Functional Analysis Of Chaperonin Gene PDM1 In Arabidopsis

PDM1 encodes chloroplast chaperonin CPN60?1,which plays an important role in plant growth and development.It has been reported that when PDM1 is completely absent,plants will not be able to complete the normal life cycle,resulting in embryo death or seedling etiolation,which seriously hinder the functional research of PDM1.A yellow leaf mutant was found and isolated from WT(Col-0)ethyl methanesulfonate(EMS)mutagenic library of Arabidopsis in the early stage of this study.By means of gene location,cloning and genetic compensation,PDM1 was identified as the mutant gene of the yellow leaf mutant.The discovery of weak allele pdm1 provides a good experimental material for further analysis of the function of PDM1.The results are as follows:1.PDM1 is involved in the regulation of chloroplast development and rRNA processing.Under normal growth conditions,the phenotype of Arabidopsis pdm1 mutant was smaller and its leaves were yellow.Compared with wild type,the chlorophyll content of Arabidopsis pdm1 mutant decreased significantly.Microscopical observation showed that the chloroplast development of pdm1 mutant was abnormal,and the number of chloroplasts in mesophyll cells was significantly reduced.It should be noted that the chloroplast of pdm1 mutant accumulated plastid globules significantly,and starch granules decreased relatively.We designed specific probes to detect 23S,16S,5S and 4.5S rRNA of chloroplast by Northern blot.Four major bands of 0.5,1.1,1.3 and 1.8 kb can be detected by WT 23S rRNA specific probes.However,there are three extra rRNAs with the corresponding sizes of 3.1,2.9 and 2.4 kb in pdm1,and the normal 23S rRNA level is significantly reduced,indicating that the mutant has significant precursor accumulation during 23S rRNA processing and maturation.When using 16S or 5S rRNA specific probes for immunoblotting,only one single band was detected in WT and pdm1,in which there was no significant difference in 16S rRNA level,while the content of 5S rRNA decreased significantly.In addition,the level of 4.5S rRNA in the process of maturation also decreased obviously.The above results showed that pdm1 specifically interferes with the normal development of chloroplast and the formation of ribosomal 50S large subunit functional rRNA,but does not affect the processing and maturity of chloroplast 16S rRNA.2.The dominant negative effect of pdm1.In the clarification of T-DNA insertion,only heterozygotes of mutant ptcpn60?1 can be identified,and homozygous progenies died.The results of genetic hybridization and PCR showed that the heterozygotes pdm1/ptcpn60?1-2 and pdm1/ptcpn60a1-3 showed the intermediate phenotypes of WT and pdm1 mutant.The chlorophyll content of leaves was measured,and the result trend was consistent with the observed leaf color.The northern imprinting analysis of mutants and hybrid strains was then carried out,and the results were also associated with the observed plant growth patterns,and the yellower the leaf color,the more serious the rRNA processing defects.These results indicate that pdm1 may have a competitive effect on other members of the ptCPN60 family at the cellular level,and its relative increase in the total endogenous ptCPN60 protein has a negative impact on plant growth and development.In order to further verify this view,we constructed a transgenic line containing pdml genomic DNA driven by CaMV 35S promoter under the background of WT,and also carried out northern immunoblotting analysis.The experimental results support the view that pdml competitively takes over the function of CPN60 located in chloroplast in a dose-dependent manner,thus causing a dominant negative effect on plant growth and development.3.Functional redundancy of ptCPN60 protein family.After analyzing the homology of the ptCPN60 protein family,we found that PDM1 has high homology with other proteins in the ptCPN60 protein family,suggesting that there is potential functional redundancy between the family members and PDM1 protein.In order to illustrate this point,we overexpressed ptCPN60a2,?1,?2 and ?3 in pdm1 by transgenic technology,which was verified by Northern blot analysis.These transgenic lines can partially restore the phenotype of pdm1,but the recovery effect of ptCPN60?1 and ?2 is better than that of ptCPN60a2 and ?3.The results showed that ptCPN60 protein family have functional redundancy in chloroplast development and rRNA processing.