Singlet oxygen formation and chlorophyll a triplet excited state deactivation in the cytochrome b6f complex from Bryopsis?corticulans

We have attempted to investigate the correlation between the detergent-perturbed structural integrity of the Cyt b ₆ f complex from the marine green alga Bryopsis corticulans and its photo-protective properties, for which the nonionic detergents n-octyl-β-d-glucopyranoside (β-OG) and n-dodecyl-β-d-maltoside (β-DM), respectively, were used for the preparation of Cyt b ₆ f, and the singlet oxygen (¹O₂*) production as well as the triplet excited-state chlorophyll a (³Chl a*) formation and deactivation were examined by spectroscopic means. Near-infrared luminescence of ¹O₂ * (~1,270 nm) on photo-irradiation was detected for the β-OG preparation where the complex is mainly in oligomeric state, but not for the β-DM one in which the complex exists in dimeric form. Under anaerobic condition, photo-excitation of Chl a in the β-DM preparation generated ³Chl a* with a lower quantum yield of Φ_T ~ 0.02 and a longer lifetime of ~600 μs with respect to those as in the case of β-OG preparation, Φ_T ~ 0.12 and 200–300 μs. These results prove that the enzymatically active and intact Cyt b ₆ f complex on photo-excitation tends to produce little ³Chl a* or ¹O₂ *, which implies that the pigment–protein assembly of Cyt b ₆ f complex per se is crucial for photo-protection. F. Ma and X.-B. Chen contributed equally to this work.     

The Rieske/cytochrome b complex of Heliobacteria

Heliobacteria have a Rieske/cytochrome b complex composed of a Rieske protein, a cytochrome b_6, a subunit IV and a di-heme cytochrome c. The overall structure of the complex seems close to the b₆f complex from cyanobacteria and chloroplasts to the exception of the di-heme cytochrome. We show here by biochemical and biophysical studies that a heme c_i is covalently attached to the Rieske/cytochrome b complex from Heliobacteria. We studied the EPR signature of this heme in two different species, Heliobacterium modesticaldum and Heliobacillus mobilis. In contrast to the case of b₆f complex, a strong axial ligand to the heme is present, most probably a protonatable amino acid residue.     

The effects of detergents DDM and β-OG on the singlet excited state lifetime of the chlorophyll a in cytochrome b6f complex from spinach chloroplasts

The singlet excited state lifetime of the chlorophyll a (Chi a) in cytochrome b6f (Cyt b6f) complex was reported to be shorter than that of free Chl a in methanol, but the value was different for Cyt b6f complexes from different sources (～200 and ～600 ps are the two measured results). The present study demonstrated that the singiet excited state lifetime is associated with the detergents n-dodecyl-β-D-maltoside (DDM) and n-octyl-β-D-glucopyranoside (β-OG), but has nothing to do with the different sources of Cyt b6f complexes. Compared with the Cyt b6f dissolved in β-OG, the Cyt b6f in DDM had a lower fluorescence yield, a lower photodegradation rate of Chl a, and a shorter lifetime of Chl a excited state. In short, the singlet excited state lifetime, ～200 ps, of the Chl a in Cyt b6f complex in DDM is closer to the true in vivo.     

Energy and electron transfer in the photosynthetic reaction center complex of Acidiphilium rubrum containing Zn-bacteriochlorophyll a studied by femtosecond up-conversion spectroscopy

A photosynthetic reaction center (RC) complex was isolated from a purple bacterium, Acidiphilium rubrum. The RC contains bacteriochlorophyll a containing Zn as a central metal (Zn-BChl a) and bacteriopheophytin a (BPhe a) but no Mg-BChl a. The absorption peaks of the Zn-BChl a dimer (P_Zn), the accessory Zn-BChl a (B_Zn), and BPhe a (H) at 4 K in the RC showed peaks at 875, 792, and 753 nm, respectively. These peaks were shorter than the corresponding peaks in Rhodobacter sphaeroides RC that has Mg-BChl a. The kinetics of fluorescence from P_Zn^*, measured by fluorescence up-conversion, showed the rise and the major decay with time constants of 0.16 and 3.3 ps, respectively. The former represents the energy transfer from B_Zn^* to P_Zn, and the latter, the electron transfer from P_Zn to H. The angle between the transition dipoles of B_Zn and P_Zn was estimated to be 36° based on the fluorescence anisotropy. The time constants and the angle are almost equal to those in the Rb. sphaeroides RC. The high efficiency of A. rubrum RC seems to be enabled by the chemical property of Zn-BChl a and by the L168HE modification of the RC protein that modifies P_Zn.     

Excitation relaxation dynamics and energy transfer in fucoxanthin–chlorophyll a/c-protein complexes,probed by time-resolved fluorescence

In algae, light-harvesting complexes contain specific chlorophylls (Chls) and keto-carotenoids; Chl a, Chl c, and fucoxanthin (Fx) in diatoms and brown algae; Chl a, Chl c, and peridinin in photosynthetic dinoflagellates; and Chl a, Chl b, and siphonaxanthin in green algae. The Fx–Chl a/c-protein (FCP) complex from the diatom Chaetoceros gracilis contains Chl c₁, Chl c₂, and the keto-carotenoid, Fx, as antenna pigments, in addition to Chl a. In the present study, we investigated energy transfer in the FCP complex associated with photosystem II (FCPII) of C. gracilis. For these investigations, we analyzed time-resolved fluorescence spectra, fluorescence rise and decay curves, and time-resolved fluorescence anisotropy data. Chl a exhibited different energy forms with fluorescence peaks ranging from 677 nm to 688 nm. Fx transferred excitation energy to lower-energy Chl a with a time constant of 300 fs. Chl c transferred excitation energy to Chl a with time constants of 500–600 fs (intra-complex transfer), 600–700 fs (intra-complex transfer), and 4–6 ps (inter-complex transfer). The latter process made a greater contribution to total Chl c-to-Chl a transfer in intact cells of C. gracilis than in the isolated FCPII complexes. The lower-energy Chl a received excitation energy from Fx and transferred the energy to higher-energy Chl a. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

The inter-monomer interface of the major light-harvesting chlorophyll a/b complexes of photosystem II (LHCII) influences the chlorophyll triplet distribution

Under strong light conditions, long-lived chlorophyll triplets (³Chls) are formed, which can sensitize singlet oxygen, a species harmful to the photosynthetic apparatus of plants. Plants have developed multiple photoprotective mechanisms to quench ³Chl and scavenge singlet oxygen in order to sustain the photosynthetic activities. The lumenal loop of light-harvesting chlorophyll a/b complex of photosystem II (LHCII) plays important roles in regulating the pigment conformation and energy dissipation. In this study, site-directed mutagenesis analysis was applied to investigate triplet–triplet energy transfer and quenching of ³Chl in LHCII. We mutated the amino acid at site 123 located in this region to Gly, Pro, Gln, Thr and Tyr, respectively, and recorded fluorescence excitation spectra, triplet-minus-singlet (TmS) spectra and kinetics of carotenoid triplet decay for wild type and all the mutants. A red-shift was evident in the TmS spectra of the mutants S123T and S123P, and all of the mutants except S123Y showed a decrease in the triplet energy transfer efficiency. We propose, on the basis of the available structural information, that these phenomena are related to the involvement, due to conformational changes in the lumenal region, of a long-wavelength lutein (Lut2) involved in quenching ³Chl.     

Conservation of lipid functions in cytochrome bc complexes

Lipid binding sites and properties are compared in two sub-families of hetero-oligomeric membrane protein complexes known to have similar functions in order to gain further understanding of the role of lipid in the function, dynamics, and assembly of these complexes. Using the crystal structure information for both complexes, we compared the lipid binding properties of the cytochrome b₆f and bc₁ complexes that function in photosynthetic and respiratory membrane energy transduction. Comparison of lipid and detergent binding sites in the b₆f complex with those in bc₁ shows significant conservation of lipid positions. Seven lipid binding sites in the cyanobacterial b₆f complex overlap three natural sites in the Chlamydomonas reinhardtii algal complex and four sites in the yeast mitochondrial bc₁ complex. The specific identity of lipids is different in b₆f and bc₁ complexes: b₆f contains sulfoquinovosyldiacylglycerol, phosphatidylglycerol, phosphatidylcholine, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol, whereas cardiolipin, phosphatidylethanolamine, and phosphatidic acid are present in the yeast bc₁ complex. The lipidic chlorophyll a and β-carotene (β-car) in cyanobacterial b₆f, as well as eicosane in C. reinhardtii, are unique to the b₆f complex. Inferences of lipid binding sites and functions were supported by sequence, interatomic distance, and B-factor information on interacting lipid groups and coordinating amino acid residues. The lipid functions inferred in the b₆f complex are as follows: (i) substitution of a transmembrane helix by a lipid and chlorin ring, (ii) lipid and β-car connection of peripheral and core domains, (iii) stabilization of the iron-sulfur protein transmembrane helix, (iv) n-side charge and polarity compensation, and (v) β-car-mediated super-complex with the photosystem I complex.     

The effects of detergents DDM and β-OG on the singlet excited state lifetime of the chlorophyll a in cytochrome b6f complex from spinach chloroplasts

The singlet excited state lifetime of the chlorophyll a (Chl a) in cytochrome b ₆ f (Cyt b ₆ f) complex was reported to be shorter than that of free Chl a in methanol, but the value was different for Cyt b ₆ f complexes from different sources (∼200 and ∼600 ps are the two measured results). The present study demonstrated that the singlet excited state lifetime is associated with the detergents n-dodecyl-β-D-maltoside (DDM) and n-octyl-β-D-glucopyranoside (β-OG), but has nothing to do with the different sources of Cyt b ₆ f complexes. Compared with the Cyt b ₆ f dissolved in β-OG, the Cyt b ₆ f in DDM had a lower fluorescence yield, a lower photodegradation rate of Chl a, and a shorter lifetime of Chl a excited state. In short, the singlet excited state lifetime, ∼200 ps, of the Chl a in Cyt b ₆ f complex in DDM is closer to the true in vivo.     

The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy

The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b₆f with the electron transfer proteins plastocyanin and cytochrome c₆. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c₆ from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c₆ were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge–charge interactions pre-orient cytochrome c₆ with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process.     

Salt-induced redox-independent phosphorylation of light harvesting chlorophyll a/b proteins in Dunaliella salina thylakoid membranes

This study investigated the regulation of the major light harvesting chlorophyll a/b protein (LHCII) phosphorylation in Dunaliella salina thylakoid membranes. We found that both light and NaCl could induce LHCII phosphorylation in D. salina thylakoid membranes. Treatments with oxidants (ferredoxin and NADP) or photosynthetic electron flow inhibitors (DCMU, DBMIB, and stigmatellin) inhibited LHCII phosphorylation induced by light but not that induced by NaCl. Furthermore, neither addition of CuCl₂, an inhibitor of cytochrome b₆f complex reduction, nor oxidizing treatment with ferricyanide inhibited light- or NaCl-induced LHCII phosphorylation, and both salts even induced LHCII phosphorylation in dark-adapted D. salina thylakoid membranes as other salts did. Together, these results indicate that the redox state of the cytochrome b₆f complex is likely involved in light- but not salt-induced LHCII phosphorylation in D. salina thylakoid membranes.     

Single-step immunoaffinity purification and characterization of dodecylmaltoside-solubilized human neutrophil flavocytochrome b

Flavocytochrome b (Cyt b) is a heterodimeric, integral membrane protein that serves as the central component of an electron transferase system employed by phagocytes for elimination of bacterial and fungal pathogens. This report describes a rapid and efficient single-step purification of Cyt b from human neutrophil plasma membranes by solubilization in the nonionic detergent dodecylmaltoside (DDM) and immunoaffinity chromatography. A similar procedure for isolation of Cyt b directly from intact neutrophils by a combination of heparin and immunoaffinity chromatography is also presented. The stability of Cyt b was enhanced in DDM relative to previously employed solubilizing agents as determined by both monitoring the heme spectrum in crude membrane extracts and assaying resistance to proteolytic degradation following purification. Gel filtration chromatography and dynamic light scattering indicated that DDM maintains a predominantly monodisperse population of Cyt b following immunoaffinity purification. The high degree of purity obtained with this isolation procedure allowed for direct determination of a 2:1 heme to protein stoichiometry, confirming previous structural models. Analysis of the isolated heterodimer by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry allowed for accurate mass determination of p22^phox as indicated by the gene sequence. Affinity-purified Cyt b was functionally reconstituted into artificial bilayers and demonstrated that catalytic activity of the protein was efficiently retained throughout the purification procedure.     

Control of cytochrome b6f at low and high light intensity and cyclic electron transport in leaves

The light-dependent control of photosynthetic electron transport from plastoquinol (PQH₂) through the cytochrome b₆f complex (Cyt b₆f) to plastocyanin (PC) and P700 (the donor pigment of Photosystem I, PSI) was investigated in laboratory-grown Helianthus annuus L., Nicotiana tabaccum L., and naturally-grown Solidago virgaurea L., Betula pendula Roth, and Tilia cordata P. Mill. leaves. Steady-state illumination was interrupted (light-dark transient) or a high-intensity 10 ms light pulse was applied to reduce PQ and oxidise PC and P700 (pulse-dark transient) and the following re-reduction of P700⁺ and PC⁺ was recorded as leaf transmission measured differentially at 810-950 nm. The signal was deconvoluted into PC⁺ and P700⁺ components by oxidative (far-red) titration (V. Oja et al., Photosynth. Res. 78 (2003) 1-15) and the PSI density was determined by reductive titration using single-turnover flashes (V. Oja et al., Biochim. Biophys. Acta 1658 (2004) 225-234). These innovations allowed the definition of the full light response curves of electron transport rate through Cyt b₆f to the PSI donors. A significant down-regulation of Cyt b₆f maximum turnover rate was discovered at low light intensities, which relaxed at medium light intensities, and strengthened again at saturating irradiances. We explain the low-light regulation of Cyt b₆f in terms of inactivation of carbon reduction cycle enzymes which increases flux resistance. Cyclic electron transport around PSI was measured as the difference between PSI electron transport (determined from the light-dark transient) and PSII electron transport determined from chlorophyll fluorescence. Cyclic e⁻ transport was not detected at limiting light intensities. At saturating light the cyclic electron transport was present in some, but not all, leaves. We explain variations in the magnitude of cyclic electron flow around PSI as resulting from the variable rate of non-photosynthetic ATP-consuming processes in the chloroplast, not as a principle process that corrects imbalances in ATP/NADPH stoichiometry during photosynthesis.     

Methylviologen and dibromothymoquinone treatments of pea leaves reveal the role of photosystem I in the Chl a fluorescence rise OJIP

The effects of dibromothymoquinone (DBMIB) and methylviologen (MV) on the Chl a fluorescence induction transient (OJIP) were studied in vivo. Simultaneously measured 820-nm transmission kinetics were used to monitor electron flow through photosystem I (PSI). DBMIB inhibits the reoxidation of plastoquinol by binding to the cytochrome b₆/f complex. MV accepts electrons from the FeS clusters of PSI and it allows electrons to bypass the block that is transiently imposed by ferredoxin-NADP⁺-reductase (FNR) (inactive in dark-adapted leaves). We show that the IP phase of the OJIP transient disappears in the presence of DBMIB without affecting F_m. MV suppresses the IP phase by lowering the P level compared to untreated leaves. These observations indicate that PSI activity plays an important role in the kinetics of the OJIP transient. Two requirements for the IP phase are electron transfer beyond the cytochrome b₆/f complex (blocked by DBMIB) and a transient block at the acceptor side of PSI (bypassed by MV). It is also observed that in leaves, just like in thylakoid membranes, DBMIB can bypass its own block at the cytochrome b₆/f complex and donate electrons directly to PC⁺ and P700⁺ with a donation time τ of 4.3 s. Further, alternative explanations of the IP phase that have been proposed in the literature are discussed.     

Localization of Pcb antenna complexes in the photosynthetic prokaryote Prochlorothrix hollandica

The freshwater filamentous green oxyphotobacterium Prochlorothrix hollandica is an unusual oxygenic photoautotrophic cyanobacterium differing from most of the others by the presence of light-harvesting Pcb antenna binding both chlorophylls a and b and by the absence of phycobilins. The pigment-protein complexes of P. hollandica SAG 10.89 (CCAP 1490/1) were isolated from dodecylmaltoside solubilized thylakoid membranes on sucrose density gradient and characterized by biochemical, spectroscopic and immunoblotting methods. The Pcb antennae production is suppressed by high light conditions (> 200 μmol photons m⁻² s⁻¹) in P. hollandica. PcbC protein was found either in higher oligomeric states or coupled to PS I (forming antenna rings around PS I). PcbA and PcbB are most probably only very loosely bound to photosystems; we assume that these pigment-protein complexes function as low light-induced mobile antennae. Further, we have detected α-carotene in substantial quantities in P. hollandica thylakoid membranes, indicating the presence of chloroplast-like carotenoid synthetic pathway which is not present in common cyanobacteria.     

Restoration of the high potential form of cytochrome b-559 through the photoreactivation of Tris-inactivated oxygen-evolving center

Restoration of a high potential (HP) form of cytochrome b-559 (Cyt b-559) from a low potential (LP) form was the primary process in the reconstitution of O₂-evolving center during the photoreactivation of Tris-inactivated chloroplasts. In normal chloroplasts, about 0.5 to 0.7 mol of Cyt b-559 was present in the HP form per 400 chlorophyll molecules. However, the HP form was converted to the LP form when the O₂-evolving center was inactivated by 0.8 M alkaline Tris-washing (pH 9.1). The inactivation was reversible and both the Cyt b-559 HP form and the O₂-evolving activity were restored by incubating the inactivated chloroplasts with weak light, Mn²⁺, Ca²⁺ and an electron donor (photoreactivation). The recovery of the HP form preceded the recovery of O₂-evolving activity. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) did not inhibit the recovery of the HP form. Thus, the recovery of Cyt b-559 HP form was the primary reaction in the photoreactivation, which was stimulated by the light-induced redox reaction of the PS-II core center.Abbreviations ASC ascorbate - BSA bovine serum albumin - Chl chlorophyll - Cyt b-559 HP form high potential form of cytochrome b-559 - Cyt b-559 LP form low potential form of cytochrome b-559 - Cyt b-559 VLP form very low potential form of cytochrome b-559 - Cyt f cytochrome f - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenol indophenol - Hepes N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid - HQ hydroquinone - SHN chloroplast-preparation medium containing 0.4 M sucrose, 50 mM Hepes-Na (pH 7.8) and 20 mM NaCl - PS-II Photosystem II     

Isolation and characterization of oxygen-evolving thylakoid membranes and Photosystem II particles from a marine diatom Chaetoceros gracilis

Thylakoid membranes retaining high oxygen-evolving activity (about 250 μmol O₂/mg Chl/h) were prepared from a marine centric diatom, Chaetoceros gracilis, after disruption of the cells by freeze-thawing. We also succeeded in purification of Photosystem II (PSII) particles by differential centrifugation of the thylakoid membranes after treatment with 1% Triton X-100. The diatom PSII particles showed an oxygen-evolving activity of 850 and 1045 μmol O₂/mg Chl/h in the absence and presence of CaCl₂, respectively. The PSII particles contained fucoxanthin chlorophyll a/c-binding proteins in addition to main intrinsic proteins of CP47, CP43, D2, D1, cytochrome b559, and the antenna size was estimated to be 229 Chl a per 2 molecules of pheophytin. Five extrinsic proteins were stoichiometrically released from the diatom PSII particles by alkaline Tris-treatment. Among these five extrinsic proteins, four proteins were red algal-type extrinsic proteins, namely, PsbO, PsbQ', PsbV and PsbU, whereas the other one was a novel, hypothetical protein. This is the first report on isolation and characterization of diatom PSII particles that are highly active in oxygen evolution and retain the full set of extrinsic proteins including an unknown protein.     

对氢键网络的干扰降低细胞色素b_6f复合体中Chla的光稳定性

细胞色素b6f蛋白复合体(Cyt b6f)参与光合膜上电子传递和跨膜质子转移,在体内以二体形式存在,每个单体只结合1分子叶绿素a(Chla).该Chla性质独特,光照条件下十分稳定,是甲醇中游离Chla的120~130倍,然而其光稳定性的机制仍未彻底阐明.Cyt b6f 2.7的晶体结构显示,Chla中心的Mg离子和H2O分子配位,并且该H2O分子通过氢键与复合体亚基Ⅳ的氨基酸G136和T137相互作用.本研究基于这一结构特点,对上述2个氨基酸进行了定点突变,以干扰、破坏氢键网络.结果发现,突变不仅导致蛋白与Chla结合能力下降,而且显著降低了Chla的光稳定性,这一结果表明,Cyt b6f中Chla相关的氢键网络对其稳定性具有重要的作用.     

18O Labeling of Chlorophyll d in Acaryochloris marina Reveals That Chlorophyll a and Molecular Oxygen Are Precursors

The cyanobacterium Acaryochloris marina was cultured in the presence of either H₂¹⁸O or ¹⁸O₂, and the newly synthesized chlorophylls (Chl a and Chl d) were isolated using high performance liquid chromatography and analyzed by mass spectroscopy. In the presence of H₂¹⁸O, newly synthesized Chl a and d, both incorporated up to four isotopic ¹⁸O atoms. Time course H₂¹⁸O labeling experiments showed incorporation of isotopic ¹⁸O atoms originating from H₂¹⁸O into Chl a, with over 90% of Chl a ¹⁸O-labeled at 48 h. The incorporation of isotopic ¹⁸O atoms into Chl d upon incubation in H₂¹⁸O was slower compared with Chl a with ∼50% ¹⁸O-labeled Chl d at 115 h. The rapid turnover of newly synthesized Chl a suggested that Chl a is the direct biosynthetic precursor of Chl d. In the presence of ¹⁸O₂ gas, one isotopic ¹⁸O atom was incorporated into Chl a with approximately the same kinetic incorporation rate observed in the H₂¹⁸O labeling experiment, reaching over 90% labeling intensity at 48 h. The incorporation of two isotopic ¹⁸O atoms derived from molecular oxygen (¹⁸O₂) was observed in the extracted Chl d, and the percentage of double isotopic ¹⁸O-labeled Chl d increased in parallel with the decrease of non-isotopic-labeled Chl d. This clearly indicated that the oxygen atom in the C3¹-formyl group of Chl d is derived from dioxygen via an oxygenase-type reaction mechanism.     

Involvement of plastoquinone bound within the isolated cytochrome b6-f complex from chloroplasts in oxidant-induced reduction of cytochrome b6

(1) Oxidant-induced reduction of cytochrome b₆ is completely dependent on a reduced component within the isolated cytochrome b₆-f complex. This component can be reduced by dithionite or by NADH/N-methylphenazonium methosulfate. It is a 2H⁺/2e⁻ carrier with a midpoint potential of 100 mV at pH 7.0, which is very similar to the midpoint potential of the plastoquinone pool in chloroplasts. (2) Oxidant-induced reduction of cytochrome b₆ is stimulated by plastoquinol-1 as well as by plastoquinol-9. The midpoint potential of the transient reduction of cytochrome b₆, however, was not shifted by added plastoquinol. (3) Quinone analysis of the purified cytochrome b₆-f complex revealed about one plastoquinone per cytochrome f. The endogenous quinone is heterogeneous, a form more polar than plastoquinone-A, probably plastoquinone-C, dominating, This is different from the thylakoid membrane where plastoquinone-A is the main quinone. (4) The endogenous quinone can be extracted from the lyophilized cytochrome b₆-f complex by acetone, but not by hydrocarbon solvents. Oxidant-induced reduction of cytochrome b₆ was observed in the lyophilized and hexane-extracted complex, but was lost in the acetone-extracted complex. Reconstitution was achieved either with plastoquinol-1 or plastoquinol-9, suggesting that a plastoquinol molecule is involved in oxidant-induced reduction of cytochrome b₆.     

The ascl1a and dlx genes have a regulatory role in the development of GABAergic interneurons in the zebrafish diencephalon

During development of the mouse forebrain interneurons, the Dlx genes play a key role in a gene regulatory network (GRN) that leads to the GABAergic phenotype. Here, we have examined the regulatory relationships between the ascl1a, dlx, and gad1b genes in the zebrafish forebrain. Expression of ascl1a overlaps with dlx1a in the telencephalon and diencephalon during early forebrain development. The loss of Ascl1a function results in a loss of dlx expression, and subsequent losses of dlx5a and gad1b expression in the diencephalic prethalamus and hypothalamus. Loss of Dlx1a and Dlx2a function, and, to a lesser extent, of Dlx5a and Dlx6a, impairs gad1b expression in the prethalamus and hypothalamus. We conclude that dlx1a/2a act downstream of ascl1a but upstream of dlx5a/dlx6a and gad1b to activate GABAergic specification. This pathway is conserved in the diencephalon, but has diverged between mammals and teleosts in the telencephalon.