Abstract
Background: Populus hopeiensis, a significant afforestation species, faces substantial growth constraints due to cold stress. The IPT gene, a pivotal rate-limiting enzyme in cytokinin synthesis, plays a crucial role in controlling plant reactions to both biotic and abiotic pressures. In this study, we isolated the PhIPT5 gene from Populus hopeiensis and analyzed its biological characteristics and cold tolerance with the aim of providing guidance for the production of cold-resistant poplars. Results: The coding sequence (CDS) of the PhIPT5 gene spans 981 bp, encoding 333 amino acid residues with a molecular weight of 37.07 kDa. The PhIPT5 protein has alkaline stability and hydrophilicity. Phylogenetic analysis revealed that Populus hopeiensis IPT5 is closely related to Populus alba. Subcellular localization studies revealed the chloroplastic localization of PhIPT5. We constructed an overexpression vector for PhIPT5 and transformed it into Populus hopeiensis, resulting in improved cold tolerance in transgenic seedlings. Analysis of cytokinin metabolites revealed significantly greater levels in leaves harboring the PhIPT5 gene than in those harboring the CK gene even after exposure to cold. Furthermore, our findings suggest that the PhIPT5 gene primarily regulates the isoamyl pyrophosphate cytokinin metabolism pathway, leading to the synthesis of tZ, iP, and DZ cytokinins. Conclusion: Our isolation of PhIPT5 from Populus hopeiensis demonstrated that its overexpression enhances resistance to cold stress in transgenic plants. This work provides a foundation for further elucidating the function of IPT genes and has significant implications for advancing research on enhancing cold tolerance in Populus hopeiensis.