Proteomic and Carbonylation Profile Analysis at the Critical Node of Seed Ageing in Oryza sativa

Abstract

The critical node (CN), which is the transition from the plateau phase to the rapid decreasing phase of seed ageing, is extremely important for seed conservation. Although numerous studies have investigated the oxidative stress during seed ageing, information on the changes in protein abundance at the CN is limited. In this study, we aimed to investigate the abundance and carbonylation patterns of proteins at the CN of seed ageing in rice. The results showed that the germination rate of seeds decreased by less than 20% at the CN; however, the abundance of 112 proteins and the carbonylation levels of 68 proteins markedly changed, indicating oxidative damage. The abundance and activity of mitochondrial, glycolytic, and pentose phosphate pathway proteins were reduced; consequently, this negatively affected energy production and germination. Proteins related to defense, including antioxidant system and heat shock proteins, also reduced in abundance. Overall, energy metabolism was reduced at the CN, leading to a decrease in the antioxidant capacity, whereas seed storage proteins were up-regulated and carbonylated, indicating that the seed had a lower ability to utilize seed storage proteins for germination. Thus, the significant decrease in metabolic activities at the CN might accelerate the loss of seed viability.

Figure 1

Representative isoelectric focusing (IEF)/dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation gels of proteins from 0 d (A), 3 d (B) and 4 d (C) aged rice seeds after imbibition for 48 h. Total 500 μg protein were separated by immobilized pH gradient (IPG) strips and 12% (w/v) SDS-PAGE gels. Protein codes correspond to those in Tables 1, 2 and 3. Number on the left represents the apparent molecular mass. Number above the gels represents the pI of separated protein spot. U, upregulation; D, downregulation.

Figure 2

Classification of downregulated (A) and upregulated (B) proteins in 0 d, 3 d, and 4 d aged rice seeds after imbibition for 48 h.

Figure 3

Two-dimensional (2D) immunoblots using antidinitrophenyl hydrazone antibody to detect carbonylated embryo proteins in 0-d (A), 3-d (B), and 4-d (C) aged rice seeds after imbibition for 48 h. Total 500 μg protein were numbered in a preparative 2D electrophoresis gel and excised for MS/MS analysis, corresponding to the proteins in Tables 1, 2 and 3. Number on the left represents the apparent molecular mass. Number above the gels represents the pI of separated protein spot. (C) Carbonylated spot.

Figure 4

The activity of malate dehydrogenase (MDH, (A)) pyruvate decarboxylase (PDC, (B)) 6-phosphogluconate dehydrogenase (6PGD, (C)) ascorbate peroxidase (APX, (D)) and glutathione S-transferase (GST, (E)) in 0-d, 3-d, and 4-d aged rice seeds after imbibition for 48 h. Data represent the mean ± standard deviation of three independent experiments. All treatments significantly differed from the control at p < 0.05 (n = 3).

Figure 5

Relative levels of malate dehydrogenase 1 (MDH1, (A)) succinate dehydrogenase 1 (SDH1, (B)) βATP synthase (C)) pyruvate decarboxylase 1 (PDC1, (D)) 6-phosphogluconate dehydrogenase 1 (6PGD1, (E)) and ascorbate peroxidase 1 (APX1, (F)) and abundance of beta subunit of ATP synthase (βATP) and ascorbate peroxidase (APX) (G) in 0-d, 3-d, and 4-d aged rice seeds after imbibition for 48 h. Transcript levels in 3-d and 4-d aged seeds were calculated in relation to a value of 1.0 that assigned to 0-d aged seeds after imbibition for 48 h. Data represent the mean ± standard deviation of three independent experiments. All treatments significantly differed from the control at p < 0.05 (n = 3).