The U-Box E3 Ubiquitin Ligase PpPUB20 Positively Regulates Low-Temperature Tolerance by Targeting Sorbitol Dehydrogenase (PpSDH) in Peaches

Low temperatures are a major environmental factor limiting the growth and productivity of peaches (Prunuspersica). U-box E3 ubiquitin ligases (PUBs) are known to play a role in plant responses to abiotic stresses; however, their role in cold tolerance of peaches is unclear. In this study, transcriptome data revealed that a U-box E3 ubiquitin ligase gene, PpPUB20, was significantly activated by low temperatures. Expression analysis showed that PpPUB20 was rapidly upregulated under cold stress, with a more pronounced early response in cold-tolerant cultivars than in the cold-sensitive cultivars. Phylogenetic analysis revealed that PpPUB20 is evolutionarily conserved across plant species, and domain prediction identified a highly conserved U-box domain. Functional assays demonstrated that overexpressing PpPUB20 gene significantly enhanced low-temperature tolerance in Arabidopsis thaliana, whereas silencing it reduced low-temperature tolerance in peach calli. Physiological studies suggested that PpPUB20 positively regulates cold tolerance by enhancing antioxidant enzyme activity and minimizing membrane damage. Yeast two-hybrid screening identified sorbitol dehydrogenase (PpSDH) as a potential PpPUB20-interacting protein, which was further confirmed by luciferase complementation and co-immunoprecipitation assays. In vitro ubiquitination assays demonstrated that PpPUB20 functions as an E3 ubiquitin ligase mediating the polyubiquitination of PpSDH. This study found that an alternatively spliced transcript of PpPUB20 was present in the non-cold-resistant variety ‘21 Shiji’ but absent in the cold-resistant variety ‘Shijizhixing’. These splicing variants show functional deficiencies in their response to low-temperature stress and its substrate protein interaction due to an absent ARM domain. Full-length and structurally intact PpPUB20 was ubiquitinated by targeting PpSDH, thereby regulating sorbitol metabolism and enhancing the low-temperature tolerance of peaches. This study reveals a new molecular mechanism of peach response to low-temperature stress and further emphasizes the important role of ubiquitination-mediated post-translational regulation in the environmental adaptability of fruit trees.