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

High temperature poses a serious threat to agriculture and plant growth. Ammonium (NH₄ ⁺) and nitrate (NO₃ ^-) 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 NH₄ ⁺/NO₃ ^- 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 NH₄ ⁺/NO₃ ^- ratio, whereas CsNPF4.4-silenced plants exhibited enhanced thermotolerance and an increased NH₄ ⁺/NO₃ ^- 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 NH₄ ⁺, while overexpression of CsAMT1 showed opposite effects. Importantly, CsNPF4.4 influenced the expression of CsAMT1 and inhibited activity of CsAMT1 to suppress NH₄ ⁺ 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 NH₄ ⁺/NO₃ ^- 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.