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Abstract

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.