Functional Study Of AtRsmD Regulating Gene Expression In Chloroplasts Of Higher Plants

The modification of ribosomal RNA is widespreadly present in all organisms,and the modification types are mainly divided into three types:base methylation,pseudouracilation,and 2'-O-methylation modification.In Escherichia coli,Rsm D is a methyltransferase encoded by the Rsm D(yhh F)gene and it acts on the m~2G966 site on 16S r RNA.Nevertheless,its deletion did not affect the growth of bacteria,which indicates that it is not essential for E.coli.In the model plant Arabidopsis thaliana,the locus AT3G28460 encodes a homologous protein of E.coli Rsm D,At Rsm D.A previous report has shown that the At Rsm D protein has a methyltransferase activity responsible for the methylation of 16S r RNA m~2G915;however,its regulatory function in chloroplast gene expression and chloroplast development in higher plants still remains unclear.This dissertation further characterized the At Rsm D gene and the corresponding mutant atrsmd-2 in detail.The results from RT-PCR and RT-q PCR analysis showed that the transcription of At Rsm D gene was detected in roots,stems,leaves,fruit pods and seedlings,and the expression levels are highly in green tissues.Green fluorescent protein observation and biochemical analysis showed that the At Rsm D protein is located in the chloroplast and is associated with the thylakoid.Under the normal growth conditions,no visible phenotype could be observed in the atrsmd-2 mutant,compared with that of the wild type.However,the Fv/Fm value of this mutant plant was lower than that of the wild type.This indicates that the deletion of this gene reduced the photosynthetic efficiency.BN-PAGE showed that the assembly of macromolecular complexes on the thylakoid membranes in the atrsmd-2mutant was less than that of the wild type.Western blotting showed that the accumulation of chloroplast-encoded photosynthetic-related proteins,D1,D2,CP43,CP47 and Atp F was significantly less than that of the wild type;in contrast,their transcription levels of these genes in the atrsmd-2 mutant were slightly higher than those of the wild type.The amounts of other chloroplast-encoded photosynthetic-related proteins,Atp A,Atp B,Rbc L,Pet A,Pet D,accumulated to that of the wild type.Additionally,the accumulation of chloroplast ribosomal proteins,PRPS1,PRPS5,PRPL2 and PRPL4 in the atrsmd-2 mutant was close to that of the wild type,respectively.Northern blotting showed that the abundance of the mature transcript of 16S r RNA in the atrsmd-2 mutant chloroplast was reduced than that in the wild type,while the mature transcripts of other chloroplast ribosomal r RNA,including 23S r RNA,5S r RNA,and 4.5S r RNA,were increased,compared to the wild type.This indicates that the absence of the At Rsm D protein affects the accumulation of 16S r RNA and the biogenesis of chloroplast ribosomes.In agreement with it,the atrsmd-2 mutant exhibited the sensitivity to cold stress and the prokaryotic translation inhibitors,lincomycin and chloramphenicol.All these data combined with a previous report showed that the deletion of 16S r RNA m~2G915 methylation probably affects the translation function of chloroplast ribosomes to some chloroplast proteins,and this effect is exacerbated by cold stress.This work would enrich the understanding of chloroplast ribosomal RNA methylation and chloroplast gene expression regulation in higher plant.