Comprehensive Profiling and Inheritance Patterns of Metabolites in Foxtail Millet

Abstract

Metabolomics aims at determining a sample's metabolites profile and hence provides a straight functional statement of an organism's physiological condition. Here, we investigated comprehensive profiling, natural variation and species-specific accumulation of both primary and secondary metabolites in foxtail millet using LC-MS, and inheritance patterns of metabolome in millet hybrids. The application of a broad target metabolomics method facilitated the simultaneous identification and quantification of more than 300 metabolites. The metabolic analysis of these compounds, such as flavonoids, phenolamides, hydrocinnamoyl derivatives, vitamins and LPCs, revealed their developmentally controlled accumulation, and natural variation in different tissues/varieties. Species-specific accumulation of secondary metabolites was observed based on a comparative metabolic analysis between millet and rice, such as flavonoid O-rutinosides/neohesperidosides and malonylated flavonoid O-glycosides. In analyzing the metabolic variations between hybrid progenies and their parental lines, including a photothermo-sensitive genic male sterility line and five Zhangzagu varieties, metabolic overdominant, and dominant patterns of inheritance could be observed. For example, hydrocinnamoyl derivatives and feruloylated flavonoids were identified as over-parent heterosis (overdominant) metabolites in milet hybrids. Our work paves the way for developing predictors of hybrid performance and the future analysis of the biosynthesis and regulation of relevant metabolic pathways in millet.

Keywords: Setaria italica; flavonoids; heterosis; metabolic profiling; phenolamides.

Figure 1

Statistical analysis of metabolic accumulation at different development all stages. (A,B) Score plot of PCA and PLS-DA for datasets from the three-leaf and five-leaf stage groups. The green color represents the sample at the three-leaf stage, the blue color represents the sample at the five-leaf stage under the short daylight condition, the red color represents the samples at the five-leaf stage under the long daylight condition, and the black color represents the quality control samples; (C) A volcano plot of the metabolic differences between the three-leaf and five-leaf stages (the red dot represents a metabolite with a fold change ≥1.2 or ≤ 0.83, P-value ≤ 0.05); and (D) a heat map of the relative differences in metabolites between the three-leaf stage and the five-leaf stage. The content value of each metabolite was normalized to complete the linkage hierarchical clustering. Red indicates high abundance, whereas the low relative abundance metabolites are green.

Figure 2

Change rate of metabolites altered at the three-leaf and five-leaf stages. (A) flavone C-aglycones; (B) flavone O-aglycones and flavonolignan; (C) amino acids; (D) lipids, nucleotide and its derivates. The identification of differential accumulation of metabolites between different tissue were determined by PLS-DA with the VIP values>1, followed by both ANOVA (P ≤ 0.05). Ace, acetyl; Ade, adenosine; Api, apigenin; Chr, chrysoeriol; Dim, dimethyl; Fer, feruloyl; GE, β-guaiacylglyceryl ether; Glu, glucoside; Gly, glycerin; Gua, guanosine; Hex, hexoside; Ino, Inosine; Lut, luteolin; mal, malonyl; oct, octadecatetraenoic acid; Phe, phenylformic acid; Pen, pentosyl; Sac, saccharopine; Sin, sinapoyl; Tricin, Tri; SE, syringyl alcohol ether.

Figure 3

Differential accumulation of metabolites among the PTGMS A2 and Zhangzagu lines at the five-leaf stage. (A) The score plot of PCA for datasets at five-leaf stage under the long daylight condition. The different shape represents the different variety sample; and (B,C) a volcano plot of metabolic differences between PTGMS A2 and Zhangzagu (B), and between PTGMS A2 and Zhangzagu No.5 (C) at the five-leaf stage. (the red dot represents a metabolite with a fold change ≥1.2 or ≤ 0.83, P-value ≤ 0.05). (D) Venn plot of the metabolic profiles of PTGMS A2 vs. Zg (group I) and PTGMS A2 vs. Zg5m (group II) at the five-leaf stage.

Figure 4

Metabolic profile analysis among hybrid progenies and the parental lines at the five-leaf stage. (A) The PCA score plot for datasets at the five-leaf stage. The green color represents the progenies, the red color represents the female parental line, and the yellow color represents the male parental lines, (B) A volcano plot of metabolic differences between the progenies and male parental lines at the five-leaf stage (the red dot represents a metabolite with a fold-change ≥1.2 or ≤ 0.83, P-value ≤ 0.05). (C) MS/MS spectra for tricin O-rutinoside; (D) MS/MS spectra for apigenin O-malonylhexoside. Glu, glucose; Hex, hexoside; mal, malonic acid; Rha, rhamnose.