WRKY46 promotes ammonium tolerance in Arabidopsis by repressing NUDX9 and indole-3-acetic acid-conjugating genes and by inhibiting ammonium efflux in the root elongation zone

Abstract

Ammonium (NH4+) is toxic to root growth in most plants, even at moderate concentrations. Transcriptional regulation is one of the most important mechanisms in the response of plants to NH4+ toxicity, but the nature of the involvement of transcription factors (TFs) in this regulation remains unclear. Here, RNA-seq analysis was performed on Arabidopsis roots to screen for ammonium-responsive TFs. WRKY46, the member of the WRKY transcription factor family most responsive to NH4+, was selected. We defined the role of WRKY46 using mutation and overexpression assays, and characterized the regulation of NUDX9 and indole-3-acetic acid (IAA)-conjugating genes by WRKY46 via yeast one-hybrid and electrophoretic mobility shift assays and chromatin immunoprecipitation-quantitative real-time polymerase chain reaction (ChIP-qPCR). Knockout of WRKY46 increased, while overexpression of WRKY46 decreased, NH4+-suppression of the primary root. WRKY46 is shown to directly bind to the promoters of the NUDX9 and IAA-conjugating genes (GH3.1, GH3.6, UGT75D1, UGT84B2) and to inhibit their transcription, thus positively regulating free IAA content and stabilizing protein N-glycosylation, leading to an inhibition of NH4+ efflux in the root elongation zone (EZ). We identify TF involvement in the regulation of NH4+ efflux in the EZ, and show that WRKY46 inhibits NH4+ efflux by negative regulation of NUDX9 and IAA-conjugating genes.