Identification and Evolution of the WUSCHEL-Related Homeobox Protein Family in Bambusoideae

Abstract

Bamboos (Bambusoideae) are fast-growing species due to their rapid growth rate and ability to reproduce annually via cloned buds produced on the rhizome. WUSCHEL-related homeobox (WOX) genes have been reported to regulate shoot apical meristem organization, lateral organ formation, cambium and vascular proliferation, and so on, but have rarely been studied in bamboos. In this study, the WOXs of both herbaceous bamboo species (12 OlaWOXs and nine RguWOXs) and woody bamboo species (18 GanWOXs, 27 PheWOXs, and 26 BamWOXs) were identified and categorized into three clades based on their phylogenetic relationship-ancient, intermediate, or WUS clade. Polyploidy is the major driver of the expansion of the bamboo WOX family. Eight conserved domains, besides the homeodomain, were identified by comparatively analyzing the WOXs of dicot and monocot species. Intensive purifying selection pressure in the coding region of specific domains explained the functional similarity of WOXs between different species. For Bambusoideae WOXs, polyploidy is the major driver of the expansion of the WOX family. Stronger purifying selection was found in orthologous WOXs of Bambusoideae, especially for WOX4s and WOX5s, which are conserved not only at the translational levels, but also at the genome level. Several conserved cis-acting elements were discovered at similar position in the promoters of the orthologous WOXs. For example, AP2/ERF protein-binding elements and B3 protein-binding elements were found in the promoters of the bamboo WOX4, while MYB protein-binding elements and Dof protein-binding elements were found in the promoters of bamboo WOX5, and MADS protein-binding sites was found in the promoters of bamboo WUS, WOX3, and WOX9. These conserved positions may play an important role in regulating the expression of bamboo WOXs. Our work provides insight into the origin and evolution of bamboo WOXs, and will facilitate functional investigations of the clonal propagation of bamboos.

Keywords: WOXs; bambusoideae; polyploidization; selection pressure.

Figure 1

The conserved domains identified in the WUSCHEL-related homeobox (WOX) family. Eleven conserved domains were identified among 15 AtWOXs, 18 PotriWOXs, 13 OsWOXs, 20 ZmWOXs, 27 PheWOXs, 13 BradiWOXs, 12 OlaWOXs, 9 RguWOXs, 18 GanWOXs, and 26 BamWOXs by Multiple Em for Motif Elicitation (MEME). Motifs 4-6 were identified in the WOXs belonging to the ancient clade. Motifs 7-10 were identified in the WOXs belonging to the intermediate clade. Motif 11 was found in the WOX belonging to the WUS clade.

Figure 2

The phylogenetic tree of WOXs from dicots and monocots. The neighbor-joining tree was constructed using amino acid sequences of WOXs from Arabidopsis thaliana (At), Populus trichocarpa (Potri), Oryza sativa (Os), Zea mays (Zm), Brachypodium distachyon (Bradi), Micromonas pusilla (Micpu), Micromonas sp. (Micsp), Ostreococcus tauri, (Osta), and Bathycoccus prasinos (Bathy), with bootstrap assessment of 1000 replicates. Each clade was assigned a different colored background. Leaf colors of black, orange, and cyan stand for WOXs of chlorophytes, monocots, and dicots, respectively. The graphic symbols of the conserved motifs are colored and termed the same as in Figure 1.

Figure 3

The phylogenetic tree of Bambusoideae WOXs. The neighbor-joining tree was constructed by the full-length CDS of Bambusoideae WOXs with bootstrap assessment of 1000 replicates. Each clade was assigned a different colored background. Green leaves of the phylogenetic tree stand for WOXs of herbaceous bamboos. Brown leaves of the phylogenetic tree stand for WOXs of woody bamboos. The protein domains and gene structures are drawn following each WOX. The name of each domain is the same as in Figure 1.

Figure 4

Sliding window of the WOX13 genes of the ancient clade. (a) The sliding window of orthologous WOX13s between P. edulis and Oryza sativa. (b) The sliding window of orthologous WOX13s between P. edulis and Brachypodium distachyon. (c–e) The sliding window of paralogous WOX13s of P. edulis, Zea mays, and Populus trichocarpa, respectively.

Figure 5

Sliding window of the WOX8/9 genes of the intermediate clade. (a) The sliding window of paralogous genes of WOX8/9. (b) The sliding window of orthologous genes of WOX8s. (c) The sliding window of orthologous genes of WOX9s.

Figure 6

The duplications of the Bambusoideae WOXs of herbaceous and woody bamboos. The colored symbols represent different members of the WOX family. Each symbol indicates a homologous gene pair on the Ks distribution plot. The y-axis indicates the calculated Ks value of each gene pair.

Figure 7

The conserved cis-element distribution in the promoters of Bambusoideae WOX4 (a), WOX5 (b), and WOX3 and WUS (c). The 1500-bp-long promoters were analyzed by PlantRegMap. The ID of the cis-elements was in accordance with the transcription factor (TF) binding motif dataset of PlantTFDB and is shown with different colored rectangles. The TFs were predicted by comparing homologous genes with TF datasets of Arabidopsis (http://planttfdb.cbi.pku.edu.cn/index.php?sp=Ath).