Developmental Physiology of Bean Leaf Plastids II. Negative Contrast Electron Microscopy of Tubular Membranes in Prolamellar Bodies

Proplastids and prolamellar bodies with tubular membranes were isolated from the dark grown primary leaves of bean seedlings (Phaseolus vulgaris L.). The combination of fluorescence microscopy and negative contrast electron microscopy provided the tentative identification of protochlorophyll holochrome as a constituent of prolamellar body membranes and new evidence for solution-filled channels within the tubular membrane systems of prolamellar bodies.     

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.     

CHANGES INDUCED BY LOW LIGHT INTENSITIES ON THE PROLAMELLAR BODY OF 8-DAY,DARK-GROWN SEEDLINGS

Etioplasts of 8-day-old, dark-grown seedlings of Phaseolus vulgaris contain large, crystalline prolamellar bodies. The basic structural unit within the prolamellar body is a six-pointed star (star module) with four tubules fusing at each of the nodes. With sufficient illumination some of the tubules are withdrawn and the crystalline prolamellar body transforms to a complex tangle of tubules, the reacted prolamellar body. In vivo spectrophotometry and electron microscopic observations were carried out on portions of the same leaves after varying periods of illumination with low light intensity. Protochlorophyllide transformation was normal. However, the structural changes are not closely tied to protochlorophyllide conversion. The pigment conversion is complete after 20 sec of illumination, but 80% of the prolamellar bodies are still in the crystalline form after 20 min of illumination. After 1 and 2 hr of illumination all prolamellar bodies are reacted. After 4 hr of continuous illumination 35%, and by 12 hr 60%, of the prolamellar bodies returned to the crystalline form. Spectrophotometric evidence and presence of grana show chlorophyll synthesis during this period. The coexistence of grana and the crystalline prolamellar body indicates that when insufficient photosynthetic membrane constituents are provided by the photo-reactions, under low light intensity, the membranes of the reacted prolamellar body will be forced to reform a crystalline prolamellar body.     

MACROMOLECULAR PHYSIOLOGY OF PLASTIDS : VIII. Pigment and Membrane Formation in Plastids of Barley Greening under Low Light Intensity

Sequential changes occurring in the etioplasts of the primary leaf of 7-day-old dark-grown barley seedlings upon continuous illumination with 20 lux have been investigated by electron microscopy, in vivo spectrophotometry, and thin-layer chromatography. Following photoconversion of the protochlorophyllide pigment to chlorophyllide and the structural transformation of the crystalline prolamellar bodies, the tubules of the prolamellar bodies are dispersed into the primary lamellar layers. As both chlorophyll a and b accumulate, extensive formation of grana takes place. After 4 hr of greening, protochlorophyllide starts to reaccumulate, and concomitantly both large and small crystalline prolamellar bodies are formed. This protochlorophyllide is rapidly photoconverted upon exposure of the leaves to high light intensity, which also effects a rapid reorganization of the recrystallized prolamellar bodies into primary lamellar layers.     

The greening process in plastids

Plastids in etiolatedAvena leaves were studied by electron microscopy of thin sectioned material fixed in glutaraldehyde and osmium tetroxide and embedded in Epon. Each plastid contains one—three prolamellar bodies. These are highly ordered systems, the membraneous component of which consists of interconnected tubules lying in the three major axes of a cubic lattice. Where three tubules (one in each axis of the lattice) meet and fuse at the corners of each unit cell, their unit membranes are smoothly confluent so that the principal curvatures of the membrane surface are of opposite sign at every point. A face view of a unit cell shows four tubules delimiting a circular opening of diameter 380 Å. The diameter of the tubules is 210 Å at their narrowest point, i. e. half way along the edges of the unit cells. The plastid stroma penetrates the prolamellar body via the 380 Å openings, and contributes ribosome—like particles to the system. These particles are centrally located, one in each unit cell. The literature on prolamellar bodies is reviewed, it is concluded that this type of organisation is widespread in plants. Structures with similar geometry are described, and it is suggested that the unit membranes of the lattice are laid down on contours of uniform “field” strength centred on the lattice of ribosome-like particles. The surface area of membrane in a prolamellar body is estimated.     

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.     

The NADPH-protochlorophyllide oxidoreductase is the major protein constituent of prolamellar bodies in wheat (Triticum aestivum L.)

A fraction of highly purified prolamellar bodies was isolated from etioplasts of wheat (Triticum aestivum L. cv. Starke II, Weibull), as previously described by Ryberg and Sundqvist (1982, Physiol. Plant., 56, 125–132). Studies on the protein composition revealed that only one major polypeptide of an apparent molecular weight of 36000 is present in the fraction of prolamellar bodies. This polypeptide was identified as the NADPH-protochlorophyllide oxidoreductase. The highest specific activity of the enzyme in etiolated leaf tissue was confirmed to be in the fraction of prolamellar bodies.Abbreviations PChlide protochlorophyllide - PLB prolamellar body - PT prothylakoid     

Lipid composition of envelopes,prolamellar bodies and other plastid membranes in etiolated,green and greening wheat leaves

Summary Comparative studies of lipid composition were made on prolamellar bodies, envelopes and other plastid membranes separately extracted from etiolated, green or greening (intermittent or continuous light) wheat (Triticum sativum L.) leaves. The different membrane fractions were examined by electron microscopy.The major lipid was digalactosyldiglyceride in the envelopes and prolamellar bodies and monogalactosyldiglyceride in stroma lamellae and grana. Phosphatidylcholine represented 60% of total phospholipids in the envelopes, 30% in prolamellar bodies and 14% in grana. All types of envelopes had the same lipid proportions.For all lipids the lowest fatty acid unsaturation was always found in the envelope membranes. The relative amount of {ie193-1} acid in the phosphatidylglycerol of envelopes increased from 4% (etioplasts) to an average of 15% (etiochloroplasts and chloroplasts).Abbreviations DGDG digalactosyldiglyceride - MGDG monogalactosyldiglyceride - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - PI phosphatidylinositol - PS phosphatidylserine - SL sulfolipid     

Binding properties of NADPH-protochlorophyllide oxidoreductase as revealed by detergent and ion treatments of isolated and immobilized prolamellar bodies

Prolamellar bodies were isolated from dark-grown leaves of 6.5-day-old wheat ( Triticum aestivum L. cv. Walde). The prolamellar bodies were immobilized in agarose beads to get a material suitable for studies on pigment and protein release, and to protect the membranes from mechanical breakage. The beads were treated with detergents and salt solutions of different ionic strengths and the eluates collected. Protochlorophyllide in the eluate was determined by fluorescence spectroscopy. Dot-blot tests were used to estimate the amount of released NADPH-protochlorophyllide oxidoreductase (E.C. 1.6.99.1.). Changes in ultrastructure of the treated prolamellar bodies were analysed. Release of both membrane constituents increased by treatment with detergents. With 0.2% (w/v) Triton X-100, 60% of the fluorescence from the immobilized prolamellar bodies was eluted within 30 min. Salt solutions with increasing ionic strength increased the release from 3 to 7%. The detergent treatment resulted in a complete (Triton X-100) or partial ([3-(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, CHAPS; 1-octyl β- d -glucopyranoside, octylglucoside) loss of the highly regular structure of the prolamellar bodies. Immunogold labelling of ultrathin sections revealed the absence of NADPH-protochlorophyllide oxidoreductase when the regular structure was dissolved into single membranes. The regular appearance of the prolamellar bodies was altered by treatment with 0.1 M CaCl₃ and 0.1 M KSCN, respectively, but not with 0.1 M KCl. Immunogold labelling showed that that enzyme was still present in the prolamellar bodies after these treatments. Despite the ultrastructural changes, the spectral properties were unchanged. Thus we conclude that NADPH-protochlorophyllide oxidoreductase is firmly attached to the prolamellar body membranes and that the regular ultrastructure of the prolamellar body is partly controlled by the ionic environment.     

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).     

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.     

Appearance of photochemical function in prothylakoids during plastid development

1. A method to separate the vesicles of prothylakoids from prolamellar body preparations obtained from etiolated and rapidly greening Avena laminae (0.25–4 h illumination) is described. The prothylakoid preparations were found to be free from contaminating prolamellar bodies but enriched prolamellar body preparations (enriched prolamellar body preparations) still contained some adhering prothylakoid material.2. Only existing β-carotene appears to be transferred from the prolamellar bodies to the prothylakoids during early development and this ceases when freshly synthesized β-carotene becomes available.3. Prolamellar body structures proper show no positive association of existing or developing photochemical activities; these are only to be found in the developing prothylakoids.4. Using methylviologen-linked electron transport-dependent oxygen consumption, Photosystem I activities may be detected with added diaminodurene within 15 min of illumination and within 30 min and 1 h with added tetramethylphenylenediamine and dichlorophenolindophenol, respectively.5. During the 2nd. and 3rd. h of greening, proton-pumping capability and later ATP formation increased in prothylakoids in the presence of diaminodurene.6. The first indications of Photosystem II activity using diphenylcarbazide as electron donor are shown at a similar time (2 h) with prothylakoids. The last photochemical activity to appear is the capacity to split water (3 h) and consequently the diphenylcarbazide activity diminishes to zero before 8 h of illumination have passed.7. The lack of effect of uncouplers such as NH⁺₄ prior to 2 h suggests that in spite of some proton-pumping ability there is the possibility of proton-leaky areas existing within prothylakoids. This. lack of a persistent proton gradient before 2 h of illumination may explain the different starting times of phenazine methosulfate- and diaminodurene-dependent photophosphorylation (0.25 and 2 h, respectively).     

Appearance of photochemical function in prothylakoids during plastid development.

1. A method to separate the vesicles of prothylakoids from prolamellar body preparations obtained from etiolated and rapidly greening Avena laminae (0.25--4 h illumination ) is described. The prothylakoid preparations were found to be free from contaminating prolamellar bodies but enriched prolamellar body preparations (enriched prolamellar body preparations) still contained some adhering prothylakoid material. 2. Only existing beta-carotene appears to be transferred from the prolamellar bodies to the prothylakoids during early development and this ceases when freshly synthesized beta-carotene becomes available. 3. Prolamellar body structures proper show no positive association of existing or developing photochemical activities; these are only to be found in the developing prothylakoids. 4. Using methylviologen-linked electron transport-dependent oxygen consumption, Photosystem I activities may be detected with added diaminodurene within 15 min of illumination and within 30 min and 1 h with added tetramethylphenylenediamine and dichlorophenolindophenol, respectively. 5. During the 2nd, and 3rd. h of greening, proton-pumping capability and later ATP formation increased in prothylakoids in the presence of diaminodurene. 6. The first indications of Photosystem II activity using diphenylcarbazide as electron donor are shown at a similar time (2 h) with prothylakoids. The last photochemical activity to appear is the capacity to split water (3 h) and consequently the diphenylcarbazide activity diminished to zero before 8 h of illumination have passed. 7. The lack of effect of uncouplers such as NH4+ prior to 2 h suggests that in spite of some proton-pumping ability there is the possibility of proton-leaky areas existing within prothylakoids. This lack of a persistent proton gradient before 2 h of illumination may explain the different starting times of phenazine methosulfate- and diaminodurene-dependent photophosphorylation (0.25 and 2 h, respectively).     

The ultrastructural development of plastids in leaves of maize plants exposed to continuous illumination

Summary Chloroplast differentiation in relation to increasing leaf age has been investigated in maize plants exposed to continuous illumination. In the young leaves the proplastids differentiate into chloroplasts containing well organized grana as well as prolamellar bodies. In the older leaves, while plastids differentiate, the prolamellar bodies are no longer detectable. Chloroplast ability to build up prolamellar bodies does not seems so much a light dependent process as it is affected by cell differentiation rate.Supported by a grant of C.N.R.     

Localization of NADPH-protochlorophyllide oxidoreductase in dark-grown wheat (Triticum aestivum) by immuno-electron microscopy before and after transformation of the prolamellar bodies

The localization of NADPH-protochlorophyllide oxidoreductase (PChlide reductase, EC 1.6.99.–) in dark-grown and in irradiated dark-grown leaves of wheat ( Triticum aestivum L. cv. Walde) was investigated by subjecting thin sections of Lowicryl K4M-embedded leaf pieces to a monospecific antiserum raised against PChlide reductase followed by protein A-gold. A well-preserved antigenicity of the tissue was achieved by polymerizing the resin under UV-light at low temperature. In dark-grown leaves PChlide reductase was found in prolamellar bodies only. In leaves irradiated for 30 min with white light PChlide reductase was found not only in the transformed prolamellar bodies but also to a large extent in connection with the prothylakoids. The localization of PChlide reductase is discussed in relation to fluorescence emission spectra of the dark-grown and greening leaves. We conclude that the light-dependent transformation of protochlorophyllide to chlorophyllide initiates a translocation of PChlide reductase from the prolamellar bodies to the prothylakoids.     

Characterization of prolamellar bodies and prothylakoids fractionated from wheat etioplasts

Prolamellar bodies and prothylakoids from etioplasts of wheat ( Triticum aestivum L. cv. Starke II, Weibull) were separated by sucrose density gradient centrifugation. Top-loaded and bottom-loaded sucrose gradients were compared. As a consequence of avoiding long time exposure of the membranes to low sucrose concentrations, separation in bottom-loaded gradients, as compared to separation in top-loaded gradients, resulted in a sharper and more narrow band of prothylakoids, and in better preservation of phototransformable protochlorophyllide, especially in the prothylakoids. In bottom-loaded gradients, the prothylakoids were found concentrated in a band at a density of 1.20 g'ml⁻¹. The prolamellar bodies were found at a density of 1.17 g'ml⁻¹. In top-loaded gradients the prothylakoids were found at a lower density than the prolamellar bodies. The prothylakoid fraction contained about 60% of the recovered protochlorophyllide and about 85% of the recovered protein. Absorption and fluorescence emission spectra revealed a higher amount of phototransformable protochlorophyllide, in relation to non-phototransformable, in the prolamellar body fraction than in the prothylakoid fraction. Polyacrylamide gel electrophoresis indicated a high proportion of protochlorophyllide reductase in the prolamellar bodies. Chloroplast ATPase (CF₁) was found predominantly in the prothylakoid fraction. Thus, our results strongly indicate the presence of phototransformable protochlorophyllide in the prolamellar bodies proper, while the main bulk of proteins are located in the prothylakoids.     

Lipid composition of prolamellar bodies and prothylakoids of wheat etioplasts

Prolamellar bodies and prothylakoids were fractionated from etioplasts of wheat ( Triticum aestivum L., cv. Starke II, Weibull) and characterized with emphasis on lipid composition. The two fractions contained the same lipid classes. Glycolipids (monogalactosyl diacylglycerol, digalactosyl diacylglycerol, and sulphoquinovosyl diacylglycerol) were the dominating complex lipids. Phospholipids (mainly phosphatidyl choline and phosphatidyl glycerol) constituted between 10 and 15 mol% of the total amounts of polar lipids. Free sterols and sterol esters were present in low amounts (ca 6 mol%). Saponins could not be detected. The contents of glycolipids and protochlorophyllide were higher in the prolamellar body fraction than in the prothylakoid fraction on a protein basis, as was the protochlorophyllide content on a glycolipid basis. The molar ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol was higher in the prolamellar body fraction (1.8) than in the prothylakoid fraction (1.2).
Since the same chemical constituents were found in the two membrane fractions we propose that the difference in ultrastructure between prolamellar bodies and prothylakoids is due to different relative amounts of lipids (glycolipids), protochlorophyllide, and proteins in the two membrane systems.     

Chlorophyll synthetase is latent in well preserved prolamellar bodies of etiolated wheat

Membrane fractions containing intact etioplasts, etioplast inner membranes, prolamellar bodies or prothylakoids from wheat ( Triticum aestivum L. cv. Walde) were assayed for chlorophyll synthetase activity. Calculated on a protein basis, the etioplast inner membrane fraction showed a higher activity than the intact etioplasts. The activity was higher in the prolamellar body fraction than in the prothylakoid fraction. However, when the fractions were incubated in isolation medium with 50% (w/w) sucrose and 0.3 m M NADPH, chlorophyll synthetase activity could not be detected in the prolamellar body fraction, while the prothylakoid fraction maintained a high activity. The spectral shift to a shorter wavelength of the newly formed endogenous chlorophyllide was very rapid in the prothylakoid fraction but slow in the prolamellar body fraction. The relation between the spectral shift of chlorophyllide and the esterification activity in the fractions is discussed. Even exogenous short-wavelength chlorophyllide could not be esterified in well preserved prolamellar bodies. This indicates that chlorophyll synthetase is present in an inactive state in the prolamellar body structure. A large-scale method for the synthesis of geranylgeranylpyrophosphate, one of the substrates of the chlorophyll synthetase reaction, is also presented.     

A Comparison between Prolamellar Bodies and Prothylakoid Membranes of Etioplasts of Dark-Grown Wheat Concerning Lipid and Polypeptide Composition

The aim of the present investigation was to find factors critical for the co-existence of prolamellar bodies and prothylakoids in etioplasts of wheat (Triticum aestivum L. cv Starke II). The lipid composition of the prolamellar body and prothylakoid fractions was qualitatively similar. However, the molar ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol was higher in the prolamellar body fraction (1.6 ± 0.1), as was the lipid content on a protein basis. Protochlorophyllide was present in both fractions. The dominating protein of the prolamellar body fraction was protochlorophyllide oxidoreductase. This protein was present also in prothylakoid fractions. The other major protein of the prothylakoid fraction was the coupling factor 1, subunit of the chloroplast ATPase. From the lipid and protein data, we conclude that prolamellar bodies are formed when monogalactosyl diacylglycerol is present in larger amounts than can be stabilized into planar bilayer prothylakoid membranes by lamellar lipids or proteins.     

Ultrastructural changes in isolated plastids

Summary Etio-chloroplasts were isolated from greening maize leaves and their ultrastructure was investigated immediately after isolation, as well as at intervals of several hours after their exposure to light or darkness. The following ultrastructural changes have been observed:In plastids isolated from etiolated leaves illuminated for 1–2 hours, the crystalline structure of the prolamellar bodies is partly restored during the isolation. In some plastids, regions with a regular, crystalline structure of densely packed tubules are even observed. The prolamellar bodies do not change further, either in light or in darkness.In young chloroplasts—i.e., in plastids isolated from etiolated leaves, illuminated for 6 or 15 hours—many prolamellar bodies, usually lying between the grana, appearde novo during isolation. These prolamellar bodies do not disappear in light either. They do not develop at all, however, if the isolation is performed at low temperature (4 °C).The results of the present paper indicate that in isolated etio-chloroplasts some tubular structures are newly formed, but that the conversion of this material into the thylakoids is not possible under the experimental conditions used.