Plant Physiol. Journal of Pharmacology and Experimental Therapeutics
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Published on January 24, 2008; 10.1104/pp.107.112946

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Received November 8, 2007
Accepted January 12, 2008

The structure of chloroplast DNA molecules and the effects of light on the amount of chloroplast DNA during development in Medicago truncatula

Jeffrey M. Shaver , Delene J. Oldenburg , and Arnold J. Bendich *

Department of Biology, University of Washington, Seattle, Washington 98195-5325 USA

* Corresponding author; email: bendich{at}u.washington.edu.

We used pulsed-field gel electrophoresis and restriction fragment mapping to analyze the structure of Medicago truncatula chloroplast DNA (cpDNA). We find most cpDNA in genome-sized linear molecules, head-to-tail genomic concatemers and complex branched forms with ends at defined sites, rather than at random sites as expected from broken circles. Our data suggest that cpDNA replication is initiated predominantly on linear DNA molecules with one of five possible ends serving as putative origins of replication. We also used 4',6-diamidino-2-phenylindole-staining of isolated plastids to determine the DNA content per plastid for seedlings grown in the dark for three days and then transferred to light before being returned to the dark. The cpDNA content in cotyledons increased after three hours of light, decreased with nine hours, and decreased sharply with 24 hours of light. In addition, we used real-time quantitative PCR to determine cpDNA levels of cotyledons in dark- and light-grown (low white, high white, blue and red light) seedlings, as well as in cotyledons and leaves from plants grown in a greenhouse. In white, blue and red light cpDNA increased initially and then declined, but cpDNA declined further in white and blue light while remaining constant in red light. The initial decline in cpDNA occurred more rapidly with increased white light intensity, but the final DNA level was similar to that in less intense light. The patterns of increase and then decrease in cpDNA level during development were similar for cotyledons and leaves. We conclude that the absence in M. truncatula of the prominent inverted repeat cpDNA sequence found in most plant species does not lead to unusual properties with respect to the structure of plastid DNA molecules, cpDNA replication, or the loss of cpDNA during light-stimulated chloroplast development.






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