Light-harvesting Polypeptides Research Articles

The decreased PG content of pgp1 inhibits PSI photochemistry and limits reaction center and light-harvesting polypeptide accumulation in response to cold acclimation.

Decreased PG constrains PSI activity due to inhibition of transcript and polypeptide abundance of light-harvesting and reaction center polypeptides generating a reversible, yellow phenotype during cold acclimation of pgp1. Cold acclimation of the Arabidopsis pgp1 mutant at 5°C resulted in a pale-yellow phenotype with abnormal chloroplast ultrastructure compared to its green phenotype upon growth at 20°C despite a normal cold-acclimation response at the transcript level. In contrast, wild type maintained its normal green phenotype and chloroplast ultrastructure irrespective of growth temperature. In contrast to cold acclimation of WT, growth of pgp1 at 5°C limited the accumulation of Lhcbs and Lhcas assessed by immunoblotting. However, a novel 43kD polypeptide of Lhcb1 as well as a 29kD polypeptide of Lhcb3 accumulated in the soluble fraction which was absent in the thylakoid membrane fraction of cold-acclimated pgp1 which was not observed in WT. Cold acclimation of pgp1 destabilized the Chl-protein complexes associated with PSI and predisposed energy distribution in favor of PSII rather than PSI compared to the WT. Functionally, in vivo PSI versus PSII photochemistry was inhibited in cold-acclimated pgp1 to a greater extent than in WT relative to controls. Greening of the pale-yellow pgp1 was induced when cold-acclimated pgp1 was shifted from 5 to 20°C which resulted in a marked decrease in excitation pressure to a level comparable to WT. Concomitantly, Lhcbs and Lhcas accumulated with a simultaneous decrease in the novel 43 and 29kD polypeptides. We conclude that the reduced levels of phosphatidyldiacylglycerol in the pgp1 limit the capacity of the mutant to maintain the structure and function of its photosynthetic apparatus during cold acclimation. Thus, maintenance of normal thylakoid phosphatidyldiacylglycerol levels is essential to stabilize the photosynthetic apparatus during cold acclimation.

Chlorella vulgaris integrates photoperiod and chloroplast redox signals in response to growth at high light.

Photoacclimation to variable light and photoperiod regimes in C. vulgaris represents a complex interplay between "biogenic" phytochrome-mediated sensing and "operational" redox sensing signaling pathways. Chlorella vulgaris Beijerinck UTEX 265 exhibits a yellow-green phenotype when grown under high light (HL) in contrast to a dark green phenotype when grown at low light (LL). The redox state of the photosynthetic electron transport chain (PETC) as estimated by excitation pressure has been proposed to govern this phenotypic response. We hypothesized that if the redox state of the PETC was the sole regulator of the HL phenotype, C. vulgaris should photoacclimate in response to the steady-state excitation pressure during the light period regardless of the length of the photoperiod. As expected, LL-grown cells exhibited a dark green phenotype, low excitation pressure (1 - qP = 0.22 ± 0.02), high chlorophyll (Chl) content (375 ± 77fg Chl/cell), low Chl a/b ratio (2.97 ± 0.18) as well as high photosynthetic efficiency and photosynthetic capacity regardless of the photoperiod. In contrast, C. vulgaris grown under continuous HL developed a yellow-green phenotype characterized by high excitation pressure (1 - qP = 0.68 ± 0.01), a relatively low Chl content (180 ± 53fg Chl/cell), high Chl a/b ratio (6.36 ± 0.54) with concomitantly reduced light-harvesting polypeptide abundance, as well as low photosynthetic capacity and efficiency measured on a per cell basis. Although cells grown under HL and an 18h photoperiod developed a typical yellow-green phenotype, cells grown at HL but a 12h photoperiod exhibited a dark green phenotype comparable to LL-grown cells despite exhibiting growth under high excitation pressure (1 - qP = 0.80 ± 0.04). The apparent uncoupling of excitation pressure and phenotype in HL-grown cells and a 12h photoperiod indicates that chloroplast redox status cannot be the sole regulator of photoacclimation in C. vulgaris. We conclude that photoacclimation in C. vulgaris to HL is dependent upon growth history and reflects a complex interaction of endogenous systems that sense changes in photoperiod as well as photosynthetic redox balance.

Exposure of high-light-grown cultures of Chlorella vulgaris to darkness inhibits the relaxation of excitation pressure: uncoupling of the redox state of the photosynthetic electron transport chain and phenotypic plasticity

Chlorella vulgaris acclimated to high light (HL) conditions exhibited a pale-green phenotype characterized by reduced chlorophyll and light harvesting polypeptide abundance compared with the dark green phenotype of the control, low-light-grown (LL) cultures. We hypothesized that if chloroplast redox status was the sole regulator of phenotype, exposure to darkness should cause reversion of the HL to LL phenotype. Surprisingly, HL cells transferred to darkness or dim light failed to green. Thus, phenotypic reversion is light-dependent with an optimal photon flux density (PFD) of 110 μmol photons·m−2·s−1. HL cells shifted to this PFD exhibited increased chlorophyll and light harvesting polypeptide abundance, which were inhibited by 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone but not by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea. We conclude that photoacclimation of HL-grown cells to LL is governed by the redox state of the intersystem photosynthetic electron transport chain (PETC) at this PFD. At lower light levels, cells maintained the HL phenotype, despite an oxidized status of the PETC. Because 110 μmol photons·m−2·s−1 was the optimal PFD for protochlorophyllide oxidoreductase accumulation, we suggest that stabilization of light-harvesting polypeptides by chlorophyll binding may also govern photoacclimation in C. vulgaris. The possible role of the metabolic balance between respiration and photosynthesis is also discussed.

PufQ regulates porphyrin flux at the haem/bacteriochlorophyll branchpoint of tetrapyrrole biosynthesis via interactions with ferrochelatase.

SummaryFacultative phototrophs such as Rhodobacter sphaeroides can switch between heterotrophic and photosynthetic growth. This transition is governed by oxygen tension and involves the large‐scale production of bacteriochlorophyll, which shares a biosynthetic pathway with haem up to protoporphyrin IX. Here, the pathways diverge with the insertion of Fe2+ or Mg2+ into protoporphyrin by ferrochelatase or magnesium chelatase, respectively. Tight regulation of this branchpoint is essential, but the mechanisms for switching between respiratory and photosynthetic growth are poorly understood. We show that PufQ governs the haem/bacteriochlorophyll switch; pufQ is found within the oxygen‐regulated pufQBALMX operon encoding the reaction centre–light‐harvesting photosystem complex. A pufQ deletion strain synthesises low levels of bacteriochlorophyll and accumulates the biosynthetic precursor coproporphyrinogen III; a suppressor mutant of this strain harbours a mutation in the hemH gene encoding ferrochelatase, substantially reducing ferrochelatase activity and increasing cellular bacteriochlorophyll levels. FLAG‐immunoprecipitation experiments retrieve a ferrochelatase‐PufQ‐carotenoid complex, proposed to regulate the haem/bacteriochlorophyll branchpoint by directing porphyrin flux toward bacteriochlorophyll production under oxygen‐limiting conditions. The co‐location of pufQ and the photosystem genes in the same operon ensures that switching of tetrapyrrole metabolism toward bacteriochlorophyll is coordinated with the production of reaction centre and light‐harvesting polypeptides.

Molecular Assembly of Zinc Chlorophyll Derivatives by Using Recombinant Light-Harvesting Polypeptides with His-tag and Immobilization on a Gold Electrode

LH1-α and -β polypeptides, which make up the light-harvesting 1 (LH1) complex of purple photosynthetic bacteria, along with bacteriochlorophylls, have unique binding properties even for various porphyrin analogs. Herein, we used the porphyrin analogs, Zn-Chlorin and the Zn-Chlorin dimer, and examined their binding behaviors to the LH1-α variant, which has a His-tag at the C-terminus (MBP-rubα-YH). Zn-Chlorin and the Zn-Chlorin dimer could bind to MBP-rubα-YH and form a subunit-type assembly, similar to that from the native LH1 complex. These complexes could be immobilized onto Ni-nitrilotriacetic acid-modified Au electrodes, and the cathodic photocurrent was successfully observed by photoirradiation. Since Zn-Chlorins in this complex are too far for direct electron transfer from the electrode, a contribution of polypeptide backbone for efficient electron transfer was implied. These findings not only show interesting properties of LH1-α polypeptides but also suggest a clue to construct artificial photosynthesis systems using these peptide materials.

Effects of a cationic PAMAM dendrimer on photosynthesis and ROS production of Chlamydomonas reinhardtii

Poly(amidoamine) (PAMAM) dendrimers hold great promises for biomedicine. This study sought to examine the toxicity of generation 4 (G4) cationic PAMAM dendrimer to the green microalga, Chlamydomonas reinhardtii, using physiological and molecular biomarkers. Results revealed that the G4 dendrimer at 15 and 25 nM stimulated the photosynthetic process and the production of reactive oxygen species (ROS) in algae. However, the over-production of ROS did not induce the expression of antioxidant enzyme genes, catalase and glutathione peroxidase. In addition, genes encoding light-harvesting proteins (lhca and lhcb), a ferredoxin (fdx) and an oxygen-evolving enhancer protein (psb) involved in photosynthesis were repressed after treatment. Nevertheless, the expression of the lhcbm9 gene, encoding a major light harvesting polypeptide, was increased. These results suggest that the strong modulation of photosynthesis induced by the dendrimer could lead to elevated ROS levels in microalgae.

Absence of the major light-harvesting antenna proteins alters the redox properties of photosystem II reaction centres in thechlorina F2mutant of barley

Although the chlorina F2 mutant of barley specifically exhibits reduced levels of the major light-harvesting polypeptides associated with photosystem II (PSII), thermoluminescence measurements of photosystem reaction centre photochemistry revealed that S2/S3QB- charge recombinations were shifted to lower temperatures, while the characteristic peak of S2QA- charge recombinations was shifted to higher temperatures compared with wild-type (WT) barley. Thus, we show that the absence of the major light-harvesting polypeptides affects the redox properties of PSII reaction centres. Radiolabeling studies in vivo and in vitro with [32P]orthophosphate or [gamma-32P]ATP, respectively, demonstrated that the D1 PSII reaction centre polypeptide is phosphorylated in both the WT and the F2 mutant. In contrast with the radiolabeling results, phosphorylation of D1 and other PSII proteins, although detected in WT barley, was ambiguous in the F2 mutant when the phosphothreonine antibody method of detection was used. Thus, caution must be exercised in the use of commercially available phosphothreonine antibodies to estimate thylakoid polypeptide phosphorylation. Furthermore, in membrano, the D1 polypeptide of the F2 mutant was less susceptible to trypsin treatment than that of WT barley. The role of the light-harvesting complex in modulating the structure and function of the D1 polypeptide of PSII reaction centers is discussed.

Transcriptome for Photobiological Hydrogen Production Induced by Sulfur Deprivation in the Green Alga Chlamydomonas reinhardtii

Photobiological hydrogen production using microalgae is being developed into a promising clean fuel stream for the future. In this study, microarray analyses were used to obtain global expression profiles of mRNA abundance in the green alga Chlamydomonas reinhardtii at different time points before the onset and during the course of sulfur-depleted hydrogen production. These studies were followed by real-time quantitative reverse transcription-PCR and protein analyses. The present work provides new insights into photosynthesis, sulfur acquisition strategies, and carbon metabolism-related gene expression during sulfur-induced hydrogen production. A general trend toward repression of transcripts encoding photosynthetic genes was observed. In contrast to all other LHCBM genes, the abundance of the LHCBM9 transcript (encoding a major light-harvesting polypeptide) and its protein was strongly elevated throughout the experiment. This suggests a major remodeling of the photosystem II light-harvesting complex as well as an important function of LHCBM9 under sulfur starvation and photobiological hydrogen production. This paper presents the first global transcriptional analysis of C. reinhardtii before, during, and after photobiological hydrogen production under sulfur deprivation.

STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF PUTATIVE REGULATORY DNA SEQUENCES OF FCP GENES IN THE CENTRIC DIATOM CYCLOTELLA CRYPTICA

In order to investigate the transcriptional regulation of light harvesting proteins in diatoms we cloned and sequenced the regions encoding the N-termini together with the respective upstream DNA stretches of 11 different fucoxanthin chlorophyll a/c binding polypeptides (FCP) of Cyclotella cryptica. The deduced N-terminal amino acid sequences almost completely matched those deduced earlier from cDNA clones. No introns were found in the regions coding for the N-terminal plastid targeting sequences or shortly downstream of them. A PLACE search for cis-acting DNA elements which are known from higher plants to be involved in the light-dependent or circadian expression of Lhc genes encoding chlorophyll a/b binding light-harvesting polypeptides in green plants resulted in the detection of several motifs. A comparative analysis with DNA stretches, upstream of the Lhc, LhcaR and Fcp genes of the green alga Chlamydomonas reinhardtii, the red alga Cyanidioschyzon merolae and the diatom Thalassiosira pseudonana, however, did not show any conservation of these elements. This indicates that green plants, red algae and diatoms most probably do not share the same conserved elements for light-regulated gene expression. Furthermore a 924 bp DNA fragment habouring a stretch of 292 bp upstream DNA and the entire Fcp6 gene was amplified from genomic DNA of Cyclotella cryptica, cloned into a modified derivative of the transformation vector pPha-T1 and expressed in the pennate diatom Phaeodactylum tricornutum. Reverse transcriptase polymerase chain reaction using RNA of the transformants as template showed that the introduced Fcp6 gene indeed was transcribed, indicating that the regulatory sequence may be generally functional in diatoms.

Phototropin involvement in the expression of genes encoding chlorophyll and carotenoid biosynthesis enzymes and LHC apoproteins in Chlamydomonas reinhardtii

Phototropin (PHOT) is a photoreceptor involved in a variety of blue-light-elicited physiological processes including phototropism, chloroplast movement and stomatal opening in plants. The work presented here tests whether PHOT is involved in expression of light-regulated genes in Chlamydomonas reinhardtii. When C. reinhardtii was transferred from the dark to very low-fluence rate white light, there was a substantial increase in the level of transcripts encoding glutamate-1-semialdehyde aminotransferase (GSAT), phytoene desaturase (PDS) and light-harvesting polypeptides (e.g. LHCBM6). Increased levels of these transcripts were also elicited by low-intensity blue light, and this blue-light stimulation was suppressed in three different RNAi strains that synthesize low levels of PHOT. The levels of GSAT and LHCBM6 transcripts also increased following exposure of algal cells to low-intensity red light (RL). The red-light-dependent increase in transcript abundance was not affected by the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, implying that the influence of RL on transcript accumulation was not controlled by cytoplasmic redox conditions, and that a red-light photoreceptor(s) may be involved in regulating the levels of transcripts from specific photosynthesis-related genes in C. reinhardtii. Interestingly, elevated GSAT and LHCBM6 transcript levels in RL were significantly reduced in the PHOT RNAi strains, which raises the possibility of co-action between blue and RL signaling pathways. Microarray experiments indicated that the levels of several transcripts for photosystem (PS) I and II polypeptides were also modulated by PHOT. These data suggest that, in C. reinhardtii, (i) PHOT is involved in blue-light-mediated changes in transcript accumulation, (ii) synchronization of the synthesis of chlorophylls (Chl), carotenoids, Chl-binding proteins and other components of the photosynthetic apparatus is achieved, at least in part, through PHOT-mediated signaling, and (iii) a red-light photoreceptor can also influence levels of certain transcripts associated with photosynthetic function, although its action requires normal levels of PHOT.

DNA macroarray and real-time PCR analysis of two nuclear photosystem I mutants from Chlamydomonas reinhardtii reveal downregulation of Lhcb genes but different regulation of Lhca genes

In photoautotrophic organisms, the expression of nuclear genes encoding plastid proteins is known to be regulated at various levels. In this study, we present the analysis of two non-photosynthetic mutants (CC1051 and TR72) from the unicellular green alga Chlamydomonas reinhardtii. Both mutant strains show a defect in the processing of chloroplast psaA mRNA, and therefore they are assumed to be defective in photosystem I (PSI) assembly. We have performed macroarray experiments with trans-splicing mutants CC1051 and TR72 in order to analyse putative pleiotropic effects of nuclear-located mutations leading to a non-functional PSI. To the best of our knowledge, this is the first example of Chlamydomonas cDNA macroarray analysis comparing the transcriptional regulation of nuclear genes in wild-type and photosystem I mutants. The macroarray results demonstrated a transcriptional downregulation of members of the Lhcb gene family more than 2-fold in both mutant strains. In addition, real-time RT-PCR experiments found a 4- to 16-fold reduction in transcript levels of several Lhca genes in TR72; whereas in CC1051, no significant change in transcript levels was observed. Taken together, our data suggest that a signal is transmitted from the chloroplast to the nucleus that serves to regulate the level of light harvesting polypeptides in the organelle.

Localisation of fucoxanthin chlorophyll a/c-binding polypeptides of the centric diatom Cyclotella cryptica by immuno-electron microscopy

Antisera were raised against the C termini of three fucoxanthin chlorophyll a/c-binding polypeptides, Fcp2, Fcp4, and Fcp6, of the centric diatom Cyclotella cryptica. Immunogold electron microscopy of ultrathin-sectioned cells indicated that Fcp2 and Fcp4 are present in almost the same amounts, whereas approximately 8- to 10-fold less gold label was registered for Fcp6. Immunogold electron microscopy of freeze-fracture replicas of thylakoid membranes showed that the C termini of at least Fcp2 and Fcp4 were located in the thylakoid lumen, thus demonstrating a 3-dimensional structure similar to that already described for the chlorophyll a/b-binding light-harvesting polypeptides of higher plants.

Selective Organization of an α-Helical Hydrophobic Polypeptide/Pigment Complex in a Lipid Domain of Binary Lipid Bilayers

We present here a novel approach to organization of an α-helical hydrophobic polypeptide complex in lipid bilayers in a 'lipid-domain-selective' manner. The key strategy is to use disulfide linkage between a cysteine-bearing polypeptide and a disulfide-based phospholipid, the latter of which forms a phase-separated lipid domain in a binary lipid bilayer with an incompatible phospholipid lacking disulfide group. The cross-linkage between the polypeptide and the phospholipid can be expected that the polypeptide acquires a higher affinity to the disulfide-based lipid domain than to the other one, resulting in selective distribution of the polypeptide in the former domain. The polypeptide analogous to a photosynthetic bacterial light-harvesting polypeptide binds a pigment, zinc-substituted bacteriochlorophyll a ([Zn]-BChl a), to form the light-harvesting polypeptide/pigment complex. The complex was incorporated into an incompatible binary lipid bilayer combination either the disulfide-based phospholipids bearing oleoyl chains (DO-DO)/1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/DSPC. The lipid-domain-selective organization was confirmed by fluorescence resonance energy transfer (FRET) from a rhodamine-labeled lipid (Lissamine rhodamine B 1,2-diacyl-sn-glycero-3-phosphoethanolamine: either N-Rh-DOPE or N-Rh-DSPE) to the assembled [Zn]-BChl a. Considering the relative affinity of these fluorescent lipid probes, N-Rh-DOPE and N-Rh-DSPE prefer to fluid and gel domains, respectively, because of the similarity of their acyl chains. When the fluorescent lipid probe, N-Rh-DOPE, was applied to the binary lipid system, DO-DO/DSPC, the higher FRET efficiency was observed compared to the counterpart FRET assay using N-Rh-DSPE. In contrast, for the DOPC/DSPC bilayer, the difference in the FRET efficiency between the fluorescent lipid probe systems was negligible. These results suggest that the polypeptide/pigment complex is preferentially incorporated into the disulfide-based lipid domain. Total internal reflection fluorescence (TIRF) microscopic observation of the assembly also evidenced heterogeneous structure by detection of fluorescence from [Zn]-BChl a. [DOI: 10.1380/ejssnt.2005.145]

Insights into the survival of Chlamydomonas reinhardtii during sulfur starvation based on microarray analysis of gene expression.

Responses of photosynthetic organisms to sulfur starvation include (i) increasing the capacity of the cell for transporting and/or assimilating exogenous sulfate, (ii) restructuring cellular features to conserve sulfur resources, and (iii) modulating metabolic processes and rates of cell growth and division. We used microarray analyses to obtain a genome-level view of changes in mRNA abundances in the green alga Chlamydomonas reinhardtii during sulfur starvation. The work confirms and extends upon previous findings showing that sulfur deprivation elicits changes in levels of transcripts for proteins that help scavenge sulfate and economize on the use of sulfur resources. Changes in levels of transcripts encoding members of the light-harvesting polypeptide family, such as LhcSR2, suggest restructuring of the photosynthetic apparatus during sulfur deprivation. There are also significant changes in levels of transcripts encoding enzymes involved in metabolic processes (e.g., carbon metabolism), intracellular proteolysis, and the amelioration of oxidative damage; a marked and sustained increase in mRNAs for a putative vanadium chloroperoxidase and a peroxiredoxin may help prolong survival of C. reinhardtii during sulfur deprivation. Furthermore, many of the sulfur stress-regulated transcripts (encoding polypeptides associated with sulfate uptake and assimilation, oxidative stress, and photosynthetic function) are not properly regulated in the sac1 mutant of C. reinhardtii, a strain that dies much more rapidly than parental cells during sulfur deprivation. Interestingly, sulfur stress elicits dramatic changes in levels of transcripts encoding putative chloroplast-localized chaperones in the sac1 mutant but not in the parental strain. These results suggest various strategies used by photosynthetic organisms during acclimation to nutrient-limited growth.

A genome?s-eye view of the light-harvesting polypeptides of Chlamydomonas reinhardtii

Chlamydomonas reinhardtii is a valuable model system for defining the structure and function of polypeptides of the photosynthetic apparatus and the dynamic aspects of photosynthesis. Recently, a genome-wide analysis of cDNAs and a draft genome sequence that covers approximately 90% of the genome were made available, providing a clear picture of the composition of specific gene families, the relationships among the gene family members, and the location of each member on the genome. We used the available sequence information to analyze the extensive family of light-harvesting genes in C. reinhardtii. There are nine genes encoding polypeptides of the major light-harvesting complex of photosystem II, two genes encoding the minor light-harvesting polypeptides of photosystem II, and nine genes encoding polypeptides predicted to comprise the photosystem I light-harvesting complex. Furthermore, there are five genes encoding early light-induced proteins and two genes encoding LI818 polypeptides. A candidate for the PsbS gene has also been found in the raw genome sequence data (Niyogi, personal communication), although no genes encoding homologues of the Sep, or Hli polypeptides have been identified. In this manuscript, we identify and classify the family of light-harvesting polypeptides encoded on the C. reinhardtii genome. This is an important first step in designing specific genetic, biochemical, and physiological studies aimed at characterizing the composition, function, and regulation of the light-harvesting complexes.

Temperature-induced greening of Chlorella vulgaris. The role of the cellular energy balance and zeaxanthin-dependent nonphotochemical quenching.

When cells of the green alga Chlorella vulgaris Beij. are transferred from growth at 5 degrees C and an irradiance of 150 micromol photons m(-2) s(-1) to 27 degrees C and the same irradiance, they undergo what is normally considered a high-light to low-light phenotypic change. This involves a 3-fold increase in cellular chlorophyll content with a concomitant increase in light-harvesting complex polypeptide levels. This process appears to occur in response to the cellular capacity to utilize the products of photosynthesis, with the redox state of the plastoquinone pool sensing the cellular energy balance. The phenotypic adjustment can be enhanced or blocked using chemical inhibitors that modulate the redox state of the plastoquinone pool. The functional changes in the photosynthetic apparatus that occurred during the high-light to low-light acclimation were examined with special consideration paid to the paradox that 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated cells, with non-functional photosystem II (PSII), accumulate light-harvesting polypeptides. At the structural and basic functional levels, the light-harvesting complex of the cells treated with DCMU was indistinguishable from that of the untreated, control cells. To examine how PSII was protected in the DCMU-treated cells, we measured the content of xanthophyll-cycle pigments. It appeared that a zeaxanthin-dependent nonphotochemical quenching process was involved in PSII protection during greening in the presence of DCMU. Metabolic inhibitors of mitochondrial respiration were used to examine how the change in cellular energy balance regulates the greening process. Apparently, the mitochondrion acts to supply energy to the chloroplast during greening, and inhibition of mitochondrial respiration diminishes chlorophyll accumulation apparently through an increase in the redox state of the plastoquinone pool.

Characterization of expressed pigmented core light harvesting complex (LH 1) in a reaction center deficient mutant of Blastochloris viridis.

The utility of photosynthetically defective mutants in the purple photosynthetic bacterium Blastochloris viridis (formerly Rhodopseudomonas viridis)was demonstrated with construction of a reaction-center deficient mutant, LH 1-H. This LH 1-H mutant has a photosynthetic apparatus in which most of the puf operon genes were deleted, resulting in an organism containing only the genes for the light harvesting polypeptides and the H subunit of the reaction center. This B. viridisstrain containing a truncation of the puf operon was characterized by gel electrophoresis, lipid-to-protein ratio analysis, optical spectroscopy, electron paramagnetic resonance and transmission electron microscopy. Optical and electron paramagnetic resonance spectroscopies revealed no photoactivity in this LH 1-H mutant consistent with the absence of intact reaction centers. Electron paramagnetic resonance evidence for assembled LH 1 complexes suggested that the interactions between light harvesting polypeptide complexes in membranes were largely unchanged despite the absence of their companion reaction center cores. The observed increase in the lipid-to-protein ratio was consistent with modified interactions between LH 1s, a view supported by transmission electron microscopy analysis of membrane fragments. The results show that B. viridis can serve as a practical system for investigating structure-function relationships in membranes and photosynthesis through the construction of photosynthetically defective mutants.

Investigations on Transcript Sizes, Steady State mRNA Concentrations and Diurnal Expression of Genes Encoding Fucoxanthin Chlorophyll a/c Light Harvesting Polypeptides in the Centric Diatom Cyclotella cryptica

Abstract: The transcript sizes of fcp genes of Cyclotella cryptica, belonging to different fcp gene clusters and encoding different fucoxanthin chlorophyll a/c light harvesting polypeptides (Fcps), were determined. Furthermore, the diurnal expression of the fcp1/fcp2/fcp3/fcp5 gene cluster and the steady state mRNA concentrations of all gene clusters were investigated in response to light quality and quantity. The mRNAs of the gene cluster consisting of fcp1/fcp2/fcp3/fcp5 are approx. 950 bases in length, whereas those of the fcp4 and the fcp6/fcp7/fcp12 clusters are approx. 1050 (fcp4), 880 (fcp6/fcp7) and 1150 (fcp12) bases in lengths. The transcript levels of the fcp1/fcp2/fcp3/fcp5 gene cluster increased with the onset of light, reached a maximum around noon and dropped in the afternoon. The steady state mRNA concentrations of the fcp1/fcp2/fcp3/fcp5 gene cluster and of the fcp4 gene cluster were higher, when Cyclotella cryptica was grown in low light, while the steady state mRNA concentration of the fcp6/fcp7/fcp12 gene cluster increased under high light growth conditions. The steady state mRNA concentrations of all gene clusters were highest when Cyclotella cryptica was grown in red light, intermediate in green light, and lowest in blue light.

Molecular Assembly of Bachteriochlorophyll a Using Light-harvesting Model 1α-Helix Polypeptides and 2α-Helix Polypeptide with Disulfide-linkage

Abstract UV-vis. absorption and CD spectral data indicate that the minimum amino acid sequence in the N-terminal segment of the light-harvesting (LH) polypeptides of photosynthetic bacteria necessary for the LH1-type complex-formation was that of the type 2, of our synthetic hydrophobic polypeptides (Scheme 2). Stable BChl a complex in the presence of a 2α-helix polypeptide with disulfide linkage, was obtained in lipid bilayers as well as in n-octyl-β-D-glucopyranoside (OG) micelles, analogous to the subunit complex, B820, of the LH I complex of photosynthetic bacteria.

Rapid and highly specific monitoring of reversible thylakoid protein phosphorylation by polyclonal antibody to phosphothreonine-containing proteins

Reversible thylakoid protein phosphorylation in plant chloroplast probably plays an important role in acclimation of the photosynthetic apparatus to changes in environmental conditions. Studies of regulation and the significance of these reactions have in vivo greatly benefited from recent availability of phosphothreonine antibodies. To verify the specificity of these polyclonal antibodies, leaves were treated in conditions expected to induce either phosphorylation or dephosphorylation of thylakoid proteins D1, D2 and CP43 of the photosystem II core as well as light harvesting polypeptides of 27 and 25 kDa; subsequently, the proteins were isolated to homogeneity. Immunoreactions of these purified proteins with phosphothreonine antibodies were very similar to those observed with intact thylakoids. Moreover, their positive immunoresponse could be totally abolished by treating the thylakoid samples or purified photosystem II core preparations with acid phosphatase before immunoblotting. We conclude that the analytical method of using polyclonal phosphothreonine antibodies will turn out to be a highly specific and valuable tool in monitoring changes in the phosphorylation patterns of individual thylakoid phosphoproteins, both in vivo and in vitro.