Peter Sitte

Plastiden-metamorphose und chromoplasten bei Chrysosplenium

Summary 1. The leaves of the Golden Saxifrage, Chrysosplenium alternifolium and Chr. oppositifolium , are deeply green, whereas the bracts and the small leaflets of the calyx (a corolla is lacking) appear (greenish-)yellow during flowering time. Correspondingly, leaf cells contain chloroplasts, and bract cells chloro-chromoplasts. The sepals contain chromoplasts, especially in their epidermal cells. 2. The sepals exhibit remarkable changes in color. They are green when the flower buds break open, and yellow during anthesis. During seed development they enlarge and turn green again. A reversible plastid metamorphosis is responsible for these color changes. 3. The fine structure of leaf and calyx plastids, and metamorphosis of plastids in calyx leaflets in particular, has been investigated (mainly in Chr. alternifolium) by conventional electron microscopy. The pictures have been analyzed by morphometric methods (for results, compare fig. 6). 4. Mesophyll chloroplasts of green leaves are uneven-shaped. The numerous grana are oriented in different planes and comprise up to 30 thylakoids in one granum (figs. 11 a, b). Starch grains are always present. Osmiophilic plastoglobules are numerous but rather small (Fig. 9). 5. The sepals contain, in every state of their development, typical chromoplasts in their upper epidermis. These chromoplasts are small and flat (figs 3, 4 and 8). They contain, in a central layer, a few thylakoids and rudimentary grana with but a few thylakoids per granum (fig. 7, see also table 1). The periphery of the chromoplasts is filled with large-size, osmiophilic plastoglobules. They are mostly transformed into polyhedral crystals in the open flower (fig. 4). After anthesis, these carotenoid-containing crystals partly enlarge. Simultaneously, new spherical and often less osmiophilic plastoglobules are formed in a more central region of the chromoplasts (fig. 8). On the whole, metamorphosis of plastids is “monotropic” in the upper sepal epidermis. 6. Macroscopic color changes of the sepals are due to a reversible chloroplast-to-chromoplast transformation in the mesophyll. At the outset of anthesis, the plastids of these cells are typical lens-shaped, starch-containing chloroplasts (fig. 2). During anthesis, chromoplasts differentiate by enlargement of plastoglobules and concomitant reduction of the thylakoid and grana system as well as of stroma matrix material and starch (fig. 5). These chromoplasts are considerably smaller than the chloroplasts from which they originate (fig. 1). Later, during seed formation, these mesophyll chromoplasts of the sepals revert to chloroplasts by reforming larger grana und starch grains, and by reducing the size of the chlotenoid-carrying plastoglobules. Hence, these plastids behave quite differently from the epidermal plastids of the very same leaflet, and thus present a good example of a “non-monotropic”, reversible transformation. 7. Regarding the other components of sepal cells, no conspicuous changes have been observed. 8. These results are discussed in relation to some data of the chromoplast literature, and a contemporary classification of different chromoplast fine structure types is given.

Structure, composition, and distribution of plastid nucleoids in Narcissus pseudonarcissus.

The size, frequency and distribution of the nucleoids of chloroplasts (cl-nucleoids) and chromoplasts (cr-nucleoids) of the daffodil have been investigated in situ using the DNA-specific fluorochrome 4′6-diamidino-2-phenylindole. Chromoplasts contain fewer nucleoids (approx. 4) than chloroplasts (> 10), and larger chromoplasts (cultivated form, approx. 4) contain more than smaller ones (wild type, approx. 2). During chromoplast development the nucleoid number decreases in parallel with the chlorophyll content. Each nucleoid contains 2–3 plastome copies on average. In chloroplasts the nucleoids are evenly distributed, whereas they are peripherally located in chromoplasts. The fine structure of isolated cl-and cr-nucleoids, purified either by Sepharose 4B-CL columns or by metrizamide gradients, was investigated electron microscopically. The cl-nucleoids consist of a central protein-rich core with ‘naked’ DNA-loops protruding from it. In cr-nucleoids, on the other hand, the total DNA is tightly packed within the proteinaceous core. The protein-containing core region of the nucleoids is made up of knotty and fibrillar sub-structures with diameters of 18 and 37 nm, respectively. After proteinase treatment, or incressing ion concentration, most of the proteins are removed and the DNA is exposed even in the case of cr-nucleoids, the stability of which proved to be greater than that of cl-nucleoids. The chemical composition of isolated plastid nucleoids has been determined qualitatively and quantitatively. Chromoplast-nucleoids contain, relative to the same DNA quantity, about six times as much protein as cl-nucleoids. Accordingly the buoyant density of cr-nucleoids in metrizamide gradients is higher than that of cl-nucleoids. In addition to DNA and protein, RNA could be found in the nucleoid fraction. No pigments were present. The cr-and cl-nucleoids have many identical proteins. There are, however, also characteristic differences in their protein pattern which are possibly related to the different expression of the genomes of chloroplasts and chromoplasts. Nucleoids of both plastid types contain some proteins which also occur in isolated envelope membranes (probably partly in the outer membrane) and thus possibly take part in binding the DNA to membranes.

Chromoplasts of Palisota barteri, and the molecular structure of chromoplast tubules

Ripe, deep-red fruits of Palisota barteri contain tubulous chromoplasts which develop from unpigmented leucoplasts. These plastids contain, besides large spherical inclusion bodies, numerous osmiophilic globules which, in the course of ripening, frequently show transition states to tubular structures. The tubules contain, in all stages of their development, acylated β-citraurin, which is also the main pigment of Citrus fruits. The tubular structures have been isolated, fragmented by French-pressure treatment, and separated into three fractions on sucrose gradients. The lightest fraction (1.044 g·cm-3) contained thick fragments (‘saccules’) with diameters of 50–60 nm, whereas the heaviest (1.083 g·cm-3) consisted of tubules 20–25 nm in diameter. The relative amounts of polar lipids, proteins, and carotenoids of the different fractions are consistent with a molecular structure model of tubules and saccules, according to which a wick of longitudinally oriented carotenoid molecules of variable thickness is coated by a monolayer of polar lipids and proteins. High-resolution ‘negative-stainings’ showed the surface of the tubules to be covered with globular particles of about 6 nm diameter. The main protein of all fractions is a 30-kDa polypeptide; it is assumed that the particles are oligomers of this specific protein.

Composition and molecular structure of chromoplast globules of Viola tricolor.

Plastoglobules have been isolated in pure form from petals of the pansy, Viola tricolor L. Their chemical composition has been determined up to a recovery of 96% dry weight. Triacyl glycerols (57%) as well as carotenoids and their esters (23%) are the main constituents. Polar lipids, proteins, alkanes, phytyl esters, plastid quinones, and steryl esters have been detected in smaller amounts (cf. Table 1). The mean diameter of chromoplast globules is 280±70 nm (corresponding to a volume of 11.7×106 nm3), their buoyant density 0.93 g cm−3. The plastoglobules are devoid of a surrounding unit membrane. However, electron microscopical evidence and analytical data are consistent with a structural model envisaging the globules to consist mainly of an apolar core, covered by a ‘half unit membrane’ of polar constituents.

Chromoplasts of Tropaeolum majus L.: Isolation and characterization of lipoprotein elements.

SummaryChromoplasts of unfolding petals of Tropaeolum majus contain large amounts of filaments (which, in sections, appear as tubules), and unevenshaped, isodiametric to elongated bodies (IBs). These structural elements are the major sites of the chromoplast pigments. They were freed from isolated chromoplasts and subjected to sucrose density gradient centrifugation. At a density of 1.080 g cm-3 a distinct orange band contained almost exclusively fine filaments of 15–20 nm in diameter as shown after negative staining. Other filaments and most of the IBs were heterogeneous in size, shape, and density and were collected in two fractions of buoyant densities of 1.025 and 1.055 g cm-3. The three fractions thus obtained comprise 15–33% protein, large amounts of carotenoids and their esters, glyco- and phospholipids, as well as minor amounts of tocopherols. A higher buoyant density of particles is correlated with a higher relative content of protein and glyco- and phospholipids and a lower relative content of carotenoids. The polypeptide pattern, as shown by SDS-polyacrylamide gel electrophoresis, is very similar in all three fractions. There is one main polypeptide, with a MW of about 30,000, accounting for up to 80% of the protein of each fraction.

Primary and secondary structure of the nuclear small subunit ribosomal RNA of the cryptomonadPyrenomonas salina as inferred from the gene sequence: Evolutionary implications

SummaryThe cryptomonadPyrenomonas salina presumably has arisen from a symbiotic event involving a flagellated phagotrophic host cell and a photosynthetic eukaryote as the symbiont. Correspondingly, in this unicellular alga there are four different genomes, e.g., the nuclear and the mitochondrial genomes of the host cell as well as the plastid genome and the genome contained in the vestigial nucleus of the endocytobiont (nucleomorph). To analyze the orgin of one of the symbiotic partners the small subunit rRNA gene sequence of the host cell nucleus was determined, and a secondary structure model has been constructed. This sequence is compared to those of 40 other eukaryotes. A phylogenetic tree constructed using the neighborliness method revealed a close relationship between the host cell ofP. salina and the chlorophytes, whereas the rhodophytes diverge more deeply in the tree.

Plastid DNA from Pyrenomonas salina (cryptophyceae) : physical map, genes,and evolutionary implications

SummaryCryptomonads are thought to have arisen from a symbiotic association between a eukaryotic flagellated host and a eukaryotic algal symbiont, presumably related to red algae. As organellar DNAs have proven to be useful tools in elucidating phylogenetic relationships, the plastid (pt) DNA of the cryptomonad alga Pyrenomonas salina has been characterized in some detail. A restriction map of the circular 127 kb ptDNA from Pyrenomonas salina was established. An inverted repeat (IR) region of about 5 kb separates two single-copy regions of 15 and 102 kb, respectively. It contains the genes for the small and large subunit of rRNA. Ten protein genes, coding for the large subunit of ribulose-1,5-bisphosphate carboxylase, the 47 kDa, 43 kDa and 32 kDa proteins of photosystem II, the ribosomal proteins L2, S7 and S11, the elongation factor Tu, as well as the α- and β-subunits of ATP synthase, have been localized on the restriction map either by hybridization of heterologous gene probes or by sequence homologies. The gene for the plastidal small subunit (SSUr) RNA has been sequenced and compared to homologous SSU regions from the cyanobacterium Anacystis nidulans and plastids from rhodophytes, chromophytes, euglenoids, chlorophytes, and land plants. A phylogenetic tree constructed with the neighborliness method and indicating a relationship of cryptomonad plastids with those of red algae is presented.

DNA in the Nucleomorph of Cryptomonas Demonstrated by DAPI Fluorescence

Abstract DNA has definitely been demonstrated in the nucleomorph of a marine Cryptomonas species by combining thin sectioning of Lowicryl K4M-embedded material and DAPI-induced DNA fluorescence.

FREEZE-FRACTURE STUDY OF THE SINGLE MEMBRANE BETWEEN HOST CELL AND ENDOCYTOBIONT IN THE DINOFLAGELLATES GLENODINIUM FOLIACEUM AND PERIDINIUM BALTICUM1

The dinoflagellates Glenodinium foliaceum Stein and Peridinium balticum (Levander) Lemmermann harbor a chrysophytic endocytobiont which is bounded by only a single membrane. This unique membrane is of particular interest because it could correspond to an intermediate stage in the evolution of “complex” plastids found in many Plastids of this type are surrounded by three or membranes instead of the usual two. With freeze‐fracture techniques, we show that the single membrane in P. balticum has a pronounced polarity with respect to the distribution of intramembrane particles (IMPs) on the two corresponding fracture faces. The inner face exhibited more IMPs than the outer. We suggest that this stdedness identifies the separating membrane as the plasma membrane of the endocytobiont. A symbiontophoric vacuole with a separate membrane apparently is lacking. In the endocytobiosis of G. foliaccum, the single membrane separating host and endocylobiont exhibits a symmetrical particle partition. Nevertheless, from the size distribution of the IMPs it appears likely that this membrane, too, corresponds to the plasma membrane of the symbiont.

Circular DNA in isolated chromoplasts.

Abstract Flower chromoplasts of Narcissus pseudonarcissus have been isolated in high purity and inte grity by a new procedure. From these chromoplasts circular DNA molecules in supercoiled form have been isolated and demonstrated by electron microscopy to have a contour length of about 44 μm (molecular weight about 92 x 106 daltons)