DNA-binding one zinc finger (DOF) transcription factors are crucial plant-specific regulators involved in growth, development, signal transduction, and abiotic stress response generation. However, the genome-wide identification and characterization of AcDOF genes and their regulatory elements in kiwifruit (Actinidia chinensis) has not been thoroughly investigated. In this study, we screened the kiwifruit genome database and identified 42 AcDOF genes (AcDOF1 to AcDOF42). Phylogenetic analysis facilitated the categorization of these genes into five subfamilies (DOF-a, DOF-b, DOF-c, DOF-d, and DOF-e). We further analyzed the motifs, conserved domains, gene structures, and collinearity of the AcDOFgene family. Gene ontology (GO) enrichment analysis indicated significant enrichment in the “flower development” term and the “response to abiotic stress” category. Promoter prediction analysis revealed numerous cis-regulatory elements related to responses to light, hormones, and low-temperature and drought stress in AcDOF promoters. RNA-seq expression profiles demonstrated the tissue-specific expression of AcDOF genes. Quantitative real-time PCR results showed that six selected genes (AcDOF04, AcDOF09, AcDOF11, AcDOF13, AcDOF21, and AcDOF22) were differentially induced by abscisic acid (ABA), methyl jasmonate (MeJA), and cold, salt, and drought stresses, with AcDOF22 specifically expressed at high levels in drought-tolerant cultivars. Further experiments indicated that transient AcDOF22 overexpression in kiwifruit leaf disks reduced water loss and chlorophyll degradation. Additionally, AcDOF22 was localized to the nucleus and exhibited transcriptional activation, enhancing drought resistance by activating the downstream drought marker gene AcDREB2A. These findings lay the foundation for elucidating the molecular mechanisms of drought resistance in kiwifruit and offer new insights into drought-resistant breeding.

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.

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.

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.