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.     

Spectral forms of protochlorophyllide in prolamellar bodies and prothylakoids fractionated from wheat etioplasts

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 PChide_628–632 could not re-form PChlide_650–657 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 PClide_650–657. Only minor amounts of PChlide_628–632 were found in these membranes. The prothylakoids had a higher content of PChlide_628–632, relative to PChlide_650–657, 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 PChlide_628–632 and PChlide_650–657 before irradiation.     

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.     

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.     

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.     

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.     

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.     

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.     

Separation and Characterization of Prolamellar Bodies and Prothylakoids from Squash Etioplasts

Intact etioplasts of squash cotyledons, which had been preparedby Percoll density gradient centrifugation, were ruptured hypotonicallyin the presence of deoxyribonuclease I then fractionated intoprolamellar bodies and prothylakoids by differential and Percolldensity gradient centrifugations. This procedure provided ahighly purified prolamellar body fraction that was composedmainly of a 36,000-dalton protein. This protein was identifiedas NADPH:protochIorophyllideoxidoreductase [Ikeuchi and Murakami(1982) Plant & Cell Physiol. 23: 1089]. The fraction alsohad a high content of protochlorophyllide that absorbed at 648nm and its NADPH:protochlorophyllide oxidoreductase had highactivity. When the fraction was illuminated, a chlorophyllidethat absorbed at 684–685 nm formed. In contrast, the prothylakoid fraction, which showed high activityfor the Ca²⁺-dependent ATPase of coupling factor 1, containedonly a small amount of the 36,000-dalton protein and showedvery low NADPH:protochlorophyllide oxidoreductase activity.The protochlorophyllide content of this fraction also was low,and the ratio of protochlorophyll to protochlorophyll(ide) high.The absorption peak in the prothylakoids was at 633–635nm, and after a brief illumination a chlorophyllide that absorbedat 672–673 nm formed. These results indicate that thephotoactive protochlorophyllide-NADPHreductase complex in etioplastsis concentrated in the prolamellar body and that the physicalstate of protochlorophyll(ide) in the prolamellar body differsfrom that of the prothylakoid. (Received April 28, 1982; Accepted November 15, 1982)     

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.     

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.     

ADP/ATP and protein phosphorylation dependence of phototransformable protochlorophyllide in isolated etioplast membranes

The effects of modulated ADP/ATP and NADPH/NADP⁺ ratios, and of protein kinase inhibitors, on the in vitro reformation of phototransformable protochlorophyllide, i.e. the aggregated ternary complexes between NADPH, protochlorophyllide, and NADPH-protochlorophyllide oxidoreductase (POR, EC 1.3.1.33), in etioplast membranes isolated from dark-grown wheat (Triticum aestivum) were investigated. Low temperature fluorescence emission spectra (–196 °C) were used to determine the state of the pigments. The presence of spectral intermediates of protochlorophyllide and the reformation of phototransformable protochlorophyllide were reduced at high ATP, but favoured by high ADP. Increased ADP level partly prevented the chlorophyllide blue-shift. The protein kinase inhibitor K252a prevented reformation of phototransformable protochlorophyllide without showing any effect on the chlorophyllide blue-shift. Addition of NADPH did not overcome the inhibition. The results indicate that protein phosphorylation plays a role in the conversion of the non-phototransformable protochlorophyllide to POR-associated phototransformable protochlorophyllide. The possible presence of a plastid ADP-dependent kinase, the activity of which favours the formation of PLBs, is discussed. Reversible protein phosphorylation is suggested as a regulatory mechanism in the prolamellar body formation and its light-dependent dispersal by affecting the membrane association of POR. By the presence of a high concentration of phototransformable protochlorophyllide, prolamellar bodies can act as light sensors for plastid development. The modulation of plastid protein kinase and protein phosphatase activities by the NADPH/NADP⁺ ratio is suggested. This revised version was published online in June 2006 with corrections to the Cover Date.     

Breakdown of photosynthetic pigments and lipids in spinach leaves with ozone fumigation: Role of active oxygens

Dark-grown wheat leaves ( Triticum L. cv. Starke II Weibull) were illuminated repeatedly with light flashes giving partial phototransformation of protochlorophyllide to chlorophyllide. After short flashes (e.g. 15 ms red light, 250 W m⁻²), transforming only a minor part of the protochlorophyllide present, the first more stable chlorophyll(ide) measured ca 15 s after the phototransformation had its absorption maximum in the red around 672 nm. It stayed there during the following 30 min in darkness. After longer flashes (e.g. 125 ms), transforming a larger portion of the protochlorophyllide, the chlorophyll(ide) formed had its maximum absorption more towards 684 nm and shifted to 672 nm during a subsequent period in darkness. Thus, in this case a Shibata shift took place.
The conditions which produce the "stable" 672 nm form, without a Shibata shift, are discussed. The presence of large amounts of non-transformed protochlorophyllide remaining after the phototransformation seems to be important. Under such conditions it is possible that the Shibata shift is completed within a very short time.
Also the possible existence of two kinds of phototransformable protochlorophyllide is discussed. According to this idea one of the two protochlorophyllide forms produces a chlorophyllide absorbing at 672 nm shortly after phototransformation without having passed a Shibata shift. The other protochlorophyllide form photo-transforms to a chlorophyllide which proceeds through the Shibata shift.     

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 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     

The regular ultrastructure of isolated prolamellar bodies depends on the presence of membrane-bound NADPH-protochlorophyllide oxidoreductase

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.     

Mutagenic analysis of putative domain II and surface residues in mosquitocidal Bacillus thuringiensis Cry19Aa toxin

The growing resistance against antibiotics demands the search for alternative treatment strategies. Photodynamic therapy is a promising candidate. The natural intermediate of chlorophyll biosynthesis, protochlorophyllide, was produced, purified and tested as a novel photosensitizer for the inactivation of five model organisms including Staphylococcus aureus, Listeria monocytogenes and Yersinia pseudotuberculosis , all responsible for serious clinical infections. When microorganisms were exposed to white light from a tungsten filament lamp (0.1 mW cm⁻²), Gram-positive S. aureus, L. monocytogenes and Bacillus subtilis were photochemically inactivated at concentrations of 0.5 mg L⁻¹ protochlorophyllide. Transmission electron microscopy revealed a disordered septum formation during cell division and the partial loss of the cytoplasmic cell contents. Gram-negative Y. pseudotuberculosis and Escherichia coli were found to be insensitive to protochlorophyllide treatment due to the permeability barrier of the outer membrane. However, the two bacteria were rendered susceptible to eradication by protochlorophyllide (10 mg L⁻¹) upon addition of polymyxin B nonapeptide at 50 and 20 mg L⁻¹, respectively. The release of DNA and a detrimental rearrangement of the cytoplasm were observed.     

Comparative investigation of the appearance of primary chlorophyllide forms in etiolated leaves,prolamellar bodies and prothylakoids

A comparative investigation of the first steps of chlorophyllide formation from protochlorophyllide in the etiolated leaves, prolamellar bodies and prothylakoids was performed by measuring fluorescence emission spectra. It was shown that the formation of the first fluorescent chlorophyllide forms from non-fluorescent intermediates is a complex process including several dark reactions with different temperature dependencies. When the temperature of samples which had been illuminated at 77 K was increased to 190 K, four primary chlorophyllide forms were found by Gaussian deconvolution of the 77 K emission spectra. They had fluorescence emission maxima at 690, 696, 684 and 706 nm, respectively. Two new forms of chlorophyllide - Chlide690 and Chlide706 - were found in addition to the major known forms. A prolonged exposure to 190 K as well as rise of the temperature to 253 K led to a disappearance of Chlide690. The fate of this form is not clear. Chlide696 and Chlide706 were transformed into Chld673 and Chld684, respectively, during the prolonged dark exposure at 253 K. The existence of two pathways of native short wavelength chlorophyllide forms formation was proposed with different temperature dependencies.