The immunophilin CYCLOPHILIN28 affects PSII-LHCII supercomplex assembly and accumulation in Arabidopsis thaliana.

In plant chloroplasts, photosystem II (PSII) complexes, together with light-harvesting complex II (LHCII), form various PSII-LHCII supercomplexes (SCs). This process likely involves immunophilins, but the underlying regulatory mechanisms are unclear. Here, by comparing Arabidopsis thaliana mutants lacking the chloroplast lumen-localized immunophilin CYCLOPHILIN28 (CYP28) to wild-type and transgenic complemented lines, we determined that CYP28 regulates the assembly and accumulation of PSII-LHCII SCs. Compared to the wild type, cyp28 plants showed accelerated leaf growth, earlier flowering time, and enhanced accumulation of high molecular weight PSII-LHCII SCs under normal light conditions. The lack of CYP28 also significantly affected the electron transport rate. Blue native-polyacrylamide gel electrophoresis analysis revealed more Lhcb6 and less Lhcb4 in M-LHCII-Lhcb4-Lhcb6 complexes in cyp28 versus wild-type plants. Peptidyl-prolyl cis/trans isomerase (PPIase) activity assays revealed that CYP28 exhibits weak PPIase activity and that its K113 and E187 residues are critical for this activity. Mutant analysis suggested that CYP28 may regulate PSII-LHCII SC accumulation by altering the configuration of Lhcb6 via its PPIase activity. Furthermore, the Lhcb6-P139 residue is critical for PSII-LHCII SC assembly and accumulation. Therefore, our findings suggest that CYP28 likely regulates PSII-LHCII SC assembly and accumulation by altering the configuration of P139 of Lhcb6 via its PPIase activity.

Integrating on-grid immunogold labeling and cryo-electron tomography to reveal photosystem II structure and spatial distribution in thylakoid membranes.

A long-standing challenge in cell biology is elucidating the structure and spatial distribution of individual membrane-bound proteins, protein complexes and their interactions in their native environment. Here, we describe a workflow that combines on-grid immunogold labeling, followed by cryo-electron tomography (cryoET) imaging and structural analyses to identify and characterize the structure of photosystem II (PSII) complexes. Using an antibody specific to a core subunit of PSII, the D1 protein (uniquely found in the water splitting complex in all oxygenic photoautotrophs), we identified PSII complexes in biophysically active thylakoid membranes isolated from a model marine diatom Phaeodactylum tricornutum. Subsequent cryoET analyses of these protein complexes resolved two PSII structures: supercomplexes and dimeric cores. Our integrative approach establishes the structural signature of multimeric membrane protein complexes in their native environment and provides a pathway to elucidate their high-resolution structures.

Circular dichroism and resonance Raman spectroscopies of bacteriochlorophyll b-containing LH1-RC complexes.

The core light-harvesting complexes (LH1) in bacteriochlorophyll (BChl) b-containing purple phototrophic bacteria are characterized by a near-infrared absorption maximum around 1010 nm. The determinative cause for this ultra-redshift remains unclear. Here, we present results of circular dichroism (CD) and resonance Raman measurements on the purified LH1 complexes in a reaction center-associated form from a mesophilic and a thermophilic Blastochloris species. Both the LH1 complexes displayed purely positive CD signals for their Qy transitions, in contrast to those of BChl a-containing LH1 complexes. This may reflect differences in the conjugation system of the bacteriochlorin between BChl b and BChl a and/or the differences in the pigment organization between the BChl b- and BChl a-containing LH1 complexes. Resonance Raman spectroscopy revealed remarkably large redshifts of the Raman bands for the BChl b C3-acetyl group, indicating unusually strong hydrogen bonds formed with LH1 polypeptides, results that were verified by a published structure. A linear correlation was found between the redshift of the Raman band for the BChl C3-acetyl group and the change in LH1-Qy transition for all native BChl a- and BChl b-containing LH1 complexes examined. The strong hydrogen bonding and π-π interactions between BChl b and nearby aromatic residues in the LH1 polypeptides, along with the CD results, provide crucial insights into the spectral and structural origins for the ultra-redshift of the long-wavelength absorption maximum of BChl b-containing phototrophs.

Incorporation of spheroidene and spheroidenone into light-harvesting complexes from purple sulfur bacteria.

Spheroidene and spheroidenone from the non-sulfur bacterium Rhodobacter (Rba.) sphaeroides were incorporated into diphenylamine (DPA) LH1-RC and LH2 complexes from sulfur bacteria Allochromatium (Alc.) minutissimum and Ectothiorhodospira (Ect.) haloalkaliphila in which carotenoid (Car) biosynthesis was inhibited by ~95%. A series of biochemical characteristics of the modified LH2 complexes was studied (electrophoretic mobility, absorption and CD spectra, Car composition, Car-to-BChl energy transfer and thermal stability). It was found that the electrophoretic mobility of the complexes with incorporated Cars did not change compared to that of the control and DPA-complexes, indicating the absence of any significant change in the structure of LH complexes upon DPA-treatment and subsequent incorporation of Cars. The analysis of fluorescence excitation spectra of the spheroidene-incorporated LH2 complex (LH2:sph) and the spheroidenone-incorporated LH2 complex (LH2:sph-ne) showed that spheroidene and spheroidenone exhibited relatively low efficiencies of energy transfer to BChl, when incorporated into the LH2 DPA-complexes from Alc. minutissimum and Ect. haloalkaliphila, although, they showed high efficiencies, being in their natural state in the LH2 complexes from Rba. sphaeroides. A significant increase in thermostability observed for the LH2:sph and LH2:sph-ne complexes with respect to the LH2 DPA-complexes indicated that the two incorporated Cars stabilized the structure of the LH2 complexes.

Determination of the Molar Extinction Coefficients of the B800 and B850 Absorption Bands in Light-harvesting Complexes 2 Derived from Three Purple Photosynthetic Bacteria Rhodoblastus acidophilus, Rhodobacter sphaeroides, and Phaeospirillum molischianum by Extraction of Bacteriochlorophyll a.

The molar extinction coefficients of light-harvesting complex 2 (LH2) have been ambiguous in spite of its fame and wide utilization. Herein we determine the molar extinction coefficients of the LH2 proteins derived from the three purple photosynthetic bacteria Rhodoblastus acidophilus, Rhodobacter sphaeroides and Phaeospirillum molischianum at 298 K by direct extraction of bacteriochlorophyll (BChl) a from the lyophilized proteins, followed by estimation of BChl a amounts from their electronic absorption spectra.

Light-Induced Infrared Difference Spectroscopy in the Investigation of Light Harvesting Complexes.

Light-induced infrared difference spectroscopy (IR-DS) has been used, especially in the last decade, to investigate early photophysics, energy transfer and photoprotection mechanisms in isolated and membrane-bound light harvesting complexes (LHCs). The technique has the definite advantage to give information on how the pigments and the other constituents of the biological system (proteins, membranes, etc.) evolve during a given photoreaction. Different static and time-resolved approaches have been used. Compared to the application of IR-DS to photosynthetic Reaction Centers (RCs), however, IR-DS applied to LHCs is still in an almost pioneering age: very often sophisticated techniques (step-scan FTIR, ultrafast IR) or data analysis strategies (global analysis, target analysis, multivariate curve resolution) are needed. In addition, band assignment is usually more complicated than in RCs. The results obtained on the studied systems (chromatophores and RC-LHC supercomplexes from purple bacteria; Peridinin-Chlorophyll-a-Proteins from dinoflagellates; isolated LHCII from plants; thylakoids; Orange Carotenoid Protein from cyanobacteria) are summarized. A description of the different IR-DS techniques used is also provided, and the most stimulating perspectives are also described. Especially if used synergically with other biophysical techniques, light-induced IR-DS represents an important tool in the investigation of photophysical/photochemical reactions in LHCs and LHC-containing systems.

Reconstruction of phyletic trees by global alignment of multiple metabolic networks.

BACKGROUND: In the last decade, a considerable amount of research has been devoted to investigating the phylogenetic properties of organisms from a systems-level perspective. Most studies have focused on the classification of organisms based on structural comparison and local alignment of metabolic pathways. In contrast, global alignment of multiple metabolic networks complements sequence-based phylogenetic analyses and provides more comprehensive information. RESULTS: We explored the phylogenetic relationships between microorganisms through global alignment of multiple metabolic networks. The proposed approach integrates sequence homology data with topological information of metabolic networks. In general, compared to recent studies, the resulting trees reflect the living style of organisms as well as classical taxa. Moreover, for phylogenetically closely related organisms, the classification results are consistent with specific metabolic characteristics, such as the light-harvesting systems, fermentation types, and sources of electrons in photosynthesis. CONCLUSIONS: We demonstrate the usefulness of global alignment of multiple metabolic networks to infer phylogenetic relationships between species. In addition, our exhaustive analysis of microbial metabolic pathways reveals differences in metabolic features between phylogenetically closely related organisms. With the ongoing increase in the number of genomic sequences and metabolic annotations, the proposed approach will help identify phenotypic variations that may not be apparent based solely on sequence-based classification.

High-light vs. low-light: effect of light acclimation on photosystem II composition and organization in Arabidopsis thaliana.

The structural response of photosystem II (PSII) and its light-harvesting proteins (LHCII) in Arabidopis thaliana after long-term acclimation to either high or low light intensity was characterized. Biochemical and structural analysis of isolated thylakoid membranes by electron microscopy indicates a distinctly different response at the level of PSII and LHCII upon plant acclimation. In high light acclimated plants, the C(2)S(2)M(2) supercomplex, which is the dominating form of PSII in Arabidopsis, is a major target of structural re-arrangement due to the down-regulation of Lhcb3 and Lhcb6 antenna proteins. The PSII ability to form semi-crystalline arrays in the grana membrane is strongly reduced compared to plants grown under optimal light conditions. This is due to the structural heterogeneity of PSII supercomplexes rather than to the action of PsbS protein as its level was unexpectedly reduced in high light acclimated plants. In low light acclimated plants, the architecture of the C(2)S(2)M(2) supercomplex and its ability to form semi-crystalline arrays remained unaffected but the density of PSII in grana membranes is reduced due to the synthesis of additional LHCII proteins. However, the C(2)S(2)M(2) supercomplexes in semi-crystalline arrays are more densely packed, which can be important for efficient energy transfer between PSII under light limiting conditions.

HPLC-DAD-ESI/MS identification of light harvesting and light screening pigments in the lake sediments at Edmonson Point.

The composition of sedimentary pigments in the Antarctic lake at Edmonson Point has been investigated and compared with the aim to provide a useful analytical method for pigments separation and identification, providing reference data for future assessment of possible changes in environmental conditions. Reversed phase high performance liquid chromatography (HPLC) with electrospray-mass spectrometry (ESI-MS) detection and diode array detection (DAD) has been used to identify light screening and light harvesting pigments. The results are discussed in terms of local environmental conditions.

Increased thermostability of thylakoid membranes in isoprene-emitting leaves probed with three biophysical techniques.

Three biophysical approaches were used to get insight into increased thermostability of thylakoid membranes in isoprene-emittingplants.Arabidopsis (Arabidopsis thaliana) plants genetically modified to make isoprene and Platanus orientalis leaves, in which isoprene emission was chemically inhibited, were used. First, in the circular dichroism spectrum the transition temperature of the main band at 694 nm was higher in the presence of isoprene, indicating that the heat stability of chiral macrodomains of chloroplast membranes, and specifically the stability of ordered arrays of light-harvesting complex II-photosystem II in the stacked region of the thylakoid grana, was improved in the presence of isoprene. Second, the decay of electrochromic absorbance changes resulting from the electric field component of the proton motive force (ΔA515) was evaluated following single-turnover saturating flashes. The decay of ΔA515 was faster in the absence of isoprene when leaves of Arabidopsis and Platanus were exposed to high temperature, indicating that isoprene protects the thylakoid membranes against leakiness at elevated temperature. Finally, thermoluminescence measurements revealed that S2Q(B)⁻ charge recombination was shifted to higher temperature in Arabidopsis and Platanus plants in the presence of isoprene, indicating higher activation energy for S2Q(B)⁻ redox pair, which enables isoprene-emitting plants to perform efficient primary photochemistry of photosystem II even at higher temperatures. The data provide biophysical evidence that isoprene improves the integrity and functionality of the thylakoid membranes at high temperature. These results contribute to our understanding of isoprene mechanism of action in plant protection against environmental stresses.

Impact of moderate Fe excess under Cd stress on the photosynthetic performance of poplar (Populus jacquemontiana var. glauca cv. Kopeczkii).

Cadmium interference with Fe nutrition has a strong impact on the development and efficiency of the photosynthetic apparatus. To shed more light on the interaction between Fe and Cd, it was studied how iron given in moderate excess under Cd stress affects the development and functioning of chlorophyll-protein complexes. Poplar plants grown in hydroponics up to four-leaf stage were treated with 10 µM Cd(NO3)2 in the presence of 50 µM Fe([III])-citrate as iron supply (5xFe + Cad) for two weeks. Though leaf area growth was inhibited similarly to that of Cad (10 µM Cd(NO3)2 + 10 µM Fe([III])-citrate) plants, chlorophyll content, ¹4CO2 fixation and quenching parameters calculated from PAM fluorescence induction measurements were control-like in 5xFe+Cad leaves. Increased chloroplast iron content (measured photometrically by the bathophenanthroline disulfonate method) without changes in the iron and cadmium content of leaves (determined by inductively coupled plasma mass spectrometry) pointed out that a key factor in the observed protection of photosynthesis is the iron-excess-induced redistribution of iron in the leaf. However, the chlorophyll a/b ratio and the chlorophyll-protein pattern obtained by Deriphat PAGE remained similar to that of Cad leaves. The decreased amount of PSII core and PSI in mature and developing leaves, respectively, refers to developmental stage-dependent remodelling of thylakoids in the presence of Cd. The results underline not only the beneficial effect of iron excess under Cd stress, but also refer to the importance of a proper Fe/Cd ratio and light environment to avoid its possible harmful effects.

Use of single-molecule spectroscopy to tackle fundamental problems in biochemistry: using studies on purple bacterial antenna complexes as an example.

Optical single-molecule techniques can be used in two modes to investigate fundamental questions in biochemistry, namely single-molecule detection and single-molecule spectroscopy. This review provides an overview of how single-molecule spectroscopy can be used to gain detailed information on the electronic structure of purple bacterial antenna complexes and to draw conclusions about the underlying physical structure. This information can be used to understand the energy-transfer reactions that are responsible for the earliest reactions in photosynthesis.

Proteotypic profiling of LHCI from Chlamydomonas reinhardtii provides new insights into structure and function of the complex.

We used isotope dilution MS to measure the stoichiometry of light-harvesting complex I (LHCI) proteins with the photosystem I (PSI) core complex in the green alga Chlamydomonas reinhardtii. Proteotypic peptides served as quantitative markers for each of the nine gene products (Lhca1-9) and for PSI subunits. The quantitative data revealed that the LHCI antenna of C. reinhardtii contains about 7.5 +/- 1.4 subunits. It further demonstrated that the thylakoid LHCI population is heterogeneously composed and that several lhca gene products are not present in 1:1 stoichiometries with PSI. When compared with vascular plants, LHCI of C. reinhardtii possesses a lower proportion of proteins potentially contributing to far-red fluorescence emission. In general, the strategy presented is universally applicable for exploring subunit stoichiometries within the C. reinhardtii proteome.

[Assembly of LH2 light-harvesting complexes in Rhodopseudomonas palustris cells illuminated by blue and red light].

We investigated the formation of the B800-850 complex in cells of the bacterium Rhodopseudomonas palustris AB illuminated by red and blue light under anaerobic growth conditions. Under red illumination, the B800-850 complex was assembled with a reduced absorption band at 850 nm. The results of re-electrophoresis of the B800-850 complex and oxidation in the presence of potassium iridate suggest its heterogeneity. It may be a mixture of two complexes (B800 and B800-850). The B800-850 complex lacks the capacity for conformational transitions if assembled under blue illumination. Accordingly, the light-harvesting complex assembled in the blue light contains polypeptides that are not synthesized under normal conditions or at increased or decreased light intensities. The mechanism of regulation of the synthesis of the polypeptides of light-harvesting B800-850 complex and its dependence on the spectral composition of the light is discussed.

Redox-state dynamics of ubiquinone-10 imply cooperative regulation of photosynthetic membrane expression in Rhodospirillum rubrum.

It is now well established that, for photosynthetic bacteria, the aerobic-to-microaerophilic transition activates the membrane-bound sensor kinase RegB, which subsequently phosphorylates the transcriptional activator RegA, thereby inducing elevated levels of intracellular photosynthetic membranes. The mechanism of RegB activation--in particular, the role of ubiquinone-10--is controversial at present. One problem here is that very limited quantitative in vivo data for the response of the ubiquinone redox state to different cultivation conditions exist. Here, we utilize Rhodospirillum rubrum to study the correlation of the quinone redox state to the expression level of photosynthetic membranes and determine an effective response function directly. Our results show that changes in the photosynthetic membrane levels between 50 and 95% of that maximally attainable are associated with only a twofold change in the ubiquinol/ubiquinone ratio and are not necessarily proportional to the total levels of either quinone or [NAD(+) + NADH]. There is no correlation between the redox potentials of the quinone and pyridine nucleotide pools. Hill function analysis of the photosynthetic membrane induction in response to the quinone redox state suggests that the induction process is highly cooperative. Our results are probably generally applicable to quinone redox regulation in bacteria.

Assessment of chlorophyll-a concentration and trophic state for Lake Chagan using Landsat TM and field spectral data.

In this study, we present the digital evaluation of Landsat TM data and field spectral measurements for retrieving chlorophyll-a (chl-a) concentration and trophic state index in Lake Chagan of Northeast China. Chl-a concentration of the lake can be estimated from the band ratio (TM4/TM3) and the field spectral data at 670 nm (absorption peak) and at 700 nm (reflectance peak). The results show that the best determination coefficient (R (2)) is 0.67 from the TM data, by which chl-a distribution can be mapped. Based on chl-a determination from laboratory analysis, field spectral and TM data, the modified trophic state index (TSI(M)) was applied to assess the lake's trophic state. With the available data in Lake Chagan, each algorithm demonstrates the similar result for assessing the lake's chl-a and trophic state. Our results indicate that Landsat TM and field spectral data could be used effectively to determine chl-a concentration and evaluate the trophic state of Lake Chagan in the study.

Lack of the light-harvesting complex CP24 affects the structure and function of the grana membranes of higher plant chloroplasts.

The photosystem II (PSII) light-harvesting antenna in higher plants contains a number of highly conserved gene products whose function is unknown. Arabidopsis thaliana plants depleted of one of these, the CP24 light-harvesting complex, have been analyzed. CP24-deficient plants showed a decrease in light-limited photosynthetic rate and growth, but the pigment and protein content of the thylakoid membranes were otherwise almost unchanged. However, there was a major change in the macroorganization of PSII within these membranes; electron microscopy and image analysis revealed the complete absence of the C(2)S(2)M(2) light-harvesting complex II (LHCII)/PSII supercomplex predominant in wild-type plants. Instead, only C(2)S(2) supercomplexes, which are deficient in the LHCIIb M-trimers, were found. Spectroscopic analysis confirmed the disruption of the wild-type macroorganization of PSII. It was found that the functions of the PSII antenna were disturbed: connectivity between PSII centers was reduced, and maximum photochemical yield was lowered; rapidly reversible nonphotochemical quenching was inhibited; and the state transitions were altered kinetically. CP24 is therefore an important factor in determining the structure and function of the PSII light-harvesting antenna, providing the linker for association of the M-trimer into the PSII complex, allowing a specific macroorganization that is necessary both for maximum quantum efficiency and for photoprotective dissipation of excess excitation energy.

Stimulation of pigment accumulation in Anabaena azollae strains: effect of light intensity and sugars.

The influence of high light intensity on the growth and pigment accumulating ability of Anabaena azollae was investigated. A. azollae responded positively to high light intensity (6 klx) and was further evaluated at higher intensities (10 and 15 klx), in the presence of glucose, sucrose and jaggery +/- DCMU. Significant enhancement in phycobiliproteins and carotenoids was observed in the sugar supplemented cultures at high light intensities. SDS-PAGE profiles of whole cell proteins revealed the presence of unique bands in such treatments. Sucrose supplementation induced a 30-90 % increase in carotenoids, phycocyanin and phycoerythrin content at 10 klx. Molecular analysis of the stimulatory and interactive role of sugars on pigment enhancement at high light intensity may aid in better exploitation of cyanobacteria as a source of pigments.

Morphological, physiochemical and molecular characterization of Anabaena strains.

A set of 30 Anabaena strains, isolated from diverse geographical regions of India, were characterized using morphological and physiochemical attributes as well as molecular marker profiles. Significant differences were observed among the Anabaena strains with regard to the shape and size of trichomes and individual cells within a filament, besides qualitative and quantitative aspects of phycobiliprotein accumulation and activities of enzymes involved in nitrogen metabolism. Analyses of molecular polymorphisms in a selected set of 13 Anabaena strains, using primers based on repetitive sequences in the genome, led to unambiguous differentiation of the strains as well as understanding of their genetic relationships. Informative morphological, physio-chemical and molecular characters have been identified that could aid in differentiation and utilization of Anabaena strains as bioinoculants or as sources of pigments.