Preliminary Study On The Effect Of Waterlogging Stress And Exogenous Inhibitors On Wheat Endosperm Cytoskeleton And PCD Characteristics Process

Wheat(Triticum aestivum L.)is one of the three major food crops in the world,and moisture damage severely hinders the yield and quality of wheat.Wheat endosperm cell development is accompanied by programmed cell death(programmed cell death,PCD),and enhanced water spillage will lead to an overall advancement of its process.In order to infiltrate the internal mechanism of the advancement of PCD progress of wheat endosperm cells under water submergence,this study used the moisture-tolerant variety Huamai 8(Huamai 8)and the non-humidity-tolerant variety Huamai 9(Huamai 9)as materials,on the day of flowering The treatment of overlapping water was carried out,and the changes of endosperm cytoskeleton and related binding proteins were studied by immunofluorescence and other methods.At the same time,the treatment of embedding microtubules and microfilaments with two inhibitors explored the relationship between cytoskeleton and PCD.In addition,the related physiological indexes of wheat endosperm in submerged water were studied.The main performance is as follows:1.During development,after field flooding treatment,Huamai No.8 and Huamai No.9 wheat corrugated fruit shrunk significantly,compared with the normal growing wheat cortex during the same period,plumpness decreased,and 100-kernel weight decreased significantly.Compared with the normal growth group,the 1000-grain weight of wheat in the flooding treatment group at maturity decreased significantly.The detection results of the physiological and biochemical indexes of wheat endosperm showed that the APX activity of the endosperm of the two varieties increased after flooding,but the APX activity of Huamai 9 decreased after 15 DAF and 18 DAF.Flooding can also lead to increased PPO activity in the endosperm of the two varieties of wheat,but the upward trend of the moisture-tolerant variety Huamai 8is more significant.After flooding,the content of non-enzyme antioxidant GSH in wheat endosperm of both varieties increased,but the change trend in Huamai No.8endosperm was not obvious,and Huamai No.9 endosperm showed a gradual increase trend.Therefore,flooding will lead to an overall increase in the content of antioxidant substances.2.Immunofluorescence detection of wheat endosperm cytoskeleton showed thatunder normal growth conditions,with the development of endosperm,the amount of tubulin synthesis in Huamai 8 and Huamai 9 endosperm increased.After flooding stress,the changes in endosperm microtubules of the two varieties first increased and then decreased,and the difference between the varieties was not obvious.Similar to tubulin,the green fluorescent signal of microfilament actin(actin)in the endosperm of Huamai No.8 and Huamai No.9 that grow normally will also become stronger with the development of endosperm.The effect of flooding on Huamai 9 was similar to that of Huamai 8.The actin content showed a gradual increase trend,and reached the maximum at 15 DAF,and then decreased.3.Immunofluorescence detection of microtubule-binding protein in wheat endosperm cells showed that under normal growth conditions,the green fluorescence signal of microtubule-binding protein MAP65-1 in Huamai 8 and Huamai 9endosperm gradually increased,indicating that there was a large amount of MAP65-1The protein is synthesized.The response of Huamai 8 and Huamai 9 endosperm MAP65-1 protein to flooding is different.Flooding will promote the synthesis of Huamai 8 MAPA65-1 protein,but the content of MAP65 protein in Huamai 9endosperm The impact is not obvious.Immunofluorescence detection of microfilament binding protein showed that under normal growth conditions,with the increase of flowering days,the content of profilin of microfilament binding protein in the two wheat endosperms will gradually decrease.The effect of flooding on the changes of profilin protein in Huamai 8 and Huamai 9 endosperm was also different.Flooding will lead to further reduction of profilin protein synthesis in Huamai 8endosperm;however,Huamai 9 endosperm profilin protein has no obvious response to flooding treatment.4.Under flooding conditions,the microtubule inhibitor Fluoryl was applied to two varieties of wheat to promote microtubule depolymerization,which was tested by TUNEL and supplemented by Evans blue staining,DNA content and DNA hydrolase activity It was found that the DNA in the endosperm cells accelerated degradation;DNase increased;the number of nuclei decreased and the deformation was severe,and the activity of caspase-like protease increased.The detection of other indirect indicators found that the content of PCD-related signal molecule ROS increased,thecontent of dehydrogenase in cells decreased and the mitochondrial membrane potential changed.It is inferred that the assembly and disassembly of microtubules under flooded conditions will further interfere with the endosperm PCD process.5.After applying the microfilament inhibitor Lachunkulin B to the two varieties of wheat under flooding conditions,after the microfilament polymerization was broken,its PCD-related indicators also changed to varying degrees.The number of nuclei and DNA content decreased,the activity of DNA hydrolase increased significantly,and the number of dead endosperm cells increased significantly.The activity of caspase-like protease also increased accordingly.The change of the intolerant variety Huamai 9 was particularly strong.Other PCD-related indicators such as ROS content,dehydrogenase and mitochondrial membrane potential were more significantly changed than the microtubule treatment group.The above indicates that the assembly and disassembly of microfilaments under flooding conditions will also have a greater impact on the PCD process of the endosperm and the effect is more significant.In summary,wheat endosperm cytoskeleton will be synthesized in large amounts to resist adversity under flooding treatment,and when the synthesis of cytoskeleton is affected,it will accelerate the occurrence of PCD in cells.The effect of changes in microfilaments in the cytoskeleton on PCD is more obvious than that in microtubules,and the PCD indicators in the endosperm of Huamai 9 are also more significant than Huamai 8 in this experiment.Finally,it is preliminarily concluded that the cytoskeleton plays a crucial role in the entire PCD signaling pathway under flooding stress.