Chloroplast biogenesis 76. Regulation of 4-vinyl reduction during conversion of divinyl Mg-protoporphyrin IX to monovinyl protochlorophyllide a is controlled by plastid membrane and stromal factors

Most of the chlorophyll (Chl) a of green plants is formed via two biosynthetic routes, namely the carboxylic divinyl and monovinyl chlorophyll biosynthetic routes. These two routes are linked by (4-vinyl) reductases that convert divinyl tetrapyrroles to monovinyl tetrapyrroles by reduction of the vinyl group at position four of the macrocycle to ethyl. The activities of these two routes are very sensitive to cell disruption. For example in barley leaves, cell disruption, a mandatory step during plastid isolation, results in partial inactivation of the carboxylic divinyl route. Investigations with subplastidic fractions revealed that the carboxylic divinyl and monovinyl biosynthetic routes were regulated by a delicate interaction that involved plastid membranes, stroma, and reduced pyridine nucleotides. While the monovinyl biosynthetic route was very active in isolated plastid membranes, activation of the divinyl biosynthetic route required the joint presence of plastid membranes and stroma. Contrary to expectation, activity of the carboxylic divinyl biosynthetic route was greatly enhanced by addition of NADPH to the lysing buffer used during plastid membranes and stroma preparation. NADPH in cooperation with the plastid stroma may play an important regulatory role during the biosynthesis of divinyl and monovinyl protochlorophyllide a. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chloroplast Biogenesis 60 : Conversion of Divinyl Protochlorophyllide to Monovinyl Protochlorophyllide in Green(ing) Barley, a Dark Monovinyl/Light Divinyl Plant Species

In higher plants, most of the chlorophyll a is formed via the divinyl and monovinyl chlorophyll monocarboxylic biosynthetic routes. These two routes are strongly interconnected prior to protochlorophyllide formation in barley (Hordeum vulgare L. cv Morex), a dark monovinyl-light divinyl plant species, but not in cucumber (Cucumis sativus L. cv Beit Alpha MR), a dark divinyl-light divinyl plant species (BC Tripathy, CA Rebeiz, 1986 J Biol Chem 261: 13556-13564). It is shown that in dark monovinyl-light divinyl plant species such as barley, the divinyl and monovinyl monocarboxylic routes become interconnected at the level of protochlorophyllide during transition from the divinyl to the monovinyl protochlorophyllide biosynthetic mode. In cucumber, a dark divinyl-light divinyl plant species, in which the monovinyl monocarboxylic biosynthetic route becomes preponderant only after an abnormally long sojourn in darkness, the conversion of divinyl to monovinyl protochlorophyllide does not take place on the barley time-scale of incubation.     

Chloroplast biogenesis. Demonstration of the monovinyl and divinyl monocarboxylic routes of chlorophyll biosynthesis in higher plants

It is shown that barley (Hordeum vulgare), a dark monovinyl/light divinyl plant species, and cucumber (Cucumis sativus L.) a dark divinyl/light divinyl plant species synthesize monovinyl and divinyl protochlorophyllide in darkness from monovinyl and divinyl protoporphyrin IX via two distinct monovinyl and divinyl monocarboxylic chlorophyll biosynthetic routes. Evidence for the operation of monovinyl monocarboxylic biosynthetic routes consisted (a) in demonstrating the conversion of delta-aminolevulinic acid to monovinyl protoporphyrin and to monovinyl Mg-protoporphyrins, and (b) in demonstrating the conversion of these tetrapyrroles to monovinyl protochlorophyllide by both isolated barley and cucumber etiochloroplasts. Likewise, evidence for the operation of divinyl monocarboxylic chlorophyll biosynthetic routes consisted (a) in demonstrating the biosynthesis of divinyl protoporphyrin and divinyl Mg-protoporphyrins from delta-aminolevulinic acid, and (b) in demonstrating the conversion of the latter tetrapyrroles to divinyl protochlorophyllide. Finally, it was shown that the divinyl tetrapyrrole substrates were metabolized differently by barley and cucumber. For example, divinyl protoporphyrin, divinyl Mg-protoporphyrin, and divinyl Mg-protoporphyrin monoester were converted predominantly to monovinyl protochlorophyllide and to smaller amounts of divinyl protochlorophyllide by barley etiochloroplasts. In contrast, cucumber etiochloroplasts converted the above substrates predominantly to divinyl protochlorophyllide, although smaller amounts of monovinyl protochlorophyllide were also formed. Furthermore, it was shown that monovinyl protochlorophyllide was not formed from divinyl protochlorophyllide either in barley or in cucumber etiochloroplasts. These metabolic differences are explained by the presence of strong biosynthetic interconnections between the divinyl and monovinyl monocarboxylic routes, prior to divinyl protochlorophyllide formation, in barley but not in cucumber.     

Chloroplast biogenesis: [4-vinyl] chlorophyllide a reductase is a divinyl chlorophyllide a-specific, NADPH-dependent enzyme.

Some properties of [4-vinyl] chlorophyllide a reductase are described. This enzyme converts divinyl chlorophyllide a to monovinyl chlorophyllide a. The latter is the immediate precursor of monovinyl chlorophyll a, the main chlorophyll in green plants. [4-Vinyl] chlorophyllide a reductase plays an important role in daylight during the conversion of divinyl protochlorophyllide a to monovinyl chlorophyll a. [4-Vinyl] chlorophyllide a reductase was detected in isolated plastid membranes. Its activity is strictly dependent on the availability of NADPH. Other reductants such as NADH and GSH were ineffective. The enzyme appears to be specific for divinyl chlorophyllide a, and it does not reduce divinyl protochlorophyllide a to monovinyl protochlorophyllide a. The conversion of divinyl protochlorophyllide a to monovinyl protochlorophyllide a has been demonstrated in barley and cucumber etiochloroplasts and appears to be catalyzed by a [4-vinyl] protochlorophyllide a reductase [Tripathy, B.C., & Rebeiz, C.A. (1988) Plant Physiol. 87, 89-94]. On the basis of reductant requirements and substrate specificity, it is possible that two different 4-vinyl reductases may be involved in the reduction of divinyl protochlorophyllide a and divinyl chlorophyllide a to their respective 4-ethyl analogues.     

Chloroplast Biogenesis 49 : Differences among Angiosperms in the Biosynthesis and Accumulation of Monovinyl and Divinyl Protochlorophyllide during Photoperiodic Greening

Various angiosperms differed in their monovinyl and divinyl protochlorophyllide biosynthetic capabilities during the dark and light phases of photoperiodic growth. Some plant species such as Cucumis sativus L., Brassica juncea (L.) Coss., Brassica kaber (DC.) Wheeler, and Portulaca oleracea L. accumulated mainly divinyl protochlorophyllide at night. Monocotyledonous species such as Avena sativa L., Hordeum vulgare L., Triticum secale L., Zea mays L., and some dicotyledonous species such as Phaseolus vulgaris L., Glycine max (L.) Merr., Chenopodium album L., and Lycopersicon esculentum L. accumulated mainly monovinyl protochlorophyllide at night.

Under low light intensities meant to simulate the first 60 to 80 minutes following daybreak divinyl protochlorophyllide appeared to contribute much more to chlorophyll formation than monovinyl protochlorophyllide in species such as Cucumis sativus L. Under the same light conditions, species which accumulated mainly monovinyl protochlorophyllide at night appeared to form chlorophyll preferably via monovinyl protochlorophyllide.

These results were interpreted in terms of: (a) a differential contribution of monovinyl and divinyl protochlorophyllide to chlorophyll formation at daybreak in various plant species; and (b) a differential regulation of the monovinyl and divinyl protochlorophyllide biosynthetic routes by light and darkness.

     

Chloroplast biogenesis: quantitative determination of monovinyl and divinyl Mg-protoporphyrins and protochlorophyll(ides) by spectrofluorometry

General equations which permit the determination of the amounts of any two closely related fluorescent compounds which can be distinguished by 77 degrees K but not by 293 degrees K spectrofluorometry have been described. This was achieved in the presence or absence of a third interfering compound, without prior separation of the fluorescent species. The adaptation of the generalized equations to the determination of the amounts of monovinyl (MV) and divinyl (DV) Mg-protoporphyrins or of MV and DV protochlorophyll(ides) in the presence or absence of Mg-Protos [Mg-protoporphyrin IX (Mg-Proto), Mg-Proto monoester, Mg-Proto diester or a mixture of those three tetrapyrroles] interference, was then demonstrated over a wide range of MV/DV tetrapyrrole proportions. These equations are likely to be very useful for the study of the intermediary metabolism of the monovinyl and divinyl chlorophyll biosynthetic routes in plants.     

Differential distribution of chlorophyll biosynthetic intermediates in stroma, envelope and thylakoid membranes in Beta vulgaris

Stroma, envelope and thylakoid membranes were prepared from chloroplasts isolated from leaves of Beta vulgaris. Out of total plastidic protochlorophyllide, envelope membranes contained 1.5%, thylakoids had the maximum 98.48% and stroma had a trace fraction of 0.02%. Distribution of the Mg-protoporphyrin IX and its monoester was 89.0% in thylakoids, 10.0% in stroma and 1.0% in envelope. A substantial fraction (33.77%) of plastidic protoporphyrin IX was partitioned into stroma. Envelope contained 0.66% and thylakoids had 65.57% of the total plastidic protoporphyrin IX pool. The proportion of monovinyl and divinyl forms of protochlorophyllide was almost similar in intact plastid, thylakoids, and outer and inner envelope membranes suggesting a tight regulation of vinyl reductase enzyme. The significance of differential distribution of chlorophyll biosynthetic intermediates among thylakoids, envelope and stroma is discussed. This work was supported by a grant from the Council of Scientific and Industrial Research (38/1079/03/EMRII) to BCT.     

Chloroplast biogenesis 51 : modulation of monovinyl and divinyl protochlorophyllide biosynthesis by light and darkness in vitro

It is shown that the monovinyl and divinyl protochlorophyllide biosynthetic patterns of etiolated maize (Zea mays L.), and cucumber (Cucumis sativus L.) seedlings and of their isolated etiochloroplasts can be modulated by light and darkness as was shown for green photoperiodically grown plants (E. E. Carey, C. A. Rebeiz 1985 Plant Physiol. 79: 1-6). In etiolated corn and cucumber seedlings and isolated etiochloroplasts poised in the divinyl protochlorophyllide biosynthetic mode by a 2 hour light pretreatment, darkness induced predominantly the biosynthesis of monovinyl protochlorophyllide in maize and of divinyl protochlorophyllide in cucumber. When etiolated seedlings and their isolated etiochloroplasts were poised in the monovinyl protochlorophyllide biosynthetic mode by a prolonged dark-pretreatment, light induced mainly the biosynthesis of divinyl protochlorophyllide in both maize and cucumber.     

Spectral properties of a divinyl chlorophyll a harboring mutant of Synechocystis sp. PCC6803

A divinyl chlorophyll (DV-Chl) a harboring mutant of Synechocystis sp. PCC 6803, in which chlorophyll species is replaced from monovinyl(normal)-Chl a to DV-Chl a, was characterized. The efficiency of light utilization for photosynthesis was decreased in the mutant. Absorption spectra at 77 K and their fourth derivative analyses revealed that peaks of each chlorophyll forms were blue-shifted by 1–2 nm, suggesting lowered stability of chlorophylls at their binding sites. This was also true both in PSI and PSII complexes. On the other hand, fluorescence emission spectra measured at 77 K were not different between wild type and the mutant. This indicates that the mode of interaction between chlorophyll and its binding pockets responsible for emitting fluorescence at 77 K is not altered in the mutant. P700 difference spectra of thylakoid membranes and PSI complexes showed that the spectrum in Soret region was red-shifted by 7 nm in the mutant. This is a characteristic feature of DV-Chl a. Microenvironments of iron–sulfur center of a terminal electron acceptor of PSI complex, P430, were practically the same as that of wild type.     

Mechanisms of regulation and interplastid localization of chlorophyll biosynthesis.

The current concepts of chlorophyll biosynthesis, its interplastid localization, biosynthetic and biochemical heterogeneity, mechanisms of regulation of the key reactions, formation of 5-aminolevulinic acid and incorporation of magnesium into protoporphyrin IX, are reviewed. The literature and author's data demonstrate the existence of in vivo multienzyme systems synthesizing chlorophyll and its precursors as monovinyl and divinyl chemical species. Both types of the multienzyme systems synthesize 5-aminolevulinic acid and regulate this process independently. A hypothesis is considered that the function of the magnesium branch of chlorophyll biosynthesis in vivo is controlled by a mechanism through inhibition of the enzymes by their products because of the limitation of the binding sites for them in the membrane. An additional influence of light on the Mg-chelatase activity not only via the photosynthetic supply with ATP but also through the light-induced synthesis of the enzyme molecules de novo is described. Efficient energy migration from protoporphyrin IX and Mg-protoporphyrin IX (monomethyl ester) molecules to the protochlorophyllide active form detected by the author is discussed considering a close location of these pigments in plastid membranes and the enzymes participating in their formation.     

Identification of a vinyl reductase gene for chlorophyll synthesis in Arabidopsis thaliana and implications for the evolution of Prochlorococcus species

Chlorophyll metabolism has been extensively studied with various organisms, and almost all of the chlorophyll biosynthetic genes have been identified in higher plants. However, only the gene for 3,8-divinyl protochlorophyllide a 8-vinyl reductase (DVR), which is indispensable for monovinyl chlorophyll synthesis, has not been identified yet. In this study, we isolated an Arabidopsis thaliana mutant that accumulated divinyl chlorophyll instead of monovinyl chlorophyll by ethyl methanesulfonate mutagenesis. Map-based cloning of this mutant resulted in the identification of a gene (AT5G18660) that shows sequence similarity with isoflavone reductase genes. The mutant phenotype was complemented by the transformation with the wild-type gene. A recombinant protein encoded by AT5G18660 was expressed in Escherichia coli and found to catalyze the conversion of divinyl chlorophyllide to monovinyl chlorophyllide, thereby demonstrating that the gene encodes a functional DVR. DVR is encoded by a single copy gene in the A. thaliana genome. With the identification of DVR, finally all genes required for chlorophyll biosynthesis have been identified in higher plants. Analysis of the complete genome of A. thaliana showed that it has 15 enzymes encoded by 27 genes for chlorophyll biosynthesis from glutamyl-tRNA(glu) to chlorophyll b. Furthermore, identification of the DVR gene helped understanding the evolution of Prochlorococcus marinus, a marine cyanobacterium that is dominant in the open ocean and is uncommon in using divinyl chlorophylls. A DVR homolog was not found in the genome of P. marinus but found in the Synechococcus sp WH8102 genome, which is consistent with the distribution of divinyl chlorophyll in marine cyanobacteria of the genera Prochlorococcus and Synechococcus.     

Chloroplast biogenesis 80. Proposal of a unified multibranched chlorophyll a/b biosynthetic pathway

A unified multibranched chlorophyll (Chl) biosynthetic pathway is proposed. The proposed pathway takes into account the following considerations: (a) that the earliest putative precursor of monovinyl Chl b that has been detected in higher plants is monovinyl protochlorophyllide b, (b) that in most cases, Chl b biosynthesis has its roots in the Chl a biosynthetic pathway, (c) that the Chl a biosynthetic pathway exhibits extensive biosynthetic heterogeneity, (d) that Chl biosynthesis may proceed differently at different stages of greening and in different greening groups of plants. Integration of the Chl a and b biosynthetic pathways into a unified multibranched pathway offers the functional flexibility to account for the structural and biosynthetic complexity of photosynthetic membranes. In this context, it is proposed that the unified, multibranched Chl a/b biosynthetic pathway represents the template of a Chl-protein biosynthesis center where photosystem (PS) 1, PS2, and light-harvesting Chl-protein complexes are assembled into functional photosynthetic units. The individual biosynthetic routes or groups of two to three adjacent biosynthetic routes may constitute Chl-protein biosynthesis subcenters, where specific Chl-protein complexes are assembled. This revised version was published online in September 2006 with corrections to the Cover Date.     

Biosynthesis of Protoheme and Heme a Precursors Solely from Glutamate in the Unicellular Red Alga Cyanidium caldarium

Two biosynthetic routes to the heme, chlorophyll, and phycobilin precursor, δ-aminolevulinic acid (ALA) are known: conversion of the intact five-carbon skeleton of glutamate, and ALA synthase-catalyzed condensation of glycine plus succinyl-coenzyme A. The existence and physiological roles of the two pathways in Cyanidium caldarium were assessed in vivo by determining the relative abilities of [2-¹⁴C]glycine and [1-¹⁴C]glutamate to label protoheme and heme a. Glutamate was incorporated to a much greater extent than glycine into both protoheme and heme a, even in cells that were unable to form chlorophyll and phycobilins. The small incorporation of glycine could be accounted for by transfer of label to intracellular glutamate pools, as determined from amino acid analysis. It thus appears that C. caldarium makes all tetrapyrroles, including mitochondrial hemes, solely from glutamate, and there is no contribution by ALA synthase in this organism.     

Photodynamic herbicides: 1. Concept and phenomenology

A new approach to the design of conceptually and phenomenologically new herbicides is described. It involves the joint utilization of tetrapyrrole precursors, such as δ-aminolaevulinic acid (a biodegradable amino acid) and activators of the chlorophyll biosynthetic pathway, such as 2,2′-dipyridyl, in order to induce treated plants to biosynthesize and accumulate massive amounts of tetrapyrrole intermediates of the chlorophyll biosynthetic pathway in the dark (i.e. at night). During the subsequent light period (daylight) the accumulated tetrapyrroles act as potent photodynamic sensitiziers, which in turn result in the death of susceptible plants in a matter of hours. We have therefore proposed to name herbicides that act via this mechanism as photodynamic herbicides, or more pictorially as laser herbicides. From a limited survey of agricultural plant and weed species it appears that photodynamic herbicides exhibit a very pronounced organ, age and species-dependent selectivity. For example, dicotyledonous weeds such as mustard, red-root pigweed, common purslane and lambsquarter are very susceptible while monocotyledonous plants such as corn, wheat, barley and oats are not. The biochemical basis of this selectivity seems to lie, among other things, in the rates of tetrapyrrole turnover and in a differential enhancement by the applied chemicals of the monovinyl and divinyl tetrapyrrole biosynthetic pathways in the various species. A survey of various groups of chemicals (herbicides and other selected biochemicals) that are likely to exhibit photodynamic herbicidal properties is currently under investigation.     

Chloroplast biogenesis 92: In situ screening for divinyl chlorophyll(ide) a reductase mutants by spectrofluorometry

Chlorophyll biosynthetic heterogeneity is rooted mainly in parallel divinyl (DV) and monovinyl (MV) biosynthetic routes interconnected by 4-vinyl reductases (4VRs) that convert DV tetrapyrroles to MV tetrapyrroles by conversion of the vinyl group at position 4 of the macrocycle to ethyl. What is not clear at this stage is whether the various 4VR activities are catalyzed by one enzyme of broad specificity or by a family of enzymes encoded by one gene or multiple genes with each enzyme having narrow specificity. Additional research is needed to identify the various regulatory components of 4-vinyl reduction. In this undertaking, Arabidopsis mutants that accumulate DV chlorophyllide a and/or DV chlorophyll [Chl(ide)] a are likely to provide an appropriate resource. Because the Arabidopsis genome has been completely sequenced, the best strategy for identifying 4VR and/or putative regulatory 4VR genes is to screen Arabidopsis Chl mutants for DV Chl(ide) a accumulation. In wild-type Arabidopsis, a DV plant species, only MV chlorophyllide (Chlide) a is detectable. However in Chl mutants lacking 4VR activity, DV Chl(ide) a may accumulate in addition to MV Chl(ide) a. In the current work, an in situ assay of DV Chl(ide) a accumulation, suitable for screening a large number of mutants lacking 4-vinyl Chlide a reductase activity with minimal experimental handling, is described. The assay involves homogenization of the tissues in Tris-HCl:glycerol buffer and the recording of Soret excitation spectra at 77K. DV Chlide a formation is detected by a Soret excitation shoulder at 459 nm over a wide range of DV Chlide a/MV Chl a ratios. The DV Chlide a shoulder became undetectable at DV Chlide a/MV Chl a ratios less than 0.049, that is, at a DV Chlide a content of less than 5%.     

Chloroplast biogenesis 84: solubilization and partial purification of membrane-bound [4-vinyl]chlorophyllide a reductase from etiolated barley leaves

[4-Vinyl] chlorophyllide a reductase (4VCR) is a key enzyme of the chlorophyll (Chl) biosynthetic pathway. It catalyzes the conversion of divinyl chlorophyllide (Chlide) a to monovinyl Chlide a by reduction of the vinyl group at position 4 of the macrocycle to ethyl. 4VCR is a membrane-bound enzyme, embedded in etioplast and etiochloroplast membranes. A study of the regulation and properties of this enzyme is mandatory for a comprehensive understanding of the biosynthetic heterogeneity of Chl biosynthesis. Solubilization and partial purification of 4VCR are described for the first time. The enzyme was solubilized with 5 mM Chaps and was partially purified by chromatography on DEAE-Sephacel and Cibacron Blue 3GA-1000 agarose. An overall 20-fold purification was achieved. The partially purified enzyme was stable for several months at -80 degrees C.     

Studies on the expression of NDH-H,a subunit of the NAD(P)H-plastoquinone-oxidoreductase of higher-plant chloroplasts

The plastid genomes of higher plants contain eleven reading frames (ndhA-K) that are homologous to genes encoding subunits of the mitochondrial NADH-ubiquinone-oxidoreductase (complex I). The carboxyterminal end of the NDH-H subunit from rice (Oryza sativa L.) was expressed as a fusion protein in Escherichia coli and antibodies against the fusion protein were generated in rabbits. The antibody was used to study the expression of NDH-H, and the following results were obtained: (i) NDH-H is expressed in mono- and dicotyledonous plants, (ii) NDH-H is localized on the stroma lamellae of the thylakoid membrane and (iii) NDH-H is expressed in etioplasts. Together with the finding that two other ndh genes (ndhI and ndhK) are expressed in plastids, these results point to the existence of an NAD(P)H-plastoquinone-oxidoreductase on the thylakoid membrane. The possible function of the enzyme in plastids is discussed and it is suggested that it works in balancing the ATP/ADP and the NADPH/NADP ratios during changing external (i.e. light) or internal (i.e. ATP and NADPH demands of biosynthetic pathways of the plastid) conditions.Abbreviations PVDF polyvinylidene difluoride - SDS sodium dodecyl sulfate We thank Professor M. Sugiura for the rice plastid DNA clone bank, Oliver Buchholz for Sorghum plastid membranes, Pioneer Hi-Bred Inc. for maize and Sorghum seeds and the Deutsche Forschungsgemeinschaft for financial support (SFB189).     

Polyethylene-based high-performance liquid chromatography of chloroplast pigments: resolution of mono- and divinyl chlorophyllides and other pigment mixtures

In addition to most chlorophylls and their derivatives, monovinyl and divinyl chlorophyll species were separated by high-performance liquid chromatography, using a polyethylene column and a simple elution with aqueous acetone. Peak retention and resolution of the pigment separation were greatly increased by increasing the polarity of the mobile phase and also by decreasing the column temperature. Polyethylene chromatography showed chlorophyll separation behavior similar to that of the octadecyl silica column, but it showed no adsorption of the pigment species containing free carboxylic acid groups, enabling the complete separation of chlorophylls and their derivatives. Polyethylene is a superior alternative stationary phase to the known reversed-phase materials for chlorophyll separation and analysis.     

With chlorophyll pigments from prolamellar bodies to light-harvesting complexes

The biosynthetic chain leading from 5-aminolevulinic acid to chlorophyll is localised to the plastid. Many of the enzymes are nuclear-encoded. NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33) is one such enzyme which is encoded by two different genes and can exist in an A and a B form. Its import into the plastid seems to be facilitated when protochlorophyllide is present in the chloroplast envelope. Within the plastid the reductase is assembled to thylakoids or prolamellar bodies. The specific properties of the reductase together with the specific properties of the lipids present in the etioplast inner membranes promote the formation of the three-dimensional regular network of the prolamellar bodies. The reductase forms a ternary complex with protochlorophyllide and NADPH that gives rise to different spectral forms of protochlorophyllide. Light transforms protochlorophyllide into chlorophyllide and this photoreaction induces a conformational change in the reductase protein which leads to a process of disaggregation of enzyme, pigment aggregates and membranes, which can be followed spectroscopically and with electron microscopy. The newly formed chlorophyllide is esterified by a membrane-bound nuclear-encoded chlorophyll synthase and the chlorophyll molecule is then associated with proteins into active pigment protein complexes in the photosynthetic machinery.     

Chloroplast biogenesis: quantitative determination of monovinyl and divinyl chlorophyll(ide) a and b by spectrofluorometry

Simultaneous equations for the determination of monovinyl (MV) and divinyl (DV) Chl a and b by spectrofluorometry in unsegregated mixtures of these tetrapyrroles were derived. The same equations can also be used for the quantitative determination of MV and DV chlorophyllide (Chlide) a and b in unpurified mixtures, after extraction of the Chls in hexane. The equations used differences in the Soret excitation maxima of these tetrapyrroles in ether at 77 degrees K, in order to correct the Soret excitation overlap between MV and DV Chl(ide) a and between MV and DV Chl(ide) b.