The polypeptide composition of highly purified prolamellar bodies and prothylakoids from wheat (Triticum aestivum) as revealed by silver staining

The inner membranes from wheat ( Triticum aestivum L. cv. Walde, Weibull) etioplasts were separated by density centrifugation. The etioplasts were broken by osmotic shock and the inner membranes were split by the sheering forces when pressed through a syringe needle. Membrane fractions representative of prolamellar bodies and prothylakoids, respectively, were achieved by separation on a 20–50% continuous sucrose density gradient followed by different purification procedures. The membrane contents of the isolated fractions were characterized by low temperature fluorescence spectra, sodium dodecyl sulphate polyacrylamide gel electrophoresis and electron micrographs. The prolamellar body and the prothylakoid fractions had a fluorescence emission ratio 657/633 nm of 18 and 0.9, respectively. The main part of the total amount of PChlide was found in the prolamellar body fraction. The electrophoretograms stained with Coomassie Blue showed the presence of mainly two polypeptides. The NADPH-protochlorophyllide oxidoreductase was the dominating polypeptide in the prolamellar body fraction, and the α and β subunits of the coupling factor 1 of chloroplast ATP synthase the dominating polypeptides in the prothylakoid fraction. Silver staining revealed at least 4 additional prominent bands with molecular weights of 86, 66, 34 and 28 kDa. The polypeptide composition of the prolamellar body is thus more complex than earlier judged after Coomassie Blue staining. The function of these polypeptides is unknown, but the knowledge of their presence is important in understanding the formation and function of the prolamellar body.     

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 With sufficient illumination some of the tubules are withdrawn and the crystalline prolamellar body transforms to a complex tangle of tubules, the reacted prolamellar body.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.     

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.     

CHLOROPLAST DEVELOPMENT IN THE GERMINATING SAFFLOWER (CARTHAMUS TINCTORIUS) COTYLEDON

Proplastids in the mesophyll cells of the cotyledons of mature seeds of safflower are irregular in shape and compressed in narrow corners between the large inclusion bodies, oil vacuoles and protein bodies. The proplastids contain a few irregular internal membranes. During dark germination, sheets or sac-like membranes are produced by the activity of the inner component of the proplastid envelope. These continuous membranes become reticulate and aggregate to the center of the proplastid to form after seven days' germination a quasicrystalline prolamellar body. The membranes are at first irregularly arranged and are of two sorts: those found in the interior of the developing prolamellar body, composed of laterally connected spherical profiles, and those on the periphery of the prolamellar body, which are continuous smooth sheets. The prolamellar body in these dark-germinated proplastids reverts after 3 hr of illumination to the irregularly arranged membranous structure of the 5-day dark germination stage. After 6 hr of illumination membranes grow from the prolamellar body forming concentric loops which, in cross section, appear as concentric circles. These membranes must be nested semi-spheroids. Small grana appear immediately on these looped membranes close to the prolamellar body. With further illumination additional grana develop along the looped membranes in close proximity to the slowly disappearing prolamellar body. Grana increase in size and number along the looped intergranal membranes. The prolamellar body disappears after 15 hr of illumination. The interconnecting fret membranes, sparse at the 15-hr stage, increase and after 24-hr illumination result in the typical grana fretwork system of the mature chloroplast. Membranes are continuously being produced by the invagination of the inner member of the plastid envelope.     

FORMATION OF THE PROLAMELLAR BODY IN 8-DAY,DARK-GROWN SEEDLINGS

The development of the prolamellar body in etioplasts of dark-grown seedlings of Phaseolus vulgaris is followed through the 8th day. From 2 to 6 days there is an increase in plastid size and starch content and synthesis of a system of porous lamellae which appear to arise, as such, from the inner component of the plastid envelope. From 6 to 8 days much of the starch disappears accompanied by rapid membrane synthesis resulting in an extensive prolamellar body. A model of the prolamellar body is discussed in which the basic structural unit is a six-pointed star with four tubules joining at each node. Observation of face views of the porous peripheral lamellae at their juncture with the prolamellar body suggests the origin of the prolamellar body by the continued contraction of the porous lamellae and the formation of interconnecting tubules between adjacent lamellae. The pores of the peripheral lamellae appear to correspond to the areas of stroma within each star module. Short lengths of membranes of individual peripheral lamellae fuse, forming short overlaps which resemble small, two-compartmented grana. It is postulated that this is the initial step in grana formation.     

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.     

Characterization of prolamellar bodies, from dark-grown seedlings of Scots pine, containing light- and NADPH-dependent protochlorophyllide oxidoreductase

Cotyledons of conifers have a light-independent pathway for chlorophyll biosynthesis. To investigate whether the prolamellar body of Scots pine ( Pinus sylveslris L.) is similar to the better known prolamellar body of wheat, etioplast membrane fractions were isolated from cotyledons of dark-grown Scots pine. Dark-grown cotyledons contained both chlorophyll and protochlorophyllide, 158 and 10 nmol (g fresh weight)'respectively, and had a chlorophyll a to b ratio of 4.2. The content of glyco- and phospholipids was 7.1 μmol (g fresh weight)¹. About 40 mol % of these lipids were the specific plastid lipids – monogalactosyl diacylglycerol. digalactosyl diacylglycerol and sulfoquinovosyl diacylglycerol in the relative amounts 50, 35 and 7 mol %. The mol ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol was 1.7. Low temperature fluorescence emission spectra of intact cotyledons and homogenate showed maxima at 633, 657, 686, 696 nm and a broad peak at 725–735 nm. The maxima at 633 and 657 nm represented different forms of protochlorophyllide and the other emission maxima represented chlorophyll protein complexes. The 657 nm form of protochlorophyllide was phototransformable both in vivo and in the isolated membranes. The phototransformable protochlorophyllide was substantially enriched in the prolamellar body fraction.
The specific activity of light dependent protochlorophyllide oxidoreductase in the prolamellar body fraction was found to be 2 nmol chlorophyllide formed [(mg protein)⁻¹ min⁻¹]. The molecular weight of the enzyme polypeptide was determined as 38 000 dalton with sodium dodecylsulphate-polyacrylamide gel electrophoresis.     

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.     

The role of chloroplast membranes in the location of chloroplast DNA during the greening ofPhaseolus vulgaris etioplasts

Summary The location of DNA containing nucleoids has been studied in greening bean (Phaseolus vulgaris L.) etioplasts using electron microscopy of thin sections and the staining of whole leaf cells with the fluorochrome DAPI. At 0 hours illumination a diffuse sphere of cpDNA surrounds most of the prolamellar body. It appears to be made up of a number of smaller nucleoids and can be asymmetric in location. The DNA appears to be attached to the outside of the prolamellar body and to prothylakoids on its periphery. With illumination the nucleoid takes on a clear ring-like shape around the prolamellar body. The maximum development of the ring-like nucleoid at 5 hours illumination is associated with the outward expansion of the prolamellar body and the outward growth of the prothylakoids. At 5 hours the electron transparent areas lie in between the prothylakoids radiating out from the prolamellar body. Between 5 hours and 15 hours observations are consistent with the growing thylakoids separating the nucleoids as the prolamellar body disappears and the chloroplast becomes more elongate. At 15 hours the fully differentiated chloroplast has discrete nucleoids distributed throughout the chloroplast with evidence of thylakoid attachment. This is the SN (scattered nucleoid) distribution ofKuroiwa et al. (1981) and is also evident in 24 hours and 48 hours chloroplasts which have more thylakoids per granum. The changes in nucleoid location occur without significant changes in DNA levels per plastid, and there is no evidence of DNA or plastid replication.The observations indicate that cpDNA partitioning in dividing SN-type chloroplasts could be achieved by thylakoid growth and effectively accomplish DNA segregation, contrasting with envelope growth segregating nucleoids in PS-type (peripheral scattered nucleoids) chloroplasts. The influence of plastid development on nucleoid location is discussed.     

On the periodic minimal surface structure of the plant prolamellar body

An evaluation of minimal surface structures formed in lipid-water systems and in lipid-protein-water systems in relation to electron microscopic data on the prolamellar body are reported. It is suggested that the characteristic square and hexagonal patterns seen in electron micrographs of prolamellar bodies constitute minimal surfaces of the P- and D-type, respectively. The existence of the G- and the H-surface in the membrane systems of prolamellar bodies is discussed.     

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.     

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.     

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     

CHLOROPLAST DEVELOPMENT IN NICOTIANA TABACUM ‘MARYLAND MAMMOTH’

The development of chloroplasts in light-grown and in previously etiolated tissues of tobacco has been studied. A single membrane-bound body is found in the developing plastids of both light- and dark-grown tissue. The contents of the body appear homogeneous, becoming progressively granular as the chloroplast develops. In the mature chloroplast the body contains a fibrillar network resembling strands shown to be DNA by other workers. The prolamellar body persists even in moderately well developed chloroplasts in light-grown plants. Frequently the prolamellar body is connected to the membrane-bound body as well as to the grana. Relatively mature chloroplasts are seen to divide in this tissue. The membrane-bound body may have a role in the formation of lamellae, but the nature of its contents is yet to be determined.     

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.     

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).     

A comparison of prolamellar bodies from wheat, Scots pine and Jeffrey pine. Pigment spectra and properties of protochlorophyllide oxidoreductase

Inner etioplast membrane fractions were isolated from wheat ( Triticum aestivum L. cv. Starkell), Scots pine ( Pinus sylvestris L.) and Jeffrey pine ( Pinus jeffreyi Murr), in order to investigate whether cotyledons of dark-grown conifers have protochlorophyllide associated to protochlorophyllide oxidoreductase (EC 1.6.99.–) in the pro-lamellar body in the same way as angiosperms. Protochlorophyllide was found to be present in dark-grown seedlings of Scots pine and Jeffrey pine to the same extent as in dark-grown wheat, 10–15.8 nmol (g fresh weight)⁻¹. Fluorescence emission spectra at 77 K showed accumulation of protochlorophyllide with emission maximum at 657 nm in the prolamellar body fractions of the three species studied. Also the light- and NADPH-dependent activity of protochlorophyllide oxidoreductase was consistently localized in the prolamellar body fractions. The three prolamellar body fractions were dominated by the same polypeptide. Its molecular weight was estimated to be 38 000 by sodium dodecylsulphate polyacrylamide gel electrophoresis.     

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.     

On the aggregational states of protochlorophyllide and its protein complexes in wheat etioplasts

The inner membranes from wheat ( Triticum aestivum L. cv. Walde) etioplasts were separated into membrane fractions representative of prolamellar bodies and prothylakoids by differential and gradient centrifugations. The isolated fractions were characterized by absorption-, low-temperature fluorescence-, and circular dichroism (CD) spectroscopy, by high performancy liquid chromatography and by sodium dodecyl sulphate polyacrylamide gel electrophoresis.
The prolamellar body fraction was enriched in NADPH-protochlorophyllide oxidoreductase (E.C. 1.6.99.1), and in protochlorophyllide showing an absorption maximum at 650 nm and a fluorescence emission maximum at 657 nm. Esterified protochlorophyllide was mainly found in the prothylakoid fraction. The carotenoid content was qualitatively the same in the two fractions. On a protein basis the carotenoid content was about three times higher in the prolamellar body fraction than in the prothylakoid fraction. The CD spectra of the membrane fractions showed a CD couplet with a positive band at 655 nm, a zero crossing at 643–644 nm and a negative band at 623–636 nm. These results differ from earlier CD measurements on protochlorophyllide holochrome preparations. The results support the interpretation that protochlorophyllide is present as large aggregates in combination with NADPH and NADPH-protochlorophyllide oxidoreductase in the prolamellar bodies.