Identification and functional analysis of transcription factor MhWRKY71-like from Malus halliana in response to low temperature

Late spring coldness and late frost during bud burst are major low-temperature disasters in apple production, which seriously hinder the sustainable development of the apple industry. WRKY transcription factors play central regulatory roles in plant responses to abiotic stresses. However, the response patterns and molecular mechanisms of Class II WRKY family genes involved in low-temperature responses remain poorly understood in apple. In this study, Malus halliana was used as the experimental material. The low-temperature response characteristics of apple Class II WRKY genes were elucidated through genome-wide identification and screening, gene cloning, Agrobacterium-mediated genetic transformation, and physiological index determination. A total of 71 Class II WRKY genes were identified across the apple genome and classified into five subgroups (IIa-IIe). Among them, subgroup IIc exhibited the most prominent evolutionary expansion. Members of this gene family possessed conserved evolutionary structures, and their promoter regions contained numerous cis-acting elements associated with stress and hormone responses. RT-qPCR results revealed that MhWRKY71-like , a gene belonging to subgroup IIc, was most strongly induced by low temperature. The protein encoded by this gene was localized in the nucleus, and phylogenetic analysis indicated that it shared the closest genetic relationship with Pyrus x bretschneideri . Subsequently, the sense sequence, antisense sequence and VIGS silencing fragment of MhWRKY71-like were cloned using cDNA of Malus halliana as the template, and the corresponding expression vectors were constructed. Agrobacterium infiltration was applied to obtain MhWRKY71-like -overexpressing Arabidopsis lines, apple calli lines with overexpression or antisense suppression, as well as Malus hupehensis var. pingyiensis plants with root overexpression and VIGS-mediated gene silencing. After low-temperature stress treatment, overexpression of MhWRKY71-like markedly enhanced plant cold tolerance. Specifically, overexpression lines showed significant increases in chlorophyll content and activities of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), while relative electrical conductivity (REC), malondialdehyde (MDA) content and reactive oxygen species (ROS) accumulation decreased obviously. In contrast, lines with antisense suppression and transient silencing displayed aggravated cold sensitivity. In summary, the IIc subgroup WRKY gene MhWRKY71-like from Malus halliana is a nuclear-localized cold-inducible transcription factor. Functional verification in Arabidopsis , apple calli and Malus hupehensis via overexpression, antisense inhibition and VIGS silencing systems confirmed that MhWRKY71-like may serve as a positive regulator involved in the apple cold stress response process by alleviating oxidative damage, maintaining membrane integrity and stabilizing chlorophyll.