On the nature of the two pathways in chlorophyll formation from protochlorophyllide

The kinetics of formation of esterified chlorophyll in etiolated barley (Hordeum vulgare L.) leaves after illumination with a single flash was studied. It was found that after partial (14–24%) and after full photoreduction of protochlorophyllide, the same quantity of esterified products appear during the first 5 s after the flash. The rest of formed chlorophyllide was esterified in a slow process during at least 30 min at 15 °C. The product of fast esterification can be correlated with ‘short-wavelength’ chlorophyll, characterized by a fluorescence emission peak at 673–675 nm. This is the only chlorophyll form detectable within 20 s after partial (14%) photoconversion, and it appears at the same time as the shoulder of the chlorophyll(ide) fluorescence after full photoconversion. The main product after full photoconversion shows a fluorescence at 689 nm shifting in darkness within 15 s to 693 nm and then within 30 min to 682 nm (Shibata shift). The slow esterification proceeds with similar kinetics as the Shibata shift. We propose that the fast esterification of only part of total chlorophyllide after full photoconversion of protochlorophyllide in etiolated leaves reflects the restricted capacity of the esterifying system. The slow esterification of the residual chlorophyllide may be time-limited by its release from protochlorophyllide oxidoreductase, by disaggregation of prolamellar bodies and by diffusion of tetraprenyl diphosphates towards chlorophyll synthase. This revised version was published online in June 2006 with corrections to the Cover Date.     

Correlation between chlorophyllide esterification, Shibata shift and regeneration of protochlorophyllide650 in flash-irradiated etiolated barley leaves

The pigments of etiolated leaves of barley ( Hordeum vulgare L.) were analysed during dark periods after flash illumination, and the results were compared with in vivo spectroscopy of the leaves. Pretreatment of the leaves with kinetin slightly stimulated and pretreatment with NaF and anaerobiosis inhibited the esterification of chlorophyllide a (Chlide) at 10–40 min after the flash, whereas the rapid esterification within 30 s after the flash remained unchanged. Irrespective of pretreatment, the amount of esterified pigment was, at any time, identical with the amount of pigment that had shifted its absorption from 684 to 672 nm (Shibata shift). Cycloheximide (CHI) had only a small inhibitory effect on esterification, but drastically inhibited the hydrogenation of geranylgeraniol to phytol, bound to Chlide. The regeneration of long-wavelength protochlorophyllide a (Pchlide650) was stimulated by kinetin and inhibited by CHI and NaF. During the rapid phase (0–30 s after the flash), the esterification was faster than the regeneration of Pchlide650, and this, in turn, was faster than the formation of photoactive Pchlide. The kinetics changed after pretreatment with 5-aminolaevulinic acid: regeneration of Pchlide650 was the fastest reaction and the Shibata shift preceded the esterification of Chlide. The results are discussed as pigment exchange reactions at NADPH:protochlorophyllide oxidoreductase (POR; EC 1.6.99.1).     

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.     

Photoreduction of Protochlorophyllide to Chlorophyllide in 2-d-old Dark-Grown Bean (Phaseolus vulgaris cv. Commodore) Leaves. Comparison with 10-d-old Dark-Grown (Etiolated) Leaves

Two-d-old leaves which do not contain prolamellar bodies synthesizeactive protochlorophyllide in darkness. When protochlorophyllideis photoreduced by one intense white flash, a main chlorophyllidespecies emitting at 690 nm is formed. After the photoreduction,the emission maximum is shifted to 675 nm within 5s. This resultsuggests that in young leaves, chlorophyllide formed after oneflash is quickly released from the active site of NADPH: protochlorophyllideoxidoreductase. This interpretation is strenghtened by time-resolvedfluorescence measurements at room temperature, showing that675 nm emitting chlorophyllide does not transfer excitationenergy to the 696 nm emitting chlorophyllide which is formedin very low amount. In 10-d-old bean leaves, the 690 nm chlorophyllideemitting species formed after one short flash undergoes thewellknown rapid and Shibata spectral shifts. The 675 nm emittingchlorophyllide appears only as a shoulder. At both ages, thefluorescence intensity of the active protochlorophyllide stronglydecreases during and after photoreduction, suggesting rapidmodifications in the close environment of the pigment. Key words: Bean, chlorophyllide, etioplast, proplastid, protochlorophyllide     

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.     

The Shibata Shift and the Transformation of Etioplasts to Chloroplasts in Wheat with Clomazone (FMC 57020) and Amiprophos-Methyl (Tokunol M)

The Shibata shift is a change in the absorption maximum of chlorophyllide from 684 to 672 nanometers that occurs within approximately 0.5 hour of phototransformation of protochlorophyllide to chlorophyllide. Two compounds, clomazone and amiprophos-methyl, which previously have been shown to inhibit the Shibata shift in vivo, were used to look for correlations between the Shibata shift and other processes that occur during etioplast to chloroplast transformation. Leaf sections from 6-day-old etiolated wheat seedlings (Triticum aestivum L. cv Walde) were treated with 0.5 millimolar clomazone or 0.1 millimolar amiprophos-methyl in darkness. In addition to the Shibata shift, the esterification of chlorophyllide to chlorophyll and the relocation of protochlorophyllide reductase from the prolamellar bodies to the developing thylakoids were inhibited by these treatments. Prolamellar body transformation did not appear to be affected by amiprophos-methyl and was only slightly affected by clomazone. The results indicate that: (a) there is a strong correlation between the occurrence of the Shibata shift and esterification activity; (b) transformation of the prolamellar bodies does not depend on the Shibata shift; and (c) the occurrence of the Shibata shift may be a prerequisite to the relocation of protochlorophyllide reductase from prolamellar bodies to thylakoids.     

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).     

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.     

FINE STRUCTURAL CHANGES IN PROPLASTIDS DURING PHOTODESTRUCTION OF PIGMENTS

Etiolated bean leaves supplied δ-amino-levulinic acid in the dark synthesize large amounts of protochlorophyllide which is not converted to chlorophyllide upon illumination of the leaves. The fine structure of the proplastids is not affected by the treatment. When leaves containing "inactive" protochlorophyllide are exposed to light of 700 ft-c for 3 hours, they lose practically all their green pigments. During this period large stacks of closed membrane structures are built up in the region of the prolamellar body. These lamellar structures remain even when no or only traces of pigment are left in the leaves. In untreated control leaves the pigment content remained constant during similar illumination and the structural changes in the plastids consisted of a rearrangement of the vesicles from the prolamellar bodies into strands dispersed through the stroma; lamellae and grana formation occurred later.     

Energy transfer from protochlorophyllide to chlorophyllide during photoconversion of etiolated bean holochrome

The photoconversion of protochlorophyllide to chlorophyllide in etiolated bean leaves or leaf extracts exhibits complicated kinetics that are neither simple first-order nor second-order with respect to the reactant. By comparing the chlorophyllide absorbance with the intensity of chlorophyllide fluorescence excited at wavelengths where both pigments absorb, we demonstrate that the kinetic complexity results from the transfer of electronic excitation from protochlorophyllide to chlorophyllide. Measurements of the polarization of chlorophyllide fluorescence indicate that efficient excitation transfer occurs at room temperature over pigment aggregates containing at least four molecules. The relative quantum efficiency of chlorophyllide-excited chlorophyllide fluorescence remains constant during photoconversion of holochrome or etioplast preparations. This result does not support the proposal of increasing exciton interaction between chlorophyllides during the course of photoconversion.     

Analysis of the subunit structure of protochlorophyllide holochrome by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

The subunit structures of protochlorophyllide holochrome (PCH) and chlorophyllide holochrome (CH) were studied by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. PCH from leaves of dark-grown (Phaseolus vulgaris var. red kidney) is a polymeric pigment-protein complex of approximately 600,000 daltons. It is composed of 12 to 14 polypeptides of 45,000 daltons, when examined prior to and immediately following photoconversion. The protochlorophyllide or chlorophyllide pigment molecules are associated with these polypeptides. Subsequent to photoconversion, the absorption maximum of newly formed chlorophyllide shifts from 678 nm to 674 nm upon standing in darkness. Following the 678 to 674 spectral shift, the chlorophyllide is associated with a polypeptide with a molecular weight of 16,000 daltons. In addition, sucrose gradient centrifugation of PCH and CH under nondenaturing conditions indicates that during the course of the dark spectroscopic shift, the 600,000 dalton CH undergoes dissociation into a small chlorophyllide protein. The dissociation of CH, the change in the molecular weight of the chlorophyllide polypeptide from 45,000 to 16,000 daltons, as well as the dark spectroscopic shift are temperature-dependent and blocked below 0 C. It was also found that each holochrome molecule of 600,000 daltons contains at least four protochlorophyllide pigment molecules.     

Esterification and Spectral Shifts of Chlorophyll(ide) in Wildtype and Mutant Seedlings Developed in Darkness

Spectral changes and esterification (presumably with phytol) of newly formed chlorophyllide a in dark-grown leaves of wildtype bean (Phaseolus vulgaris) and barley (Hordeum vulgare) and a number of chloroplast mutants in barley, were studied by spectrofluorimetry on leaves and on solvent extracts. The shift of the fluorescence emission maximum from 692–694 to 678 nm (excitation shift: 682–684 to 672 nm) and esterification of chlorophyllide a have a similar time course, and both processes are temperature dependent in a similar manner. After completion of the spectral shift and esterification, the fluorescence efficiency of chlorophyll a increases with a subsequent reaccumulation of protochlorophyllide. In leaves of mutants where the shift of fluorescence from 692 to 678 nm is lacking, esterification and the subsequent processes are also blocked. In leaves of mutants with a rapid shift of the fluorescence from 692 to 678 nm, or with direct photoconversion to chlorophyllide a with the fluorescence at 678 nm, esterification is also rapid. The results are interpreted as a sequence of molecular events involving a conformational relaxation of the chlorophyllide holochrome and a translocation of chlorophyll a to reaction centers of the photosystems.     

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.     

Chlorophyll Formation in Greening Bean Leaves during the Early Stages

In the evolution of the absorption spectrum of etiolated bean leaves (Phaseolus vulgaris L.) following illumination, a rapid photoconversion of 50% or more of the active protochlorophyllide at room temperature is followed by a shift of the chlorophyll(ide) absorption maximum: C₆₇₈→ →C₆₈₄→C_{672 nm}. Kinetic studies at 2 C and the absence of an isosbestic point provide evidence for an intermediate between C₆₇₈ and C₆₈₄. A dramatically different evolution is observed following the photoconversion of only 5 to 30% of the active protochlorophyllide at room temperature. C₆₇₂ appears within 30 seconds, and no subsequent dark shift occurs during the following 90 minutes. At 0 C, conversion of 5% of the active protochlorophyllide produces a new species, C₆₇₆, which converts progressively to C₆₇₂ within 10 minutes. We interpret the results in terms of two photochemical steps operating in series for the complete conversion of active protochlorophyllide. Furthermore, there appears to be competition between an irreversible, terminal dark shift and the second light reaction. We propose a scheme based on dimers of protochlorophyllide reduced stepwise to dimers of chlorophyllide in two successive light reactions. The intermediate mixed protochlorophyllide-chlorophyllide dimer absorbs at 676 nm and displays a much faster dissociation to monomers than does the chlorophyllide-chlorophyllide dimer.     

Aggregation of the 636 nm emitting monomeric protochlorophyllide form into flash-photoactive, oligomeric 644 and 655 nm emitting forms in vitro

Artificial formation of flash-photoactive oligomeric protochlorophyllide complexes was found in etiolated pea (Pisum sativum L. cv. Zsuzsi) epicotyl homogenates containing glycerol (40% v/v) and sucrose (40% m/v). The 77 K fluorescence emission spectra indicated that the ratio of the 644 and 655 nm emitting forms to the 636 nm form increased during 3 to 5-day incubation in the dark at -14 degrees C. Electron micrographs showed the presence of well-organized prolamellar bodies in the homogenates. The same phenomena were found when the homogenates were frozen into liquid nitrogen and thawed to room temperature in several cycles. Similar treatments of intact epicotyl pieces caused significant membrane destructions. In homogenates, the in vitro produced 644 and 655 nm emitting protochlorophyllide forms were flash-photoactive; the extent of phototransformation increased compared to that in native epicotyls. The newly appeared 692 nm chlorophyllide band showed a blue shift (similar to the Shibata shift in leaves), however this process took place only partially due to the effect of the isolation medium. These results prove that the in vitro accumulated 644 and 655 nm protochlorophyllide forms were produced from the flash-photoactive 636 nm emitting monomeric NADPH:protochlorophyllide oxidoreductase units via aggregation, in connection with structure stabilization properties of glycerol and sucrose.     

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.     

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.     

Developmental Physiology of Bean Leaf Plastids III. Tube Transformation and Protochlorophyll (ide) Photoconversion by a Flash Irradiation

A light flash of about 1 millisecond duration elicits tube transformation in paracrystalline prolamellar bodies as well as maximal protochlorophyll(ide) photoconversion in etiolated bean leaves (Phaseolus vulgaris L.). These findings support a more detailed hypothesis on the linkage between tube transformation and protochlorophyll(ide) photoconversion than has been offered previously.     

Tentoxin effects on infrastructure and greening of ivyleaf morningglory (Ipomoea hederacea var. hederacea) cotyledons

The effects of 20 μM tentoxin on mesophyll chloroplast ultra-structural development, chlorophyll organization and accumulation, and pigment transformations in cotyledons of dark-grown, 4-day-old ivyleaf morningglory [Ipomoea hederacea (L.) Jacq. var. hederacea]were monitored. After 6 h of white light (200 μEm^?2T.s^?1), many plastids of tentoxin-treated tissues contained prolamellar bodies or inconsistent internal membrane orientation in contrast to the uniform internal membrane orientation and absence of prolamellar bodies in controls. Grana stacking did not progress beyond three to four disc loculi in tentoxin-treatments, and fret membranes were usually discontinuous and reduced. Cylindrical or cupped grana appeared in many chloroplasts after 3 days of light, while other chloroplasts in which disruption was more pronounced had few grana except for remnants, but usually did possess vesicles or structures resembling prolamellar bodies. Tentoxin had no apparent effect on stroma density or plastoglobuli size and number. No starch grains appeared in any of the tentoxin treatments, whereas they appeared after 24 h in controls. Initial protochlorophyllide content and its photoconversion to chlorophyllide and subsequent Shibata shift were not affected by tentoxin. Chlorophyll accumulation rates in tentoxin-treated cotyledons were about 10% of control rates during the first 24 h of greening and about 20% of controls from 48 to 72 h of greening. Chlorophyll alb ratio and PSU size (total Chl/P700) were not significantly affected by tentoxin.     

Kinetics of photoconversion of protochlorophyllide 649 to chlorophyllide 676 at low temperature in etiolated cotyledons of Pharbitis nil.

The kinetics of the photoconversion of protochlorophyllide 649 to chlorophyllide 676 were studied spectrophotometrically over the temperature range of -15 -- -80 degrees C under light-saturating conditions in etiolated cotyledons of Pharbitis nil. Photoconversion obeyed the sum of two first-order kinetics over this low temperature range. Activation energies obtained from the rate constants were about 5000 cal; this suggests that these two processes may be physical processes not chemical reactions. The results indicate that photoconversion involves two main steps. One is the step dependent on both light intensity and temperature that has been well studied. The other, which is concerned in this study, is the step dependent on temperature only, which may be the requisite for photoconversion. This latter step seems to be related to the binding mode of protochlorophyllide to a holochrome protein or to conformational changes in the protochlorophyllide-holochrome.