Global identification, structural analysis and expression characterization of cytochrome P450 monooxygenase superfamily in rice

Abstract

Background: The cytochrome P450 monooxygenases (CYP450, CYP, P450) catalyze numerous monooxygenation/hydroxylation reactions in biochemical pathways. Although CYP superfamily has been systematically studied in a few species, the genome-scale research about it in rice has not been done.

Results: In this study, a total of 355 CYPs encoded by 326 genes were identified in japonica genome. The OsCYP genes are classified into 10 clans including 45 families according to phylogenetic analysis. More than half of the genes are distributed in 53 tandem duplicated gene clusters. Intron-exon structure of OsCYPs exhibits highly conserved and specificity within a family, and divergences of duplicate genes in gene structure result in non-functionalization, neo-functionalization or sub-functionalization. Selection pressure analysis showed that rice CYPs are under purifying selection. The microarray data analysis shows that some genes are tissue-specific expression, such as OsCYP710A5 and OsCYP71X14 in endosperm, OsCYP99A3 and OsCYP78A16 in root and OsCYP93G2 and OsCYP97D7 in leaf. Analysis of RNA-seq data derived from rice leaf developmental gradient indicates that some OsCYPs exhibit zone-specific expression patterns. OsCYP87C2, OsCYP96B5, OsCYP96B8 and OsCYP84A5 were specifically expressed in leaf base and transitional zone. The transcripts of lineages II and IV-1 members were highly abundant in maturing zone. Eighty three OsCYPs are differentially expressed in response to drought stress, of which OsCYP51G3, OsCYP709C9, OsCYP709C5, OsCYP81A6, OsCYP72A18 and OsCYP704A5 are strongly induced and OsCYP78A16, OsCYP89C9 and OsCYP704A5 are down-regulated significantly, and some of the results were validated by qPCR. And 23 up-regulated and 17 down-regulated genes are specific to Osbhlh148 mutation under drought stress. Compared to those in wild type, the changes in transcript levels of several genes are slight in the mutant, such as OsCYP51G3, OsCYP94C2, OsCYP709C9 and OsCYP709C5.

Conclusion: The whole-genomic analysis of rice P450 superfamily provides a clue to understanding biological function of OsCYPs in development regulation and drought stress response, and is helpful to rice molecular breeding.

Keywords: Cytochrome P450; Development; Drought stress; Expression profile; Gene duplication; Rice.

Fig. 1

Superposition of three dimensional homology models of rice CYPs and template structures. a Structures of HmCYP51 (dark khaki), OsCYP51G1 (sky blue) and OsCYP51G3 (gray). Heme molecule is shown as sphere representation. Two VFV molecules are shown by ball-stick model. b Close-up view of the substrate (two VFV molecules surrounded with cyan mesh) environment. The substrate recognition sites were identified based on the alignment with template structure and shown by stick model. c Structures of AtCYP74A1 (dark khaki), OsCYP74A4 (sky blue) and OsCYP74A5 (gray). Heme molecule is shown as sphere representation. 13(S)-HPOT molecule is shown by ball-stick model. d Close-up view of the substrate (13(S)-HPOT molecule surrounded with forest green mesh) environment. The substrate recognition sites were identified based on the alignment with template structure and shown by stick model

Fig. 2

Chromosomal localization of OsCYP genes. All putative OsCYPs are shown on the chromosomes and indicated by CYP names omitting “CYP” root. The scale is in megabases (Mb). Chromosome numbers are indicated at the top of each bar, and ellipse on each chromosome shows the rough position of centromere. The rectangle filled with the colour of corresponding chromosome is superposed on chromosome to show the duplicated chromosomal segment. The segmentally duplicated genes are connected by gray dashed lines, and tandemly duplicated genes are joined with black vertical lines. The genes within metabolic gene clusters are marked with red characters

Fig. 3

The expression profiling of OsCYPs throughout life cycle of rice grown. Hierarchial clustering heatmap displays expression patterns of 156 OsCYP genes in 62 distinct tissues representing 12 major organ systems in Nipponbare, which are divided into three groups. The CV values are added on the right side of CYP labels

Fig. 4

Spatially and temporally specific expression of OsCYPs in leaf. a Hierarchical clustering shows the similar and distinct expression patterns of 148 OsCYP genes in 11 continuous leaf sections. The CV values are shown on the right side of the CYP labels. b A co-expression network constructed using OsCYP87C2 as “guide gene” and visualized using Cytoscape software. The three triangles represent for the transcription factor genes. c The schematic of phenylpropanoid pathway

Fig. 5

Differential expression of OsCYP genes and the drought-responsive network. a Expression profiles of 142 OsCYP genes in WT and Osbhlh148 mutant under drought conditions. The line charts show the dynamic changes of gene expression levels in drought stress response. The genes with no expression values in well-watered condition are presented by black boxes. b Overveiw of drought-responsive network. The red solid line depict synthetic route; the blue solid line depict catabolic route. c Venn diagram showing the number of transcripts enhanced or decreased in response to drought stress. d Expression profiles of 22 candidate genes in drought stress signal pathway. e The expression levels of 10 representative drought-responsive OsCYPs were determined by qPCR

Fig. 6

In silico promoter analyses of 17 OsCYP genes. Different regulatory elements are indicated by different geometric shape with different colour in their relative position in promoter regions. TSS: Transcriptional Start Site