The Existence of Chlorophyll c in the Chl b-Containing,Light-Harvesting Complex of the Green Alga Mantoniella squamata (Prasinophyceae)

The prasinophycean alga Mantoniella contains, in addition to Chl a and b, at least a third green pigment which is functionally active in the light-harvesting antenna. This third Chl was isolated in order to elucidate its chemical structure. The absorption and fluorescence spectra were measured not only from the purified pigment but also from its pheophytin and its methylpheophorbide. The spectra were compared with those of authentic Chl c-1 and c-2, which were isolated from the diatom Nitzschia sp. and with Mg-DVPP (purified from Rhodobacter). The results show that the pigment from Mantoniella compares best with Chl c-1. In order to clarify the spectral data, Chl c-1 and c-2, Mg-DVPP, and the pigment from Mantoniella were subjected to a chromatographic system that is able to separate these porphyrins. The chromatographic analysis clearly shows that the pigment from Mantoniella co-migrates with Chl c-1 and not with the bacterial pigment. Mantoniella is the first organism which has been demonstrated to contain Chl a, b, and authentic c.     

Non-photochemical chlorophyll fluorescence quenching and structural rearrangements induced by low pH in intact cells of Chlorella fusca (Chlorophyceae) and Mantoniella squamata (Prasinophyceae)

We have used circular dichroism (CD) spectroscopy and chlorophyll fluorescence induction measurements in order to examine low-pH-induced changes in the chiral macro-organization of the chromophores and in the efficiency of non-photochemical quenching of the chlorophyll a fluorescence (NPQ) in intact, dark-adapted cells of Chlorella fusca (Chlorophyceae) and Mantoniella squamata (Prasinophyceae). We found that: (i) high proton concentrations enhanced the formation of chiral macrodomains of the complexes, i.e. the formation of large aggregates with long-range chiral order of pigment dipoles; this was largely independent of the low-pH-induced accumulation of de-epoxidized xanthophylls; (ii) lowering the pH led to NPQ; however, efficient energy dissipation, in the absence of excess light, could only be achieved if a substantial part of violaxanthin was converted to zeaxanthin and antheraxanthin in Chlorella and Mantoniella, respectively; (iii) the low-pH-induced changes in the chiral macro-organization of pigments were fully reversed by titrating the cells to neutral pH; (iv) at neutral pH, the presence of antheraxanthin or zeaxanthin did not bring about a sizeable NPQ. Hence, low-pH-induced NPQ in dark adapted algal cells appears to be associated both with the presence of de-epoxidized xanthophylls and structural changes in the chiral macrodomains. It is proposed that the macrodomains, by providing a suitable structure for long-distance migration of the excitation energy, in the presence of quenchers associated with de-epoxidized xanthophylls, facilitate significantly the dissipation of unused excitation energy. This revised version was published online in June 2006 with corrections to the Cover Date.     

Organization of the pigment molecules in the chlorophyll a/b/c containing alga Mantoniella squamata (Prasinophyceae) studied by means of absorption, circular and linear dichroism spectroscopy

In order to obtain information on the organization of the pigment molecules in chlorophyll (Chl) a/b/c-containing organisms, we have carried out circular dichroism (CD), linear dichroism (LD) and absorption spectroscopic measurements on intact cells, isolated thylakoids and purified light-harvesting complexes (LHCs) of the prasinophycean alga Mantoniella squamata. The CD spectra of the intact cells and isolated thylakoids were predominated by the excitonic bands of the Chl a/b/c LHC. However, some anomalous bands indicated the existence of chiral macrodomains, which could be correlated with the multilayered membrane system in the intact cells. In the red, the thylakoid membranes and the LHC exhibited a well-discernible CD band originating from Chl c, but otherwise the CD spectra were similar to that of non-aggregated LHC II, the main Chl a/b LHC in higher plants. In the Soret region, however, an unusually intense (+) 441 nm band was observed, which was accompanied by negative bands between 465 and 510 nm. It is proposed that these bands originate from intense excitonic interactions between Chl a and carotenoid molecules. LD measurements revealed that the Q(Y) dipoles of Chl a in Mantoniella thylakoids are preferentially oriented in the plane of the membrane, with orientation angles tilting out more at shorter than at longer wavelengths (9 degrees at 677 nm, 20 degrees at 670 nm and 26 degrees at 662 nm); the Q(Y) dipole of Chl c was found to be oriented at 29 degrees with respect to the membrane plane. These data and the LD spectrum of the LHC, apart from the presence of Chl c, suggest an orientation pattern of dipoles similar to those of higher plant thylakoids and LHC II. However, the tendency of the Q(Y) dipoles of Chl b to lie preferentially in the plane of the membrane (23 degrees at 653 nm and 30 degrees at 646 nm) is markedly different from the orientation pattern in higher plant membranes and LHC II. Hence, our CD and LD data show that the molecular organization of the Chl a/b/c LHC, despite evident similarities, differs significantly from that of LHC II.     

Both spillover and light absorption cross-section changes are involved in the regulation of excitation energy distribution between the two photosystems during state transitions in wheat leaf

Weak red light-induced changes in chlorophyll fluorescence parameters and in the distribution of PS I and PS II in thylakoid membranes were measured in wheat leaves to investigate effective ways to alter the excitation energy distribution between the two photosystems during state transition in vivo. Both the chlorophyll fluorescence parameter Fm/Fo and F685/F735, the ratio of fluorescence yields of the two photosystems at low temperature (77 K), decreased when wheat leaves were illuminated by weak red light of 640 nm, however, Fm/Fo decreased to its minimum in a shorter time than F685/F735. When Photosystem (PS II) thylakoid (BBY) membranes from adequately dark-adapted leaves (control) and from red light-illuminated leaves were subjected to SDS-polyacrylamide gel electrophoresis under mildly denaturing conditions, PS I was almost absent in the control, but was present in the membranes from the leaves preilluminated with the weak red light. In consonance with this result, the content of Cu, measured by means of the energy dispersive X-ray microanalysis (EDX), increased in the central region, but decreased in the margin of the grana stacks from the leaves preilluminated by the red light as compared with the control. It is therefore suggested that: (i) both spillover and absorption cross-section changes are effective ways to alter the excitation energy distribution between the two photosystems during state transitions in vivo, and the change in spillover has a quicker response to the unbalanced light absorption of the two photosystems than the change in light absorption cross-section, and (ii) the migration of PS I towards the central region of grana stack during the transition to state 2 leads to the enhancement of excitation energy spillover from PS II to PS I.     

Distribution of excitation energy among photosystem I and photosystem II in red algae. I. Action spectra of light reactions I and II

Action spectra of light reaction I and light reaction II from red algae (marine members of Florideae and Bangiales) were measured with 550 nm (light 2) or 699 nm (light 1) background light, using a Teflon-covered platinum electrode for O₂ measurement. Care was taken to ensure that maximum enhancement was reached by the background light.The action spectra of light reaction I, we found under these conditions, are very similar to the thallus absorption, whilst the action spectra of light reaction II show, besides strong bands of the phycobilins, only minor bands of chlorophyll a, which account for only 10–20% of the total chlorophyll.The spectra are discussed on the basis of two main types of models of energy distribution over both photosynthetic systems. If this distribution is considered to be invariable (models 1a and b), one has to assume that almost exactly half of the total chlorophyll is not involved in the supply of the non-cyclic electron transport with excitation energy. This part, however, has to be thought of as incorporated in the thylakoid membrane in a similar manner to the chlorophyll in photosystem I. However, if one supposes an almost complete equilibration in the energy distribution over both systems as long as the primary absorption in photosystem II prevails (models 2a and b), there is no need for the assumption of such photosynthetically ‘inactive’ or less active chlorophyll. Some evidence is shown that strongly supports model 2.     

Relationships between the photon distribution between the two photosystems, the concentration of system II reaction centers and the intersystem equilibrium constant in Chlorella pyrenoidosa

From Emerson enhancement measurements of O₂ evolution in Chlorella pyrenoidosa, it was possible to establish a relationship between the concentration of photosystem II open reaction centers (E) and the distribution of photons between photosystems I and II [(1 − )/] during steady state. The superposition of lights of two different wavelengths (1 and 2) gives concentrations of E and intermediate between those obtained with light 1 and 2 separately. This relationship extends a previous one based on quantum yield measurements. It has been expressed here by a curve corresponding to a fixed value of the intersystem apparent equilibrium constant (K). Up to 700 nm, K remains equal to 6. Above this wavelength, although the margin of error is rather great, K apparently increases to 12 or more.

The possibility of “spill-over” of light absorbed by System II to System I was studied. There is no probability that this spill-over, if any, exceeds 25% in Chlorella.

The apparent equilibrium constant is decreased by 3(3,4-dichlorophenyl)-1,1-dimethylurea. This is not in favor of the hypothesis of fully independent electron-transfer chains in photosynthesis; it is therefore likely that some communication between those chains exists.     

Light-induced increase in initial chlorophyll fluorescence Fo level and the reversible inactivation of PS II reaction centers in soybean leaves

With a portable PAM-2000 fluorometer it was observed that responses of initial chlorophyll fluorescence F_o level to strong light were different in various plant species examined. When the photochemical efficiency of Photosystem II, F_v/F_m, declined, F_o increased significantly in leaves of some plants such as soybean and cotton, while F_o decreased remarkably in other plants such as wheat and barley. In order to explore the mechanism of the increase in F_o in soybean leaves, the change in D1 protein amount and effects of lincomycin and far-red light on these fluorescence parameters were observed by SDS–PAGE combined with gel scanning and chlorophyll fluorescence analysis. The following results were obtained. (1) The amount of inactive PS II reaction centers increased under strong light and decreased during subsequent dark recovery [Hong and Xu (1997) Chinese Sci Bull 42(8): 684–689]. (2) No net loss of D1 protein occurred after strong light treatment. (3) Lincomycin taken up through petioles following strong light treatment had no significant effect on D1 protein level and the decay of F_o in the dark. (4) Far-red light applied after strong light treatment could largely attenuate the increase in F_o and accelerate F_o decay in the dark. Based on these results, it is deduced that the increase in F_o under strong light is mainly due to reversible inactivation of part of PS II reaction centers, rather than the net loss of D1 protein and that reversible inactivation of PS II is prevalent in some plants.     

Differential thermal effects on the energy distribution between photosystem II and photosystem I in thylakoid membranes of a psychrophilic and a mesophilic alga

Sensitivity of the photosynthetic thylakoid membranes to thermal stress was investigated in the psychrophilic Antarctic alga Chlamydomonas subcaudata. C. subcaudata thylakoids exhibited an elevated heat sensitivity as indicated by a temperature-induced rise in F_o fluorescence in comparison with the mesophilic species, Chlamydomonas reinhardtii. This was accompanied by a loss of structural stability of the photosystem (PS) II core complex and functional changes at the level of PSI in C. reinhardtii, but not in C. subcaudata. Lastly, C. subcaudata exhibited an increase in unsaturated fatty acid content of membrane lipids in combination with unique fatty acid species. The relationship between lipid unsaturation and the functioning of the photosynthetic apparatus under elevated temperatures is discussed.     

The distribution of Photosystem I and Photosystem II polypeptides between the cytoplasmic and thylakoid membranes of cyanobacteria

Abstract In a previous study we found that the 33 kDa extrinsic polypeptide of Photosystem II is present in both the cytoplasmic and thylakoid membranes of cyanobacteria, but forms part of a functional complex only in the latter [Smith et al. (1987) Mol. Microbiol. 6, 1821–1827]. In order to determine if this phenomenon is restricted to the 33 kDa polypeptide we have extended this study in Anacystis nidulans to include a number of other polypeptides of Photosystem I and Photosystem II. We have found that D1 and possibly PsaC are present in both membranes, CP43 and CP47 are confined to the thylakoid membranes, and the distribution of PsaD and PsaE is dependent upon the growth stage of the cyanobacteria.     

Measurement of degree of chlorophyll fluorescence polarization in relation to the regulation of excitation energy transfer between Photosystems I and II in pea chloroplasts

The degree of chlorophyll fluorescence polarization (p) at 740 nm was measured at room temperature for pea chloroplasts subjected to various conditions. (1) In agreement with previous published observations, p decreased when the Photosystem (PS) II traps were closed by illumination in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. (2) Under these conditions, the magnitude of p was also sensitive to the presence of salts. Under conditions when ‘spillover’ of the excitation energy from PS II to PS I was low, p was also low, being consistent with increased migration of energy between the PS II light-harvesting chlorophylls. In contrast, when spillover was at a maximum p increased. (3) The change in p in the presence of salts was dependent on the concentration and valency of the cations in such a way as to suggest the changes were mediated through electrostatic forces. The dependency of p on ionic composition of the experimental medium was closely related to the associated changes in fluorescence yield. (4) Membrane stacking, caused by lowering pH of the chloroplast suspension, did not induce a significant change in p, suggesting that this pH-induced process is different from the membrane stacking brought about by manipulating the salt levels. (5) Incubation of thylakoids with ATP induces light-dependent phosphorylation of the light-harvesting chlorophyll-protein complexes, and regulates excitation energy transfer between PS I and PS II (Bennett, J., Steinback, K.R. and Arntzen, C.J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5253–5257). Under conditions which bring about this phosphorylation it was found that p increased to a value indicative of spillover.     

Electron transfer in reaction centers of Rhodopseudomonas sphaeroides. II. Free energy and kinetic relations between the acceptor states Q(-)A Q(-)B and QAQ(2-)B

Thermodynamic equilibria and electron transfer kinetics involving the quinone acceptor complex in reaction centers from Rhodopseudomonas sphaeroides were investigated. We focussed on reactions involving the two-electron states QA Qn and QAQ~-, described by the scheme DQAQa~-D +X,~~A- , ~~a- ~k~ .~D+ "r~~ AK~'La2- - k~2~ k~lk O~ (2)D+~D The equilibrium partitioning between QA Q n and QAQ 2n- was determined spectroscopically from either the concentration of oxidized cytochrome c or the concentration of semiquinone after successive flashes of light.At pH < 9.5, QAQ2n - is stabilized relative to QAQn, while for pH > 9.5, QAQB is energetically favored.The reduction of QA, to form QAQ~, is not associated with a protonation step (pK< 8). However, the reduction of Q~, to form the final state QAQ~-, is accompanied by an uptake of a proton (pK >/10.7). The preferential interaction of a proton with QAQ2n - provides the driving force for the forward electron transfer.The shift toward the photochemically inactive state QAQa with increasing pH may serve as a feedback mechanism in photosynthetic organisms to limit the rise in intracellular pH. The electron-transfer rate constants were determined from the observed kinetics and the equilibria between the states QAQ2n - and QA Q n. The forward rate constant z-.A~2n~ was approximately proportional to the proton concentration, whereas kta2A~ depended only weakly on pH. The recombination kinetics of D +QAQ2n- was biphasic. The slow rate agreed with the predicted charge recombination via the intermediate state D +QAQff; the fast rate may be due to the recombination from a separate (conformational) state. The results of this work were combined with those of a previous study on reactions involving the one-electron precursor states QAQa and QAQn(Kleinfeld, D., Okamura, M.Y., and Feher, G. (1984) Biochim. Biophys. Acta 766, 126-140). The overall sequence for the protonation of the reaction center in response to successive reductions of the accept or complex involves the uptake of one proton for each electron transferred to QB- This sequential uptake initiates the formation of a proton gradient across the cell membrane.     

Electron transfer in reaction centers of Rhodopseudomonas sphaeroides. I. Determination of the charge recombination pathway of D+QAQ(-)B and free energy and kinetic relations between Q(-)AQB and QAQ(-)B

The electron-transfer reactions and thermodynamic equilibria involving the quinone acceptor complex in bacterial reaction centers from R. sphaeroides were investigated. The reactions are described by the scheme: (Formula: see text). We found that the charge recombination pathway of D+QAQ(-)B proceeds via the intermediate state D+Q(-)AQB, the direct pathway contributing less than approx. 5% to the observed recombination rate. The method used to obtain this result was based on a comparison of the kinetics predicted for the indirect pathway (given by the product kAD-times the fraction of reaction centers in the Q-AQB state) with the observed recombination rate, kobsD+----D. The kinetic measurements were used to obtain the pH dependence (6.1 smaller than or equal to pH smaller than or equal to 11.7) of the free energy difference between the states Q(-)AQB and QAQ(-)B. At low pH (less than 9) QAQ(-)B is stabilized relative to Q(-)AQB by 67 meV, whereas at high pH Q(-)AQB is energetically favored. Both Q(-)A and Q(-)B associate with a proton, with pK values of 9.8 and 11.3, respectively. The stronger interaction of the proton with Q(-)B provides the driving force for the forward electron transfer.     

Pleiotropy in the melanocortin system: expression levels of this system are associated with melanogenesis and pigmentation in the tawny owl (Strix aluco)

The adaptive function of melanin‐based coloration is a long‐standing debate. A recent genetic model suggested that pleiotropy could account for covariations between pigmentation, behaviour, morphology, physiology and life history traits. We explored whether the expression levels of genes belonging to the melanocortin system (MC1R, POMC, PC1/3, PC2 and the antagonist ASIP), which have many pleiotropic effects, are associated with melanogenesis (through variation in the expression of the genes MITF, SLC7A11, TYR, TYRP1) and in turn melanin‐based coloration. We considered the tawny owl (Strix aluco) because individuals vary continuously from light to dark reddish, and thus, colour variation is likely to stem from differences in the levels of gene expression. We measured gene expression in feather bases collected in nestlings at the time of melanin production. As expected, the melanocortin system was associated with the expression of melanogenic genes and pigmentation. Offspring of darker reddish fathers expressed PC1/3 to lower levels but tended to express PC2 to higher levels. The convertase enzyme PC1/3 cleaves the POMC prohormone to obtain ACTH, while the convertase enzyme PC2 cleaves ACTH to produce α‐melanin‐stimulating hormone (α‐MSH). ACTH regulates glucocorticoids, hormones that modulate stress responses, while α‐MSH induces eumelanogenesis. We therefore conclude that the melanocortin system, through the convertase enzymes PC1/3 and PC2, may account for part of the interindividual variation in melanin‐based coloration in nestling tawny owls. Pleiotropy may thus account for the covariation between phenotypic traits involved in social interactions (here pigmentation) and life history, morphology, behaviour and physiology.     

Characterization of primary reactants in bacterial photosynthesis. II. Kinetic studies of the light-induced EPR signal (g = 2.0026) and the optical absorbance changes at cryogenic temperatures

We have shown that the rise and decay kinetics of the light-induced EPR signal are identical to the kinetics of the optical changes at 80 °K. This identity provides independent evidence that the EPR signal is due to the oxidized primary electron donor which is bacteriochlorophyll. The EPR and optical changes could be described by a model photochemical reaction scheme that takes into account spin-lattice relaxation. The optical decay rate was found to be temperature independent between 1.5 and 80 °K and to obey approximately first order kinetics. These results are consistent with the hypothesis that the charge recombination occurs via tunneling through a potential barrier. The decay constants at these temperatures were found to be the same for different bacterial species and strains. No differences were found between purified reaction centers of R. spheroides R-26 and whole cells. Reaction centers treated with sodium dodecylsulfate or urea were still photochemically active but showed a markedly different kinetic behavior. The decay constant may, therefore, serve as a probe to investigate the molecular environment of the primary reactants.     

The endomembrane system and mechanism of membrane flow in the green alga,Gloeomonas kupfferi (Volvocales,Chlorophyta) II. A cytochemical analysis

Summary Cytochemical analysis of the endomembrane system of the chlamydomonad flagellate,Gloeomonas kupfferi (Chlorophyta), reveals distinct compartmentalization. Phosphatase localization shows that: IDPase is located throughout all cisternae of the dictyosome and vesicles associated with the contractile vacuole. Other alkaline phosphatases like TPPase, ATPase and ITPase were localized within the trans-face cisternae and vesicles of the contractile vacuole. IMPase was localized at the plasmamembrane and not within the endomembrane system. Acid phosphatases, incl. CMPase, NADPase and -glycerophosphatase, were localized in vesicles emerging from the central terminus of the trans-face of the dictyosome and in the peripheral vacuolar network. Silver proteinate labeling was noted in the dictyosome, contractile vacuole and on the anterior plasmamembrane. A summary of endomembrane compartmentalization and a putative interpretation of membrane flow and economy are presented.Abbreviations ER endoplasmic reticulum - IDPase inosine 5-diphosphatase - ITPase inosine 5-triphosphatase - ATPase adenosine 5-triphosphatase - TPPase thiamine pyrophosphatase - CMPase cytidine 5-monophosphatase - NADPase -nicotinamide adenine diphosphatase - AcPase acid phosphatase     

The endomembrane system and mechanism of membrane flow in the green alga,Gloeomonas kupfferi (Volvocales,Chlorophyta) I. An ultrastructural analysis

Summary The endomembrane system of the chlamydomonad flagellate,Gloeomonas kupfferi (Chlorophyta), is complex. It consists of a proliferating ER network, a perinuclear complex of 14–18 dictyosomes and 8–12 vacuoles and an anterior contractile vacuole complex. The ER network extends from the nuclear envelope outwards, ensheafhs a dictyosome, extends out through a lobe of the chloroplast and terminates in the thin zone of peripheral cytoplasm between the chloroplast and plasmamembrane. The individual dictyosome is polar with distinct cis- and trans-faces. The cis-face is closely associated with transition vesicles emerging from the adjacent ER. Large vesicles emerge from peripheral swellings of terminal cisternae. The dictyosome-associated ER is connected to the peripheral vacuolar system. During cell division and cytokinesis, changes in the endomembrane system occur. Dictyosomes divide and quickly separate to form perinuclear complexes around the daughter nuclei. Each dictyosome undergoes morphological changes during this wall precursor-producing stage. ER lines the furrow zone and is closely associated with phycoplast microtubules. A discussion of the endomembrane system in membrane flow mechanics is provided.Abbreviations ER endoplasmic reticulum - OsFeCN Osmium ferricyanide