Functional identification of ANR genes in apple (Malus halliana) that reduce saline–alkali stress tolerance

As one of the major abiotic stresses restricting the development of global agriculture, saline-alkali stress causes osmotic stress, ion poisoning, ROS damage and high pH damage, which seriously restrict sustainable development of fruit industry. Therefore, it is essential to develop and cultivate saline-alkali-resistant apple rootstocks to improve the yield and quality of apples in China. Based on transcriptome data, MhANR (LOC114827797), which is significantly induced by saline-alkali stress, was cloned from Malus halliana. The physicochemical properties, evolutionary relationships and cis-acting elements were analysed. Subsequently, the tolerance of MhANR overexpression in Arabidopsis thaliana, tobacco, and apple calli to saline-alkali stress was verified through genetic transformation. Transgenic plants contained less Chl a, Chl b and proline, SOD, POD and CAT activity, and higher relative electrical conductivity (REC) compared to WT plants under saline-alkali stress. In addition, expression of saline-alkali stress-related genes in overexpressed apple calli were also lower than in WT calli, including the antioxidant genes (MhSOD and MhCAT^), the Na⁺ transporter genes (MhCAX5, MhCAX5, MhSOS1, MhALT1), and the H⁺ -ATPase genes (MhAHA2 and MhAHA8), while expression of the K⁺ transporter genes (MhSKOR and MhNHX4) were higher. Expression of MhANR reduced tolerance of A. thaliana, tobacco, and apple calli to saline-alkali stress by regulating osmoregulatory substances, chlorophyll content, antioxidant enzyme activity, and expression of saline-alkali stress-related genes. This research provides a theoretical basis for cultivating apple rootstocks with effective saline-alkali stress tolerance.