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Notes: Low-temperature fluorescence emission spectra of epicotyls of 6.5-day-old dark-grown seedlings of pea (Pisum sativum L.) showed the dominance of short-wavelength protoch lorophyllide forms with emission maxima at 629 and 636 nm, respectively. The presence of long-wavelength protochlorophyllide with emission maxima around 650 nm was just detectable. Accordingly, irradiation with millisecond flashes gave a minute formation of chlorophyllide. The chlorophyll(ide) formation varied along the epicotyl. Irradiation with continuous light for 1.5 h resulted in an evident accumulation of chlorophyll(ide) in the upper part of the epicotyl. Only small amounts accumulated in the middle section. The conversion of protochlorophyllide to chlorophyllide was temperature dependent and almost arrested at 0°C. The chlorophyll(ide) formed had one dominating fluorescence peak at 681 nm. Irradiation for 24 h gave almost 100 times more chlorophyll in the upper part of the epicotyl than in the lower part. Electron micrographs from the upper part of the epicotyl irradiated for 6 h showed plastids with several developing thylakoids, while the plastids in the lower part of the epicotyl had only a few thylakoids. The dominance of short-wavelength protochlorophyllide forms indicated the presence of protochlorophyllide not bound to the active site of NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33). The inability of the short-wavelength form to transform into chlorophyllide with flash light denotes a dislocation from the active site. The time and temperature dependence of the chlorophyll(ide) formation in continuous light indicates that a relocation is required of the short-wavelength protochlorophyllide before chlorophyllide formation can occur.

Notes: The effects of microtubule inhibitors on the spectral properties of leaves of wheat (Triticum aestivum L. cv. Walde) and on the presence of plastid microtubule–like structures (MTLS) during etioplast to chloroplast transformation were examined. Amiprophos-methyl (APM, 0.1 mM), fed to leaf sections of 7-day-old dark-grown wheat, reduced the ration of phototransformable to non-phototransformable proto-chlorophyllide (PChlide), decreased the rate of the Shibata shift, and inhibited chlorophyll accumulation and grana stacking. The spectral properties of isolated etioplasts were not affected by APM. Colchicine (10 mM), fed to leaf sections, inhibited greening but had no effect on the PChlide ratio or the Shibata shift. MTLS were still visible on electron micrographs after treatment with APM or colchicine at frequencies similar to controls. A third inhibitor, vinblastine, had no effect on the spectral properties of non-irradiated or irradiated etiolated leaves except at concentrations that produced visible tissue damage before the irradiation. The effects of APM and colchicine may reflect inhibitions of respiration and protein synthesis, respectively. It is concluded that MTLS are insensitive to microtubule inhibitors and thus are probably not composed of tubulin.

Notes: 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 mM 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.

Notes: 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.

Notes: Isolated prolamellar bodies from the etioplasts of dark-grown wheat (Triticum aestivum L. cv. Walde, Weibull) contain the enzyme NADPH-protochlorophyllide oxidoreductase. The organisation of this enzyme in a pigment-protein complex results in fluorescence emission maxima at 633 and 657 nm. Isolated prolamellar bodies stored in darkness for 24 or 48 h at 4°C (pH 7.2) in the presence of NADPH showed a fluorescence emission ratio 657/633 nm around 4 at −196°C. With acidic conditions this fluorescence ratio increased, with an optimum at pH 5.5. Such an increase was even more pronounced in the presence of ATP and NADPH with ratios up to 8, but was completely blocked when the sulfhydryl inhibitor, dithiobis-nitrobenzoic acid, was added. As shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis the amount of NADPH-protochlorophyllide oxidoreductase in the prolamellar bodies did not change during storage for 24 or 48 h.The total amount of protochlorophyllide measured in acetone extracts did not change significantly during storage for 48 h. The values were similar for storage at pH 7.2 and 5.5, but at lower pH (around 5) the pigment content decreased to a third.The most plausible explanation for the increase in fluorescence ratio is that low pH and ATP give rise to a change in conformation, which results in transformation of the short wavelength (633 nm) fluorescing protochlorophyllide to the long wavelength (657 nm) fluorescing form.

Notes: 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.

Notes: 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.

Notes: 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.

Notes: The relation between the different protochlorophyllide (PChlide) forms in isolated etioplast inner membranes was dependent on the concentration of sucrose and NADPH in the isolation media. Etioplasts were prepared from wheat (Triticum aestivum L. cv. Starke II, Weibull) by differential centrifugation. The etioplasts were freed of envelope and stroma and the etioplast inner membranes were exposed to a concentration series of sucrose. Fluorescence emission spectra revealed a positive correlation between the emission ratio 657/633 nm and the sucrose concentration in which the membranes were suspended. Addition of NADPH prevented the degradation of 657 nm emission caused by low sucrose concentrations. PChlide already altered to PChide628–632 could not re-form PChlide650–657 after the addition of NADPH in darkness. Prolamellar bodies and prothylakoids were separated in a bottom-loaded sucrose density gradient in the presence of NADPH. The dominating PChlide-protein complex in the prolamellar bodies was PClide650–657. Only minor amounts of PChlide628–632 were found in these membranes. The prothylakoids had a higher content of PChlide628–632, relative to PChlide650–657, than the prolamellar bodies, as judged from absorption and fluorescence spectra. After phototransformation the fluorescence emission at 633 nm increased relative to the emission from phototransformed PChlide indicating an efficient energy transfer between PChlide628–632 and PChlide650–657 before irradiation.

Notes: Light-induced alterations of isolated prolamellar bodies (PLBs) were studied in flash-irradiated suspensions of a PLB-enriched fraction and a mixed membrane fraction isolated from dark-grown seedlings of wheat (Triticum aestivum L. cv. Walde). The mixed membrane fraction consisted of PLB fragments and membrane vesicles originating from the prothylakoids. Ultrastructural and spectral properties, as well as pigment and protein composition of non-irradiated and of flash-irradiated suspensions were studied. The addition of 0.3 mM NADPH prevented spectral shifts towards shorter wavelengths in irradiated as well as in non-irradiated PLB-fractions. as measured by fluorescence emission at – 196°C. In non-irradiated PLB-fractions the amount of phototransformable protochlorophyllide (PChlide) as compared to nonphototransformable PChlide decreased when NADPH was not added. The emission maximum due to chlorophyll(ide) shifted from 696 nm to 680 um in the flashirradiated fractions where no NADPH was added. The amount of chlorophyllous pigments, as well as the amount of NADPH-protochlorophyllide oxidoreductase, decreased during the experimental period of 4 h in the suspensions without added NADPH. especially in the irradiated ones. The ultrastructure of the pelletable material in the different suspensions was analyzed by transmission and scanning electron microscopy. The non-irradiated PLBs appeared as cottonball-like structures in the scanning electron microscope. Without NADPH added more PLBs with an irregular tubular appearance were seen. After irradiation and storage for 1 h in darkness the surface was covered with vesicles. These vesicles were still present after 4 h. In the presence of NADPH no vesicle-formation occurred and the regular network of the PLBs was preserved also after an irradiation which caused transformation of PChlide to chlorophyllide. Thus, the regular structure seems to depend on an ample supply of NADPH. which in turn may be necessary to stabilize the pigment-protein complex in the lipid moiety of the PLB membranes. The formation of vesicles may thus be caused by a loss of this pigment-protein complex in suspensions with a low level of NADPH. The possible significance of an NADPH-dependence in vivo is discussed.