A Reversible Conversion of Phototransformable Protochlorophyll(ide)(656) to Photoinactive Protochlorophyll(ide)(656) by Hydrogen Sulfide in Etiolated Bean Leaves

The relationship of phototransformable protochlorophyll-(ide) to photoinactive protochlorophyll(ide) has been studied in the primary leaves of 7- to 9-day-old dark-grown bean (Phaseolus vulgaris L. var. Red Kidney) seedlings. Subjecting the leaves to an atmosphere of H₂S causes an immediate loss of phototransformable protochlorophyll(ide)₆₅₀ and a simultaneous increase in photoinactive protochlorophyll(ide)₆₃₃. When such leaves are returned to air or N₂, the absorbance at 650 nm increases, whereas the absorbance at 633 nm decreases and photoactivity is restored. The reversion of protochlorophyll-(ide)₆₃₃ to protochlorophyll(ide)₆₅₀ is one-half complete in 3 minutes at 22 C in 8-day-old leaves. Ninety-five per cent recovery of protochlorophyll(ide)₆₅₀ is obtained when exposure to H₂S is less than 3 minutes in duration; longer periods reduce the reversion capacity proportionately. The leaves are relatively undamaged by brief exposures to H₂S, as judged by electron microscopy and by their ability to synthesize chlorophyll under continuous illumination. Hydrogen sulfide has no immediate effect upon the absorption properties of a partially purified preparation of the protochlorophyll(ide) holochrome, an etioplast suspension, or leaves subjected to freezing and thawing. Compounds such as HCN and HN₃ cause an irreversible conversion of protochlorophyll(ide)₆₅₀ to protochlorophyll(ide)₆₃₃ with total loss of photoactivity. Sulfhydryl agents, such as β-mercaptoethanol and cysteine, cause a slow, irreversible transformation of the photoactive pigment to the photoinactive form and inhibit the ability of the leaves to synthesize chlorophyll under continuous illumination. The results obtained suggest that H₂S may alter the interaction between the source of hydrogens on the protein moiety of the holochrome and the chromophore in vivo by reducing a disulfide bond in the protein, thereby causing a reversible conformational change in the complex.     

Relationship between Photoconvertible and Nonphotoconvertible Protochlorophyllides

Two forms of protochlorophyllide are found in dark-grown bean (Phaseolus vulgaris, var. Black Velentine) leaves, one (protochlorophyllide₆₅₀) which is directly photoconvertible to chlorophyllide and another (protochlorophyllide₆₃₂) which is not. Dark-grown leaves placed in solutions of δ-aminolevulinic acid accumulate protochlorophyllide₆₃₂. Protochlorophyllide₆₅₀ and protochlorophyllide₆₃₂ can be partially separated on sucrose density gradients. A nitrogen atmosphere blocks chlorophyll synthesis in light or the regeneration of protochlorophyllide₆₅₀ in the dark, even in the presence of excess δ-aminolevulinic acid, except when a stockpile of protochlorophyllide₆₃₂ is present in the leaf. Under the latter conditions chlorophyll synthesis or protochlorophyllide₆₅₀ regeneration is accompanied by a decrease in protochlorophyllide₆₃₂. These experiments suggest that protochlorophyllide₆₃₂ may be converted to protochlorophyllide₆₅₀.     

Rapid regeneration of protochlorophyllide(650)

The rate of regeneration of protochlorophyllide₆₅₀ was examined spectrophotometrically after a saturating light flash using 8- to 9-day-old dark-grown bean leaves. The regeneration occurred to the extent of 15% with a half rise time of about 20 seconds. Feeding δ-aminolevulinic acid to the excised leaves in the dark increased protochlorophyllides₆₃₅ but not the absorption at 650 nanometers, suggesting that the holochrome was normally saturated with protochlorophyllide and that the holochrome protein was not controlled by the level of protochlorophyllide. After a light flash, the excess protochlorophyllide, formed from exogenous δ-aminolevulinic acid, readily combined to regenerate the 650 nanometer absorbing species; the regeneration occurred to the extent of 60 to 80% with a half rise time of about 50 seconds. Regeneration was blocked at 0°, suggesting that there was some enzymic process required for regeneration, possibly the formation of a reductant component of the protochlorophyllides₆₅₀ holochrome.     

The Influence of Varying Light Intensities on the Photo-transformation of Protochlorophyllide636 in Dark Grown Wheat Leaves Treated with δ-Aminolevulinic Acid

Leaves treated with δ-aminoievulinic acid accumulate protochlorophyllide₆₃₆ in large amounts. Due to a continuous conversion of protochlorophyllide₆₃₆ (nonphototransformable) into protochlorophyllide₆₅₀ (phototransformable) in weak red light, the photoreduction of protochlorophyllide to chlorophyllide can proceed for at least 20 minutes and results in a chlorophyllide content of the leaves three times higher than that in untreated leaves. The half time for this chlorophyllide accumulation is 55 seconds. A photodestruction of the pigments takes place at high light intensities or if the content of protochlorophyllide₆₃₆ is high. The conversion of protochlorophyllide₆₃₆ to chlorophyllide is dependent on the light intensity used for phototransformation of protochlorophyllide₅₅₀ The conversion of PChlide₆₄₆ was not limiting for chlorophyllide formation within the range of the light intensity used. The extrapolation of a double reciprocal plot of chlorophyllide formation, rate versus light intensity gives a maximal value of 8.7 μg chlorophyllide per g fresh weight and min. The conversion of protochlorophyllide₃₆₃ to protochlorophyllide₆₅₀ is believed to depend on the available sites of an apophotoenzyme.     

A Short-lived Intermediate Form in the in Vivo Conversion of Protochlorophyllide 650 to Chlorophyllide 684

When dark-grown leaves of Phaseolus vulgaris, Hordeum vulgare, Zea mays and Pisum sativum were irradiated for 3 sec at 2° the first product of protochlorophyllide 650 conversion had an absorption maximum at 678 nm. This form was then converted in a dark reaction to chlorophyllide 684, the form generally observed and regarded as the in vivo product of the photoreaction. The dark conversion at 2° was complete in 6 to 10 min in the various plants. The time course of the dark reaction was followed at 690 nm near the maximum of the difference spectrum for the conversion. There was a constant relationship between the initial amount of chlorophyllide 678 and the final amount of chlorophyllide 684. The rates of the dark reaction at 2° varied 3-fold among the plants treated. The reaction was not first order. At 25° the reaction followed at 690 nm was complete in 20 to 60 sec. Q₁₀'s varied from 2.8 to 3.7 between 2° and 25°. Phytochrome absorbancy changes were shown to be too low to interfere with these measurements except in pea leaves. In a subsequent stage of greening newly regenerated protochlorophyllide went through the same sequence upon photoconversion. Chlorophyllide 678 probably corresponds to the product formed in vitro from the protochlorophyllide holochrome. The dark reaction appears to represent the first interaction between the photoconverted holochrome and other elements of the proplastid. The lack of this dark reaction could also account for the spectral properties of certain albino mutants.     

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     

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).     

In Vivo Measurements of the Photo-Oxidation of Chlorophyll Pigments in Dark Grown Wheat Leaves after Treatment with δ-Aminolevulinic Acid

Dark-grown leaves of wheat fed with δ-aminolevulinic acid accumulate protochlorophyllide₆₃₆ in excess. After the leaves had been illuminated with high intensity red light (154 W × m^?2) for half a minute, a treatment which blocks the phototrans-formation protochlorophyllide chlorophyllide, the sensitivity of chlorophyllide and protochlorophyllide to light was examined. The decrease in pigment content, caused by photo-oxidation was found to be very close to a second order reaction. The second order “rate constant” for decrease in absorbance was found to be eight times greater for the formed chlorophyllide than for protochlorophyllide. The light intensity dependence of the decomposition was found to be linear within the intensity range used (E= 25 – 154 W × m^?2). In samples in which the pigments had been heat denatured, it was possible to photodecompose the chlorophyllide without affecting the protochlorophyllide. The results are discussed in connection with the theory of a photodynamic action involving oxygen in the singlet state (¹ΔO₂).     

Measurement and Identification of NADPH:Protochlorophyllide Oxidoreductase Solubilized with Triton X-100 from Etioplast Membranes of Squash Cotyledons

Etioplast membranes were solubilized with 1 mM Triton X-100in the presence of excess NADPH and protochlorophyllide to isolateNADPH:protochlorophyllide oxidoreductase. The activity of thisreductase was assayed as the formation of chlorophyllide bya single flash and was equivalent to the amount of photoactiveprotochlorophyllide-NADPH-enzyme complex present before illumination.The rate of regeneration of the phtoactive complex was estimatedfrom the time course of chlorophyllide formation under a longflash. The highest rate was 651 nmol chlorophyllide formed min^–1mg^–1 protein. Photoconversion of protochlorophyllide to chlorophyllide andregeneration of the photoactive protochlorophyllide-NADPH-enzymecomplex were not much affected in a pH range from 6 to 8, atleast for several minutes. The apparent dissociation constantsof the photoactive complex were 0.039 µM for protochlorophyllideand 0.44 µM for NADPH. Triton-solubilized etioplast membraneswere fractionated by glycerol density gradient centrifugationto isolate the NADPH:protochlorophyllide oxidoreductase. Mostof the 36,000-dalton protein, the major protein of the prolamellarbody was recovered in the fraction enriched by NADPH:protochlorophyllideoxidoreductase and protochlorophyllide. Protochlorophyll andcarotenoids were present in different fractions. This is evidencethat the 36,000-dalton protein has the activity of NADPH:protochlorophyllideoxidoreductase and specifically binds protochlorophyllide. Themost highly purified fraction of the enzyme showed an activityof 7.8 nmol chiorophyllide formed flash^–1 mg^–1 proteinand bound 11.1 nmol protochlorophyllide mg^–1 of protein. (Received April 28, 1982; Accepted June 29, 1982)     

Protochlorophyllide transformations and chlorophyll accumulation in epicotyls of pea (Pisum sativum)

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.     

Photochemical reaction of chlorophyll biosynthesis at 4.2 K

Phototransformation of protochlorophyllide in etiolated leaves was shown to take place at 4.2 K. Upon illumination of the sample a photoactive form PChlide₆₅₀ was transformed into a non-fluorescent intermediate which was then transformed into fluorescent forms of chlorophyllide (Chlide) upon increase of temperature in the dark. Reported transformations are similar to those observed under illumination at 77 K.     

The Conversion of Protochlorophyllide636 to Protochlorophyllide630 in Leaves Treated with δ-Aminolevulinic Acid

Absorbancy changes in dark-grown, excised wheal leaves fed with δ-aminolevulinic acid are measured in vivo. The treatment with σ-aminolevulinic acid caused accumulation of protochlorophyllide, absorbing at 636 nm. After flashlight this form is found to convert in darkness to protochlorophyllide, absorbing at 650 nm. The conversion starts instantly after the leaves have been exposed to the flashlight, and the pre-existent pool of protocholorophyllidc absorbing at 650 nm will become emptied. The conversion is completed after 15–20 minutes, when a new pool of protochlorophyllide has been filled up. This new pool is transformed to chlorophyllide by a second flash and the sequence is repeated. The conversion may be composed of two reactions, a conclusion which can be drawn from the behaviour at different temperatures. One of these reactions is fairly temperature independent while the other is temperature dependent. The action of the protochlorophyllide holochrome is discussed.     

Chlorophyll Synthesis in Dark-Grown Pine Primary Needles

The pigment content of dark-grown primary needles of Pinus jeffreyi L. and Pinus sylvestris L. was determined by high-performance liquid chromatography. The state of protochlorophyllide a and of chlorophylls during dark growth were analyzed by in situ 77 K fluorescence spectroscopy. Both measurements unambiguously demonstrated that pine primary needles are able to synthesize chlorophyll in the dark. Norflurazon strongly inhibited both carotenoid and chlorophyll synthesis. Needles of plants treated with this inhibitor had low chlorophyll content, contained only traces of xanthophylls, and accumulated carotenoid precursors. The first form of chlorophyll detected in young pine needles grown in darkness had an emission maximum at 678 nm. Chlorophyll-protein complexes with in situ spectroscopic properties similar to those of fully green needles (685, 695, and 735 nm) later accumulated in untreated plants, whereas in norflurazon-treated plants the photosystem I emission at 735 nm was completely lacking. To better characterize the light-dependent chlorophyll biosynthetic pathway in pine needles, the 77 K fluorescence properties of in situ protochlorophyllide a spectral forms were studied. Photoactive and nonphotoactive protochlorophyllide a forms with emission properties similar to those reported for dark-grown angiosperms were found, but excitation spectra were substantially red shifted. Because of their lower chlorophyll content, norflurazon-treated plants were used to study the protochlorophyllide a photoreduction process triggered by one light flash. The first stable chlorophyllide photoproduct was a chlorophyllide a form emitting at 688 nm as in angiosperms. Further chlorophyllide a shifts usually observed in angiosperms were not detected. The rapid regeneration of photoactive protochlorophyllide a from nonphotoactive protochlorophyllide after one flash was demonstrated.     

Detection of the photoactive protochlorophyllide-protein complex in the light during the greening of barley.

A photoactive protochlorophyllide-protein complex with absorbance and fluorescence maxima at 648 and 653 nm was detected in greening barley leaves without any re-darkening. The variations of the amplitudes of the absorbance and the fluorescence of the photoactive protochlorophyllide with greening time at two different light intensities indicate a close relationship between the rate of chlorophyll synthesis and the amount of the complex during the first hours. The chlorophyllide resulting from photoreduction during greening has an absorbance maximum at 684 nm, which shifts towards a shorter wavelength within a few seconds, indicating rapid liberation of the pigment from the enzyme. We conclude that chlorophyll accumulation proceeds through continuous regeneration and phototransformation of the photoactive complex.     

The Pool Size of Protochlorophyllide during Different Stages of Greening of Dark Grown Wheat Leaves

The pool size of protochlorophyllide in wheat leaves irradiated for 5 minutes to 6 hours was studied. Protochlorophyllide then accumulated in the dark, but the pool size of regenerated protochlorophyllide was considerably smaller in leaves irradiated for six hours than in leaves irradiated for 5 minutes. The decrease in pool size of regenerated protochlorophyllide was found to take place at the time when the chlorophyll formation had accelerated and reached the linear phase. The protochlorophyllide accumulated is the form with absorption maximum at 650 nm, which is phototransformed to chlorophyllide with maximum absorption at 684 nm. This species goes through the Shibata shift when formed even after 6 hours of irradiation. If leaves, irradiated for 1 or 6 hours, were fed with δ-amino-levulinic acid the protochlorophyllide synthesis was only 1.2 times faster in the leaves irradiated for 6 hours than in those irradiated for 1 hour. In the case of leaves fed with δ-amino-levulinic acid the absorption maximum of protochlorophyllide is at 636 nm and the absorption maximum of the chlorophyllide formed is at 672 nm.     

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.     

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.     

The Relationship between Chlorophyllide Accumulation, the Amount of Protochlorophyllide636 and Protochlorophyllide650 in Dark Grown Wheat Leaves Treated with δ-Aminolevulinic Acid

The influence of different amounts of protochlorophyl-lide₆₃₆ and protochlorophyllide₃₅₀ on light Induced chloro-phyllide formation was. studied in wheat leaves treated with δ-aminolevulinic acid. The phototransformation of proto-chlorophyllide was performed with weak red light. This transformation is unaffected by the δ-aminolevulinic acid treatment, whilst the accumulation of chlorophyllide, both the rate and the amount, is greatly stimulated by moderate amounts of protochlorophyllide₆₃₆. The presence of large amounts of protochlorophyllide₆₃₆ decreases the rate of chlorophyllide formation, but increases the final amount of chloro-phyllide formed. A decreased level of protochlorophyllide₆₅₀, obtained by treatment with N_aN₃, results in a decreased transformation rate. Inhibitors; of protein synthesis do not seem to influence the transformation of protochlorophyllide₆₃₆ to chlorophyllide, suggesting that no new synthesis of protein is required. The experimental results indicate that the final steps in chlorophyll biosynthesis are protochlorpnyllide₆₃₆→ protochlorophyllide₆₅₀→ chlorophyllide → chlorophyll.     

Low temperature phototransformations of protochlorophyll(ide) in etiolated leaves

By methods of difference and derivative spectroscopy it was shown that in etiolated leaves at 77 K three photoreactions of P650 protochlorophyllide take place which differ in their rates and positions of spectral maxima of the intermediates formed in the process: P650R668, P650R688, and P650R697. With an increase of temperature up to 233 K, in the dark, R688 and R697 are transformed into the known chlorophyllide forms C695/684 and C684/676, while R668 disappears with formation of a shorter wavelength form of protochlorophyllide with an absorption maximum at 643–644 nm.Along with these reactions, at 77 K phototransformations of the long-wave protochlorophyllide forms with absorption maxima at 658–711 nm into the main short-wave forms of protochlorophyllide are observed. At 233 K in the dark this reaction is partially reversible. This process may be interpreted as a reversible photodisaggregation of the pigment in vivo.The mechanism of P650 reactions and their role in the process of chlorophyll photobiosynthesis are discussed.Abbreviations P650 protochlorophyll(ide) with absorption maximum at 650 nm - C697/684 chlorophyllide with fluorescence maximum at 695 nm and absorption maximum at 684 nm - R697 intermediate with absorption maximum at 697 nm     

Photoconversion of Photochlorophyllide in the y-1 Mutant of Chlamydomonas reinhardtii

Dark-grown y-1 mutant cells of Chlamydomonas reinhardtii accumulate protochlorophyllide (Pchlide) in both 635 nanometers (P635) and 650 nanometers (P650) forms. Plastids in these cells lack the normal thylakoid membrane structure except some remnants of membrane vesicles. Using difference spectrophotometry, P635 is shown to be photoconverted to chlorophyllide at 672 nanometers (C672) and P650 is photoconverted to C688 followed by a rapid shift to C672 (Shibata shift) and regeneration of P650. Some of the Pchlide is not photoconverted despite repeated illumination. Although P650 is destroyed by freezing and thawing, it is not transformed into P635. Freezing and thawing treatment also made Pchlide no longer photoactive.