One thousand to 1,700 copies of plastid DNA are present per cell in Arabidopsis thaliana leaves (Zoschke et al. 2007) whilst five thousand to over ten thousand copies of plastid DNA per cell are present in leaves of Pisum sativum (Lamppa and Bendich 1979), Triticum aestivum (Day and Ellis 1984), Spinacia oleracea (Lawrence and Possingham 1986), and Hordeum vulgare (Baumgartner et al. 1989). Fewer plastid genomes per cell are found in other organs containing non-green plastids, such as the roots of P. sativum (~500 copies per cell, Lamppa and Bendich 1979) and T. aestivum (~300 copies per cell, Day and Ellis 1984). An increase in plastid genome copies is associated with the development of chloroplasts from precursor plastids. Copy number estimates based on quantifying the DNA present in purified plastids from leaf cells of different ages indicate the number of genomes per chloroplast reaches a maximum value in young leaves and then decreases in older cells well before senescence. For example, in the developing primary leaf of four-day-old H. vulgare seedlings, plastids in the basal meristem were estimated to contain ~130 genomes, this increased to ~210 genomes in chloroplasts in older cells located one to three cm above the meristem, and decreased to ~50 genomes per chloroplast in the oldest cells in the leaf tip
(Baumgartner et al. 1989). More recent publications also report decreases in genomes per chloroplasts in mature leaves compared to young leaves. Decreases observed include 225 to 106 genomes per chloroplast in Zea mays (Oldenburg and Bendich 2004a; Shaver et al. 2006), 135 to 53 genomes per chloroplast in P. sativum, 122 to 47 genomes per chloroplast in Medicago truncatula, and 190 to 70 genomes per chloroplast in Nicotiana tabacum (Shaver et al. 2006). These results are consistent with the idea that replication of plastid DNA takes place predominantly in meristematic cells and leaf primordia (Kuroiwa 1991; Fujie et al. 1994; see Section 13.1) and as plastids divide during leaf development the number of genomes per plastid falls. We know very little about the replication mechanisms regulating the copy number of plastid DNA. Some progress has been made in this field with the recent finding that copy number is influenced by specific plastid DNA sequences. Deletion of the OriA plastid DNA sequence implicated in DNA replication (Section 4.1 below) reduces the copy number of plastid DNA in developing leaves of N. tabacum (Scharff and Koop 2007).
Recent publications detailing two to threefold reductions in DNA levels per plastid during leaf maturation also suggest the apparent absence of DNA in some mature chloroplasts: DNA was not observed in approximately 11% of M. trunca-tula, 9% of P. sativum, 80-90% of Z. mays (Shaver et al. 2006) and 29% of A. thaliana chloroplasts (Rowan et al. 2004). Loss of DNA from chloroplasts during leaf ageing was not observed in N. tabacum (Shaver et al. 2006). Based on the results obtained in Z. mays and A. thaliana a mechanism that actively degrades DNA in maturing chloroplasts was proposed by the authors (Oldenburg and Bendich 2004a; Rowan et al. 2004). These results appear to suggest that chloroplasts lacking DNA retain photosynthetic activity for long periods (Oldenburg and Bendich 2004a; Rowan et al. 2004), which conflicts with our current understanding of the importance of plastid gene expression for maintaining chloroplast functions. An alternative explanation for the apparent absence of DNA in isolated plastids is that it is an artefact resulting from the experimental approaches used (Li et al. 2006). In particular, degradation of plastid DNA during the purification of plastids or during treatment of plastids with DNase I (to remove contaminating extra-plastidic DNA outside plastids) will give rise to low copy number estimates. This might be more problematic for old leaf cells of some species where the release of DNA nucleases during homogenisation and changes in plastid porosity might allow nucleases to enter plastids. DNA fluoresces when stained with the DNA-binding dye 4',6-diamidino-2-phenylindole (DAPI). DAPI stained chloroplasts in leaf sections of A. thaliana and N. tabacum fixed immediately after sectioning appeared to show the same pattern of DNA reduction or loss obtained with isolated chloroplasts supporting the data with isolated chloroplasts (Shaver et al. 2006). Quantitation of plastid DNA levels in total DNA preparations from liquid nitrogen frozen leaves using Southern blot analysis is less sensitive to plastid DNA degradation during sample preparation and provides a robust method for estimating plastid DNA levels. Such an approach using rapidly extracted total DNA can be used to confirm or dismiss the findings based on isolated chloroplasts. Copy number estimates can also be obtained using quantitative real-time PCR on total DNA with controls to rule out contamination or amplification of 'promiscuous' plastid
DNA sequences present in mitochondria and nuclei (Zoschke et al. 2007). Using these methods it appears that once chloroplast development is completed the levels of plastid DNA in total DNA appear to remain relatively constant during further leaf development in A. thaliana (Zoschke et al. 2007; Li et al. 2006) and N. tabacum (Li et al. 2006). These results do not support the idea of a dramatic reduction in plastid DNA levels during leaf development in A. thaliana.
Replication of plastid and nuclear DNA do not appear to be tightly co-ordinated in N. tabacum (Heinhorst et al. 1985) or the green alga Chlamydomonas reinhard-tii (Chiang and Sueoka 1967). In contrast to the stringent controls restricting nuclear DNA synthesis to one round of replication during the S phase of each cell cycle, plastid DNA replication appears to be less stringent and is not limited to the S phase (Heinhorst and Cannon 1993). Moreover, plastid genomes appear to be chosen randomly for replication (Birky 1994). The prereplication factor CDT1 appears to affect both nuclear DNA replication and plastid division in A. thaliana and provides a possible link between the cell cycle and plastid division (Raynaud et al. 2005). In synchronous cultures of C. reinhardtii, duplication of plastid DNA could be localised to a particular time period (Chiang and Sueoka 1967) whereas plastid DNA synthesis, monitored by 32P incorporation, was observed throughout the cell cycle (Grant et al. 1978). The 32P-incorporation was suggested to be due to DNA repair activities which were required to maintain plastid genomes throughout the cell cycle (Grant et al. 1978).
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