Identification of Metabolomic Biomarkers of Seed Vigor and Aging in Hybrid Rice

Abstract

Seed deterioration during rice seed storage can lead to seed vigor loss, which adversely affects agricultural production, the long-term preservation of germplasm resources, and the conservation of species diversity. However, the mechanisms underlying seed vigor maintenance remain largely unknown. In this study, 16 hybrid rice combinations were created using four sterile lines and four restorer lines that have been widely planted in southern China. Following artificial aging and natural aging treatments, germination percentage and metabolomics analysis by gas chromatography-mass spectrometry was used to identify the metabolite markers that could accurately reflect the degree of aging of the hybrid rice seeds. Significant differences in the degree of seed deterioration were observed among the 16 hybrid rice combinations tested, with each hybrid combination having a different germination percentage after storage. The hybrid rice combination with the storage-resistant restorer line Guanghui122 exhibited the highest germination percentage under both natural and artificial storage. A total of 89 metabolic peaks and 56 metabolites were identified, most of which were related to primary metabolism. Interestingly, the content of galactose, gluconic acid, fructose and glycerol in the seeds increased significantly during the aging process. Absolute quantification indicated that galactose and gluconic acid were highly significantly negatively correlated with the germination percentage of the seeds under the different aging treatments. The galactose content was significantly positively correlated with gluconic acid content. Additionally, glycerol showed a significant negative correlation with the germination percentage in most hybrid combinations. Based on the metabolomics analysis, metabolite markers that could accurately reflect the aging degree of hybrid rice seeds were identified. Galactose and gluconic acid were highly significantly negatively correlated with the germination percentage of the seeds, which suggested that these metabolites could constitute potential metabolic markers of seed vigor and aging. These findings are of great significance for the rapid and accurate evaluation of seed aging degree, the determination of seed quality, and the development of molecular breeding approaches for high-vigor rice seeds.

Keywords: Galactose; Gluconic acid; Metabolomic biomarkers; Oryza sativa; Seed aging; Seed vigor.

Fig. 1

Changes in seed germination percentage of 16 hybrid rice combinations (BIIIY-122, TY-122, IIY-122, QY-122, BIIIY-998, TY-998, IIY-998, QY-998, BIIIY-368, TY-368, IIY-368, QY-368, BIIIY-3618, TY-3618, IIY-3618 and QY-3618) after 24 months of natural aging or 8 days of artificial aging. Data represent the mean ± SE of three biological replicates of 100 seeds each. Means denoted by the same letter did not significantly differ at p < 0.05 according to Fisher’s least significant difference test

Fig. 2

Changes in rice seed germination of 4 sterile lines (BIIIA, II-32A, Tianfeng A, Qiu A) (A) and 4 restorer lines (Guanghui122, Guanghui3618, Guanghui368, Guanghui998) (B) after 24 months of natural aging or 8 days of artificial aging. Data represent the mean ± SE of three biological replicates of 100 seeds each. Means denoted by the same letter did not significantly differ at p < 0.05 according to Fisher’s least significant difference test

Fig. 3

The score plots (A–D) and S-plots of the different metabolites (E–H) generated from pairwise OPLS-DAGC-MS data showing the distinct metabolomic changes among the BIIIY-122, BIIIY-998, BIIIY-368, BIIIY-3618 (A, E),IIY-122, IIY-998, IIY-368, IIY-3618 (B, F), QY-122, QY-998, QY-368, QY-3618 (C, G), and TY-122, TY-998, TY-368, TY-3618 (D, H). The significantly different metabolites with VIP > 0.5 and |p(corr)| > 0.5 have been labeled in the S-plot (B, D, F, H)

Fig. 4

Changes in the relative contents of fructose (A), glycerol (B), galactose (C), gluconic acid (D), oxaloacetic acid (E) and glucopyranoside (F) in the seeds of 16 hybrid rice combinations by natural aging for 24 months and 0 mouths. Data represent the mean ± SE of six biological replicates of 100 mg seeds each. Means denoted by the same letter did not significantly differ at p < 0.05 according to Fisher’s least significant difference test

Fig. 5

Changes in germination percentage and six metabolites in seeds of two hybrid rice combination after 0, 6 or 15 days of artificial aging. A Seed germination percentage of G8Y2156; B Seed germination percentage of G8Y169; C Relative content of six metabolites in seed endosperm of G8Y2156; D Relative content of six metabolites in seed embryo of G8Y2156; E Relative content of six metabolites in seed endosperm of G8Y169; F Relative content of six metabolites in seed embryo of G8Y169. Data represent the mean ± SE of six biological replicates of 100 mg seeds (endosperm or embryo) each. Means denoted by the same letter did not significantly differ at p < 0.05 according to Fisher’s least significant difference test

Fig. 6

Regression equation between the absolute content of galactose (A) and gluconic acid (B) and different germination percentages of 26 rice materials with different seed vigor. Y represent germination percentage; R² represent the determination coefficient