Preliminary Analysis Of The Function And Composition Of Arabidopsis Mitochondrial HSP60/HSP10 Chaperone

Plants are sessile organisms that are subjected to both abiotic and biotic stressors during their life cycle.A number of environmental stresses cause damage to protein structure leading to protein aggregation,which affects life of all living organisms.Mitochondrial chaperone HSP60/HSP10 belongs to the chaperone family.Nascent or misfolded proteins are often assisted to achieve their native structures by molecular chaperones.Chaperones could be divided into the type I chaperone and the type II chaperone,and mitochondrial HSP60/HSP10 belongs to the type I chaperone.In Arabidopsis thaliana,there are three HSP60-2,HSP60-3A,HSP60-3b protein subunits of HSP60,and two Co-chaperones,HSP10-1 and HSP10-2.At present,there are plenty of studies on the function of HSP60/HSP10 in mammals,which play a very important role in innate immunity and adaptive immunity.However,there are relatively few studies on the function of HSP60/HSP10 in plants,and its substrate and working mechanism are still unclear.Therefore,in order to explore its functions and effects in plants,the main content of this project is to purchase corresponding mutants for identification,and conduct preliminary observation and analysis of their phenotypes.The main findings are as follows:(1)In this study,five homozygous mutants hsp10-1,hsp10-2,hsp60-2,hsp60-3a and hsp60-3b were obtained from the ABRC mutant library.Compared with the wild type,hsp10-1 homozygous mutant showed slower growth,fewer rosette leaves,shorter roots,late moss extraction and reproductive defects.The homozygous mutants of hsp10-2,hsp60-2,hsp60-3a and hsp60-3b showed no obvious phenotypic changes compared to wild type plants.(2)The homozygous diploids of hsp60-2hsp60-3a and hsp60-3ahsp60-3b were obtained by hybridization experiments.Compared to the wild type,hsp60-2hsp60-3a had no obvious phenotypic changes.hsp60-3ahsp60-3b showed phenotypic changes of slow growth,short roots and late moss extraction.hsp60-2hsp60-3b showed a lethal phenotype.Combined with the results of evolutionary tree and relative expression level,it was found that HSP60-3B plays a dominant role in gene function,followed by HSP60-2 and HSP60-3A in Arabidopsis thaliana.In the hybridization experiment of hsp10-1 and hsp10-2,only AAbb,Aa Bb and aa BB plants were found.The experiment verified that the cross exchange was prevented during the process of gamete biogenesis in the heterozygous Aa Bb plants,which only produce Ab and a B gametes,not AB or ab gametes,thus the offspring carrying other genotypes were not available.(3)The phenotype of hsp10-1 homozygous mutant was restored to the level of wild type by transgenic complementation experiment,indicating that the phenotype of hsp10-1homozygous mutant is indeed caused by the deficiency of gene HSP10-1.Both HSP10-1 and HSP10-2 were found to be localized in mitochondria and expressed in roots,stems,leaves,flowers and seedlings by subcellular localization and GUS staining.The content of superoxide anion in hsp10-1,hsp10-2,hsp60-2,hsp60-3a,hsp60-3b,hsp60-2hsp60-3a,hsp60-3ahsp60-3b homozygous mutants increased by 6,1.1,1.1,1.3,2,2,3.5 times compared with the wild type.(4)RNA-seq analysis of hsp10-1 homozygous mutant revealed a total of 1079 differentially expressed genes.Compared with wild-type plants,786 genes were up-regulated and 293 genes were down-regulated.These genes were mainly involved in transcriptional regulation,protein folding and oxidative phosphorylation.Response to external stimuli(hypoxia,dehydration,oxidation,salt,injury)and plant hormone signaling pathways(ethylene,abscisic acid,salicylic acid,brassinosteroid,abscisic acid,jasmonic acid,gibberellin,auxin);In i TRAQ data analysis,a total of 1459 differential proteins were identified,including 688up-regulated proteins and 771 down-regulated proteins.These proteins are mainly involved in RNA metabolism,protein folding,protein phosphorylation,oxidative phosphorylation,Response to external stimuli(stimulation,hypoxia,dehydration,oxidation,salt,heat)and plant hormone signaling(abscisic acid,cytokinin,brassinosteroids,salicylic acid).These results indicate that the co-chaperone protein HSP10-1 plays an important role in these pathways.