Peach (Prunus persica) production and the northward expansion of its cultivation boundaries are severely constrained by recurrent extreme climatic events. Based on time-series transcriptomic analysis and qRT-PCR validation, multiple candidate transcription factor genes responsive to abscisic acid (ABA) were identified (PpERF48, PpERF017, PpMYB6, PpWRKY46, PpWRKY40, and PpbHLH35). Among these, PpMYB6 was further characterized through bioinformatic analysis, and transgenic peach callus and Arabidopsis thaliana lines overexpressing PpMYB6 were generated, revealing its dual role in modulating plant growth and conferring low-temperature stress tolerance. Yeast one-hybrid and dual-luciferase reporter assays confirmed that PpMYB6 directly binds to the PpCBF2 promoter to activate its transcription, thereby enhancing cold resistance. Furthermore, yeast two-hybrid library screening identified DWARF8 as a candidate interacting protein, pointing to a putative mechanism by which PpMYB6 may negatively regulate vegetative growth via the gibberellin pathway. Together, this study elucidates a novel molecular framework governing cold tolerance and growth balance in peach, providing a promising target gene for molecular breeding of cold-resistant cultivars and rootstocks.

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.

Peach (Prunus persica) production and the northward expansion of its cultivation boundaries are severely constrained by recurrent extreme climatic events. Based on time-series transcriptomic analysis of two independent parallel treatments (cold stress and ABA application) and qRT-PCR validation, multiple candidate transcription factor genes co-responsive to both cold stress and ABA were identified (PpERF48, PpERF017, PpMYB6, PpWRKY46, PpWRKY40, and PpbHLH35). Among these, PpMYB6 was further characterized through bioinformatic analysis, and transgenic peach callus and Arabidopsis thaliana lines overexpressing PpMYB6 were generated, revealing its dual role in modulating plant growth and conferring low-temperature stress tolerance. Yeast one-hybrid and dual-luciferase reporter assays confirmed that PpMYB6 directly binds to and activates the PpCBF2 promoter. Yeast two-hybrid library screening identified DWARF8 as a candidate interacting protein, pointing to a working hypothesis by which PpMYB6 may negatively regulate vegetative growth via the gibberellin pathway. Together, this study characterizes a novel molecular module associated with cold tolerance and growth balance in peach, providing a promising candidate gene for molecular breeding of cold-resistant cultivars and rootstocks.