Salicylic acid improves chilling tolerance via CsNPR1–CsICE1 interaction in grafted cucumbers

Salicylic acid (SA) plays a role in the regulation of grafting-induced cold tolerance. However, the molecular mechanism behind it is still unknown. Here, we established that the phenylalanine ammonia-lyase (PAL) pathway-dependent elevate in SA content in grafted cucumber leaves was not only synthesized in the leaves but also transported from the roots under chilling stress. RNAi-<i>CsPAL</i> with low SA content as rootstock reduced SA accumulation in grafted seedling leaves while decreasing rootstock-induced cold tolerance, as evidenced by higher electrolyte leakage (EL), hydrogen peroxide (H₂O₂), and superoxide anion (O₂ ^·-) contents and lower expression of cold-responsive genes (<i>CsICE1</i>, Cs<i>DREB1A</i>, <i>CsDREB1B</i>, and <i>CsCOR47</i>), whereas OE-<i>CsPAL</i> with high SA content as rootstock improved the cold tolerance of grafted plants in comparison with the wild type (WT). In addition, <i>CsNPR1</i> was significantly upregulated in grafted cucumber under chilling stress, with exogenous and endogenous overexpressed SA inducing its transcriptional expression and protein stability, which exhibited higher expression in grafted plants than in self-root plants. While <i>CsNPR1</i>-overexpression (<i>OE</i>-<i>CsNPR1</i>) seedlings as scions were more tolerant to chilling stress than WT seedlings, <i>CsNPR1</i>-suppression (<i>Anti</i>-<i>CsNPR1</i>) seedlings as scions were more vulnerable to chilling stress. Notably, CsNPR1-CsICE1 interactions alleviated ROS accumulation and activated the expression of <i>CsDREB1A, CsDREB1B, CsCOR47, CsCOR15, CsCOR413,</i> and <i>CsKIN1</i> to enhance SA-mediated chilling tolerance in grafted cucumber. Overall, our findings reveal that SA enhances chilling tolerance in grafted cucumbers via the model of the CsNPR1-CsICE1 transcriptional regulatory cascade.