Evolutionary Insight and Expression Pattern of <scp>WUSCHEL</scp>‐Related Homebox Genes of <i>Dendrobium huoshanense</i>

The <i>WOX</i> (<i>WUSCHEL-related homebox</i>) gene family is critical for plant growth, development, and the regulation of stress responses; however, the function of <i>Dendrobium huoshanense</i> WOX has not been extensively studied. Nine <i>WOX</i> genes have been identified in the <i>D. huoshanense</i> genome. These <i>WOX</i> genes were unequally distributed on five chromosomes, with four <i>WOX</i> genes occupying chr1. A phylogenetic tree of <i>D. huoshanense</i> and five other species was built based on the maximum likelihood method, suggesting that these WOX proteins could be grouped into three classical clades. Structural variation analysis of the three <i>Dendrobium</i> relatives revealed that <i>D. huoshanense</i> had more translocations and inversions with <i>D. nobile</i> than <i>D. chrysotoxum</i>. Intraspecific collinearity analysis of <i>D. huoshanense</i> based on the MCScanX revealed no large-scale WGD and segmental duplications between these <i>WOX</i> genes. The Ka/Ks ratio and calculated timeline (MYA), indicate that all <i>DhWOX</i> genes were subject to purifying selection. Interspecies microsynteny analysis revealed that <i>D. huoshanense</i> shares more gene pairs with two closely related species, consistent with their genetic relationships. Gene structure analysis showed that <i>DhWOX1</i>, <i>DhWOX2</i>, <i>DhWOX6</i>, and <i>DhWOX8</i> contained 3' or 5' UTR. Conserved motif analysis revealed that these WOX contain conserved homeodomain domains and similar components. Multiple sequence alignments showed that the homeodomain near the C-terminus has a typical helix-turn-helix structure and amino acid composition, while DhWOX3 uniquely contains WUS-box motif. Protein-protein interaction analysis showed that DhWOX5 may be co-expressed with DhWOX6 and DhWOX9. A large number of cis-acting elements in the promoter regions associated with hormone signaling, vegetation development stages, and stress responses. Differential gene expression was analyzed after MeJA treatment. <i>DhWOX4</i> showed high expression at 2 and 4 h, whereas <i>DhWOX6</i> and <i>DhWOX7</i> showed dynamic fluctuations. <i>DhWOX8</i> showed consistently low expression, whereas DhWOX9 expression was highest at 2 and 4 h. <i>DhWOX1</i> and <i>DhWOX3</i> showed no detectable expression levels. The subcellular localization results indicated that <i>DhWOX2</i> and <i>DhWOX6</i> genes were expressed in the nucleus. These findings contribute to our understanding of the roles of these genes in <i>D. huoshanense</i>.