Genome-Wide Identification and Analyses of Drought/Salt-Responsive Cytochrome P450 Genes in Medicago truncatula

Abstract

Cytochrome P450 monooxygenases (P450s) catalyze a great number of biochemical reactions and play vital roles in plant growth, development and secondary metabolism. As yet, the genome-scale investigation on P450s is still lacking in the model legume Medicago truncatula. In particular, whether and how many MtP450s are involved in drought and salt stresses for Medicago growth, development and yield remain unclear. In this study, a total of 346 MtP450 genes were identified and classified into 10 clans containing 48 families. Among them, sixty-one MtP450 genes pairs are tandem duplication events and 10 MtP450 genes are segmental duplication events. MtP450 genes within one family exhibit high conservation and specificity in intron-exon structure. Meanwhile, many Mt450 genes displayed tissue-specific expression pattern in various tissues. Specifically, the expression pattern of 204 Mt450 genes under drought/NaCl treatments were analyzed by using the weighted correlation network analysis (WGCNA). Among them, eight genes (CYP72A59v1, CYP74B4, CYP71AU56, CYP81E9, CYP71A31, CYP704G6, CYP76Y14, and CYP78A126), and six genes (CYP83D3, CYP76F70, CYP72A66, CYP76E1, CYP74C12, and CYP94A52) were found to be hub genes under drought/NaCl treatments, respectively. The expression levels of these selected hub genes could be induced, respectively, by drought/NaCl treatments, as validated by qPCR analyses, and most of these genes are involved in the secondary metabolism and fatty acid pathways. The genome-wide identification and co-expression analyses of M. truncatulaP450 superfamily genes established a gene atlas for a deep and systematic investigation of P450 genes in M. truncatula, and the selected drought-/salt-responsive genes could be utilized for further functional characterization and molecular breeding for resistance in legume crops.

Keywords: M. truncatula; co-expression analysis; cytochrome P450 monooxygenase; drought stress; expression profile; salt stress.

Figure 1

Phylogenetic relationships of MtP450 proteins. An unrooted NJ tree of MtP450s was constructed using the MEGA-X software, bootstrap used was 1000 replicates. The entire MtP450 family members are shown, for each clan, with different colors.

Figure 2

Chromosomal localization and gene duplication analysis of MtP450 genes. (A) All 346 MtP450s are shown on the chromosomes and indicated by their names. Chromosome numbers are indicated on the left side of each bar. Tandem duplicated genes are joined with blue arc lines. (B) The gray lines in the background indicate the collinear blocks within M. truncatula, while the red lines highlight the segmental duplication of MtP450 gene pairs. The yellow background on the chromosomes represents the gene density at each chromosomal position, and the vertical red line inside represents relatively higher gene density. The horizontal red line also represents gene density at each chromosomal position, with peak values representing relatively higher gene density and valley values representing relatively lower gene density.

Figure 3

Synteny analysis of MtP450 genes between M. truncatula and two representative plant species (A. thaliana and O. sativa). Gray lines in the background indicate the collinear blocks within M. truncatula, and A. thaliana/O. sativa, while the red lines highlight the syntenic MtP450 gene pairs.

Figure 4

Gene structural features and conserved motifs of MtP450 proteins. (A) Gene structural features of MtP450s. “p450” indicate the conserved domain of Cytochrome p450 family, “CDS” indicated the remaining partial coding sequence. “UTR” indicates untranslated regions. The numbers 0, 1, and 2 stand for the types of intron phase. The length of the intron, exon and UTR can be estimated from the scale. (B) Analysis of the conserved motifs of MtP450 proteins. Each motif is represented by a colored box. Box length corresponds to motif length.

Figure 4

Gene structural features and conserved motifs of MtP450 proteins. (A) Gene structural features of MtP450s. “p450” indicate the conserved domain of Cytochrome p450 family, “CDS” indicated the remaining partial coding sequence. “UTR” indicates untranslated regions. The numbers 0, 1, and 2 stand for the types of intron phase. The length of the intron, exon and UTR can be estimated from the scale. (B) Analysis of the conserved motifs of MtP450 proteins. Each motif is represented by a colored box. Box length corresponds to motif length.

Figure 5

Expression profiles of MtP450 genes. (A) Expression profiles of MtP450 genes in eight different tissues from microarray data. (B,C) are hierarchical clustering of gene expression profiles under drought (B) and NaCl (C) treatments from microarray data. Each column indicates a sampling time point, and each row indicates an MtP450 gene, wherein each treatment was normalized in the same row, the relative expressions levels are log2-transformed and visualized by heatmap. The colors vary from blue to red, and circles from small to large represent the scale of relative expression levels. Those genes with deeper background color showed that their expression was significantly increased after stress induction. “SHT” and “RT” stand for “shoot” and “root” materials, respectively, used for drought treatment; “Hyd” and “Sdl” stand for hydroponic treatment with 200 mM NaCl, and in vitro culture with 180 mM NaCl, respectively.

Figure 5

Expression profiles of MtP450 genes. (A) Expression profiles of MtP450 genes in eight different tissues from microarray data. (B,C) are hierarchical clustering of gene expression profiles under drought (B) and NaCl (C) treatments from microarray data. Each column indicates a sampling time point, and each row indicates an MtP450 gene, wherein each treatment was normalized in the same row, the relative expressions levels are log2-transformed and visualized by heatmap. The colors vary from blue to red, and circles from small to large represent the scale of relative expression levels. Those genes with deeper background color showed that their expression was significantly increased after stress induction. “SHT” and “RT” stand for “shoot” and “root” materials, respectively, used for drought treatment; “Hyd” and “Sdl” stand for hydroponic treatment with 200 mM NaCl, and in vitro culture with 180 mM NaCl, respectively.

Figure 6

Co-expression network analysis and hierarchical clustering of expression profiles of MtP450 genes under drought treatments from microarray data. (A) Weighted gene co-expression network analysis of genes in shoots; (B) weighted gene co-expression network analysis of genes in roots; (C) Venn diagram showing MtP450 genes in shoots and roots under drought treatments through co-expression; (D) hierarchical clustering of expression profiles of MtP450 genes under drought treatments from microarray data.

Figure 7

Co-expression network analysis and hierarchical clustering of expression profiles of MtP450 genes under NaCl stresses. Co-expression network analysis (A) and hierarchical clustering of expression profiles (B) of MtP450 genes under NaCl treatments from microarray data.

Figure 8

Analysis of the 14 hub genes under drought/salt treatments by qPCR. (A) Relative expression levels of eight hub MtP450 genes under drought treatment at 3h, 24h, 48 h and 72 h, with that of 0h as control (set as value of 1). (B) Relative expression levels of six hub MtP450 genes under salt treatment at 3 h, 24 h, 48 h and 72 h, with that of 0h as control (set as value of 1). The differential expression analysis was conducted based on the 2^−∆∆ct method. Data are from three biological replicates and three technical replicates. * p < 0.05, ** p < 0.01.