Plants encounter numerous adversities during growth, necessitating the identification of common stress activators to bolster their resistance. However, the current understanding of these activators’ mechanisms remains limited. This study identified three anti-stress activators applicable to apple trees, all of which elevate plant proline content to enhance resistance against various adversities. The results showed that the application of these sugar substitutes increased apple proline content by two to three times compared to the untreated group. Even at a lower concentration, these activators triggered plant stress resistance without compromising apple fruit quality. Therefore, these three sugar substitutes can be exogenously sprayed on apple trees to augment proline content and fortify stress resistance. Given their effectiveness and low production cost, these activators possess significant application value. Since they have been widely used in the food industry, they hold potential for broader application in plants, fostering apple industry development.

Calcineurin B-like (CBL) proteins are plant-specific calcium sensors critical for ion homeostasis and stress tolerance. Here, seven MdCBL genes were genome-wide identified in apple (Malus domestica). We conducted systematic bioinformatic profiling of their physicochemical features, subcellular localization, cis-regulatory elements, phylogeny, secondary structures, phosphorylation sites, and functional annotations and further verified the salt-stress regulatory function of MdCBL5 via transgenic tests. MdCBL proteins contain 210–246 amino acids, with molecular weights of 24,196.73–28,283.39 Da, pI values of 4.63–4.97 and instability indices of 37.12–49.20. Localization prediction placed these proteins in nuclei, cytosol and chloroplasts. Their promoter regions are rich in hormone-responsive (auxin, ABA, salicylic acid) and stress-responsive (cold, drought, salt) cis-elements. Phylogenetic clustering divided MdCBLs into five subgroups (A–E) with high homology to Arabidopsis CBLs. Random coils and α-helices dominate their secondary structures, and serine residues constitute most phosphorylation sites. Functional annotation supports their involvement in calcium signaling, ion transport and diverse stress adaptation. Salt stress experiments have confirmed that MdCBL5 may enhance apple salt resistance by promoting MdSOS gene expression. Meanwhile, transient transformation in apple fruit showed that MdCBL5 can effectively enhance fruit quality traits. Collectively, this study establishes a theoretical foundation for further elucidating the biological functions of the apple MdCBL5 gene and provides valuable insights for genetically improving stress resistance and fruit quality in apple via molecular breeding strategies.

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.