Although it might be expected that chloroplast DNA (cpDNA) would be stably maintained in mature leaves, I report the surprising observation that cpDNA levels decline during plastid development in Arabidopsis thaliana (Col.) until many of the chloroplasts contain little or no DNA long before the onset of senescence. I measured cpDNA in shoot tissues at different times during development using several methods: staining leaf sections as well as individual isolated chloroplasts with 4'6-diamidino-2-phenylindole (DAPI), real-time quantitative PCR with DNA prepared from total tissue as well as from isolated chloroplasts, fluorescence microscopy of ethidium-stained DNA molecules prepared in gel from isolated plastids, and blot-hybridization of restriction-digested total tissue DNA. I observed a developmental decline in DNA per chloroplast and the fraction of cellular DNA represented by cpDNA. The reduction in cpDNA content could not be attributed to an artifact of chloroplast isolation, and thus the DNA within chloroplasts was degraded in vivo as leaves matured. Molecules of cpDNA consisted primarily of branched, linear, multigenomic forms that became progressively fragmented during leaf development. That fragmentation might reflect the developmental degradation of cpDNA that is damaged but not repaired. Since almost nothing is known about the DNA repair process in chloroplasts, I investigated the roles of genes that encode chloroplast-targeted RecA, a DNA repair protein in E. coli. Mutation of the Arabidopsis cpRecA gene resulted in altered structural forms of cpDNA molecules, a reduced amount of cpDNA, and an increased amount of single-stranded cpDNA. The plants showed signs of reduced chloroplast function: variegation, necrosis and impaired growth. We conclude that the process by which damaged DNA is repaired in bacteria has been retained in their endosymbiotic descendent, the chloroplast. I also developed a method for analyzing and sorting chloroplasts on the basis of DNA amount, which should facilitate identification of the environmental and genetic factors that contribute to the loss or retention of cpDNA during development.
|Advisor:||Bendich, Arnold J.|
|School:||University of Washington|
|School Location:||United States -- Washington|
|Source:||DAI-B 70/01, Dissertation Abstracts International|
|Subjects:||Molecular biology, Plant biology, Cellular biology|
|Keywords:||Chloroplast DNA, DNA repair, Leaf development, Nucleoid, Recombinational repair|
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