Integrated Metabolomics and Transcriptomics Analyses Reveal the Metabolic Differences and Molecular Basis of Nutritional Quality in Landraces and Cultivated Rice

Abstract

Rice (Oryza sativa L.) is one of the most globally important crops, nutritionally and economically. Therefore, analyzing the genetic basis of its nutritional quality is a paramount prerequisite for cultivating new varieties with increased nutritional health. To systematically compare the nutritional quality differences between landraces and cultivated rice, and to mine key genes that determine the specific nutritional traits of landraces, a seed metabolome database of 985 nutritional metabolites covering amino acids, flavonoids, anthocyanins, and vitamins by a widely targeted metabolomic approach with 114 rice varieties (35 landraces and 79 cultivars) was established. To further reveal the molecular mechanism of the metabolic differences in landrace and cultivated rice seeds, four cultivars and six landrace seeds were selected for transcriptome and metabolome analysis during germination, respectively. The integrated analysis compared the metabolic profiles and transcriptomes of different types of rice, identifying 358 differentially accumulated metabolites (DAMs) and 1982 differentially expressed genes (DEGs), establishing a metabolite-gene correlation network. A PCA revealed anthocyanins, flavonoids, and lipids as the central differential nutritional metabolites between landraces and cultivated rice. The metabolite-gene correlation network was used to screen out 20 candidate genes postulated to be involved in the structural modification of anthocyanins. Five glycosyltransferases were verified to catalyze the glycosylation of anthocyanins by in vitro enzyme activity experiments. At the same time, the different mechanisms of the anthocyanin synthesis pathway and structural diversity in landrace and cultivated rice were systematically analyzed, providing new insights for the improvement and utilization of the nutritional quality of rice landrace varieties.

Keywords: UDP-glucosyltransferase; anthocyanins; landrace; metabolome; rice (Oryza sativa L.); transcriptome.

Figure 1

Comparative metabolic differences in cultivar rice and landrace seeds. (A) The seed colors between landraces and cultivated rice. The cultivar was represented by white, and the landrace was represented by red and black. (B) Principal components analysis (PCA) of the metabolite profiling in different types of rice seeds. Green represents cultivated rice, and red represents quality control, while blue represents landraces. (C) Neighbor-joining tree of 114 rice varieties based on 985 metabolites. (D) Heatmap based on the metabolome data of cultivated and landrace rice seeds. Red indicates a high abundance, and blue indicates low relative abundance metabolites.

Figure 2

Comparative metabolic analysis in cultivar rice and landrace seeds during germination. (A) PCA of the metabolite profiling in seeds of landraces and cultivated rice during germination. (B) Venn diagram depicting the number of differential metabolites in the seeds of landraces and cultivated rice during germination. (C) The composition and proportion of different metabolites in the seeds of landraces and cultivated rice during germination. (D) Phylogenetic tree based on differential accumulated metabolites in seeds of landraces and cultivated rice during germination.

Figure 3

Construction of a metabolite–gene regulatory network in cultivar rice and landrace seeds during germination. (A) A PCA analysis based on the transcriptome in the seeds of landraces and cultivated rice during germination. (B) KEGG analysis of DEGs in the seeds of landraces and cultivated rice during germination. Y-axis represents KEGG pathways, and X-axis indicates the gene ratio. (C) GO enrichment analysis of DEGs in the seeds of landraces and cultivated rice during germination. MF, molecular function; BP, biological process; CC, cellular component. (D) KEGG analysis of DAMs and DEGs in the seeds of landraces and cultivated rice during germination.

Figure 4

Correlation network analysis based on metabolites and genes in the seeds of landraces and cultivated rice during germination. (A) Construction of a co-expression gene module. The same color represents the same module. If the module feature genes between two different modules are similar, they will be merged automatically. (B) Heatmap between metabolites and 35 gene modules. Y-axis represents each module, and the X-axis represents each trait. Red shows upregulated transcripts, and blue shows downregulated transcripts. (C) Co-expression network of genes in modules. (D) Dynamic of metabolite accumulation and gene expression in co-expression clusters. Metabolite and gene clusters are represented by red. The numbers of the X-axis indicate the germination stage. (E) The expression level of PAL in the seeds of landraces and cultivated rice during germination.

Figure 5

Analysis of the in vitro enzyme activity of OsGTs. (A) MS/MS spectra information of cyanidin-3-O-glucoside with the authentic standard. The reactions were performed with 5 purified OsGTs, UDP-glucose, and cyanidin as substrates: GT1, glucosyltransferase 1 (B); GT2, glucosyltransferase 2 (C); GT3, glucosyltransferase 3 (D); GT4, glucosyltransferase 4 (E); GT5, glucosyltransferase 5 (F).

Figure 6

Integrated transcriptomic and metabolomic data to exhibit the anthocyanin synthesis pathway in the germination seeds of cultivar rice and landrace. A heatmap represents the folds of the expression levels of the corresponding structural genes in the seeds of landraces and cultivated rice, and from red to green in the heatmap indicates the expression levels of structural genes ranging from low to high. Genes in anthocyanin synthesis are shown as follows: PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′,5′-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; flavonoid 3-O-glucosyltransferase; 3GT, flavonoid 3-O-glucosyltransfers; GT1, glucosyltransferase 1; GT4, glucosyltransferase 4.