Ultrastructural changes in isolated plastids

Summary Etioplasts were isolated from maize leaves and the changes in their ultrastructure were followed in light and in darkness for several hours. It has been shown that the regular crystalline structures of prolamellar bodies, present after the isolation in darkness, disappear after 30 to 60 minutes of illumination, and long straight tubules appear within prolamellar bodies. Their appearance is influenced by the molarity of the isolation medium used, by light intensity, duration of illumination and by the temperature at which the isolates are kept. Long tubules appear, however, also in isolated etioplasts incubated for several hours in complete darkness.In isolates illuminated for 2–3 hours long tubules disappear again, and prolamellar bodies produced eventually consist of irregularly connected short tubules. In prolamellar bodies, regions with regular and very dense arrangement of tubules sometimes develop at this stage. The thylakoids (usually perforated) are now arranged concentrically in the plastids. True grana or poly-thylakoids can never be found in isolated etioplasts, not even when the etioplasts have been illuminated for 6 hours or more (up to 24).The present investigations have indicated that in isolated etioplasts in light, tubular elements, which build up the prolamellar bodies, cannot normally be transformed into thylakoids as is the case with intact tissue.The survival of isolated etioplasts is limited at present, and for this reason changes in their fine structure could be followed successfully for as long as 6 hours (in light at 15 °C), although a certain percentage of plastids survive up to 24 hours.     

Properties of reformed prolamellar bodies from illuminated and redarkened etiolated wheat plants

Prolamellar bodies were isolated from etiolated leaves of wheat ( Triticum aestivum L. cv. Walde, Weibull), which were illuminated for 4 h and then grown in darkness for 16 h. The inner etiochloroplast membranes were isolated by differential centrifugation, and prolamellar bodies and thylakoids were separated on a 10–50% continuous sucrose density gradient. The reformed prolamellar bodies contained phototransformable protochlorophyllide as the main pigment as shown by low temperature fluorescence spectra and high performance liquid chromatography. After illumination with 3 flashes of white light almost all of the protochlorophyllide was transformed to chlorophyllide. In the thylakoids, however, most of the protochlorophyllide was not phototransformed. The reformed prolamellar bodies and the thylakoids showed a fluorescence emission ratio 657/633 nm of 5.6 and 0.5, respectively. Both membrane systems contained also chlorophyllide and chlorophyll synthesized during the illumination. Polyacrylamide gel electrophoresis showed the main chlorophyllide oxidoreductasse.
Teransmission and scanning electron micrographs indicated that the reformed prolamellar bodies are mainly of the "narrow" type and that the prolamellar body fraction had only a minor contamination with thylakoid membranes.
The results obtained showed that reformed prolamellar bodies isolated from illuminated redarkened etiolated wheat leaves had features very similar to the prolamellar bodies isolated from etiolated leaves. This provides support for the idea that prolamellar bodies are an important natural membrane system which plays a dynamic role in the development of the etio-chloroplasts in light.     

Chloroplast Development in Rye Coleoptiles

In the parenchyma cells of 1-d-old dark-grown rye coleoptiles (Secale cereale) proplastids occurred which sometimes contained starch grains. During coleoptile growth in darkness starch-filled amyloplasts are formed from the preexisting proplastids. No prolamellar bodies were observed in the stroma of the plastids of the etiolated coleoptile. After irradiation of 3-d-old etiolated coleoptiles with continuous white light three different types of plastids occurred. In the epidermal cells proplastids were observed. The parenchyma cells below the stomata of the outer epidermis (above the two vascular bundles) contained mature, spindle-shaped chloroplasts with a well-developed thylakoid system. In the parenchyma cells that surround the vascular bundles amyloplasts with some thylakoid membranes (chloroamyloplasts) occurred. The mesophyll cells of the primary leaves of dark-grown seedlings contained etioplasts with large prolamellar bodies. In the primary leaves of irradiated plants chloroplasts similar to those of the parenchyma cells of the coleoptile were observed. Our results show that the rye coleoptile, which grows underground as a heterotrophic organ, is capable of developing mature chloroplasts upon reaching the light above the soil surface. The significance of this expression of photosynthetic capacity for the carbon economy of the developing seedling is discussed.     

EARLY STAGES IN THE DEVELOPMENT OF PLASTID FINE STRUCTURE IN RED AND FAR-RED LIGHT

Developmental changes in fine structure were studied in plastids of etiolated bean leaves during the time required for the protochlorophyllide-chlorophyllide transformation and the following lag phase prior to chlorophyll accumulation. In agreement with some other workers, two distinct stages of change in the fine structure of proplastids were found to occur upon illumination during this period. The first involves a dissociation of the previously fused units in the prolamellar bodies of the proplastids and occurs simultaneously with the protochlorophyllide-chlorophyllide conversion in light of 655 mµ, but not of 682, 700, or 730 mµ. The effect of the red light could not be reversed by a simultaneously supplied stronger far-red irradiation. The energy requirements for these structural changes parallel those for the pigment conversion. During the following step the vesicles which arose from the fused units of the prolamellar body were dispersed in rows through the stroma, and the prolamellar bodies themselves disappeared. For these changes to occur, higher light energies were required and the leaves had to be illuminated for longer periods. A red preillumination seemed to accelerate the development somewhat. The structural changes could be induced by light of 655 mµ, but also, to a lesser degree, of 730 mµ. No measurable additional chlorophyll accumulated during this period. Thus, the structural changes observed were independent of major changes in pigment content.     

The effects of inhibitors of protein synthesis on the differentiation of plastids in etiolated bean seedlings

Summary It has been shown that inhibitors of protein synthesis do not influence the breakdown of the crystal-lattice-like structure of the prolamellar bodies in the plastids when etiolated plants are exposed to light. The formation of grana and the greening of leaves are however considerably inhibited, depending on the concentration of the inhibitor used.     

Osservazioni Anatomo-Fisiologiche Sulla Kleinia Articulata Haworth

Abstract

Ultrastructural modifications of plastids in leaflets of Larix decidua and Picea excelsa during sprouting of buds.—Ultrastructural modifications of plastids in leaflets of Larix decidua and Picea excelsa during sprouting of buds kept in different light conditions were observed.

While in quiescent buds of both species typical plastids with magnograna are present, fully expanded leaflets kept in the light have plastids with an organized lamellar apparatus.

When the buds are kept in darkness the cells of the fully expanded, etiolated leaflets have hardly differentiated plastids with prolamellar bodies partially modified into short tubules and vesicles.

Plastids of Picea and Larix buds, in their development, behave almost identically both in darkness and in the light.

The differences previously observed in dark grown seedlings of the two species are not to be found in buds.     

Ultrastructural localization of diaminobenzidine photooxidation in etiochloroplasts

Summary Photooxidation of diaminobenzidine (DAB) has been used to detect ultrastructurally the photosynthetic activity (photosystem I) in the thylakoids of etiochloroplasts in greening bean leaves. The result of this photooxidation is a dark (osmiophilic) deposit which accumulates at first in certain portions of the primary thylakoids. In the course of further greening this area enlarges more and more and at last all the thylakoids become uniformly dark. It has been shown that the beginning of the appearance of the DAB deposits and the speed of their accumulation in the thylakoids largely depends on the experimental conditions of the plants: in leaves maintained in damp atmosphere the first DAB deposits appear between the first and the second hour of greening, while in those kept in dry air this does not happen until three or more hours in light.The tubules of the transformed prolamellar bodies in etiochloroplast—at least at the beginning of their dispersion — do not react with DAB. The tubules formedde novo after a period of darkness in young chloroplasts remain also without DAB deposits.     

Light-induced structural changes during incubation of isolated maize etioplasts

Summary Etioplasts were isolated from leaves of 9-day-old etiolated maize (Zea mays L.) seedlings and incubated in a relatively simple medium in light and in the dark. During the first 5 h no changes occurred in the fine structure of the isolated etioplasts in the dark. In light the size of the prolamellar bodies decreased and significantly more plastid sections without prolamellar bodies were counted. The total length of the thylakoids per plastid section increased, but there was no evidence for bi- and polythylakoid formation. It is concluded that light induces the structural transformation of the prolamellar body membranes into primary thylakoids also in isolated etioplasts.     

Electron microscope studies on the morphogenesis of plastids

Streptomycin sulphate (2 mg/ml) did not affect the formation of proplastids or the elaboration of prolamellar bodies. The plastids of the streptomycin (SM)-treated cotyledons contained both crystalline prolamellar bodies and ribosomes, and were undistinguishable from the plastids of the water-grown cotyledon. However, plastids from dark-grown SM-treated cotyledons were no longer able to differentiate to more advanced stages of development, even after exposure to light. The plastids of light and dark-grown SM-treated cotyledons often contained prolamellar bodies and abnormal giant grana. Variegation developed in the cotyledons germinated in Hoagland's solution plus SM. The plastids in pale green tissue contained stroma-lamellae and one or two giant grana, whereas in those of pale yellow tissue, many osmiophilic globules, large vacuoles and crystal bodies were observed. It is suggested that the formation of prolamellar bodies may depend on cytoplasmic protein synthesis whereas functional stroma- and grana-lamellae may depend on protein synthesis within the plastids. The inhibitory effects of SM on protein synthesis were used as a tool to test this hypothesis. This work was carried out in the Department of Botany, University of California, Davis, by Grant-GB-11906 from National Science Foundation of U.S.A.     

Changes in the plastid ultrastructure duringSedum rotundifolium leaf development

The ultrastructure of plastids was investigated in succulent leaves ofSedum rotundifolium to examine their changes during development. Leaves were categorized as etiolated, immature, young, and mature, based on their developmental stage and size. Of particular interest were the features of the tubular inclusion bodies (TIBs) and starch grains. These, along with vacuole size, showed remarkable changes over time. Etioplasts of unexposed leaves had prolamellar bodies, abundant starch grains, large TIB, few plastoglobuli, and thylakoid systems. Membranes of the thylakoids were still continuous with those of the prolamellar body. The plastids were often influenced by the presence and profile of inclusion bodies and starch grains throughout the early stages. Morphology was highly variable in the etioplasts but consistently hemispherical or ovoid in mature chloroplasts. TIB was most abundant in the etiolated leaves, but disappeared completely with development. Starch grains also became significantly reduced in size. Both young and mature mesophyll cells exhibited a normal chloroplast ultra-structure and huge central vacuoles, with an extremely thin peripheral cytoplasm. Grana were extensive and comprised a large portion of the chloroplasts. Traces of peripheral reticulum were also discovered in the chloroplasts of expanded leaves. The implications of these ultrastructural changes in the tubular inclusions and starch grains are discussed with relevance to Crassulacean acid metabolism (CAM).     

FINE STRUCTURE AND PIGMENT CONVERSION IN ISOLATED ETIOLATED PROPLASTIDS

Proplastids containing a prolamellar body were isolated from leaves of etiolated bean plants. The isolation methods do not necessarily lead to destruction of their submicroscopic structure and most of the isolated proplastids show well preserved outer membranes, lamellar strands, and the prolamellar body. Morphological intactness of the proplastids varies; certain leaf fractions contain single prolamellar bodies as well as proplastids. Since pellets after centrifugation between 350 g and 1000 to 3000 g contain intact proplastids and, as was shown by quantitative experiments, the same fractions show photoconversion of protochlorophyll to chlorophyll, it is supposed that the isolated particles probably retain many of the properties which are characteristic of them in situ. Isolated proplastids may thus be a valuable tool in investigations on the development of the photosynthetic apparatus.     

Protoplasts as a means of studying chloroplast development in vitro

Protoplasts obtained enzymically from etiolated primary leaves of oat were illuminated in vitro, and the process of etioplast chloroplast transformation followed. Chloroplast development proceeded up to 6 hours of incubation in the light (20 C). During this period, complete photosynthetic light and dark reactions were constituted, in addition to prolamellar body-degrading protease activity.     

FINE STRUCTURAL CHANGES IN PROPLASTIDS DURING PHOTODESTRUCTION OF PIGMENTS

Etiolated bean leaves supplied δ-amino-levulinic acid in the dark synthesize large amounts of protochlorophyllide which is not converted to chlorophyllide upon illumination of the leaves. The fine structure of the proplastids is not affected by the treatment. When leaves containing "inactive" protochlorophyllide are exposed to light of 700 ft-c for 3 hours, they lose practically all their green pigments. During this period large stacks of closed membrane structures are built up in the region of the prolamellar body. These lamellar structures remain even when no or only traces of pigment are left in the leaves. In untreated control leaves the pigment content remained constant during similar illumination and the structural changes in the plastids consisted of a rearrangement of the vesicles from the prolamellar bodies into strands dispersed through the stroma; lamellae and grana formation occurred later.     

Osservazioni Citologiche Su Peschi Clorotici E Rinverditi in Seguito A Trattamenti Con Soluzioni Minerali

Abstract

A Cytological study of peach leaves, chlorotic or regreening after treatment with salt solutions. — Whitening and regreening (for treatment with solutions containing Fe) leaves of chlorotic (Fe deficient) peaches were examined both at the optical and the electron microscope. The nuclei, as seen at the optical microscope, and the plastids infrastructures of chlorotic leaves sharply differ from the same structure of the leaves of virused plants. The differentiation of plastids of peach chlorotic leaves is arrested at a very early stage comparable to that of proplastids in etiolated plants before a vescicolar body is formed. In peach plastids a prolamellar body is never formed, not even during the greening of plastids. This is a further confirmation that such a structure, although normal in etiolated and genetically variegated plants, does not represent a fixed stage during differentiation of the lamellar system.

The present observations put into evidence that, even when the formation of lamellae is not immediate, the formation of the prolamellar body is not a necessary condition for the further development of the lamellar apparatus.     

A POSSIBLE MECHANISM FOR THE MORPHOGENESIS OF LAMELLAR SYSTEMS IN PLANT CELLS

A mechanism for the formation of lamellar systems in the plant cell has been proposed as a result of electron microscope observations of young and mature cells of Nitella cristata and the plastids of Zea mays in normal plants, developing plants, and certain mutant types. The results are compatible with the concept that lamellar structures arise by the fusion or coalescence of small vesicular elements, giving rise initially to closed double membrane Structures (cisternae). In the chloroplasts of Zea, the cisternae subsequently undergo structural transformations to give rise to a compound layer structure already described for the individual chloroplast lamellae. During normal development, the minute vesicles in the young chloroplast are aggregated into one or more dense granular bodies (prolamellar bodies) which often appear crystalline. Lamellae grow out from these bodies. In fully etiolated leaves lamellae are absent and the prolamellar bodies become quite large, presumably because of inhibition of the fusion step which appears to require chlorophyll. Lamellae develop rapidly on exposure of the plant to light, and subsequent development closely parallels that seen under normal conditions. The plastids of white and very pale green mutants of Zea similarly lack lamellae and contain only vesicular elements. A specialized peripheral zone immediately below the double limiting membrane in Zea chloroplasts appears to be responsible for the production of vesicles. These may be immediately converted to lamellae under normal conditions, but accumulate to form a prolamellar body if lamellar formation is prevented, as in the case of etiolation and chlorophyll-deficient mutation, or when the rate of lamellar formation is slower than that of the production of precursor material (as appears to be the case in the early stages of normal development).     

Prolamellar body transformation with increasing cell age in the maize leaf

Summary The fine structure of the plastids in the leaf's basal meristem and in the leaf tissues 2 cm immediately above has been studied in maize leaves of different ages. In the young leaves the proplastids of the basal meristem differentiate, in the tissues within 2 cm above the meristem, into chloroplasts containing two or more prolamellar bodies, indipendently whether the tissues have been fixed 11 hours after a period of illumination or of darkness. In the oldest leaves, in the tissues immediately above the basal meristem, no prolamellar body is present in the plastids, and the proplastids differentiate directly into chloroplasts, without passing through an etio-chloroplast stage.Supported by a grant of C.N.R.     

Biochemical differentiation of plastids and other organelles in rye leaves with a high-temperature-induced deficiency of plastid ribosomes

Summary 1. In developing rye (Secale cereale L.) leaves the formation of plastidic ribosomes was selectively prevented in light as well as in darkness, when the seedlings were grown at an elevated temperature of 32° instead of 22° where normal development ocurred. Plastid ribosome deficient parts of lightgrown leaves were chlorotic at 32°. — 2. At both temperatures the leaves contained under all conditions (light or dark, on H₂O or nutrient solution) equal or very similar amounts of total amino nitrogen. In light, the contents of total protein and dry weight were lower at 32° than at 22°, especially when the plants were grown on nutrient solution. — 3. Mitochondrial marker enzymes had normal or even higher activities in 32°-grown leaves. Respiration rates were similar for segments of leaves grown on water in light either at 32° or at 22° but by 20–30% lower for 32°-grown plants when they had been raised in darkness or on nutrient solution. In contrast to 22°-grown tissue, respiration of 32°-grown leaf segments was rather insensitive to KCN. Comparative inhibitor studies indicated the presence of both the cyanide-sensitive and the cyanide-insensitive pathway of respiration in 32°-grown leaves. — 4. Leaf microbody marker enzymes were present in leaves grown at 32°. From chlorotic parts of 32°-light-grown leaves a typical microbody fraction was isolated on sucrose densitygradients. — 5. Leaves of seedlings grown at 32° contained only very low levels of ribulosediphosphate carboxylase activity and of fraction I protein. Photosynthetic ¹⁴CO₂-fixation of such leaves was only a few per cent of that observed in normal leaves, and no photosynthetic oxygen evolution was observed in chlorotic leaf segments. However, ten other soluble enzymes which are exclusively or partially localized in chloroplasts reached high activities under all conditions at 32° (Table 4). — 6. From chlorotic parts of 32°-light-grown leaves as well as from etiolated 32°-grown leaves a fraction of intact plastids was isolated and purified by sucrose gradient centrifugation which contained several soluble chloroplast enzymes. From the results we conclude that cytoplasmic protein synthesis must contribute a functional chloroplast envelope including the mechanism for the recognition and uptake of chloroplast proteins which are synthesized on cytoplasmic ribosomes.     

Changes induced on the prolamellar body of pea seedlings by white,red and blue low intensity light

Summary We analyzed transformation, recrystallization, splitting and dispersion of prolamellar bodies during chloroplast development in pea seedlings illuminated by white, red and blue light of low intensity. With the help of a stereometric method we determined that there was a significant increase of prolamellar body number and a sharp decrease of their volume in differentiating chloroplast even in the first 2 hours of illumination. Decrease of prolamellar body dimensions was due both to gradual dispersion of its elements into primary thylakoids (indicated by the decrease of total volume of prolamellar bodies in plastid) and to splitting of prolamellar bodies (indicated by the increase of number of promellar bodies in plastid). Red light was more effective in transformation, splitting and dispersion of prolamellar bodies than blue light during the first 8–12 hours. Longer treatment with blue light had a stronger influence on these processes and on complete recrystallization than other light treatments.     

PLASTID DEVELOPMENT IN IOJAP- AND CHLOROPLAST MUTATOR-AFFECTED MAIZE PLANTS

Plastids affected by either iojap or chloroplast mutator fail to green, and altered plastids are maternally transmitted to subsequent generations. The ultrastructure of iojap-affected plastids indicates that these plastids contain no ribosomes and are capable of supporting little internal membrane organization in either light or dark-grown plants. Chloroplast mutator-affected plastids of light-grown plants contain some organized internal membrane structures. In dark-grown plants, chloroplast mutator-aftected plastids contain a crystalline prolamellar body, numerous vesicles, and osmiophilic granules. The chloroplast mutator-affecled etioplasts display an abnormal distribution of lamellar membranes; these membranes, rather than radiating in a spokelike pattern from the prolamellar body, are condensed into a portion of the organelle. Light causes disruption of the prolamellar body in chloroplast mutator-affected plastids without promoting the organization of a normal thylakoid membrane system. The effects of iojap and chloroplast mutator are cell autonomous and apparently influence the individual plastid, as evidenced by the persistence of heteroplastidic cells containing normal and affected plastids.     

MORPHOLOGY OF MARSILEA VESTITA. III. MORPHOGENESIS OF THE LEAVES OF ETIOLATED PLANTS

The laminae of etiolated Marsilea vestita leaves develop by means of marginal meristems. Unlike light-grown plants, the form of the etiolated plant is not affected by growth on a solid medium. All of the young leaves isolated from light-grown submerged plants will elongate in darkness. The smallest, etiolated, uncoiled leaves develop into land leaves when they are placed in light, and this development occurs regardless of whether the leaves remain on the plant or are isolated on nutrient agar. Only these smallest leaves (2.3 mm average length) are actually capable of being converted from a submerged leaf form to a land leaf form by darkness.