Genome-Wide Identification and Characterization of the Trehalose-6-Phosphate Synthetase Gene Family in Chinese Cabbage (Brassica rapa) and Plasmodiophora brassicae during Their Interaction

Abstract

Trehalose is a nonreducing disaccharide that is widely distributed in various organisms. Trehalose-6-phosphate synthase (TPS) is a critical enzyme responsible for the biosynthesis of trehalose, which serves important functions in growth and development, defense, and stress resistance. Although previous studies have found that the clubroot pathogen Plasmodiophora brassicae can lead to the accumulation of trehalose in infected Arabidopsis organs, it has been proposed that much of the accumulated trehalose is derived from the pathogen. At present, there is very little evidence to verify this view. In this study, a comprehensive analysis of the TPS gene family was conducted in Brassica rapa and Plasmodiophora brassicae. A total of 14 Brassica rapa TPS genes (BrTPSs) and 3 P. brassicae TPS genes (PbTPSs) were identified, and the evolutionary characteristics, functional classification, and expression patterns were analyzed. Fourteen BrTPS genes were classified into two distinct classes according to phylogeny and gene structure. Three PbTPSs showed no significant differences in gene structure and protein conserved motifs. However, evolutionary analysis showed that the PbTPS2 gene failed to cluster with PbTPS1 and PbTPS3. Furthermore, cis-acting elements related to growth and development, defense and stress responsiveness, and hormone responsiveness were predicted in the promoter region of the BrTPS genes. Expression analysis of most BrTPS genes at five stages after P. brassicae interaction found no significant induction. Instead, the expression of the PbTPS genes of P. brassicae was upregulated, which was consistent with the period of trehalose accumulation. This study deepens our understanding of the function and evolution of BrTPSs and PbTPSs. Simultaneously, clarifying the biosynthesis of trehalose in the interaction between Brassica rapa and P. brassicae is also of great significance.

Keywords: Brassica rapa; Plasmodiophora brassicae; TPS; Trehalose; clubroot.

Figure 1

Phenotypic investigation and trehalose contents in the roots of Chinese cabbage after plasmodiophora brassicae inoculation. CK: inoculation-distilled water; Pb: inoculation with P. brassicae. (A) Investigation of clubroot disease of roots at 4 and the 5 weeks post-inoculation (wpi) with P. brassicae. The red arrow points to the location of the clubroot disease. The white scale range represents 2 cm. (B) The trehalose contents in the roots of Chinese cabbage after P. brassicae inoculation. The abscissa represents the time post-inoculation, and the ordinate represents the value of sugar content. The data represent mean values ± SDs. The asterisks indicate p-values (** p < 0.01) according to Student’s t test. Horizontal axis is the time point from 2 weeks to 5 weeks post-inoculation (wpi).

Figure 2

Phylogenetic relationship of BrTPS and PbTPS. All TPS proteins were divided into three subgroups, represented by three colors. The green color represents Class I proteins, the blue color represents Class II proteins. The yellow circle symbol represents the BrTPS proteins. The unrooted phylogenetic tree was constructed using the neighbor-joining (NJ) method with 1000 bootstrap replications by MEGA6 software.

Figure 3

Gene structures, domains, and motifs of the BrTPS family. (A) Exon/intron organization of BrTPS genes. Pink boxes represent exons and black lines of same length represent introns. The length of exons can be inferred by the scale at the bottom. (B) The conserved domain analysis of BrTPS protein. Trehalose-6-phosphate synthase (TPS) domain (Glyco_transf_20), trehalose-6-phosphate phosphatase (TPP) domain (Trehalose_PPase), and haloacid dehalogenase-like hydrolase domain-containing 3 (Hydrolase 3) are shown by green, yellow, and red, respectively. (C) Conserved motifs of BrTPS proteins. Ten putative motifs are indicated in different colored boxes. The details of the motifs are listed at the bottom.

Figure 4

Gene structures, domains, and motifs of the PbTPS family. (A) Exon/intron organization of PbTPS genes. Pink boxes represent exons and black lines with same length represent introns. The length of exons can be inferred by the scale at the bottom. (B) The conserved domain analysis of PbTPS protein. Trehalose-6-phosphate synthase (TPS) domain (Glyco_transf_20), trehalose-6-phosphate phosphatase (TPP) domain (Trehalose_PPase) are shown by green and yellow, respectively. (C) Conserved motifs of PbTPS. Ten putative motifs are indicated in different colored boxes. The details of the motifs are listed on the right.

Figure 5

The TPS gene locations in B. rapa chromosomes. The chromosomes are represented by yellow bars. The chromosomal position of each BrTPS gene was mapped according to the B. rapa genome. The chromosome number is indicated at the top of each chromosome. The scale is in megabases (Mb).

Figure 6

Predicted cis-elements in BrTPS gene promoters. Promoter sequences (2000 bp) of 14 BrTPS genes were analyzed using the PlantCARE database. (A) Kind and position of cis-acting elements in BrTPSs. (B) numbers of cis-acting elements. The gradient blue colors and numbers in the grid indicate the number of different cis-acting elements in BrTPSs. (C) Number of cis-acting elements in 14 BrTPS genes containing three category; histograms with different colors represent different categories. (D) Proportion of different cis-acting elements in each category.

Figure 7

Relative Expression profiles of BrTPS genes in the Chinese cabbage root after of P. brassicae. infection. CK represents inoculation with water; Pb represents inoculation with P. brassicae. Expression of each BrTPS gene was measured by qRT-PCR from 1 week to 5 weeks post-inoculation. The expression levels of genes are presented using fold change values transformed to Log2 format. The data indicate the relative expression levels normalized to that of the internal control 18srRNA. Red and green colors correspond to up- and downregulations of the BrTPS gene expressions, respectively.

Figure 8

Analysis PbTPS genes in the Chinese cabbage root after P. brassicae infection. (A) Reverse-transcription polymerase chain reaction analysis of the expression of PbTPS in infected and healthy plants roots, the template of PCR reaction was root cDNA mixture from 1 week to 5 weeks post-inoculation (wpi). CK: heathy plants; Pb: infected plants. (B) Real-time PCR analysis of the expression of PbTPSs in infected plants roots. 1W–5W represent five time points from 1 week to 5 weeks post-inoculation with P. brassicae. (C) qRT-PCR analysis of the expression of PbTPSs in infected plants roots at 4 wpi and 5 wpi. Values represent the mean and standard deviation of triplicate results. “ND” means not detected. Asterisks indicate values that are statistically significantly different from the 4 wpi using Student’s t test. (* p < 0.05; ** p < 0.01).