Function And Mechanism Of Catalase Interacting Protein NCA In Rice

Hydrogen peroxide is an important signal molecule in plants.It plays an important role in plant growth and development.However,excess accumulation of hydrogen peroxide can cause toxicity to plants when plants are exposed to environmental stresses.Therefore,timely removal of hydrogen peroxide from plants and adjustment of cell redox levels are very important.Plant contains many proteins related to H₂O₂ metabolism.Catalase?CAT?can decompose H₂O₂ into H₂O and O₂.It is a very important active oxygen scavenging enzyme.A major change under many environmental stresses,such as salinity,high light,and pathogen infection,is the accumulation of reactive oxygen species in the plant and the altered intracellular redox state.Rice is widely grown in tropical and subtropical areas.These places are often exposed to stresses.Therefore,it is of theoretical and practical significance to study the regulatory mechanisms of CAT activity in rice and how it participates in stress response.Previous studies have shown that CAT enzyme activation in plants requires the help of NCA1?No Catalase Activity 1?protein.There are two OsNCA1 genes in the rice genome,here named as OsNCA1a and OsNCA1b,both of which are located on chromosomes 1 and 2,respectively,with a similarity of up to 94.5%.Ourprevious work have demonstarated that both of them can activate CAT activity in rice and are located in the cytoplasm.Further studies are conducted in this thesis and following results have been obtained:1.The CRISPR/Cas9 technique was used to generate nca1a,or nca1b single mutant and nca1a/nca1b double mutant.Phenotypic observation and enzyme activity determination of the above mutant plants revealed that the phenotype and enzyme activity of the nca1a and nca1b homozygous lines were not significantly different from those of the wild type,whereas nca1a/nca1b homozygous lines had less than 10%CAT activity in leaves and showed white spot lesions.The above result impicates that OsNCA1a and OsNCA1b are functionally redundant under normal growth conditions.2.Yeast three-hybrid analysis demonstrated that either OsNCA1a protein or OsNCA1b protein could interact with CATC alternatively,but both NCA1 proteins could not interact with CATC simultaneously.OsNCA1a and OsNCA1b can not interact with each other.The above result indicates that there is only one NCA binding site on CATC.3.In order to further explore the function and mechanism of two OsNCA1 in rice,we determined the enzyme activity and phenotypic traits of the mutant plants under stress conditions.The results showed that under salt stress,change in enzyme activity of single mutant was not different from that of WT plants.The result means that either of the two genes in rice can meet the requirement of CAT activation in rice under salinity stress.In addition,association/dissociation of CAT with glycolate oxidase?GLO?,another interaction protein of CAT,was further investigated in this study.Our previous work has showed that GLO and CAT can interact in rice,and a GLO-CAT dynamic interaction dissociation model was proposed to explain how CAT scavenges H₂O₂efficiently even if its affinity to H₂O₂ is very low.Further analysis was conducted in this study:?1?rice protoplast BiFC analysis found that glutathione?GSH?can significantly reduce the BiFC fluorescence generation efficiency of GLO-CAT,meaning that GSH is also able to inhibit the interaction of GLO with CAT,like salicylic acid?SA?;?2?The BiFC transgenic rice plants were generated in this study,which sets essential foundation for determingGLO-CAT interaction/dissociation pattern at the whole plant level in the future.