Jinmei Zhang

Reduced mitochondrial and ascorbate–glutathione activity after artificial ageing in soybean seed

The effect of artificial ageing on the relationship between mitochondrial activities and the antioxidant system was studied in soybean seeds (Glycine max L. cv. Zhongdou No. 27). Ageing seeds for 18d and 41d at 40°C reduced germination from 99% to 52% and 0%, respectively. In comparison to the control, malondialdehyde content and leachate conductivity in aged seeds increased and were associated with membrane damage. Transmission electron microscopy and Percoll density gradient centrifugation showed that aged seeds mainly contained poorly developed mitochondria in which respiration and marker enzymes activities were significantly reduced. Heavy mitochondria isolated from the interface of the 21% and 40% Percoll were analyzed. Mitochondrial antioxidant enzymes activities including superoxide dismutase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase were significantly reduced in aged seeds. A decrease in total ascorbic acid (ASC) and glutathione (GSH) content as well as the reduced/oxidized ratio of ASC and GSH in mitochondria with prolonged ageing showed that artificial ageing reduced ASC-GSH cycle activity. These results suggested an elevated reactive oxygen species (ROS) level in the aged seeds, which was confirmed by measurements of superoxide radical and hydrogen peroxide levels. We conclude that mitochondrial dysfunction in artificially aged seeds is due to retarded mitochondrial and ASC-GSH cycle activity and elevated ROS accumulation.

Activity levels and expression of antioxidant enzymes in the ascorbate–glutathione cycle in artificially aged rice seed

Reactive oxygen species are the main contributors to seed deterioration. In order to study scavenging systems for reactive oxygen species in aged seed, we performed analyses using western blotting, real-time quantitative reverse-transcription polymerase chain reaction, high-performance liquid chromatography, and antioxidant enzyme activity analyses in artificially aged rice seeds (Oryza sativa L. cv. wanhua no.11). Aging seeds by storing them at 50 °C for 1, 9, or 17 months increased the superoxide radical and hydrogen peroxide levels and reduced the germination percentage from 99% to 92%, 55%, and 2%, respectively. The activity levels of superoxide dismutase (SOD), glutathione reductase (GR), and dehydroascorbate reductase (DHAR) did not change in aged seeds. In contrast, the activity levels of catalase (CAT), ascorbate peroxidase (APX), and monodehydroascorbate reductase (MDHAR) were significantly decreased in aged seeds, as were the expression of catalase and cytosolic ascorbate peroxidase protein. Transcript accumulation analysis showed that specific expression patterns were complex for each of the antioxidant enzyme types in the rice embryos. Overall, the expression of most genes was down-regulated, along with their protein expression. In addition, the reduction in the amount of ascorbate and glutathione was associated with the reduction in scavenging enzymes activity in aged rice embryos. Our data suggest that the depression of the antioxidant system, especially the reduction in the expression of CAT1, APX1 and MDHAR1, may be responsible for the accumulation of reactive oxygen species in artificially aged seed embryos, leading to a loss of seed vigor.

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

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.

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

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.