We examined the DNA from chloroplasts obtained from young and fully expanded leaves of tobacco (Nicotiana tabacum L.), Medicago truncatula, pea (Pisum sativum L.), and maize (Zea mays L.). The changes in plastid DNA content and structure were monitored by four independent methods: 4',6-diamidino-2-phenylindole (DAPI) staining with intact chloroplasts, in situ DAPI staining of cytological sections, ethidium bromide staining at the single-molecule level after exhaustive deproteinization of lysed chloroplasts, and pulsed-field gel electrophoresis. During leaf development, we found a decline of chloroplast DNA (cpDNA) in all four plants. For tobacco, for which plants can readily be regenerated from somatic cells, cpDNA persisted longer than in the other three plants. We also found a striking progression from complex multigenomic DNA molecules to simple subgenomic molecules during plastid development. Although the decrease in molecular size and complexity paralleled the decrease in DNA content per plastid, 6% of the chloroplasts in a fully expanded tobacco leaf still contained DNA in complex branched structure, whereas no such complex structures were found in mature leaves for the hard-to-regenerate maize.

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 3 d and then transferred to light before being returned to the dark. The cpDNA content in cotyledons increased after 3 h of light, decreased with 9 h of light, and decreased sharply with 24 h of light. In addition, we used real-time quantitative polymerase chain reaction to determine cpDNA levels of cotyledons in dark-and lightgrown (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.