Research On The Mechanism Of Photooxidative Damage Of Rice Mutant 812HS

812HS is a photooxidation mutant of wild-type rice variety 812S.It has a typical"yellowing"phenotype of leaf tip under high light conditions in the field,specifically in the middle and late tillering stage,812HS is subjected to intense sunlight radiation,resulting in photooxidation damage,resulting in leaf yellowing and a decrease in photosynthetic capacity.In this study,through the comparison of wild type and mutant under field natural light and shading treatment,it was clear that light condition was the decisive factor of mutant phenotype.Compare the changes of physiological indexes,photosynthetic performance parameters and related protein abundance in different stages of photooxidation process under different light conditions,clarify the photosynthetic characteristics and physiological mechanism of 812HS from the aspects of physiology,biochemistry,antioxidant system and protein,and explain the mechanism of photooxidation damage.The purpose of this study is to provide experimental help and theoretical reference for further comprehensive analysis of the regulation mechanism of rice photooxidation and mitigation of rice photooxidation.The specific results are as follows:1.The effect of photooxidation on the photosynthetic performance of rice:the leaf"yellowing"photooxidation phenomenon occurred in 812HS under the natural light with an average light intensity of about 40000Lux,while 812S could keep the leaves from chlorosis and yellowing at the same light intensity.The detection of physiological indexes during yellowing period showed that strong light could lead to the rapid decrease of net photosynthetic rate（P_N）,transpiration rate（Tr）,stomatal conductance（Gs）and chlorophyll content（SPAD）of 812HS,and the increase of intercellular CO₂concentration（Ci）.Shading treatment can avoid the decline of photosynthetic performance by weakening light intensity.The results of chlorophyll a fluorescence kinetic parameters analysis showed that F_mand F_v/F_mdecreased,electron transfer efficiencyψEo,ψRo and quantum yield parametersφP_O,φE_O,φR_Odecreased significantly.The closing rate M₀of the reaction center（RC）increased,RC decreased,ABS/RC and TRo/RC increased,and the increase of DIo/RC reflected the loss of excess light energy through a large amount of heat dissipation,while the light energy absorption,capture and electron transfer energy（ABS/CSm,TRo/CSm and ETo/CSm）on the excitation surface of each reaction center decreased.There was almost no difference between shading treatment wild type 812S and mutant 812HS,exceptδR_O,ψRo andφR_Odecreased slightly,other performance parameters had no significant change.The results showed that excessive light seriously damaged the integrity of the photosystem II（PSII）reaction center and electron transport chain of the mutant 812HS,the ability of electron transport decreased and the photosynthetic performance was weakened.Shading treatment can reduce the light intensity and effectively reduce the degree of photooxidative damage to 812HS.2.Effects of photooxidation on oxidation degree and antioxidant system of rice:under high light intensity,the ability of light energy utilization in leaves is low,and excess light energy will lead to excessive production of reactive oxygen species and accumulation in leaves.In this study,it was found that under the light of 40000Lux,the contents of O₂^·-and H₂O₂in 812HS leaves were significantly higher than those in812S,and the activities of superoxide dismutase（SOD）,peroxidase（POD）and catalase（CAT）in 812HS were significantly lower than those in 812S.Under shading treatment,the ROS content of the two kinds of rice was lower,and the activity of antioxidant enzymes maintained a high level,and there was no significant difference between 812S and 812HS.And under strong light,the content of malondialdehyde（MDA）,the product of membrane lipid peroxidation of 812HS,was much higher than that of shading group,and the soluble protein of leaves was degraded greatly.The reaction of ROS and antioxidant enzymes showed that the photorepair ability of812HS under strong light was much lower than that of 812S,when the photoinhibition damage was always greater than the photorepair ability,excess light could stimulate a large number of reactive oxygen species and could not be removed in time,resulting in serious peroxidation damage to leaves.Shading can reduce the light from the growth environment,reduce the formation and accumulation of reactive oxygen species,and avoid photoinhibition.3.Effects of photooxidation on chloroplast photosynthetic proteins:the results of Blue-Native PAGE electrophoresis showed that strong light degraded a large number of membrane complexes such as PSI and PSII complex core subunits,monomers,trimers,cytochrome b6f and light-harvesting pigment LHCII in 812HS chloroplast thylakoid membrane,and down-regulated 19 protein spots related to photosynthesis.However,the thylakoid proteins and complexes were not degraded after shading.Western-Blot bands showed that the contents of 812HS OEC protein,ATP-βsubunit,Ru Bis Co large subunit,PSI core subunit Psa A and Psa B,PSII core subunit Psb A（D1）,Psb B and Psb O were significantly lower than those of 812S under strong light.Shading can protect the above photosynthetic proteins,and the content of these protein subunits is higher in wild types and mutants,thus maintaining normal photosynthesis.These results show that strong light can damage or degrade some membrane proteins and complexes closely related to 812HS and photosynthesis,and photosynthesis can not be carried out in an orderly manner.To sum up,by dynamically comparing the changes of photochemical reaction activity,photoprotection mechanism,photosynthetic pigment and thylakoid membrane protein between mutant 812HS and wild-type 812S rice under different light intensity,we can deepen the understanding of the mechanism of photooxidative damage in rice leaves and lay a theoretical foundation for the cultivation of high-light-tolerant rice varieties.