International Network of Plant Abiotic Stress

Short Term Scientific Mission COST-STSM-FA0605-010911-008440: “Subcellular localization of water stress induced H₂O₂ production and pyrroline-5-carboxylate synthase accumulation in Arabidopsis thaliana seedlings”

STSM Applicant:     Kilani Ben Rejeb, PhD student,

Extremophile Plants laboratory (LPE),

Centre of Biotechnology of Borj-Cédria (CBBC), BP 901, Hammam-lif, 2050, Tunisia.

kilanib@yahoo.com

Host:                          Pr Arnould Savouré

Plant Cellular and Molecular Physiology laboratory (PCMP), Pierre & Marie Curie University of Paris (UPMC), France.

arnould.savoure@upmc.fr

Period of STSM:       1^st to 30^th of September, 2011.

Purpose of the visit: The aim of this STSM was to investigate a possible link between H₂O₂, P5CS protein and proline accumulation. The subcellular localization of H₂O₂ production upon water stress and reverse genetic approach to investigate the origin of this H₂O₂ synthesis were developed. This work is part of my PhD thesis.

Main results obtained

To investigate the subcellular localization of H₂O₂, accumulation of H₂O₂ in leaves of A. thaliana seedlings exposed to water stress was investigated using a cytochemical approach with cerium chloride (CeCl₃), which reacts with H₂O₂ to produce electron-dense deposits of cerium perhydroxides detectable by transmission electron microscopy. My results showed that the cell wall and intercellular spaces revealed the highest increase in H₂O₂ accumulation. Diphenylene iodonium (DPI), an inhibitor of the plasma membrane-bound NADPH oxidase called Rboh (Respiratory Burst Oxidase Homologs), which is one of the main sources of H₂O₂ formation in the apoplast after stress induction in plants, inhibited NaCl or mannitol-induced H₂O₂ and proline accumulation in plants after 24h of treatment. Similarly, the expression level of Δ¹-pyrroline-5-carboxylate synthetase (P5CS), a key enzyme involved in the biosynthesis of proline, was also measured. The involvement of NADPH oxidase in the accumulation of H₂O₂ and proline upon water stress was also investigated using genetic mutants defective in different NADPH oxidase isoforms.

My visit was very fruitful. I have had the opportunity to get familiar with several techniques (fluorescence and transmission electron microscopy, western blot and qPCR).

Acknowledgements

I would like to thank my host Pr Arnould Savouré. I would like to also thank the European COST project, FA0605 for support of this Short Term Scientific Mission (STSM).