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.     

7-Hydroxymethyl chlorophyll a reductase functions in metabolic channeling of chlorophyll breakdown intermediates during leaf senescence

During natural or dark-induced senescence, chlorophyll degradation causes leaf yellowing. Recent evidence indicates that chlorophyll catabolic enzymes (CCEs) interact with the photosynthetic apparatus; for example, five CCEs (NYC1, NOL, PPH, PAO and RCCR) interact with LHCII. STAY-GREEN (SGR) and CCEs interact with one another in senescing chloroplasts; this interaction may allow metabolic channeling of potentially phototoxic chlorophyll breakdown intermediates. 7-Hydroxymethyl chlorophyll a reductase (HCAR) also acts as a CCE, but HCAR functions during leaf senescence remain unclear. Here we show that in Arabidopsis, HCAR-overexpressing plants exhibited accelerated leaf yellowing and, conversely, hcar mutants stayed green during dark-induced senescence. Moreover, HCAR interacted with LHCII in in vivo pull-down assays, and with SGR, NYC1, NOL and RCCR in yeast two-hybrid assays, indicating that HCAR is a component of the proposed SGR-CCE-LHCII complex, which acts in chlorophyll breakdown. Notably, HCAR and NOL are expressed throughout leaf development and are drastically down-regulated during dark-induced senescence, in contrast with SGR, NYC1, PPH and PAO, which are up-regulated during dark-induced senescence. Moreover, HCAR and NOL are highly up-regulated during greening of etiolated seedlings, strongly suggesting a major role for NOL and HCAR in the chlorophyll cycle during vegetative stages, possibly in chlorophyll turnover.     

Endolithic chlorophyll d-containing phototrophs

Cyanobacteria in the genus Acaryochloris are the only known oxyphototrophs that have exchanged chlorophyll a (Chl a) with Chl d as their primary photopigment, facilitating oxygenic photosynthesis with near infrared (NIR) light. Yet their ecology and natural habitats are largely unknown. We used hyperspectral and variable chlorophyll fluorescence imaging, scanning electron microscopy, photopigment analysis and DNA sequencing to show that Acaryochloris-like cyanobacteria thrive underneath crustose coralline algae in a widespread endolithic habitat on coral reefs. This finding suggests an important role of Chl d-containing cyanobacteria in a range of hitherto unexplored endolithic habitats, where NIR light-driven oxygenic photosynthesis may be significant.     

The appearance of chlorophyll derivatives in senescing tissue

Chlorophylls a-1 and b′, which are breakdown products of chlorophylls a and b respectively, were found in senescing leaves of Phaseolus vulgaris and Hordeum vulgare following excision from the plant. Chlorophyll a-1 was not detected in healthy plants, in senescing attached leaves or in chlorophyll-proteins isolated from senescent tissue. Chlorophyll a-1 formation in excised leaves increased with time for up to 10 days as chlorophyll a levels fell.     

Peroxidase-mediated chlorophyll degradation in horticultural crops

One of the symptoms of senescence in harvested horticultural crops is the loss of greenness that comes with the degradation of chlorophyll (Chl). With senescence, peroxidase, which is involved in Chl degradation, increased greatly in stored horticultural crops. C13²-hydroxychlorophyll a, an oxidized form of Chl a, is formed in vitro through Chl oxidation by peroxidase. Peroxidase mediates Chl degradation in the presence of phenolic compounds such as p-coumaric acid and apigenin, which have a hydroxyl group at the p-position. Apparently, not all phenolic compounds are able to degrade Chl in this system, and their effectiveness appears to depend on their molecular configuration. In peroxidase-mediated Chl degradation, peroxidase oxidizes the phenolic compounds with hydrogen peroxide and forms phenoxy radical; then, the phenoxy radical oxidizes Chl and its derivatives to colorless low molecular weight compounds through the formation of C13²-hydroxychlorophyll a,a fluorescent Chl catabolite and a bilirubin-like compound as an intermediate. In addition to the phenoxy radical, superoxide anion, which is formed in the peroxidase-catalyzed reaction, might be involved in Chl oxidation. Moreover, Chl degradation by peroxidase seems to occur in the chloroplast and/or the vacuole. The involvement of peroxidase in Chl degradation in senescing horticultural crops is also discussed.     

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 photosynthetic response to a shift in the chlorophyll a to chlorophyll B ratio of chlorella

The chlorophyll a:b ratio was shifted in Chlorella vannielii by varying the illuminance under which the cells were cultured—the ratio increased from 2.9, 3.0, 4.0, and 4.8 to 6.2, respectively, at 100, 300, 900, 2,700 and 6,000 foot candles. The 6,000-foot candle cells retained an optimal growth rate at the chlorophyll a:b ratio of 6.2 which was the upper limit of normal growth. Comparisons were made between the 300-and 6,000-foot candle cultures to determine the significance to the photosynthetic mechanism of a shift in the chlorophyll a:b ratio.     

Use of the atLEAF+ chlorophyll meter for a nondestructive estimate of chlorophyll content

At present, chlorophyll meters are widely used for a quick and nondestructive estimate of chlorophyll (Chl) contents in plant leaves. Chl meters allow to estimate the Chl content in relative units - the Chl index (CI). However, using such meters, one can face a problem of converting CI into absolute values of the pigment content and comparing data acquired with different devices and for different plant species. Many Chl meters (SPAD-502, CL-01, CCM-200) demonstrated a high degree of correlation between the CI and the absolute pigment content. A number of formulas have been deduced for different plant species to convert the CI into the absolute value of the photosynthetic pigment content. However, such data have not been yet acquired for the atLEAF+ Chl meter. The purpose of the present study was to assess the applicability of the atLEAF+ Chl meter for estimating the Chl content. A significant species-specific exponential relationships between the atLEAF value (corresponding to CI) and extractable Chl a, Chl b, Chl (a+b) for Calamus dioicus and Cleistanthus sp. were shown. The correlations between the atLEAF values and the content of Chl a, Chl b, and Chl (a+b) per unit of leaf area was stronger than that per unit of dry leaf mass. The atLEAF value- Chl b correlation was weaker than that of atLEAF value-Chl a and atLEAF value-Chl (a+b) correlations. The influence of light conditions (Chl a/b ratio) on the atLEAF value has been also shown. The obtained results indicated that the atLEAF+ Chl meter is a cheap and convenient tool for a quick nondestructive estimate of the Chl content, if properly calibrated, and can be used for this purpose along with other Chl meters.     

Peroxidase-catalysed oxidation of chlorophyll by hydrogen peroxide

Chlorophyll is effectively bleached by H₂O₂ in the presence of certain phenols and peroxidase (EC 1.11.1.7) extracted from acetone powders of orange flavedo (Citrus sinensis). Optimal conditions for chlorophyll: hydrogen peroxide oxidoreductase include: pH, 5.9; [H₂O₂] 222 μM; ionic strength 0.11. A phenol is required and resorcinol is the most effective. Catechol and hydroquinone are inhibitory. Chlorophyll a, chlorophyllide a, and chlorophyll b all have similar V_max but K_m for chlorophyll a is about one-third that of chlorophyll b, while the K_m for chlorophyllide a is about one-half that of chlorophyll a. Pheophytin a was much less reactive than chlorophyll a, and Mg²⁺ included in the reaction system did not affect rates of pheophytin destruction.     

Redox potential of chlorophyll d in vitro

Chlorophyll (Chl) d is a major chlorophyll in a novel oxygenic prokaryote Acaryochloris marina. Here we first report the redox potential of Chl d in vitro. The oxidation potential of Chl d was + 0.88 V vs. SHE in acetonitrile; the value was higher than that of Chl a (+ 0.81 V) and lower than that of Chl b (+ 0.94 V). The oxidation potential order, Chl b > Chl d > Chl a, can be explained by inductive effect of substituent groups on the conjugated π-electron system on the macrocycle. Corresponding pheophytins showed the same order; Phe b (+ 1.25 V) > Phe d (+ 1.21 V) > Phe a (+ 1.14 V), but the values were significantly higher than those of Chls, which are rationalized in terms of an electron density decrease in the π-system by the replacement of magnesium with more electronegative hydrogen. Consequently, oxidation potential of Chl a was found to be the lowest among Chls and Phes. The results will help us to broaden our views on photosystems in A. marina.     

Enzymatic degradation of chlorophyll a by marine phytoplankton in vitro

The enzymatic degradation of chlorophyll a and the formation of chlorophyllide a, phaeophytin a, and phaeophorbide a were detected in vitro in several species of marine phytoplankton. Loss of phytol and Mg²⁺ were found to be catalysed by chlorophyllase and a magnesium-releasing enzyme, respectively. The activities of the two enzymes could be distinguished from each other by inhibiting with Mg²⁺ and/or p-chloromercurobenzoate. Both enzymes are activated by cell disintegration. Degradation products were not detected spectrophotometrically in vivo. Additionally, in some species, chlorophyll a was degraded to products which do not absorb visible light.     

Energy transfer by chlorophyll a in detergent micelles

The process of energy transfer was studied in the chlorophyll a-containing detergent micelle, serving as a possible model of the photosynthetic unit. Chlorophyll a was added to aqueous solutions of the detergent Triton X-100 and incorporated into the micelles. The energy transfer process was studied by investigating the concentration depolarization of fluorescence of chlorophyll a. On the basis of the experimental depolarization curves as well as the value of the Förster parameter $\text{R}{}_0\text{= 56}\text{A}\text{?}$ calculated from the overlap of absorption and fluorescence spectra it was concluded that energy transfer between chlorophyll a molecules in this model follows the Förstertype mechanism of inductive resonance. Furthermore it was found that the local concentration of chlorophyll a in the micelles is higher by 1–3 orders of magnitude than its overall concentration in the solution and by choosing the appropriate ratio between the concentration of chlorophyll a and the detergent it is possible to reach the in vivo chlorophyll concentration of 0.1 M within the micelles. Thus the chlorophyll-detergent micelle model may be applied as a model of the separate package-type photosynthetic unit.     

Water content of chlorophyll hydrate

Thermogravimetry shows that polycrystalline chlorophyll a is a chlorophyll dihydrate. Neither thermogravimetry nor differential thermal analysis indicates the existence of a stable chl a monohydrate. Moreover it is found that larger amounts of water-free chlorophyll a (>50 mg) cannot be prepared by application of vacuum and heat in a reasonable time, if chemical decomposition is to be avoided. Thin layers of polycrystalline chlorophyll a undergo spectroscopic changes depending on temperature and vacuum.     

Protoheme turnover and chlorophyll synthesis in greening barley tissue

Studies in which ¹⁴C-labeled precursors were fed to etiolated barley leaves (Hordeum vulgare L. var. Proctor) yielded chlorophyll and protoheme having similar specific radioactivities. These findings indicate: (a) there appears to be a rapid turnover of protoheme in the absence of net synthesis; (b) both pigments probably originate from a single 5-aminolevulinic acid pool; (c) the efficient utilization of glutamate-1-¹⁴C and the relatively poor utilization of glycine-2-¹⁴C suggest that 5-aminolevulinic acid is probably synthesized by a pathway other than 5-aminolevulinic acid synthetase (succinyl CoA-glycine succinyltransferase) in agreement with previously published work; (d) protoheme turnover appears to be faster under conditions which allow for rapid chlorophyll accumulation; (e) difference spectra indicate that mitochondrial cytochromes make a relatively minor contribution to the total heme in barley leaves. These findings are discussed in the light of current knowledge about tetrapyrrole regulation in photosynthetic organisms.     

Intramembranous particles and chlorophyll complexes in chloroplasts

The size and population density of large and small particles from freeze-fractured chloroplasts of three wild-type algae and of normal spinach were determined.Computer analyses of low-temperature absorption spectra of chloroplast preparations from these species were performed, and a possible correlation between the occurrence of seven chlorophyll complexes and the aforementioned properties of the intramembranous particles was studied.It was found that only single-sized particles occur in a species containing neither chlorophyll b nor chlorophyll a-685 complexes. The three remaining species carry particles of two sizes, termed large and small particles. However, from quantitative considerations it is concluded that the chlorophyll content of none of the various pigment complexes is related to the size and the population density of the studied particles. If such a relationship exists, it seems likely to be due to the carrier moiety of the chlorophyll b · chlorophyll a-685 complex.     

Dinitrogen fixation in a unicellular chlorophyll d-containing cyanobacterium

Marine cyanobacteria of the genus Acaryochloris are the only known organisms that use chlorophyll d as a photosynthetic pigment. However, based on chemical sediment analyses, chlorophyll d has been recognized to be widespread in oceanic and lacustrine environments. Therefore it is highly relevant to understand the genetic basis for different physiologies and possible niche adaptation in this genus. Here we show that unlike all other known isolates of Acaryochloris, the strain HICR111A, isolated from waters around Heron Island, Great Barrier Reef, possesses a unique genomic region containing all the genes for the structural and enzymatically active proteins of nitrogen fixation and cofactor biosynthesis. Their phylogenetic analysis suggests a close relation to nitrogen fixation genes from certain other marine cyanobacteria. We show that nitrogen fixation in Acaryochloris sp. HICR111A is regulated in a light–dark-dependent fashion. We conclude that nitrogen fixation, one of the most complex physiological traits known in bacteria, might be transferred among oceanic microbes by horizontal gene transfer more often than anticipated so far. Our data show that the two powerful processes of oxygenic photosynthesis and nitrogen fixation co-occur in one and the same cell also in this branch of marine microbes and characterize Acaryochloris as a physiologically versatile inhabitant of an ecological niche, which is primarily driven by the absorption of far-red light.     

Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromothymoquinone

The quenching action of dibromothymoquinone on fluorescence and on primary photochemistry was examined in chloroplasts at ?196 °C. Both the initial (F₀) and final (F_M) levels of fluorescence as well as the fluorescence of variable yield (F_v = F_M ? F₀) were quenched at ?196 °C to a degree which depended on the concentration of dibromothymoquinone added prior to freezing. The initial rate of photoreduction of C-550 at — 196 °C, which was assumed to be proportional to maximum yield for primary photochemistry, ?_Po, was also decreased in the presence of dibromothymoquinone. Simple theory predicts that the ratio $\text{F}{}_{\mathrm{<mtext>V</mtext>}}\text{F}{}_{\mathrm{<mtext>M</mtext>}}$ should equal ?_Po. Excellent agreement was found in a comparison of relative values of ?_Po with relative values of $\text{F}{}_{\mathrm{<mtext>V</mtext>}}\text{F}{}_{\mathrm{<mtext>M</mtext>}}$ at various degrees of quenching by dibromothymoquinone. These results are taken to indicate that F₀ and F_V are the same type of fluorescence, both emanating from the bulk chlorophyll of Photosystem II.Dibromothymoquinone appears to create quenching centers in the bulk chlorophyll of Photosystem II which compete with the reaction centers for excitation energy. The rate constant for the quenching of excitation energy by dibromothymoquinone is directly proportional to the concentration of the quencher. Rate constants for the de-excitation of excited chlorophyll molecules by fluorescence, k_F, by nonradiative decay processes, k_D, by photochemistry, k_P, and by the specific quenching of dibromothymoquinone, k_Q, were calculated assuming the absolute yield of fluorescence at F₀ to be either 0.02 or 0.05.