Photoinduced formation of pheophytin/chlorophyll-containing complexes during the greening of plant leaves

Illumination of etiolated maize leaves with low-intensity light produces a chlorophyll/pheophytin-containing complex. The complex contains two native chlorophyll forms Chl 671/668 and Chl 675/668 as well as pheophytin Pheo 679/675 (with chlorophyll/pheophytin ratio of 2/1). The complex is formed in the course of two successive reactions: reaction of protochlorophyllide Pchlde 655/650 photoreduction resulted in chlorophyllide Chlde 684/676 formation, and the subsequent dark reaction of Chlde 684/676 involving Mg substitution by H₂ in pigment chromophore and pigment esterification by phytol. Out data show that the reaction leading to chlorophyll/pheophytin-containing complex formation is not destructive. The reaction is in fact biosynthetic, and is competitive with the known reactions of biosynthesis of the bulk of chlorophyll molecules. The relationship between chlorophyll and pheophytin biosynthesis reactions is controlled by temperature, light intensity and exposure duration.The native complex containing pheophytin a and chlorophyll a is supposed to be a direct precursor of the PS II reaction centre in plant leaves.Abbreviations Chl chlorophyll - Chlde chlorophyllide - Pchl protochlorophyll - Pchlde protochloropyllide - Pheo pheophytin - PS II RC Photosystem II reaction centres. Abbreviations for native pigment forms: the first number after pigment symbol corresponds to the maximum position of low-temperature fluorescence band (nm); the second number corresponds to the maximum position of long wave absorption band     

Biosynthesis of non-fluorescent chlorophyll of Photosystem II core in greening plant leaves. Effect of etiolated plants growing under heat shock conditions (38 °C)

Preliminary dark incubation of etiolated pea and maize plants at 38 °C allowed to observe a new dark reaction of Chl biosynthesis occuring after photoconversion of protochlorophyllide Pchld 655/650 into chlorophyllide Chld 684/676. This reaction was accompanied by chlorophyllide esterification and by the bathochromic shift of pigment spectra: Chld 684/676 Chl 688/680. After completion of the reaction, a rapid (20–30 s at 26 °C) quenching of Chl 688/680 low-temperature fluorescence was observed. The reaction Chld 684/676 Chl 688/680 was inhibited under anaerobic conditions as well as in the presence of KCN; the reaction accompanied by Chl fluorescence quenching was inhibited in the leaves of pea mutants with impaired function of Photosystem II reaction centers. The spectra position of newly formed Chl, effects of Chl fluorescence quenching allowed to assume that the new dark reaction is responsible for biosynthesis of P–680, the key pigment of Photosystem II reaction centres.     

Photoconversion of long-wavelength protochlorophyll native form Pchl 682/672 into chlorophyll Chl 715/696 in Chlorella vulgaris B-15

By spectral methods, the final stages of chlorophyll formation from protochlorophyll (ide) were studied in heterotrophic cells of Chlorella vulgaris B-15 mutant, where chlorophyll dark biosynthesis is inhibited. It was shown that during the dark cultivation, in the mutant cells, in addition to the well-known protochlorophyll (ide) forms Pchlide 655/650, Pchl(ide) 640/635, Pchl(ide) 633/627, a long-wavelength protochlorophyll form is accumulated with fluorescence maximum at 682 nm and absorption maximum at 672 nm (Pchl 682/672). According to the spectra measured in vivo and in vitro, illumination of dark grown cells leads to the photoconversion of Pchl 682/672 into the stable long wavelength chlorophyll native form Chl 715/696. This reaction was accompanied by well-known photoreactions of shorter-wavelength Pchl (ide) forms: Pchlide 655/650Chlide 695/684 and Pchl (ide) 640/635Chl (ide) 680/670. These three photoreactions were observed at room temperature as well as at low temperature (203–233 K).Abbreviations Chl chlorophyll - Chlide chlorophyllide - Pchlide protochlorophyllide - Pchl protochlorophyll - PS I RC Photosystem I reaction centres. Abbreviations for native pigment forms: the first number after the pigment symbol corresponds to maximum position of low-temperature (77 K) fluorescence band (nm), second number to maximum position of long-wavelength absorption band     

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     

A comparative study of the terminal stages of chlorophyll biosynthesis before and after heavy water (D2O) introduction into greening plant leaves

By spectral methods, the final stages of chlorophyll formation from protochlorophyllide proceeding in intact greening maize leaves were studied before and after the introduction of heavy water (D₂O) into etiolated leaves. Three effects of D₂O introduction were observed: 1) a complete inhibition of the reaction pathway leading to pheophytin biosynthesis and formation of pheophytin/chlorophyll-containing complexes (presumably, direct precursors of Photosystem II reaction centers): 2) 5-fold inhibition of the reaction of the Shibata shift ; 3) appearance of a new dark reaction of the primary chlorophyllide native form Chld 684/676 'Chld 690/680'. It was shown that the intermediate Chld 684/676 presents the point of a triple branching of chlorophyllide transformation; activities of these three parallel pathways of Chld 684/676 transformation can be regulated by light intensity as well as by temperature.     

Biosynthesis of chlorophyll P-680 of photosystem II in greening leaves of plants adapted to heat shock

In etiolated pea and maize leaves illuminated after incubation at 38 degreesC, a new dark reaction was shown manifested in the bathochromic shift of spectral bands and accompanied by esterification of the product of protochlorophyllide photochemical reduction--Chld 684/676: Chld 684/676 --> Chl 688/680. After completion of the reaction a rapid (20-30 sec) quenching of the fluorescence of the reaction product (Chl 688/680) was observed. The reaction Chld 684/676 --> Chl 688/680 is inhibited under anaerobic conditions and in the presence of cyanide; the reaction accompanied by Chl 688/680 fluorescence quenching is not observed in pea mutants with impaired function of photosystem II reaction centers. The spectral properties of the formed Chl form with the absorption maximum at 680 nm, fluorescence quenching, and simultaneous synthesis of pheophytin suggest that the reaction is connected with the chlorophyll of photosystem II reaction center--P-680.     

Spectroscopic analysis of desiccation-induced alterations of the chlorophyllide transformation pathway in etiolated barley leaves

Effects of water deficit on the chlorophyllide (Chlide) transformation pathway were studied in etiolated barley (Hordeum vulgare) leaves by analyzing absorption spectra and 77-K fluorescence spectra deconvoluted in components. Chlide transformations were examined in dehydrated leaves exposed to a 35-ms saturating flash triggering protochlorophyllide (Pchlide) and Chlide transformation processes. During the 90 min following the flash, we found that dehydration induced modifications of Chlide transformations, but no effect on Pchlide phototransformation into Chlide was observed. During this time, content of NADPH-Pchlide oxydoreductase in leaves did not change. Chlide transformation process in dehydrated leaves was characterized by the alteration of the Shibata shift process, by the appearance of a new Chlide species emitting at 692 nm, and by the favored formation of Chl(ide) A(668)F(676). The formation of Chl(ide) A(668)F(676), so-called "free Chlide," was probably induced by disaggregation of highly aggregated Chlide complexes. Here, we offer evidence for the alteration of photoactive Pchlide regeneration process, which may be caused by the desiccation-induced inhibition of Pchlide synthesis.     

A new pathway of chlorophyll biosynthesis from long-wavelength protochlorophyllide Pchlide 686/676 in juvenile etiolated plants

By spectral methods, the final stages of chlorophyll formation from protochlorophyllide were studied using etiolated pea, bean, barley, wheat and maize plants in early stages (4 days) of growth. For these juvenile plants, along with the reaction chain known for mature (7–9-day-old) plants, a new reaction chain was found, which started with phototransformation of the long-wavelength form Pchlide 686/676(440) into Pchlide 653/648(440). (Pchlide 653/648(440) differs from the main known precursor form Pchlide 655/650(448)). The subsequent photoreduction of Pchlide 653/648(440) leads to the formation of Chlide 684/676(440), which is transformed into Chl 688/680(440) in the course of a dark reaction. After completion of this reaction, fast (20–30 s) quenching of the low-temperature fluorescence of the reaction product is observed with the formation of non-fluorescent Chl 680. The reaction accompanied by pigment fluorescence quenching is absent in pea mutants with depressed function of Photosystem II reaction centers. This suggests that the newly found reaction chain leads to the formation of chlorophyll of the Photosystem II core. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Isolation and classification of chlorophyll-deficient xantha mutants of Arabidopsis thaliana

Mutant lines of Arabidopsis thaliana that are either blocked at various steps of the biosynthetic pathway of chlorophyll (Chl) or that are disturbed in one of the subsequent steps leading to the assembly of an active photosynthetic membrane were isolated by screening for Chl-deficient xantha (xan) mutants. Only mutants that segregated in a 31 ratio, that contained the same carotenoid spectrum as etiolated wild-type seedlings and less than 2% of the Chl of wild-type control seedlings, and whose Chl content was not affected by the addition of sucrose to the growth medium were selected for a more detailed analysis. As a final test for the classification of the selected mutants, light-grown xan mutants were vacuum-infiltrated and incubated with the common precursor of tetrapyrroles, -aminolevulinic acid (ALA), in the dark. Two major groups of mutants could be distinguished. Some of the mutants were blocked at various steps of the Chl pathway between ALA and protochlorophyllide (Pchlide) and did not accumulate the latter in the dark. The other mutants accumulated Pchlide in the dark regardless of whether exogenous ALA was added. This latter group could be subdivided into mutants with a biochemical lesion in a recently discovered second light-dependent Pchlide reduction step that occurs in green plants and mutants that have blocks in the assembly of Chl protein complexes. In the present work a total of seven different loci could be defined genetically in Arabidopsis that affect the synthesis of Chl and its integration into the growing photosynthetic membrane.Abbreviations ALA -aminolevulinic acid - Chl chlorophyll - Chlide chlorophyllide - Pchlide protochlorophyllide - POR NADPH-Protochlorophyllide oxidoreductase - xan xantha This study was initiated while one of the authors (K.A.) was on sabbatical leave in the laboratory of Dr. C. Somerville (MSU, East Lansing, Mich., USA). We are extremely grateful to Dr. Somerville and his coworkers for advice and support during this time. This research was supported by the Deutsche Forschungsgemeinschaft and the Schweizerischer Nationalfonds.     

The regulation of NADPH-protochlorophyllide oxidoreductases A and B in green barley plants kept under a diurnal light/dark cycle

In etiolated barley (Hordeum vulgare L.) seedlings the light-induced accumulation of chlorophyll is controlled by two light-dependent NADPH-proto-chlorophyllide oxidoreductase (POR; EC 1.6.99.1) enzymes. While the concentration of one of these enzymes (POR A) and its mRNA rapidly decline during illumination, the second POR protein (POR B) and its mRNA remain at an approximately constant level during the transition from dark growth to the light. These results may suggest that only one of the enzymes, POR B, operates throughout the greening process and in light-adapted mature plants while the second enzyme, POR A, is active only in etiolated seedlings at the beginning of illumination. The fate of the two POR proteins and their mRNAs in fully green plants, however, has not been studied yet. In the present work we determined changes in the level of POR A and POR B proteins and mRNAs in green barley plants kept under a diurnal 12 h light/12 h dark cycle. In green barley plants, not only POR B is present but also trace amounts of POR A continue to reappear transiently at the end of a night period and seem to be involved in the synthesis and accumulation of chlorophyll at the beginning of each day.Abbreviations Chl chlorophyll - Chlide chlorophyllide - Lhcb light-harvesting chlorophyll a/b protein - Pchlide protochlorophyllide - POR NADPH-protochlorophyllide oxidoreductase Dedicated to Horst Senger on the occasion of his 65th birthday.We thank Dr. Dieter Rubli for photography and Renate Langjahr for typing. This work was supported by the Swiss National Science Foundation and the ETH-Zürich.     

Immunodetection and photostability of NADPH-protochlorophyllide oxidoreductase in Pinus pinea L.

Antibody against the light-dependent NADPH-protochlorophyllide oxidoreductase of oat was used to detect a protein of the same molecular weight in cotyledons of 40-day-old dark-grown seedlings of Pinus pinea L. Exposure of the seedlings to light resulted in a rapid decrease in protochlorophyllide content without the concomitant decrease in 38 kDa protein which is observed on transfer of dark-grown angiosperm seedlings to light. The stability of the light-dependent NADPH-protochlorophyllide oxidoreductase in pine in the absence of accumulated substrate is consistent with either (1) a different mechanism of regulation of chlorophyll synthesis in gymnosperms or (2) a higher proportion of stable extra-plastidic protein reacting with the antibody to the light-dependent NADPH-protochlorophyllide oxidoreductase than is the case in angiosperms.Abbreviations Chl chlorophyll - Chlide chlorophyllide - NADPH-Pchlide oxidoreductase NADPH protochlorophyllide oxidoreductase - NC nitrocellulose - PBS phosphate buffered saline - Pchlide protochlorophyllide - SDS sodum dodecyl sulphate - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Biosynthesis of Chlorophyll of Photosystem II Reaction Centers in Plant Leaves in Early Stages of Etiolation

Spectral methods were used to study the sequences of chlorophyll biosynthesis reactions in etiolated pea, bean, and maize plants in early stages (3-4 days) of growth. For these juvenile plants, along with the reaction chain known for mature (7-9 day-old) plants, a new reaction chain was found which started with phototransformation of the long-wavelength form PChld 686/676 into PChld 653/648. (PChld 653/648 differs from the main known precursor form PChld 655/650). The subsequent photoreduction of PChld 653/648 leads to the formation of Chld 684/676, which is transformed into Chl 688/680 in the course of a dark reaction. After completion of this reaction, fast (20-30 sec) quenching of the fluorescence of the reaction product is observed with the formation of non-fluorescent Chl 680. The reaction accompanied by pigment fluorescence quenching is absent in pea mutants with depressed function of Photosystem II reaction centers. This suggests that the newly found reaction chain leads to the formation of chlorophyll of the Photosystem II reaction center.     

Phosphorescence of protochlorophyll(ide) and chlorophyll(ide) in etiolated and greening bean leaves

The assignment is presented for the principal phosphorescence bands of protochlorophyll(ide), chlorophyllide and chlorophyll in etiolated and greening bean leaves measured at -196°C using a mechanical phosphoroscope. Protochlorophyll(ide) phosophorescence spectra in etiolated leaves consist of three bands with maxima at 870, 920 and 970 nm. Excitation spectra show that the 870 nm band belongs to the short wavelength protochlorophyll(ide), P627. The latter two bands correspond to the protochlorophyll(ide) forms, P637 and P650. The overall quantum yield for P650 phosphorescence in etiolated leaves is near to that in solutions of monomeric protochlorophyll, indicating a rather high efficiency of the protochlorophyll(ide) triplet state formation in frozen plant material. Short-term (2–20 min) illumination of etiolated leaves at the temperature range from -30 to 20°C leads to the appearance of new phosphorescence bands at about 990–1000 and 940 nm. Judging from excitation and emission spectra, the former band belongs to aggregated chlorophyllide, the latter one, to monomeric chlorophyll or chlorophyllide. This indicates that both monomeric and aggregated pigments are formed at this stage of leaf greening. After preillumination for 1 h at room temperature, chlorophyll phosphorescence predominates. The spectral maximum of this phosphorescence is at 955–960 nm, the lifetime is about 2 ms, and the maximum of the excitation spectrum lies at 668 nm. Further greening leads to a sharp drop of the chlorophyll phosphorescence intensity and to a shift of the phosphorescence maximum to 980 nm, while the phosphorescence lifetime and a maximum of the phosphorescence excitation spectrum remains unaltered. The data suggest that chlorophyll phosphorescence belongs to the short wavelength, newly synthesized chlorophyll, not bound to chloroplast carotenoids. Thus, the phosphorescence measurement can be efficiently used to study newly formed chlorophyll and its precursors in etiolated and greening leaves and to address various problems arising in the analysis of chlorophyll biosynthesis.Abbreviations Pchl protochlorophyll and protochlorophyllide - Chld chlorophyllide - Chl chlorophyll     

Control of chlorophyll synthesis by phytochrome

Summary The rate of chlorophyllide esterification in mustard cotyledons can be increased by a pretreatment with 5 min red light applied 24 h prior to the protochlorophyll(ide)chlorophyll(ide) photoconversion at 60 h after sowing. Simultaneously the red light pulse pretreatment leads to a decrease of the total amount of chlorophyll(ide) a in darkness. It has been proven that phytochrome (P_fr) is the photoeffector for both. Since the amounts of esterified chlorophyllide are determined by the ratio [chlorophyll a]/[chlorophyllide a+chlorophyll a] it is assumed that P_fr increases the rate of esterification indirectly via stimulating the decrease of chlorophyll(ide) a. The regulation of chlorophyll synthesis by P_fr does not seem to involve a control of esterification. The duration of the chlorophyllide esterification differs from the duration of the Shibata shift although both are greatly shortened by the red light pulse pretreatment. The effect of 5 min red light on the duration of the esterification is fully reversible by 5 min far-red light while the reversibility with respect to the Shibata shift is lost within 2 min [Jabben, M. and H. Mohr, Photochem. Photobiol. 22, 55–58 (1975)]. We conclude that the control of the chlorophyllide esterification and the control of the Shibata shift cannot be traced back to the same initial action of P_fr.Abbreviations Chl chlorophyll - Chlide chlorophyllide - Chl(ide) sum of Chl and Chlide - PChl protochlorophyll - PChlide protochlorophyllide - PChl(ide) sum of PChl and PChlide - P_fr far-red absorbing form of the phytochrome system     

The photostability of different chlorophyll forms in dark grown leaves of wheat

Formulae were developed for calculation of the relative amount of different pigment forms of dark grown leaves of wheat, present before and after photoreduction of the protochlorophyllide. Three pigment forms were calculated from in vivo absorption spectra: the photoreducible protochlorophyllide with absorption maximum at 650 nm and the two chlorophyll(ide) forms with absorption maximum at 684 nm and 673 nm, respectively. The formulae were used to study the changes of the pigment forms at repeated photoreduction of the protochlorophyllide, and at a repeated treatment involving photoreduction of the protochlorophyllide followed by partial photo-decomposition of the chlorophyllide formed. Five consecutive photoreductions and reaccumulations of protochlorophyllide were carried out by high intensity irradiations of one second (red light, 700 W m^-2) given at intervals of 3 h. The results show that the pool size of reaccumulated protochlorophyllide decreased sharply with the number of photoreductions performed. The absorption spectrum of the chlorophyllide formed at each photoreduction proceeded through the Shibata shift (transformation of the 684-form to the 673-form) and the late red-shift (transformation of the 673-form to other pigment form(s) in the dark). High intensity irradiation for ten minutes (red light, 700 W m^-2) immediately after each phototransformation caused a photodecomposition of about three quarters of the newly formed chlorophyllide (which was in the 684-form) while the earlier formed chlorophyll(ide) (in the 673-form) appeared not to be decomposed. This partial photodecomposition of the chlorophyllide had no effect on further accumulation of protochlorophyllide in the dark, and the absorption spectrum of the remaining chlorophyllide proceeded through the Shibata shift. The partial photodecomposition caused an inhibition of the late red-shift, and the accumulated chlorophyll(ide) remained in the 673-form.     

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.     

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).     

The biosynthesis of chlorophyll in greening barley (Hordeum vulgare). Is there a light-independent protochlorophyllide reductase?

Recently, some evidence for the occurence of a light-independent protochlorophyllide-reducing enzyme in greening barley plants has been presented. In the present work this problem was reinvestigated. -[¹⁴C] Aminolevulinic acid was fed to isolated barley shoots from plants which had been preilluminated for various lengths of time. Porphyrins which had been synthesized during the dark incubation were analyzed by high-performance liquid chromatography. There was no evidence for a light-independent synthesis of chlorophyll(ide). The ¹⁴C-labelled precursor was incorporated almost exclusively into protochlorophyllide. The reduction of labelled protochlorophyllide to chlorophyllide was strictly light-dependent. These results are not consistent with the existence of a light-independent protochlorophyllide-reductase in barley as proposed previously.Abbreviation HPLC high-performance liquid chromatography     

Incorporation of phytol precursors into chlorophylls of tobacco cell cultures

The incorporation of [1-³H] geranylgeranyl diphosphate (GGPP), [1-³H] geranylgeranyl monophosphate (GGMP) and [U-¹⁴C] phytyl diphosphate (PhPP) into chlorophylls a and b in growing tobacco cell cultures was investigated. The substrates were effectively incorporated into chlorophylls a and b, 3.2% of the total activity of applied GGPP or GGMP and 12.4% of the total activity of applied PhPP being found in chlorophylls a and b after 24 h incubation. The radioactivity was found in phytyl chlorophyllide through-out which means effective hydrogenation of the alcohol moiety in the case of GGPP and GGMP. With increasing substrate concentration, the specific radioactivity of chlorophyll increased up to a saturation level which was reached either at 20–40 M PhPP or at 60 M GGPP and GGMP. The specific radioactivity of the chlorophyll formed during the 24-h incubation period was the same as that of the applied substrate at saturating substrate concentration. The specific radioactivity of chlorophyll a was higher than that of chlorophyll b only in the case of PhPP.Abbreviations Chlide chlorophyllide a - Chl_Ph phytyl chloro-phyllide - Chl_GG geranylgeranyl chlorophyllide a - GGPP geranylgeranyl diphosphate - GGMP geranylgeranyl monophosphate - HPLC high-performance liquid chromatography - PhPP phytyl diphosphate Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday