# Evolutionary Insight and Expression Pattern of WUSCHEL‐Related Homebox Genes of Dendrobium huoshanense

## TL;DR

This study identifies nine WOX genes in Dendrobium huoshanense, analyzing their evolutionary patterns, expression, and roles in development and stress response.

## Contribution

The study provides the first comprehensive analysis of WOX genes in Dendrobium huoshanense, including phylogenetic classification and expression patterns.

## Key findings

- Nine WOX genes were identified and classified into three classical clades.
- All DhWOX genes are under purifying selection with evolutionary events dating back to 363 MYA.
- Promoter cis-elements are linked to hormone signaling, development, and stress responses.

## Abstract

The WOX (WUSCHEL‐related homebox) gene family is critical for plant growth, development, and the regulation of stress responses; however, the function of Dendrobium huoshanense WOX has not been extensively studied. Nine WOX genes have been identified in the D. huoshanense genome. These WOX genes were unequally distributed on five chromosomes, with four WOX genes occupying chr1. A phylogenetic tree of D. huoshanense 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 Dendrobium relatives revealed that D. huoshanense had more translocations and inversions with D. nobile than D. chrysotoxum. Intraspecific collinearity analysis of D. huoshanense based on the MCScanX revealed no large‐scale WGD and segmental duplications between these WOX genes. The Ka/Ks ratio and calculated timeline (MYA), indicate that all DhWOX genes were subject to purifying selection. Interspecies microsynteny analysis revealed that D. huoshanense shares more gene pairs with two closely related species, consistent with their genetic relationships. Gene structure analysis showed that DhWOX1, DhWOX2, DhWOX6, and DhWOX8 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. DhWOX4 showed high expression at 2 and 4 h, whereas DhWOX6 and DhWOX7 showed dynamic fluctuations. DhWOX8 showed consistently low expression, whereas DhWOX9 expression was highest at 2 and 4 h. DhWOX1 and DhWOX3 showed no detectable expression levels. The subcellular localization results indicated that DhWOX2 and DhWOX6 genes were expressed in the nucleus. These findings contribute to our understanding of the roles of these genes in D. huoshanense.

Nine WOX genes were identified in D. huoshanense. The phylogenetic tree suggests they belong to three classical clades. All DhWOX genes are subject to purifying selection, with replication times dating back as early as 363 MYA. D. huoshanense has more gene pairs with its relative species, consistent with their genetic relationship. The cis‐regulatory elements in promoter regions are mainly associated with hormone signaling, vegetation development stages, and stress responses.