Biohybrid antennas built upon chromophore–polypeptide conjugates show promise for the design of efficient light-capturing modules for specific purposes. Three new designs, each of which employs analogs of the ?-polypeptide from Rhodobacter sphaeroides, have been investigated. In the first design, amino acids at seven different positions on the polypeptide were individually substituted with cysteine, to which a synthetic chromophore (bacteriochlorin or Oregon Green) was covalently attached. The polypeptide positions are at –2, –6, –10, –14, –17, –21, and –34 relative to the 0-position of the histidine that coordinates bacteriochlorophyll a (BChl a). All chromophore–polypeptides readily formed LH1-type complexes upon combination with the ?-polypeptide and BChl a. Efficient energy transfer occurs from the attached chromophore to the circular array of 875 nm absorbing BChl a molecules (denoted B875). In the second design, use of two attachment sites (positions –10 and –21) on the polypeptide affords (1) double the density of chromophores per polypeptide and (2) a highly efficient energy-transfer relay from the chromophore at –21 to that at –10 and on to B875. In the third design, three spectrally distinct bacteriochlorin–polypeptides were prepared (each attached to cysteine at the –14 position) and combined in an ~1:1:1 mixture to form a heterogeneous mixture of LH1-type complexes with increased solar coverage and nearly quantitative energy transfer from each bacteriochlorin to B875. Collectively, the results illustrate the great latitude of the biohybrid approach for the design of diverse light-harvesting systems.
Surfactants play important roles in the preparation, structural, and functional research of membrane proteins, and solubilizing and isolating membrane protein, while keeping their structural integrity and activity intact is complicated. The commercial n-Dodecyl-?-D-maltoside (DDM) and Triton X-100 (TX) were used as solubilizers to extract and purify trimeric photosystem I (PSI) complex, an important photosynthetic membrane protein complex attracting broad interests. With an optimized procedure, TX can be used as an effective surfactant to isolate and purify PSI, as a replace of the much more expensive DDM. A mechanism was proposed to interpret the solubilization process at surfactant concentrations lower than the critical solubilization concentration. PSI-TX and PSI-DDM had identical polypeptide bands, pigment compositions, oxygen consumption, and photocurrent activities. This provides an alternative procedure and paves a way for economical and large-scale trimeric PSI preparation.
Algae have been used for food and nutraceuticals for thousands of years, and the large-scale cultivation of algae, or algaculture, has existed for over half a century. More recently algae have been identified and developed as renewable fuel sources, and the cultivation of algal biomass for various products is transitioning to commercial-scale systems. It is crucial during this period that institutional frameworks (i.e., policies) support and promote development and commercialization and anticipate and stimulate the evolution of the algal biomass industry as a source of renewable fuels, high value protein and carbohydrates and low-cost drugs. Large-scale cultivation of algae merges the fundamental aspects of traditional agricultural farming and aquaculture. Despite this overlap, algaculture has not yet been afforded a position within agriculture or the benefits associated with it. Various federal and state agricultural support and assistance programs are currently appropriated for crops, but their extension to algal biomass is uncertain. These programs are essential for nascent industries to encourage investment, build infrastructure, disseminate technical experience and information, and create markets. This review describes the potential agricultural policies and programs that could support algal biomass cultivation, and the barriers to the expansion of these programs to algae.
Glycogen synthesis initiated by glucose-1-phosphate adenylyltransferase (glgC) represents a major carbon storage route in cyanobacteria which could divert a significant portion of assimilated carbon. Significant growth retardation in cyanobacteria with glgC knocked out (?glgC) has been reported in high light conditions. Here, we knocked out the glgC gene and analyzed its effects on carbon distribution in an isobutanol-producing strain of Synechococcus elongatus PCC7942 and its parental wild-type strain. We showed that isobutanol production was able to partially rescue the growth of ?glgC mutant where the growth rescue effect positively correlated with the rate of isobutanol production. Using NaH14CO3 incorporation analysis, we observed a 28 % loss of total carbon fixation rate in the ?glgC mutant compared to the wild-type. Upon expression of the isobutanol production pathway in ?glgC mutant, the total carbon fixation rate was restored to the wild-type level. Furthermore, we showed that 52 % of the total carbon fixed was redirected into isobutanol biosynthesis in the ?glgC mutant expressing enzymes for isobutanol production, which is 2.5 times higher than that of the wild-type expressing the same enzymes. These results suggest that biosynthesis of non-native product such as isobutanol can serve as a metabolic sink for replacing glycogen to rescue growth and restore carbon fixation rate. The rescue effect may further serve as a platform for cyanobacteria energy and carbon metabolism study.
The carboxylase activities of crude carboxysome preparations obtained from the wild-type Synechococcus elongatus strain PCC 7942 strain and the mutant defective in the carboxysomal carbonic anhydrase (CA) were compared. The carboxylation reaction required high concentrations of bicarbonate and was not even saturated at 50 mM bicarbonate. With the initial concentrations of 50 mM and 25 mM for bicarbonate and ribulose-1,5-bisphosphate (RuBP), respectively, the initial rate of RuBP carboxylation by the mutant carboxysome (0.22 ?mol mg?1 protein min?1) was only 30 % of that observed for the wild-type carboxysomes (0.71 ?mol mg?1 protein min?1), indicating the importance of the presence of CA in efficient catalysis by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). While the mutant defective in the ccmLMNO genes, which lacks the carboxysome structure, could grow under aeration with 2 % (v/v) CO2 in air, the mutant defective in ccaA as well as ccmLMNO required 5 % (v/v) CO2 for growth, indicating that the cytoplasmically localized CcaA helped utilization of CO2 by the cytoplasmically localized Rubisco by counteracting the action of the CO2 hydration mechanism. The results predict that overexpression of Rubisco would hardly enhance CO2 fixation by the cyanobacterium at CO2 levels lower than 5 %, unless Rubisco is properly organized into carboxysomes.
In recent years, temperate bamboo species have been introduced in Europe for multiple uses such as renewable bio-based materials (wood, composites, fibres, biochemicals…) and numerous ecological functions (soil and water conservation, erosion control, phytoremediation…). Despite their interesting potential, little is known on the ecophysiology of these plants in their new habitat. Therefore, we studied gas exchange parameters on a full soil bamboo plantation of Phyllostachys humilis on a test field in Ireland (Europe). We evaluated the seasonal, diurnal and vertical variation of the parameters of two commonly used photosynthetic models, i.e. the light response curve (LRC) model and the model of Farquhar, von Caemmerer and Berry (FvCB). Furthermore, we tested if there were environmental effects on the photosynthetic parameters of these models and if a correlation between photosynthetic parameters and fluorescence parameters was present, fluorescence parameters can be easily and fast determined. Our results show that the gas exchange parameters do not vary diurnally or vertically. Only seasonal variations were found and should, therefore, be taken into account when using the LRC or FvCB model when modelling canopy growth. Therefore, a big-leaf model or a sunlit-shade model can be used for modelling bamboo growth in Western Europe. There is no straightforward relation between environmental variables and the photosynthetic parameters. Although fluorescence parameters showed a correlation with the photosynthetic parameters, application of such correlation may be limited.
Oxygen dictates the catabolic “lifestyle” of Rhodobacter sphaeroides. When it is present, the bacteria are fully equipped for aerobic respiration. When it is absent, the cells outfit themselves to make use of energy-gathering options that do not require oxygen. Thus, while respiring on alternate electron acceptors in the absence of oxygen even in the dark, the cells are fully enabled for phototrophy. PrrA, PpsR, and FnrL are global regulatory proteins mediating oxygen control of gene expression in this organism. For each of these, regulon members include a subset of a cluster of genes known as the photosynthesis genes, which encode the structural proteins and enzymes catalyzing biosynthesis of the pigments of the light-harvesting and reaction center complexes. The complexes are housed in a specialized structure called the intracytoplasmic membrane (ICM). Although details are emerging as to the differentiation process leading to fully formed ICM, little is known of necessary regulatory events beyond changes in photosynthesis gene transcription. This study used transmission electron microscopy toward gaining additional insights into potential roles of PrrA, PpsR, and FnrL in the formation of ICM. The major findings were (1) the absence of either PrrA or FnrL negatively affects ICM formation, (2) the lack of ICM in the absence of PrrA is partially, but not fully reversed by removing PpsR from the cell, (3) unlike R. sphaeroides, ICM formation in Rhodobacter capsulatus does not require FnrL. New avenues these findings provide toward identifying additional genes involved in ICM formation are discussed.
In this study, we demonstrate the selective in vivo detection of diadinoxanthin (DD) and diatoxanthin (DT) in intact Cyclotella cells using resonance Raman spectroscopy. In these cells, we were able to assess both the content of DD and DT carotenoids relative to chlorophyll and their conformation. In addition, the sensitivity and selectivity of the technique allow us to discriminate between different pools of DD on a structural basis, and to follow their fate as a function of the illumination conditions. We report that the additional DD observed when cells are grown in high-light conditions adopts a more twisted conformation than the lower levels of DD present when the cells are grown in low-light (LL) conditions. Thus, we conclude that this pool of DD is more tightly bound to a protein-binding site, which must differ from the one occupied by the DD present in LL conditions.
The Fenna–Matthews–Olson (FMO) complex from the green sulfur bacterium Chlorobaculum tepidum was studied with respect to its stability. We provide a critical assessment of published and recently measured optical spectra. FMO complexes were found to destabilize over time producing spectral shifts, with destabilized samples having significantly higher hole-burning efficiencies; indicating a remodeled protein energy landscape. Observed correlated peak shifts near 825 and 815 nm suggest possible correlated (protein) fluctuations. It is proposed that the value of 35 cm?1 widely used for reorganization energy (E?), which has important implications for the contributions to the coherence rate (Kreisbeck and Kramer 3:2828–2833, 2012), in various modeling studies of two-dimensional electronic spectra is overestimated. We demonstrate that the value of E? is most likely about 15–22 cm?1 and suggest that spectra reported in the literature (often measured on different FMO samples) exhibit varied peak positions due to different purification/isolation procedures or destabilization effects.
The effect of the inhibitor of carotenoid (Car) biosynthesis, diphenylamine (DPA), on the cells of the purple sulfur bacterium Ectothiorhodospira (Ect.) haloalkaliphila has been studied. There occurs an inhibition of the biosynthesis of colored Cars (?99 %) at 71 ?M DPA. Considering “empty” Car pockets (Moskalenko and Makhneva 2012) the content of Cars in the DPA-treated samples is first calculated more correctly. The total content of the colored Cars in the sample at 71 ?M DPA does not exceed 1 % of the wild type. In the DPA-treated cells (membranes) a complete set of pigment-protein complexes is retained. The LH2 complex at 71 ?M DPA is isolated, which is identical to the LH2 complex of the wild type in near IR absorption spectra. This suggests that the principles for assembling this LH2 complex in vivo in the absence of colored Cars remain the same. These results are in full agreement with the data obtained earlier for Allochromatium (Alc.) minutissimum (Moskalenko and Makhneva 2012). They are as follows: (1) DPA almost entirely inhibits the biosynthesis of the colored Cars in Ect.haloalkaliphila cells. (2) In the DPA-treated samples non-colored Cars are detected at 53.25 ?M DPA (as traces) and at 71 ?M DPA. (3) DPA may affect both phytoene synthase (at ?71 ?M DPA) and phytoene desaturase (at ?53.25 ?M DPA). (4) The assembly of LH2 complex does occur without any colored Cars.
In this study, we have compared photosynthetic performance of barley leaves (Hordeum vulgare L.) grown under sun and shade light regimes during their entire growth period, under field conditions. Analyses were based on measurements of both slow and fast chlorophyll (Chl) a fluorescence kinetics, gas exchange, pigment composition; and of light incident on leaves during their growth. Both the shade and the sun barley leaves had similar Chl a/b and Chl/carotenoid ratios. The fluorescence induction analyses uncovered major functional differences between the sun and the shade leaves: lower connectivity among Photosystem II (PSII), decreased number of electron carriers, and limitations in electron transport between PSII and PSI in the shade leaves; but only low differences in the size of PSII antenna. We discuss the possible protective role of low connectivity between PSII units in shade leaves in keeping the excitation pressure at a lower, physiologically more acceptable level under high light conditions.
The aim of this study was to characterize the roles of sulphur (S) nutrition in modulating the responses to iron (Fe) deficiency in the photosynthetic organelles of oilseed rape. Eight-week-old plants grown hydroponically were fed with S-sufficient or S-deprived solution with or without FeIII–EDTA. Responses to four S and Fe combined treatments were analysed after 5 and 10 days. Leaf chlorosis was generated by either S- or Fe-deprivation, with a decrease in chlorophyll and carotenoid content. These negative effects were more severe in the absence of S. The expression of Fe2+ transporter (IRT1) and Fe(III) chelate reductase (FRO1) gene was induced for the first 5 days and decreased after 10 days in the S-deprived roots, but largely improved by S supply even in the absence of Fe. Lack of ferric chelate reducing activity in the Fe-deprived roots in the absence of S was largely improved by S supply. The activity of photosynthesis, RuBisCO and sucrose synthase was closely related to S status in leaves. Electron microscopic observation showed that the Fe-deficiency in the absence of S greatly resulted in a severe disorganisation of thylakoid lamellae with loss of grana. However, these impacts of Fe-deficiency were largely restored in the presence of S. The present results indicate that S nutrition has significant role in ameliorating the damages in photosynthetic apparatus caused by Fe-deficiency.
Photosynthetic energy consumption and non-photosynthetic energy quenching processes are inherently linked. Both processes must be controlled by the cell to allow cell maintenance and growth, but also to avoid photodamage. We used the chlorophyte algae Dunaliella tertiolecta to investigate how the interactive regulation of photosynthetic and non-photosynthetic pathways varies along dissolved inorganic carbon (DIC) and photon flux gradients. Specifically, cells were transferred to DIC-deplete media to reach a CO2 compensation before being re-supplied with DIC at various concentrations and different photon flux levels. Throughout these experiments we monitored and characterized the photophysiological responses using pulse amplitude modulated fluorescence, oxygen evolution, 77 K fluorescence emission spectra, and fast-repetition rate fluorometry. O2 uptake was not significantly stimulated at DIC depletion, which suggests that O2 production rates correspond to assimilatory photosynthesis. Fluorescence-based measures of relative electron transport rates (rETRs) over-estimated oxygen-based photosynthetic measures due to a strong state-transitional response that facilitated high effective quantum yields. Adoption of an alternative fluorescence-based rETR calculation that accounts for state-transitions resulted in improved linear oxygen versus rETR correlation. This study shows the extraordinary capacity of D. tertiolecta to maintain stable effective quantum yields by flexible regulation of state-transitions. Uncertainties about the control mechanisms of state-transitions are presented.
In the present study, the influence of Mg2+ ions and low pH values on the aggregation state of the diatom FCP and the LHCII of vascular plants was studied. In addition, the concentration of thylakoid membrane lipids associated with the complexes was determined. The results demonstrate that the FCP, which contained a significantly higher concentration of the negatively charged lipids SQDG and PG, was less sensitive to Mg2+ and low pH values than the LHCII which was characterized by lower amounts of SQDG and a higher concentration of MGDG. High MgCl2 concentrations and pH values below pH 6 induced significant changes of the absorption and 77K fluorescence emission spectra of the LHCII, indicating a strong aggregation of the light-harvesting complex. This aggregation was also visible as a pellet after centrifugation on a sucrose cushion. Although the FCP responded with changes of the absorption and fluorescence spectra to low pH and Mg2+ incubation, these spectral changes were less pronounced than those observed for the LHCII. In addition, the FCP complexes did not show a visible pellet after incubation with either low pH values or high Mg2+ concentrations. Only the combined action of Mg2+ and pH 5 led to FCP aggregates of a size that could be pelleted by centrifugation. The decreased sensitivity of FCP aggregation to Mg2+ and low pH is discussed with respect to the differences in the concentration of the lipids surrounding the FCP and LHCII and the different thylakoid membrane organizations of diatoms and vascular plants.
Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes carboxylation of ribulose-1,5-bisphosphate, the first in a series of reactions leading to the incorporation of atmospheric CO2 into biomass. Rubisco requires Rubisco activase (RCA), an AAA+ ATPase that reactivates Rubisco by remodelling the conformation of inhibitor-bound sites. RCA is regulated by the ratio of ADP:ATP, with the precise response potentiated by redox regulation of the alpha-isoform. Measuring the effects of ADP on the activation of Rubisco by RCA using the well-established photometric assay is problematic because of the adenine nucleotide requirement of 3-phosphoglycerate (3-PGA) kinase. Described here is a novel assay for measuring RCA activity in the presence of variable ratios of ADP:ATP. The assay couples the formation of 3-PGA from ribulose 1,5-bisphosphate and CO2 to NADH oxidation through cofactor-dependent phosphoglycerate mutase, enolase, PEP carboxylase and malate dehydrogenase. The assay was used to determine the effects of Rubisco and RCA concentration and ADP:ATP ratio on RCA activity, and to measure the activation of a modified Rubisco by RCA. Variations of the basic assay were used to measure the activation state of Rubisco in leaf extracts and the activity of purified Rubisco. The assay can be automated for high-throughput processing by conducting the reactions in two stages.
Chlorosomes from green photosynthetic bacteria belong to the most effective light-harvesting antennas found in nature. Quinones incorporated in bacterichlorophyll (BChl) c aggregates inside chlorosomes play an important redox-dependent photo-protection role against oxidative damage of bacterial reaction centers. Artificial BChl c aggregates with and without quinones were prepared. We applied hole-burning spectroscopy and steady-state absorption and emission techniques at 1.9 K and two different redox potentials to investigate the role of quinones and redox potential on BChl c aggregates at low temperatures. We show that quinones quench the excitation energy in a similar manner as at room temperature, yet the quenching process is not as efficient as for chlorosomes. Interestingly, our data suggest that excitation quenching partially proceeds from higher excitonic states competing with ultrafast exciton relaxation. Moreover, we obtained structure-related parameters such as reorganization energies and inhomogeneous broadening of the lowest excited state, providing experimental ground for theoretical studies aiming at designing plausible large-scale model for BChl c aggregates including disorder.
Effects of ocean acidification on Emiliania huxleyi strain RCC 1216 (calcifying, diploid life-cycle stage) and RCC 1217 (non-calcifying, haploid life-cycle stage) were investigated by measuring growth, elemental composition, and production rates under different pCO2 levels (380 and 950 ?atm). In these differently acclimated cells, the photosynthetic carbon source was assessed by a 14C disequilibrium assay, conducted over a range of ecologically relevant pH values (7.9–8.7). In agreement with previous studies, we observed decreased calcification and stimulated biomass production in diploid cells under high pCO2, but no CO2-dependent changes in biomass production for haploid cells. In both life-cycle stages, the relative contributions of CO2 and HCO3? uptake depended strongly on the assay pH. At pH values ? 8.1, cells preferentially used CO2 (? 90 % CO2), whereas at pH values ? 8.3, cells progressively increased the fraction of HCO3? uptake (~45 % CO2 at pH 8.7 in diploid cells; ~55 % CO2 at pH 8.5 in haploid cells). In contrast to the short-term effect of the assay pH, the pCO2 acclimation history had no significant effect on the carbon uptake behavior. A numerical sensitivity study confirmed that the pH-modification in the 14C disequilibrium method yields reliable results, provided that model parameters (e.g., pH, temperature) are kept within typical measurement uncertainties. Our results demonstrate a high plasticity of E. huxleyi to rapidly adjust carbon acquisition to the external carbon supply and/or pH, and provide an explanation for the paradoxical observation of high CO2 sensitivity despite the apparently high HCO3? usage seen in previous studies.
Cytochrome c553 of Heliobacterium modesticaldum is the donor to P800+, the primary electron donor of the heliobacterial reaction center (HbRC). It is a membrane-anchored 14-kDa cytochrome that accomplishes electron transfer from the cytochrome bc complex to the HbRC. The petJ gene encoding cyt c553 was cloned and expressed in Escherichia coli with a hexahistidine tag replacing the lipid attachment site to create a soluble donor that could be made in a preparative scale. The recombinant cytochrome had spectral characteristics typical of a c-type cytochrome, including an asymmetric ?-band, and a slightly red-shifted Soret band when reduced. The EPR spectrum of the oxidized protein was characteristic of a low-spin cytochrome. The midpoint potential of the recombinant cytochrome was +217 ± 10 mV. The interaction between soluble recombinant cytochrome c553 and the HbRC was also studied. Re-reduction of photooxidized P800+ was accelerated by addition of reduced cytochrome c553. The kinetics were characteristic of a bimolecular reaction with a second order rate of 1.53 × 104 M?1 s?1 at room temperature. The rate manifested a steep temperature dependence, with a calculated activation energy of 91 kJ mol?1, similar to that of the native protein in Heliobacillus gestii cells. These data demonstrate that the recombinant soluble cytochrome is comparable to the native protein, and likely lacks a discrete electrostatic binding site on the HbRC.
Aquatic microalgae induce a carbon-concentrating mechanism (CCM) to maintain photosynthetic activity in low-CO2 (LC) conditions. Although the molecular mechanism of the CCM has been investigated using the single-cell green alga Chlamydomonas reinhardtii, and several CCM-related genes have been identified by analyzing high-CO2 (HC)-requiring mutants, many aspects of the CO2-signal transduction pathways remain to be elucidated. In this study, we report the isolation of novel HC-requiring mutants defective in the induction of CCM by DNA tagging. Growth rates of 20,000 transformants grown under HC and LC conditions were compared, and three HC-requiring mutants (H24, H82, and P103) were isolated. The photosynthetic CO2-exchange activities of these mutants were significantly decreased compared with that of wild-type cells, and accumulation of HLA3 and both LCIA and HLA3 were absent in mutants H24 and H82, respectively. Although the insertion of the marker gene and the HC-requiring phenotype were linked in the tetrad progeny of H82, and a calcium-sensing receptor CAS was disrupted by the insertion, exogenous expression of CAS alone could not complement the HC-requiring phenotype.
Chlorophyll f is a photosynthetic pigment that was discovered in 2010. In this study, we present investigations on spectral and dynamic characteristics of singlet-excited and triplet states of Chl f with the application of ultrafast time-resolved absorption and fluorescence spectroscopies. The pigment was studied at room temperature in two organic solvents: pyridine and diethyl ether that have different characters of coordination of the chlorophyll magnesium (Mg) atom (hexa- and penta-coordination, respectively). Cryogenic measurements (77 K) were performed in 2-methyltetrahydrofuran (hexa-coordination). The singlet-excited state lifetime was measured to be 5.6 ns at room temperature regardless of Mg coordination and 8.1 ns at 77 K. The fluorescence quantum yield of Chl f was also determined in pyridine to be 0.16. The triplet state lifetime was studied in detail in pyridine at room temperature, and the inherent lifetime was estimated to ~150 ?s. Selective measurements at 77 K demonstrated that the metastability of the triplet state greatly enhances, and its lifetime increases by a factor of more than three.
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