Dynamic Metabolomic Changes in the Phenolic Compound Profile and Antioxidant Activity in Developmental Sorghum Grains

Abstract

Phenolic compounds (PC) were analyzed by UHPLC-ESI-QTOF-MS^E in two sorghum genotypes, harvested in two growing seasons (GS) at five distinct days after flowering (DAF) to evaluate how genotype/GS influences the PC synthesis and antioxidant capacity during grain growth. Total phenolic contents were strongly correlated with antioxidant capacity (r > 0.9, p < 0.05). Globally, 97 PC were annotated, including 20 PC found irrespective of the grain developmental stage and genotype/GS. The phenolic profile clearly differs between stages: phenolic acids were the most abundant class in early stages (50%), and flavonoid accumulation becomes predominant in late ones (3/5 of total ion abundance). Dimeric and trimeric tannins were identified even in 10DAF grains. Chemometry revealed great PC variability between genotypes (27%) and important biomarkers of GS differentiation (e.g., ferulic acid). This work can input open databases of PC and paves the way to understand biosynthetic pathways of PC in sorghum and future sorghum selection.

Keywords: Sorghum bicolor; UHPLC-MSE; antioxidant compounds; polyphenols.

Figure 1

Evaluation of days after flowering (DAF) in total reducing capacity (TRC) and antioxidant capacity (DPPH and FRAP methods) in free (FPC), bound (BPC), and total (TPC) phenolic compound extracts in different genotypes and growing seasons (GS) of sorghum grains. The ratio between the FPC and BPC values in each analysis is shown in the last column. Results are expressed as mean ± standard deviation (n = 3). Different letters indicate a significant difference between DAF (Tukey, p < 0.05).

Figure 2

Metabolomic analysis. (A) Venn diagram with the number of identification distribution in grains from different development stages. (B) Total relative ion abundance of phenolic compounds by class during grain development. (C) Total relative ion abundance of phenolic compounds in each sample during DAF. (D) Venn diagram with the number of identification distribution in each genotype/growing season (GS). Σ = sum of the total group value. Different lowercase and uppercase letters mean a significant difference (p < 0.05 by one-way ANOVA and Tukey post-test) between DAF and samples/genotypes, respectively. Bars represent standard deviation (n = 3).

Figure 3

Principal component analysis (PCA) biplot: (A) of all sorghum samples and (B–D) in each genotype/growing season (GS). The samples (symbols) are distributed according to relative intensity of phenolic compounds (red empty circles). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Figure 4

Hierarchical clustering (HCA) heat map of metabolomic data. Three cluster groups (G1–G3) and subclusters were generated using a Pearson correlation (ANOVA, p < 0.05) on the differentially abundant phenolic compounds during grain development. Different clusters and subclusters are expressed by the mean of the group total abundance. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Figure 5

Variable importance in projection (VIP) scores generated from orthogonal partial least-squares discriminant analysis (OPLS-DA). The 20 top important phenolic compounds (VIP score >1.0) contributing to the separation of phenolic profile in early vs mature stages. The relative abundance of phenolic compounds is indicated by a colored scale from blue to red representing the low and high, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Figure 6

S-plot of orthogonal partial least-squares discriminant analysis (OPLS-DA) between growing season influence (Macia GS1 vs Macia GS2; A, B) and genotype influence (Macia GS1 vs IS15752 GS1; C, D) in developing grains. In the x-axis, the relative magnitude of variables (phenolic compounds) is represented, and in the y axis, it is the confidence/reliability. Compounds in bold represent phenolics annotated at both immature and mature stages for each treatment (GS or genotype). Variables farthest from the origin in the plot have higher covariance (p[1]) and deemed significant markers. Inset tables show the phenolic compound name in ascending order of covariance. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).