Genome-Wide Identification and Evolution Analysis of Trehalose-6-Phosphate Synthase Gene Family in Nelumbo nucifera

Abstract

Trehalose-6-phosphate synthase (TPS) plays a key role in plant carbohydrate metabolism and the perception of carbohydrate availability. In the present work, the publicly available Nelumbo nucifera (lotus) genome sequence database was analyzed which led to identification of nine lotus TPS genes (NnTPS). It was found that at least two introns are included in the coding sequences of NnTPS genes. When the motif compositions were analyzed we found that NnTPS generally shared the similar motifs, implying that they have similar functions. The d_N /d_S ratios were always less than 1 for different domains and regions outside domains, suggesting purifying selection on the lotus TPS gene family. The regions outside TPS domain evolved relatively faster than NnTPS domains. A phylogenetic tree was constructed using all predicted coding sequences of lotus TPS genes, together with those from Arabidopsis, poplar, soybean, and rice. The result indicated that those TPS genes could be clearly divided into two main subfamilies (I-II), where each subfamily could be further divided into 2 (I) and 5 (II) subgroups. Analyses of divergence and adaptive evolution show that purifying selection may have been the main force driving evolution of plant TPS genes. Some of the critical sites that contributed to divergence may have been under positive selection. Transcriptome data analysis revealed that most NnTPS genes were predominantly expressed in sink tissues. Expression pattern of NnTPS genes under copper and submergence stress indicated that NNU_014679 and NNU_022788 might play important roles in lotus energy metabolism and participate in stress response. Our results can facilitate further functional studies of TPS genes in lotus.

Keywords: Nelumbo nucifera; expression pattern; molecular evolution; protein properties; trehalose-6-phosphate synthase.

Figure 1

Structure and phylogenetic analysis of NnTPS proteins. The unrooted phylogenetic tree resulting from the full-length amino acid alignment of all the NnTPS proteins is shown on the left side of the figure. Exon-intron structures of the identified NnTPS genes are shown on the right side. The graphic representation of the optimized gene model displayed using GSDS.

Figure 2

Pairwise sequence identity of full-length TPS proteins. Pairwise sequence identities between TPS domain, TPP domain, full length protein sequence, and sequence outside domain were calculated. The boxplot shows the median (black line), interquartile range (box), and maximum and minimum scores (whiskers) for each data set.

Figure 3

Schematic diagram of amino acid motifs of TPS protein. Motif analysis was performed using MEME 4.11.2 as described in the methods. The different-colored boxes represent different motifs and their position in each TPS sequence. The sequences of key motifs that located in TPS and TPP domain were indicated.

Figure 4

The d_N/d_S ratios for different regions of lotus TPS proteins.

Figure 5

Phylogenetic relationships of the TPS from lotus, Arabidopsis (AtTPS), poplar (PtTPS), soybean (GmTPS), and rice (OsTPS). Red circles indicate the most recent common ancestral TPS genes among dicots and rice.

Figure 6

Type I functional divergence among the plant TPS gene members. Posterior probability (PP) profiles of the site-specific type I functional divergence. The line indicates cutoff = 0.95.

Figure 7

Expression profiles of the nine NnTPS genes in different tissues (A) and upon submergence and copper stresses (B). The color scale represents RPKM normalized log₂ transformed data. Red indicates high expression level and blue indicate low expression level. OL, leaf; RTZ, Combined Rhiz Tip Zone; Rhiz, Rhizome internode; R, Root; P, Petiole; T, Rhizome apical tip; Z, Rhizome elongation zone.

Figure 8

Validation of expression of selected NnTPS genes in various tissues and in rhizome upon exogenous stimuli. Tissues including leaf, petiole, and rhizome were sampled from 2 month old lotus seedlings. Meanwhile, lotus seedlings were treated with submergence stress or copper stress for 24 h and then rhizome was sampled. The sample without 5 mm copper treatment or submergence treatment was the control (Con). Gene expression was analyzed by real-time RT-PCR. The relative transcript abundance was normalized using lotus actin gene. Values are means ± SE of at least four independent experiments. Bars with different letters are significantly different at P < 0.05 according to Turkey's multiple range test. Fold changes of genes under copper stress and submergence treatment were values relative to control samples. Asterisks indicate significant differences compared with control sample at P < 0.05 levels according to t-test.