Identification of WUSCHEL-related homeobox gene in Macadamia and functional analysis revealed three WUS clade members linked to regeneration
Macadamia (Macadamia spp.), a high-value woody crop, faces significant challenges due to inefficient clonal propagation, which severely limits industry development. This study focused on the WUSCHEL-related homeobox (WOX) gene family, key regulators of plant regeneration, to systematically identify WOX members across four Macadamia cultivars. A total of 19 WOX genes were characterized in the elite cultivar M. integrifolia GUIRE1. Expression profiling revealed that WUS clade members MiWOX1c, MiWOX4a, and MiWOX4b were highly expressed in meristematic and callus tissues, suggesting their functional relevance to regeneration. Functional validation demonstrated that overexpression of MiWOX1c and MiWOX4a/b significantly enhanced adventitious root regeneration in Nicotiana benthamiana and induced spontaneous ectopic shoot formation in hypocotyls. Transcriptomic analysis further revealed that these genes promote regeneration by activating auxin biosynthesis pathways (TAR1, TIR1, YUC2/4) and upregulating regeneration-associated genes (GRFs, BBM). Crucially, MiWOX1c successfully induced transgenic shoot regeneration in the woody oil crop Plukenetia volubilis, validating its cross-species applicability. This work provides novel insights into the molecular mechanisms of regeneration in woody plants and offers candidate genes for genetic improvement of recalcitrant species like Macadamia.