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 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.     

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.     

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.     

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.     

Chlorophyll synthetase activity is relocated from transforming prolamellar bodies to developing thylakoids during irradiation of dark-grown wheat

Analyses of the esterification of newly formed chlorophyllide in irradiated dark-grown leaves of wheat ( Triticum aestivum L. cv. Kosack) suggest a translocation of chlorophyll synthetase activity from transforming prolamellar bodies to developing thylakoids. We have fractionated plastid inner membranes from dark-grown leaves and from leaves irradiated for 5, 10, or 20 min and compared the in vitro esterification of chlorophyllide in two fractions, corresponding (in density) to the prolamellar body and the prothylakoid fraction of dark-grown leaves. The relative amounts of chlorophyllide, and total protein, as well as the specific esterification activity, increased with irradiation time in the prothylakoid fraction. The esterification of chlorophyllide seems to depend on a transformation of the prolamellar body structure. The results are discussed also in relation to other events initiated by irradiation, such as the Shibata-shift and the altered distribution of NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33).     

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.     

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.     

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     

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.     

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 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.     

Variation of plastid types in spinach

Summary During growth in the light the plastids of cultured leaf discs of spinach divide, increase in size, and differentiate in a similar manner to those in intact leaves. By contrast when l'eaf discs are grown in the dark prolamellar bodies begin to develop in partially differentiated chloroplasts within 2 hours. After 7 days growth in the dark the plastids contain many vesicles which appear to arise from swelling of thylakoids. These vesicles often contain large crystals. When dark grown discs are illuminated they regreen and fully differentiated chloroplasts are reformed.Proplastids are found in the stem apex of whole plants; these develop into the partially differentiated chloroplasts of young leaves, which divide and increase in size to form the fully differentiated chloroplasts of mature leaves. The cotyledons of dormant seeds contain proplastids; these develop into amyloplasts during germination and after exposure to white light differentiate into chloroplasts.     

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.     

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.     

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.     

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.     

Prolamellar-body structure,composition of molecular species and amount of galactolipids in etiolated,greening and reetiolated primary leaves of oat,wheat and rye

The greening and reetiolation process of etiolated leaves of oat, wheat and rye, possessing different types of prolamellar bodies (PLBs), was observed by electron microscopy. Oat is known to possess unusual crystalline PLBs (so-called narrow type). Rye and what, which normally show PLBs with more loosely packed tubules (wide type) during etiolation, exhibited PLBs of the narrow type after illumination and subsequent reincubation in the dark (=reetiolation). Thus the reetiolated PLBs of wheat and rye did not differ from etiolated or reetiolated oat PLBs. In parallel with the microscopic analysis, intact leaves of all developmental stages were analysed for their galactolipid content and composition of molecular species using a newly developed high-performance liquid chromatography procedure. When oat, wheat and rye were compared, differences in the molecular species and the molar ratio of the two galactolipids monogalactosyldiacylglyceride (MGDG) and digalactosyldiacylglyceride (DGDG) were found. However, no parameter showed a correlation with PLB construction, disintegration or reconstruction. The results presented in this paper are not consistent with the hypothesis that the molar ratio of MGDG/DGDG is responsible for the tubular structure of prolamellar bodies in etioplasts.Abbreviations DGDG digalactosyl diacylglyceride - HPLC high-performance liquid chromatograpyh - MGDG monogalactosyl diacylglyceride - PLB prolamellar body     

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.     

The localization of magnesium-protoporphyrin and protochlorophyllide in separated prolamellar bodies and prothylakoids of wheat treated with 8-hydroxyquinoline and δ-aminolevulinic acid

Dark-grown leaves of wheat ( Triticum aestivum L. cv. Starke II, Weibull) were treated in darkness with 8-hydroxyquinoline and δ-aminolevulinic acid in order to accumulate magnesium-protoporphyrin and/or magnesium-protoporphyrin monomethylester. Prolamellar bodies and prothylakoids were separated from the treated leaves by sucrose density gradient centrifugation. About 90% of the recovered magnesium-protoporphyrin/magnesium-protoporphyrin monomethylester and about 75% of the recovered protochlorophyllide was found in the prothlakoid fraction. The significance of the distribution pattern of the chlorophyll precursors between the prolamellar bodies and the prothylakoids is discussed. The results indicate that the prothylakoids are the site for synthesis of membrane-bound chlorophyll precursors and that phototransformable protochlorophyllide is a constituent of prolamellar bodies as well as of prothylakoids.