chlorophyll-a and 2-4-divinylprotochlorophyllide

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.

Topics: Amino Acid Sequence; Arabidopsis; Arabidopsis Proteins; Base Sequence; Chlorophyll; Evolution, Molecular; Gene Dosage; Gene Expression Regulation, Plant; Genome, Plant; Molecular Sequence Data; Oxidoreductases; Phylogeny; Prochlorococcus; Protochlorophyllide; Synechococcus

We characterized the pcb2 (pale-green and chlorophyll b reduced 2) mutant. We found through electron microscopic observation that chloroplasts of pcb2 mesophyll cells lacked distinctive grana stacks. High-performance liquid chromatography (HPLC) analysis showed that the pcb2 mutant accumulated divinyl chlorophylls, and the relative amount of divinyl chlorophyll b was remarkably less than that of divinyl chlorophyll a. The responsible gene was mapped in an area of 190 kb length at the upper arm of the 5th chromosome, and comparison of DNA sequences revealed a single nucleotide substitution causing a nonsense mutation in At5g18660. Complementation analysis confirmed that the wild-type of this gene suppressed the phenotypes of the mutation. Antisense transformants of the gene also accumulated divinyl chlorophylls. The genes homologous to At5g18660 are conserved in a broad range of species in the plant kingdom, and have similarity to reductases. Our results suggest that the PCB2 product is divinyl protochlorophyllide 8-vinyl reductase.

Topics: Arabidopsis; Arabidopsis Proteins; Chlorophyll; Chloroplasts; Chromosome Mapping; Codon, Nonsense; DNA, Plant; Gene Expression Regulation, Plant; Genome, Plant; Microscopy, Electron, Transmission; Molecular Sequence Data; Oxidoreductases; Protochlorophyllide; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Suppression, Genetic

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.

Topics: Chlorophyll; Chlorophyll A; Chloroplasts; Intracellular Membranes; NADP; Plants; Protochlorophyllide; Subcellular Fractions; Substrate Specificity

Rhodobacter sphaeroides grown in the presence of nicotinamide excreted bacteriochlorophyll precursors, 2,4-divinyl protochlorophyllide (DV-Pchlide) and a small amount of 2-monovinyl protochlorophyllide (MV-Pchlide). Accumulation of these pigments indicates that nicotinamide inhibited the bacteriochlorophyll biosynthetic pathway site-specifically between DV-Pchlide and MV-Pchlide. This phenomenon is also observed in an aerobic photosynthetic bacterium, Erythrobacter sp. OCh 114. Among 12 nicotinamide derivatives and isomers tested, only nicotinamide was effective, indicating that in addition to the completeness of the pyridine ring skeleton at positions 1 to 3, the carboxylic acid amide group is essential for this inhibition. The technique described in this report permits the simple preparation of large quantities of DV-Pchlide.

Topics: Bacteriochlorophylls; Chlorophyll; Chromatography, High Pressure Liquid; Niacinamide; Protochlorophyllide; Rhodobacter sphaeroides

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.

Topics: Chlorophyll; Chloroplasts; Mathematics; Photosynthesis; Plants; Porphyrins; Protochlorophyllide; Protoporphyrins; Pyrroles; Spectrometry, Fluorescence; Temperature; Tetrapyrroles

The major product of an aerobic reaction mixture containing developing chloroplasts, Mg-protoporphyrin IX, S-adenosylmethionine, and other cofactors was isolated and purified. Structural studies using nuclear magnetic resonance confirmed earlier reports, based on fluorescence and absorption spectra, that this compound is Mg-2,4-divinyl pheoporphyrin a5. The molecular weight determined by secondary-ion mass spectroscopy further confirmed the assigned structure. Absorption and fluorescence spectra indicate that this compound is identical to that reported previously by various workers in less-purified biological extracts. The nuclear magnetic resonance spectrum of the Mg-free base also supports the assigned structure.

Topics: Aerobiosis; Chlorophyll; Chloroplasts; Mass Spectrometry; Micromonosporaceae; Plants; Protochlorophyllide; Rhodobacter sphaeroides; Spectrometry, Fluorescence; Spectrophotometry

It is shown that the protochlorophyllide ester pool of etiolated higher plants is a faithful copy of the protochlorophyllide pool. It is made up of both monovinyl- and divinylprotochlorophyllide esters. Although the two tetrapyrroles exhibited similar emission maxima, they were distinguishable by their Soret excitation maxima, which were found at 436-437 and 443-444 nm, respectively, in ether at 77K. The two pigments were partially separated on thin layers of polyethylene. They were accompanied by two unknown fluorescent compounds. It was also shown that during greening, the protochlorophyllide ester pool maintained a constant qualitative composition. This was in sharp contrast with the drastic qualitative changes undergone by the protochlorophyllide pool of etiolated tissues grown under identical conditions.

Topics: Chlorophyll; Chloroplasts; Esters; Photochemistry; Plants; Protochlorophyllide; Spectrometry, Fluorescence

It is shown that the protochlorophyllide pool of etiolated higher plants is made up of both monovinyl and divinyl protochlorophyllide. Although the two pigments exhibited similar emission maxima, they were distinguishable by their Soret excitation maxima, which were found at 436 to 437 and 443 to 444 nm, respectively, in ether at 77 K. The two pigments were partially separated on thin layers of polyethylene. They were shown to be accompanied by two unknown fluorescent compounds. The latter were designated compound (E451 F626) and compound (E453 F640) where E refers to the Soret excitation maxima and F to the fluorescence emission maxima of the two unknown compounds. Furthermore, it was shown under several different growth conditions that divinyl protochlorophyllide constituted the major component of the protochlorophyllide pool.

Topics: Chlorophyll; Chloroplasts; Plant Development; Protochlorophyllide; Species Specificity; Spectrometry, Fluorescence; Spectrophotometry