Changes in chloroplast DNA during development in tobacco, Medicago truncatula, pea, and maize

Abstract

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.

Chloroplast DNA in Mature and Senescing Leaves: A Reappraisal

“…Our findings cannot be reconciled with those of Bendich and coworkers, who have claimed that ptDNA sharply declines or even vanishes completely from chloroplasts long before the onset of senescence in several species, notably including Arabidopsis and maize Rowan et al, 2004Rowan et al, , 2009Oldenburg et al, 2006;Shaver et al, 2006). The reasons for these differences are not entirely clear, but in our opinion, the reported findings are not conclusive because of artifact-prone methods and the lack of appropriate controls checking the biochemistry used.…”

“…It was concluded that, in fully expanded leaves, most chloroplasts contain no or only insignificant amounts of DNA long before the onset of leaf senescence Rowan et al, 2004;Oldenburg et al, 2006;Shaver et al, 2006;Rowan et al, 2009). Retention of ptDNA was proposed to be dispensable after the photosynthetic machinery was established in that the plastome-encoded photosynthesis genes were no longer needed in adult leaves.…”

“…However, since contradictory data were reported for the same species that were grown under comparable, if not identical, conditions (Rowan et al, 2004(Rowan et al, , 2009Li et al, 2006;Oldenburg et al, 2006;Shaver et al, 2006;Zoschke et al, 2007;Evans et al, 2010;Udy et al, 2012), it is apparent that some of them must reflect methodological insufficiencies of the experimental approaches employed.…”

Effects of Reduced Chloroplast Gene Copy Number on Chloroplast Gene Expression in Maize

“…2). Because organelle DNA copy number changes during leaf cell differentiation (Baumgartner et al, 1989;Shaver et al, 2006), we sampled different developmental stages by taking advantage of the developmental gradient in the leaves of maize seedlings (Leech et al, 1973). The second leaf of seedlings at the early three-leaf stage was divided into basal, middle, and apical sections (Fig.…”

“…It is generally agreed that the ratio of chloroplast to nuclear gene copy number decreases as leaf cells mature, although the magnitude of this decrease has been a subject of debate (Baumgartner et al, 1989;Oldenburg and Bendich, 2004a;Rowan et al, 2004;Li et al, 2006;Shaver et al, 2006;Zoschke et al, 2007;Rowan et al, 2009).…”