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Summary Many plants accumulate proline, a compatible osmolyte, in response to various environmental stresses such as water deficit and salinity. In some stress responses, plants generate hydrogen peroxide (H2O2) that mediates numerous physiological and biochemical processes. The aim was to study the relationship between stress‐induced proline accumulation and H2O2 production. Using pharmacological and reverse genetic approaches in Arabidopsis thaliana, we investigated the role of NADPH oxidases, Respiratory burst oxidase homologues (Rboh), in the induction of proline accumulation was investigated in response to stress induced by either 200 mM NaCl or 400 mM mannitol. Stress from NaCl or mannitol resulted in a transient increase in H2O2 content accompanied by accumulation of proline. Dimethylthiourea, a scavenger of H2O2, and diphenylene iodonium (DPI), an inhibitor of H2O2 production by NADPH oxidase, were found to significantly inhibit proline accumulation in these stress conditions. DPI also reduced the expression level of Δ1‐pyrroline‐5‐carboxylate synthetase, the key enzyme involved in the biosynthesis of proline. Similarly, less proline accumulated in knockout mutants lacking either AtRbohD or AtRbohF than in wild‐type plants in response to the same stresses. Our data demonstrate that AtRbohs (A. thaliana Rbohs) contribute to H2O2 production in response to NaCl or mannitol stress to increase proline accumulation in this plant.

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In addition to its role in primary metabolism as a component of proteins, proline is one of the most widely distributed compatible solutes that accumulates in plants during adverse environmental constraints and plays an important role in plant stress tolerance. Proline was proposed to act as stabilizer for proteins and macromolecular complexes, scavenger of free radicals and regulator of cellular redox potential. Intracellular proline concentration depends on a tight regulation between its biosynthesis and catabolism. However the exact role of proline and the signaling pathways involved in the regulation of its metabolism are not completely known yet. Investigation of proline metabolism in model plants would allow to acquire information about the diversity of the mechanisms developed by plants to overcome environmental constraints and to establish some reliable tools for the improvement of crop tolerance.