Chlorophyll c(CS-170) isolated from Ostreococcus sp. is [7-methoxycarbonyl-8-vinyl]protochlorophyllide a.

The controversial molecular identification of the so-called chlorophyll cCS-170 has been settled. Despite its relevance as a potential biomarker in the study of eukaryotic picophytoplankton, the structure of this chlorophyll remained so far uncertain. A full characterization by NMR, UV-vis, and ESI-MS is reported, revealing this chlorophyll as [7-methoxycarbonyl-8-vinyl]-protochlorophyllide a.

Methods for analysis of photosynthetic pigments and steady-state levels of intermediates of tetrapyrrole biosynthesis.

Tetrapyrroles and carotenoids are required for many indispensable functions in photosynthesis. Tetrapyrroles are essential metabolites for photosynthesis, redox reaction, and detoxification of reactive oxygen species and xenobiotics, while carotenoids function as accessory pigments, in photoprotection and in attraction to animals. Their branched metabolic pathways of synthesis and degradation are tightly controlled to provide adequate amounts of each metabolite (carotenoids/tetrapyrroles) and to prevent accumulation of photoreactive intermediates (tetrapyrroles). Many Arabidopsis mutants and transgenic plants have been reported to show variations in steady-state levels of tetrapyrrole intermediates and contents of different carotenoid species. It is a challenging task to determine the minute amounts of these metabolites to assess the metabolic flow and the activities of both pigment-synthesising and degrading pathways, to unravel limiting enzymatic steps of these biosynthetic pathways, and to characterise mutants with accumulating intermediates. In this chapter, we present a series of methods to qualify and quantify anabolic and catabolic intermediates of Arabidopsis tetrapyrrole metabolism, and describe a common method for quantification of different plant carotenoid species. Additionally, we introduce two methods for quantification of non-covalently bound haem. The approach of analysing steady-state levels of tetrapyrrole intermediates in plants, when applied in combination with analyses of transcripts, proteins, and enzyme activities, enables the biochemical and genetic elucidation of the tetrapyrrole pathway in wild-type plants, varieties, and mutants. Steady-state levels of tetrapyrrole intermediates are only up to 1/1,000 of the amounts of the accumulating end-products, chlorophyll, and haem. Although present in very low amounts, the accumulation and availability of tetrapyrrole intermediates have major consequences on the physiology and activity of chloroplasts due to their additional photoreactive and possible signalling functions. Although adjusted for Arabidopsis tetrapyrrole metabolites, the presented methods can also be applied for analysis of cyanobacterial and other plant tetrapyrroles.

EXECUTER1- and EXECUTER2-dependent transfer of stress-related signals from the plastid to the nucleus of Arabidopsis thaliana.

Shortly after the release of singlet oxygen ((1)O2), drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. In contrast to retrograde control of nuclear gene expression by plastid signals described earlier, the primary effect of (1)O2 generation in the flu mutant is not the control of chloroplast biogenesis but the activation of a broad range of signaling pathways known to be involved in biotic and abiotic stress responses. This activity of a plastid-derived signal suggests a new function of the chloroplast, namely that of a sensor of environmental changes that activates a broad range of stress responses. Inactivation of the plastid protein EXECUTER1 attenuates the extent of (1)O2-induced up-regulation of nuclear gene expression, but it does not fully eliminate these changes. A second related nuclear-encoded protein, dubbed EXECUTER2, has been identified that is also implicated with the signaling of (1)O2-dependent nuclear gene expression changes. Like EXECUTER1, EXECUTER2 is confined to the plastid. Inactivation of both EXECUTER proteins in the ex1/ex2/flu triple mutant is sufficient to suppress the up-regulation of almost all (1)O2-responsive genes. Retrograde control of (1)O2-responsive genes requires the concerted action of both EXECUTER proteins within the plastid compartment.

Fluorescence lifetimes and spectral properties of protochlorophyllide in organic solvents in relation to the respective parameters in vivo.

In this work, absorption and fluorescence spectra of protochlorophyllide (Pchlide), as well as its fluorescence lifetime, were investigated in organic solvents having different physical properties. The obtained Pchlide spectral features are discussed in relation to the parameters describing solvent properties (refractive index and dielectric constant) and taking into account the specific solvent-Pchlide interaction. The correlation of Pchlide Qy and Soret absorption bands with solvent polarizability function ((n2 - 1)/(n2 + 2)) has been found; however, the dispersion of the observed points was rather high. A small Stokes shift of a magnitude between 50 and 300 cm(-1) was found, which indicates low sensitivity of Pchlide to nonspecific solvation. The fluorescence decay of Pchlide was single exponential in all the investigated solvents, with the lifetime value ranging from 5.2 ns for dioxane to 3.5 ns for methanol. Dependence of the obtained fluorescence lifetimes on the solvent orientation polarizability, a parameter being the function of both refractive index and dielectric constant, was discussed. In water-methanol mixtures, a further decrease of the fluorescence lifetime was observed, giving values of 2.9 ns for 25% methanol. Double-exponential decay of Pchlide fluorescence was found for Pchlide in a solution of 15% methanol with the lifetimes of 4.5 +/- 0.5 ns and 1.2 +/- 0.3 ns and in pure water with the lifetimes of 2.5 +/- 0.5 ns and 0.4 +/- 0.1 ns. The obtained results are discussed in relation to spectroscopic properties of Pchlide in vivo.

Tyr275 and Lys279 stabilize NADPH within the catalytic site of NADPH:protochlorophyllide oxidoreductase and are involved in the formation of the enzyme photoactive state.

Fluorescence spectroscopic and kinetic analysis of photochemical activity, cofactor and substrate binding, and enzyme denaturation studies were performed with highly purified, recombinant pea NADPH:protochlorophyllide oxidoreductase (POR) heterologously expressed in Escherichia coli. The results obtained with an individual stereoisomer of the substrate [C8-ethyl-C13(2)-(R)-protochlorophyllide] demonstrate that the enzyme photoactive state possesses a characteristic fluorescence maximum at 646 nm that is due to the presence of specific charged amino acids in the enzyme catalytic site. The photoactive state is converted directly into an intermediate having fluorescence at 685 nm in a reaction involving direct hydrogen transfer from the cofactor (NADPH). Site-directed mutagenesis of the highly conserved Tyr275 (Y275F) and Lys279 (K279I and K279R) residues in the enzyme catalytic pocket demonstrated that the presence of these two amino acids in the wild-type POR considerably increases the probability of photoactive state formation following cofactor and substrate binding by the enzyme. At the same time, the presence of these two amino acids destabilizes POR and increases the rate of enzyme denaturation. Neither Tyr275 nor Lys279 plays a crucial role in the binding of the substrate or cofactor by the enzyme. In addition, the presence of Tyr275 is absolutely necessary for the second step of the protochlorophyllide reduction reaction, "dark" conversion of the 685 nm fluorescence intermediate and the formation of the final product, chlorophyllide. We propose that Tyr275 and Lys279 participate in the proper coordination of NADPH and PChlide in the enzyme catalytic site and thereby control the efficiency of the formation of the POR photoactive state.

Compositional heterogeneity of protochlorophyllide ester in etiolated leaves of higher plants.

The formation and degradation of protochlorophyllide esters, i.e., protochlorophylls, were studied in etiolated leaves of kidney bean in relation to their aging. By the sensitive analysis of the pigments using high-performance liquid chromatography, the presence of four protochlorophylls esterified with phytol, tetrahydrogeranylgeraniol (THGG), dihydrogeranylgeraniol (DHGG), and geranylgeraniol (GG) was detected in kidney bean grown in the dark. Similar components were also observed in the etiolated seedlings of cucumber, sunflower, and corn. The content of each protochlorophyll species changed with the plant species and age of plants. In the case of kidney bean, the content of protochlorophyll phytol reached a maximal level at 9 days, then decreased rapidly during the subsequent development, in spite of the total protochlorophyll content remaining unchanged. In contrast to the degradation of protochlorophyll phytol, the other three protochlorophylls esterified with THGG, DHGG, and GG accumulated. These results may indicate that (i) protochlorophyll phytol is formed from the first esterified protochlorophyll GG through the next three hydrogenation steps as in the case of chlorophyll a phytol formation; (ii) the esterification reaction stops at 9 days and then reaction proceeds in sequence in the reverse direction, leading to the dehydrogenation of the alcohol moiety of protochlorophyll phytol to protochlorophylls THGG, DHGG, and GG.

Some properties of purified fractions from bean etioplast membranes.

Distribution of proteins, protochlorophyllide and photoactivity was studied in fractions obtained from bean etioplast membranes purified by linear or discontinuous sucrose density gradient centrifugation. The polypeptide pattern of two selected membrane fractions was examined by means of polyacrylamide gel electrophoresis. Both fractions contained the doublet of molecular weights 34.000-35.000 characteristic of NADPH-Protochlorophyllide oxido-reductase but differed by accompanying proteins. When both fractions were illuminated, protochlorophyllide was photoreduced in chlorophyllide fluorescing at 688 nm. During a subsequent 30 min. dark period, the emission peak shifted rapidly up to 678 nm in one of the fractions while it moved slowly to 683 nm in the other one. The possible occurrence of two phototransformable protochlorophyllide-complexes is discussed. Delta-aminolevulinic acid pretreatment of etiolated leaves led to an increase in the amount of proteins in the etioplast membranes.

[Isolation and properties of the pigment-protein complex (protochlorophyllide - holochrome) from etiolated leaves of corn sprouts]. / Poluchenie i svoistva pigment-belkovogo kompleksa (protokhlorofillid-golokhroma) iz list'ev etiolirovannykh prorostkov kukuruzy.

A pigment -- protein complex was isolated from etiolated leaves of corn sprouts using treatment by the detergent Triton X-100, ultracentrifugation and gel-filtration. The complex has a molecular weight of 50000 and upon illumination can convert protochlorophyllide (Pchld) into chlorophyllide (Chld). The low temperature fluorescence spectra of dark preparations reveal maxima of Pchld 632 (photo-inactive form) and Pchld 653 (photo-active form). Upon illumination Pchld 653 is converted into Chld 685. The kinetics of the precursor photoconversion into Chld are coincident with those for photoconversion in native etiolated corn leaves. The complex is completely inactivated at a decrease of pH down to 6.5 or during incubation at high temperature (30 degrees).

Chloroplasts biogenesis: detection of divinylprotochlorophyllide ester in higher plants.

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.