DNA in Chromoplasts: Ultrastructure and Nucleoid Fibrils in Plastids

Chromoplast DNA is the genetic material found inside chromoplasts, the pigmented plastids responsible for the yellow, orange, and red colours of many flowers and fruits. Electron-microscopy and biochemical studies confirm that chromoplasts, like chloroplasts, mitochondria, and bacteria, carry their own DNA, localised in electron-transparent regions as thin fibrils. This DNA is present in fewer copies per organelle than in chloroplasts, yet it links chromoplasts to the shared endosymbiotic ancestry of all plant plastids.

What did microscopy reveal about the DNA-containing electron-transparent regions of chromoplasts?

Studies specifically examining the DNA-containing, electron-transparent zones inside chromoplasts remain few in number. K. Kawallik and R.G. Herrmann investigated the plastids of the petals of yellow Narcissus and found fibrils 25–30 Å thick located within the electron-transparent areas of the plastids.

Fibril structure and the DNase experiments on Narcissus petals

To test the deoxyribonucleic origin of the fibrils, sections of the corona tissue were treated with DNase in an attempt to destroy the fibrils and demonstrate their DNA nature. The fibrils, however, were not broken down after exposure to the enzyme.

Treatment with proteases (trypsin and pronase), which normally improves the visibility of DNA strands in such regions, was likewise unsuccessful. Even so, given the strong resemblance between the electron-transparent regions and fibrils of chromoplasts and the analogous structures of chloroplasts, mitochondria, and bacteria, the authors concluded that chromoplasts contain DNA localised in the electron-transparent regions in the form of fibrils.

How glutaraldehyde fixation affected DNA visibility

The negative enzyme results are attributed to steric hindrance caused by glutaraldehyde fixation of the material rather than to an absence of DNA. Glutaraldehyde cross-links proteins and can shield DNA fibrils from enzymatic access, which explains why DNase and protease treatments failed on the Narcissus preparations while succeeding on differently fixed tissue. This methodological detail matters for interpreting any electron-microscopy study of plastid DNA: the way a specimen is prepared can conceal genetic material that is in fact present.

The ultrastructure of chromoplasts in cucumber cotyledons

E. Mikulska and colleagues, studying the ultrastructure of chromoplasts in cucumber cotyledons treated with the growth retardant 2-chloroethyltrimethylammonium chloride, also observed electron-transparent regions containing fibrils. At high magnification these were shown to consist of a central body of varying shape and size, with thin fibrils radiating in all directions; the smallest measured 10–15 Å in diameter. After DNase treatment these structures disappeared completely, or only isolated fragments were barely discernible, so the enzyme experiments confirmed that the observed structures contain DNA.

Comparison with leucoplasts and chloroplasts

The DNA-containing electron-transparent regions in cucumber cotyledon chromoplasts are few in number, and their structure does not differ from the analogous regions of leucoplasts and chloroplasts. Morphologically these regions more closely resemble those found in the matrix of leucoplasts: they are not surrounded by thylakoid membranes and show no sharp demarcation from the surrounding stroma. This shared organisation across plastid types reinforces the view that chromoplasts, leucoplasts, and chloroplasts are developmental variants of a single organelle lineage.

Chromoplast studies in the Cucurbitaceae and Solanaceae

Electron-transparent regions displaying structures similar to fibrillar DNA formations, contrasted with uranyl acetate, have been reported by other researchers as well, including B. T. Matienko and E. M. Chebanu in their studies of chromoplast ultrastructure in Cucurbitaceae and Solanaceae. The convergence of findings across the Narcissus, cucumber, gourd, and nightshade families supports the general presence of DNA in chromoplasts regardless of the plant family.

How is chromoplast DNA isolated and measured?

Chromoplast DNA is characterised by first isolating a pure chromoplast fraction, counting the organelles, and then quantifying the DNA using the diphenylamine (Dische) reaction. Because chromoplasts must be separated cleanly from chloroplasts, nuclei, and cytoplasmic debris, the reliability of every DNA-per-organelle figure depends on the purity of the fraction. The two central steps are:

• Isolating a pure chromoplast fraction — gently homogenising the pigmented tissue and separating intact chromoplasts by density gradient centrifugation so that contaminating organelles do not inflate the DNA reading.
• The diphenylamine method for DNA determination — a colourimetric assay in which the deoxyribose of DNA reacts with diphenylamine, allowing the DNA content to be calculated per counted organelle.

How much DNA does a chromoplast contain?

A single chromoplast contains only a fraction of the DNA held by a chloroplast of the same plant, corresponding to roughly 8 to 36 genome copies per organelle depending on the species and developmental stage. B. Liedvogel was the first to estimate the DNA content of a pure chromoplast fraction from Narcissus petals, measuring the amount by the diphenylamine method after counting the organelles.

The DNA estimate in Narcissus (B. Liedvogel)

Narcissus chromoplasts contained 0.13 × 10⁻¹⁴ g of DNA per organelle (the mean of three independent measurements). Under identical conditions the chloroplasts of the same plant held 0.8 × 10⁻¹⁴ g of DNA per chloroplast — six times more than a single chromoplast. The ratio of chloroplast to chromoplast volume was likewise 6:1, so the DNA scales with organelle size.

The number of DNA copies per organelle

Combining the measured DNA content with the genome size established by physical methods, B. Liedvogel calculated that Narcissus leaf chloroplasts contained on average 50 DNA copies per organelle, whereas the flower chromoplasts held about 8 copies. Chromoplasts therefore keep a functional but reduced complement of their genome relative to the photosynthetic chloroplast.

DNA content in the chromoplasts of garden nasturtium

Chromoplasts of garden nasturtium (Tropaeolum majus) contained 0.6 × 10⁻¹⁴ g of DNA per plastid, or 36 DNA copies per organelle. This is consistent with the DNA copy number of higher-plant chloroplasts, which ranges from 20 to 60 copies per chloroplast, and shows that some chromoplasts retain a copy number comparable to that of chloroplasts.

DNA content in carrot chromoplasts and amyloplasts

DNA content was determined in the chromoplasts of red carrot roots of the "Kharkovskaya Nantskaya" cultivar and in the amyloplasts of white carrot roots of the "Belaya Zelenogolovaya" cultivar. The data were expressed relative to the dry weight of the plastids, per organelle, and per unit mass of protein. The DNA content of both plastid types is not constant but changes during the ontogeny of the root (Table 1).

Table 1 — DNA content in the chromoplasts of red carrot roots of the "Kharkovskaya Nantskaya" cultivar and in the amyloplasts of white carrot roots of the "Belaya Zelenogolovaya" cultivar.

During the early stages of red carrot root formation the DNA content of the chromoplasts increased gradually, reached a maximum in mid-season, and then declined. Although the overall pattern of change in chromoplast DNA content was the same whether calculated per plastid dry weight, per plastid protein, or per organelle — rising at first and falling somewhat toward the end of the growing season — the magnitude of change differed among these measures.

In the sample with the maximum DNA content (root diameter 9–12 mm), the DNA in the chromoplasts rose relative to the starting value about threefold when calculated per plastid dry weight, somewhat more than twofold per protein, and almost fourfold per organelle. These differences were even more pronounced in the amyloplast DNA determinations of white carrot.

Calculated per plastid dry weight, amyloplast DNA in the early stages of root development rose 1.5- to 2-fold and peaked at a root diameter of 5–10 mm, after which it gradually fell back to the starting level. Calculated per organelle it also peaked at a root diameter of 5–10 mm but declined only slightly thereafter. Calculated per plastid protein it increased continuously, exceeding the starting level 10- to 12-fold by the end of the season. These differences reflect changes in the protein and dry-matter content of the plastids during the ontogeny of white and red carrot roots.

The protein content of amyloplasts and chromoplasts changed substantially over the carrot growing season. Whereas the change in protein content was modest for chromoplasts, the amyloplasts showed a 6- to 8-fold decrease in protein per organelle by the end of the growing period (Table 2).

Table 2 — Protein content in the chromoplasts of red carrot roots of the "Kharkovskaya Nantskaya" cultivar and in the amyloplasts of white carrot roots of the "Belaya Zelenogolovaya" cultivar.

The comparative analysis of DNA content in carrot chromoplasts and amyloplasts showed that the most objective results are obtained when the calculation is made per organelle. On this basis the DNA content of red carrot chromoplasts ranged from 0.10–0.11 × 10⁻¹⁴ g in the early stages of root formation to 0.41 × 10⁻¹⁴ g at its mid-season maximum.

The DNA content of the amyloplasts was somewhat lower, at 0.09–0.21 × 10⁻¹⁴ g per organelle. The genome size of carrot plastids, established by reassociation kinetics, is 103.5–105.7 × 10⁶ Da (1 Da = 1.67 × 10⁻¹⁴ g). From these figures, the DNA copy number per amyloplast ranged from 5–6 in the early stages of root development to 12–13 at maximum, and for chromoplasts from 6–7 to 23–24 copies respectively. These values are somewhat lower than the DNA copy numbers of chloroplasts in the photosynthetic tissues of higher plants and of nasturtium chromoplasts, and close to the copy number of yellow Narcissus chromoplasts.

The change in nucleic acid content per organelle followed a similar pattern in both amyloplasts and chromoplasts of carrot roots: DNA content rose in mid-season and fell toward the end of the growing period, the difference between the two plastid types being only quantitative.

What are the structure and composition of chromoplast DNA?

Chromoplast DNA is a circular, double-stranded molecule of the same physical class as chloroplast DNA, distinguished from nuclear DNA by its buoyant density, its base composition, and its DNA methylation pattern. The reassociation kinetics of carrot plastid DNA place its genome at roughly 104 × 10⁶ Da, consistent with the compact genome of a plastid rather than the far larger nuclear genome.

The buoyant density of plant organellar DNA

The buoyant density of plant organellar DNA is measured by centrifugation to equilibrium in a caesium chloride (CsCl) density gradient, where each DNA species forms a band at the point matching its own density. Because chloroplast and chromoplast DNA usually band at a density distinct from the main nuclear DNA peak, CsCl analysis is a standard tool for separating and identifying plastid DNA, and it complements the reassociation-kinetics estimates of genome size and DNA renaturation used to define plastid genomes.

5-methylcytosine content in chromoplast DNA

The base composition of chromoplast DNA includes 5-methylcytosine, a methylated form of cytosine that arises from DNA methylation and can be measured as part of the base-composition analysis of organellar DNA. The proportion of 5-methylcytosine is a marker used to compare the DNA of different plastid types and to distinguish plastid DNA from nuclear and mitochondrial DNA, since methylation patterns differ across the plant cell's genetic compartments.

The genome size of chromoplasts and chloroplasts

Chromoplast DNA and chloroplast DNA share the same underlying genome, so their genome size is essentially identical within a species; what differs is the number of copies retained per organelle. In higher plants the plastid genome is typically 120,000–160,000 base pairs and encodes on the order of 100–120 genes. Carrot plastid DNA, at about 104 × 10⁶ Da measured by reassociation kinetics, falls within the size range expected for a higher-plant plastid genome, confirming that a ripening or storage plastid does not shed its genome but simply maintains fewer copies of it.

How does chromoplast DNA compare with chloroplast DNA?

Chromoplast DNA and chloroplast DNA are the same genetic molecule housed in two developmental states of one organelle, so their sequence and structure match while their copy number and expression differ. The measurements above make the contrast concrete: a Narcissus chloroplast holds about 50 DNA copies and six times the DNA mass of a chromoplast (about 8 copies) from the same plant.

Properties and structure of chloroplast DNA

Chloroplast DNA (cpDNA) is a circular, double-stranded molecule located in the stroma of the chloroplast, replicated and transcribed independently of the nucleus. Chloroplasts are the double-membraned, green plastids where photosynthesis occurs, packed with the thylakoid membranes that hold chlorophyll and drive light capture. The genetic autonomy of the chloroplast was foreshadowed by Karl Erich Correns, whose early work on non-Mendelian, maternal inheritance in Mirabilis jalapa — extending the framework of Gregor Mendel's genetics — pointed to hereditary factors residing outside the nucleus.

Gene number and genome size of chloroplasts

The chloroplast genome encodes roughly 100–120 genes across about 120–160 kilobases, including genes for the photosynthetic machinery such as the large subunit of RuBP carboxylase (Rubisco) encoded by the rbcL gene, as well as ribosomal RNAs, transfer RNAs, and a number of open reading frames of still-unidentified function. The rbcL gene is widely used in plant phylogenetics and DNA-based classification, because its slowly evolving sequence allows evolutionary relationships between species to be reconstructed. Many chloroplast proteins are not encoded in the plastid at all but in the nucleus, then imported through the TOC and TIC translocon complexes of the outer and inner envelope membranes, a cooperation of nuclear and chloroplast genes that also underlies retrograde signalling from the plastid back to the nucleus.

Chromoplasts as organelles of the plant cell

Chromoplasts are pigment-bearing plastids that synthesise and store carotenoids, giving colour to many flowers, fruits, roots, and ageing leaves. Along with chloroplasts (green, photosynthetic) and leucoplasts (colourless storage plastids such as the starch-storing amyloplasts), chromoplasts belong to the interconvertible plastid family, all bounded by a double membrane and all descended from a single kind of plastid within the plant cell. Chromoplasts are grouped into five main structural categories based on how they store pigment — globular, crystalline (as in tomato, rich in lycopene), fibrillar, tubular, and membranous forms.

The function of chromoplasts in flowers and fruits

The function of chromoplasts is to produce and hold the carotenoid pigments that colour flowers and fruits, and that colour serves the plant biologically. Bright petals and ripe fruit attract pollinators and animals that disperse seeds, so the pigment stored in chromoplasts is directly tied to pollinator attraction and seed dispersal. Unlike chloroplasts, chromoplasts are non-photosynthetic; their role is signalling through colour rather than energy capture.

Chromoplast location and distribution in tissues

Chromoplasts are located in the coloured tissues of the plant — the petals of flowers, the flesh and skin of ripe fruits, and certain storage roots such as the carrot. In green organs the pigmented plastids are chloroplasts of the leaf mesophyll, whereas in colourless tissues leucoplasts predominate; chromoplasts appear wherever a tissue accumulates yellow, orange, or red carotenoid rather than green chlorophyll.

Carotenoid pigments and coloration

The colours of chromoplasts come from carotenoids — a family of lipid-soluble pigments that includes the orange carotenes such as lycopene, which reddens the tomato and the Valencia orange, and the yellow xanthophylls. These carotenoid pigments produce the yellow-to-red range of plant colours, in contrast to the water-soluble anthocyanins that give many blue, purple, and red hues in the cell vacuole. Where the carotenoid biosynthetic pathway is disrupted by mutation, pigment fails to accumulate — one route by which white-flowered variants arise.

Pigment synthesis and storage in chromoplasts

Pigment synthesis and storage are the defining activities of the chromoplast: carotenoids are manufactured within the organelle and then deposited in the plastid's globules, crystals, fibrils, tubules, or membranes according to its structural type. The scale of accumulation is under gene regulation of carotenoid biosynthesis and hormonal control, which is why fruit colour intensifies at a specific ripening stage rather than continuously. The lycopene crystals of the tomato (Solanum lycopersicum) make it a favoured model for research into how chromoplasts build and store their pigment.

The conversion of chloroplasts into chromoplasts during ripening

Chromoplasts most often form from chloroplasts during fruit ripening: as a green fruit ripens, its chloroplasts dismantle the thylakoid membranes and chlorophyll of photosynthesis and re-differentiate into carotenoid-filled chromoplasts, turning the fruit from green to red, orange, or yellow. This chloroplast-to-chromoplast transition is a leading example of plastid interconvertibility, and under certain conditions it can even reverse, with chromoplasts reverting toward chloroplasts. Molecular regulators such as the SP1 ubiquitin ligase, which acts on the plastid protein-import machinery, help control this remodelling of plastid identity.

Autumn leaf colour change and senescence

The yellow and orange colours of autumn leaves reveal the same pigment shift within the plastid. As leaves senesce, chlorophyll is broken down while the more stable carotenoids remain, so the underlying yellow and orange of xanthophylls and carotenes becomes visible as the green fades. This seasonal transformation of the mesophyll plastids, alongside chlorophyll synthesis and its variation, illustrates how leaf colour tracks the changing balance of pigments during senescence.

The evolution of chromoplasts and the endosymbiotic theory

Chromoplasts, like all plastids, trace their ancestry to a free-living prokaryote engulfed by an early eukaryotic cell — the core claim of the endosymbiotic theory (symbiogenesis). The presence of a genome inside the chromoplast, the very DNA discussed throughout this page, is direct evidence for this origin: an organelle keeps its own DNA precisely because it descends from an organism that once lived on its own.

The origin of plastids from prokaryotes

Plastids arose when an ancestral eukaryote took up a photosynthetic cyanobacterium that was not digested but retained as an internal partner, an event known as endosymbiosis. Over evolutionary time the endosymbiont's genome shrank as many genes moved to the host nucleus — the plastid genome reduction that leaves modern chloroplasts and chromoplasts with only about 100–120 genes and dependent on nuclear-encoded, imported proteins. The double membrane of every plastid, its circular DNA, and its own ribosomes are the relics of this cyanobacterial ancestry, and the same lineage is seen in single-celled algae such as Euglena gracilis and Chlamydomonas.

The agronomic and horticultural significance of chromoplast DNA research

Research into chromoplast and chloroplast DNA underpins practical work in agriculture and horticulture, from breeding more colourful, more nutritious crops to engineering plastid genomes for improved traits. Because carotenoid content determines both the visual appeal and the nutritional value (provitamin A) of crops such as tomato, carrot, and citrus, understanding the plastid genome and the genes that govern pigment synthesis guides efforts to enhance fruit and root colour, extend shelf life, and improve dietary quality. Plastid engineering — inserting genes directly into the high-copy plastid genome — offers a route to crop improvement that keeps transgenes out of pollen, while comparative plastid-DNA sequencing, using markers like the rbcL gene, supports plant classification and the reconstruction of crop lineages for breeding programmes.

Source: V. P. Lobov, I. A. Petrov, "Chromoplasts".