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.     

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.     

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.     

Organization of protochlorophyllide oxidoreductase in prolamellar bodies isolated from etiolated carotenoid-deficient wheat leaves as revealed by fluorescence probes

Carotenoid importance for membrane organization of NADPH protochlorophyllide oxidoreductase (POR) was studied by comparing interaction of two membrane fluorescent probes with proteins in prolamellar bodies isolated from norflurazon-treated wheat plants (cdPLBs) to those isolated form plants with normal carotenoid amount (oPLBs). The tryptophan fluorescence quenching by 1-anilino-8-naphthalene sulfonate (attached to the surface of membrane lipid phase) and pyrene (situated deep into the fatty acid region of membrane lipids) was used to locate the position of POR molecules toward lipid phase, to analyze their supramolecular organization and the light-induced structural transitions. Our results showed that the pigment-protein complexes of cdPLBs were larger than those of oPLBs. Upon flash irradiation the aggregates of both types of PLB dissociated into smaller units but in cdPLBs this process was accompanied by reorientation of the POR molecules closer to the lipid surface and/or dissociation from the lipids. These results revealed that carotenoid deficiency led to a looser attachment of POR to the lipid phase and its early (in comparison with oPLBs) dissociation from the membranes during the light-induced transformation of cdPLBs. This might be one of the reasons for the inability of carotenoid-deficient plants to form functional plastids.     

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.     

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.     

Isoelectric focusing of pigment-protein complexes solubilized from non-irradiated and irradiated prolamellar bodies

Preparative isoelectric focusing was employed to compare the association of protochlorophyllide and chlorophyllide with the enzyme NADPH-protochlorophyllide oxidoreductase (PCR; EC 1.3.1.33). Photoactive protochlorophyllide-PCR complexes were solubilized with 1-O- n -octyl-β- d -glucopyranoside from non-irradiated prolamellar bodies of wheat ( Triticum aestivum ). Also, chlorophyllide-PCR complexes were solubilized from prolamellar bodies irradiated under conditions either preventing or favouring a spectral shift of chlorophyllide to shorter wavelengths. Independently of the treatment prior to the solubilization, the pigments and the PCR focused together at pHs of 4 to 5. The results indicate that protochlorophyllide-PCR complexes are conformationally similar to chlorophyllide-PCR complexes. The results support the hypothesis that the spectral shift, referred to as the Shibata shift, reflects a breaking-up of large chlorophyllide-PCR aggregates to smaller chlorophyllide-PCR units, rather than a dissociation of the chlorophyllide from the enzyme protein.     

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.     

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.     

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 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.     

Protochlorophyllide-NADP+ and protochlorophyllide- NADPH complexes and their regeneration after flash illumination in leaves and etioplast membranes of dark-grown wheat

The fast (1 min) regeneration process of the photoactive Pchlide forms after a light flash was studied in etiolated wheat leaves, and this process was simulated in vitro by incubating etioplast inner membranes of wheat with excess NADPH or NADP+. The 77 K fluorescence spectra were recorded after flash illumination, dark incubation and a subsequent flash illumination of the samples. A non-photoactive Pchlide form with an emission maximum at 650 nm was transiently detected in leaves during regeneration of a photoactive Pchlide form with an emission maximum at 654 nm. Gaussian deconvolution of fluorescence spectra of isolated membranes showed that this 650 nm form appeared in conditions of excess NADP+, as suggested in previous studies. Additionally a Pchlide form emitting at 638.5 nm was detected in the same conditions. The analysis of the spectra of leaves at different times after a flash indicated that these two non-photoactive forms are involved as intermediates in the regeneration of photoactive Pchlide. This regeneration is in correlation with the production of the Chlide form emitting at 676 nm. The results demonstrate that, in vivo, part of the NADPH:protochlorophyllide oxidoreductase is reloading with nonphotoactive Pchlide on a fast time-scale and that the 676 nm Chlide form is the released product of the phototransformation in this process.     

Association of the NADPH:protochlorophyllide oxidoreductase (POR) with isolated etioplast inner membranes from wheat

Membrane association of NADPH:protochlorophyllide oxidoreductase (POR, EC: 1.6.99.1) with isolated prolamellar bodies (PLBs) and prothylakoids (PTs) from wheat etioplasts was investigated. In vitro-expressed radiolabelled POR, with or without transit peptide, was used to characterize membrane association conditions. Proper association of POR with PLBs and PTs did not require the presequence, whereas NADPH and hydrolysable ATP were vital for the process. After treating the membranes with thermolysin, sodium hydroxide or carbonate, a firm attachment of the POR protein to the membrane was found. Although the PLBs and PTs differ significantly in their relative amount of POR in vivo, no major differences in POR association capacity could be observed between the two membrane systems when exogenous NADPH was added. Experiments run with only an endogenous NADPH source almost abolished association of POR with both PLBs and PTs. In addition, POR protein carrying a mutation in the putative nucleotide-binding site (ALA06) was unable to bind to the inner membranes in the presence of NADPH, which further demonstrates that the co-factor is essential for proper membrane association. POR protein carrying a mutation in the substrate-binding site (ALA24) showed less binding to the membranes as compared to the wild type. The results presented here introduce studies of a novel area of protein-membrane interaction, namely the association of proteins with a paracrystalline membrane structure, the PLB.     

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).     

Plastid development in germinating wheat (Triticum aestivum) is enhanced by gibberellic acid and delayed by gabaculine

Etioplast development and protochlorophyllide (Pchlide) accumulation was studied in wheat seedlings ( Triticum aestivum L. cv. Walde, Weibull) grown in darkness on gibberellic acid (GA₃), gabaculine (3-amino-2,3-dihydrobenzoic acid), or on a combination of the two. The results were compared with the features of seedlings grown on water only. GA₃ enhanced shoot growth and promoted etioplast development. A correlation was observed between the appearance of prolamellar bodies (PLBs) and of phototransformable Pchlide. Gabaculine, a known tetrapyrrole biosynthesis inhibitor, delayed growth, slowed down the rate of PLB formation and caused structural alterations of the etioplasts up to 48 h of germination. Gabaculine also delayed the formation of phototransformable Pchlide as well as overall Pchlide biosynthesis, as determined by low-temperature fluorescence emission in vivo. The spectral blue-shift of newly formed chlorophyllide (Chlide) was delayed in irradiated dark-grown gabaculine-grown seedlings, indicating an inhibited dissociation of Chlide and NADPH-Pchlide oxidoreductase (Pchlide reductase: EC 1.3.1.33). Thus there is a close correlation between accumulation of Pchlide and etioplast development, also under conditions when development is enhanced or delayed.     

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.     

Protochlorophyllide photo-transformation and chlorophyll(ide) formation in barley etioplast fractions

Localization of protochlorophyll(ide) (Pchlide) forms and chlorophyllide (Chlide) transformation process were studied by using comparative analyses of de-convoluted 77 K fluorescence spectra of barley etioplast stroma and different membrane fractions obtained by sucrose gradient centrifugation. Non-photoactive 633 nm Pchlide form was mainly located in the envelope-prothylakoid membrane mixture while the photoactive 657 nm Pchlide was dominant pigment in the prolamellar body membrane and in the soluble etioplast fraction (stroma). When these fractions were exposed to a saturating flash, conversion of photoactive Pchlide into 697 nm Chlide was preferential in the prolamellar body and in the stroma, while the 676 nm Chlide was dominant pigment form in the envelope-prothylakoid fraction. These spectral characteristics are considered to reflect molecular composition and organization of the pigment-protein complexes specific for each etioplast compartment.     

Chlorophyll synthesis in green leaves and isolated chloroplasts of barley (Hordeum vulgare)

The photoreduction of protochlorophyllide was studied in leaves and isolated chloroplasts of barley. Leaves of plants which had been preilluminated for varying lengths of time were incubated with [¹⁴C]-δ- aminolevulinic acid for 2 h in the dark. The subsequent photoreduction of [¹⁴C]-protochlorophyllide was analyzed by high performance liquid chromatography of pigments extracted from illuminated leaves and plastids. The plastids used in this study were isolated in the dark from leaves at the end of the 2 h labelling period. Three major results were obtained:

• 1

  The extent of protochlorophyllide reduction in vivo was rapidly reduced as a function of the preillumination period. In 24 h preilluminated plants only a small fraction of the radioactively labelled protochlorophyllide was reduced during the subsequent light period.
• 2

  The amount of NADPH-protochlorophyllide oxidoreductase (EC 1.6.99.-) present in plastids of fully-green plants was drastically reduced relative to levels in plastids of dark-grown plants as estimated by the methods of immunoblotting of plastid proteins and immunogold labelling of ultrathin sections of the leaf tissue.
• 3

  In etiolated plants light seemed to affect the reduction of protochlorophyllide directly through the excitation of protochlorophyllide. In fully green plants, however, light also affected chlorophyll formation indirectly by the supply of NADPH via photosynthetic electron transport.

     

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.