during germination and investigated the possibility for ROS being signalling molecules in this process.
Reactive oxygen species (ROS, e.g. superoxide (O2•-), hydrogen peroxide (H2O2), hydroxyl radical (.OH•)) are by-products constantly produced during normal metabolic processes and the level of these cell-damaging compounds need to be controlled. ROS can also be produced for a specific purpose such as oxidative burst during plant defence. In particular, ROS released by the embryo through the seed coat have an important role in protecting the emerging embryo against pathogen attacks (Schopfer et al., 2001). They also play a major role in physiological processes such as root elongation through their action in cell wall loosening (Dunand et al., 2007) but also during seed germination and dormancy break (Ogawa and Iwabuchi, 2001; Sarath et al., 2007). Interestingly, several data suggest that ROS act as signalling molecules in plants during defence responses, stress responses and programmed cell death (Gechev and Hille, 2005; Laloi et al., 2007; Miller et al., 2007; Neill et al., 2002)
We showed that ROS are released prior to endosperm rupture, that ROS production is required for germination, and that class III peroxidases could be implicated in the spacio-temporal regulation of ROS production. H2O2 modifies the expression of genes specifically active during germination encoding for enzymes regulating ROS levels, pointing out a regulatory feed back loop for ROS production. Measurements of endogenous levels of ROS following application of GA and ABA suggested that ABA inhibits germination by repressing ROS accumulation, and that, conversely, GA triggers germination by promoting an increase of ROS levels.

Objectives
Using Arabidopsis thaliana as model, the main objectives of the projects are (i) to quantify the ROS production during early steps of germination (testa and endosperm ruptures) with the luminol assays, (ii) to determine the role of calcium during the early steps with specific Arabidopsis mutants containing calcium probe, (iii) to identify which NADPH oxidases are necessary for the ROS production (mutants, et (iv) to study the NADPH oxidase transcripts following various chemical treatments affecting germination.

References

Dunand, C., Crevecoeur, M. and Penel, C. (2007). Distribution of superoxide and hydrogen peroxide in Arabidopsis root and their influence on root development: possible interaction with peroxidases. New Phytol 174, 332-41.
Gechev, T. S., Van Breusegem, F., Stone, J. M., Denev, I. and Laloi, C. (2006). Reactive oxygen species as signals that modulate plant stress responses and programmed cell death. Bioessays 28, 1091-101.
Laloi, C., Stachowiak, M., Pers-Kamczyc, E., Warzych, E., Murgia, I. and Apel, K. (2007). Cross-talk between singlet oxygen- and hydrogen peroxide-dependent signaling of stress responses in Arabidopsis thaliana. Proc Natl Acad Sci U S A 104, 672-7.
Miller, G., Suzuki, N., Rizhsky, L., Hegie, A., Koussevitzky, S. and Mittler, R. (2007). Double mutants deficient in cytosolic and thylakoid ascorbate peroxidase reveal a complex mode of interaction between reactive oxygen species, plant development, and response to abiotic stresses. Plant Physiol 144, 1777-85.
Neill, S. J., Desikan, R., Clarke, A., Hurst, R. D. and Hancock, J. T. (2002). Hydrogen peroxide and nitric oxide as signalling molecules in plants. J Exp Bot 53, 1237-47.
Ogawa, K. and Iwabuchi, M. (2001). A mechanism for promoting the germination of Zinnia elegans seeds by hydrogen peroxide. Plant Cell Physiol 42, 286-91.
Sarath, G., Hou, G., Baird, L. M. and Mitchell, R. B. (2007). Reactive oxygen species, ABA and nitric oxide interactions on the germination of warm-season C4-grasses. Planta 226, 697-708.
Schopfer, P. (2001). Hydroxyl radical-induced cell-wall loosening in vitro and in vivo: implications for the control of elongation growth. Plant J 28, 679-88.