Conjugation of the small ubiquitin-like modifier (SUMO) peptide to target proteins is an important post-translational modification. SAP AND MIZ1 DOMAIN-CONTAINING LIGASE1 (MdSIZ1) is an apple (Malus domestica Borkh). SUMO E3 ligase that mediates sumoylation of its targets during plant growth and development under adverse environmental conditions. However, it is unclear how MdSIZ1 senses the various environmental signals and whether sumoylation is regulated at the transcriptional level. In this study, we analyzed the MdSIZ1 promoter and found that it contained an MYB binding site (MBS) motif that was essential for the response of MdSIZ1 to low temperature (LT) and drought. Subsequently, we used yeast one-hybridization screening to demonstrate that a MYB transcription factor, MdMYB2, directly bound to the MBS motif in the MdSIZ1 promoter. Phenotypic characterization of MdMYB2 and MdSIZ1 suggested that the expression of both MdMYB2 and MdSIZ1 substantially improved cold tolerance in plants. MdMYB2 was induced by LT and further activated the expression of MdSIZ1, thereby promoting the sumoylation of MdMYB1, a key regulator of anthocyanin biosynthesis in apple. MdMYB2 promoted anthocyanin accumulation in apple fruits, apple calli, and Arabidopsis (Arabidopsis thaliana) in an MdSIZ1-dependent manner. In addition, the interaction of MdMYB2 and the MdSIZ1 promoter substantially improved plant tolerance to cold stress. Taken together, our findings reveal an important role for transcriptional regulation of sumoylation and provide insights into plant anthocyanin biosynthesis regulation mechanisms and stress response.

Apple (Malus domestica Borkh.) is the most widely cultivated deciduous fruit tree with the largest industrial scale and the highest economic value in China. Fruit surface glossiness and plant stress tolerance are two core traits that determine the economic benefits and sustainable development of the apple industry. The plant epidermal cuticle is not only the core material basis for determining fruit glossiness but also the first barrier for plants to resist abiotic and biotic stresses. Glycosylphosphatidylinositol-anchored lipid transfer proteins (LTPGs) are the core functional factors mediating trans-cell wall lipid transport in plants. At present, the functions and action mechanisms of LTPG family members that simultaneously regulate fruit appearance quality and stress tolerance in apple remain largely unclear. In this study, we took the MdLTPG17 gene as the research object, clarified its biological function of stress resistance under drought stress, and dissected the molecular mechanism by which it mediates fruit glossiness formation via regulating fruit cuticle thickening. The results of this study provide important genetic resources and a theoretical basis for molecular breeding of stress resistance and targeted improvement of fruit appearance quality in apple.

Nitrogen assimilation is vital for apple growth, yield, and quality, with nitrate reductase (NIA), nitrite reductase (NIR), glutamine synthetase (GS), and glutamate synthase (GOGAT) serving as key regulatory enzymes. This study systematically identified these four gene families in apple (Malus domestica) through genome-wide analysis and examined their expression patterns under nitrate treatment. In total, 13 genes were identified, 2 MdNIAs, 1 MdNIR, 7 MdGSs, and 3 MdGOGATs, with gene lengths ranging from 2577 to 27736 base pairs (bp); MdGLT1A had the longest coding sequence (6627 bp). The encoded proteins contained 355–2208 amino acids, with predicted isoelectric points (pIs) between 5.55 and 6.63. Subcellular localization analysis predicted distinct compartmentalization: MdNIA1A in peroxisomes; MdGS1 in the cytosol; MdNIR1, MdGS2, and MdGLU1 in chloroplasts; and MdGLT1 in mitochondria/chloroplasts. Functional site prediction revealed multiple phosphorylation and glycosylation sites, with ATP/GTP-binding motifs present only in certain MdGOGAT proteins. Protein interaction analysis suggested close associations among these genes and possible interactions with NRT2.1/2.2. Chromosomal mapping showed their distribution across eight chromosomes, while promoter analysis identified diverse cis-acting regulatory elements (e.g., ABRE and G-box). Under nitrate treatment (0–12 h), these genes exhibited distinct expression dynamics: MdNIA1A and B were rapidly induced (0–6 h) and maintained high expression; MdNIR1 peaked at 6 h and then declined; MdGS1.1B was activated after 6 h; and MdGS2A, MdGLU1, and MdGLT1A/B peaked at 6 h before decreasing. Therefore, these results elucidate the structural and functional divergence of nitrogen assimilation genes in apple and provide a basis for understanding nitrogen utilization mechanisms and developing nitrogen-efficient breeding strategies.