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

Abstract

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.

Keywords: APX; ASC; ASH; ATP; Artificial ageing; Ascorbate–glutathione cycle; BSA; COX; DHA; DHAR; GR; GSH; GSSG; MDA; MDHAR; Mitochondria; N-Tris (hydroxymethyl)-methyl-2-aminoethanesulfonic acid; RCR; ROS; Reduced ascorbate; SOD; Soybean seed; TCA; TEM; TES; adenosine triphosphate; ascorbate peroxidase; ascorbic acid; bovine serum albumin; cytochrome c oxidase; dehydroascorbate (oxidized ascorbate); dehydroascorbate reductase; glutathione; glutathione disulfide; glutathione reductase; malondialdehyde; monodehydroascorbate reductase; reactive oxygen species; respiration control rate; superoxide dismutase; transmission electron microscopy; tricarboxylic acid.