Physiological and Molecular Mechanisms of ABA and CaCl₂ Regulating Chilling Tolerance of Cucumber Seedlings

Cold stress is a limiting factor to the growth and development of cucumber in the temperate regions; hence, improving the crop's tolerance to low temperature is highly pertinent. The regulation of low-temperature tolerance with exogenous ABA and CaCl₂ was investigated in the cucumber variety Zhongnong 26. Under low-temperature conditions (day/night 12/12 h at 5 °C), seedlings were sprayed with a single application of ABA, CaCl₂, or a combination of both. Our analysis included a calculated chilling injury index, malondialdehyde (MDA) content, relative electrical conductivity, antioxidant enzyme activities (SOD, CAT, and APX), leaf tissue structure, and expression of cold-related genes by transcriptome sequencing. Compared with the water control treatment, the combined ABA + CaCl₂ treatment significantly improved the superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) of the seedlings by 34.47%, 59.66%, and 118.80%, respectively (p < 0.05), and significantly reduced the chilling injury index, relative electrical conductivity, and MDA content, by 89.47%, 62.17%, and 44.55%, respectively (p < 0.05). Transcriptome analysis showed that compared with the water control treatment, 3442 genes were differentially expressed for the combined treatment, 3921 for the ABA treatment, and 1333 for the CaCl₂ treatment. KEGG enrichment analysis for both the ABA and combined ABA + CaCl₂ treatments (as compared to the water control) showed that it mainly involves genes of the photosynthesis pathway and metabolic pathways. Differentially expressed genes following the CaCl₂ treatment were mainly involved in plant hormone signal transduction, plant-pathogen interaction, MAPK signaling pathway-plant, phenylpropanoid biosynthesis, and circadian rhythm-plant. qRT-PCR analysis and RNA-seq results showed a consistent trend in variation of differential gene expression. Overall, this study demonstrated that although all three treatments provided some protection, the combined treatment of ABA (35 mg/L) with CaCl₂ (500 mg/L) afforded the best results. A combined ABA + CaCl₂ treatment can effectively alleviate cold-stress damage to cucumber seedlings by inducing physiological changes in photosynthesis and metabolism, and provides a theoretical basis and technical support for the application of exogenous ABA and CaCl₂ for low-temperature protection of cucumber seedlings.