Effect Of Artificial Aging On Structure, Function And Antioxidant System Of Soybean Seed Mitochondria

Mitochondria are the organelle providing energy and intermediate materials for seed germination and previous studies have shown that mitochondria are the first organelle of seed aging damage. Therefore, carrying out studies on the effect of ageing on mitochondrial structure, function and antioxidant systems helps to reveal the mechanism of seed aging, propose the theory and methods to delay and predict seed aging, for ensuring the safe conservation of germplasm. In this study, "zhongdou 27"was used as materials, aged to different germination percentage under 40°C condition after moisture content rehydrated to 14 %, electron microscopy, respiration rate determination, enzymes activity determination and other methods were used to analyze the effect of artificial aging on mitochondria structure, function and antioxidant system. Main results are as follows:1. The structure of mitochondria was affected by aging, and aging inhibited the development and repairmen of mitochondria during germination. Electron microscopy observed that control seeds axes mitochondria had a large number of well-developed crista and well-defined inner and outer membrane, which showed classical morphology; while aged seeds mitochondria swelled, outer membrane and crista degraded gradually until disappear, inner membrane blurred and the matrix became thin, which showed severely altered morphology. Percoll density gradient separate analysis showed that CK seeds axes mainly contained heavy mitochondria, while with aging, heavy mitochondria decreased while light mitochondria increased gradually; the results showed that aging inhibited the development and repairmen of light mitochondria to heavy mitochondria during germination. Determination of mitochondria integrity indicated that aging reduced the integrity of soybean axes mitochondria.2. The function of mitochondria was reduced by aging. Determination of COX and MDH activities showed that aging reduced the activities of COX and MDH, indicating that aging leads to the reduction of the activity of mitochondria and the function of TCA. Oxygen consumption capacity analysis suggested that aging reduced the NADH-dependent and succinate-dependent respiration rate and RCR, which indicating that the respiration capacity and oxidative phosphorylation efficiency of mitochondria were reduced by aging. The damage of mitochondria structure and function may lead to the disruption of electron transfer in electron transport chain, then increase electron leak, on the one hand, it may lead to the outbreak of ROS and increase oxidative damage, on the other hand, may lead to the shortage of energy and intermediate materials produced in mitochondria for seed germination.3. The antioxidant ability of mitochondria was reduced by aging. The results showed, by measurement of activities of antioxidant enzymes and antioxidants content, that the activities of SOD, APX, MDHAR and GR decreased quickly, while the activity of MDHAR decreased more slowly in mitochondria during accelerated aging; and the content and proportion of antioxidants were also affected by aging, as aging time, ASC content, ASC/DHA, GSH content and GSH/GSSG decreased sharply, DHA content increases first and then remained constant, while the GSSG content decreased first and then remained unchanged. The reduction of mitochondria antioxidant ability would not inhibited the accumulation of ROS effectively caused by damage to mitochondrial structure and function, then increase oxidative damage. The raise of O₂^·–production rate and H2O2 content in the early stage of aging verified the accumulation of ROS.4. The accumulation of ROS caused by oxidative damage may be one of the reasons of the loss of seed vigor. Aging increased MDA content significantly by determination of MDA content of soybean axes after imbibitions for 24 h, indicating that lipid peroxidation caused by the accumulation of ROS increased. Determination of electrical conductivity showed that seed electrical conductivity increased gradually with the reduction of germination percentage, the lower germination percentage, the larger electrical conductivity. The results suggesting that aging destroyed cell membrane structure, increased the membrane permeability, leaded to electrolyte leakage, which may due to the increase of lipid peroxidation. With the aging time, ROS would further accumulate, result in serious damage to the membrane structure that cannot be repair, and then lead to the leakage of a variety of substances involved in metabolism, which may be one of the reasons of the loss of seed vigor.