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PubMed1996

Substrate specificity of chlorophyll(ide) b reductase in etioplasts of barley (Hordeum vulgare L.).

Enzyme activity of chlorophyll(ide) b reductase is present in etioplasts. Recently the conversion of chlorophyllide b to chlorophyll a via 7(1)-hydroxychlorophyll a was demonstrated in barley etioplasts. We used zinc pheophorbide b for a detailed investigation of the reduction of the 7-formyl group to the 7(1)-hydroxy compound in intact barley etioplasts. The reaction proceeded likewise before esterification and after esterification with phytyl diphosphate. The metal-free pheophorbide b, that is not accepted by chlorophyll synthase for esterification, is reduced to 7(1)-hydroxypheophorbide a to a small extent. The zinc (13(2)S)-pheophorbide b is at least equally well accepted for reduction as the epimer with the 13(2)R configuration of natural chlorophyll b. The reaction requires NADPH or NADH, although the latter is less effective. ATP is not required for the first step to the 7(1)-hydroxy compound. The significance of chlorophyll b reduction for acclimation from shade to sun leaves and for chlorophyll degradation is discussed.

Adenosine Triphosphate↗
PubMed1992

A chloroplast gene is required for the light-independent accumulation of chlorophyll in Chlamydomonas reinhardtii.

The light-independent pathway of chlorophyll synthesis which occurs in some lower plants and algae is still largely unknown. We have characterized a chloroplast mutant, H13, of Chlamydomonas reinhardtii which is unable to synthesize chlorophyll in the dark and is also photosystem I deficient. The mutant has a 2.8 kb deletion as well as other rearrangements of its chloroplast genome. By performing particle gun mediated chloroplast transformation of H13 with defined wild-type chloroplast DNA fragments, we have identified a new chloroplast gene, chlN, coding for a 545 amino acid protein which is involved in the light-independent accumulation of chlorophyll, probably at the step of reduction of protochlorophyllide to chlorophyllide. The chlN gene is also found in the chloroplast genomes of liverwort and pine, but is absent from the chloroplast genomes of tobacco and rice.

Algal Proteins↗
PubMed1983

The light-dependent control of chloroplast development in barley (Hordeum vulgare L).

The light-induced greening of etiolated barley plants is used as a model to study the light-dependent control of plastid development. Upon illumination a rapid transformation of etioplasts to chloroplasts is induced. The effect of illumination does not only include the light-dependent chlorophyll synthesis but also the appearance or decline of specific proteins within the plastid membrane fractions. So far two of these proteins have been studied in detail. The light-harvesting chlorophyll a/b protein (LHCP) is one of the major protein constituents of the thylakoid membrane of chloroplasts. However, this protein is not detectable among the membrane polypeptides of etioplasts. Illumination of dark-grown barley plants induces a massive insertion of the LHCP. The appearance of the protein is controlled by the cooperation of at least two distinct photoreceptors: protochlorophyllide and phytochrome. In dark-grown barley plants not only the LHCP but also its mRNA is not detectable. The light-dependent appearance of mRNA activity for the LHCP is under the control of phytochrome (Pfr). Even though the appearance of mRNA activity is induced via Pfr by a single red light pulse, the assembly of the complete LHCP takes place only under continuous illumination, which allows chlorophyll synthesis. The second protein analyzed so far is the NADPH-protochlorophyllide-oxidoreductase. This enzyme catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide and thus controls one of the first detectable light-dependent reactions during the greening period. It is generally assumed that this enzyme is responsible for the overall chlorophyll synthesis and accumulation during the greening period.(ABSTRACT TRUNCATED AT 250 WORDS)

Chlorophyll↗
PubMed2004

Semiempirical PM5 molecular orbital study on chlorophylls and bacteriochlorophylls: comparison of semiempirical, ab initio, and density functional results.

The semiempirical PM5 method has been used to calculate fully optimized structures of magnesium-bacteriochlorin, magnesium-chlorin, magnesium-porphin, mesochlorophyll a, chlorophylls a, b, c(1), c(2), c(3), and d, and bacteriochlorophylls a, b, c, d, e, f, g, and h with all homologous structures. Hartree-Fock/6-31G* ab initio and density functional B3LYP/6-31G* methods were used to optimize structures of methyl chlorophyllide a, chlorophyll c(1), and methyl bacteriochlorophyllides a and c for comparison. Spectroscopic transition energies of the chromophores and their 1:1 or 1:2 solvent complexes were calculated with the Zindo/S CIS method. The self-consistent reaction field model was used to estimate solvent shifts. The PM5 calculations predict planar structure of the porphyrin ring and central position of the four coordinated magnesium atoms in all pigments studied, in accord with the experimental, ab initio, and density functional results, a significant improvement as compared to the older semiempirical PM3 approach. Only small differences in PM5 and B3LYP/6-31G* or Hartree-Fock/6-31G* minimum energy geometries of the reference molecules were observed. Calculations show that in 1:1 solvent complexes, where the magnesium atom is five coordinated, the magnesium atom is shifted out of the plane of the porphyrin ring towards the solvent molecule, while the hexa coordinated 1:2 complexes are again planar. The PM5 method gives atomic charges that are comparable with those obtained from the Hartree-Fock/6-31G* and B3LYP/6-31G* calculations. The single point ZINDO/S CIS calculations with PM5 minimum energy structure gave excellent correlations between calculated and experimental transition energies of the chlorophylls and bacteriochlorophylls studied. Such correlations may be used for prediction of transition energies of the chromophores in protein binding sites. Calculations also predict existence of dark electronic states below the main Soret absorption band in all chromophores studied. The results suggest that the semiempirical PM5 method is a fairly reliable and computationally efficient method in predicting molecular parameters of porphyrin-like molecules.

Bacteriochlorophylls↗
PubMed1995

Light-enhanced gene expression of NADPH-protochlorophyllide oxidoreductase in cucumber.

The NADPH-protochlorophyllide oxidoreductase (NPR) is a well known enzyme catalyzing photoconversion of protochlorophyllide to chlorophyllide a and its expression is negatively regulated by light. We cloned a NPR cDNA from cucumber (Cucumis sativus L.). The cDNA contained an open reading frame encoding 398 amino acids and amino acid sequence showed high similarity to those of NPR from other plants. Contrasting with most other plants, the levels of the NPR mRNA increased in etiolated cotyledons of cucumber when they were illuminated with continuous light, suggesting that the expression of this NPR gene was strongly related to chlorophyll synthesis in green leaves.

Amino Acid Sequence↗
PubMed2002

A 3-D structural model of solid self-assembled chlorophyll a/H(2)O from multispin labeling and MAS NMR 2-D dipolar correlation spectroscopy in high magnetic field.

Magic angle spinning (MAS) NMR with Lee-Goldburg cross-polarization (LG-CP) is used to promote long-range heteronuclear transfer of magnetization and to constrain a structural model for uniformly labeled chlorophyll a/H(2)O. An effective maximum transfer range d(max) can be determined experimentally from the detection of a gradually decreasing series of intramolecular correlations with the (13)C along the molecular skeleton. To probe intermolecular contacts, d(max) can be set to approximately 4.2 A by choosing an LG-CP contact time of 2 ms. Long-range (1)H-(13)C correlations are used in conjunction with carbon and proton aggregation shifts to establish the stacking of the chlorophyll a (Chl a) molecules. First, high-field (14.1 T) 2-D MAS NMR homonuclear ((13)C-(13)C) dipolar correlation spectra provide a complete assignment of the carbon chemical shifts. Second, proton chemical shifts are obtained from (1)H-(13)C heteronuclear dipolar correlation spectroscopy in high magnetic field. The shift constraints and long-range (1)H-(13)C intermolecular correlations reveal a 2-D stacking homologous to the molecular arrangement in crystalline solid ethyl-chlorophyllide a. A doubling of a small subset of the carbon resonances, in the 7-methyl region of the molecule, provides evidence for two marginally different well-defined molecular environments. Evidence is found for the presence of neutral structural water molecules forming a hydrogen-bonded network to stabilize Chl a sheets. In line with the microcrystalline order observed for the rings, the long T(1)'s, and absence of conformational shifts for the (13)C in the phytyl tails, it is proposed that the Chl a form a rigid 3-D space-filling structure. Probably the only way this can be realized with the sheets is by forming bilayers with interpenetration of elongated tails. Such a 3-D space-filling organization of the aggregated Chl a from MAS NMR would match existing models inferred from electron microscopy and low-resolution X-ray powder diffraction, while a micellar model based on neutron diffraction and antiparallel stacking observed in solution can be discarded.

Carbon Isotopes↗
PubMed1996

Transcripts of the two NADPH protochlorophyllide oxidereductase genes PorA and PorB are differentially degraded in etiolated barley seedlings.

The light-dependent reduction of protochlorophyllide to chlorophyllide in higher plants is catalyzed by two closely related enzymes, the NADPH-Pchlide oxidoreductases A and B that are encoded by the nuclear genes PorA and PorB, respectively. The expression of the PorA gene is negatively regulated by light. It has formerly been reasoned that, apart from the well-studied transcriptional down-regulation, a post-transcriptional mechanism may exist that contributes markedly to the light-induced decline of PorA mRNA steady-state levels. We investigated the degradation kinetics of the PorA messenger after inhibiting RNA synthesis with cordycepin. The PorA mRNA was found to be inherently unstable. In contrast, the PorB mRNA was shown to be stabilized in the presence of cordycepin, suggesting degradation by a mechanism different from that of PorA mRNA degradation. The PorA messenger instability is postulated to be conferred by a previously described plant-specific DST element in its 3'UTR.

Base Sequence↗
PubMed1996

Identification of three cDNA clones expressed in the leaf extension zone and with altered patterns of expression in the slender mutant of barley: a tonoplast intrinsic protein, a putative structural protein and protochlorophyllide oxidoreductase.

Three cDNA clones have been isolated on the basis of altered patterns of expression in the leaf extension zone of the developmental mutant, slender barley, compared with the wild type. mRNAs corresponding to two of the cDNAs, 7s and 8s, are increased in slender compared with normal. 7s encodes a putative gamma-TIP and is expressed throughout the elongation zone. gamma-TIPs form transmembrane channels which allow the passive transfer of water. Although expression of 7s was increased in slender leaf tissue, the increase was much less extreme than that shown by Phillips and Huttly (1994) following the application of GA to an extreme dwarf of Arabidopsis. 8s is maximally expressed in the region of early cell elongation and has 66% encoded protein identity with MFS18, a cDNA encoding a putative cell wall structural protein isolated from male flowers of maize. Both 8s and MFS18 encode small (128 amino acids) basic proteins rich in glycine, alanine, proline and serine. mRNA corresponding to the third cDNA, 24n, is present at a greatly reduced level in slender compared with normal and encodes protochlorophyllide oxidoreductase (POR). POR catalyses the conversion of protochlorophyllide into chlorophyllide. The reduced level of POR mRNA is not correlated with a similar reduction in expanded leaf blade chlorophyll levels. Western analysis identified two POR proteins present in light-grown seedlings. Whilst the larger of the proteins is present throughout most of the leaf, the smaller protein mimics the mRNA results, being both maximally present in the elongation tissue and present at a reduced level in slender. An antagonistic relationship between chlorophyll biosynthesis and extension growth is suggested.

Amino Acid Sequence↗
PubMed1995

The in vitro assembly of the NADPH-protochlorophyllide oxidoreductase in pea chloroplasts.

The NADPH-protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) is the major protein in the prolamellar bodies (PLBs) of etioplasts, where it catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide during chlorophyll synthesis in higher plants. The suborganellar location in chloroplasts of light-grown plants is less clear. In vitro assays were performed to characterize the assembly process of the pchlide reductase protein in pea chloroplasts. Import reactions employing radiolabelled precursor protein of the pchlide reductase showed that the protein was efficiently imported into fully matured green chloroplasts of pea. Fractionation assays following an import reaction revealed that imported protein was targeted to the thylakoid membranes. No radiolabelled protein could be detected in the stromal or envelope compartments upon import. Assembly reactions performed in chloroplast lysates showed that maximum amount of radiolabelled protein was associated to the thylakoid membranes in a thermolysin-resistant conformation when the assays were performed in the presence of hydrolyzable ATP and NADPH, but not in the presence of NADH. Furthermore, membrane assembly was optimal at pH 7.5 and at 25 degrees C. However, further treatment of the thylakoids with NaOH after an assembly reaction removed most of the membrane-associated protein. Assembly assays performed with the mature form of the pchlide reductase, lacking the transit peptide, showed that the pre-sequence was not required for membrane assembly. These results indicate that the pchlide reductase is a peripheral protein located on the stromal side of the membrane, and that both the precursor and the mature form of the protein can act as substrates for membrane assembly.

Adenosine Triphosphate↗
PubMed1999

Chlorophyll b reduction during senescence of barley seedlings

During senescence of flowering plants, only breakdown products derived from chlorophyll a were detected although b disappears, too (Matile et al., 1996, Plant Physiol 112: 1403-1409). We investigated the possibility of chlorophyll b reduction during dark-induced senescence of barley (Hordeum vulgare L.) leaves. Plastids isolated from senescing leaves were lysed and incubated with NADPH. We found 7(1)-hydroxy-chlorophyll a, 7(1)-hydroxy-chlorophyllide a, and, after incubation with Zn-pheophorbide b, also Zn-7(1)-hydroxy-pheophorbide a, indicating activity of chlorophyll(ide) b reductase. The highest activity was found at day 2 of senescence when chlorophyll breakdown reached its highest rate. Chlorophyllase reached its highest activity under the same conditions only at days 4-6 of senescence. Based on the chlorophyll b reductase activity of plastids at day 2.5 of senescence (=100%), the bulk of activity (83%) was found in the thylakoids and only traces (5%) in the envelope fraction. Chlorophyll b reduction is considered to be an early and obligatory step of chlorophyll b breakdown.

Journal Article↗
PubMed1986

Preparation and properties of the water-soluble chlorophyll-bovine serum albumin complexes.

By mixing chlorophyll (Chl) a or b with a dense bovine serum albumin solution, the water-soluble Chl-bovine serum albumin complexes were prepared. These complexes, eluted near the void volume on a gel filtration, were separated well from unreacted bovine serum albumin, indicating an aggregation of such molecules in the complexes. Preparation of chlorophyllide (Chlide) a- or Chlide b-bovine serum albumin complex was unsuccessful, while the phytol-, and beta-carotene-bovine serum albumin complexes could be obtained. Chls in the Chl-bovine serum albumin complexes had the following characteristics. Main absorption peak of Chl a or b in the red region occurred at 675 nm or 652 nm, respectively. The Chl a-bovine serum albumin complex having absorption peak at 740 nm was also prepared. As compared with the stabilities of Chl a and b in Triton X-100. Both Chls in the bovine serum albumin-complexes were stable against oxidative stresses, such as photobleaching, Fenton reagent, peroxidase-H2O2 system. But they were easily hydrolyzed by chlorophyllase. These properties of Chls in the bovine serum albumin-complexes were similar to those of Chls in the isolated light-harvesting Chl a/b protein complex. A possible localization of Chls within the bovine serum albumin complexes was suggested that the porphyrin moiety of Chl was buried in bovine serum albumin; however, the hydrophilic edge of porphyrin ring, adjacent to the phytol group, occurred in the hydrophilic region of a bovine serum albumin molecule.

Chlorophyll↗
PubMed1992

Detection of the photoactive protochlorophyllide-protein complex in the light during the greening of barley.

A photoactive protochlorophyllide-protein complex with absorbance and fluorescence maxima at 648 and 653 nm was detected in greening barley leaves without any re-darkening. The variations of the amplitudes of the absorbance and the fluorescence of the photoactive protochlorophyllide with greening time at two different light intensities indicate a close relationship between the rate of chlorophyll synthesis and the amount of the complex during the first hours. The chlorophyllide resulting from photoreduction during greening has an absorbance maximum at 684 nm, which shifts towards a shorter wavelength within a few seconds, indicating rapid liberation of the pigment from the enzyme. We conclude that chlorophyll accumulation proceeds through continuous regeneration and phototransformation of the photoactive complex.

Darkness↗
PubMed1996

Stabilization of the chlorophyll binding apoproteins, P700, CP47, CP43, D2, and D1, by synthesis of Zn-pheophytin a in intact etioplasts from barley.

Chlorophyll a was compared with Zn-pheophytin a for stabilization of chlorophyll binding apoproteins, P700, CP47, CP43, D2, and D1, in intact etioplasts from barley (Hordeum vulgare L.). Intact etioplasts were shown to effectively translate the chlorophyll apoproteins, to take up and esterify the exogenously added substrates, chlorophyllide a and Zn-pheophorbide a, with geranylgeraniolpyrophosphate. For stabilization of P700, CP47, D2, and D1, the product, Zn-pheophytin a, was shown to substitute for chlorophyll a. Stabilization of CP43 was selectively increased in the presence of Zn-pheophytin a. The degree of stabilization was shown to depend on the amount of newly synthesized Zn-pheophytin a and on the central atom of the chlorophyll molecule.

Chlorophyll↗
PubMed1993

Phototransformation of monovinyl and divinyl protochlorophyllide by NADPH:protochlorophyllide oxidoreductase of barley expressed in Escherichia coli.

The chlorophyll precursors monovinyl protochlorophyllide (MV-PChlide) and divinyl protochlorophyllide (DV-PChlide) were extracted from mutant C-2A' of the unicellular green alga Scenedesmus obliquus which accumulates both protochlorophyllide derivatives in the dark. The two pigments were characterized by absorption and fluorescence spectroscopy and by plasma desorption mass spectrometry. The molecular masses of MV-PChlide and DV-PChlide were determined as 612 and 610 atomic mass units (amu) respectively. Both MV-PChlide and DV-PChlide were accepted as substrates and photoconverted to chlorophyllides in vitro by NADPH:protochlorophyllide oxidoreductase of barley expressed in Escherichia coli.

Chlorophyta↗
PubMed2005

15N-labeling to determine chlorophyll synthesis and degradation in Synechocystis sp. PCC 6803 strains lacking one or both photosystems.

Rates of chlorophyll synthesis and degradation were analyzed in Synechocystis sp. PCC 6803 wild type and mutants lacking one or both photosystems by labeling cells with ((15)NH(4))(2)SO(4) and Na(15)NO(3). Pigments extracted from cells were separated by HPLC and incorporation of the (15)N label into porphyrins was subsequently examined by MALDI-TOF mass spectrometry. The life time (tau) of chlorophyll in wild-type Synechocystis grown at a light intensity of 100 micromol photons m(-2) s(-1) was determined to be about 300 h, much longer than the cell doubling time of about 14 h. Slow chlorophyll degradation (tau approximately 200-400 h) was also observed in Photosystem I-less and in Photosystem II-less Synechocystis mutants, whereas in a mutant lacking both Photosystem I and Photosystem II chlorophyll degradation was accelerated 4-5 fold (tau approximately 50 h). Chlorophyllide and pheophorbide were identified as intermediates of chlorophyll degradation in the Photosystem I-less/Photosystem II-less mutant. In comparison with the wild type, the chlorophyll synthesis rate was five-fold slower in the Photosystem I-less strain and about eight-fold slower in the strain lacking both photosystems, resulting in different chlorophyll levels in the various mutants. The results presented in this paper demonstrate the presence of a regulation that adjusts the rate of chlorophyll synthesis according to the needs of chlorophyll-binding polypeptides associated with the photosystems.

Chlorophyll↗
PubMed2006

Aggregation of the 636 nm emitting monomeric protochlorophyllide form into flash-photoactive, oligomeric 644 and 655 nm emitting forms in vitro.

Artificial formation of flash-photoactive oligomeric protochlorophyllide complexes was found in etiolated pea (Pisum sativum L. cv. Zsuzsi) epicotyl homogenates containing glycerol (40% v/v) and sucrose (40% m/v). The 77 K fluorescence emission spectra indicated that the ratio of the 644 and 655 nm emitting forms to the 636 nm form increased during 3 to 5-day incubation in the dark at -14 degrees C. Electron micrographs showed the presence of well-organized prolamellar bodies in the homogenates. The same phenomena were found when the homogenates were frozen into liquid nitrogen and thawed to room temperature in several cycles. Similar treatments of intact epicotyl pieces caused significant membrane destructions. In homogenates, the in vitro produced 644 and 655 nm emitting protochlorophyllide forms were flash-photoactive; the extent of phototransformation increased compared to that in native epicotyls. The newly appeared 692 nm chlorophyllide band showed a blue shift (similar to the Shibata shift in leaves), however this process took place only partially due to the effect of the isolation medium. These results prove that the in vitro accumulated 644 and 655 nm protochlorophyllide forms were produced from the flash-photoactive 636 nm emitting monomeric NADPH:protochlorophyllide oxidoreductase units via aggregation, in connection with structure stabilization properties of glycerol and sucrose.

Freezing↗
PubMed2005

Occurrence of chlorophyll precursors in leaves of cabbage heads--the case of natural etiolation.

In the interior of cabbage (Brassica oleracea) heads (kale, white cabbage, Brussels sprouts), natural leaf etiolation takes place due to a limited light access and chlorophyll biosynthesis is inhibited in a consequence. Instead, apart from carotenoids, whose biosynthesis is not light-dependent, chlorophyll precursors accumulate, mainly protochlorophyllide and to a smaller extent also chlorophyllides a and b. Protochlorophyllide was also detected in green, light-exposed leaves of heads of all the investigated cabbage varieties. Protochlorophyll was not found in the investigated leaves. The analysis of xanthophylls composition showed that the central leaves of kale and white cabbage heads contain relatively high amounts of trans-neoxanthin and lutein epoxide which are found only in trace amounts in green leaves. This is the first systematic study on natural occurrence of chlorophyll biosynthesis precursors in different cabbage varieties.

Brassica↗
PubMed2005

Green genes gleaned.

A recent paper by Ayumi Tanaka and colleagues identifying an Arabidopsis thaliana gene for 3,8-divinyl(proto)chlorophyllide 8-vinyl reductase brings a satisfying conclusion to the hunt for genes encoding enzymes for the steps in the chlorophyll biosynthetic pathway. Now, at least in angiosperm plants represented by Arabidopsis, genes for all 15 steps in the pathway from glutamyl-tRNA to chlorophylls a and b have been identified.

Arabidopsis↗