Metabolite Profiling Reveals Distinct Modulation of Complex Metabolic Networks in Non-Pigmented, Black, and Red Rice (Oryza sativa L.) Cultivars

Abstract

Comprehensive profiling of primary and secondary metabolites was performed to understand metabolic differences associated with color formation in pigmented rice (Oryza sativa L.). Overall, 110 metabolites from non-pigmented, black, and red rice cultivars were identified. Black and red rice contained high levels of flavonoids associated with plant color. Black rice also contained high levels of terpenoids (carotenoids, tocopherols, phytosterols, and monoterpenes). The non-pigmented rice contained relatively low levels of secondary metabolites. Multivariate and pathway analyses were performed to data-mine the metabolite profiles. Hierarchical clustering analysis of correlation coefficients revealed metabolite clusters based on nitrogen and carbon sources. These clusters suggested a negative correlation between nitrogen and carbon. Pathway analysis revealed that black rice was rich in carbon-based secondary metabolites, with relatively low levels of primary metabolites compared with other rice cultivars. These data highlight the complex interactions between nitrogen and carbon metabolism of primary and secondary metabolites in rice. For the first time, the relationships and metabolic differences in terpenoid content (monoterpenes, triterpenes, and tetraterpenes) of non-pigmented and pigmented rice cultivars were analyzed. These findings should greatly contribute to the understanding of pigmented rice metabolome and inform breeding programs for new rice cultivars.

Keywords: PathVisio 3; carbon; metabolite profiling; metabolomics; multivariate analysis; nitrogen; pigmented rice; terpenoid.

Figure 1

Partial least squares-discriminant analysis (PLS-DA) score (A) and loading plots (B) derived from 110 metabolites of black, red, and non-pigmented (white) rice cultivars. The ellipse represents the Hotelling T2 with 95% confidence in the score plot. Plot annotation 1, C20-ol (Eicosanol); 2, C21-ol (Heneicosanol); 3, C22-ol (Docosanol); 4, C24-ol (Tetracosanol); 5, C26-ol (Hexacosanol); 6, β-Tocopherol; 7, γ-Tocopherol; 8, C27-ol (Heptacosanol); 9, C28-ol (Octacosanol); 10, γ-Tocotrienol; 11, α-Tocopherol; 12, Cholesterol; 13, α-Tocotrienol; 14, Campesterol; 15, C30-ol (Triacontanol); 16, Stigmasterol; 17, β-Sitosterol; 18, Lutein; 19, Zeaxanthin; 20, β-Carotene; 21, Cyanidin-3-O-glucoside; 22, Peonidin-3-O-glucoside; 23, Catechin; 24, Epicatechin; 25, C16:0 (Palmitic acid); 26, C18:0 (Stearic acid); 27, C18:1 (Oleic acid); 28, C18:2 (Linoleic acid); 29, C18:3 (α-Linolenic acid); 30, C20:0 (Arachidonic acid); 31, Pyruvic acid; 32, Lactic acid; 33, Alanine; 34, Oxalic acid; 35, Valine; 36, Ethanolamine; 37, Leucine; 38, Glycerol; 39, Phosphoric acid; 40, Isoleucine; 41, Proline; 42, Glycine; 43, Succinic acid; 44, Fumaric acid; 45, Serine; 46, Threonine; 47, β-Alanine; 48, Malic acid; 49, Salicylic acid; 50, Methionine; 51, Aspartic acid; 52, Pyroglutamic acid; 53, γ-Aminobutyric acid; 54, Cysteine; 55, Threonic acid; 56, Glutamic acid; 57, p-Hydroxy benzoic acid; 58, Phenylalanine; 59, Asparagine; 60, Xylose; 61, Vanillic acid; 62, Glutamine; 63, Protocatechuic acid; 64, Shikimic acid; 65, Citric acid; 66, Fructose; 67, Galactose; 68, Lysine; 69, Glucose; 70, p-Coumaric acid; 71, Tyrosine; 72, Mannitol; 73, Ferulic acid; 74, Inositol; 75, Caffeic acid; 76, Tryptophan; 77, Sinapinic acid; 78, Sucrose; 79, Raffinose 80, 1-Butanol; 81, Pentanal; 82, 1-Pentanol; 83, Toluene; 84, Hexanal; 85, 1-Hexanol; 86, Ethylbenzene; 87, p-Xylene; 88, 2-Heptanone; 89, 2-Butyl furan; 90, Styrene; 91, Heptanal; 92, 2-Acetyl-1-pyrroline; 93, α-Pinene; 94, 1-Heptanol; 95, Benzaldehyde; 96, 1-Octen-3-ol; 97, 2-Pentyl furan; 98, Octanal; 99, p-Cymene; 100, D-Limonene; 101, 3-Octen-2-one; 102, γ-Terpinene; 103, 1-Octanol; 104, Linalool; 105, o-Cymene; 106, Nonanal; 107, 1-Nonanol; 108, Naphthalene; 109, Decanal; 110, 1H-Indole.

Figure 2

Orthogonal partial least squares-discriminant analysis (OPLS-DA) score and variable importance in the projection (VIP) plots derived from 110 metabolites of black, red, and non-pigmented (white) rice cultivars (A,B). A is comparison of non-pigmented rice and pigmented rice. B is comparison of black and red pigmented rice. The ellipse represents the Hotelling T2 with 95% confidence in the score plot. Plot annotation 1, C20-ol (Eicosanol); 2, C21-ol (Heneicosanol); 3, C22-ol (Docosanol); 4, C24-ol (Tetracosanol); 5, C26-ol (Hexacosanol); 6, β-Tocopherol; 7, γ-Tocopherol; 8, C27-ol (Heptacosanol); 9, C28-ol (Octacosanol); 10, γ-Tocotrienol; 11, α-Tocopherol; 12, Cholesterol; 13, α-Tocotrienol; 14, Campesterol; 15, C30-ol (Triacontanol); 16, Stigmasterol; 17, β-Sitosterol; 18, Lutein; 19, Zeaxanthin; 20, β-Carotene; 21, Cyanidin-3-O-glucoside; 22, Peonidin-3-O-glucoside; 23, Catechin; 24, Epicatechin; 25, C16:0 (Palmitic acid); 26, C18:0 (Stearic acid); 27, C18:1 (Oleic acid); 28, C18:2 (Linoleic acid); 29, C18:3 (α-Linolenic acid); 30, C20:0 (Arachidonic acid); 31, Pyruvic acid; 32, Lactic acid; 33, Alanine; 34, Oxalic acid; 35, Valine; 36, Ethanolamine; 37, Leucine; 38, Glycerol; 39, Phosphoric acid; 40, Isoleucine; 41, Proline; 42, Glycine; 43, Succinic acid; 44, Fumaric acid; 45, Serine; 46, Threonine; 47, β-Alanine; 48, Malic acid; 49, Salicylic acid; 50, Methionine; 51, Aspartic acid; 52, Pyroglutamic acid; 53, γ-Aminobutyric acid; 54, Cysteine; 55, Threonic acid; 56, Glutamic acid; 57, p-Hydroxy benzoic acid; 58, Phenylalanine; 59, Asparagine; 60, Xylose; 61, Vanillic acid; 62, Glutamine; 63, Protocatechuic acid; 64, Shikimic acid; 65, Citric acid; 66, Fructose; 67, Galactose; 68, Lysine; 69, Glucose; 70, p-Coumaric acid; 71, Tyrosine; 72, Mannitol; 73, Ferulic acid; 74, Inositol; 75, Caffeic acid; 76, Tryptophan; 77, Sinapinic acid; 78, Sucrose; 79, Raffinose 80, 1-Butanol; 81, Pentanal; 82, 1-Pentanol; 83, Toluene; 84, Hexanal; 85, 1-Hexanol; 86, Ethylbenzene; 87, p-Xylene; 88, 2-Heptanone; 89, 2-Butyl furan; 90, Styrene; 91, Heptanal; 92, 2-Acetyl-1-pyrroline; 93, α-Pinene; 94, 1-Heptanol; 95, Benzaldehyde; 96, 1-Octen-3-ol; 97, 2-Pentyl furan; 98, Octanal; 99, p-Cymene; 100, D-Limonene; 101, 3-Octen-2-one; 102, γ-Terpinene; 103, 1-Octanol; 104, Linalool; 105, o-Cymene; 106, Nonanal; 107, 1-Nonanol; 108, Naphthalene; 109, Decanal; 110, 1H-Indole.

Figure 3

Correlation matrix and cluster analysis of data for 110 metabolites of black, red, and non-pigmented (white) rice cultivars (A). Detailed correlation matrix of each compound cluster (B). Each square indicates a Pearson’s correlation coefficient for a pair of compounds. The value for the correlation coefficient is represented by the intensity of the blue or red color, as indicated on the color scale. Hierarchical clusters are presented as a cluster tree. C20-ol, Eicosanol; C21-ol, Heneicosanol; C22-ol, Docosanol; C24-ol, Tetracosanol; C26-ol, Hexacosanol; C27-ol, Heptacosanol; C28-ol, Octacosanol; C30-ol, Triacontanol; C16:0, Palmitic acid; C18:0, Stearic acid; C18:1, Oleic acid; C18:2, Linoleic acid; C18:3, alpha-Linolenic acid; C20:0, Arachidic acid.

Figure 4

Metabolic pathway diagrams visualized by PathVisio 3. Detailed metabolic pathway diagrams of amino acids, organic acids, sugars, phenylpropanoids, and fatty acids (A); terpenoids and volatiles (B). The expression data consist of log₂-transformed fold change (FC) values (log₂FC). A log₂FC value range is −1 < log₂FC < 1. If log₂FC value is higher than zero (indicated in red), metabolite content is higher in pigmented rice than in non-pigmented rice (WR: white rice). If log₂FC value is less than zero (indicated in green), metabolite content is lower in pigmented rice than in non-pigmented rice. If log₂FC value is zero (indicated in white), metabolite content is identical in pigmented and non-pigmented rice. (BR: Black Rice, RR: Red Rice).

Figure 5

Metabolic pathway diagrams visualized by PathVisio 3. Detailed metabolic pathway diagrams of amino acids, organic acids, sugars, phenylpropanoids, and fatty acids (A); terpenoids and volatiles (B). The expression data consist of log₂-transformed fold change (FC) values (log₂FC). A log₂FC value range is −1 < log₂FC < 1. If log₂FC value is higher than zero (indicated in red), metabolite content is higher in black rice than in red rice. If log₂FC value is less than zero (indicated in green), metabolite content is higher in red rice than in black rice. If log₂FC value is zero (indicated in white), metabolite content is identical in black and red rice. (BR: Black Rice, RR: Red Rice).