Cytochrome P450 CYP89A9 Is Involved in the Formation of Major Chlorophyll Catabolites during Leaf Senescence in Arabidopsis

Nonfluorescent chlorophyll catabolites (NCCs) were described as products of chlorophyll breakdown in Arabidopsis thaliana. NCCs are formyloxobilin-type catabolites derived from chlorophyll by oxygenolytic opening of the chlorin macrocycle. These linear tetrapyrroles are generated from their fluorescent chlorophyll catabolite (FCC) precursors by a nonenzymatic isomerization inside the vacuole of senescing cells. Here, we identified a group of distinct dioxobilin-type chlorophyll catabolites (DCCs) as the major breakdown products in wild-type Arabidopsis, representing more than 90% of the chlorophyll of green leaves. The molecular constitution of the most abundant nonfluorescent DCC (NDCC), At-NDCC-1, was determined. We further identified cytochrome P450 monooxygenase CYP89A9 as being responsible for NDCC accumulation in wild-type Arabidopsis; cyp89a9 mutants that are deficient in CYP89A9 function were devoid of NDCCs but accumulated proportionally higher amounts of NCCs. CYP89A9 localized outside the chloroplasts, implying that FCCs occurring in the cytosol might be its natural substrate. Using recombinant CYP89A9, we confirm FCC specificity and show that fluorescent DCCs are the products of the CYP89A9 reaction. Fluorescent DCCs, formed by this enzyme, isomerize to the respective NDCCs in weakly acidic medium, as found in vacuoles. We conclude that CYP89A9 is involved in the formation of dioxobilin-type catabolites of chlorophyll in Arabidopsis.     

Isolation and Characterization of a Light-Harvesting Chlorophyll a/b Protein Complex Associated with Photosystem I

A chlorophyll a/b protein complex has been isolated from a resolved native photosystem I complex by mildly dissociating sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chlorophyll a/b protein contains a single polypeptide of molecular weight 20 kilodaltons, and has a chlorophyll a/b ratio of 3.5 to 4.0. The visible absorbance spectrum of the chlorophyll a/b protein complex showed a maximum at 667 nanometers in the red region and a 77 K fluorescence emission maximum at 681 nanometers. Alternatively, by treatment of the native photosystem I complex with lithium dodecyl sulfate and Triton, the chlorophyll a/b protein complex could be isolated by chromatography on Sephadex G-75. Immunological assays using antibodies to the P₇₀₀-chlorophyll a-protein and the photosystem II light-harvesting chlorophyll a/b protein show no cross-reaction between the photosystem I chlorophyll a/b protein and the other two chlorophyll-containing protein complexes.     

Lipoate metabolism in Pseudomonas putida LP: thiolsulfinates of lipoate and bisnorlipoate.

Lipoate thiolsulfinate and two bisnorlipoate thiolsulfinates, as well as the previously identified products of β-oxidation (bisnorlipoate, tetranorlipoate, and β-hydroxybisnorlipoate), were isolated and identified as catabolites of [¹⁴C]lipoate from cultures of Pseudomonas putida LP, an organism capable of growth on lipoic acid as a sole source of carbon and sulfur. The newly identified metabolites were characterized by ion-exchange and paper chromatography and infrared, ultraviolet, and mass spectroscopies. Comparison of the isolated catabolites with synthetic standards implies that the lipoic thiolsulfinate isolated is the S-1 monoxide of 1,2-dithiolane-3-valeric acid; one bisnorlipoic thiolsulfinate isolated is the S-1 monoxide, the other apparently the S-2 monoxide. Metabolic studies with P. putida show that lipoate thiolsulfinate is taken up by this microorganism in an energy-dependent process, but less readily than lipoate; lipoate thiolsulfinate supports oxygen consumption in short-term experiments but does not support growth. These results are interpreted as meaning that the thiolsulfinates are “dead-end” metabolites, not intermediates in the sulfur metabolism of this organism. Lipoate thiolsulfinate is not detectably β-oxidized to bisnorlipoate thiolsulfinate under the usual culture conditions.     

Chlorophyll Catabolites in Senescent Leaves of the Plum Tree (Prunus domestica)

In cold extracts of senescent leaves of the plum tree (Prunus domestica ssp. domestica), six colorless non‐fluorescent chlorophyll catabolites (NCCs) were characterized, named Pd‐NCCs. In addition, several minor NCC fractions were tentatively classified. The structure of the most polar one of the NCCs, named Pd‐NCC‐32, featured an unprecedented twofold glycosidation pattern. Three of the NCCs are also functionalized at their 3²‐position by a glucopyranosyl group. In addition, two of these glycosidated NCCs carry a dihydroxyethyl group at their 18‐position. In the polar Pd‐NCC‐32, the latter group is further glycosidated at the terminal 18²‐position. Four other major Pd‐NCCs and one minor Pd‐NCC were identified with five NCCs from higher plants known to belong to the ‘epi’‐series. In addition, tentative structures were derived for two minor fractions, classified as yellow chlorophyll catabolites, which represented (formal) oxidation products of two of the observed Pd‐NCCs. The chlorophyll catabolites in leaves of plum feature the same basic structural pattern as those found in leaves of apple and pear trees.     

Chlorophyll breakdown in spinach: on the structure of five nonfluorescent chlorophyll catabolites*

In extracts of senescent leaves of spinach (Spinacia oleracea), five colourless compounds with UV/Vis-characteristics of nonfluorescent chlorophyll catabolites (NCCs) were detected and tentatively named So-NCCs. The most abundant polar NCC in the leaves of this vegetable, So-NCC-2, had been isolated earlier and its constitution was determined by spectroscopic means. The catabolite So-NCC-2 was found to be an epimer of a polar NCC from barley (Hordeum vulgare), the first non-green chlorophyll catabolite from a higher plant to be structurally analyzed. Here, we report on the isolation of four additional So-NCCs from the extracts of senescent leaves of Sp. oleracea by two- (or multi-)stage chromatographic purification and on their structural characterization. The constitution of So-NCC-3 could be determined by spectroscopic analysis in combination with chemical correlation with a known NCC from Cercidiphyllum japonicum (Cj-NCC): So-NCC-3 was identified as the hydrolysis product of the methyl ester function of Cj-NCC. The less polar catabolite So-NCC-4 could be directly identified with Cj-NCC. Two further So-NCCs, So-NCC-1 and So-NCC-5, were detected only in trace amounts. Five structurally related nonfluorescent chlorophyll catabolites (So-NCCs) are thus present in senescent leaves of spinach. The structures of this set of So-NCCs indicate several peripheral refunctionalization reactions and inform on the late catabolic transformations during leaf senescence. The transformation of the tetrapyrrolic skeleton in chlorophyll catabolism in spinach and in C. japonicum is revealed to exhibit a common stereochemical pattern. This revised version was published online in June 2006 with corrections to the Cover Date.     

A new form of chlorophyll C involved in light-harvesting

A new form of chlorophyll c has been isolated from the pyrmnesiophyte Pavlova gyrans Butcher. This pigment is spectrally similar to chlorophyll c₂, but all the absorption maxima (454, 583, and 630 nm in diethyl ether) are shifted 4 to 6 nanometers to longer wavelengths. The new pigment can be separated from other chlorophyll c-type pigments by reversed-phase high performance liquid chromatography and thin layer chromatography. Both chlorophylls c₁ and c₂ are found with the new chlorophyll c pigment in P. gyrans, and it has also been detected in the chrysophyte Synura petersenii Korsh. The light-harvesting function of the new chlorophyll c pigment is indicated by its presence along with chlorophyll c₁ and c₂ in a light-harvesting pigment-protein complex isolated from P. gyrans in which chlorophyll c pigments efficiently transfer absorbed light energy to chlorophyll a.     

The effect of light on four protochlorophyllide-binding polypeptides of barley (Hordeum vulgare)

Protochlorophyllide-binding proteins were investigated and possible changes in the pigment-protein association during light-induced chlorophyll synthesis were analyzed. Three major results were obtained. (1) Four protochlorophyllide-binding polypeptides were separated electrophoretically on polyacrylamide gels and visualized by their fluorescence. The number and size of these protochlorophyllide-binding proteins isolated from darkgrown and illuminated plants were not affected by light. (2) The association of pigment with these proteins was studied using exogenous [³H]protochlorophyllide as substrate in an NADPH-dependent in vitro chlorophyll synthesis assay. It was found that a constant exchange of protein-bound and free pigment occurs and that freshly synthesized chlorophyllide does not accumulate on any of the four pigment-binding proteins in vitro. (3) NADPH does not affect the amount of pigment bound to protein in vitro, even during chlorophyll synthesis which occurred only in the presence of NADPH.     

Organization of chlorophyll a in bilayer membranes. The chlorophyll a/dimyristoylphosphatidylcholine system

In order to investigate the relative importance of the hydrophobic and headgroup interactions of chlorophyll a in phospholipid bilayers, we have carried out differential scanning calorimetry (DSC) and deuterium (²H) and phosphorus (³¹P) nuclear magnetic resonance (NMR) experiments on the multilamellar system of chlorophyll a in dimyristoylphosphatidylcholine (DMPC). Compared to the phytol chain of chlorophyll and the previously reported distearoylphosphatidylcholine (DSPC), the acyl chains of DMPC are shorter in length by three and four carbons, respectively. A lowering in the phase-transition temperature was observed for the DMPC multilayers in the presence of chlorophyll a in the DSC thermograms and in the ³¹P chemical shift anisotropy measurements. These results, together with data on hydrophobic interactions as measured by ²H-NMR and on headgroup interactions as evidenced from ³¹P-NMR, suggest a phase diagram for the chlorophyll a/DMPC system in which phase separation readily occurs between a chlorophyll-rich compound phase and a chlorophyll-poor phospholipid phase. Compound formation appears to be important in the stabilization of chlorophyll a in bilayers with shorter chains.     

Crystal Structures of the Substrate-Bound Forms of Red Chlorophyll Catabolite Reductase: Implications for Site-Specific and Stereospecific Reaction

Red chlorophyll catabolite reductase (RCCR) catalyzes the ferredoxin-dependent reduction of the C20/C1 double bond of red chlorophyll catabolite (RCC), the catabolic intermediate produced in chlorophyll degradation. The crystal structure of substrate-free Arabidopsis thaliana RCCR (AtRCCR) demonstrated that RCCR folds into a characteristic α/β/α sandwich, similar to that observed in the ferredoxin-dependent bilin reductase (FDBR) family. Here we have determined the crystal structures of RCC-bound AtRCCR, RCC-bound F218V AtRCCR, and substrate-free F218V AtRCCR, a mutant protein that produces the stereoisomer of primary fluorescent chlorophyll catabolites at the C1 position. RCC is bound to the pocket between the β-sheet and the C-terminal α-helices, as seen in substrate-bound FDBRs, but RCC binding to RCCR is much looser than substrate binding to FDBRs. The loose binding seems beneficial to the large conformational change in RCC upon reduction. Two conserved acidic residues, Glu154 and Asp291, sandwich the C20/C1 double bond of RCC, suggesting that these two residues are involved in site-specific reduction. The RCC in F218V AtRCCR rotates slightly compared with that in wild type to fill in the space generated by the substitution of Phe218 with valine. Concomitantly, the two carboxy groups of Glu154 and Asp291 move slightly away from the C20/C1 double bond. The geometrical arrangement of RCC and the carboxy groups of Glu154 and Asp291 in RCCR would appear to be essential for the stereospecificity of the RCCR reaction.     

Chlorophyll breakdown in oilseed rape

Chlorophyll catabolism accompanying leaf senescence is one of the most spectacular natural phenomena. Despite this fact, the metabolism of chlorophyll has been largely neglegted until recently. Oilseed rape has been used extensively as a model plant for the recent elucidating of structures of chlorophyll catabolites and for investigation of the enzymic reactions of the chlorophyll breakdown pathway. The key reaction which causes loss of green color is catalyzed in a two-step reaction by pheophorbide a oxygenase and red chlorophyll catabolite reductase. In this Minireview, we summarize the actual knowledge about catabolites and enzymes of chlorophyll catabolism in oilseed rape and discuss the significance of this pathway in respect to chlorophyll degradation during Brassica napus seed development. This revised version was published online in June 2006 with corrections to the Cover Date.     

Involvement of AtNAP1 in the regulation of chlorophyll degradation in Arabidopsis thaliana

In plants, chlorophyll is actively synthesized from glutamate in the developmental phase and is degraded into non-fluorescent chlorophyll catabolites during senescence. The chlorophyll metabolism must be strictly regulated because chlorophylls and their intermediate molecules generate reactive oxygen species. Many mechanisms have been proposed for the regulation of chlorophyll synthesis including gene expression, protein stability, and feedback inhibition. However, information on the regulation of chlorophyll degradation is limited. The conversion of chlorophyll b to chlorophyll a is the first step of chlorophyll degradation. In order to understand the regulatory mechanism of this reaction, we isolated a mutant which accumulates 7-hydroxymethyl chlorophyll a (HMChl), an intermediate molecule of chlorophyll b to chlorophyll a conversion, and designated the mutant hmc1. In addition to HMChl, hmc1 accumulated pheophorbide a, a chlorophyll degradation product, when chlorophyll degradation was induced by dark incubation. These results indicate that the activities of HMChl reductase (HAR) and pheophorbide a oxygenase (PaO) are simultaneously down-regulated in this mutant. We identified a mutation in the AtNAP1 gene, which encodes a subunit of the complex for iron–sulfur cluster formation. HAR and PaO use ferredoxin as a reducing power and PaO has an iron-sulfur center; however, there were no distinct differences in the protein levels of ferredoxin and PaO between wild type and hmc1. The concerted regulation of chlorophyll degradation is discussed in relation to the function of AtNAP1.     

Light-Harvesting Function in the Diatom Phaeodactylum tricornutum: I. Isolation and Characterization of Pigment-Protein Complexes

Three pigment-protein complexes were isolated from the marine diatom Phaeodactylum tricornutum (Bohlin) by treatment of thylakoid membrane fragments with 1% Triton X-100 at 4°C followed by centrifugation on sucrose density gradients. The major complex contains chlorophyll a, c₁, c₂, and the carotenoid fucoxanthin (chlorophyll a: c₁: c₂: fucoxanthin = 1.0: 0.09: 0.28: 2.22) bound to an apoprotein doublet of 16.4 and 16.9 kilodaltons. This complex accounts for >70% of the total pigment and 20 to 40% of the protein in the thylakoid membranes. Efficient coupling of chlorophyll c and fucoxanthin absorption to chlorophyll a fluorescence supports a light-harvesting function for the complex. A minor light-harvesting complex containing chlorophyll a, c₁, and c₂ but no fucoxanthin (chlorophyll a: c₁: c₂ = 1.0: 0.23: 0.26) was also isolated at Triton: chlorophyll a ratios between 20 and 40. These pigments are bound to a similar molecular weight apoprotein doublet. The third complex isolated was the P700-chlorophyll a protein, the reaction center of photosystem I, which showed characteristics similar to those isolated from other plant sources. The yield of the chlorophyll a/c-fucoxanthin complex was shown to respond strongly to changes in light intensity during growth, accounting for most of the changes in cellular pigmentation.     

Chlorophyll Turnover in Skeletonema costatum, a Marine Plankton Diatom

[³H]- and δ-[¹⁴C]Aminolevulinic acids were incorporated into the chlorophylls of Skeletonema costatum, a marine plankton diatom. In the stationary phase of growth, the tetrapyrrole-based pigments reached steady-state labeling after 10 hours. Under conditions of exponential cell division and chlorophyll accumulation, ³H was rapidly lost from the labeled chlorophylls and was replaced with ¹⁴C derived from δ-[4−¹⁴C]aminolevulinic acid. The kinetics of isotope dilution suggests recycling of tetrapyrrole precursors and/or two pigment pools, containing both chlorophyll a and chlorophyllide c, one which turns over rapidly (10 hours) and another which turns over more slowly (100 hours). Calculation of turnover times varied from 3 to 10 hours for chlorophyll a and from 7 to 26 hours for chlorophyllide c. The data suggest the dynamics of chlorophyll metabolism in S. costatum and explain the diatom's ability to undergo light-shade adaptation within a generation time.     

Chloroplast culture: The chlorophyll repair potential of mature chloroplasts incubated in a simple medium

The chlorophyll repair potential of mature Cucumis chloroplasts incubated in a simple Tris-HCI/sucrose medium is described. The chloroplasts were isolated from green, fully expanded Cucumis cotyledons which were capable of chlorophyll repair. This was evidenced by a functional chlorophyll biosynthetic pathway in the mature tissue. The biosynthesis of protochlorophyllide from exogenous δ-aminolevulinic acid was used as a marker for the operation of the chlorophyll biosynthetic chain between δ-aminolevulinic acid and protochlorophyllide. The conversion of exogenous protochlorophyllide into chlorophyll a was used as a marker for the operation of the chlorophyll pathway beyond protochlorophyllide. It appeared from these studies that contrary to published reports, unfortified fully developed Cucumis chloroplasts incubated in Tris-HCl/sucrose without the addition of cofactors exhibited a partial and limited chlorophyll repair capability. Their net tetrapyrrole biosynthetic competence from δ-aminolevulinic acid was confined to the accumulation of coproporphyrin. No net tetrapyrrole biosynthesis beyond coproporphyrin was observed. However, the plastids were capable of incorporating small amounts of δ-amino-[4-¹⁴C]levulinic acid into [¹⁴C] protochlorophyllide but were incapable of converting exogenous protochlorophyllide into chlorophyll. After prolonged incubation of the unfortified chloroplasts in the dark, a fluorescent protochlorophyllide-like compound accumulated. This compound [Cp (E₄₃₀-F₆₃₁)] exhibited a soret excitation maximum at 430 nm (E₄₃₀) and a fluorescence emission maximum at 631 nm (F₆₃₁) in methanol/acetone (4 : 1, v/v). Cp (E₄₃₀-F₆₃₁) was shown to be neither protochlorophyllide nor zinc-protochlorophyllide but an enzymatic degradation product of chlorophyll. The exact chemical identity of this compound has not yet been determined.     

The P700-chlorophyll a-protein. Isolation and some characteristics of the complex in higher plants

A P700-chlorophyll a-protein complex has been purified from several higher plants by hydroxylapatite chromatography of Triton X-100-dissociated chloroplast membranes. The isolated material exhibits a red wavelength maximum at 677 nm, major spectral forms of chlorophyll a at 662, 669, 677, and 686 nm, a chlorophyll/P700 ratio of $\text{40–45}\text{1}$, and contains only chlorophyll a and β-carotene of the photosynthetic pigments present in the chloroplast. The spectral characteristics and composition of the higher plant material are homologous to those of the P700-chlorophyll a-protein previously isolated from blue-green algae; however, unlike the blue-green algal component, cytochromes f and b₆ are associated with the higher plant material. Evidence is presented that a chlorophyll a-protein termed “Complex I” which can be isolated from sodium dodecyl sulfate extracts of chloroplast membranes is a spectrally altered form of the eucaryotic P700-chlorophyll a-protein. The isolation procedure described in this paper is a more rapid technique for obtaining the heart of photosystem I than presently exists; furthermore, the P700 photooxidation and reduction kinetics in the fraction are improved over those in other isolated components showing the same enrichment of P700. It appears very probable that the heart of photosystem I is organized in the same manner in all chlorophyll a-containing organisms.     

Crystal Structure of Red Chlorophyll Catabolite Reductase: Enlargement of the Ferredoxin-Dependent Bilin Reductase Family

The key steps in the degradation pathway of chlorophylls are the ring-opening reaction catalyzed by pheophorbide a oxygenase and sequential reduction by red chlorophyll catabolite reductase (RCCR). During these steps, chlorophyll catabolites lose their color and phototoxicity. RCCR catalyzes the ferredoxin-dependent reduction of the C20/C1 double bond of red chlorophyll catabolite. RCCR appears to be evolutionarily related to the ferredoxin-dependent bilin reductase (FDBR) family, which synthesizes a variety of phytobilin pigments, on the basis of sequence similarity, ferredoxin dependency, and the common tetrapyrrole skeleton of their substrates. The evidence, however, is not robust; the identity between RCCR and FDBR HY2 from Arabidopsis thaliana is only 15%, and the oligomeric states of these enzymes are different. Here, we report the crystal structure of A. thaliana RCCR at 2.4 Å resolution. RCCR forms a homodimer, in which each subunit folds in an α/β/α sandwich. The tertiary structure of RCCR is similar to those of FDBRs, strongly supporting that these enzymes evolved from a common ancestor. The two subunits are related by noncrystallographic 2-fold symmetry in which the α-helices near the edge of the β-sheet unique in RCCR participate in intersubunit interaction. The putative RCC-binding site, which was derived by superimposing RCCR onto biliverdin-bound forms of FDBRs, forms an open pocket surrounded by conserved residues among RCCRs. Glu154 and Asp291 of A. thaliana RCCR, which stand opposite each other in the pocket, likely are involved in substrate binding and/or catalysis.     

Molecular cloning and characterization of a chlorophyll degradation regulatory gene from bamboo

Leaf senescence constituted the final stage of leaf development and it is always accompanied by the leaf yellowing. The non-yellowing gene (NYE1), initially identified from Arabidopsis in our laboratory, is a key regulatory gene responsible for chlorophyll degradation during senescence. In this study, an orthologue of AtNYE1 was isolated from the bamboo (Bambusa emeiensis cv. Viridiflavus) and tentatively named BeNYE1. The full length sequence of 1 386 bp contains an open reading frame of 801 bp. The protein encoded by BeNYE1 consists of 266 amino acids. Sequence analysis revealed that BeNYE1 had high similarity with other NYE/SGR proteins from various monocotyledon species. BeNYE1 was strongly induced by natural senescence and dark-induced senescence in bamboo. Driven by a 1.5 kb upstream fragment of AtNYE1, BeNYE1 could rescue the stay-green phenotype of nye1-1. The constitutive over-expression of BeNYE1 could accelerate the chlorophyll degradation. These results indicated that BeNYE1 might play an important role in the regulation of chlorophyll degradation during leaf senescence in bamboo.     

Absorption and fluorescence spectra of chlorophyll-proteins isolated from Euglena gracilis

A spectroscopic study of chlorophyll-protein complexes isolated from Euglena gracilis membranes was carried out to gain information about the state of chlorophyll in vivo and energy transfer in photosynthesis. The membranes were dissociated by Triton X-100 and separated into fractions by sucrose gradient centrifugation and hydroxyapatite chromatography. Four different types of chlorophyll-protein complexes were distinguished from each other and from detergent-solubilized chlorophyll in these fractions by examination of their absorption, fluorescence excitation (400–500 nm) and emission spectra at low temperature. These types were: (1). A mixture of antenna chlorophyll a- and chlorophyll ab-proteins with an absorption maximum at 669 and emission at 682 nm; (2) a P-700-chlorophyll a-protein (chlorophyll: P-700 = 30 : 1), termed CPI with an absorption maximum at 676 nm and emission maxima at 698 and 718 nm; (3) a second chlorophyll a-protein (CPI-2) less enriched in P-700, with an absorption maximum at 676 nm and emission maxima at 680, 722 and 731 nm; (4) a third chlorophyll a-protein (CPa₁) with no P-700, absorption maxima at 670 and 683 nm, and an unusually sharp emission maximum at 687 nm. Treatment of CPa₁ with sodium dodecyl sulfate drastically altered its spectroscopic properties indicating that at least some chlorophyll-proteins isolated with this detergent are partially denatured. The results suggest that the complex absorption spectra of chlorophyll in vivo are caused by varying proportions of different chlorophyll-protein complexes, each with different groups of chlorophyll molecules bound to it and making up a unique entity in terms of electronic transitions.     

Isolation and characterization of a major chlorophyll ac2 light-harvesting protein from a Chroomonas species (Cryptophyceae)

Three chlorophyll-protein complexes of a Chroomonas species (Cryptophyceae) have been separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The two bands at 100 and 42 kDa are Complex I (CP I) and Complex IV (CP IV), the ubiquitous chlorophyll a-proteins associated with Photosystems I and II, respectively. The third 55 kDa band, which had two peptide subunits (24 and 20 kDa), contained both chlorophyll a and chlorophyll c₂ in a molar ratio of 1.4 chlorophyll a to 1 chlorophyll c₂ ($\text{chlorophyll}\text{a}\text{chlorophyll}\text{c}{}_2$ ratio in whole cells = 4). A chlorophyll $\text{a}\text{c}{}_2$ fraction with similar spectral and electrophoretic properties was isolated by digitonin-sucrose density gradient centrifugation. This fraction had no photochemical activity and contained only a single carotenoid species with absorbance maxima in methanol at 424, 448 and 476 nm. Efficient energy transfer from chlorophyll c₂ to chlorophyll a occurred in the complex.