The carotenoid zeaxanthin and ‘high-energy-state quenching’ of chlorophyll fluorescence

The possibility that zeaxanthin mediates the dissipation of an excess of excitation energy in the antenna chlorophyll of the photochemical apparatus has been tested through the use of an inhibitor of violaxanthin de-epoxidation, dithiothreitol (DTT), as well as through the comparison of two closely related organisms (green and blue-green algal lichens), one of which (blue-green algal lichen) naturally lacks the xanthophyll cycle. In spinach leaves, DTT inhibited a major component of the rapidly relaxing high-energy-state quenching' of chlorophyll fluorescence, which was associated with a quenching of the level of initial fluorescence (F₀) and exhibited a close correlation with the zeaxanthin content of leaves when fluorescence quenching was expressed as the rate constant for radiationless energy dissipation in the antenna chlorophyll. Green algal lichens, which possess the xanthophyll cycle, exhibited the same type of fluorescence quenching as that observed in leaves. Two groups of blue-green algal lichens were used for a comparison with these green algal lichens. A group of zeaxanthin-free blue-green algal lichens did not exhibit the type of chlorophyll fluorescence quenching indicative of energy dissipation in the pigment bed. In contrast, a group of blue-green algal lichens which had formed zeaxanthin slowly through reactions other than the xanthophyll cycle, did show a very similar response to that of leaves and green algal lichens. Fluorescence quenching indicative of radiationless energy dissipation in the antenna chlorophyll was the predominant component of high-energy-state quenching in spinach leaves under conditions allowing for high rates of steady-state photosynthesis. A second, but distinctly different type of high-energy-state quenching of chlorophyll fluorescence, which was not inhibited by DTT (i.e., it was zeaxanthin independent) and which is possibly associated with the photosystem II reaction center, occurred in addition to that associated with zeaxanthin in leaves under a range of conditions which were less favorable for linear photosynthetic electron flow. In intact chloroplasts isolated from (zeaxanthin-free) spinach leaves a combination of these two types of rapidly reversible fluorescence quenching occurred under all conditions examined.Abbreviations DTT dithiothreitol - F₀ (or F₀) yield of instantaneous fluorescence at open PS II reaction centers in the dark (or during actinic illumination) - F_M (or F_M) yield of maximum fluorescence induced by a saturation pulse of light in the dark (or during actinic illumination) - F_V (or F_V) yield of variable fluorescence induced by a saturating pulse of light in the dark (or during actinic illumination) - k _D rate constant for radiationless energy dissipation in the antenna chlorophyll - SV Stern-Volmer equation - PFD photon flux density - PS I photosystem I - PS II photosystem II - Q_A acceptor of photosystem II - q_N coefficient of nonphotochemical chlorophyll fluorescence quenching - q_P coefficient of photochemical chlorophyll fluorescence quenching     

Photoactive pigment—enzyme complexes of chlorophyll precursor in plant leaves

This review summarizes contemporary data on structure and function of photoactive pigment--enzyme complexes of the chlorophyll precursor that undergoes photochemical transformation to chlorophyllide. The properties and functions of the complex and its principal components are considered including the pigment (protochlorophyllide), the hydrogen donor (NADPH), and the photoenzyme protochlorophyllide oxidoreductase (POR) that catalyzes the photochemical production of chlorophyllide. Chemical variants of the chlorophyll precursor are described (protochlorophyllide, protochlorophyll, and their mono- and divinyl forms). The nature and photochemical activity of spectrally distinct native protochlorophyllide forms are discussed. Data are presented on structural organization of the photoenzyme POR, its substrate specificity, localization in etioplasts, and heterogeneity. The significance of different POR forms (PORA, PORB, and PORC) in adaptation of chlorophyll biosynthesis to various illumination conditions is considered. Attention is paid to structural and functional interactions of three main constituents of the photoactive complex and to possible existence of additional components associated with the pigment-enzyme complex. Historical aspects of the problem and the prospects of further investigations are outlined.     

Quest for minor but key chlorophyll molecules in photosynthetic reaction centers – unusual pigment composition in the reaction centers of the chlorophyll d-dominated cyanobacterium Acaryochloris marina

A short overview, based on our own findings, is given of the minor pigments that function as key components in photosynthesis. Recently, we found the presence of chlorophyll a, chlorophyll d′ and pheophytin a as minor pigments in the chlorophyll d-dominated cyanobacterium Acaryochloris marina. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of the peridinin–chlorophyll a-protein complex in the dinoflagellate Symbiodinium

The water-soluble peridinin–chlorophyll a-proteins (PCPs) are one of the major light harvesting complexes in photosynthetic dinoflagellates. PCP contains the carotenoid peridinin as its primary pigment. In this study, we identified and characterized the PCP protein and the PCP gene organization in Symbiodinium sp. CS-156. The protein molecular mass is 32.7 kDa, revealing that the PCP is of the monomeric form. The intronless PCP genes are organized in tandem arrays. The PCP gene cassette is composed of 1095-bp coding regions and spacers in between. Despite the heterogeneity of PCP gene tandem repeats, we identified a single form of PCP, the sequence of which exactly matches the deduced sequence of PCP gene clone 7 (JQ395030) by LC–MS/MS analysis of tryptic digested PCP, revealing the mature PCP apoprotein is 312 amino acids in length. Pigment analysis showed a peridinin-to-Chl a ratio of 4. The peridinin-to-Chl a Q_y energy transfer efficiency is 95% in this complex.     

Supramolecular organization of fucoxanthin–chlorophyll proteins in centric and pennate diatoms

Fucoxanthin–chlorophyll proteins (FCP) are the major light-harvesting proteins of diatom algae, a major contributor to marine carbon fixation. FCP complexes from representatives of centric (Cyclotella meneghiniana) and pennate (Phaeodactylum tricornutum) diatoms were prepared by sucrose gradient centrifugation and studied by means of electron microscopy followed by single particle analysis. The oligomeric FCP from a centric diatom were observed to take the form of unusual chain-like or circular shapes, a very unique supramolecular assembly for such antennas. The existence of the often disputed oligomeric form of FCP in pennate diatoms has been confirmed. Contrary to the centric diatom FCP, pennate diatom FCP oligomers are very similar to oligomeric antennas from related heterokont (Stramenopila) algae. Evolutionary aspects of the presence of novel light-harvesting protein arrangement in centric diatoms are discussed.     

Is dimethylsulfoxide a reliable solvent for extracting chlorophyll under field conditions?

Dimethylsulfoxide (DMSO) appears to be a reliable solvent for extracting chlorophyll (Chl), however, modification of standard methods may be necessary for some species under field conditions. We found that Chl extraction of whole leaf tissue with DMSO incubated at between 25 and 40 °C was generally similar to the 80% acetone method, except for one graminoid species that required maceration. There was little effect of incubation temperature or duration of incubation beyond 7 h on extraction efficiency, but DMSO extracts were less stable than acetone extracts during one week of cold storage, especially if they thawed during this period. Since Chl extraction methods may provide variable results, particularly in the field, studies using different solvents should be compared cautiously unless specific methods have been calibrated. This revised version was published online in August 2006 with corrections to the Cover Date.     

‘Low-waves>’ in chlorophyll fluorescence kinetics indicate deprivation of bicarbonate*

A brief reversible lowering of chlorophyll fluorescence yield (so called low-waves) immediately after application of a saturating light pulse in parallel with a short-time enhancement of the P700 oxidation level was observed in the green alga Haematococcus pluvialis. The phenomenon occurred in the steady-state time region of fluorescence induction kinetics under mild acidic conditions, and was eliminated by bicarbonate. Shortly after expression of low-waves, the photosynthetic oxygen evolution rate decreased and the non-photochemical chlorophyll fluorescence quenching component increased. The enhancement of the non-photochemical chlorophyll fluorescence quenching component was nigericin-sensitive indicating its dependence on the transthylakoid proton gradient. On the other hand, the formation of low-waves was not removed by the uncoupler. Only when bicarbonate was applied additionally, the reversible short-term decrease in fluorescence yield following each saturating light flash was abolished. Dimethyl-4-nitroso-aniline as an artificial electron acceptor of Photosystem I did not limit the brief drops in fluorescence. However, formate as a competitive inhibitor of bicarbonate binding in Photosystem II induced low-wave formation. Therefore, our results suggest that low-waves in chlorophyll fluorescence kinetics indicate deprivation of bicarbonate in the reaction centre of Photosystem II. This revised version was published online in June 2006 with corrections to the Cover Date.     

Pavel Siffel (1954–2003) or Life full of chlorophyll

Life and research results of Pavel Siffel, a talented but untimely deceased Czech scientist in photosynthesis, are reviewed. He studied biophysics and physiology of chlorophyll, its complexes with proteins, their absorption and fluorescence spectra, activities in mutants and transformants, dealt with chlorophyll biosynthesis and protochlorophyllide photoreduction, pigments in plants grown at CO₂ deficiency and under simulated acid rain, with changes accompanying leaf and plant development, photobleaching, etc. He participated in construction of specialised spectrofluorometers, finally he built the kinetic spectrophotometer SpeKin.     

Left- and right-handed LHC II macroaggregates revealed by circularly polarized chlorophyll luminescence

Circularly polarized chlorophyll luminescence (CPL) may serve as a measure of chiral macroaggregates of the light-harvesting chlorophyll-protein complexes (LHC II) in both isolated chloroplasts and intact leaves (Gussakovsky et al (2000) Photosynth Res 65: 83–92). In the present work, we applied the CPL approach to study the effect of fast (1–2 min) thermal impacts on LHC II macroaggregates. The results revealed unexpected temperature-response kinetics, composed of initial bell-shaped changes in the CPL signal, followed by degradation down to a steady state (equilibrium). The bell-shape effect was dependent upon illumination, and vanished in the dark. A mathematical analysis of the temperature-response kinetics uniquely indicated that LHC II chiral macroaggregates may persist in both left- and right-handed forms. These forms differ in their response to high temperatures. Both forms are more thermostable in leaves than in isolated chloroplasts. The cooperative degradation of LHC II macroaggregates, which is induced by the thermal impact, is irreversible. It is therefore suggested that the native LHC II macroaggregates are stable, stationary, non-equilibrium, spatially heterogeneous (dissipative) structures. The dissipative properties probably allow the interconversion between left- and right-handed forms under perturbation by certain factors. Illumination probably serves as one such perturbation factor, initiating the interconversion of dark-adapted, left-handed to light-dependent, right-handed LHC II macroaggregates. The chiral heterogeneity of the LHC II macroaggregates is a newly revealed aspect which needs to be taken into consideration in future circular dichroism or CPL studies.     

Seasonality of chlorophyll and nutrients in Lake Erken – effects of weather conditions

The effect of submerged macrophytes on interactions among epilimnetic phosphorus, phytoplankton, and heterotrophic bacterioplankton has been acknowledged, but remains poorly understood. Here, we test the hypotheses that the mean summer phytoplankton biomass (chlorophyll a): phosphorus ratios decrease with increased macrophyte cover in a series of nine lakes. Further, we test that both planktonic respiration and bacterioplankton production increase with respect to phytoplankton biomass along the same gradient of increasing macrophyte cover. Increased macrophyte cover was associated with a lower fraction of particulate phosphorus in epilimnia, with total particulate phosphorus declining from over 80% of total phosphorus in a macrophyte free lake to less than 50% in a macrophyte rich lake. Phytoplankton biomass (chlorophyll a) too was lower in macrophyte dominated lakes, despite relatively high levels of total dissolved phosphorus. Planktonic respiration and bacterioplankton production were higher in macrophyte rich lakes than would be expected from phytoplankton biomass alone, pointing to a subsidy of bacterioplankton metabolism by macrophyte beds at the whole lake scale. The results suggest that the classical view of pelagic interactions, which proposes phosphorus determines phytoplankton abundance, which in turn determines bacterial abundance through the production of organic carbon, becomes less relevant as macrophyte cover increases.     

Are syn-ligated (bacterio)chlorophyll dimers energetic traps in light-harvesting systems?

A recent study of the stereochemical details of chlorophyll ligation in photosystem I [Balaban et al., Biochim. Biophys. Acta 1556 (2002) 197-207] has revealed that only 14 chlorophylls out of the total 96 are ligated from the same side (syn) as the 17-propionic acid residue which is esterified with phytol. The syn chlorophylls are carefully surrounding the reaction center forming the inner core antenna system and their ligands have been strongly conserved in several species during evolution. We hypothesize here that the two dimers of closely spaced syn chlorophylls which are encountered within roughly 2 nm of P700 are the ultimate energetic traps of this light-harvesting system. Structurally very similar bacteriochlorophyll a dimers are encountered within the Fenna-Matthews-Olson protein complex and within the B850 ring of the LH2 complex of purple bacteria. The non-random disposal of these dimers lends support to our hypothesis that the syn ligation coupled with a strong excitonic interaction leads to the most red-shifted pigments in light-harvesting systems. We would like to encourage both theoretical and experimental studies to either prove or disprove this intriguing structure-function conjecture in view of designing efficient artificial light-harvesting systems.     

Uncoupling of chlorophyll and nutrients in lakes – possible reasons,expected consequences

Total phosphorus and total nitrogen explained a low percentage of summer chlorophyll variability in epilimnia of the Great Masurian Lakes. Division of the whole data set into two subgroups of lakes improved approximation of the chlorophyll nutrient relationship but revealed also functional differences between the lakes distinguished in that way. Chlorophyll in eutrophic lakes correlated well with nitrogen and phosphorus, that in mesotrophic lakes (those with summer chlorophyll <=22 mg m^–3 as calculated in the model) was related to none of the nutrients. Higher summer chlorophyll content in epilimnetic waters was accompanied by higher chl:PP and chl:PN ratios. Algal adaptation to poor light conditions in eutrophic lakes is postulated as a possible reason for that difference.Chlorophyll – nutrient relationships varied with the trophic status of lakes. Epilimnetic chlorophyll strictly followed phosphorus changes in eutrophic lakes but did not do so in mesotrophic ones. Detailed comparison of selected meso- and eutrophic lakes showed marked differences in the seasonal changes of chlorophyll and nutrient concentrations and in sedimentation rates, especially in spring. Nutrient limitation rather than zooplankton grazing is suggested as a possible mechanism of controlling algal abundance and the sequence of spring events in a eutrophic lake. It is hypothesised that phosphorus turnover in eutrophic lakes is dominated by seasonal vertical fluxes, while in mesotrophic lakes it is more conservative with consumption and regeneration restricted mostly to metalimnion. Possible consequences of such conclusion are discussed in the paper.     

Polypeptide profiles of chlorophyll · protein complexes and thylakoid membranes of spinach chloroplasts

In addition to the major chlorophyll · protein complexes I and II, two minor chlorophyll proteins have been observed in sodium dodecyl sulfate (SDS)-polyacrylamide gels of spinach chloroplast membranes. These minor pigmented zones appeared to be derived from the light-harvesting chlorophyll $\text{a}\text{b}$ · protein and from the reaction centre complex of Photosystem II.Data are presented on the polypeptide profiles of purified digitonin-subchloroplast particles, with special regard to the effect of solubilization temperature and extraction of lipids. The results are compared with the SDS-polypeptide pattern of spinach thylakoids obtained under exactly the same conditions with respect to electrophoresis technique, solubilization method and presence of lipid. In addition, the effects of temperature and lipid extraction on the distinct chlorophyll · protein complexes appearing in SDS gel electrophoretograms of chloroplast membranes were studied by slicing the chlorophyll-containing regions and subjecting them to a second run with or without heating or extraction with acetone. By supplementing these data with an examination of the polypeptide composition of cytochrome f and coupling factor, it has been possible to identify most of the major chloroplast membrane polypeptides.     

Secchi disk — chlorophyll relationships in a lake with highly variable phytoplankton biomass

In meseutrophic Lake Constance mean euphotic phytoplankton chlorophyll concentrations vary about 100-fold over the year. Concomitant fluctuations in euphotic depth (Z_eu) and Secchi depth (Z_s) are related to each other in a non-linear fashion that as a rough approximation can be expressed by Z_eu 5 Z_s.Secchi depth is to a great extent a function of beam attenuation of light which depends on the inherent optical properties of the water and is highly sensitive to light scattering from particles. Euphotic depth, by contrast, is a function of the vertical light attenuation coefficient which also depends on absorption and scattering, but is less sensitive to the latter than beam attenuation. Algal cells both absorb and scatter light and therefore influence Secchi depth and euphotic depth, however, in different fashions.Whenever the lake is clear due to scarce phytoplankton, scattering is small and beam attenuation only exceeds vertical light attenuation by a relatively small factor. As a consequence, the ratio of euphotic depth to Secchi depth is small (1.5–2.5). When the lake is turbid due to high algal density, enhanced scattering from algal cells and detrital particles causes beam attenuation to rise more than vertical light attenuation, thus leading to high ratios of euphotic depth to Secchi depth (3–5). The relatively close relationships between Secchi depth and chlorophyll in Lake Constance are due to (1) high influence of chlorophyll concentration on water transparency, (2) co-variation of phytoplankton and other suspended particles, and (3) limited variation of cellular chlorophyll contents.     

Changes with age in the absorption spectrum of chlorophyll α in a diatom

Summary Absorption spectra of a young and an old culture of the diatom Pheodactylum tricornutum were measured in thin layers between two opal glass sheets. The spectra at 24° and at -196°C were replotted to give equal areas from 730–625 m to allow direct comparison. At 24°C the spectrum for the difference between the two cultures had a negative component of 18 m half width centered at 675 m and a positive region of W_0.5=26 m near 700 m.The spectra at -196°C may be somewhat distorted by clumping of the cells during freezing but nevertheless the 16 day culture clearly showed a smaller proportion of Ca 670 to Ca 680. This older culture has a shoulder due to a 707 m component. The difference curve at -196°C shows the decrease of an unsymmetrical band peaking at 669 m and an increase at 695 m in addition to the 707 m component. Due to the possibility of distortion, the presence of an actual component at 695 is doubtful in these particular cultures.The room temperature spectrum in the chloropyhll a region for the 5 day culture can be closely fitted by a single probability curve at 675 m having a half-width of 31 m. The sum of two components, with widths more reasonable for chlorophylls, also matched the data well enough. These two probability curves, of 22 m half width, centered on 669 and 683.2 m and had a height ratio, h₆₆₉/h₆₈₃ of 1.18. In the 16 day culture the ratio for these bands changed to 1.11 and there was extra absorption around 700 m.Dedicated to Professor C. B. van Niel on the occasion of his 70th birthday     

Normal-phase HPLC quantitation of chlorophyll a′ and phylloquinone in Photosystem I particles

Fractionated Photosystem (PS) I particles consisting of six, five or two core proteins were analyzed by HPLC for chlorophyll (Chl) a and phylloquinone (PhQ). Each particle had a Chl a/P700 molar ratio of 50–55 and contained ca. 2 molecules of Chl a per P700. Deliberate control of eluent composition led to isolated elution of PhQ and -carotene in the normal-phase chromatogram. Based on these a simple HPLC procedure has been established to determine the PhQ/P700 molar ratio, which was ca. 2 for the larger two PS I particles and ca. 1 for the smallest particle, in line with previous reports.     

Correction to: Why is chlorophyll b only used in light-harvesting systems?

Journal of Plant Research - The article Why is chlorophyll b only used in light-harvesting systems.