Comprehensive Mitochondrial Metabolic Shift during the Critical Node of Seed Ageing in Rice

Abstract

The critical node (CN) in seed aging in rice (Oryza sativa) is the transformation from Phase I (P-I) to Phase II (P-II) of the reverse S-shaped curve (RS-SC). Although mitochondrial dysfunction plays a key role in seed ageing, the metabolic shift in the CN remains poorly understood. Here, we investigated the mitochondrial regulatory mechanisms during the CN of rice seed ageing. We showed that during the CN of seed ageing, the mitochondrial ultrastructure was impaired, causing oxygen consumption to decrease, along with cytochrome c (cyt c) oxidase and malate dehydrogenase (MDH) activity. In addition, the transcript levels for the alternative pathway of the electron transport chain (ETC) were significantly induced, whereas the transcripts of the cytochrome oxidase (COX) pathway were inhibited. These changes were concomitant with the down-regulation of mitochondrial protein levels related to carbon and nitrogen metabolism, ATP synthase (ATPase) complex, tricarboxylic acid cycle (TCA) cycle, mitochondrial oxidative enzymes, and a variety of other proteins. Therefore, while these responses inhibit the production of ATP and its intermediates, signals from mitochondria (such as the decrease of cyt c and accumulation of reactive oxygen species (ROS)) may also induce oxidative damage. These events provide considerable information about the mitochondrial metabolic shifts involved in the progression of seed ageing in the CN.

Fig 1. Influence of the artificial ageing treatment on rice germination percentage and potential.

Data represent the mean ± standard deviation of three independent experiments. The results of all treatments differed significantly from the control (p< 0.05, one-way ANOVA, n = 3).

Fig 2. Transmission electron micrographs of rice embryos aged 0 d (A–C), 3 d (D–F), 4 d (G–I), and 7 d (J–L).

N, nucleus; CW, cell wall; St, starch granule; V, vacuole; M, mitochondria; Cr, cristae; OM, outer membrane; IM, inner boundary membrane.

Fig 3. Abundance of the transcripts of mitochondrial components in rice embryos during germination.

For each of the 13 transcripts investigated, a value of 1.0 was assigned to 0 d aged seed after imbibition 48 h and message abundance at the 3 d, 4 d, 7 d and 11 d aged seed was calculated relative to it. Data represent the mean ± standard deviation of 3 independent experiments. Asterisks indicate significant differences to 0 d aged seed (Student’s t-test; *p< 0.05).

Fig 4. Cytochrome c oxidase (COX, A) and malate dehydrogenase (MDH, B) activity in crude mitochondria from 0 d, 3 d, 4 d, 7 d, and 11 d aged seeds.

Data represents mean ± standard deviation of 3 independent experiments. All treatment significantly differed from the control (p< 0.05, one-way ANOVA, n = 3).

Fig 5. Cytochrome c oxidase (COX, A) and malate dehydrogenase (MDH, B) activity in purified mitochondria from 0 d, 3 d, and 4 d aged seeds.

Data represent the mean ± standard deviation of 3 independent experiments. All treatment significantly differed from the control (p< 0.05, one-way ANOVA, n = 3).

Fig 6. Abundance of a variety of known mitochondrial proteins by the immunoblot analysis of mitochondria isolated from 0 d, 3 d, and 4 d aged rice embryos after 48 h imbibition.

Total 10 μg protein was separated by SDS gel electrophoresis and blotted to supported polyvinylidene difluoride, then probed with antibodies against beta subunit of ATP synthase (AtpB), serine hydroxymethyltransferase (SHMT), isocitrate dehydrogenase (IDH), voltage dependent anion channel 1 (VDAC1) and cytochrome c (Cyt c).

Fig 7. Western blot analysis of antioxidant enzymes in purified mitochondria from 0 d, 3 d, and 4 d aged seeds.

Total 10 μg protein was separated by SDS gel electrophoresis and blotted to supported polyvinylidene difluoride, then probed with antibodies against catalase (CAT), glutathione reductase (GR) and manganese superoxide dismutase (MnSOD).

Fig 8. Representative IEF/SDS–PAGE separation gels of mitochondrial proteins from 0 d (A), 3 d (B) and 4 d (C) aged rice seeds after 48 h imbibition.

The proteins were separated by first-dimensional pH 5–8 linear IPG strips and 12% SDS-PAGE gels in the second dimension. The proteins were numbered in a preparative 2D electrophoresis gel and excised for MS/MS analysis, corresponding to the proteins in Table 3. Number on the left represents the apparent molecular mass. Number above the gels represents the pI of separated protein spot.