ObjectiveChloroplasts are originated from endosymbiontic cyanobacteria. During cell proliferation, chloroplasts divide by binary fission, a manner like that in their bacterial ancestor, to ensure the stable inheritance. Chloroplast division requires highly coordinated actions of proteins with both prokaryotic and eukaryotic origin. Arabidopsis thaliana is a model plant for the study of chloroplast division. The chloroplast division machinery has been preliminarily studied in Arabidopsis thaliana in the past two decades. However, the function of CRL gene in chloroplast division is still unclear.
MethodIn this study, we identified two new crl mutants x17-3 and pd50 by mutant screening and genetic mapping. We compared the chloroplast phenotype, gene splicing, mRNA level and chlorophyll contents of x17-3, pd50 and the wild type by microscopic observation and molecular biology techniques, respectively. Further gene function confirmations were done by transformation complementation and RNAi.
ResultCompared with the wild type, x17-3 and pd50 showed an obviously different chloroplast phenotype. They had much larger chloroplast size and a lower number of chloroplasts per cell than those in the wild type. The chloroplast number of x17-3 was 40% of the wild type. pd50 contained only 1 to 4 chloroplasts per cell and showed a severe growth inhibition. Genetic mapping and DNA sequencing analysis revealed mutations of the CRL gene in x17-3 and pd50, which were present in intron and caused altered splicing and open reading frame shift mutations. The mRNA level of CRL decreased in pd50 compared with the wild type and x17-3 by real-time quantitative RT-PCR analysis. Complementation experiment result confirmed that the chloroplast division and plant growth defects of x17-3 and pd50 were due to the mutations in CRL gene. The CRL RNAi lines with decreased levels of the CRL mRNA also showed chloroplast division defect. In addition, the content of chlorophyll decreased in x17-3 and pd50 compared with that in the wild type.
ConclusionOur study provides new material and some useful information for the study of the function of CRL.