Identification of the DEAD-box gene family in apple (Malus domestica) and functional verification of MdRH28 under low-temperature stress

DEAD-box helicases represent the largest subfamily of RNA helicases and play a crucial role in plant stress responses. Based on the whole genome of apple, 134 members of the DEAD-box family (designated as MdRH1 to MdRH134) was identified. These members exhibit significant differences in protein physicochemical properties, which are unevenly distributed across 17 chromosomes, with segmental duplication being the main expansion mechanism. Additionally, the promoter regions of these family genes are rich in cis-elements related to hormones, stresses, and growth. Real-time fluorescence quantification-polymerase chain reaction (RT-qPCR) revealed that MdRH28 is significantly upregulated under low temperature (4 °C). To clarify its function, the MdRH28 gene was cloned and stably transformed into apple calli and transiently transformed into Malus hupehensis. After 4 °C low-temperature treatment, compared with the WT lines, the overexpression lines of MdRH28 exhibited a significantly better growth status in apple calli. The specific manifestations were as follows: higher fresh weight; lower accumulation of malondialdehyde (MDA), relative electrical conductivity (REC), and reactive oxygen species (ROS, including H₂O₂ and O₂^-); higher proline content and higher activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT); The content of abscisic acid (ABA) increased, while the contents of growth-related hormones such as indole-3-acetic acid (IAA), gibberellin A₃ (GA₃), and zeatin (ZT) decreased. Meanwhile, the expression of low-temperature response genes (CBF1/2/3, COR47, and NCED1) was upregulated. In contrast, the antisense and gene-silencing lines showed the opposite trends. Specifically, the silencing MdRH28 lines through virus-induced gene silencing (VIGS) exhibited severe wilting; the levels of REC, MDA, and ROS in their leaves increased; the chlorophyll content, net photosynthetic rate (Pn), and maximum photochemical efficiency of photosystem Ⅱ (Fv/Fm) decreased more significantly; and the expression of cold-resistant genes was downregulated. In conclusion, MdRH28 significantly enhances the low-temperature tolerance by alleviating low-temperature-induced osmotic and oxidative damage, regulating the balance of endogenous hormones, and activating genes in the low-temperature response pathway. This study provides important genetic resources and a theoretical basis for cold-resistant apple breeding.