Transcriptome and Proteome Profiling of Different Colored Rice Reveals Physiological Dynamics Involved in the Flavonoid Pathway

Abstract

Black and red rice are rich in both anthocyanin and proanthocyanin content, which belong to a large class of flavonoids derived from a group of phenolic secondary metabolites. However, the molecular pathways and mechanisms underlying the flavonoid biosynthetic pathway are far from clear. Therefore, this study was undertaken to gain insight into physiological factors that are involved in the flavonoid biosynthetic pathway in rice cultivars with red, black, and white colors. RNA sequencing of caryopsis and isobaric tags for relative and absolute quantification (iTRAQ) analyses have generated a nearly complete catalog of mRNA and expressed proteins in different colored rice cultivars. A total of 31,700 genes were identified, of which 3417, 329, and 227 genes were found specific for red, white, and black rice, respectively. A total of 13,996 unique peptides corresponding to 3916 proteins were detected in the proteomes of black, white, and red rice. Coexpression network analyses of differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) among the different rice cultivars showed significant differences in photosynthesis and flavonoid biosynthesis pathways. Based on a differential enrichment analysis, 32 genes involved in the flavonoid biosynthesis pathway were detected, out of which only CHI, F3H, ANS, and FLS were detected by iTRAQ. Taken together, the results point to differences in flavonoid biosynthesis pathways among different colored rice cultivars, which may reflect differences in physiological functions. The differences in contents and types of flavonoids among the different colored rice cultivars are related to changes in base sequences of Os06G0162500, Os09G0455500, Os09G0455500, and Os10G0536400. Current findings expand and deepen our understanding of flavonoid biosynthesis and concurrently provides potential candidate genes for improving the nutritional qualities of rice.

Keywords: Oryza sativa L.; black rice; flavonoid biosynthesis; iTRAQ; red rice; transcriptome sequencing.

Figure 1

Comparisons of transcript and protein abundance in the different rice cultivars. (A) Congruency between the detected transcripts and proteins in the rice caryopses of the three different rice cultivars. (B) Venn diagram of commonly expressed and unique genes in the caryopses of black, red, and white rice. (C) Venn diagram of the differentially expressed genes (DEGs) in the caryopses of the different cultivars as well as the endosperm and pericarp of black rice. (D) Venn diagram of differentially expressed proteins (DEPs) in the caryopses of the different rice cultivars. (E) The number of DEGs and (F) proteins (DEPs) between the caryopses of the three cultivars (and the genes of the pericarp and endosperm of black rice). The black bars in (E) denote upregulated genes, whereas the gray bars represent downregulated genes. In (F), black bars denote upregulated proteins and grey bars represent downregulated proteins. W-C = white rice caryopsis, R-C = red rice caryopsis, B-C = black rice caryopsis, B-E = black rice endosperm, and B-P = black rice pericarp. The comparative analysis between black rice and red rice, red rice as control; comparative analysis between black rice and white rice, white rice as control; and comparative analysis between red rice and white rice, white rice as control.

Figure 2

Gene ontology (GO) function depiction of the DEGs and DEPs by WEGO analysis. (A) Web Gene Ontology Annotation Plotting (WEGO) analysis of the different comparisons for the transcriptome, and (B) WEGO analysis of the different comparisons for the proteome. The abscissa represents each GO term. The left Y-axis represents the percentage of each term, whereas the right Y-axis represents the genes/proteins corresponding to each GO term. Different colored bars represent different comparisons.

Figure 2

Gene ontology (GO) function depiction of the DEGs and DEPs by WEGO analysis. (A) Web Gene Ontology Annotation Plotting (WEGO) analysis of the different comparisons for the transcriptome, and (B) WEGO analysis of the different comparisons for the proteome. The abscissa represents each GO term. The left Y-axis represents the percentage of each term, whereas the right Y-axis represents the genes/proteins corresponding to each GO term. Different colored bars represent different comparisons.

Figure 3

PageMan and cluster analyses of the DEGs in the different rice cultivars. (A) PageMan analysis for the different paired comparisons of DEGs between the rice cultivars (the term of significant enrichment is defined as p < 0.01); (B) expression profiles and cluster analysis of 211 DEGs in the photosynthesis pathway; and (C) expression profiles and cluster analysis of 132 genes in the flavonoid biosynthesis pathway.

Figure 4

Network analysis between different pathways and flavonoid synthesis. (A) Basic helix-loop-helix (bHLH) and flavonoids; (B) Myeloblastosis (MYB) and flavonoids. The solid and dotted lines indicate positive and negative correlation coefficients, respectively, and the line intensity denotes their strength. Each straight arrow, from tail to head, indicates the interaction direction. A red color indicates that the Pearson’s correlation coefficient is greater than 0.8. The gene networks were obtained using the “GeneNet” method from the R package. The top 100 largest absolute correlations are displayed.

Figure 4

Network analysis between different pathways and flavonoid synthesis. (A) Basic helix-loop-helix (bHLH) and flavonoids; (B) Myeloblastosis (MYB) and flavonoids. The solid and dotted lines indicate positive and negative correlation coefficients, respectively, and the line intensity denotes their strength. Each straight arrow, from tail to head, indicates the interaction direction. A red color indicates that the Pearson’s correlation coefficient is greater than 0.8. The gene networks were obtained using the “GeneNet” method from the R package. The top 100 largest absolute correlations are displayed.

Figure 5

Correlation analysis between the expressed genes and proteins. (A) Analysis for black and red rice; (B) analysis for black and white rice; and (C) analysis for red and white rice. The red dot represents genes and proteins for flavonoid, photosynthesis, sugar, phenylalanine, peroxide, MYB, and bHLH transcriptome factors. The black dot represents the other genes and proteins. The blue dotted lines express the thresholds of differential expression (the fold-change of the RNA is log2|FC| > 1; the fold-change of a protein is log2|FC > 1.5 or FC < 0.667).

Figure 6

Pathway map visualizing the changes involved in genes, proteins, and metabolites for the flavonoid biosynthesis pathway among the three rice cultivars. (A) Pathway map for the three cultivars; and (B) pathway map for the pericarp and endosperm of black rice. ANR, anthocyaninidin reductase; ANS, anthocyanin synthase; AS, aurone synthase; C4H, cinnamate 4-hydroxylase; CHI, chalcone isomerase; 4CL, 4-coumarate coenzyme A ligase; CHR, chalcone reductase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; F3′,5′H-hydroxylase; FLS, flavonol synthase; HID, 2-hydroxyisoflavanone dehydratase; IFS, 2-hydroxyisoflavanone synthase; LAR, leucoanthocyanidin reductase; PAL, L-phenylalanine ammonia-lyase; and TAL, L-tyrosine ammonia-lyase. Red font represents the key proteins identified by iTRAQ. Blue font represents the key metabolites.

Figure 7

Quantitative analysis of the genes involved in the flavonoid biosynthesis pathway by qRT-PCR. Bars with identical superscripts letters denote relative expression of flavonoid biosynthesis pathway gene is not significantly different (p < 0.01) in different pigmented rice cultivars.