The Structure of Chloroplast DNA Molecules and the Effects of Light on the Amount of Chloroplast DNA during Development in Medicago truncatula      

Abstract

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.

On the Fate of Plastid DNA Molecules during Leaf Development: Response to the Golczyk et al. Commentary

“…We classified the chloroplasts into three types with respect to DAPI-DNA signal and then scored the number of cells (Supplemental Figure 2; see Methods), protoplasts, and isolated chloroplasts in each category (Table 1). These results demonstrate that plastids without detectable DAPI-DNA are present in the mature leaf blade and indicate that our inability to detect punctate forms of ptDNA in some chloroplasts cytologically is not attributable to a DNase artifact, as suggested by Golczyk et al, but to a decrease in ptDNA content and/or molecular integrity as leaves develop, as we concluded previously Rowan et al, 2004Oldenburg et al, 2006;Shaver et al, 2006Shaver et al, , 2008Zheng et al, 2011). This progression of proplastid-to-chloroplast development is shown in Supplemental Figure 3, illustrating a visible reduction in DAPI-DNA intensity and decreased genome equivalents per plastid for the mature chloroplasts (Table 2).…”

“…(Reprinted from Rowan and Bendich [2009], Figure 1. ) to pale-green chloroplast to fully green chloroplast (as well as the etioplast-tochloroplast transition upon illumination of etiolated maize seedlings) Rowan et al, 2004Oldenburg et al, 2006;Shaver et al, 2006Shaver et al, , 2008Zheng et al, 2011). Those data (summarized in Figure 1) show that after the decrease to the level in mature chloroplasts, ptDNA remains essentially constant as leaves reach maturity, in agreement with the conclusion of Golczyk et al As for gerontoplasts in senescing leaves, although Golczyk et al concluded that the ptDNA level remains unchanged, their description may, in fact, reflect a decline in ptDNA, as they noted that "DNA fluorescence became more and more diffuse and difficult to visualize."…”

“…For maize, we found undetectable DAPI-DNA in 85 and 82% of the plastids treated with and without DNase, respectively; for Arabidopsis, the corresponding values were 7 and 7% Rowan et al, 2004). In subsequent work, we omitted the DNase and used a high-salt buffer to remove DNA/chromatin and still found plastids with no DAPI-DNA: 9% for pea, 11 to 34% for M. truncatula, 2 to 36% for maize from DAPI-DNA quantification, and 80 to 87% for maize from visual scoring Shaver et al, 2006Shaver et al, , 2008Zheng et al, 2011). These data indicate that the absence of detectable DAPI-DNA fluorescence in some plastids is not due to an artifact arising from DNA degradation by nucleases during plastid isolation.…”

Teores de pigmentos fotossintéticos e estrutura de cloroplastos de Alfavaca-cravo cultivadas sob malhas coloridas

“…SHAVER et al (2008) verificaram que modulações na fluência de luz azul alteram a estrutura e as taxas de degradação de DNAs cloroplastídicos e a divisão plastídica em Medicago truncatula.…”

Chloroplast DNA Replication Is Regulated by the Redox State Independently of Chloroplast Division in Chlamydomonas reinhardtii

“…Chloroplasts also contain multiple copies of DNA (approximately 1,000 copies; Boffey and Leech, 1982;Miyamura et al, 1986;Baumgartner et al, 1989;Oldenburg and Bendich, 2004;Oldenburg et al, 2006;Shaver et al, 2008). In algae, chloroplast DNA is replicated in a manner that keeps pace with chloroplast and cell division in order to maintain the proper DNA content per chloroplast (i.e.…”