Genome-wide Analysis of the WUSCHEL-related Homeobox Gene Family and Functional Characterization of VcWOX4b Regarding the Inhibition of Adventitious Root Formation in Blueberry (Vaccinium Spp.)

Background: Blueberry (Vaccinium corymbosum L.) is one of the most important commercial fruit tree species. The development of high-quality seedlings is a prerequisite for fruit production. Stem cutting and tissue culture methods are widely applied for propagating blueberry seedlings. Both methods require adventitious roots (ARs), indicating ARs are critical for vegetative propagation. However, the underlying factors and molecular mechanisms regulating blueberry AR formation remain relatively unknown. Results: In this study, the rooting abilities of differentially lignified cuttings from various cultivars or the same cultivars cultured differently were evaluated following an indole-3-butyric acid (IBA) treatment. Field-grown semi-lignified and tissue culture-grown cuttings formed ARs, but the latter had more pericycle and secondary xylem cells and formed ARs more easily and faster. WUSCHEL-related homeobox genes are commonly involved in vascular tissue development and early root meristem maintenance. On the basis of the available Vaccinium corymbosum genome data, 29 putative WOX genes with conserved homeodomains were identified and divided into three major clades (modern/WUS, intermediate, and ancient). These 29 WOX genes were differentially expressed in the root, shoot, leaf, flower bud, and fruit. Additionally, a qRT-PCR analysis revealed that five selected VcWOX genes were responsive to an IBA treatment during AR formation. Accordingly, VcWOX4b was functionally characterized. The overexpression of VcWOX4b in transgenic tobacco inhibited AR formation by altering vascular cell division and differentiation and the indole-3-acetic acid (IAA):cytokinin (CTK) ratio. These observations suggest that VcWOX4b regulates the IAA:CTK ratio to promote primary xylem cell differentiation, thereby inhibiting AR formation. However, an IBA treatment can induce AR formation by inhibiting VcWOX4b expression. Conclusions: Current study elucidates the rooting abilities of various cultivars and the cytological characters of influence on AR formation of blueberry cuttings, which may provide novel insights into the selection of high-quality blueberry cuttings. VcWOX4b, VcWOX8/9a, VcWOX11/12c, and VcWOX13b might regulate blueberry AR formation in an IBA-dependent manner. Ectopic expression of VcWOX4b modulated the IAA:CTK ratio to promotes primary xylem cell differentiation, but inhibit secondary xylem cell differentiation, ultimately leading to decreased AR formation.