The Changes Of ROS And Antioxidant Enzymes, And Its Influence On Programmed Cell Death In Endosperm Cells Of Winter Wheat (Triticum Aestivum L.) Under Waterlogging

Previous studies had proved that programmed cell death of endosperm cells happened ahead of time under waterlogging. In order to find the cytobiology mechanism behind this phenomenon, highly waterlogging tolerant eultivar Hua 8 and waterlogging susceptible eultivar Hua 9 were employed in this dissertation. These materials first had been under different waterlogging treatment during flowering, and then dynamic changes of reactive oxygen species (ROS) levels in the endosperm cells of these materials were detected by micro and ultracytochemical localization. Real time PCR technique was employed to study the gene expression of key antioxidant enzymes—NADPH thioredoxin reductase (NTR), Mn superoxide dismutase (MnSOD) and Catalase (CAT)—in endosperm cells. At the same time, total SOD and CAT activities were determined. Moreover, the influence of ascorbic acid and mannitol on materials under waterlogging treatment, and exogenous H2O2 on materials without waterlogging were researched. The main results were as follows:1. In endosperm cells of highly waterlogging tolerant eultivar Hua 8 and waterlogging susceptible eultivar Hua 9 which without waterlogging treatment, ROS content which detected by fluorescence probe raised at 12 DAF (12 days after flowing, 12 DAF) and 15 DAF, then declined. The accumulation of H2O2 and O2" were mainly in cell membrane, cell wall and mitochondria by ultracytochemical localization.7 days waterlogging treatment (7 DWT) induced the increase of ROS in endosperm cells of different waterlogging tolerant wheat cultivars compared with that without waterlogging treatment, and culminated at 12 DAF, and reduced gradually after 12 DAF. With longer waterlogging treatment time, the ROS accumulation of Hua 8 and Hua 9 culminated at 12 DAF as well. Then Hua 8 decreased gradually, but Hua 9 still kept at a very high level until 18 DAF. And the accumulation of H2O2 and O2" increased in cell membrane, cell wall and mitochondria. And the deformation, rupture of cytomembrane and swelling, distortion of mitochondrium were also dectected.2. In endosperm cells of Hua 8 and Hua 9 which omitted waterlogging treatment, the gene expression level of antioxidant enzymes including NTR, MnSOD and CAT decreased gradually, and the total enzyme activity of total SOD and CAT decreased gradually too. Under 7 days waterlogging treatment, CAT gene expression in Hua 8 increased in comparison with the endosperm cells in control group of the same period, but the activity decreased, which implied that CAT had been suppressed possibly at the stage of post-transcription or post-translation. The total SOD activity and MnSOD gene expression were raised, and NTR expression quantity changed with great increase. At the same condition, in endosperm cells of Hua 9, the activity and gene expression of CAT were greatly decreased, moreover, NTR gene expression raised with less scope. Under longer waterlogging treatment time (12 DWT), the activity of CAT and total SOD in endosperm of Hua 9 declined with more scope; and the expression of NTR gene began to decline at 15 DAF. The activity of total SOD in Hua 8 also increased, and the increase extent of NTR gene expression was larger than that in Hua 9.3.10,100, or 200 mmol/L exogenous H2O2 treatment could increase the intensity of Evans Blue staining and numbers of PCD cells in endosperm. DNA fragmentation was observed in the endosperm cells under 100 or 200 mmol/L exogenous H2O2. DNA fragmentation was more obvious in 200 mmol/L H2O2 treatment group. These results showed that exogenous H2O2 could intensify the PCD processes of endosperm cells.4. ROS scavengers could inhibit the intensifying of PCD processes in endosperm cells induced by waterlogging. The spray of 0.1 mol/L ascorbic acid and mannitol onto waterlogging wheat plants showed that Evans Blue staining became light and DNA fragmentation extent reduced comparing to the group without ascorbic acid and mannitol treatment.Conclusions:ROS played roles in the normal development of endosperm cells. Under waterlogging treatment, the increase level of ROS and the decrease of antioxidant enzyme activity might explain why the PCD processes in the endosperm cells shifting to an earlier date. ROS might not only play as signal molecule regulating expression level of genes related to metabolism of endosperm cells, but also being responsible for the cellular structure integrity and oxidation damage of key antioxidant enzymes, therefore, caused the PCD processes ahead of time.