The finding that plastid genomes map as circular molecules and the identification of circular ptDNA molecules by electron microscopy (the contour length of which corresponded to the determined size of the plastome) in many independent studies led to the long-held belief that the structural conformation of the plastome in vivo is a simple circle of double-stranded DNA. However, more recent investigations have revealed that the ptDNA displays a surprisingly great structural plasticity with only a minority of the genome molecules being circular (25 to 45% in developing leaf tissue; Lilly et al. 2001). In addition to circles, both electron microscopic investigations and pulsed-field gel electrophoretic analyses have identified various linear genome conformations, including plastome multimers (resembling concatemers as arising during rolling-circle replication of bacteriophage genomes) and branched multimers (Bendich and Smith 1990; Lilly et al. 2001; Oldenburg and Bendich 2004). Linear ptDNA molecules are unlikely to originate just from randomly broken circles: they were demonstrated to possess defined ends, some of which correspond to known origins of DNA replication (Oldenburg and Bendich 2004; Scharff and Koop 2006).
Interestingly, the fraction of genome molecules that is circular also shows a variety of different conformations. A substantial amount of the circles (>30% in tobacco; Lilly et al. 2001) is multimeric in that two or more copies of the genome form a single large circle (Deng et al. 1989). Multimers can come, for example, from rolling-circle replication and/or fusion of monomeric circles by homologous recombination. In addition to circles, lasso-like structures and suspected partially single-stranded molecules (showing D-loop-like bubbles) have been seen (Lilly et al. 2001). Similar to other circular genomes and episomes found in nature, the ptDNA can also adopt various supercoiled conformations and form catenanes (interlocking circles; Mukherjee et al. 1994; Kumar et al. 1995; Ahlert et al. 2003; Cho et al. 2004; Bendich 2004). Another structural peculiarity of the plastid genome is its presence in two isoforms due to flip-flop recombination of the two inverted repeat regions (3.1).
The functional relevance of most of the many different conformations of the ptDNA is still unclear. Some of them may simply represent replication intermediates; others may lack any functional significance. An interesting exception may be the degree of ptDNA supercoiling: studies in the unicellular green alga Chlamy-domonas reinhardtii have revealed that DNA topology fluctuates in dependence on the diurnal rhythm and that these fluctuations correlate with changes in the transcriptional activity (Salvador 1998). This finding may suggest that conformational changes of the ptDNA are involved in the regulation of plastid gene expression.
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