Pan-genome Analysis of WOX Gene Family and Function Exploration of CsWOX9 in Cucumber

Cucumber is an economically important vegetable crop, and the warts (composed of spines and Tubercules) of cucumber fruit are an important quality trait that influences its commercial value. WOX transcription factors are known to have pivotal roles in regulating various aspects of plant growth and development, but their studies in cucumber are limited. Here, genome-wide identification of cucumber WOX genes was performed using the pan-genome analysis of 12 cucumber varieties. Our findings revealed diverse CsWOX genes in different cucumber varieties, with variations observed in protein sequences and lengths, gene structure, and conserved protein domains, possibly resulting from the divergent evolution of CsWOX genes as they adapt to diverse cultivation and environmental conditions. Expression profiles of the CsWOX genes demonstrated that CsWOX9 was significantly expressed in unexpanded ovaries, especially in the epidermis. Additionally, analysis of the CsWOX9 promoter revealed two binding sites for the C2H2 zinc finger protein. We successfully executed a yeast one-hybrid assay (Y1H) and a dual-luciferase (LUC) transaction assay to demonstrate that CsWOX9 can be transcriptionally activated by the C2H2 zinc finger protein Tu, which is crucial for fruit Tubercule formation in cucumber. Overall, our results indicated that CsWOX9 is a key component of the molecular network that regulates wart formation in cucumber fruits, and provide further insight into the function of CsWOX genes in cucumber.