Organization of plastid genomes in nucleoids

The plastid genomes do not swim around as naked DNA in the plasmatic compartment (stroma) of the organelle. Instead, several copies of the plastome are densely packed together in large nucleoprotein bodies called plastid nucleoids (Kuroiwa 1989, 1991). Nucleoids can be visualized by fluorescence microscopy after staining of cells or tissues with the DNA-intercalating fluorochrome DAPI (4',6-diamidino-2-phenylindole; Kuroiwa 1991). Number, shape, and size of the nucleoids as well as their distribution in the chloroplast vary depending on the species. In algae and higher plants, five different subtypes of nucleoid morphology have been described, ranging from spherical to ring-like structures (Kuroiwa 1989). Likewise, plastome copy numbers per nucleoid are variable between species and in dependence on plastid differentiation. Proplastids, for example, often contain only a single nucleoid, whereas mature chloroplasts can easily contain several or even dozens of nucleoids.

The nucleoid, and probably each individual plastid genome, is membrane bound. In higher plants, evidence has been provided for both an association with thylakoid membranes and an anchoring to the inner envelope of the chloroplast (Liu and Rose 1992; Sato et al. 1993). Isolated nucleoids retain transcriptional activity in vitro (Sakai et al. 1991) suggesting that the transcriptional apparatus (RNA polymerases and sigma factors) is tightly associated with the plastid genome (Krause and Krupinska 2000). Little is known about the molecular processes and mechanisms that organize plastid nucleoids. Notably, it is entirely unclear, how a defined number of genome copies are packed into one nucleoid and how higher-order structures of the plastid genome are built and regulated (Salvador et al. 1998). It is known, however, that nucleoid size and number per cell are controlled by nuclear genes. In the unicellular green alga Chlamydomonas reinhardtii, mutants were obtained that had increased or drastically decreased numbers of nu-cleoids (Ikehara et al. 1996; Misumi et al. 1999). While wild type cells contain on average seven nucleoids, mutants with increased nucleoid number had 14-23 nu-cleoids. At the other extreme, a mutant called moc (for 'monokaryotic chloroplast') had only a single huge nucleoid per chloroplast (Misumi et al. 1999). How ever, the genes and mutations responsible for these interesting nucleoid pheno-types have not been identified to date.

Recently, several DNA-binding nucleoid proteins have been identified and/or biochemically characterized to some extent (Nakano et al. 1997; Sekine et al. 2002; Jeong et al. 2003; Cannon et al. 1999). The arguably best-studied nucleoid constituent is Hlp (also called HU), a histone-like DNA-binding protein that, similar to Hlp homologs in eubacteria, is believed to serve as a general architectural nucleoid protein (Kobayashi et al. 2002). Recently, a first step towards determining the proteome of chloroplast nucleoids has been taken in Arabidopsis (Pfalz et al. 2006). Nucleoid preparations obtained by a two-step chromatographic purification were subjected to mass spectrometric protein identification. Although the preparations were not absolutely pure, several good candidates for genuine nu-cleoid proteins could be identified, including RNA polymerase subunits, topoi-somerases and DNA polymerase subunits (along with a number of novel proteins of unknown function; Pfalz et al. 2006). Interestingly, Hlp, the most abundant and major architectural nucleoid protein in bacteria and algae (Kobayashi et al. 2002) was not identified, possibly suggesting that nucleoid organization in higher plants is fundamentally different from that in eubacteria and algae. The systematic identification of nucleoid proteins and their functional characterization using the power of Arabidopsis nuclear genetics should pave the way to a better understanding of the higher-order structure of plastid DNA, its dynamics and impact on the regulation of plastid gene expression.

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