Beta-Cyanoalanine synthase action in root hair elongation is exerted at early steps of the root hair elongation pathway and is independent of direct cyanide inactivation of NADPH oxidase

Abstract

In Arabidopsis thaliana, cyanide is produced concomitantly with ethylene biosynthesis and is mainly detoxified by the ß-cyanoalanine synthase CAS-C1. In roots, CAS-C1 activity is essential to maintain a low level of cyanide for proper root hair development. Root hair elongation relies on polarized cell expansion at the growing tip, and we have observed that CAS-C1 locates in mitochondria and accumulates in root hair tips during root hair elongation, as shown by observing the fluorescence in plants transformed with the translational construct ProC1:CASC1-GFP, containing the complete CAS-C1 gene fused to GFP. Mutants in the SUPERCENTIPEDE (SCN1) gene, that regulate the NADPH oxidase RHD2/AtrbohC, are affected at the very early steps of the development of root hair that do not elongate and do not show a preferential localization of the GFP accumulation in the tips of the root hair primordia. Root hairs of mutants in CAS-C1 or RHD2/AtrbohC, which catalyzes the generation of ROS and the Ca2+ gradient, correctly start to grow out but they do not elongate either. Genetic crosses between the cas-c1 mutant and scn1 or rhd2 mutants were performed and the detail phenotypic and molecular characterization of the double mutants demonstrate that scn1 mutation is epistatic to cas-c1 and cas-c1 is epistatic to rhd2 mutation, indicating that CAS-C1 acts in early steps of the root hair development process. Moreover, our results show that the role of CAS-C1 in root hair elongation is independent of H2O2 production and of a direct NADPH oxidase inhibition by cyanide