Comparative functional genomics analysis of cytochrome P450 gene superfamily in wheat and maize

Abstract

Background: The cytochrome P450s (CYP450s) as the largest enzyme family of plant metabolism participate in various physiological processes, whereas no study has demonstrated interest in comprehensive comparison of the genes in wheat and maize. Genome-wide survey, characterization and comparison of wheat and maize CYP450 gene superfamily are useful for genetic manipulation of the Gramineae crops.

Results: In total, 1285 and 263 full-length CYP450s were identified in wheat and maize, respectively. According to standard nomenclature, wheat CYP450s (TaCYP450s) were categorized into 45 families, while maize CYP450s (ZmCYP450s) into 43 families. A comprehensive analysis of wheat and maize CYP450s, involved in functional domains, conserved motifs, phylogeny, gene structures, chromosome locations and duplicated events was performed. The result showed that each family/subfamily in both species exhibited characteristic features, suggesting their phylogenetic relationship and the potential divergence in their functions. Functional divergence analysis at the amino acid level of representative clans CYP51, CYP74 and CYP97 in wheat, maize and rice identified some critical amino acid sites that are responsible for functional divergence of a gene family. Expression profiles of Ta-, ZmCYP450s were investigated using RNA-seq data, which contribute to infer the potential functions of the genes during development and stress responses. We found in both species CYP450s had preferential expression in specific tissues, and many tissue-specific genes were identified. Under water-deficit condition, 82 and 39 significantly differentially expressed CYP450s were respectively detected in wheat and maize. These genes may have some roles in protecting plants against drought damage. Thereinto, fourteen CYP450s were selected to validate their expression level through qRT-PCR. To further elucidating molecular mechanisms of CYP450 action, gene co-expression network was constructed. In total, 477 TaCYP450s were distributed in 22 co-expression modules, and some co-expressed genes that likely take part in the same biochemical pathway were identified. For instance, the expression of TaCYP74A98_4D was highly correlated with TaLOX9, TaLOX36, TaLOX39, TaLOX44 and TaOPR8, and all of them may be involved in jasmonate (JA) biosynthesis. TaCYP73A201_3A showed coexpression with TaPAL1.25, TaCCoAOMT1.2, TaCOMT.1, TaCCR1.6 and TaLAC5, which probably act in the wheat stem and/or root lignin synthesis pathway.

Conclusion: Our study first established systematic information about evolutionary relationship, expression pattern and function characterization of CYP450s in wheat and maize.

Keywords: Cytochrome P450; Drought stress; Expression regulation; Gramineae crops; Growth and development.

Fig. 1

Site-specific profiles for two types of functional divergence (Type-I and Type-II), measured by the posterior ratio. θ_I and θ_II, the coefficients of Type-I and Type-II functional divergence between two gene clusters; LRT, Likelihood Ratio Statistic

Fig. 2

Overview of structures. Key residues are shown in stick presentation, substrate molecule in ball-and-stick model, and heme molecule as spheres. Single-letter abbreviations for the amino acid residues are as follows: C, Cys; H, His; T, Thr; Q, Gln; R, Arg; E, Glu; and F, Phe. a Superposition of 4LXJ, TaCYP51G3_2D and ZmCYP51G35. Lanosterol molecule is shown in ball-and-stick model. The RMSD between the 4LXJ and TaCYP51G3_2D is 0.122 Å. The RMSD between the 4LXJ and ZmCYP51G35 is 0.162 Å. b Superposition of 2RCH, TaCYP74A98_4A and ZmCYP74A39. The RMSD between the 2RCH and TaCYP74A98 is 0.107 Å. The RMSD between the 2RCH and ZmCYP74A39 is 0.082 Å. c Superposition of 2X2N, TaCYP97A59_6B and ZmCYP97A16. The RMSD between the 2X2N and TaCYP97A59 is 0.178 Å. The RMSD between the 2X2N and ZmCYP97A16 is 0.151 Å. POSACONAZOLE molecule is shown in ball-and-stick model. SRS1–6: six putative SRS (substrate recognition sites) regions which are involved in recognition and binding of substrates according to Gotoh’s predicted models

Fig. 3

Expression analysis of CYP450s during development. a Box plot of TaCYP450s expression. RCE: Root at cotyledon emergence stage; RLP.03: Root at three leaves visible stage; RSE.99: Root at maximum stem length reached stage; SSE.00:Stem at stem elongation begins stage; SSE.02: Stem at two nodes or internodes visible stage; SFL.02:Stem at 1/2 of flowers open stage; LCE: Leaf at cotyledon emergence stage; L3N:Leaf at main shoot and axillary shoots visible at three nodes stage; LF1: Leaf at whole plant fruit formation stage 30 to 50%; ISE.02: Inflorescence at two nodes or internodes visible stage; ISE.99: Inflorescence at maximum stem length reached stage; IFL.02: Inflorescence at 1/2 of flowers open stage; FF1: Fruit at whole plant fruit formation stage 30 to 50%; FF2: Fruit at whole plant fruit formation stage 70% to final size; FR: Fruit at whole plant fruit ripening stage. b Box plot of ZmCYP450s expression. c The diagram of metabolic pathway involved in JA biosynthesis and co-expression analysis of TaCYP450s. d The diagram of lignin biosynthesis pathway and expression patterns of TaCYP73As in three sets. e Co-expression analysis of TaCYP450s involved in lignin biosynthesis.

Fig. 4

Analysis of the CYP450s expression under drought stress. a Violin plots showing expression levels of TaCYP450s. DS1h: after drought treatment at 1 h; DS6h: after drought treatment at 1 h. b Violin plots showing expression levels of ZmCYP450s. c Validation of the expression level of 14 CYP450s by qRT-PCR analysis. d A schematic diagram of CYP450-mediated signaling pathways under drought stress

Fig. 5

Co-expression modules of TaCYP450s. a Radar charts of expression values of TaCYP450s in 10 modules with the most obvious tissue-specific expression trends. The radar chart displays over a gray polygonal layout. Each axis represents one kind of tissue and each point in gray polygonal layout is labeled with the tissue. But we only showed part of the tissues with tissue-specific genes. The larger the radius of a ring represents the higher level of gene expression. Each ring is labeled with a number that represents gene expression level. The expression values of each gene in the corresponding module along each axis of the radar chart are connected linearly to visualize the data set as a polygon, and the different color polygons represent different genes. RCE: Root at cotyledon emergence stage; RLP.03: Root at three leaves visible stage; RSE.99: Root at maximum stem length reached stage; SSE.00: Stem at stem elongation begins stage; SSE.02: Stem at two nodes or internodes visible stage; LCE: Leaf at cotyledon emergence stage; L3N: Leaf at main shoot and axillary shoots visible at three nodes stage; LF1: Leaf at whole plant fruit formation stage 30 to 50%; ISE.99: Inflorescence at maximum stem length reached stage; IFL.02: Inflorescence at 1/2 of flowers open stage; FR: Fruit at whole plant fruit ripening stage. b Top 20 most significantly enriched pathways in purple module. c Top 20 most significantly enriched pathways in lightgreen module. d Top 70 most significantly enriched GO terms in biological process category in a two-dimensional semantic space of purple module. Color intensity reflects the significance of enrichment test. Circle radiuses depict the sizes of the aggregated GO terms. e Top 70 most significantly enriched GO terms in biological process category in a two-dimensional semantic space of lightgreen module