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.

澳洲坚果（Macadamia spp.）作为一种高价值木本经济作物，因无性繁殖效率低下面临诸多严峻挑战，严重制约了其产业发展。本研究以植物再生关键调控因子WUSCHEL相关同源框（WUSCHEL-related homeobox, WOX）基因家族为研究对象，对4个澳洲坚果品种中的WOX家族成员开展系统鉴定。在优良品种‘桂热1号’（M. integrifolia GUIRE1）中，共鉴定得到19个WOX基因。表达谱分析结果显示，WUS进化枝成员MiWOX1c、MiWOX4a和MiWOX4b在分生组织与愈伤组织中高表达，提示其与植株再生过程存在功能关联。功能验证实验表明，过表达MiWOX1c与MiWOX4a/b可显著提升本氏烟草（Nicotiana benthamiana）的不定根再生能力，并能诱导其下胚轴自发形成异位芽。转录组分析进一步揭示，上述基因可通过激活生长素生物合成通路（TAR1、TIR1、YUC2/4）并上调再生相关基因（GRFs、BBM）的表达，进而促进植株再生。尤为关键的是，MiWOX1c可成功在木本油料作物星油藤（Plukenetia volubilis）中诱导出转基因植株再生，验证了其跨物种应用潜力。本研究为解析木本植物再生的分子机制提供了全新视角，并为澳洲坚果等顽拗型物种的遗传改良提供了候选基因资源。