Chromoplast DNA Size and Conformation: Circular and Supercoiled Plastid Molecules

Native plastid DNA — including the DNA of chromoplasts — exists in a circular form. This conclusion was first established by isolating circular molecules of chromoplast DNA, and subsequent comparative studies have shown that the chromoplast genome closely matches the genomes of other plastid types. Before going further, it helps to read the companion article Isolation of chromoplast DNA.

Who first isolated circular molecules of chromoplast DNA?

Circular molecules of chromoplast DNA were first isolated by H. Falk and co-workers, who used the chromoplasts from the flowers of the yellow daffodil (narcissus) as their research object. In the preparations obtained, the bulk of the DNA appeared as linear molecules up to 44.5 µm long, together with several supercoiled circular molecules. The mean length of eight supercoiled chromoplast DNA molecules was 42.6 ± 1.2 µm.

The small spread in these measurements indicated the absence of any marked heterodispersity among individual chromoplast DNA molecules. Applying a supercoiling correction of 3.7%, the authors concluded that the contour length of the circular chromoplast DNA molecules of the yellow daffodil is 44 µm — equal to the size of the longest linear molecules found in the DNA preparations studied. The molecular mass of the circular molecules was 92–106 kDa.

The flowers of the yellow daffodil served as the object for studying the circular molecule of chromoplast DNA. H. Wuttke later detected circular molecules in DNA preparations extracted from purified tulip chromoplasts. In those preparations linear DNA predominated, among which a few circular molecules could be identified. Unlike the chromoplast DNA of the daffodil corona, however, the circular plastid DNA from tulip petals had an open configuration.

To determine contour length, the authors measured six such tulip molecules, obtaining individual values of 42.4, 42.5, 43.1, 44.1, 44.1 and 45.3 µm. From these data the mass of the circular tulip chromoplast DNA molecules was calculated as 91.6–106 kDa — indistinguishable from the equivalent value for the chromoplast DNA of the yellow daffodil.

Circular DNA molecules were also isolated from the chromoplasts of garden nasturtium (Tropaeolum majus). In the resulting preparations, 48% of the DNA was found as linear molecules and 52% as open or supercoiled circles. The contour length of the circular DNA molecules in nasturtium chromoplast preparations was somewhat greater than in the plastids of tulip and daffodil petals, and their molecular mass was 99.35 kDa.

How do chromoplast, amyloplast and chloroplast DNA compare by thermal denaturation and reassociation kinetics?

Comparative analysis by thermal denaturation and reassociation kinetics shows that the DNA of chromoplasts, amyloplasts and chloroplasts is essentially identical, which points to a single shared plastid genome. This matters because the eukaryotic genome — including that of plants — is not fixed: under various environmental influences, and within specialised organs, its structure can change.

Cytophotometric methods, for example, established that when ivy plants pass from the juvenile to the adult stage the DNA content per haploid genome rises by 70%. In the flowering buds of elder, the DNA content per haploid genome was 40% higher than in vegetative buds. Thermal denaturation and reassociation kinetics data revealed a noticeable difference between the DNA structure of generative buds and that of vegetative buds — meaning that the formation of generative plant organs is accompanied by differential amplification of specific DNA sequences.

Because the formation of specialised plastids results from the differentiation of initially uniform organelles, the question of whether the DNA structure of plastids performing different functions can change is of particular interest. As noted above, the molecular masses of the circular chromoplast DNA molecules of yellow daffodil, tulip and nasturtium are 92, 91.6 and 99.35 kDa respectively. On this measure, chromoplast DNA does not differ from the chloroplast DNA of the higher plants studied.

Buoyant density of nuclear, plastid and mitochondrial DNA

R. Herrmann compared DNA preparations isolated from the chromoplasts, chloroplasts, nuclei and mitochondria of daffodil. The buoyant densities of chloroplast and chromoplast DNA (ρ = 1.697) were practically indistinguishable from nuclear DNA (ρ = 1.698) and clearly differed from mitochondrial DNA (ρ = 1.707). Heat-denatured chromoplast and chloroplast DNA likewise did not differ from each other (ρ = 1.711–1.712) or from nuclear DNA (ρ = 1.713), and again differed markedly from mitochondrial DNA (ρ = 1.722).

So neither native nor denatured chromoplast and chloroplast DNA differed from one another in buoyant density. When the denatured DNA samples were renatured for 4 hours and centrifuged in a caesium chloride density gradient, however, a clear distinction emerged between the plastid and nuclear DNA. Nuclear DNA reassociated weakly under these conditions and had a buoyant density of 1.708–1.711, whereas chromoplast and chloroplast DNA reassociated almost completely and had a buoyant density of about 1.700 g/cm³.

The chloroplast and chromoplast DNA of the yellow daffodil also did not differ in melting temperature (T_m = 86.0 °C) or in dispersion (7 °C), although on both of these measures they differed from nuclear DNA (88 °C and 9 °C respectively).

Figure 1 — Thermal denaturation of the DNA of bacteriophage T2 (1), amyloplasts (2) and chromoplasts (3) of carrot of the cultivar White Green-Head; chromoplasts (4) and chloroplasts (5) of carrot of the cultivar Mshak; chromoplasts (6) and chloroplasts (7) of carrot of the cultivar Kharkiv Nantes. A₂₅ is the absorbance of native DNA at 25 °C, A_t is the absorbance of DNA at the given temperature.

The DNA of chromoplasts from nasturtium petals and the DNA of chloroplasts from nasturtium leaves had the same buoyant density, melting temperature and base composition (calculated from T_m). Some differences observed in the size of the circular DNA molecules of nasturtium chromoplasts and chloroplasts were statistically insignificant. On the basis of these data the conclusion was drawn that the chromoplast and chloroplast genomes are identical.

This conclusion received further support somewhat later from restriction analysis. The patterns of fragment distribution obtained after digestion of the DNA of daffodil chromoplasts and chloroplasts proved to be identical.

Physico-chemical study of carrot plastid DNA

To investigate whether plastid DNA structure can change, V. P. Lobov and I. A. Petrov examined the physico-chemical characteristics of the DNA of chromoplasts and amyloplasts from carrot taproots, as well as of chloroplasts from carrot leaves, in three carrot cultivars.

Figure 2 — Reassociation kinetics of the DNA of chloroplasts (a) and amyloplasts (b) of carrot of the cultivar White Green-Head (1); chloroplasts (a) and chromoplasts (b) of carrot of the cultivar Mshak (2); chloroplasts (a) and chromoplasts (b) of carrot of the cultivar Kharkiv Nantes (3); and of bacteriophage T2 (4). A(∞) is the absorbance of native DNA at 260 nm, A is the absorbance of denatured DNA at 260 nm, A_t is the absorbance of reassociating DNA t minutes after the start of reassociation. The numbers beside the curves are DNA concentrations (µg/ml).

Figure 1 shows the melting profiles of the studied plastid DNA together with that of bacteriophage T2 DNA, which served as a standard. The DNA of chloroplasts, chromoplasts and amyloplasts were similar to one another and differed slightly from bacteriophage T2 DNA in melting profile. The difference lay in the fact that the bacteriophage DNA melted over a narrow temperature range, whereas the DNA of the specialised carrot plastids was characterised by a substantial melting interval.

The melting characteristics calculated from these curves are summarised in Table 1. The T_m values of the bacteriophage and plastid DNA did not differ, amounting to 83.5–83.9 °C, which corresponds to 34.5–35.6% GC pairs in the plastome. The plastid DNAs did not differ among themselves but differed from bacteriophage T2 DNA in their doubled standard deviation, 2σ.

The 2σ value characterises the degree of intramolecular heterogeneity in GC composition. These results therefore revealed intra-plastome heterogeneity in the DNA of carrot plastids, in contrast to bacteriophage T2 DNA, where such heterogeneity is absent. Studies of the chloroplast DNA of higher plants and algae likewise indicate the presence of GC-composition heterogeneity.

V. P. Lobov and I. A. Petrov also studied the reassociation kinetics of carrot plastid DNA and established the identity of the reassociation of chloroplast, chromoplast and amyloplast DNA. The reassociation curves in Figure 2 — the linearity of the reassociation points, their extrapolation to unity, and the direct proportionality between the slope of the curves in this coordinate system and the DNA concentration — indicate the absence of highly repetitive sequences in carrot plastid DNA.

The reassociation rate constants of the plastid DNAs lay between 41.67 and 42.54 and differed somewhat from the rate constant for bacteriophage T2 DNA, which was 36.72. The kinetic complexity of the carrot plastid genome was 103.5–105.7 × 10⁶ Da, using a bacteriophage T2 genome size of 120 × 10⁶ Da. Notably, the kinetic complexities of chloroplast DNA coincide with plastome sizes calculated from electron-microscopic measurements and from summing the lengths of restriction fragments.

From this it can be concluded that the size of the carrot plastome equals its kinetic complexity and amounts to 103.5–105.7 × 10⁶ Da. This is somewhat higher than the plastid genome sizes of the flowering plants studied, which fall within 90–100 × 10⁶ Da. A recent report, however, indicates that the circular chloroplast DNA of the duckweed Spirodela has a contour length of 54.1 ± 1.8 µm, equivalent to 115–120 × 10⁶ Da — 25% larger than the plastid DNA of flowering plants studied earlier. The figure of 90–100 × 10⁶ Da for the flowering-plant plastome is thus rather provisional, given that only a comparatively small number of plastid DNAs have been examined.

Where is chromoplast DNA located within the organelle?

Chromoplast DNA, like the DNA of chloroplasts, leucoplasts, mitochondria and bacteria, is localised in electron-transparent regions of the matrix in the form of fibrils up to 25–30 Å in diameter. These electron-transparent regions occur less frequently in chromoplasts than in chloroplasts and are usually located at the centre of the organelle.

Although the structure of these regions does not differ from the analogous regions in leucoplasts and chloroplasts, morphologically they more closely resemble those in the leucoplast matrix. They are not surrounded by thylakoid membranes and show no clear demarcation from the surrounding stroma. Chromoplasts as a whole contain less DNA than chloroplasts, yet their DNA content is markedly higher than that of amyloplasts.

As in chloroplasts and amyloplasts, the DNA content of chromoplasts is not constant but changes over the course of the plant's growing season, owing to changes in the number of DNA copies per organelle. Unlike the nuclear genome, the plastid genome of plants — including the chromoplast genome — is polyploid.

Thus, carrot taproot chromoplasts contain 6–24 DNA copies per organelle, yellow daffodil petals 8, and garden nasturtium petals 36. Such plastid polyploidy nevertheless conflicts with genetic data, according to which the Chlamydomonas plastome resembles a diploid one. To explain this, it was hypothesised that during hybridisation two copies play a determining role while the rest play a subordinate one — but no experimental data confirming this hypothesis have been obtained.

Thermal denaturation of carrot plastid DNA (Table 1)

Do chromoplast DNAs from different sources differ?

Chromoplast DNAs from different sources do differ among themselves in their physico-chemical properties. The chromoplast genome size of carrot taproots is 103.5–105.7 kDa, of yellow daffodil petals 92, of tulip petals 91.6, and of garden nasturtium petals 99.35 kDa. The melting temperatures of chromoplast DNA from yellow daffodil, garden nasturtium and carrot are 86.0, 82.5 and 83.5–83.9 °C respectively.

At the same time, chromoplast DNA does not differ in its physico-chemical characteristics from the DNA of other plastid types. The chromoplast, amyloplast and chloroplast DNA of three carrot cultivars had the same thermal denaturation profiles, melting temperature, reassociation curves and genome size. The leaf chloroplast and petal chromoplast DNA of yellow daffodil shared similar buoyant densities of their native and denatured molecules, similar melting temperatures and similar reassociation rates, and differed on these measures from nuclear and mitochondrial DNA.

The chromoplast and chloroplast DNA of this plant also did not differ in the distribution patterns of restriction fragments in agarose gels. Likewise, the chromoplast DNA of garden nasturtium flowers did not differ from the leaf chloroplast DNA of that plant in melting temperature or buoyant density.

What other plastid DNA features do chromoplasts share?

Chromoplast DNAs probably possess other features characteristic of plastid — and especially chloroplast — DNA. Chloroplast DNA, like the amyloplast DNA of potato tubers, contains no 5-methylcytosine, whereas nuclear DNA is methylated at cytosine to a level of 5% and plant mitochondrial DNA to a level of 2–3%.

Another distinctive feature of plastid DNA is the presence within its structure of ribonucleotides covalently incorporated into the DNA molecule. The chloroplast DNA of pea and spinach each contained 18 ± 2 ribonucleotides, and that of lettuce 12 ± 2 ribonucleotides. It is also known that, unlike nuclear DNA, the chloroplast DNA of higher plants and algae does not form stable protein complexes resembling chromatin.