The MdWRKY17 positively regulates nitrate uptake by promoting MdNRT2.5 expression under long-term low N stress in apple

Abstract

Nitrogen (N) supply is critical for apple yield and quality. Improving nitrogen use efficiency (NUE) could reduce fertilizer application for maintaining apple yield at the cost of environmental pollution in infertile soil. The molecular mechanisms underlying nitrate (NO₃^-) uptake are foundational for breeding high NUE cultivars. The two-month low N treatment mimicking infertile soil dramatically induced the accumulation of transcription factor MdWRKY17 in apple. Overexpression of MdWRKY17 conferred enhanced long-term low nitrogen tolerance in transgenic apple plants and calli, while RNA interference of MdWRKY17 reduced this tolerance. MdNRT2.5 encoding a high-affinity nitrate transporter was identified by chromatin immunoprecipitation sequencing (ChIP-seq) as the direct target of MdWRKY17. This is confirmed by in vitro EMSA and in vivo ChIP-qPCR assay. Notably, overexpression of MdNRT2.5 increased NO₃^- uptake under long-term N-deficiency conditions. RNA interference of MdNRT2.5 in roots decreased NO₃^- uptake efficiency of MdWRKY17-OE transgenic apple plants, indicating that MdWRKY17 improves NO₃^- uptake mainly by activating MdNRT2.5 expression. Our study identified an important MdWRKY17-MdNRT2.5 module in response to long-term low N stress, which will contribute to the molecular breeding of high NUE apple cultivars.