Integrative Profiling of Phytohormones, Metabolomics, and Transcriptomics Reveals Key Regulators of Cold Tolerance in Cucumber Leaves

A low-temperature condition in a root zone is a major abiotic stress that threatens cucumber (<i>Cucumis sativus L.</i>) growth and development, yet the molecular mechanism by which the leaf reacts to root zone chilling stress remains largely unknown. In this study, we applied three temperature treatments, including room temperature (20°C-22°C), suboptimal temperature (13°C-15°C), and low temperature (8°C-10°C), to investigate how root zone chilling affects hormone dynamics, metabolomics, and transcriptomics in the leaves of the cucumber variety "Jinyou 35", the main cultivar in northwest and southwest China. Through integrative physiological and biochemical analysis, auxin emerges as the most significant accumulated hormone, accounting for 88% in room temperature-treated leaves (RL), 99% in suboptimal temperature-treated leaves (SL), and 94% in low-temperature-treated leaves (LL). Under chilling stress, flavanones were the most abundant metabolite in cucumber leaves, constituting over 50% of total metabolites, while phenolic acids showed a marked decrease. Several differentially expressed transcription factors (DETFs), such as <i>LOB</i> (<i>CsaV3_3G020650</i>), <i>MYB</i> (<i>CsaV3_3G043510</i>), and <i>bHLH</i> (<i>CsaV3_2G005070</i> and <i>CsaV3_4G029740</i>), were upregulated in SL and LL, potentially enhancing cucumber's defense against chilling injury. Additionally, terminal flower formation was observed under suboptimal and low-temperature conditions, with <i>CsFT</i> expression in SL and LL lower than in RL, and a significant negative correlation observed between <i>CsFT</i> and <i>CsNAC6</i>. These findings deepen our understanding of cucumber's resilience mechanisms to root zone chilling stress, shedding light on its cold tolerance strategies.