CNGC Gene Family in Pyrus betulaefolia: Genome-Wide Analysis and CNGC4/14 Function in Salt Tolerance via DNA Methylation

Salt stress severely restricts the growth and development of pear trees, and DNA methylation may potentially modulate the salt tolerance of Pyrus betulaefolia by regulating ion transporter genes. Cyclic nucleotide-gated channel (CNGC) family genes are essential for plant ion transport and salt stress adaptation; however, the epigenetic regulatory relationship between DNA methylation and CNGC genes underlying pear salt tolerance remains unclear. In this study, 26 PbCNGC genes were systematically identified from P. betulaefolia, which possess the conserved motifs characteristic of the CNGC family and can be classified into five subclades. PbCNGC members exhibit distinct spatiotemporal expression patterns in ordinary and salt-tolerant genotypes. Under salt stress, core members PbCNGC3, PbCNGC4, PbCNGC10, and PbCNGC14 displayed typical fluctuating up-and-down expression patterns across roots, stems, and leaves, with distinct spatiotemporal specificity in their expression peaks. In contrast, PbCNGC19 and PbCNGC20;1 were exclusively induced in roots under salt stress. Quantitative results showed marked genotypic differences in salt responsiveness. At 4 h of salt treatment, the root transcript levels of PbCNGC4 and PbCNGC14 were upregulated by 8.27-fold and 6.76-fold in the salt-tolerant genotype, respectively, which were considerably higher than those in the ordinary genotype (2.08-fold and 4.50-fold). Obvious genotypic differences in mCHH methylation modifications of PbCNGC4 and PbCNGC14 were detected after salt treatment, and pharmacological experiments confirmed that DNA methylation negatively modulates the transcription of these two genes. Yeast functional complementation assays further demonstrated that PbCNGC4 and PbCNGC14 act as functional Na+ and K+ permeable cation influx channels. Combined with methylation analysis results, this study reveals a potential regulatory cascade in which DNA methylation may inhibit the transcription of PbCNGC4 and PbCNGC14, modulating their ion-transport capacity, effectively reduces Na+ overaccumulation, sustains cellular K+ retention to alleviate salt-induced ionic toxicity, and may ultimately shape the salt tolerance of P. betulaefolia. These findings enrich the understanding of epigenetic regulation of plant salt tolerance and provide valuable candidate genes and theoretical references for salt-tolerant molecular breeding in pear trees.