Functional Analysis of CsWOX4 Gene Mutation Leading to Maple Leaf Type in Cucumber (Cucumis sativus L.)

The leaf morphology is an important agronomic trait in crop production. Our study identified a maple leaf type (<i>mlt</i>) cucumber mutant and located the regulatory gene for leaf shape changes through BSA results. Hybrid F1 and F2 populations were generated by F1 self-crossing, and the candidate <i>mlt</i> genes were identified within the 2.8 Mb region of chromosome 2 using map cloning. Through the sequencing and expression analysis of genes within the bulk segregant analysis (BSA) region, we identified the target gene for leaf shape regulation as <i>CsWOX4</i> (CsaV3_2G026510). The change from base C to T in the original sequence led to frameshift mutations and the premature termination of translation, resulting in shortened encoded proteins and conserved WUSCHEL (<i>WUS</i>) box sequence loss. The specific expression analysis of the <i>CsWOX4</i>/<i>Cswox4</i> genes in the roots, stems, leaves and other tissue types of wild-type (WT) and mutant plants revealed that <i>CsWOX4</i> was higher in the root, but <i>Cswox4</i> (mutant gene) was significantly higher in the leaf. Subcellular localization analysis revealed that CsWOX4 was localized in the nucleus. RNA-seq analysis revealed that the differentially expressed genes were mainly enriched in the mitochondrial cell cycle phase transition, nucleosome and microtubule binding pathways. Simultaneously, the quantitative analysis of the expression trends of 25 typical genes regulating the leaf types revealed the significant upregulation of <i>CsPIN3</i>. In our study, we found that the conserved domain of CsWOX4 was missing in the mutant, and the transcriptome data revealed that the expression of some genes, such as <i>CsPIN3,</i> changed simultaneously, thereby jointly regulating changes in the cucumber leaf type.