CCCH zinc-finger proteins constitute a unique class of transcription factors that play vital roles in mediating plant tolerance to biotic and abiotic stresses and regulating various physiological and developmental processes. This study systematically identified and characterized the apple (Malus domestica) CCCH (MdC3H) gene family, aiming to elucidate its evolutionary patterns, functional characteristics, and regulatory mechanisms under drought stress. Genome-wide analysis revealed 85 MdC3H genes, which were unevenly distributed across chromosomes and exhibited significant differences in physiochemical properties, suggesting functional divergence. Phylogenetic analysis classified these genes into 9 subfamilies with distinct conservation. Collinearity analysis indicated a close evolutionary relationship between apple and Malus sieversii, with 150 collinear gene pairs identified, highlighting the conservation of the C3H gene family during speciation. Cis-acting element prediction in promoter regions uncovered abundant stress-responsive elements (e.g., ABRE, DRE, MYB), implying the potential of MdC3H genes in coordinating environmental signals. Functional verification demonstrated that MdC3H49, a key member of the family, is localized in the nucleus and possesses transcriptional activation activity. Overexpression of MdC3H49 in Arabidopsis and apple calli significantly enhanced drought tolerance, characterized by reduced malondialdehyde (MDA) content, relative electrical conductivity, and increased proline accumulation. Mechanistic studies revealed that MdC3H49 directly regulates the expression of MdP5CS, a core gene in proline biosynthesis, thereby strengthening the cellular antioxidant capacity and mitigating drought-induced damage. Collectively, this study establishes MdC3H49 as a critical regulator in apple drought stress response, providing valuable insights into the molecular mechanisms underlying abiotic stress tolerance in perennial plants and laying a foundation for genetic improvement of drought resistance in apple breeding.

Seed oil content and fatty acid (FA) composition collectively determine the quality and economic value of Brassica napus. Little is known about the role of R2R3-MYB transcription factors (TFs) in regulating FA biosynthesis in B. napus. Here, BnaC04.MYB89 was found to be expressed primarily in developing seeds. Overexpression of BnaC04.MYB89 consistently decreased FA levels, as evidenced by its effect in both the Arabidopsis thaliana myb89-1 mutant and B. napus seeds. RNA-seq of developing seeds at 30 DAP (days after pollination) revealed marked suppression of FA biosynthetic genes in BnaC04.MYB89-overexpressing plants compared to the K407 control. ChIP (Chromatin immunoprecipitation) analysis revealed that BnaC04.MYB89 directly inhibited the expression of BnaA03.BCCP1 and BnaC03.HD while indirectly regulating that of BnaA09.BADC1, BnaA03.BADC3, BnaA03.MOD1, and BnaA08.FAT8, thereby reducing seed FA accumulation. Collectively, these results elucidate the role for BnaC04.MYB89 and provide new insights into the transcriptional regulatory network controlling seed oil accumulation in B. napus.

AT-hook motif nuclear-localized (AHL) proteins are pivotal in plant growth, development, and stress responses. Nevertheless, there is limited research on AHL proteins in Malus sieversii. Our study identified 25 AHL genes from the M. sieversii genome, named MsAHL1–MsAHL25. The encoded protein sequences had lengths ranging from 195 to 554 amino acids, molecular weights from 19.17 to 58.53 kDa, and isoelectric points from 4.67 to 10.09. Chromosomal mapping revealed that these 25 genes were unevenly distributed across 10 chromosomes. Collinearity analysis of AHL genes in M. sieversii implied that gene loss might have occurred during its evolution. The phylogenetic tree classified the AHL proteins of M. sieversii into two subfamilies, showing a close relationship with multiple proteins of M. domestica. Promoter analysis indicated that the AHL genes in M. sieversii harbored numerous stress- and hormone-responsive elements, suggesting their potential role in various stress responses. qRT-PCR analysis of six representative MsAHLs under biotic and abiotic stresses demonstrated that the expression of MsAHL13, MsAHL15, and MsAHL17 was significantly upregulated under salt, drought, and cold stresses, while MsAHL01 expression was inhibited under low-temperature stress. All six MsAHLs were induced by the pathogen Valsa mali. Subcellular localization analysis of the specifically expressed protein MsAHL13 showed its nuclear location. Furthermore, luciferase and yeast two-hybrid assays confirmed the in vitro physical interaction between the MsAHL13 and MsMYB1 proteins. This research offers an important theoretical basis for further exploration of the functional mechanisms of this gene family in responding to environmental stresses.

Background: Plant architecture, particularly branching patterns, plays a crucial role in plant growth, photosynthetic performance, and yield. Spur-type apple, characterized by compact growth, early fruiting, high productivity, and manageable canopy structure, represent valuable germplasm for establishing dwarf and high-density apple orchards. While hybrid breeding of spur-type varieties offers significant potential for genetic advancement, severe segregation of traits in hybrid progeny and the difficulty of combining multiple favorable traits still significantly limit breeding efficiency. Moreover, the genetic basis and molecular mechanisms of the spur-type trait remain poorly understood at the genomic level, hindering the development of precise molecular breeding approaches. Methods: To address this, we used the spur-type line ‘0301-13-14’ and the non-spur-type line ‘0301-50-32’ from hybrid progenies of the spur-type cultivars ‘Miyazaki Spur Fuji’ and ‘Starkrimson’ to elucidate the regulatory mechanisms underlying apple branch formation and spur-type trait development by characterizing their branching traits, performing whole-genome resequencing analysis, and identifying candidate genes using bioinformatics analyses. Results: Anatomical observations revealed that the spur-type line ‘0301-13-14’ possessed smaller cells with a more compact spatial arrangement compared to the non-spur-type line ‘0301-50-32’. Whole-genome resequencing generated 5,003,968 high-quality single-nucleotide polymorphisms (SNPs) and 577,886 high-quality insertions/deletions (InDels). We further identified 29,157 candidate genes harboring predicted deleterious mutations (classified as high or moderate impact). Gene Ontology (GO) enrichment analysis indicated that genes associated with the spur-type trait were mainly enriched in molecular function and biological process categories. Specifically, variant genes related to molecular function were enriched in transferase and catalytic activities, while those in biological process were mainly involved in phosphorylation and phosphorus metabolism. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that candidate genes were significantly enriched in environmental information processing and metabolic pathways. Conclusions: These results will provide a genomic foundation for identifying genes controlling spur-type branching traits and facilitate the genetic improvement of spur-type apple.

KNOTTED1-LIKE HOMEOBOX (KNOX) genes are conserved transcription factors that play crucial roles in plant growth, development, and stress responses. However, systematic characterization of the KNOX family in Malus sieversii, a valuable germplasm resource with outstanding stress tolerance and flavonoid accumulation, remains lacking. In this study, we performed a genome-wide identification of the KNOX gene family in M. sieversii and identified 21 MsiKNOX genes. Phylogenetic analysis classified these genes into three subfamilies (Class I, II, and M), with structural features and motif compositions consistent with those of their orthologs in Arabidopsis thaliana and cultivated apple. Chromosomal localization revealed an uneven distribution across 13 chromosomes, and synteny analysis indicated both conserved evolution and lineage-specific expansion of the KNOX family in M. sieversii. Promoter cis-element analysis suggested that MsiKNOX genes are potentially involved in responses to multiple abiotic stresses and hormone signaling. Expression profiling under ABA and GA treatments showed that most MsiKNOX genes responded differentially to these phytohormones. Notably, MsiKNOX09 was significantly upregulated by ABA and downregulated by GA, and was further shown to physically interact with the anthocyanin-associated MsiMYB1 in yeast two-hybrid and split-luciferase assays. These findings provide a comprehensive overview of the KNOX gene family in M. sieversii and suggest that MsiKNOX09 acts as a hormone-responsive regulator and may participate in MsiMYB1-mediated regulatory pathways.