A comparative approach to elucidate chloroplast genome replication
© Krishnan and Rao; licensee BioMed Central Ltd. 2009
Received: 22 October 2008
Accepted: 20 May 2009
Published: 20 May 2009
Electron microscopy analyses of replicating chloroplast molecules earlier predicted bidirectional Cairns replication as the prevalent mechanism, perhaps followed by rounds of a rolling circle mechanism. This standard model is being challenged by the recent proposition of homologous recombination-mediated replication in chloroplasts.
We address this issue in our current study by analyzing nucleotide composition in genome regions between known replication origins, with an aim to reveal any adenine to guanine deamination gradients. These gradual linear gradients typically result from the accumulation of deaminations over the time spent single-stranded by one of the strands of the circular molecule during replication and can, therefore, be used to model the course of replication. Our linear regression analyses on the nucleotide compositions of the non-coding regions and the synonymous third codon position of coding regions, between pairs of replication origins, reveal the existence of significant adenine to guanine deamination gradients in portions overlapping the S mall S ingle C opy (SSC) and the L arge S ingle C opy (LSC) regions between inverted repeats. These gradients increase bi-directionally from the center of each region towards the respective ends, suggesting that both the strands were left single-stranded during replication.
Single-stranded regions of the genome and gradients in time that these regions are left single-stranded, as revealed by our nucleotide composition analyses, appear to converge with the original bi-directional dual displacement loop model and restore evidence for its existence as the primary mechanism. Other proposed faster modes such as homologous recombination and rolling circle initiation could exist in addition to this primary mechanism to facilitate homoplasmy among the intra-cellular chloroplast population
The chloroplast is a vital organelle, responsible for photosynthetic metabolism in plants. Their replication is crucial in ensuring the cellular maintenance and working within plants . Understanding the mechanisms underlying the process of replication can yield important insights that can be used towards plastid engineering and transformation , an area in the growing discipline of plant biotechnology. It is, therefore, imperative to delve into and develop our understanding of replication in chloroplast genomes. In the early 1970s, electron microscopy analyses of replicating chloroplast intermediates from pea and corn drew a model of replication . This model was based on two displacement loops (D-loops) separated by some distance on the genome, where the displacement of the two D-loops occur on opposite strands of the parental DNA molecule and subsequently, move towards each other. As a result of this mechanism, half of each displaced parental strand (from either origin until the center of two origins) is left single-stranded on both sides of the pair of inverted repeats. This discovery of Cairn's replication mechanism  in pea and corn chloroplast genomes was followed by a series of studies independently confirming this model for various plant species (Euglena gracilis ; single D-loop]; Nicotiana tabacum ; Chlamydomonas reinhardtii ; Oenothera ; Zea mays ).
The rolling circle mechanism could be initiated after one round of Cairns type of replication, so as to generate multiple copies of the chloroplast genome even though replication is initiated only once (pea and corn, ). Electron microscopy analyses of certain in vitro tobacco chloroplast replication intermediates also revealed Y-arc patterns, indicative of rolling circle replication.
Bendich and colleagues recently countered the entire proposition of bi-directional replication mediated by two D-loops (see  for review) and also the possibility of any rolling circle initiation. The basis for this contrary view is their observation of a large number of molecules in linear or complex branched oligomeric forms [12, 13], which were earlier either dismissed as broken circles  or physically excluded (by virtue of scientific judgment or ultra-centrifugation methods exercised on the samples prior to electron microscopy analyses). They put forth homologous recombination as the primary mechanism of replication, explaining the generation of all chloroplast replicative intermediates such as oligomeric forms, head to tail concatemers and isomers as well as circular molecules themselves. They further re-iterate the need for a revised view of the standard dual displacement loop model.
We exploit the availability of a large number of chloroplast genomes (>100; see Additional File 1) and adopt a comparative genomics approach in our current manuscript for predicting the chloroplast DNA replication mechanism. It is well-established in animal mitochondrial genomes that replication leaves an imprint on the genome composition, by way of deaminations accumulating during the time spent single-stranded by the parental heavy strand. The adenine to guanine (A → G) deaminations accumulate linearly over the time spent single-stranded during replication while, cytosine to thymine deaminations exhibit a complex, asymptotic response [15, 16]. The A → G deamination response is simpler to detect using a linear regression model. One could, therefore, explore the presence of A → G gradients in chloroplast genome regions between mapped replication origins and infer from the direction of these gradients, the direction in which the DNA is left single-stranded during replication. For instance, an increasing gradient of A → G from point 'X' to point 'Y' of the genome indicates that the replication fork proceeded from Y to X causing Y to become single-stranded before X, thereby exposing Y to greater accumulation of A → G deaminations than X.
The tobacco chloroplast genome is best-documented in terms of replication origins . Annotation of the tobacco genome (Nicotiana tabacum: NC_001879) in NCBI reveals four origins on each of the two inverted repeats: A1 (35 nucleotides), A2 (82 nucleotides), and two copies of B (243 nucleotides), one on each strand. Formation of D-loops were observed only in those tobacco clones that contained all the origins, and these replication origins were also found to be the minimal sequences to ensure the completion of replication . Replication origins are not annotated in other complete chloroplast genomes, available in NCBI. We use the tobacco genome as a benchmark, to look for homologues of these origins in other genomes and find several matches using the NCBI pair-wise Blast tool. Linear regression analyses reveal for a majority of plant species, significant symmetric bi-directional A → G deamination gradients in the S mall S ingle C opy (SSC) and L arge S ingle C opy (LSC) regions. The single-strandedness window experienced during homologous recombination is too small to result in such nucleotide composition patterns. Secondly, the single-stranded bubble moves as strand invasion progresses during recombination and does not expand like in the case of conventional replication mechanism, thus preventing accumulation of deaminations. On the other hand, the dual displacement loop model  very well explains these symmetric A → G gradient trends, suggesting its pre-dominant existence as the mechanism of replication.
Complete Chloroplast Genomes
We analyzed complete chloroplast genomes belonging to 116 plant species, available in NCBI Genbank http://www.ncbi.nlm.nih.gov as of September 2008. The names of these plant species, their abbreviations and locus IDs are presented in a table (see Additional File 1).
Identifying homologues to known replication origin sequences
Categorization of chloroplast genomes based on the numbers of homologues to tobacco chloroplast replication origin sequences
Aco, Agr, Atr, Ath, Abe, Ahi, Bve, Bmi, Cbu, Cpa, Csp, Csi, Car, Cwa, Csa, Cre, Dca, Dne, Dgr, Egl, Fes, Gba, Ghi, Lvi, Les, Mes, Min, Nof, Nsy, Nta, Nto, Nad, Opu, Pal, Pgi, Ptr, Rma, Sbu, Sly, Stu, Sol, Vvi
Cfe, Cfl, Cex, Del, Iol, Nal, Pap, Poc
Ast, Aev, Afo, Ami, Bdi, Cde, Cta, Evi, Gab, Gma, Han, Hvu, Hlu, Ipu, Lsa, Lpe, Lja, Oar, Obi, Ogl, Oni, Opa, Osa, Pvu, Shy, Sof, Sbi, Tae, Tca, Wmi, Zma
Chgl, Cvu, Cat, Mvi, Pnu, Sob, Ota, Ppa
Cja, Lte, Spu, She, Zci
Cre, Chvu, Hsi, Nol, Ovi, Pak, Sun, Oca
Linear Regression Analyses
We wrote PERL scripts to extract sequences for each region between a pair of replication origins and further prune them to only consist of non-coding nucleotides and the synonymous third codon position nucleotide, corresponding to an 'A' or a 'G'. This was essential as we were testing for presence of A → G deamination gradients in each region of interest. These positions have relatively lower selection pressure, and hence are likelier to retain and reflect the deamination trends caused during replication. Site-specific adenine (A) to guanine (G) deaminations were estimated as the ratio: A/(A+G), where the nucleotide 'A' at each position was assigned a binary value of 0 and likewise, 'G; was assigned a value of 1. Linear regressions were performed for each extracted region, where the X axes were the positions of nucleotides on the genome, and Y axes values were the estimated site-specific A/A+G ratios. Significance was determined according to a two-tailed t-test (P < 0.05). Similar regression analyses were also performed after splitting the region of interest mid-way into two regions, for each sub-region.
Tobacco Chloroplast Replication Origin Homologues in Other Chloroplast Genomes
We found varying numbers of sequence homologues to tobacco chloroplast replication origins in 95 out of 115 other chloroplast genomes (Table 1 and Figures 2, 3, 4, 5, 6, 7 and 8). The local and genomic positions of these homologues are depicted in a table (see Additional File 2). As evident from this table, partial homologues to certain replication origins exist in some genomes, occasionally in addition to their complete homologues. The entire dataset of 115 complete chloroplast genomes could be divided into eight categories, based on the number of similarity matches (including partial matches). Representative genomes from the first seven categories are depicted in Figures 2, 3, 4, 5, 6, 7 and 8. A category often contains more then one representative genome, further depending on the type of matches. For example, Category VI is comprised of four representative genomes, bearing different pairs of replication origin homologues. Such categorization and sub-categorization for all genomes are further represented in Table 1. Among these, III [A] and V [C] sub-categories are highly populated. The eighth category corresponds to genomes (seven green algae: Chlamydomonas reinhardtii, Chlorella vulgaris, Helicosporidium sp ex Simulium jonesii, Nephroselmis olivacea, Oltmannsiellopsis viridis, Oedogonium cardiacum, Pseudendoclonium akinetum and a blue spike-moss: Selaginella uncinata), which lack similarity to any of the tobacco replication origin sequences.
Significant deamination gradients in certain regions of the genome
We explored the presence of A → G deamination gradients (linearly increasing or decreasing A/A+G ratios) in genome regions interspersed between replication origin homologues. These regions comprised of non-coding and synonymous third codon positions of coding genes. We consistently find significant negative deamination gradients in the region (A2 → A2-C) containing LSC and tail ends spanning ~15kb each of the two inverted repeats for all species belonging to the first six categories (with the exception of Medicago truncatula which bears only one inverted repeat; see Additional File 3). Dividing this region (LSC plus tail ends of inverted repeats) mid-way into the two halves, and analyzing each half separately, revealed the presence of two gradients of greater significance, increasing in one half and more strongly decreasing in the other half. Significant A → G gradients in opposite directions were also observed in each half of the genome portion overlapping the SSC region and ~7kb of the other tail end, respectively, of both the inverted repeats (A1 → A1-C) for all species from these six categories. However, the entire SSC region does not exhibit any deamination trend. This is perhaps due to the balancing effect of equally increasing and decreasing trends in both halves. Significant gradients in similar directions were observed in the complete SSC and LSC regions and also within individual halves of these regions, even after excluding the tail ends of inverted repeats suggesting that the detection of these symmetric gradients is not merely influenced by the nucleotide composition of the inverted repeats themselves (data not shown). Directions of these consensus A → G gradients, are depicted on a circular map of the Zea mays chloroplast genome (Figure 9). Such significant gradients were not observed consistently across all species in any other region of the genome (see Additional File 3) for these six categories. The seventh category of genomes bears only one replication origin homologue to tobacco origin sequences, and therefore, such symmetric gradients were not observed consistently in them. It is likelier that this category of species have other putative replication origin homologues, pertaining more to lower plant forms (see Discussion)
Model of Replication in Chloroplast Genomes
Deamination trends caused by cumulative single strandedness during replication
Accumulation of deaminations has been attributed to single-strandedness during replication in bacterial [18–21] as well as animal mitochondrial genomes [15, 16, 22–24]. Both these types of genomes also report strand asymmetry in nucleotide compositions [22, 24, 25]. A close attempt to studying strand asymmetry in nucleotide composition in chloroplast genomes failed to detect the patterns we observe, because of their approach of studying the entire strand at once . Strand switching asymmetry at the replication origins was however observed in the Euglena gracilis chloroplast genome, such that one strand is AC rich and the other is GT rich .
Chloroplast genomes are also known to undergo heavy RNA-editing, where C → U and U → C mutations occur at the first two codon positions of protein coding genes [30–32] and also certain non-coding regions  during transcription. RNA level variation is brought about as a result of such editing to a level that is complementary to DNA level variation . C to U editing is thought to be invoked by deaminations rather than specific nucleotide excision/replacement or trans-glycosylation pathway [35–37]. This suggests the existence of mechanisms to regulate RNA-editing within the chloroplast system, which could also putatively affect C → U (T) deaminations that occur during replicative single-strandedness. This speculation is strengthened by the finding of a chloroplast specific apparatus responsible for editing of Zea mays plastid mRNAs . C → T deaminations have also been reported in association with single-strandedness during transcription in E. coli, by formation of RNA-DNA hybrids and thereby, C → T mutations accumulating on the non-transcribed strand [39, 40]. In order to clearly interpret the nucleotide composition trends as those to have resulted from replication, we rely solely on A → G deamination gradient analyses to infer single-strandedness of the genome during replication.
Number of Homologues: Genome Size and Evolutionary Trends
Symmetric Gradients in Bacterial Genomes: Similarity in Replication processes
We also find symmetric A → G gradients in opposite directions in regions between replication origins for an E.coli Pola 52 plasmid carrying three replication origins (ori-alpha, ori-beta and ori-gamma; see Additional File 4), especially in the larger region between ori-beta and ori-alpha (R3). This plasmid was chosen for analyses as it carries multiple replication origins. The symmetric increasing and decreasing gradients in the larger region between ori-beta and ori-alpha very well fits with in vivo evidence for clockwise and counter-clockwise modes of replication from ori-beta and ori-alpha, respectively, known for the highly replicative R-plasmid (R6K) and its derivatives in E.coli, carrying a similar arrangement of vegetative replication origins .
Ethidium bromide stained fluorescence microscopy images of the nucleoids of Borrelia burgdorferi and Borrelia hermsii were observed to be different from that of the E. coli nucleoid [49, 50], and more similar to that observed for chloroplast DNA of maize , Arabidopsis , pea, tobacco and M. truncatula , especially when the DNA is extracted from young tissue at very early stages of development . These observations provide some evidence for the existence of developmentally regulated homologous recombination in Borrelia genomes. Strand-specific asymmetry has been shown to exist also in genomes of Borrelia species . Since replication mediated by homologous recombination does not generate enough cumulative single-strandedness to result in strand asymmetric nucleotide compositions or gradients, therefore, bi-directional replication using multiple origins could also be possible in Borrelia genomes.
Mixed Modes of Replication
The relative abundance of oligomeric forms observed for chloroplast DNA stays constant through all stages of leaf development, as found in the case of spinach , triggering the possibility of intra-molecular recombination between inverted repeats , to generate such multimeric intermediates through a process , similar to yeast 2 μm plasmid replication. These plasmids follow Cairn's mechanism of replication after initiation at a single origin. This is trailed by further amplification through intra-molecular recombination between the inverted repeats, after the replication fork passes through one of the inverted repeats , such that the two replication forks now chase one another, thereby resembling a double rolling circle, a process also referred to as copy-choice recombination during replication .
Homologous recombination cannot explain the generation of nearly symmetric consensus gradients in A → G deaminations in the SSC and LSC regions of the chloroplast genomes as observed by our approach, since the single-strandedness generated during this process is small and non-cumulative. Nevertheless, mixed modes of traditional Cairn's replication via origin firing as well as replication slippages  following recombination could indeed occur in chloroplast DNA . The high rate of homologous recombination between multiple circular chloroplast DNA molecules present in close physical proximity inside a single chloroplast can as well bring about efficient homoplasmy [58–60].
Dynamic Network of Chloroplast Genomes
With advent of approaches that monitor DNA dynamics in living cells, animal mitochondria were found to not exist as autonomous individual organelles but instead form a highly dynamic semi-tubular network. It is possible that the arrangement of ethidium-stained chloroplast DNA as clots and comets with extended fibers as observed by fluorescence microscopy visualization  resembles foci formed by ethidium-stained DNA on the dynamic tubular network arrangement of mitochondria in human living cells . Quite analogous to an average of eight genome equivalents found in these highly branched chloroplast DNA structures , the number of genomes in human mitochondrial DNA foci also vary from six to ten . In the case of human mitochondria, the model of replication where each genome acts as an individual unit and replicates independently, even while being part of a focus appears as the one most satisfying the observed kinetics. Similarities between structural-functional organizations of these organelles, predicts such independent genome replication model also for chloroplast DNA. A mixed mode of replication could be followed by chloroplast genomes, even in this dynamic network like arrangement.
Minimalist Model of Chloroplast Replication
The minimalist model of chloroplast replication presented here (Figure 11), excludes ori-B pairs on each inverted repeat and is based on A1 and A2 origins alone. This is because we observe significant symmetric gradients for genome categories V and VI, which lack ori-B homologues. Excluding the ori-B homologues from genomes belonging to the first four categories, we find similar significant symmetric gradients in regions overlapping the SSC and LSC respectively. The presence or absence of ori-B determines in vitro, whether the D-loop mode or rolling circle mode of replication is adopted as the predominant mechanism in tobacco chloroplast DNA . It is possible that the ori-B sequences act as accessory units in the strand displacement process, after replication initiation at the A2 and A1 origins. These stretches could also putatively assist intra- or inter-molecular homologous recombination, to result in branched oligomeric structures, as found by Bendich and colleagues. It is nevertheless surprising to note that the minimalist model of replication derived using an independent approach based on comparative genomics, resembles the initial model of dual displacement loop mode of replication, suggesting that it prevails at least during advanced developmental stages.
Model of chloroplast replication as inferred by analyzing local deamination gradients in regions between replication origins conforms to the bi-directional replication model put forth by the Kolodner group. Homologous recombination could exist as an alternate or additional mechanism.
We thank Dr. Santosh Noronha and K. P. Hari, for reading the manuscript and providing useful and critical inputs. We also thank two anonymous reviewers whose comments enhanced the readability of this manuscript.
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