Overexpression of <i>HmWOX8</i> promotes callus proliferation and shoot regeneration by regulating hormone signaling and shoot development-related genes

The remarkable shoot regeneration capacity is essential for prosperous genetic transformation. Previous studies have shown that WUSCHEL-related homeobox (WOX) transcription factor plays a crucial role in callus growth, shoot regeneration and root development, However, the mechanisms and functions of shoot regeneration related to WOX8 remain unclear. In the current research, the HmWOX8 gene was isolated from Hemerocallis middendorffii by RACE (Rapid-amplification of cDNA ends) technology. Overexpression of HmWOX8 improved callus proliferation and shoot regeneration ability of Arabidopsis and rice, whereas silencing HmWOX8 in H. middendorffii resulted in the inverse correlation. Transcriptome analysis revealed that HmWOX8 enhances the efficiency of callus proliferation and shoot regeneration through two different regulation ways, including hormone signaling pathways and shoot development-related genes. (I) HmWOX8 regulates crosstalk among different hormone signaling pathways by activating and inhibiting the expression of different genes in these pathways, thus ensuring signal integration for efficient callus proliferation and shoot regeneration. (II) HmWOX8 can upregulate the expression level of shoot developmental genes, including WOX5/7, BBM, AIL5/7, PLT1, PIN6, CUC3 and SCR14/30, to regulate shoot emergence and outgrowth. In addition, Yeast two-hybrid assays and Bimolecular fluorescence complementation assay suggested that HmWOX8 directly interacts with HmCUC2, thereby promoting shoot regeneration. Our research improves the understanding of molecular mechanisms for HmWOX8-mediated regeneration, and provides valuable gene resources for breeding programs to promote plant regeneration.