2004/07/01 by Arnold J. Bendich · 4 citations
Biochemistry, Genetics and Molecular Biology · Environmental Science · #Photosynthetic Processes and Mechanisms #Protist diversity and phylogeny #Microbial Community Ecology and Physiology
paper · pdf · doi:10.1105/tpc.160771
openalex publication_date 2004/07/01 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/30
If we could extract, purify, and visualize the intact DNA molecules from chloroplasts, what would those molecules look like? Most would expect to find circular DNA molecules the size of the chloroplast genome. By contrast, however, only a small fraction of the DNA obtained from chloroplasts is found as genome-sized circles. The reasons for this profound discrepancy are the subject of this article. I will trace the history of research on chloroplast DNA (cpDNA) to elucidate why it has taken more than 30 years to realize that the circle is not the principal form of DNA in chloroplasts and to examine the relationship between the chloroplast genome and the segregating genetic unit or chromosome in chloroplasts. The critical finding is that the chloroplast chromosome can contain many genome equivalents. I will also discuss the coupling between chromosomal replication and segregation in chloroplasts. To avoid confusion, I define some terms. The genome is the entire complement of genetic material in a virus, prokaryote, mitochondrion, or chloroplast or the haploid nuclear genetic complement of a eukaryotic species. A genome equivalent is the amount of DNA in a single copy of a genome. A chromosome is the segregating genetic unit that carries either a subset or all of the genome into each daughter cell or organelle during division. For example, the ant Myrmecia pilosula has a single pair of homologous chromosomes in the nucleus (Crosland and Crozier, 1986). The amount of DNA in a single chromosome equals the amount of DNA in the genome. In most ants and most eukaryotes, however, the DNA content of a single nuclear chromosome is less than that of the genome, and the DNA content of a segregating genetic unit never exceeds that of the genome. The genome in most bacteria, such as Escherichia coli, is contained on a single DNA-containing body known as the nucleoid. As a nucleoid segregates to daughter cells upon division, that nucleoid represents a bacterial chromosome. In contrast with what is found in the eukaryotic nucleus, however, the amount of DNA per bacterial chromosome in rapidly dividing cells is typically several to many times larger than one genome equivalent. As I will show, the situation in chloroplasts is similar to that in bacteria, and the size of the chloroplast chromosome is rarely as small as one genome equivalent. There is a strong belief that a chromosome from any source should contain no more DNA than one genome equivalent. The first chromosomes to be studied were those in the nucleus of eukaryotes, where cytological and genetic evidence indicated that a gamete, typically being haploid, contains a single genome. Later for bacteria, the small size of chromosomes limited cytological investigation, so only genetic evidence was informative. The monoploid nature of genetics in model prokaryotic organisms combined with the circular autoradiographic images of large E. coli DNA molecules in the early 1960s led to the conclusion that the chromosome in bacteria was contained on a single circular DNA molecule (Cairns, 1963; Bendich, 2001). It was only recently that we learned that bacterial chromosomes can consist of many copies of their genome. Rapidly dividing cells of E. coli can contain an average of ∼11 genome equivalents per cell (the range is ∼5 to 18) (Akerlund et al., 1995). Because approximately equal amounts of DNA are partitioned to daughter cells during cytokinesis (Woldringh, 1976), none of the cells in the population inherit a chromosome containing a single genome equivalent. The DNA content per cell decreases as the culture continues to grow exponentially, illustrating variability in the number of genome equivalents per chromosome. When deproteinized DNA is examined by fluorescence microscopy, most individual cells of E. coli and other bacteria contain a bunched-up form of DNA several times larger than one genome equivalent (Hinnebusch and Bendich, 1997). I conclude that because none of this information was available in 1972, when isolated cpDNA was first visualized, there was no reason then to challenge the concept of a strictly genome-sized chromosome in bacteria or to embrace the possibility of a complex and branched form of chromosomal DNA in their endosymbiotic descendant, the chloroplast. Kolodner and Tewari (1972)were the first to utilize DNase treatment of chloroplasts before DNA extraction, so that contamination of cpDNA with nuclear DNA was avoided. Their article is probably the most influential paper in cpDNA research. They found that the size of the pea chloroplast genome determined by DNA reassociation kinetics measurements matched the length of the single size class of circular cpDNA molecules observed by electron microscopy (EM). Circles represented a substantial fraction (37%) of the cpDNA molecules that they measured, and because all the linear molecules were shorter than the genome size, linear molecules were attributed to breakage of the circles during extraction. This report established the Broken Circles theory for cpDNA that has dominated the field until the present day. Using similar procedures for cpDNA isolation and EM with several plant species, Herrmann et al. (1975)found as much as 80% circular cpDNA molecules in some preparations, and they too adopted the Broken Circles theory. EM was again used to provide additional examples of plants with circular cpDNA (Kolodner and Tewari, 1975a). The results of these studies were entirely consistent with those for bacteria and seemed to definitively establish the chloroplast chromosome as a genome-sized circle. Subsequent restriction fragment mapping and genome sequencing data yielded circular genomic maps and served to solidify and generalize the conclusions in these early investigations. How then, could the early investigators have missed the bulk of the cpDNA that we now find in complex branched forms of multigenomic size (Oldenburg and Bendich, 2004)? We suspect that the very strong expectation that the chromosome in vivo should be a simple genome-sized circle, as in bacteria, may have led to the physical or conceptual exclusion from analysis of complex forms. Before examination by EM, a high-speed centrifugation step was performed, which may have removed the largest forms of cpDNA from the sample to be analyzed (Kolodner and Tewari, 1972; Herrmann et al., 1975). Another possibility is that complex forms of DNA much larger than the size of genomic circles might have been taken as nuclear DNA contamination (or uninterpretable structures) and excluded from analysis. The use of in-gel procedures for the extraction and purification of DNA coupled with pulsed-field gel electrophoresis (PFGE) permits analysis of megabase-sized chromosomal DNA molecules. The first to adapt these procedures for cpDNA were Deng et al. (1989), who found gel bands representing an oligomeric series of genomic sizes for several plants. They subscribed to the Broken Circles theory and concluded that “the chloroplast DNA exists as a mixture of circular forms, whether or not the chloroplast DNA in the pulsed-field gels is present as circular or linear molecules.” Our work then showed that the PFGE bands contained linear cpDNA molecules, and we suggested that they and the analogous bands of Deng et al. (1989)contained cpDNA that was artifactually and randomly linearized during extraction (Bendich and Smith, 1990). The Broken Circles theory was first invoked to explain the extreme difficulty in obtaining genome-sized circular forms of mitochondrial DNA (mtDNA) from fungi (Bendich, 1993). Williamson (2002)describes how dogged adherence to the Broken Circles theory impeded progress toward the realization that the genome in yeast mitochondria is actually borne on linear DNA. He bemoans his refusal to even measure the linear mtDNA molecules that filled his electron microscopic images. I suggested that the strong expectation in the late 1960s that fungal mtDNA should be circular derived chiefly from the discovery in the mid 1960s that circular molecules were the predominant form of mtDNA from many metazoan animals (Bendich, 1993). When I reported complex cpDNA forms several to many times the size of the genome, I interpreted them as representing either catenated or otherwise clustered circular molecules of genome size (Bendich, 1991), exemplifying the pernicious power of the Broken Circles theory. It was only when we produced high-resolution digital images, rather than the low-resolution images produced by image intensification, that the ethidium-stained cpDNA appeared as branched linear forms including molecules longer than the genome size (Oldenburg and Bendich, 2004). Much or most of the cpDNA subjected to PFGE remains immobile, and in 1995 the consensus was that this DNA was “trapped in the agarose as large circles which cannot move into the gel” (Backert et al., 1995). It is now clear, however, that the immobility of most of this DNA is due to its complex structure, with only a minor fraction due to relaxed circular molecules trapped by impediments (Oldenburg and Bendich, 2004). To summarize, the Broken Circles theory was formulated to provide an excuse for why we failed to find most of the DNA in the form we expected to find, both for chloroplasts and mitochondria. Furthermore, although it was long known that mapping alone cannot be used to distinguish linear from circular chromosomes (Stahl and Steinberg, 1964; Stahl, 1967; Bendich, 2001), these techniques (sequence assembly is a mapping technique) commonly provided the only data used to assign the circular form to these organellar chromosomes. The ends of linear, genome-sized cpDNA molecules could be located either at specific sites within the genome or at random sites, as expected from circles broken during extraction. Deng et al. (1989)concluded that the ends were random because they did not detect any restriction fragment length polymorphism between unfractionated spinach cpDNA and the cpDNA from PFGE bands containing one to four lengths of the genome. The absence of differences between total cpDNA and the PFGE bands, however, is also expected if all forms of cpDNA (except a minor circular form) had the same, specific ends. Specific ends were found for unfractionated maize cpDNA, as well as the linear monomeric and oligomeric forms, thus providing the evidence that linear cpDNA is the most common form of DNA within chloroplasts (Oldenburg and Bendich, 2004). Because the ends were near putative origins of replication, analysis of molecular structure can also lead to insights concerning the mechanism of cpDNA replication. It is remarkable how little is known of even the most basic aspects of cpDNA replication. The current standard model for cpDNA replication is based largely on inferences drawn from electron micrographs of cpDNA produced 30 years ago (Kolodner and Tewari, 1975b). Furthermore, that cpDNA was obtained from nonmeristematic tissues, and it is now known that cpDNA replication is largely or entirely restricted to meristematic cells (Kuroiwa, 1991; Fujie et al., 1994). It should therefore come as no surprise that the scheme presented below differs completely from the standard model. Chloroplast DNA Structure and Recombination-Dependent DNA Replication. (A) The end of a monomeric genome recombines with another molecule and initiates replication. Replication procedes to the right to generate product 1, an all-blue head-to-tail concatemer. Digestion with a restriction endonuclease that cleaves the genome once, at the site marked in red, yields a genome-size fragment. An alternative recombination initiates leftward replication to generate product 2, a head-to-tail concatemer containing an inverted (green) segment, that yields a larger-than-genome-sized fragment. Bold arrows indicate the direction of replication. The IRs are indicated by thick gray and black lines. (B) A multigenomic structure produced by recombination-dependent replication. (C) Circular forms of the genome produced by intramolecular recombination (flipping). Note that the larger-than-genome-sized fragment predicted by the map of product 2, and detected by Oldenburg and Bendich (2004), cannot be generated from genome-sized circles. A Multigenomic Chloroplast DNA Structure. This ethidium-stained structure was obtained from meristematic tissue of maize. Similarly complex cpDNA forms were found for every plant species examined, including watermelon and pea (Bendich, 1991), Arabidopsis (Rowan et al., 2004), wheat, and Medicago truncatula. No such complex forms were reported for pea and maize (Kolodner and Tewari, 1972, 1975a). It must be emphasized that the scheme in Figure 1Ais based entirely on structural analysis without benefit from genetics. It is intended merely to focus attention on linear genomes, rather than circular molecules, to guide future research. Figure 1Adoes, however, show how the two isomeric forms of the chloroplast genome in most plants can be produced without invoking flipping: intramolecular recombination between the large inverted repeat sequences (IRs) in a circular cpDNA molecule (Figure 1C). Flipping was proposed as the mechanism responsible for the equimolar alternative fragment maps for species with IRs (Palmer, 1983, 1985). The mapping data were obtained before the advent of PFGE, so that the sizes of the largest fragments could not be determined accurately. Recent mapping employing PFGE and rare-cutting enzymes revealed a larger-than-genome-size fragment for maize cpDNA that cannot be derived from the circular forms in Figure 1C(Oldenburg and Bendich, 2004). This large fragment appeared in approximately half-stoichiometric amount, relative to other fragments. Half-stoichiometric fragments were an essential observation in the older mapping experiments that led to the proposed flipping. Figure 1Ashows the linear maps, including the long half-stoichiometric fragments that render flipping untenable. The only form of the cpDNA that can lead to the observed data is the head-to-tail linear concatemer (Oldenburg and Bendich, 2004). Using EM, results with the denaturation mapping technique were interpreted to indicate a head-to-head linkage between neighboring genomic units in most (extremely rare) circular dimers of cpDNA from lettuce and spinach (Kolodner and Tewari, 1979). The same dumbbell forms, however, would result from head-to-tail dimers containing an inversion of the large single copy region in one of the genomic units (Oldenburg and Bendich, 2004). I conclude that all concatemeric forms of cpDNA are likely composed of head-to-tail units. To summarize, it is replication driven by recombination that leads to the cpDNA branching, head-to-tail concatemers, and inversion isomers. The isomers cannot result from flipping. The small amount of cpDNA found in circular form may actually serve no function that depends on circularity, but may represent an incidental byproduct of the recombination events required for cpDNA replication and repair, as we suggested for mtDNA circles (Oldenburg and Bendich, 1998). In a eukaryotic cell, the G2 phase of the cell cycle separates the replication of nuclear DNA in the S phase from segregation of the duplicated chromosomes in the M phase. Activities in the G2 phase ensure that only once-replicated and precisely replicated chromosomes are inherited by daughter cells. Such activities are absent in E. coli cells that divide every 25 min at 37° and require 40 min to replicate their genome (Helmstetter, 1996), so that chromosomal segregation occurs before DNA replication is complete and the DNA content among cells is quite variable (Akerlund et al., 1995). I suggest that it is the absence of such activities in the chloroplast that allows both the segregation to daughter organelles of chromosomes containing multigenomic amounts of DNA, including incompletely replicated genomes, and the large variability in DNA content among chloroplasts in the population. In the terms of Murray (2004), the coupling between chromosomal replication and segregation is flexible for the chloroplast and tight for the cell that harbors the chloroplast. Flexible coupling for chloroplasts is best illustrated in Scenedesmus quadricauda, a chlorococcal alga with a single chloroplast. In one daughter cell, the chloroplast contains nine nucleoids each carrying from 1 to 20 genome equivalents (assuming a 200-kb genome size), and the other daughter contains five nucleoids with 4 to 19 genomes (Zachleder et al., 1995). Only one of the 30 nucleoids among the two pairs of daughter cells analyzed contained as little as one genome equivalent. I propose the following rules for the chromosomes in chloroplasts. (1) The replication of cpDNA begins at one of several potential replication origins on linear cpDNA molecules, but most of the cpDNA is produced via recombination-dependent replication (RDR) that does not require firing of an origin. This RDR process is similar to those for herpes simplex virus (HSV) and bacteriophage T4, both of which contain a genome borne on linear DNA molecules (Kreuzer and Morrical, 1994; Jackson and DeLuca, 2003). A T4-like RDR process was also proposed for the mtDNA in plants (Oldenburg and Bendich, 1998). (2) Replicating cpDNA structures consist of branched, multigenomic forms, and it is these complex cpDNA forms that comprise the chromosomes in chloroplasts. For both HSV and T4, replication intermediates are multigenomic structures that are processed down to genome-sized units before packaging into virus particles because of the need to maximize the number of infectious particles per genome. There is no such need for chloroplasts that divide into only two daughter organelles, and so chloroplast chromosomes are inherited without processing. (3) There is usually more than one genome equivalent in a chloroplast chromosome, just as in rapidly dividing cells of E. coli. After cpDNA replication ceases, and the number of chloroplasts per cell increases to the final level in nonmeristematic cells, the number of genome equivalents per chromosome decreases. This plasticity of DNA content per chromosome is also observed for E. coli as the culture ages, but contrasts with the constancy found in the nucleus of plant cells. I now apply these rules to reinterpret cytological observations of the chloroplast nucleoid that are puzzling when a circular genome-sized molecule is assumed to represent the chloroplast chromosome. Chlamydomonas is a single-celled alga containing one chloroplast and has served as the principal organism for studying chloroplast inheritance. Vegetative cells of C. reinhardtii contain ∼75 copies of the chloroplast genome distributed unequally among 8 to 10 nucleoids (Armbrust, 1998). If the nucleoid is the segregating genetic unit, as proposed by VanWinkle-Swift (1980), we have a conundrum. How do we reconcile the small number of nucleoids (chromosomes) with the large number of genomes if each genome exists as a discrete circle (also a possible chromosome)? The problem of defining a chromosome when the 8 to 10 nucleoids to one large nucleoid in the The however, when we the nucleoid as one complex containing many genomes by recombination the that replication would the cpDNA to be partitioned among the four haploid from the A cytological is the nature of the cpDNA in nucleoids of including a that just the and a distributed the chloroplast et al., How can such structures be composed of discrete circles of I suggest that all such forms contain complex cpDNA molecules of the in Figure 2, rather than a series of circles to a (Kuroiwa, the of chloroplasts from in et al., and Arabidopsis et al., nucleoids first in size and then fragment and at the level of cpDNA molecules these cytological In one there is an in the number of genome-sized circles at clustered the circles are as the and the and chloroplast (Kuroiwa, I now an alternative scheme based on As the first of Arabidopsis from cells in the the number of genomes per increases from are to at when the is in length et al., 1994). The nucleoids in size during this and then in size and within the chloroplast by In the at the fluorescence of nucleoids is the cpDNA has from structures such as in Figure much forms, and many chloroplasts contain no DNA (Rowan et al., 2004). most chloroplasts from maize contain no DNA at all Oldenburg and Bendich, I propose that the size and of complex cpDNA forms their in As replication and the ends of DNA is from the complex and borne on the of cpDNA occurs long before the of and to be in both Arabidopsis and the of these observations for other species remains to be To summarize, the variability in chromosomal DNA molecules among and within is only if one a genome-sized chromosome and a tight coupling between chromosomal replication and as in the The variability is not at all if the coupling is as in The of cpDNA from nonmeristematic cells may be but such cells no longer need chloroplast chromosomes as of or to There are to be learned how to data that at with among them is that complex DNA structures should not be because they do not for a chromosomal DNA simple in form and genomic in The complex cpDNA should not have been removed either or before analysis. The segregating genetic units but many genomes per should have toward chromosomes of multigenomic the circular forms were a although mapping cannot establish chromosomal head-to-tail and linear DNA molecules have circular a circular map continues to be the principal or only evidence used to conclude that a chromosome is circular both for chloroplasts and I and Oldenburg for the and Oldenburg for the This work was by a from the