Adventitious shoot (AS) regeneration is a significant factor in the genetic transformation of horticultural plants. It is also a noteworthy approach to their vegetative propagation. AS regeneration remains highly dependent on the genotype or maturity of explants. We here found that the AS regeneration abilities of apple leaves were positively correlated with MdAIL5 expression. MdAIL5 overexpression dramatically increased AS regeneration efficiency. Notably, MdAIL5 overexpression could restore the AS formation ability of explants to a certain extent, which was lost with an increase in maturity. Endogenous hormone detection revealed that MdAIL5 overexpression changed the contents of auxin, cytokinin (CK), and other hormones in apple leaves. Transcriptome analysis revealed that many genes related to auxin, CK, and brassinolide signaling pathways were significantly and differentially expressed between MdAIL5-overexpressing transgenic apple and wild-type apple plants. Yeast one-hybrid assays, the electrophoretic mobility shift assay, and the dual-luciferase reporter assay revealed that MdAIL5 directly binds to MdARF9 and MdHB14 promoters and positively affects their expression. We here established a model of MdAIL5 regulating AS formation, which acts as a theoretical basis for facilitating genotype- or explant maturity-independent AS regeneration in the future.

Nutrient patterns can significantly influence the growth and development of fruit trees, especially fruit quality. In order to clarify the appropriate ratio of inorganic and organic fertilizers, six treatments with different ratios of cow manure and chemical fertilizers were set up in this study to evaluate the effects of different treatments on fruit yield, appearance quality, intrinsic quality and volatile substances. The results showed that, after replacing some chemical fertilizers with cow manure, the content of sugar, titratable acid, organic acid and aroma substance in the fruit increased, the fruit yield and colour did not change significantly, and the fruit weight per fruit decreased. Among the treatments of cow manure with chemical fertilizer, the fruits of 50% chemical fertilizer with 50% cow manure treatment had higher titratable acid (0.4%), malic acid (4.15 mg/g), sorbitol (0.51%), glucose (1.30%), fructose (5.81%) content and total aroma substance content (1047.82 μg/g) than chemical fertilizer alone. Compared to the fertilizer treatment alone, the application of cow manure instead of 50% of the chemical fertilizer had a positive effect on fruit quality.

The homeodomain–leucine zipper IV (HD-Zip IV) transcription factor subfamily plays essential roles in epidermal development, cuticle formation, lipid metabolism, and environmental adaptation in plants. Despite its biological importance, the HD-Zip IV family has not been systematically characterized in apple (Malus domestica). Here, we identified 17 apple HD-Zip IV genes and named them MdHDZIV1–MdHDZIV17 based on their locations on the chromosomes. The 17 genes showed a nonuniform distribution on eight chromosomes, while the occurrence of both tandem and segmental duplications indicated that family expansion involved more than one duplication mechanism. All MdHDZIV proteins contained the conserved HD, LZ, START, and SAD domains but lacked the MEKHLA domain, consistent with typical HD-Zip IV structural features. Phylogenetic analysis classified MdHDZIV proteins into five groups together with HD-Zip IV members from Arabidopsis thaliana and rice, indicating evolutionary conservation of this subfamily. Collinearity and Ka/Ks analyses revealed that duplicated MdHDZIV gene pairs were mainly subjected to purifying selection. Promoter scanning revealed diverse cis-regulatory motifs associated with hormonal signaling, environmental stress, light response, and epidermal regulation, including ABRE, ARE, W-box, MYC, G-box, and L1-box motifs. Integration of transcriptomic profiling with qRT-PCR validation revealed pronounced tissue-dependent differences in the expression of MdHDZIV genes in leaf, fruit skin, and branch bark. Under PEG₆₀₀₀-induced osmotic stress and NaCl-induced salt stress, 10 candidate MdHDZIV genes displayed gene-specific and stress type-specific expression patterns, with MdHDZIV3 showing strong induction under PEG₆₀₀₀ treatment. Functional validation in apple calli showed that MdHDZIV3 overexpression enhanced PEG tolerance, increased fresh weight, elevated SOD and POD activities, and reduced MDA accumulation under osmotic stress. These findings provide a genome-wide framework for understanding the apple HD-Zip IV gene family.