Study On PCD And Antioxidant Characteristics Of PTGMS Rice Anther Tapetum At Different Temperatures

As an important germplasm resource,photo-thermo sensitive genic male-sterile（PTGMS）has always been the focus of basic research on two-line hybrid rice.Peiai 64S（PA64S）is one of the photo thermo sensitive male sterile lines with large application area in Two-line Hybrid Rice in China.Its pollen fertility is regulated by light and temperature,but the mechanism of fertility regulated by temperature is not clear.In this study,PA64S near isogenic lines PA2364S and PA2864S with different fertility critical temperatures were selected as materials.Through the cytological observation,immunofluorescence staining and physiological and biochemical detection of anthers of plants with different fertility under different temperatures,the process of programmed cell death（PCD）in tapetum,the production of reactive oxygen species（ROS）,the accumulation of antioxidants and the level of gene expression were explored,Enrich the biological basis of temperature induced PTGMS rice pollen fertility transformation process.The main research results are as follows:1.The pollen fertility of the studied materials has obvious sensitivity to temperature regulation.The pollen fertility of PA2364S and PA2864S changed completely under the treatment of long light（14.0 h）and different temperatures（30℃and 21℃）:the two materials had no fertile pollen under the treatment of 30℃,the pollen abortion rate was100%,and there was no selfing;Under the treatment of 21℃,the two materials were fertile to varying degrees,and the iodine staining rate of pollen and self seed setting rate were35.0%-40.0%.2.The cytological changes and PCD characteristics of anthers treated at different temperatures were observed by toluene blue staining,transmission electron microscope and TUNEL.The DNA fragments of the tapetum treated at different temperatures from 8a to 8b disappeared obviously at the anther development stage;The DNA fragmentation degree of sterile materials treated with high temperature was low in phase 9,and there was no obvious degradation process of tapetum cells.The obvious characteristics of DNA fragmentation appeared only in phase 11,and the degradation of tapetum was delayed,and there was still tapetum in phase 12.TDR,Os PTC2,EAT1 and UDT1 which are involved in PCD of rice anther tapetum,have low expression level in anthers of sterile materials treated with high temperature and high expression level in anthers of fertile materials treated with low temperature,indicating that temperature regulates the PCD process of tapetum.3.ROS reactive dyes NBT and DAB were used to detect O₂^-and H₂O₂ in anthers of materials treated at different temperatures.It was found that the ROS signal of fertile materials treated at 21℃was stronger in the 9 th stage of anther development,while that of sterile materials treated at 30℃was stronger in the 11th stage.By measuring the content of ROS in anthers of plants with different fertility,it was found that the result was consistent with ROS staining knot.The results showed that the peak of ROS in the anther of fertile plants treated with low temperature appeared in the early stage of anther development,and the peak of ROS in the anther of sterile plants treated with high temperature moved backward.The activities of antioxidant enzymes POD,SOD and CAT in anthers were low in phase 9 and high in phase 11,which did not respond to the trend of ROS expression,while the change of APX activity was opposite to the trend of ROS expression.It suggests that APX may be the main antioxidant enzyme involved in the ROS reaction of anthers.4.The detection results of miR528,AO and As A in anther development stages 9 and11 treated with different temperatures showed that the highly expressed miR528 negatively regulated the expression of Ao,The content of ASA increased and the level of ROS decreased in the early stage of sterile materials.The above results preliminarily show that the two PA64s near isogenic line materials have differences in anther miR528-AO mediated regulation of antioxidant substances and ROS levels,anther PCD process and tapetum degradation process at different temperatures,so as to regulate rice pollen fertility.