Plastids are the characteristic organelles of photosynthetic eukaryotes. They are the sites of photosynthesis, and their biosynthetic pathways supply the plant cell with many essential compounds. Chloroplasts evolved from a cyanobacterial ancestor after a single endosymbiotic event, that was followed by an extensive reduction of the plastid genome size (Timmis et al., 2004; Bock and Timmis, 2008; Green, 2011). Among the genes still present in the 100–200 kbp plastid genomes are the ribosomal RNA genes, 27–31 genes encoding tRNAs, and a variable number of other genes, that in higher plants include about 85 encoding proteins of the photosynthetic apparatus (Green, 2011).

Within the chloroplast, multiple copies of the plastid DNA (ptDNA) together with RNA and proteins are organized in structures that are similar to bacterial nucleoids. The compact structure of DNA in such nucleoids has been compared with the chromatin in the nucleus of eukaryotic cells (Sakai et al., 2004). The fundamental difference between genome organization in plastids vs. that in bacteria is, that plastids have multiple nucleoids with a varying number of genome copies, whereas bacteria only have a single nucleoid containing a variable number of DNA molecules. Nucleoids contain all enzymes necessary for transcription, replication and segregation of the plastid genome (Sakai et al., 2004). Moreover, posttranscriptional processes including RNA splicing and editing, as well as ribosome assembly, take place in association with the nucleoid, suggesting that these processes occur co-transcriptionally (Majeran et al., 2012). However, among the many proteins found in the nucleoid and identified by proteomic analyses (Phinney and Thelen, 2005; Majeran et al., 2012; Melonek et al., 2012) only a few have been functionally characterized so far. In Table 1, proteins, that were proposed to play roles in nucleoid architecture, and which in analogy to the architectural proteins of bacterial nucleoids have been named plastid nucleoid associated proteins (ptNAPs) (Krupinska et al., 2013), are listed.

The dynamic shaping of nuclear chromatin and bacterial nucleoids is known to have profound effects on gene expression. Whereas the mechanisms underlying chromatin remodeling in the nucleus of plant cells have been investigated intensively, research on the mechanisms underlying the dynamics of the structure and organization of plastid nucleoids is still in its infancy. This is in sharp contrast with the enormous importance of chloroplast metabolism for growth and productivity of plants. Expression of plastid genes needs to be continuously coordinated with the activity of the nuclear genome. Structural changes are likely to be involved in the crosstalk between plastid and nuclear genomes.

In this article recent progress in the elucidation of the composition of plastid nucleoids is reviewed in the context of the complex DNA-protein architecture. The unique characteristics of plastid nucleoids will be highlighted by comparison with bacterial nucleoids and nuclear chromosomes. The involvement of plastid specific NAPs in regulation of DNA availability for replication and transcription and the functional significance of nucleoid association with the thylakoid membranes in chloroplasts will be discussed.

In 1962, Ris and Plaut discovered irregularly shaped bodies containing DNA in the chloroplast of Chlamydomonas by staining with acridine orange. Electron micrographs revealed microfibrils in areas of low density corresponding to DNA macromolecules similar to those that were shown before in bacteria (Robinow and Kellenberger, 1994). These microfibrils suggested that at least part of the plastid DNA is “naked” in contrast to the nuclear DNA that together with basic proteins, histones, is organized in highly compact structures known as chromatin (Kuroiwa, 1991). Images obtained by staining with 4′,6-diamidino-2-phenylindole (DAPI) or other DNA dyes such as SYBR Green revealed a quite different organization of plastid DNA. In chloroplasts, tiny compact structures associated with the thylakoids are detectable (Figure 1A). Protease treatment and reconstitution assays on such isolated structures indicated that the packaging degree of DNA is higher than in the metaphase chromosomes of animals (Nemoto et al., 1988; Kuroiwa, 1991). From these results it was concluded that ptDNA is not “naked,” but tightly packed in nucleoids by interactions with basic proteins as it is also known for the nuclear chromatin.

Indeed, the concept of “naked” DNA in plastids and bacteria was based only on conventional electron microscopy employing chemical fixation and dehydration of the tissue, known to lead to denaturing and loss of proteins. As a result, DNA filaments devoid of proteins get visualized in electron-lucent areas from which proteinous material was lost during dehydration (Figure 1B). When instead of chemical fixation, physical fixation by high pressure freezing and freeze substitution (HPF-FS) is employed, no DNA filaments are detectable (Figure 1C). Specimens prepared by HPF-FS were used for immunogold labeling with an antibody specific for single- and double-stranded DNA. Thereby regions of intensive labeling could be detected that have about the size of nucleoids as detected by epifluorescence or confocal microscopy (Figure 1D).

Genomic DNA in most eukaryotic cells is hierarchically organized within the chromatin (Campos and Reinberg, 2009; Fudenberg and Mirny, 2012). The basic unit of chromatin is the nucleosome that consists of double stranded DNA wrapped around a histone octamer. The nucleosomes organize into 11 nm fibers that resemble beads on strings. This structure is thought to further fold into so-called 30 nm fibers stabilized by the H1 linker histone. Although very little is known about the organization of chromatin beyond this stage, it is assumed that organization of the higher order chromatin structure involves formation of interacting fibers, chromatin loops and positioning to generate a distinctive spatial arrangement of the genome within the three-dimensional space of the nucleus (for a review see Li and Reinberg, 2011).

In general, the higher-order structures of nuclear chromatin inhibit DNA transaction processes, i.e., replication, repair, recombination and transcription of the DNA (Li and Reinberg, 2011). These DNA transaction processes require chromatin remodeling by mechanisms such as: (i) posttranslational modifications (acetylation and methylation) of N- and C-terminal tails of histones, (ii) exchanging histones variants, (iii) DNA methylation, (iv) non-histone architectural proteins, (v) ATP-dependent nucleosome remodelers, as well as (vi) the action of negatively charged histone chaperones.

Most eukaryotic genes are transcribed by RNA polymerase II (RNAP II). Interestingly, transcription by RNA polymerase II requires dynamic changes in the chromatin structures of the templates (Orphanides and Reinberg, 2000; Studitsky, 2005). During high rates of transcription, nucleosomes are completely disassembled and reassembled with the assistance of ATP-dependent nucleosome remodelers and histone chaperones altering contacts between DNA and histones. These remodelers are specific for certain genes in different cell types and contexts of cell differentiation (de la Serna et al., 2006). ATP-dependent nucleosome remodelers allow the DNA to “inch-worm” around the histone octamer. Acidic histone chaperones, on the other hand, “collect” the basic histones after the histone-DNA interactions have been broken by the ATP-dependent nucleosome remodelers.

Non-histone architectural proteins, such as high mobility group (HMG) proteins (Grasser, 1995) also play a role in chromatin structural dynamics, since they decrease the compactness of the chromatin fiber and enhance the accessibility of DNA to regulatory factors. Members of the HMGN family contain a functional nucleosome-binding domain (NBD) and a negatively charged C-terminus of varying length. It has been shown that the negatively charged C-terminal domain of HMGN5 interacts with the positively charged C-terminal domain of the linker histone H1 and thereby counteracts the H1-mediated compaction of a nucleosomal array. In turn, this facilitates transcriptional activation (Rochman et al., 2010).

Packaging of DNA by histones into nucleosomes is not a distinguishing feature of eukaryotes, but also occurs in some groups of archaebacteria which might have participated in the origin of eukaryotes (Bendich and Drlica, 2000). In any case, a nucleosome based packaging of DNA results in a rather closed structure, and the access of DNA by DNA transaction enzymes involves several interconnected processes modeling the chromatin.

Whereas the ability of histones to interfere with the nuclear chromatin structure and thereby to regulate transcription is rather well conserved among eukaryotes and understood in great detail, the situation in eubacteria seems to be more diverse and complicated. Research on the folding of bacterial DNA began in the 1970s, but the first systematic inventory of nucleoid associated proteins (NAP) (Azam and Ishihama, 1999) is still being extended (Dillon and Dorman, 2010). Many of these proteins are abundant basic proteins similar to histones and were found to influence chromatin structure and gene transcription. Accordingly, they were earlier named “histone like” proteins (Drlica and Rouviere-Yaniv, 1987; Dorman and Deighan, 2003). This group includes the highly conserved HU (heat unstable), the H-NS (histone-like nucleoid-structuring), IHF (integration host factor) and FIS (factor for inversion stimulation) (Dillon and Dorman, 2010). By the use of a bioinformatics approach it has been estimated that the bacterial nucleoid contains approximately one NAP per 100 bp (Li et al., 2009). According to their architectural mode of action toward DNA, three classes of architectural proteins are distinguished: wrappers, benders, or bridgers (Luijsterburg et al., 2008).

Importantly, there is no sequence or structural similarity between the prokaryotic histone-like proteins and eukaryotic histones (Macvanin and Adhya, 2012). The histone-like HU, H-NS, IHF and FIS proteins bind to AT-rich regions and shape the local structure of DNA upon binding (Browning et al., 2010). In contrast to histones that bind to both coding and non-coding DNA, the binding of these proteins occurs mostly in non-coding regulatory regions of the genome as shown by in vivo protein occupancy display (Grainger et al., 2006; Vora et al., 2009).

By electron microscopy, isolated nucleoids of Escherichia coli (E. coli) were shown to be organized as rosettes with a compact central core from which supercoiled DNA loops with an average size of 10 kbp were observed to radiate (Delius, 1974; Postow et al., 2004). The loops comprise topologically isolated domains with boundaries set by different NAPs such as H-NS and FIS, that can cross-link either different genomic loci or one locus with a membrane (Postow et al., 2004; Travers and Muskhelishvili, 2005; Luijsterburg et al., 2008). At a higher organizational level, the E. coli genome is folded into a structure containing four so-called macro-domains with specific NAPs and two less structured regions (Espeli et al., 2008). In Caulobacter crescentus, some domain specific NAPs are involved in control of replication and distribution of nucleoids (Dame et al., 2011), while others were shown to regulate the position of chromosomes and the initiation of cytokinesis (Mohl et al., 2001).

NAPs have both structural and regulatory roles. They shape the overall organization of nucleoids depending on the external conditions and growth phase (Rimsky and Travers, 2011). The composition of the NAPs is known to change during the cell cycle, in response to growth phase and external conditions such as nutrient supply and stress factors. For example, FIS is a bending NAP with high levels in growing cells, but it is absent under conditions of slow growth and in cells of the stationary phase (Dillon and Dorman, 2010). In contrast, Dps (DNA protection from starvation), whose expression is regulated by FIS and other NAPs, accumulates at the end of the stationary phase mediating the formation of stable and highly ordered nucleoprotein complexes, also termed biocrystals, that are important for the protection of DNA during stress (Wolf et al., 1999).

In addition to their dynamic functions as structural proteins most NAPs serve dual or multiple purposes and also have specific functions (Dillon and Dorman, 2010; Dame et al., 2011). The HU protein was shown to form transcription foci that are spatially confined aggregations of RNA polymerases (Berger et al., 2010). Other NAPs such as CRP (cyclic AMP regulatory protein) act as transcription factors of specific genes (Nasser et al., 2001; Rimsky and Travers, 2011). The NAP repertoire has considerable impact on global gene expression and in many cases NAPs regulate gene expression by mutually antagonistic activities (Dillon and Dorman, 2010).

Taken together, in contrast to the eukaryotic chromatin, the composition of bacterial nucleoids is more diverse and dynamic. The composition of the NAP fraction is regulated mainly at the level of NAP gene expression whereby NAPs can regulate both the transcription of genes encoding other NAPs and/or their own genes (Travers and Muskhelishvili, 2005).

The number and positions of nucleoids were shown to depend on the developmental stage of the plastids (Kuroiwa et al., 1981; Miyamura et al., 1986). Intensive remodeling of nucleoids occurs during the development of proplastids to photosynthetic competent chloroplasts and during interconversions between different plastid types (Hashimoto, 1985; Kuroiwa, 1991; Chi-Ham et al., 2002). Proplastids contain a cluster of nucleoids located in the center of the plastids. At the beginning of seed germination, these nucleoids are considered to move to the envelope, where extensive DNA amplification takes place, and eventually the enlarged nucleoids form a spherical ring (Figure 3A). Upon illumination, during transition from proplastids to chloroplasts, small nucleoids are distributed along developing thylakoid membranes (for review see Sakai et al., 2004). Sections from barley primary foliage leaves were stained with SYBR Green to show typical stages of nucleoid organization. At the border between white and green stripes of a heterozygous leaf of the mutant albostrians small undifferentiated and photosynthetically inactive plastids were found besides chloroplasts. As observed in proplastids of leaf primordial of imbibited wheat seeds (Miyamura et al., 1986), the nucleoid in the plastids of white albostrians leaves and leaf stripes appears to be ring-shaped. The ring-shaped nucleoid is typical for proplastids developing in darkness. In a basal segment from a primary foliage leaf of seedlings grown for 5 days in the light, developing chloroplasts were found to be organized as a necklace of pearls in the peripheries of the organelles indicating a light-dependent disintegration of the nucleoid ring as proposed previously (Miyamura et al., 1986). In the upper part of a leaf from 7 days old seedlings, mature chloroplasts with many tiny nucleoids attached to thylakoids were found. During development of barley seedlings in darkness, proplastids differentiate into etioplasts, where a few large nucleoids are found that might be distributed at the periphery of prolamellar bodies (Figure 3B). Temporal changes of nucleoid structure have also been studied intensively in variegated leaves of Arabidopsis mutants, e.g., var2 (Sakamoto et al., 2009). Plastids in white leaf sectors were observed to contain few large nucleoids. During chloroplast development nucleoids were observed to become smaller in size, more dense and more abundant (Sakamoto et al., 2009). The only protein so far identified to be involved in the distribution of nucleoids, YlmG1 (Table 1), is of prokaryotic origin. Overexpression or knockdown of the gene was shown to impair nucleoid partitioning (Kabeya et al., 2010).

Attachment of nucleoids to membranes was proposed to be important for organization, replication and transcription of ptDNA (Sato et al., 1993; Sato, 2001). In chloroplasts, formation of thylakoids seems to be tightly linked with nucleoid morphology and distribution (Kobayashi et al., 2012). Nucleoid structure and transcriptional activity are not affected in mutants developing residual thylakoids with altered lipid composition and impaired photosynthetic machinery (Kobayashi et al., 2012). These studies suggest that the formation of the thylakoid system and the attachment of nucleoids to these membranes precede the assembly of the photosynthetic machinery. On the other hand, ptDNA displays reduced compaction in plastids of the yellow leaf tissue of mutants with silencing of the CHLD (Mg chelatase subunit D) and CHLI (Mg chelatase subunit I) genes. These mutants possess thylakoids but lack grana stacks and are devoid of the photosynthetic complexes resulting in compromised photosynthesis (Luo et al., 2013). Taken together, these studies demonstrate that thylakoid formation during chloroplast development and alterations in shape and distribution of nucleoids are interconnected processes.

Interestingly, architectural reorganization of nucleoids during light-dependent chloroplast differentiation is correlated with a switch in polymerase usage: transcription of PEP (plastid encoded polymerase) -dependent genes increases, whereas at the same time the expression of NEP (nuclear encoded polymerase) -dependent genes decreases (Liere et al., 2011). It was recently proposed that the structural establishment of the transcriptional subdomain within the nucleoid represents a bottleneck in chloroplast development (Pfalz and Pfannschmidt, 2013). In this context it has been proposed that the assistance of DNA/RNA-protein assembly factors SVR4 and SVR4-like is required for expression of a set of chloroplast encoded genes involved in chloroplast formation (Qiao et al., 2011; Powikrowska et al., 2014).

Of particular importance for the activity of nucleoids is their association with the thylakoid membranes where the photosynthetic machinery undergoes changes in composition in response to environmental conditions. A prerequisite for remodeling of the photosynthetic apparatus is the regulation of plastid gene transcription in response to light-dependent changes in the redox state of the photosynthetic apparatus (Pfannschmidt et al., 1999). Thereby the composition of the photosynthetic apparatus can continuously be adjusted to the ever changing environmental conditions. Recent research in Chlamydomonas has clearly shown that chloroplast nucleoids are able to sense the redox state and that also the DNA replication activity can be adjusted accordingly (Kabeya and Miyagishima, 2013). SVR4 seems to be among the nucleoid proteins that are able to sense the redox state and to modulate the nucleoid architecture in response to redox changes. SVR4 was reported to possess disulfide reductase activity in vitro and to interact in vivo with thioredoxin Z (TrxZ), as well as with the two nucleoid associated superoxide dismutases FSD2 and FSD3 (Qiao et al., 2013; Yua et al., 2013). TrxZ regulates the redox state of proteins in response to light and has been shown to be associated with PEP (Steiner et al., 2011; Pfalz and Pfannschmidt, 2013) and to be required for transcriptional activity (Arsova et al., 2010; Schröter et al., 2010). FSD2 and FSD3 are two iron superoxide dismutases found in the chloroplast nucleoid associated with PEP. Both proteins were shown to act as ROS scavengers within the nucleoids (Myouga et al., 2008). It is likely that the redox state of nucleoid associated proteins is regulated by electrons provided from the photosynthetic machinery. It is interesting to note that the enzymatic activity of SiR was shown to be regulated by photoreduced ferredoxin. The DNA binding of SiR did not affect the enzymatic activity suggesting that both ferredoxin and sulfites are accessible to SiR within the nucleoids (Sekine et al., 2007). It remains, however, unknown whether the redox activity of SiR has an impact on DNA binding.

In this context, proteins found to be located at the interface between nucleoids and the thylakoid membrane are of particular interest. MFP1 was proposed to anchor nucleoids to thylakoids in chloroplasts (Jeong et al., 2003). WHIRLY1/pTAC1 is another nucleoid associated protein (Pfalz et al., 2006) associated with thylakoid membranes. Prikryl et al. (2008) showed that the attachment to thylakoid membranes is disrupted by DNaseI. WHIRLY1 was shown to form 24-mer complexes (Cappadocia et al., 2010, 2012) and was proposed to function analogously as the oligomeric NONEXPRESSOR OF PR1 (NPR1) in the cytoplasm (Foyer et al., 2014). Upon changes in the redox state of the photosynthetic machinery the complexes might get monomerized and the monomer might change gene expression in the nucleus. In accordance with this model the WHIRLY3 protein in Arabidopsis chloroplasts was identified among the redox-sensitive proteins (Ströher and Dietz, 2008). Another protein found to be distributed between thylakoids and the nucleoid is pTAC16. Its phosphorylation in response to redox-changes of the photosynthetic apparatus was suggested to regulate membrane-anchoring functions of the nucleoid (Ingelsson and Vener, 2012). It seems that the nucleoid containing besides WHIRLY1 further central proteins shown to be involved in plastid-to-nucleus signaling such as GUN1 (Koussevitzky et al., 2007) and PRIN2 (Kindgren et al., 2012; Barajas-López Jde et al., 2013), is the place where redox signals known to induce changes in nuclear gene expression are integrated.

Transgenic plants with different levels of nucleoid/thylakoid associated proteins might help to elucidate the roles of these proteins in linking the activity of the photosynthetic machinery to organization and expression of plastid genes as well as the expression of nuclear genes.

In the nucleus the availability of DNA for transcription is regulated mainly by posttranslational modifications, whereas in bacteria regulation of transcription involves the exchange of DNA binding proteins (Luijsterburg et al., 2008) and changes in the compaction of the nucleoid (Berger et al., 2010). It seems, that in plastids the architecture of the nucleoids is regulated by both kinds of mechanisms. Both in the nucleus and in plastid nucleoids, posttranslational modifications are important for nucleoid associated processes. For MFP, SiR and SWIB-4 it was shown that the binding to DNA is regulated by phosphorylation. The three proteins seem not to bind to DNA when they are phosphorylated (Chi-Ham et al., 2002; Jeong et al., 2004; Melonek et al., 2012). Several kinases were found to be associated with nucleoids, e.g., the fructo-kinase like protein FLN1/2, the casein kinase CK-II, as well as two atypical ABCK1 type kinases (Lundquist et al., 2012). Probably, other posttranslational modifications are likely to play important roles as well. Counterparts of enzymes known to be involved in histone modifications in the nucleus, such as the Arabidopsis SET-domain proteins ATXR5 and ATXR6 involved in methylation (Raynaud et al., 2006; Jacob et al., 2009) and de-acetylases were found in plastids (Chung et al., 2009).

It remains to be shown whether the different packaging of DNA in different regions of the nucleoid changes during development and in response to environmental cues. Whether, however, the central body of nucleoids with dense packaging (Sakai et al., 2004) can be compared with eukaryotic heterochromatin (Sato, 2001) remains questionable. It rather seems that DNA packaging is beneficial for a high transcriptional activity as it was described for bacteria as well (Dillon and Dorman, 2010; Krupinska et al., 2013).

The comparison of DNA organization in plastids, nucleus and bacteria shows that the shaping and organization of plastid nucleoids involves novel organelle specific mechanisms resembling those acting on eukaryotic chromatin besides mechanisms described for eubacterial nucleoids. During evolution of plants, the architectural proteins of bacterial nucleoids have been lost and replaced by new proteins. Some of these are enzymes that have acquired an additional function as DNA binding proteins. Others might have been contributed by Chlamydiae which facilitated establishment of the primary endosymbiosis between an early eukaryote and the cyanobacterial ancestor of plastids. These proteins do not exhibit sequence or structural conservation with the eukaryotic histones, but similar to the histones they might be regulated by posttranslational modifications.

In comparison to eukaryotic chromatin, nucleoids of plastids have as those of bacteria a more open structure, that allows easy access for DNA transaction enzymes. The enrichment of enzymes involved in RNA processing and translation in the nucleoid fraction suggests that transcription, RNA processing and translation are tightly connected with each other.

Similarly to bacteria, also in plastids, membranes seem to play a key role in the organization and maintenance of nucleoids. In chloroplasts, the proximity of nucleoids and photosynthetic machinery as well as the presence of several redox active proteins in nucleoids, allows for a tight coordination of photosynthesis and nucleoid function, i.e., replication and gene expression. It is striking that not only particular enzymes involved in gene expression but also architectural proteins are controlled by redox signals. Thereby these proteins might have a tremendous impact on the different enzymatic activities associated with nucleoids; in particular replication, transcription and DNA repair.

The architectural organization of the plastid genetic machinery is not well understood. Since principles underlying the dynamic shaping of genomes are uniform in all forms of life, the knowledge about DNA organization in bacteria and eukaryotes can be used in future studies on the dynamic architecture of chloroplast nucleoids.