Identification and Functional Analysis of <i>WOX</i> Genes in <i>Macadamia</i> <scp>spp.</scp> Reveal <i>WOX1</i> and <i>WOX4</i> Homologs Involved in Shoot Regeneration

Macadamia spp., high-value forest crops, face considerable challenges in increasing yields due to inefficient clonal propagation of superior varieties, which severely limits industrial development. This study focused on the WUSCHEL-related homeobox (WOX) family, which includes key regulators of plant regeneration. We systematically identified the members of the WOX family in four cultivated varieties of the "smooth-shelled" type and in one species of the "rough-shelled" type. We demonstrated the presence of 19 WOX family members in M. integrifolia "GR1." Expression analysis revealed that the WUS clade members MiWOX1b, 4a, and 4b were highly expressed in shoot apices, roots, and calli. Functional validation experiments demonstrated that overexpressing MiWOX1b, 4a, and 4b significantly enhanced de novo root regeneration (DNRR) in Nicotiana benthamiana. The hypocotyls of MiWOX1b- and MiWOX4a-overexpressing N. benthamiana seedlings spontaneously produced ectopic buds on hormone-free Murashige and Skoog medium in a dose-dependent manner, suggesting their involvement in de novo shoot regeneration (DNSR). Transcriptomic analysis further revealed that these genes promoted regeneration by activating auxin biosynthesis genes, such as TAR1 and YUC2/4, and upregulating the regeneration-associated genes GRFs and BBM. Moreover, MiWOX1b-assisted transformation of woody oil crop Plukenetia volubilis resulted in transgenic shoots. These findings highlight the potential of these genes for forest genetic engineering while providing novel insights into the molecular mechanisms of regeneration in woody plants.

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