11.2.1—
Chloroplast DNA

11.2.1.1—
Discovery

The existence of DNA in chloroplasts has been established by both cytological and biochemical criteria. Ris and Plaut (1962) provided the first convincing evidence that chloroplasts contain DNA. Electron microscopy of chloroplasts in sections of both algal and higher plant cells revealed areas of low electron density which contained fibrils 2.5–3.0 nm in diameter. These fibrils were not seen if the sections were treated with deoxyribonuclease. There are earlier reports that chloroplasts contain regions which can be stained with the Feulgen reagent for DNA, but in general these reports have not proved reproducible. It is now clear that in most species the concentration of DNA inside the chloroplast is below the limit of detection by the Feulgen method. It can however be detected by autoradiography after exposure of cells to -H-thymidine; the report of Rose et al. (1974) on the distribution of DNA in dividing spinach chloroplasts contains an excellent example of this method. It is important when evaluating such studies to consider the controls that are used. If the incorporated label is all present in DNA, it should be removed by treatment of sections with deoxyribonuclease, but not by treatment with ribonuclease. This control works well for tobacco and spinach leaves and several algae, but not or maize leaves, where deoxyribonuclease fails to remove the label from chloroplasts.

Most studies on chloroplast DNA since 1962 have been carried out using isolated chloroplasts. This approach suffers from the difficulty of distinguishing chloroplast DNA from DNA originating from nuclei, mitochondria, and microorganisms which may contaminate the chloroplast pellet. The resolution of these problems is still not complete, and depends mainly on the differing properties of the DNA from the various sources. There are four characteristic properties of chloroplast DNA that have been used to identify it: namely buoyant density, ease of renaturation, lack of histones, and the absence of 5-methylcytosine. These properties are discussed below. It must be remembered that there is a possibility some chloroplast DNA may not meet these criteria. If, for example, chloroplasts in some species contain a minor DNA component which is a nuclear transcript, contains 5-methylcytosine and renatures poorly, it is doubtful whether present techniques could identify it as chloroplast in location. In one species the problem of contamination by nuclei and micro-

organisms can be entirely avoided; DNA has been identified in chloroplasts isolated from sterile enucleated plants of Acetabularia (Gibor & Izawa, 1963; Baltus & Brachet, 1963).

11.2.1.2—
Buoyant Density

It is now clear that chloroplast DNA from the algae Euglena, Chlamydomonas, and Chlorella has a buoyant density sufficiently different from that of nuclear DNA to allow resolution by analytical ultracentrifugation, but that in many higher plants the densities of the two DNA types are often, but not always, too close to permit this. This information has been hard-won; the story of the way in which the interpretation of band patterns in neutral caesium chloride density gradients has changed since 1963 has been told by Kirk (1971) and Tewari (1971). A summary is given here since it illustrates the very real problem of establishing the identity of chloroplast DNA from higher plants by biochemical methods.

The first report of the chemical characterization of higher plant chloroplast DNA was provided by Kirk (1963). He found that chloroplast DNA from Vicia faba has a GC content (37.4 %) which is slightly but significantly different from that of nuclear DNA (39.4%). In the same year a contrasting report was published by Chun et al., (1963), who found that chloroplast preparations from Spinacia oleracea and Beta vulgaris contained two components of much higher densities than the nuclear DNA as judged by caesium chloride equilibrium density gradient centrifugation. However the bulk of the DNA in these chloroplast preparations had a density similar to that of nuclear DNA; this band was attributed to contamination of the chloroplast pellet by nuclear fragments, and thus the chloroplast DNA was regarded as the two high density components. There followed a spate of papers which supported the claim that, in higher plants, chloroplast DNA has an appreciably higher density than nuclear DNA. This picture changed when re-examination by more rigorous methods supported the earlier view of Kirk, and the higher density components are now attributed to contamination by mitochondria and bacteria.

The present position can be summarized by saying that, in all the higher plants examined, chloroplast DNA had a base composition of 37.5 ± 1% GC and a buoyant density of 1.697 ± 0.001 g cm^–3 . Table 11.1 lists some values for the buoyant densities of chloroplast and nuclear DNA which have been agreed by Kirk and Tewari. Nuclear DNA is seen to have a smaller, larger, or similar density to chloroplast DNA from the same plant, depending on the species. The GC contents in Table 11.1 have been calculated from the buoyant densities, but a more accurate method has been devised by Kirk (1967). This method releases the purine bases by gentle acid hydrolysis and separates the adenine and guanine by ion-exchange chromatography.

The moral to be drawn from this story is that the purity of subcellular fractions from higher plant tissues must be established by positive methods if

meaningful interpretations are to be made. Microbial contamination can be reduced by surface sterilization of fresh tissues and by the use of sterile solutions, while mitochondrial contamination can be assayed by succinic dehydrogenase or cytochrome oxidase activities. Nuclear contamination can be reduced by incubation of intact chloroplasts with deoxyribonuclease and phosphodiesterase. The extent of nuclear contamination is difficult to measure, but the ease of renaturation and the absence of 5-methylcytosine serve to distinguish chloroplast from nuclear DNA in all cases so far studied.

11.2.1.3—
Ease of Renaturation

A more useful criterion than the buoyant density for detecting chloroplast DNA from higher plants is the ease with which it will renature after heat or alkali denaturation. Nuclear DNA renatures only to a slight extent in a few hours,

Figure 11.1
Densitometer tracings of ultraviolet absorption photographs of  Vicia faba  DNA banded
in caesium chloride gradients. (a) Native chloroplast DNA; (b) heat-denatured chloroplast
DNA; (c) renatured chloroplast DNA; (d) native nuclear DNA; (e) heat-denatured nuclear
DNA; (f) renatured nuclear DNA. The numbers refer to densities in g cm^-3  as follows: (1)
1.696; (2) 1.699; (3) 1.712; (4) 1.728; (5) 1.696; (6) 1.709; (7) 1.712; (8) 1.728. The
peaks at 1.728 g cm^–3  represent marker DNA from  Micrococcus radiodurans.
(From Kung & Williams, 1968, by courtesy of Elsevier.)

depending on its content of reiterated sequences. Figure 11.1 illustrates this difference in the case of Vicia faba. The rapid renaturation of chloroplast DNA has encouraged attempts to estimate its genome size from measurements of kinetic complexity. The kinetic complexity of a DNA sample is a measure of the size of the unique set of nucleotide sequences it contains, as judged from the rate at which the DNA renatures. A rapid rate of renaturation implies that like sequences are present in high concentration, and therefore the number of unique sequences, or kinetic complexity, is small. Table 11.2 lists the kinetic complexity, in terms of molecular weight, for the chloroplast DNA from some algae and higher plant species. Some of these values have been corrected from the published figures since the estimate of the molecular weight of the bacteriophage T4 DNA, used as standard, has been revised from 1.3 × 10⁸ to 1.06 × 10⁸ (Dubin et al., 1970). It is striking that the corrected values for the kinetic complexity of chloroplast DNA from the few algae and higher plants examined so far are all in the range 0.9–1.0 × 10⁸ . This may be a coincidence, or it could mean the information content of chloroplast DNA is basically similar throughout the plant kingdom. All the species so far examined belong to the Chlorophyta, with the exception of Euglena. It would be interesting to make such measurements of chloroplast DNA from other plant groups, especially from algae with unusually shaped chloroplasts. Table 11.2 also lists the analytical complexities of chloroplast DNA, i.e. the amount of DNA per chloroplast. Since the kinetic complexities are always much less than the analytical complexities, there must be between 20 and 60 copies of the DNA sequences in each chloroplast. These renaturation studies cannot rule out the possibility of microheterogeneity in nucleotide sequence between the copies but, if it does exist, such heterogeneity is beyond detection by current techniques.

11.2.1.4—
Absence of Histones and 5-Methylcytosine

When released from intact isolated chloroplasts by gentle lysis, chloroplast DNA is not combined with basic proteins. This finding confirms the microscopic observations of Ris and Plaut (1962) that chloroplasts contain DNA fibrils in the same form as they appear in the nucleoplasms of prokaryotic cells. It is also characteristic of chloroplast DNA from both higher plants and algae that it contains no detectable 5-methylcytosine, whereas nuclear DNA invariably does. Whitfeld and Spencer (1968) regard the absence of this base as the most reliable criterion for establishing the purity of chloroplast DNA.

11.2.1.5—
Circularity

A recent discovery is that, when precautions are taken to minimize shearing, a proportion of chloroplast DNA can be isolated as circles. Circular DNA has been reported from chloroplasts of Euglena (Manning et al., 1971), Pisum sativum (Kolodner & Tewari, 1972), Spinacia oleracea (Manning et al., 1972), Antirrhinum majus, Oenothera hookeri, and Beta vulgaris (Hermann et al., 1975). Figure 11.2 shows a molecule of chloroplast DNA from Spinacia oleracea. The significance of circularity in DNA is not known, but it is a useful property since it implies the molecule has not been degraded on isolation. The most interesting aspect of this finding is that in all the species listed the contour length of the majority of the circles is in the range 37–45 µm; this length of double-stranded DNA has a calculated molecular weight of 0.85–1.0 × 10⁸ daltons, which is in the same range as the kinetic complexity of chloroplast DNA (Table 11.2). This correspondence of length and kinetic complexity suggests that the genetic information carried by the chloroplast DNA is accommodated by the length of the circular molecule.

Figure 11.2
Open circular DNA molecule, contour length
44.7 µm, from chloroplasts of  Spinacia oleracea.
(Reprinted from Hermann et al.,  (1975) by courtesy of Elsevier.)

11.2.1.6—
Ploidy

It is clear from the data in Table 11.2, and from the evidence for circular DNA, that each chloroplast may contain many copies of a circular genome, and thus may be genetically polyploid. It has been pointed out by Kirk (1972) that this

multiplicity increases the probability that a mutation will appear in a chloroplast in a given time, but there is likely to be a long delay before the mutation can be expressed, since a mutation in one copy will be swamped by all the other wild-type copies. It is therefore not surprising that known non-Mendelian mutations affecting chloroplasts are small in number, and difficult to induce with mutagens. In Chlamydomonas reinhardi a number of mutations are known which alter the sensitivity of chloroplast ribosomes to inhibition by antibiotics such as erythromycin and carbomycin; some of these mutations show uniparental inheritance (Sager, 1972; Mets & Bogorad, 1972). Those genes which are inherited in a uniparental fashion have been shown by recombinational analysis to form one linkage group which behaves as if it were diploid in vegetative cells. It is difficult to reconcile this diploid behaviour with the evidence which suggests that there are many copies of the genome in each chloroplast. It may be that the uniparental linkage group does not reside in chloroplast DNA, or if it does, that a reduction in the number of genomes in each chloroplast occurs at some stage in the cell cycle, so that only a 'master copy' is passed during sexual reproduction. There is as yet no evidence to resolve these questions.

11.2.1.7—
Functions

A 40 µm circle of double-stranded DNA of unique base sequence is sufficient in principle to code for about 125 proteins each of molecular weight 50,000. Since our knowledge depends ultimately on the validity of the techniques which can be used, the evidence for the functions of chloroplast DNA will be considered in terms of the four methods tried so far:

The Selective Inhibition of Chloroplast DNA Transcription

The antibiotic rifampicin is a potent inhibitor of the initiation of RNA synthesis in bacteria. This compound has been reported to inhibit the incorporation of labelled precursors into chloroplast ribosomal-RNA, but not into cytoplasmic ribosomal-RNA, in several unicellular algae, namely Chlamydomonas, Chlorella, and Acetabularia. Rifampicin has also been reported to inhibit chloroplast RNA polymerase activity in extracts of Chlamydomonas. These results suggest that the functional genes for chloroplast ribosomal-RNA are located in chloroplast DNA and are transcribed by the chloroplast RNA polymerase.

The effect of rifampicin in higher plant systems is controversial. There have been some reports that it specifically inhibits chloroplast ribosomal-RNA synthesis, but other workers could not reproduce this result (Bottomley et al., 1971). There now seems general agreement that chloroplast RNA polymerase from higher plants, as normally prepared and assayed, is insensitive to rifampicin, but this may mean only that the preparations are not initiating RNA synthesis.

Genetic Analysis of Mutants

Many mutations are known which affect chloroplast components, but the vast majority are inherited in a Mendelian fashion and are therefore presumed, to be located in nuclear genes. For example, seven different genes are known which control steps in the chlorophyll biosynthetic pathway, and nuclear genes have been shown to be involved in the synthesis of phosphoribulokinase and at least five components of the photosynthetic electron transport chain (Kirk, 1972; Kirk & Tilney-Bassett, 1967; Levine & Goodenough, 1970).

In Chlamydomonas many mutations affecting chloroplast ribosomes and photosynthetic capacity are inherited as a single linkage group in a uniparental fashion (Sager, 1972; Schlanger & Sager, 1974). In this alga, like gametes fuse completely, and the physical basis for uniparental inheritance has been suggested to be the destruction of the chloroplast DNA of one of the gametes by a modification-restriction system of the type known in bacteria. As pointed out earlier, there are difficulties in concluding that this uniparental class of mutations resides in chloroplast DNA, but it is tempting to believe that it does. A simple interpretation suggests that some of the proteins of the chloroplast ribosomes are encoded in chloroplast DNA; there is also evidence that at least one other protein of the chloroplast ribosome is encoded in a nuclear gene (Mets & Bogorad, 1972).

Rigorous proof that a mutation inherited in a maternal or uniparental fashion actually alters the amino acid sequence of a protein has been obtained in only one case. Chan and Wildman (1972) studied the inheritance of a mutation in the large subunit of Fraction I protein in Nicotiana tabacum at the tryptic peptide level; this mutation was inherited via the maternal line only. By contrast, mutations in the small subunit of Fraction I protein are inherited in a Mendelian fashion (Kawashima & Wildman, 1972). This type of combined biochemical-genetic approach is very promising and deserves further use, especially with cultivated plants where many varieties are available. It must be emphasized that this approach must be conducted at the level of the tryptic peptide analysis of a purified protein. It is not sufficient to show that particular proteins are absent in a mutant variety, since absence of a protein might result from a mutation in a chloroplast gene which controls the formation of a protein encoded in the nucleus.

DNA-RNA Hybridization Studies

If RNA isolated from chloroplasts can be shown to hybridize to chloroplast DNA but not to nuclear DNA, this is good grounds for believing that such RNA is both encoded in and transcribed from chloroplast DNA. A number of hybridization studies have been carried out with chloroplast ribosomal-RNA, which hybridizes to 0.5–6.0% of chloroplast DNA (Ellis & Hartley, 1974). The

most recent study is that of Thomas and Tewari (1974); they found that in a number of higher plants each circle of chloroplast DNA contains two genes for chloroplast ribosomal-RNA. There are several reports that chloroplast ribosomal-RNA will also hybridize to nuclear DNA. The significance of these reports awaits further study, but the possibility is raised that there are two types of chloroplast ribosomal-RNA, one encoded in chloroplast DNA and the other in nuclear DNA. This arrangement could be a means whereby the nucleus exerts control over events in the chloroplasts, especially if it is further postulated that chloroplast ribosomes containing nuclear RNA translate only messengers originating in the nucleus.

There is evidence that some of the transfer RNA species found in chloroplasts will also hybridize to chloroplast DNA. Tewari and Wildman (1970) found that tRNA from tobacco chloroplasts would hybridize to 0.4–0.7% of chloroplast DNA. This amount of DNA would be enough to code for 20 to 30 transfer RNA molecules, each of molecular weight 25,000. This work used unfractionated tRNA however, and more studies need to be done to establish the site of encoding of individual tRNA species.

Identification of RNA and Protein Molecules Synthesized by Isolated Chloroplasts

This is the most direct method; if transcription coupled to translation can be obtained in isolated chloroplasts, identification of the products would simultaneously determine both the structural genes present in chloroplast DNA and the function of chloroplast ribosomes. Work in the author's laboratory has shown that isolated intact chloroplasts from Pisum sativum and Spinacia oleracea will synthesize discrete protein and RNA molecules, but transcription and translation are not coupled (Hartley & Ellis, 1973; Ellis, 1974; Ellis & Hartley, 1974). It is therefore not possible to infer that the proteins synthesized by isolated chloroplasts are encoded in chloroplast DNA. The RNA synthesized by isolated chloroplasts has been analyzed by polyacrylamide gel electrophoresis (Fig. 11.3). The chief product is a species of molecular weight about 2.7 × 10⁶ ; this has been shown by competitive hybridization to chloroplast DNA to be a precursor to chloroplast ribosomal-RNA. Isolated chloroplasts have not been shown convincingly to synthesize ribosomal-RNA; presumably the processing system which trims the precursor molecules does not survive the trauma of isolation (see also chapter 9).

The known genes in chloroplast DNA can be summarized as follows: chloroplast transfer-RNA, chloroplast ribosomal-RNA, several chloroplast ribosomal proteins, and the large subunit of Fraction I protein. These genes account for less than 10% of the total potential coding capacity of a 40 µm circle of DNA. The elucidation of the function of the remaining 90% of chloroplast DNA is the most important problem in this field.

Figure 11.3
Analysis by polyacrylamide gel electrophoresis of RNA synthesized by
chloroplasts isolated from  Spinacia oleracea.  Isolated chloroplasts were incubated
with ³ H-uridine and 15,000 lux of red light for 20 min at 20°C. Nucleic acid was extracted
and run on polyacrylamide gels. The solid line represents the A₂₆₀ , while the histogram
shows the radioactivity. The figures are the molecular weights × 10^-6  of the RNA components.
(Reprinted from Hartley & Ellis, 1973, by courtesy of the Biochemical Journal).