Genome-Wide Analyses of the XTH Gene Family in Brachypodium distachyon and Functional Analyses of the Role of BdXTH27 in Root Elongation

Abstract

Xyloglucan endotransglucosylase/hydrolases (XTHs) are a class of cell wall-associated enzymes involved in the construction and remodeling of cellulose/xyloglucan crosslinks. However, knowledge of this gene family in the model monocot Brachypodium distachyon is limited. A total of 29 BdXTH genes were identified from the whole genome, and these were further divided into three subgroups (Group I/II, Group III, and the Ancestral Group) through evolutionary analysis. Gene structure and protein motif analyses indicate that closely clustered BdXTH genes are relatively conserved within each group. A highly conserved amino acid domain (DEIDFEFLG) responsible for catalytic activity was identified in all BdXTH proteins. We detected three pairs of segmentally duplicated BdXTH genes and five groups of tandemly duplicated BdXTH genes, which played vital roles in the expansion of the BdXTH gene family. Cis-elements related to hormones, growth, and abiotic stress responses were identified in the promoters of each BdXTH gene, and when roots were treated with two abiotic stresses (salinity and drought) and four plant hormones (IAA, auxin; GA3, gibberellin; ABA, abscisic acid; and BR, brassinolide), the expression levels of many BdXTH genes changed significantly. Transcriptional analyses of the BdXTH genes in 38 tissue samples from the publicly available RNA-seq data indicated that most BdXTH genes have distinct expression patterns in different tissues and at different growth stages. Overexpressing the BdXTH27 gene in Brachypodium led to reduced root length in transgenic plants, which exhibited higher cellulose levels but lower hemicellulose levels compared to wild-type plants. Our results provide valuable information for further elucidation of the biological functions of BdXTH genes in the model grass B. distachyon.

Keywords: Brachypodium distachyon; gene expression; genome-wide analysis; xyloglucan endotransglucosylase/hydrolase.

Figure 1

Subcellular localization of the BdXTH10 and BdXTH27 proteins. The control (35S-GFP) and fusion vectors (35S::BdXTH10-GFP and 35S::BdXTH27-GFP) were transiently expressed separately in onion epidermal cells using agrobacterium-mediated transfection. CW, cell wall; PR, protoplast. scale bar = 50 μm.

Figure 2

Evolutionary relationships among XTH proteins from Brachypodium and three other plant species. The tree was constructed with the Neighbor Joining (NJ) method as implemented in MEGA-X v10.1 software, and branch confidence was estimated by bootstrapping with 1000 replicates. The XTH proteins are classified into three major clades (Group I/II, Group III, and the Ancestral Group). Proteins in Group I/II and the Ancestral Group are shown with purple and blue backgrounds, respectively. Group III is further divided into two subclades, Group III-A and Group III-B, which are indicated with red and green backgrounds, respectively.

Figure 3

Unrooted neighbor-joining phylogenetic tree, conserved protein motifs, and structural analysis of BdXTH genes. (a) Evolutionary relationships of the XTH proteins in Brachypodium. Proteins from the four clades (Group I/II, Group III-A, Group III-B, and the Ancestral Group) are color coded as in Figure 1. (b) The structures of the 29 putative BdXTH genes. The UTRs, exons, and introns are represented by green boxes, yellow boxes, and black lines, respectively. (c) Conserved motif analysis of the BdXTH proteins. The different motifs are indicated by different colored boxes numbered from motif 1 to motif 20. The structural features of the 20 motifs are shown in Figure S1.

Figure 4

The physical locations of BdXTH genes on the five Brachypodium chromosomes. Tandemly duplicated gene pairs and segmentally duplicated genes are linked by red lines. The chromosome numbers are displayed at the top of each chromosome, and the scale in megabases (Mb) is shown on the left.

Figure 5

Structure-based sequence alignment of BdXTH proteins. The structures of two proteins (PttXET16-34, PDB id: 1UN1; TmNXG1, PDB id: 2UWA) have been experimentally determined. Proteins in Group I/II and the Ancestral Group had similar structures to 1UN1, and proteins in Group III show similar structures to 2UWA. The active site (ExDxE) and loops 1, 2, and 3 are underlined in black and green, respectively. The N-glycosylation site residues are indicated by asterisks. Proteins in Group I/II and the Ancestral Group are shown with purple and blue backgrounds, respectively. Group III is further divided into two subclades, Group III-A and Group III-B, which are indicated with red and green backgrounds, respectively.

Figure 6

Numbers of cis-acting elements in the promoter regions of the 29 BdXTH genes. Three types of cis-acting elements in the 2000 bp of DNA sequence upstream of the promoter regions are shown in the figure, including phytohormone- and environmentally responsive elements, and plant growth and development-related elements. Members of the different element classes are shown at the top of the figure in different shades of gray. Genes in Group I/II and the Ancestral Group are shown with purple and blue backgrounds, respectively. Group III is further divided into two subclades, Group III-A and Group III-B, which are indicated with orange and green backgrounds, respectively.

Figure 7

Expression analysis of BdXTH genes under different conditions. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of BdXTH gene expression in response to abiotic stresses (drought and salinity) (a), and phytohormone treatments (ABA, BR, IAA, and GA3) (b). The means ± SD of three biological replicates are present.

Figure 8

Heat map showing the expression pattern of BdXTH genes in Brachypodium. Expression profiles from various tissues and developmental stages were downloaded from the NCBI database. The relative expression levels are represented by the colored bars. Red and green boxes indicate high and low expression levels, respectively.

Figure 9

Functional analyses of the BdXTH27 gene in B. distachyon. (A) Phenotype of the wild-type (WT) and transgenic Brachypodium plants over-expressing the BdXTH27 gene (OE-1, OE-2, and OE-3), scale bar = 1 cm; (B) Root paraffin sections of the BdXTH27 over-expression and wild-type lines, scale bar = 100 µm; (C) Relative expression level of BdXTH27 gene in roots and (D) root length in transgenic lines (OE-1, OE-2, and OE-3) and wild-type (WT). (E) The contents of cellulose and (F) hemicellulose, and the expression levels of the seven cellulose synthase genes (G) of wild-type (WT) and transgenic lines. * p < 0.05, ** p < 0.01, *** p < 0.001.