Cloning and Characterization of WOX4 Gene from Vitis vinifera L. Involved in Stem Cell Regulation

Wuschel-related homeobox (WOX) genes play essential, specific, and sometimes redundant roles in plant embryo development, shoot and root meristem maintenance, and plant development. Though much information was quickly gained with members of the WOX gene family of Arabidopsis, monocotyledonous crops, and gymnospermous conifers, little is known about perennial woody plants. In this study, we isolated the first WOX gene family member from grape (Vitis vinifera L. cv. Cabernet Sauvignon), and named it VvWOX4 based on its characteristic domains and phylogenetic analysis. The identity of VvWOX4 was validated by MALDI-TOF MS and Western blot with polyclonal antibody against Arabidopsis thaliana Wuschel. Functional analysis showed that VvWOX4 markedly increased shoot primordia structures when overexpressed under CaMV 35S promoter in tobacco. A different expression pattern was found for VvVOX4 compared with AtWUCHEL and its expression was detected in unique organs of grapevines. Besides the expression in the vegetative shoot apical meristem (SAM) of grape shoot tips, VvWOX4 is expressed in dormant winter buds, inflorescence, young leaves, and tendril tips, but not in root tips. In young leaves, the expression of VvWOX4 is strongly upregulated by wounding, and also by plant growth regulators such as 2 mg L−1 2,4-D, 1 mg L−1 NAA and 1 mg L−1 BAP treatments, while downregulation was monitored by 1 mg L−1 IBA treatment, and there was no response to 0.5 mg L−1 GA3 treatment. Together, our results revealed the first member of grape WOX gene family and indicated different roles and regulation of VvWOX4 in the perennial woody crop grapevine.