Genome-Wide Identification of the DOF Gene Family in Kiwifruit (Actinidia chinensis) and Functional Validation of AcDOF22 in Response to Drought Stress

Abstract

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 <i>AcDOF</i> genes and their regulatory elements in kiwifruit (<i>Actinidia chinensis</i>) has not been thoroughly investigated. In this study, we screened the kiwifruit genome database and identified 42 <i>AcDOF</i> genes (<i>AcDOF1</i> to <i>AcDOF42</i>). Phylogenetic analysis facilitated the categorization of these genes into five subfamilies (<i>DOF</i>-a, <i>DOF</i>-b, <i>DOF</i>-c, <i>DOF</i>-d, and <i>DOF</i>-e). We further analyzed the motifs, conserved domains, gene structures, and collinearity of the <i>AcDOF</i>gene 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 <i>AcDOF</i> promoters. RNA-seq expression profiles demonstrated the tissue-specific expression of <i>AcDOF</i> genes. Quantitative real-time PCR results showed that six selected genes (<i>AcDOF04</i>, <i>AcDOF09</i>, <i>AcDOF11</i>, <i>AcDOF13</i>, <i>AcDOF21</i>, and <i>AcDOF22</i>) were differentially induced by abscisic acid (ABA), methyl jasmonate (MeJA), and cold, salt, and drought stresses, with <i>AcDOF22</i> specifically expressed at high levels in drought-tolerant cultivars. Further experiments indicated that transient <i>AcDOF22</i> overexpression in kiwifruit leaf disks reduced water loss and chlorophyll degradation. Additionally, <i>AcDOF22</i> was localized to the nucleus and exhibited transcriptional activation, enhancing drought resistance by activating the downstream drought marker gene <i>AcDREB2A</i>. These findings lay the foundation for elucidating the molecular mechanisms of drought resistance in kiwifruit and offer new insights into drought-resistant breeding.