CsRAP2 ‐1‐ CsNPF4 .4‐ CsAMT1 module regulates ammonium/nitrate ratio to mediate thermotolerance in cucumber

SUMMARY High temperature poses a serious threat to agriculture and plant growth. Ammonium (NH 4 + ) and nitrate (NO 3 − ) are the two main inorganic nitrogen sources used by plants. However, how ammonium shapes the plant response to heat stress remains a mystery. Here, we found that cucumber ( Cucumis sativus L.) plants exhibited an increase in the NH 4 + /NO 3 − ratio in response to heat stress. Overexpression of Nitrate Transporter 1/Peptide Transporter Family 4.4 ( CsNPF4.4) in cucumber compromised thermotolerance and reduced the NH 4 + /NO 3 − ratio, whereas CsNPF4.4 ‐silenced plants exhibited enhanced thermotolerance and an increased NH 4 + /NO 3 − ratio. Using yeast one‐hybrid, dual‐luciferase, and electrophoretic mobility shift assay, we found that ERF/AP2 transcription factors 2–1 ( CsRAP2‐1 ), which was highly induced by high temperature, directly bound to the promoter of CsNPF4.4 to suppress its expression. Silencing CsRAP2‐1 compromised thermotolerance, while overexpression CsRAP2‐1 showed opposite effects. Meanwhile, we identified that Ammonium transporters 1 (CsAMT1) interacted with CsNPF4.4. Silencing CsAMT1 compromised thermotolerance and decreased the accumulation of NH 4 + , while overexpression of CsAMT1 showed opposite effects. Importantly, CsNPF4.4 influenced the expression of CsAMT1 and inhibited activity of CsAMT1 to suppress NH 4 + transport. Thus, heat stress induced the expression of CsRAP2‐1 , which repressed CsNPF4.4 transcription, leading to enhanced CsAMT1 expression and activity and ultimately increasing the ratio of NH 4 + /NO 3 − to enhance high‐temperature stress tolerance. These findings not only uncover a regulatory module CsRAP2‐1‐CsNPF4.4‐CsAMT1 that fine‐tunes ammonium/nitrate homeostasis to enhance thermotolerance, but also provide actionable targets for molecular breeding or nutrient management strategies aimed at improving crop resilience under high‐temperature stress.