CAP1 regulates root hair ammonium response through RBOHD-dependent ROS homeostasis in Arabidopsis.

Abstract

Elucidating the molecular mechanisms underlying ammonium (NH4+)-mediated root system development is critical for alleviating NH4+ toxicity in plants. The Arabidopsis thaliana [Ca2+]cyt-associated protein kinase CAP1 regulates root hair growth in response to NH4+ treatment, with the cap1-1 mutant exhibiting hypersensitivity to NH4+ and elevated reactive oxygen species (ROS) levels. Based on our previous phosphoproteomic data, here we investigated the role of Respiratory Burst Oxidase Homolog D (AtRBOHD) in CAP1-mediated root hair growth under NH4+ treatment. Through yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP), and in vitro phosphorylation assays, we demonstrate that CAP1 physically interacts with and phosphorylates RBOHD, with Ser347 identified as a critical phosphorylation site in RBOHD that is required for their interaction. Genetic complementation assays revealed that expressing either wild-type RBOHD or its phospho-mimetic variant (RBOHDS347D) partially restored root hair growth in cap1-1 mutants while reducing ROS levels to those of the wild type under NH4+ treatment. Collectively, our findings establish a mechanistic model in which CAP1 phosphorylates RBOHD at Ser347 to regulate NH4+-dependent root hair growth by modulating ROS homeostasis.