| Photosynthesis is the physiological basis of plant growth and development on the earth.Plants and other organisms perform this biochemical reaction in chloroplast,to ensure their own growth.Recent studies on chloroplast proteins have shown that thylakoid lumen proteome has a function in the process of photosynthesis.Immunophilins,as an intracellular receptor of immunesuppressive agents,has been found in many different species including animals,plants,inserts and yeast,which have functions in the mechaniums of immunesupression.CYP38(Cyclophilin 38)is a member of the immunophilin family in Arabidopsis thaliana,which plays an important role in the assembly and maintenance of the photosynthesis photosystem II supercompexes,but the assembly mechanism is still not clear.We identified several interaction proteins of CYP38 by using yeast two hybrid screening,including At4g24930 and At1g21500.It is still unknown whether they can help CYP38 to participate in the assembly of PSII or not,In this study,we focus on the functions of these two genes,and try to find the relationship between them and CYP38.According to bioinformatics analysis,At4g24930 contains 162 amino acids,the size of mature protein is 17.9 k Da,the theoretical isoelectric point is 5.06.At1g21500 contains 67 amino acids,the size of mature protein is 7.3 k Da,the theoretical isoelectric point is 10.06.Structures prediction software showed that both At1g21500 and At4g24930 do not have any conserve domain.Arabidopsis e FP Brower predicts that At4g24930 and At1g21500 are mainly expressed in green tissues and organs.Evolutionary analysis shows that there were few homologous species of At4g24930 and At1g21500 in Arabidopsis thaliana,and they were all higher plants,indicating that these two genes may play an important role in the photosynthesis of higher plants.To study the function,we first obtained homozygous mutants of two different lines of At4g24930 and At1g21500 through CRISPR-Cas9 gene editing technology and screening.Subcellular localization and Western analysis of At4g24930 and At1g21500 showed that both genes were located in the thylakoid lumen of chloroplasts.The interaction between the two proteins and the C-terminal domain of CYP38 was verified by yeast two-hybrid in vivo and pull down assay.At4g24930 mutants had no significant difference from the wild type under normal light or fluctuating light conditions.However,under high light stress,the leaves show a distinctive purple phenotype compared with wildtype,and the edge of the leaves were yellow under salt stress,indicating that the mutants were sensitive to high light and salt.After high light treatment,Using Blue Native PAGE,we show that the C2S2M2 complex of the At4g24930-m1 mutant was reduced compared with the wild type,indicating that At4g24930 might have a special influence on the enrichment of the super complex during high light stress,which may be involved in the physiological mechanism of plants in response to high light.Under normal light conditions,At1g21500 mutants show 3 days later flowering than the wild type,and were more severe under fluctuating light conditions.The anthocyanin content under high light stress showed no significant difference with the wild type.The photosynthetic complex was detected by Blue Native PAGE under normal light,and no significant changes was found.These results suggest that At1g21500 may affect plant growth in some way.Mutants of At4g24930 and At1g21500 were crossed with cyp38-2 to obtain homozygous double mutants,which show that short,yellowish and stunted phenotype,both of the the mutants can not be survival under high light conditions.The above preliminary research on the functions of At4g24930 and At1g21500 indicates that At4g24930 and At1g21500 do interact with CYP38,and At4g24930 may participate in the assembly of PSII under high light,and At1g21500 affects the growth of plants.These two genes may jointly influence the photosynthetic efficiency of Arabidopsis in some way,and provide a basis for subsequent research. |
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