Analysis of the IPT gene family reveals the critical role of the JrERF113-JrIPT1 module in cold resistance of walnut (Juglans regia).

Walnut (Juglans regia), a globally significant nut-bearing tree species, holds paramount economic and ecological importance. However, it is susceptible to abiotic stress, especially low-temperature stress. Cytokinin (CTK), a central phytohormone orchestrating plant growth, developmental plasticity, and stress resilience. As the rate-limiting enzyme in CTK biosynthesis, isopentenyltransferase (IPT) mediates adaptive responses to environmental challenges. In this study, eight IPT family members were identified from the walnut genome, with phylogenetic analysis classifying them into two evolutionary clades. Notably, JrIPT1, belonging to the ATP/ADP-IPT subclass, exhibited broad tissue-specific expression and a marked response to cold treatment. Functional characterization revealed that JrIPT1-overexpressing transgenic Arabidopsis displayed an early flowering phenotype accompanied by significantly upregulated expression of CTK signaling pathway-related flowering genes. Further analysis demonstrated that JrIPT1 overexpression promoted CTK accumulation in both transgenic Arabidopsis and walnut plants, leading to substantially enhanced survival rates, photosystem activity, and reactive oxygen species (ROS) scavenging capacity under cold stress. Conversely, virus-induced gene silencing (VIGS)-mediated suppression of endogenous JrIPT1 in walnut reduced CTK levels and increased cold sensitivity. Mechanistic insights revealed that the cold-induced ERF transcription factor JrERF113 directly binds to the GCC-box motif in the JrIPT1 promoter and then activates its transcription, also endows walnuts with cold resistance. This study establishes a ‘JrERF113-JrIPT1’ regulatory module that enhances walnut cold tolerance by boosting CTK biosynthesis. The elucidated CTK metabolic regulatory network not only advances the understanding of molecular mechanisms underlying plant cold adaptation but also provides potential genetic targets and theoretical foundations for breeding cold-resistant walnut varieties.