| In this study, the changes of programmed cell death (PCD) and mitochondrial in roots of Malus Hupehensis seedlings under 200μmol/L cadmium sulfide (CdSO4) treatment were studied through the methods of transmission electron microscope, agarose gel electrophoresis, acridine orange (AO) staining, caspase3/7 activity determination and flow cytometry (FCM) technique with annual M. hupehensis seedlings as experimental material. And the effects of some messenger molecule, such as reactive oxygen species (ROS), nitric oxide (NO), polyamines and calcium on mitochondrial changes and process of programmed cell death (PCD) were also investigated. The main results were as follows:1. The mitochondrial permeability transition (MPT) was increased, while the content of mitochondrial transmembrane potential (Δψm) and cytochrome c (Cyt c) was decreased in roots of Malus Hupehensis when treated by 200μmol/L CdSO4. At the same time, chromatin condensation and aggregation side, DNA fragmentation and other features of programmed cell death (PCD) were also observed. The caspase-like 3/7 activity, cell apoptosis rate and cell death quantity were increasing with the treatment time of CdSO4 prolonged.2. Programmed cell death (PCD) in roots was promoted by the accumulation of reactive oxygen species (ROS) under CdSO4 stress. The production rate of superoxide anion (O2.-) and hydrogen peroxide (H2O2) content were raised sharply after induced for short time. With the increase of stress time, they were both decreased but still kept at the significantly higher level than those of control. And these were accompanied with higher level of root cell death quantity and cell apoptosis rate. The production rate of superoxide anion (O2.-) and hydrogen peroxide (H2O2) content under CdSO4 stress were reduced by ascorbic acid (ASA) and catalase (CAT), which were known as ROS scavenger. Consequently, the increase of MPT and decrease ofΔψm and Cyt c content were suppressed, and root cell death quantity and cell apoptotic rate were also decreased.3. The changes of nitric oxide (NO) production rate in roots and its role in cell death process induced by CdSO4 stress were similar to ROS. CdSO4-induced programmed cell death could be promoted by NO through increased mitochondrial permeability and activation of caspases. In the initial stage of CdSO4 treatment, NO production rate in roots was increased dramatically. With the prolonging of treatment, NO content was decreasing but was still significantly higher than control. Consistent with the results of CdSO4 treatment, the level of endogenous NO was also increased by SNP, which was used as the NO donor, under CdSO4 stress. The increase of mitochondria MPT and decrease ofΔψm and Cyt c were promoted. Thereby, the number of cell death, caspase-like3/7 activity and root apoptotic rate under CdSO4 stress were increased. However, the level of endogenous NO was all reduced by AG (NOS inhibitor), NaN3 (NR inhibitor) and cPTIO (NO scavenger). The increase of MPT and decrease ofΔψm and Cyt c were also suppressed. At the same time, the number of cell death, caspase-like3/7 activity and root apoptotic rate were decreased.4. The production of NO and the generation of ROS in roots under CdSO4 stress could promote each other. The increase of NO level was inhibited by ASA and CAT; the production rate of superoxide anion (O2.-) and hydrogen peroxide (H2O2) content were enhanced by SNP; the increase of superoxide anion (O2.-) production rate and hydrogen peroxide (H2O2) content could be inhibited by AG, NaN3 and cPTIO.5. Polyamines could regulate the process of programmed cell death (PCD) in roots under CdSO4 stress. The free Put content in roots was increased dramatically, while free Spm and Spd contents were decreased under CdSO4 stress. Exogenous Spm and Spd significantly reduced the accumulation of Put and increased endogenous Spm and Spd levels, inhibited MPT increasing, H2O2 accumulating,Δψm decreasing and Cyt c reducing, decreased root cell death quantity, caspase-like3/7 activity and cell apoptotic rate. In the early state of CdSO4 stress, the PAO activity was significantly increased and reached the highest level at 6h, then it was decreased gradually with the stress time prolonged, but was still kept a higher level, which was consistent with the changes of H2O2 content. The PAO activity and H2O2 content under non-stress could be improved, but were decreased by separate exogenous Spm or Spd under CdSO4 stress.6. The production of NO and the accumulation of polyamines in roots under CdSO4 stress could influence each other. The contents of Spm and Spd were further decreased by exogenous NO under CdSO4 stress, while this process could be prevented by AG, NaN3 and cPTIO. Spm and Spd could promote NO production rate in roots, the separate exogenous Spm or Spd treatment caused some increase in NO production rate, NOS and NR activity. However, exogenous Spm or Spd suppressed the increase of NO production rate under CdSO4 stress.7. Calcium was involved in the regulation of CdSO4-induced programmed cell death (PCD) and its effect was varied due to calcium concentration. Low concentration of calcium (5 mmol/L Ca2+ and 10 mmol/L Ca2+) could prevent the decrease ofΔψm and Cyt c content, the increase of MPT and H2O2, and reduce the apoptotic rate in root cells, and the efficiency of 10 mmol/L Ca+ was better than that of 5mmol/L Ca+. However, high concentration of calcium (50 mmol/L Ca2+) accelerated the increase of MPT and H2O2 content, the decrease ofΔψm and Cyt c content, and promoted the root apoptotic rate. Ca+-chelate EGTA, calpaim inhibitor CPZ and Ca2+ channel blocker LaCl3 suppressed the increase of MPT and H2O2 content, the decrease ofΔψm and Cyt c content, and reduced the root apoptotic rate. However, Ca2+-ATPase inhibitor Van played a reversed role, presenting as promoting the increase of MPT and H2O2 content, the decrease ofΔψm and Cyt c content, and accelerating the root apoptotic rate ultimately.
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