Identification and characterization of the WOX Gene Family revealed two WUS Clade Members associated with embryo development in Cunninghamia lanceolata

The WUSCHEL-related homeobox (WOX) gene family is vital for plant development and stress response. In this study, we conducted a comprehensive analysis of WOX genes in Cunninghamia lanceolata (C. lanceolata) and subsequently explored the potential roles of two ClWOX genes within the WUS clade. In total, six ClWOX genes were identified through a full-length transcriptome analysis. These genes, exhibiting conserved structural and functional motifs, were assigned to the ancient clade and Modern/WUS clade, respectively, through a phylogenetic analysis. Our expression analysis indicated that these ClWOX genes were highly expressed in the middle and late developmental stages of zygotic embryos in C. lanceolata. Moreover, only ClWOX5 and ClWOX6 within the Modern/WUS clade exhibited transcriptional activity, and their expressions were also induced in response to auxin and wounding. Overexpression of ClWOX5 and ClWOX6 in Arabidopsis caused a partially sterile phenotype, resulting in a very low seed setting rate. Transcriptomic analysis revealed that expressions of many embryo-defective (EMB) genes, phytohormone-related genes, and transcription factors (TFs) were dramatically altered in ClWOX5 and ClWOX6 transgenic plants, which suggested that ClWOX5 and ClWOX6 may play specific important roles in embryo development via complex gene networks. In addition, overexpression of ClWOX5 and ClWOX6 in leaf segments promoted shoot regeneration in tobacco, indicating that ClWOX5 and ClWOX6 can promote plant regeneration and could be used to improve genetic transformation. In conclusion, these results help to elucidate the function of the WOX gene and provide a valuable basis for future studies of the developmental regulation and applications of WOX genes in C. lanceolata.