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PubMed2006

Consistent sets of spectrophotometric chlorophyll equations for acetone, methanol and ethanol solvents.

A set of equations for determining chlorophyll a (Chl a) and accessory chlorophylls b, c2, c1 + c2 and the special case of Acaryochloris marina, which uses Chl d as its primary photosynthetic pigment and also has Chl a, have been developed for 90% acetone, methanol and ethanol solvents. These equations for different solvents give chlorophyll assays that are consistent with each other. No algorithms for Chl c compounds (c2, c1 + c2) in the presence of Chl a have previously been published for methanol or ethanol. The limits of detection (and inherent error, +/- 95% confidence limit), for chlorophylls in all organisms tested, was generally less than 0.1 microg/ml. The Chl a and b algorithms for green algae and land plants have very small inherent errors (< 0.01 microg/ml). Chl a and d algorithms for Acaryochloris marina are consistent with each other, giving estimates of Chl d/a ratios which are consistent with previously published estimates using HPLC and a rarely used algorithm originally published for diethyl ether in 1955. The statistical error structure of chlorophyll algorithms is discussed. The relative error of measurements of chlorophylls increases hyperbolically in diluted chlorophyll extracts because the inherent errors of the chlorophyll algorithms are constants independent of the magnitude of absorbance readings. For safety reasons, efficient extraction of chlorophylls and the convenience of being able to use polystyrene cuvettes, the algorithms for ethanol are recommended for routine assays of chlorophylls. The methanol algorithms would be convenient for assays associated with HPLC work.

Acetone↗
PubMed1976

Protein-protein interactions of the light-harvesting chlorophyll a/b protein. II. Evidence for two stages of cation independent association.

In a previous paper, we observed a two-stage cation-independent association of the light-harvesting chlorophyll a/b protein from spinach chloroplasts based on concentration-dependent changes in the sedimentation coefficient. The two stages of association occurred between (2-4) and (4-7) mug/ml chlorophyll. In this paper, we provide further evidence for this association. This includes: (1) A decrease in the number of divalent cation binding sites in the second stage of association. (2) A corresponding decrease in the extent of the cation-dependent association. (3) A positive deviation from Beer's law for chlorophyll b for both stages of the cation-independent association and a positive deviation for chlorophyll a for the second stage of association only. (4) A change in the fluorescence emission of both chlorophyll a and b. The change for chlorophyll b was observed for both steps of association whereas that for chlorophyll a was observed for the second step of association only. Therefore, the first stage of association affects only chlorophyll b whereas the second stage alters the environment of both chlorophyll a and b. (5) In addition, divalent cations quenched chlorophyll fluorescence. However, the quenching which required 200-300 muM divalent cation for half-maximal effects was related neither to divalent cation binding nor to the divalent cation-induced association of the protein.

Aerobiosis↗
PubMed1977

Appearance and composition of chlorophyll-protein complexes I and II during chloroplast membrane biogenesis in Chlamydomonas reinhardi y-1.

The chlorophyll-protein complexes I and II have been isolated and anlyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis during greening and degreening of Chlamydomonas reinhardi y-1. At all stages of membrane formation, the complexes, when present, have a constant composition. Chlorophyll-protein complex I consists of a major polypeptide(s) of molecular weight 64,000 synthesized in the chloroplast, to which about 29 chlorophyll a molecules are bound. The complex is not detected when other polypeptides of chloroplastic origin, related to both Photosystem I and Photosystem II activities, are not synthesized. However, Photosystem I activity can develop in membranes in which chlorophyll-protein complex I is not detectable. Chlorophyll-protein complex II consists of two polypeptides of cytoplasmic origin, molecular weights 24,000 and 22,000, which bind 12 chlorophylls (a and b). The chlorophyll-protein complex II can be detected in membranes in which the development of photosystem II activity is prevented. Clipping of a Mr = 2000 fragment(s) from the Mr = 22,000 polypeptide following trypsin digestion of membranes, does not affect the complex. The detection of the complexes is possible only in membranes in which the simultaneous synthesis of both the chlorophyll and the corresponding polypeptides occurs. The 28,000 dalton polypeptide, reported to be present in the chlorophyll-protein complex II, comigrates with the complex but apparently is not part of the complex itself. The apparent molecular weight of the chlorophyll-protein complexes I and II are 88,000 and 28,000, respectively. The minimal true value for complex I is 89,000 or 154,000 and for complex II is 56,000.

Binding Sites↗
PubMed2005

Differential changes in degradation of chlorophyll-protein complexes of photosystem I and photosystem II during flag leaf senescence of rice.

The stability of chlorophyll-protein complexes of photosystem I (PSI) and photosystem II (PSII) was investigated by chlorophyll (Chl) fluorescence spectroscopy, absorption spectra and native green gel separation system during flag leaf senescence of two rice varieties (IIyou 129 and Shanyou 63) grown under outdoor conditions. During leaf senescence, photosynthetic CO(2) assimilation rate, carboxylase activity of Rubisco, chlorophyll and carotenoids contents, and the chlorophyll a/b ratio decreased significantly. The 77 K Chl fluorescence emission spectra of thylakoid membranes from mature leaves had two peaks at around 685 and 735 nm emitting mainly from PSII and PSI, respectively. The total Chl fluorescence yields of PSI and PSII decreased significantly with senescence progressing. However, the decrease in the Chl fluorescence yield of PSI was greater than in the yield of PSII, suggesting that the rate of degradation in chlorophyll-protein complexes of PSI was greater than in chlorophyll-protein complexes of PSII. The fluorescence yields for all chlorophyll-protein complexes decreased significantly with leaf senescence in two rice varieties but the extents of their decrease were significantly different. The greatest decrease in the Chl fluorescence yield was in PSI core, followed by LHCI, CP47, CP43, and LHCII. These results indicate that the rate of degradation for each chlorophyll-protein complex was different and the order for the stability of chlorophyll-protein complexes during leaf senescence was: LHCII>CP43>CP47>LHCI>PSI core, which was partly supported by the green gel electrophoresis of the chlorophyll-protein complexes.

Carbon Dioxide↗
PubMed1988

Biogenesis of thylakoid membranes is controlled by light intensity in the conditional chlorophyll b-deficient CD3 mutant of wheat.

Biogenesis of thylakoid membranes in the conditional chlorophyll b-deficient CD3 mutant of wheat is dramatically altered by relatively small differences in the light intensity under which seedlings are grown. When the CD3 mutant is grown at 400 microE/m2 S (high light, about one-fifth full sunlight) plants are deficient in chlorophyll b (chlorophyll a/b ratio greater than 6.0) and lack or contain greatly reduced amounts of the chlorophyll a/b-binding complexes CPII/CPII (mobile or peripheral LHCII), CP29, CP24 and LHCI, as shown by mildly denaturing 'green gel' electrophoresis, by fully denaturing SDS-PAGE, and by Western blot analysis. High light CD3 chloroplasts display an unusual morphology characterized by large, sheet-like stromal thylakoids formed into parallel unstacked arrays and a limited number of small grana stacks displaced toward the edges of the arrays. Changes in the supramolecular organization of CD3 thylakoids, seen with freeze-fracture electron microscopy, include a reduction in the size of EFs particles, which correspond to photosystem II centers with variable amounts of attached LHCII, and a redistribution of EF particles from the stacked to the unstacked regions. When CD3 seedlings are grown at 150 microE/m2 S (low light) there is a substantial reversal of all of these effects. Thus, chlorophyll b and the chlorophyll a/b-binding proteins accumulate to near wild-type levels (chlorophyll a/b ratio = 3.5-4.5) and thylakoid morphology is more nearly wild type in appearance. Growth of the CD3 mutant in the presence of chloramphenicol stimulates the accumulation of chlorophyll b and its binding proteins (Duysen, M. E., T. P. Freeman, N. D. Williams, and L. L. Huckle. 1985. Plant Physiol. 78:531-536). We show that this partial rescue of the CD3 high light phenotype is accompanied by large changes in thylakoid structure. The CD3 mutant, which defines a new class of chlorophyll b-deficient phenotype, is discussed in the more general context of chlorophyll b deficiency.

Chlorophyll↗
PubMed2001

A theoretical analysis of the influence of heterogeneity in chlorophyll distribution on leaf reflectance.

Attempts to determine the vitality of vegetation and to detect vegetation stress from remotely sensed data have focused on chlorophyll concentration, because it influences the reflectance of vegetation and tends to correlate with vegetation health and stress. Pollution, pathogens and pests can cause localized regions of chlorosis and necrosis across a leaf surface, but the extent to which these patches influence the overall reflectance and spectral signature of the leaf and canopy has not been tested. A conifer leaf model (LIBERTY), which simulates the influence of leaf biochemical concentrations of chlorophyll, water, lignin, cellulose and protein on the reflectance of leaves from 400 to 2500 nm, was used to determine the effect of patches of chlorosis on leaf reflectance. A fraction of the leaf f is assumed to be chlorotic with a chlorophyll concentration C(1). The remainder of the leaf has chlorophyll concentration C(2) such that mean leaf chlorophyll concentration, C(mean) = fC(1) + (1 - f)C(2), is constant for a range of f and C(1) values. LIBERTY can be used to estimate the reflectance of a leaf with a particular chlorophyll concentration at a particular wavelength R(lambda,C) (assuming other leaf properties remain constant), thus we can estimate the reflectance of the chlorotic leaf as fR(lambda,C(1))+ (1 - f)R(lambda,C(2)). The model indicated that small areas of chlorosis have a disproportionately large influence on overall leaf reflectance. For example, a leaf with 25% of its area chlorotic can have the same reflectance (400-700 nm) as a homogeneous leaf with 60% less chlorophyll. Thus, determination of chlorophyll concentration from remotely sensed data is prone to underestimation when chlorophyll is nonuniformly distributed. Hence, attempts to model leaf and canopy reflectance using radiative transfer models will need to consider how to incorporate nonuniform chlorophyll distribution.

Chlorophyll↗
PubMed2005

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.

Amino Acid Sequence↗
PubMed1995

Light-dependent chlorophyll a biosynthesis upon chlL deletion in wild-type and photosystem I-less strains of the cyanobacterium Synechocystis sp. PCC 6803.

Part of the chlL gene encoding a component involved in light-independent protochlorophyllide reduction was deleted in wild type and in a photosystem I-less strain of Synechocystis sp. PCC 6803. In resulting mutants, chlorophyll biosynthesis was fully light-dependent. When these mutants were propagated under light-activated heterotrophic growth conditions (in darkness except for 15 min of weak light a day) for several weeks, essentially no chlorophyll was detectable but protochlorophyllide accumulated. Upon return of the chlL- mutant cultures to continuous light, within the first 6 h chlorophyll was synthesized at the expense of protochlorophyllide at a rate independent of the presence of photosystem I. Chlorophyll biosynthesized during this time gave rise to a 685 nm fluorescence emission peak at 77 K in intact cells. This peak most likely originates from a component different from those known to be directly associated with photosystems II and I. Development of 695 and 725 nm peaks (indicative of intact photosystem II and photosystem I, respectively) required longer exposures to light. After 6 h of greening, the rate of chlorophyll synthesis slowed as protochlorophyllide was depleted. In the chlL- strain, greening occurred at the same rate at two different light intensities (5 and 50 microE m-2 s-1), indicating that also at low light intensity the amount of light is not rate-limiting for protochlorophyllide reduction. Thus, in this system the rate of chlorophyll biosynthesis is limited neither by biosynthesis of photosystems nor by the light-dependent protochlorophyllide reduction. We suggest the presence of a chlorophyll-binding 'chelator' protein (with 77 K fluorescence emission at 685 nm) that binds newly synthesized chlorophyll and that provides chlorophyll for newly synthesized photosynthetic reaction centers and antennae.

Bacterial Proteins↗
PubMedDate not supplied

Chlorophyll-protein complexes of brown algae: P700 reaction centre and light-harvesting complexes.

Thylakoid membranes from several brown algae have been fragmented with the non-ionic detergent, Triton X-100. Three intrinsic chlorophyll-protein complexes with different pigment compositions have been isolated by sucrose density gradient centrifugation. Brown algae contain the photosystem 1 reaction-centre complex, a P700-chlorophyll a-protein which has similar spectroscopic and chemical properties to those of higher plants. This complex represents about 10--20% of the total chlorophyll in all species; the Acrocarpia paniculata complex has a chlorophyll/P700 ratio of 38. Two main light-harvesting complexes have also been isolated, which have properties unique to brown algae. The heavier of these, an orange fraction, is a fucoxanthin-chlorophyll a/c-protein; this complex contains most of the fucoxanthin and has only chlorophyll c2. The other, a green fraction, is a chlorophyll a/c-protein enriched in violaxanthin. Neither of these complexes possesses detectable photosystem 1 or photosystem 2 activities. Both of these complexes efficiently transfer light energy to chlorophyll a, indicating that the molecular arrangement of their pigments is similar to that in vivo. Differential extraction of thylakoid membranes indicates that the P700-chlorophyll a-protein is the complex most firmly embedded in the membrane, but the fucoxanthin-chlorophyll a/c-protein is the least firmly bound. We suggest that the fucoxanthin complex is the most variable component of the photosynthetic unit of brown algal chloroplasts.

Cell Membrane↗
PubMed1983

Chlorophyll-protein organization of membranes from the cyanobacterium Anacystis nidulans.

Six chlorophyll-containing bands were observed upon electrophoretic analysis of Anacystis nidulans thylakoid membranes. These ranged in apparent molecular weights from approximately 360 to 45 kdalton. Measurements of the light absorption and chlorophyll fluorescence properties of these bands revealed numerous differences among the aggregates. The larger chlorophyll-protein complexes had a chlorophyll absorption maximum at 676 nm while the smallest band, band VI, at approximately 45 kdalton, absorbed at 668 nm. The chlorophyll-protein organization of four submembrane particles was also examined. Digitonin and N-tetradecyl-N,N-dimethyl-3-ammonio-1-pro-panesulfonate were used to fractionate thylakoids and each treatment yielded two green fractions after sucrose density gradient centrifugation. The upper green fractions of both procedures were enriched in band VI. In addition, these fractions showed low temperature fluorescence emission at 686 nm. Conversely, the lower green fractions were enriched in the larger bands (bands I and II), and yielded fluorescence emission at 696 and 716 nm. The gel electrophoresis analysis of these chlorophyll-protein bands revealed 11 peptides ranging in size from less than 10 to 64 kdaltons. The larger CP bands contained as many as five to six polypeptides, whereas band VI contained only two species (at 45 and 48 kdalton). These data suggest that the only proteins in band V (approximately 75 kdalton) and band VI are the chlorophyll binding proteins for photosystems I and II, respectively. We present a model which correlates chlorophyll-protein organization and specific fluorescence emission peaks. Central to this model is the interaction of the larger chlorophyll-protein complexes with bands V and VI to yield fluorescence at 696 and 716 nm, respectively. In addition, the polypeptide composition of each complex allows us to construct a topological model of these complexes within the Anacystis thylakoid.

Chemical Phenomena↗
PubMed1976

Photoelectrolysis using chlorophyll electrodes.

By the conversion of sunlight to chemical energy, photoelectrolysis was carried out using two different chlorophyll-redox compound lining electrodes. These electrodes were prepared by covering platinum plates with chlorophyll and naphthoquinone or anthrahydroquinone and a conducting adhesive. These electrodes exhibit a photoexciting property. The potential was found to shift to a less noble state when the system of the chlorophyll-naphthoquinone electrode was inserted into NAD solution with illumination. On the other hand, the photoexcitation of the system of the chlorophyll-anthrahydroquinone electrode inserted into ferrocyanide solution made the potential more noble. (If the potential in the dark is in the dark is in the positive region of the scheme and the potential moves in the positive direction when the light is turned on, it can be said to be more positive region, it can be said to become less noble, but it is not suitable to say that it becomes more negative.) To make the potential difference between two electodes as big as possible, various factors such as intensity of illumination, molar ratio of chlorophyll to naphthoquinone or anthrahydroquinone and concentration of redox compound in electrolyte were examined. A cell was set up by combining the system of the chlorophyll-naphthoquinone electrode in NAD solution with that of the chlorophyll-anthrahydroquinone electode in ferrocyanide solution, and photoelectrolysis was carried out by closing the external circuit with illumination. The photovoltage between the two electrodes was 0.25 V and the current density was 8 muA/cm2. It was found that NAD was reduced to form NADH at the chlorophyll-naphthoquinone electrode and ferrocyanide was oxidized to form ferricyanide at the chlorophyll-anthrahydroquinone electrode.

Chlorophyll↗
PubMed1977

Excitation energy transfer in the light-harvesting chlorophyll a/b.protein.

The "light-harvesting chlorophyll a/b.protein" described by Thornber has been prepared electrophoretically from spinach chloroplasts. The optical properties relevant to energy transfer have been measured in the red region (i.e. 600-700 nm). Measurements of the absorption spectrum, fluorescence excitation spectrum and excitation dependence of the fluorescence emission spectrum of this protein confirm that energy transfer from chlorophyll b to chlorophyll a is highly efficient, as is the case in concentrated chlorophyll solutions and in vivo. The excitiation dependence of the fluorescence polarization shows a minimum polarization of 1.9% at 650 nm which is the absorption maximum of chlorophyll b in the protein and rises steadily to a maximum value of 13.8% at 695 nm, the red edge of the chlorophyll a absorption band. Analysis of these measurements shows that at least two unresolved components must be responsible for the chlorophyll a absorption maximum. Comparison of polarization measurements with those observed in vivo shows that most of the depolarization observed in vivo can take place within a single protein. Circular dichroism measurements show a double structure in the chlorophyll b absorption band which suggest an exciton splitting not resolved in absorption. Analysis of these data yields information about the relative orientation of the So leads to S1 transition moments of the chlorophyll molecules within the protein.

Chlorophyll↗
PubMed1978

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-HCl/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 protocholorphyllide from exogenous delta-aminolevulinic acid was used as a marker for the operation of the chlorophyll biosynthetic chain between delta-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 delta-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 delta-amino[4-(14)C]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) was shown to be neither protochlorophyllide nor zinc-prototochlorophyllide but 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-PROTOTOCHLOROPHYLLIDE BUT an enzymatic degradation product of chlorophyll. The exact chemical identity of this compound has not yet been determined.

Cells, Cultured↗
PubMed1979

Analysis of absorption spectra changes induced by temperature lowering on phycobilisomes, thylakoids and chlorophyll-protein complexes.

Using fourth derivative analysis, differences between room and low temperature absorption spectra were studied. The positions of most absorption bands of the water-soluble, accessory pigment complex, the phycobilisome, remained unchanged after cooling. The stability of the wavelength positions of chlorophyll a forms in vivo as a function of temperature (Gulyaev, B.A. and Litvin, F.F. (1967) Biofizika 12, 845--854) was generally confirmed. The wavelength positions of all chlorophyll a forms in the P-700 chlorophyll a protein complex were unchanged when the preparations were cooled to -196 degrees C. Likewise, with other chlorophyll-containing materials: the light-harvesting chlorophyll a/b protein complex and the thylakoids of higher plants, algae, and cyanobacteria, the wavelengths positions of most chlorophyll a forms were stable upon cooling. An exception was a 680 nm chlorophyll a band which was generally split at low temperature into two bands with the materials investigated. An interpretation of the multiplicity of chlorophyll spectral forms and the spectral changes induced by cooling for these forms is given using exciton theory and the energy-coupling variation of chlorophyll a molecules.

Chlorophyll↗
PubMedDate not supplied

The metabolic activation of 4-nitro-o-phenylenediamine by chlorophyll-containing plant extracts: the relationship between mutagenicity and antimutagenicity.

Chlorophyllin, the sodium and copper salt of chlorophyll, chlorophyll a, and chlorophyll b were tested for their ability to inhibit the mutagenic activity of the direct-acting mutagen 4-nitro-o-phenylenediamine (NOP) and its plant-activated mutagenic enhancement. All three forms of chlorophyll were antimutagenic against both NOP and its plant-activated product, with chlorophyllin proving most effective. Chlorophyll-containing plant extracts, however, proved very efficient at activating NOP into a mutagen of greater potency. When these extracts were assayed for total chlorophyll content it was found that they contained far less chlorophyll than was required for an antimutagenic effect to occur. Thus, the balance between chemical mutagen activation and/or enhancement by chlorophyll-containing plant extracts and the potential antimutagenicity of these plant extracts is a function of chlorophyll concentration. The data presented here indicate that this balance must be taken into consideration in future studies investigating the efficacy of complex natural plant extracts as antimutagenic substances.

Biotransformation↗
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↗
PubMed2000

Energy transfer among CP29 chlorophylls: calculated Förster rates and experimental transient absorption at room temperature.

The energy transfer rates between chlorophylls in the light harvesting complex CP29 of higher plants at room temperature were calculated ab initio according to the Förster mechanism (Förster T. 1948, Ann. Physik. 2:55-67). Recently, the transition moment orientation of CP29 chlorophylls was determined by differential linear dichroism and absorption spectroscopy of wild-type versus mutant proteins in which single chromophores were missing (Simonetto R., Crimi M., Sandonà D., Croce R., Cinque G., Breton J., and Bassi R. 1999. Biochemistry. 38:12974-12983). In this way the Q(y) transition energy and chlorophyll a/b affinity of each binding site was obtained and their characteristics supported by reconstruction of steady-state linear dichroism and absorption spectra at room temperature. In this study, the spectral form of individual chlorophyll a and b ligands within the protein environment was experimentally determined, and their extinction coefficients were also used to evaluate the absolute overlap integral between donors and acceptors employing the Stepanov relation for both the emission spectrum and the Stokes shift. This information was used to calculate the time-dependent excitation redistribution among CP29 chlorophylls on solving numerically the Pauli master equation of the complex: transient absorption measurements in the (sub)picosecond time scale were simulated and compared to pump-and-probe experimental data in the Q(y) region on the native CP29 at room temperature upon selective excitation of chlorophylls b at 640 or 650 nm. The kinetic model indicates a bidirectional excitation transfer over all CP29 chlorophylls a species, which is particularly rapid between the pure sites A1-A2 and A4-A5. Chlorophylls b in mixed sites act mostly as energy donors for chlorophylls a, whereas site B5 shows high and bidirectional coupling independent of the pigment hosted.

Biophysical Phenomena↗
PubMed2001

Assessment of degradation and intestinal cell uptake of carotenoids and chlorophyll derivatives from spinach puree using an in vitro digestion and Caco-2 human cell model.

Although numerous studies have demonstrated the health benefits of chlorophyll derivatives, information regarding the digestion, absorption, and metabolism of these phytochemicals is quite limited. To better understand the digestion of these pigments, green vegetables including fresh spinach puree (FSP), heat- and acid-treated spinach puree (HASP), and ZnCl(2)-treated spinach puree (ZnSP) were subjected to an in vitro digestion method which simulates both the gastric and small intestinal phases of the process. Native chlorophylls were converted to Mg-free pheophytin derivatives during digestion. Conversely, Zn-pheophytins were completely stable during the digestive process. Transfer of lipophilic chlorophyll derivatives, as well as the carotenoids lutein and beta-carotene, into the aqueous micellar fraction from the food matrix was quantified. Micellarization of total chlorophyll derivatives differed significantly (p < 0.05) for FSP (37.6%), HASP (17.2%), and ZnSP (8.7%). Micellarization of chlorophyll a derivatives was determined to be significantly more efficient than chlorophyll b derivatives in FSP and HASP (p < 0.01), but not in ZnSP (p > 0.05). Intestinal cell uptake of micellarized pigments was investigated using HTB-37 (parent) and clonal TC7 lines of human Caco-2 cells. Medium containing the pigment-enriched fraction generated during digestion was added to the apical surface of fully differentiated monolayers for 4 h. Pigments were then extracted from cells and analyzed by C18 HPLC with photodiode array detection. Both Caco-2 HTB-37 and TC7 clone cells accumulated 20-40% and 5-10% of micellarized carotenoid and chlorophyll derivatives, respectively. These results are the first to demonstrate uptake of chlorophyll derivatives by human intestinal cells and to support the potential importance of chlorophylls as health-promoting phytochemicals.

Biological Availability↗