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PubMed1997

hcf5, a nuclear photosynthetic electron transport mutant of Arabidopsis thaliana with a pleiotropic effect on chloroplast gene expression.

A photosynthetic mutant of Arabidopsis thaliana, hcf5, was isolated by screening M2 seedlings for high chlorophyll fluorescence. Thylakoid morphology was strikingly abnormal, with large grana stacks and almost no stroma lamellae. Fluorescence induction kinetics, activity assays, and immunoblotting showed that photosystem II was absent. Polypeptides of the photosystem I complex, the Cyt b6/f complex, coupling factor, and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase were also severely depleted. However, the nuclear-encoded chlorophyll a/b light-harvesting complex polypeptides were unaffected. The rbcL transcript was present at very low levels, the pattern of transcripts from the polycistronic psbB-psbH-petB-petD operon was abnormal, and the mature psbH message was almost completely lacking. This suggests that the hcf5 locus may encode a product required for the correct expression of several chloroplast genes.

Arabidopsis↗
PubMed1988

Complex RNA maturation in chloroplasts. The psbB operon from spinach.

The psbB operon of the spinach plastid chromosome encodes the genes for the 51-kDa chlorophyll a apoprotein (psbB), the 10-kDa phosphoprotein (psbH), both associated with photosystem II, as well as cytochrome b6 (petB) and subunit IV (petD) of the cytochrome b/f complex in the order given. These genes are not expressed coordinately. The RNA pattern of this DNA region is complex and resolves into eighteen major RNA species. Using northern and S1 protection analysis we demonstrate (a) that all RNA species derive from one DNA strand and hybridize in an overlapping fashion; and (b) that they arise by processing rather than by multiple transcription initiation/termination. (c) The operon is bordered by a single prokaryote-like promotor in front of psbB, and by a putative factor-independent terminator with characteristic sequence elements following petD. The terminator appears to function bidirectionally. (d) At least four distinct modification activities operate on the putative primary transcript of 5650 nucleotides and on the processing intermediates, including a novel endonucleolytic activity cleaving within a characteristic hexanucleotide motif, 3'-exonucleolytic activity at discrete RNA ends, 5' shortage of mRNA (psbB), and excision of class II intervening sequences (petB and petD). (e) Kinetically, maturation of the primary transcript is largely a stochastic process. (f) Processing results ultimately in the formation of monocistronic mRNAs for each of the two photosystem II polypeptides and a bicistronic mRNA encoding both subunits of the cytochrome b/f complex. We postulate that these RNA species represent the translationally active components in the non-coordinate dark/light expression of these genes. (g) Light is without any noticeable effect on posttranscriptional modification. Under our conditions it appears to operate at a translational rather than a transcriptional or posttranscriptional level indicating that the biogenesis of thylakoid membranes is regulated at various levels.

Amino Acid Sequence↗
PubMed2006

Redox modulation of cyclic electron flow around photosystem I in C3 plants.

We have investigated the occurrence of cyclic electron flow in intact spinach leaves. In particular, we have tested the hypothesis that cyclic flow requires the presence of supercomplexes in the thylakoid membrane or other strong associations between proteins. Using biochemical approaches, we found no evidence of the presence of supercomplexes related to cyclic electron flow, making previous structural explanations for the modulation of cyclic flow rather unlikely. On the other hand, we found that the fraction of photosystem I complexes engaged in cyclic flow could be modulated by changes in the redox state of the chloroplast stroma. Our findings support therefore a dynamic model for the occurrence of linear and cyclic electron flow in C3 plants, based on the competition between cytochrome b(6)f and FNR for electrons carried by ferredoxin. This would be ultimately regulated by the balance between the redox state of PSI acceptors and donors during photosynthesis, in a diffusing system.

Adaptation, Physiological↗
PubMed2001

Isolation of membrane protein subunits in their native state: evidence for selective binding of chlorophyll and carotenoid to the b(6) subunit of the cytochrome b(6)f complex.

Cytochrome (cyt) b-c complexes play a central role in electron transfer chains and are almost ubiquitous in nature. Although similar in their basic structure and function, the cyt b(6)f complex of photosynthetic membranes and its counterpart, the mitochondrial cyt bc(1) complex, show some characteristic differences which cannot be explained by the high resolution structure of the cyt bc(1) complex alone. Especially the presence of a chlorophyll molecule is a striking feature of all cyt b(6)f complex preparations described so far, imposing questions as to its structural and functional role. To allow a more detailed characterization, we here report the preparation of native subunits cyt b(6) and IV starting from a monomeric cyanobacterial cyt b(6)f complex. Spectroscopical and reversed-phase HPLC analyses of the purified cyt b(6) subunit showed that it contained not only two b-type hemes, but also one chlorophyll a molecule and a cyanobacterial carotenoid, echinenone. Evidence for selective binding of both pigments to this subunit is presented and their putative function is discussed.

Carotenoids↗
PubMed1997

Characterization of the orf31-petG gene cluster from the plastid genome of Populus deltoides.

The orf31-petG gene cluster is located approximately 1.2 kb away from the psbEFLJ operon in the chloroplast genome of Populus deltoides. The orf31 (ycf7) encodes an unidentified polypeptide while the petG gene encodes subunit V of an important component, cytochrome b6/f complex, involved in photosynthetic electron transport. We have determined the nucleotide sequence of the orf31-petG gene cluster from the plastid genome of a tree, Populus deltoides. Our sequence analysis suggests that these genes possess high homology with the published sequences of these genes from other plants. Northern analysis suggests development dependent transcription of the orf31-petG cluster in leaves.

Amino Acid Sequence↗
PubMed2002

The HCF136 protein is essential for assembly of the photosystem II reaction center in Arabidopsis thaliana.

Hcf136 encodes a hydrophilic protein localized in the lumen of stroma thylakoids. Its mutational inactivation in Arabidopsis thaliana results in a photosystem II (PHII)-less phenotype. Under standard illumination, PSII is not detectable and the amount of photosystem I (PSI) is reduced, which implies that HCF136p may be required for photosystem biogenesis in general. However, at low light, a comparison of mutants with defects in PSII, PSI, and the cytochrome b(6)f complex reveals that HCF136p regulates selectively biogenesis of PSII. We demonstrate by in vivo radiolabeling of hcf136 that biogenesis of the reaction center (RC) of PSII is blocked. Gel blot analysis and affinity chromatography of solubilized thylakoid membranes suggest that HCF136p associates with a PSII precomplex containing at least D2 and cytochrome b(559). We conclude that HCF136p is essential for assembly of the RC of PSII and discuss its function as a chaperone-like assembly factor.

Arabidopsis↗
PubMed2000

Evidence for a role of ClpP in the degradation of the chloroplast cytochrome b(6)f complex.

In the green alga Chlamydomonas reinhardtii, the ClpP protease is encoded by an essential chloroplast gene. Mutating its AUG translation initiation codon to AUU reduced ClpP accumulation to 25 to 45% of that of the wild type. Both the mature protein and the putative precursor containing its insertion sequence were present in reduced amounts. Attenuation of ClpP did not affect growth rates under normal conditions but restricted the ability of the cells to adapt to elevated CO(2) levels. It also affected the rate of degradation of the cytochrome b(6)f complex of the thylakoid membrane in two experimental situations: (1) during nitrogen starvation, and (2) in mutants deficient in the Rieske iron-sulfur protein. The ClpP level also controls the steady state accumulation of a mutated version of the Rieske protein. In contrast, attenuation of ClpP did not rescue the fully unassembled subunits in other cytochrome b(6)f mutants. We conclude that proteolytic disposal of fully or partially assembled cytochrome b(6)f is controlled by the Clp protease.

Adenosine Triphosphatases↗
PubMedDate not supplied

[A kinetic model of the cytochrome bf complex. Evaluation of kinetic parameters].

A kinetic model of the cytochrome bf complex was developed on the assumption that the Q-cycle operates. The bf complex was considered as a membrane enzyme catalyzing the electron transfer from plastoquinol to plastocyanine, which is coupled with proton translocation from the chloroplast stroma to the thylakoid lumen. The dependence of the electron transfer rates on the value of the transmembrane electric potential was taken into account. The model was applied to describe the experimental data on the flash-induced turnover of cytochromes b, plastocyanine, and the kinetics of proton deposition in the thylakoid lumen. The estimation of model parameters was performed.

Cytochrome b6f Complex↗
PubMed2006

Lipids in photosynthetic reaction centres: structural roles and functional holes.

Photosynthetic proteins power the biosphere. Reaction centres, light harvesting antenna proteins and cytochrome b(6)f (or bc(1)) complexes are expressed at high levels, have been subjected to an intensive spectroscopic, biochemical and mutagenic analysis, and several have been characterised to an informatively high resolution by X-ray crystallography. In addition to revealing the structural basis for the transduction of light energy, X-ray crystallography has brought molecular insights into the relationships between these multicomponent membrane proteins and their lipid environment. Lipids resolved in the X-ray crystal structures of photosynthetic proteins bind light harvesting cofactors, fill intra-protein cavities through which quinones can diffuse, form an important part of the monomer-monomer interface in multimeric structures and may facilitate structural flexibility in complexes that undergo partial disassembly and repair. It has been proposed that individual lipids influence the biophysical properties of reaction centre cofactors, and so affect the rate of electron transfer through the complex. Lipids have also been shown to be important for successful crystallisation of photosynthetic proteins. Comparison of the three types of reaction centre that have been structurally characterised reveals interesting similarities in the position of bound lipids that may point towards a generic requirement to reinforce the structure of the core electron transfer domain. The crystallographic data are also providing new opportunities to find molecular explanations for observed effects of different types of lipid on the structure, mechanism and organisation of reaction centres and other photosynthetic proteins.

Binding Sites↗
PubMed1990

Thylakoid membrane protein topography. Location of the termini of the chloroplast cytochrome b6 on the stromal side of the membrane.

The orientation of cytochrome b6 in the thylakoid membrane and the question of whether the number of membrane spanning helices is an even or odd number was tested through the relative trypsin susceptibility of epitopes (Asp-5 to Gln-14) and (Ile-205 to Leu-214) at the NH2 and COOH termini, respectively, of the 214-residue cytochrome b6 polypeptide. A structure of the cytochrome with an even number of helices and the NH2 and COOH termini on the stromal side of the membrane was inferred from the following: 1) cleavage of cytochrome b6 by trypsin added to thylakoids occurs by removal of both of the exposed NH2- and COOH-terminal epitopes. The epitopes at the termini were more sensitive to trypsin after prior treatment of thylakoids with carboxypeptidase A, indicating that these epitopes are shielded on the stromal side of the membrane by the COOH termini of other proteins. 2) Both epitopes were more trypsin-sensitive in thylakoid membranes than was cytochrome f that is only sensitive to trypsin acting on the lumen side of the membrane. 3) The NH2- and COOH-terminal epitopes of cytochrome b6 were also more sensitive to trypsin added to thylakoid membranes than were the oxygen-evolving complex 16- and 33-kDa proteins that are completely located on the lumen side. 4) The order of trypsin susceptibility was reversed in inside-out membranes, where the cytochrome NH2- and COOH-terminal epitopes were less sensitive than the 16- and 33-kDa proteins. The decreased relative sensitivity of the cytochrome b6 epitopes occurs in spite of a greater absolute sensitivity of these epitopes to trypsin in inside-out membranes. 5) The greater absolute sensitivity can be explained by a 4-helix model that includes trypsin-sensitive sites on the lumen side.

Amino Acid Sequence↗
PubMed1999

Purification and two-dimensional crystallization of highly active cytochrome b(6)f complex from spinach.

The purification and two-dimensional crystallization of highly active cytochrome b(6)f complex from spinach is described. The preparation shows all spectroscopic characteristics of the pure complex. The electron transfer activity of 450+/-60 electrons per s is the highest in vitro activity reported to date. Using dimethyl sulfoxide (DMSO) as a solvent for the electron donor enhanced the performance and reproducibility of the assay. The high yield and the high activity of the protein make it an ideal candidate for biophysical and structural studies. Preliminary two-dimensional crystallization experiments yielded several different forms of two-dimensional and thin three-dimensional crystals, exhibiting varying degrees of order.

Animals↗
PubMed2002

A chloroplast FKBP interacts with and affects the accumulation of Rieske subunit of cytochrome bf complex.

Immunophilins are intracellular receptors of the immunosuppressants cyclosporin A, FK506, and rapamycin. Although all immunophilins possess peptidyl-prolyl isomerase activity and are identified from a wide range of organisms, little is known about their cellular functions. We report the characterization and functional analysis of an FK506 and rapamycin-binding protein (AtFKBP13) from Arabidopsis. The AtFKBP13 protein is synthesized as a precursor that is imported into chloroplasts and processed to the mature form located in the thylakoid lumen, as shown by chloroplast import assays and Western blot analysis. Experiments show that AtFKBP13 is translocated across the thylakoid membrane by the DeltapH-dependent pathway. Yeast two-hybrid screening identified Rieske FeS protein, a subunit of the cytochrome bf complex in the photosynthetic electron transport chain, as an interacting partner for AtFKBP13. Both yeast two-hybrid and in vitro protein-protein interaction assays showed that the precursor, but not the mature form, of AtFKBP13 interacted with Rieske protein, suggesting that interaction between the two proteins occurs along the import pathway. When AtFKBP13 expression was suppressed by RNA interference method, the level of Rieske protein was significantly increased in the transgenic plants.

Amino Acid Sequence↗
PubMed2000

Photosynthetic parameters of leaves of wild type and Cyt b6/f deficient transgenic tobacco studied by CO2 uptake and transmittance at 800 nm.

Parallel measurements of CO2 assimilation and 800 nm transmission were carried out on intact leaves of wild type and cytochrome b6/f deficient transgenic tobacco grown at different light intensities and temperatures, with the aim to diagnose rate-limiting processes in photosynthesis and investigate their adaptations to growth conditions. Maximum CO2- and light-saturated photosynthetic rate, mesophyll conductance, assimilatory charge and specific carboxylation efficiency were determined from CO2 fixation measurements and postillumination P700 rereduction time constant was measured from the transient of the 800 nm signal. Results show that growth conditions continue to modulate the expression of genes in transgenic plants, interfering with the antisense modulation, but under all environmental conditions the antisense treatment to decrease Cyt b6/f complexes ensured that the control of electron/proton transport rate by proton backpressure on the PSI donor side was stronger than the control by electron backpressure on the PSI acceptor side. Coordinated control of gene expression and enzyme activation ensures that different parts of the photosynthetic machinery--components of the electron transport chain, ribulose-1,5-bisphosphate carboxylase/oxygenase, enzymes of the sucrose and starch synthesis chains-are synthesized more or less proportionally under different environmental conditions and in case of mild genetic interference.

Carbon Dioxide↗
PubMed1989

Identification of a Mr = 17,000 protein as the plastoquinone-binding protein in the cytochrome b6-f complex from spinach chloroplasts.

An azidoquinone derivative, 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]octyl)-1,4-benzoquinone (azido-Q), was used to study the plastoquinone-protein interaction and to identify the plastoquinone-binding protein in the cytochrome b6-f complex from spinach chloroplasts. When the lipid- and plastoquinone-deficient cytochrome b6-f complex is incubated with varying concentrations of azido-Q and illuminated with long wavelength UV light for 7 min at 2 degrees C, the enzymatic activity, assayed after reconstitution with lipid, decreases as the concentration of azido-Q increases. Maximum inactivation (45%) is observed when 30 mol of azido-Q is used per mol of cytochrome f. The extent of the decrease in activity upon illumination correlates with the amount of azido-Q incorporated into the protein. The 50% inactivation is in good agreement with that expected based on the amount of plastoquinone deficiency of the isolated enzyme complex. When the photolyzed, [3H]azido-Q-treated sample is extracted with organic solvent and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radioactivity is found primarily in the Mr = 17,000 subunit. When the enzyme is pretreated with the electron transfer inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone or 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole, significantly less radioactive label is observed in the Mr = 17,000 protein, suggesting that the action sites of these inhibitors are the same or near the plastoquinone-binding site. When the deficient complex is reconstituted with glycolipid prior to the addition of azido-Q, less than 5% inactivation is observed upon photolysis, and the amount of radioactive label on the Mr = 17,000 protein decreases greatly, suggesting that the plastoquinone-binding site is easily masked by glycolipid when endogenous plastoquinone is absent. Plastoquinol-2 apparently competes with azido-Q for the plastoquinone-binding site since it decreases the radioactive label on the Mr = 17,000 protein.

Azides↗
PubMed1999

Isolation and characterization of photoautotrophic mutants of Chlamydomonas reinhardtii deficient in state transition.

In photosynthetic cells of higher plants and algae, the distribution of light energy between photosystem I and photosystem II is controlled by light quality through a process called state transition. It involves a reorganization of the light-harvesting complex of photosystem II (LHCII) within the thylakoid membrane whereby light energy captured preferentially by photosystem II is redirected toward photosystem I or vice versa. State transition is correlated with the reversible phosphorylation of several LHCII proteins and requires the presence of functional cytochrome b(6)f complex. Most factors controlling state transition are still not identified. Here we describe the isolation of photoautotrophic mutants of the unicellular alga Chlamydomonas reinhardtii, which are deficient in state transition. Mutant stt7 is unable to undergo state transition and remains blocked in state I as assayed by fluorescence and photoacoustic measurements. Immunocytochemical studies indicate that the distribution of LHCII and of the cytochrome b(6)f complex between appressed and nonappressed thylakoid membranes does not change significantly during state transition in stt7, in contrast to the wild type. This mutant displays the same deficiency in LHCII phosphorylation as observed for mutants deficient in cytochrome b(6)f complex that are known to be unable to undergo state transition. The stt7 mutant grows photoautotrophically, although at a slower rate than wild type, and does not appear to be more sensitive to photoinactivation than the wild-type strain. Mutant stt3-4b is partially deficient in state transition but is still able to phosphorylate LHCII. Potential factors affected in these mutant strains and the function of state transition in C. reinhardtii are discussed.

Animals↗
PubMed2000

The sequence and secondary structure of the 3'-UTR affect 3'-end maturation, RNA accumulation, and translation in tobacco chloroplasts.

RNA maturation and modulation of RNA stability play important roles in chloroplast gene expression. In vitro and in vivo studies have shown that both the 5'- and 3'-untranslated regions (UTRs) contain sequence and structural elements that guide these processes, and interact with specific proteins. We have previously characterized the spinach chloroplast petD 3'-UTR in detail by in vitro approaches. This stem-loop forming sequence is a weak terminator but is required for RNA maturation and also exhibits sequence-specific protein binding. To test petD 3'-UTR function in vivo, tobacco chloroplast transformants were generated containing uidA reporter genes flanked by variants of the petD 3'-UTR, including one which does not form an RNA-protein complex in vitro, and one which lacks a stem-loop structure. Analysis of uidA mRNA indicated that a stable secondary structure is required to accumulate a discrete mRNA, and that changes in the 3'-UTR sequence which affect protein binding in vitro can also affect RNA metabolism in vivo. The 3'-UTR also influenced beta-glucuronidase protein accumulation, but not in proportion to RNA levels. These results raise the possibility that in tobacco chloroplasts, the 3'-UTR may influence translational yield.

3' Untranslated Regions↗
PubMed2005

Stromal over-reduction by high-light stress as measured by decreases in P700 oxidation by far-red light and its physiological relevance.

The oxidation level of P700 induced by far-red light (DeltaA(FR)) in briefly dark-treated leaves of some sun plants decreased during the daytime and recovered at night. The dark recovery of decreased DeltaA(FR) proceeded slowly, with a half-time of about 5 h. We propose that stromal over-reduction induced by sunlight was the direct cause of the depression of DeltaA(FR). The depression of DeltaA(FR) found during the daytime was reproduced by controlled illumination with saturating light of fully dark-treated leaves. Simultaneous measurement of P700 redox and chlorophyll fluorescence showed that the depression of DeltaA(FR) was associated with dark reduction of the plastoquinone pool, which represented cyclic electron transport activity. The decrease of DeltaA(FR) in the light-stressed chloroplasts was partly reversed by treatment with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, an inhibitor of electron transport at the cytochrome b6/f complex, and the subsequent addition of methyl viologen, an efficient electron acceptor from photosystem I (PSI), stimulated further recovery, showing that both cyclic electron flow around PSI and the charge recombination within PSI were responsible for the light-induced depression of DeltaA(FR). The dark level of blue-green fluorescence, an indicator of NAD(P)H concentration, from intact chloroplasts was increased by high-light stress, suggesting that NADPH accumulated in stroma as a result of the high-light treatment. Possible effects on photosynthetic activity of over-reduction and its physiological relevance are discussed.

Chlorophyll↗
PubMed1998

Steps toward constructing a cytochrome b6 f complex in the purple bacterium Rhodobacter sphaeroides: an example of the structural plasticity of a membrane cytochrome.

We have modified the cytochrome b subunit of the cytochrome bc1 complex from the purple bacterium Rhodobacter sphaeroides to introduce two distinctive features of cytochrome b6 f complexes. In the first one, we have split cyt b into two polypeptides thus mimicking the organization of cyt b6 and subunit IV in the b6 f complexes. In the second, an extra residue was added between His198 and Phe199, thus extending the span between the histidine ligands for the two b-hemes in helix D. The properties of the mutant strains were determined using thermodynamic and kinetic analysis. The two mutant enzymes were assembled and functioned so as to allow the photosynthetic growth of the mutant strains. For the split enzyme, we show that two independently translated fragments of cyt b are inserted in the membrane. Our results indicate a decrease in the stability of the semiquinone formed at the quinone reduction (Qi) site in this mutant. This property, characteristic for b6 f complexes, indicates the functional importance of the connecting span between helices D and E. The presence of the inserted threonine in helix D modified the spectrum and redox potential of the bL-heme, shifting the potential difference between the two b-hemes from 140 mV in the wild-type to 55 mV in the mutant strain. This change in the driving force of electron transfer through the membrane was reflected in an inability of the mutant strain to accumulate a large transmembrane electrical potential on successive flashes.

Amino Acid Sequence↗