Transcriptome and Metabolome Analysis of a Late-Senescent Vegetable Soybean during Seed Development Provides New Insights into Degradation of Chlorophyll

Abstract

(1) Background: Senescence represents the final stage of plant growth and development, which transfers nutrients to growing seeds and directly affects the yield and quality of crops. However, little is known about chlorophyll degradation in developing and maturing seeds, in contrast to leaf senescence; (2) Methods: RNA-Seq was used to analyze the differentially expressed genes of different late-senescent germplasms. A widely untargeted metabolic analysis was used to analyze differential metabolites. In addition, qRT-PCR was conducted to detect gene expression levels; (3) Results: Transcriptome analysis revealed that ZX12 seeds have a higher expression level of the chlorophyll synthesis genes in the early stage of maturity, compared with ZX4, and have a lower expression level of chlorophyll degradation genes in the late stage of maturity. Flavonoids were the primary differential metabolites, and ZX12 contains the unique and highest expression of three types of metabolites, including farrerol-7-O-glucoside, cyanidin-3-o-(6'-o-feruloyl) glucoside, and kaempferide-3-o-(6'-malonyl) glucoside. Among them, farrerol-7-O-glucoside and cyanidin-3-o-(6'-o-feruloyl) glucoside are flavonoid derivatives containing mono and dihydroxy-B-ring chemical structures, respectively; and (4) Conclusions: It is speculated that the two metabolites can slow down the degradation process of chlorophyll by scavenging oxygen-free radicals in the chloroplast.

Keywords: chlorophyll metabolism; flavonoids; late-senescent; senescence; soybean seed.

Figure 1

Chl content of soybean varieties ZX4 and ZX12. (A) Photographs of the phenotype of pods and seeds at different days (20 d, 25 d, 30 d, 35 d, 40 d, 45 d, and 50 d) after flowering. (B) Chl content (Chl a, Chl b, total Chl, and Rate of Chl degradation) of ZX4 and ZX12 seeds at different DAF. (C) Photographs of Chl autofluorescence intensity of ZX4 and ZX12 seeds at different DAF. (D) Chl fluorescence intensity of ZX4 and ZX12 seeds.

Figure 2

SOD, POD, and T-AOC activities of ZX4 and ZX12 seeds at different maturity stages. (A) SOD activities of ZX4 and ZX12 seeds at different maturity stages. (B) POD activities of ZX4 and ZX12 seeds at different maturity stages. (C) T-AOC activities of ZX4 and ZX12 seeds at different maturity stages.

Figure 3

Transcriptome analysis of ZX12 and ZX4 seeds at different maturity stages. (A) Number of DEGs in ZX12 and ZX4 seeds at different maturity stages. (B) Venn diagram of DEGs between ZX12 and ZX4 seeds at different maturity stages. (C) KEGG analysis on the differentially expressed genes at 20, 30, and 40 DAF.

Figure 4

Chl metabolism and photosynthesis pathway-related genes are differentially expressed in ZX12/ZX4 at 20, 30 and 40 DAF time. (A) Heat map of Chl metabolism gene expression. (B) Heat map of key gene expression in photosynthesis pathway. The value of the heat map is the log₂ (ZX12/ZX4) value of the ratio of the expression in RNA-seq.

Figure 5

Metabolome analysis of differential metabolic profile between ZX4 and ZX12 seeds at different maturity stages. (A) Classification of differential metabolites. (B) The combination of the top 10 upregulating and downregulating metabolites in three periods. (C) Cluster analysis of expression patterns of differential metabolites. (D) Group metabolite expression heat map.

Figure 6

Correlation analysis between differential metabolites and differentially expressed genes of flavonoids and anthocyanins. Black color represents undetected metabolites; Purple represents detected metabolites; and the red asterisk represents a positive correlation and green represents a negative correlation.

Figure 7

The proposed model affecting the difference of stay-green ability between ZX12 and ZX4 seeds at mature stages. The font in red indicates that they are positively regulated in ZX12; red boxes represent up-regulation; green boxes represent down-regulation; and the arrow represents promotion, and the T represents inhibition.