Electron Flow from NAD(P)H Dehydrogenase to Photosystem I is Required for Adaptation to Salt Shock in the Cyanobacterium Synechocystis sp. PCC 6803

The role of the NAD(P)H-dehydrogenase complex in adaptationto salt stress was examined in an ndhB-inacti-vated mutant ofthe cyanobacterium Synechocystis sp. PCC 6803. Wild-type cellsand ndhB-inactivated mutant cells grew at similar rates underconditions of low salinity (<0.6M NaCl) and high CO² (3%).However, when the concentration of NaCl in the culture mediumwas higher than 0.6 M, the mutant cells grew much more slowlythan the wild-type cells. Upon addition of high concentrationsof NaCl, the oxygen-evolving activity was rapidly inhibitedbut then it recovered, with the rate of recovery depending onthe concentration of NaCl. The recovery of the mutant cellswas significantly delayed when the concentration of NaCl wasabove 0.3 M. At 0.9 M NaCl, wild-type cells recovered with ahalf time of about 40 min, while mutant cells did not recover.The kinetics of changes in Chi fluorescence confirmed theseresults. In wild-type cells, input of electrons from the cytosolto PSI via the NAD(P)H-dehy-drogenase complex increased uponsalt shock. It appears, therefore, that the electron flow fromthe cytosol to PSI via NAD(P)H-dehydrogenase is essential forthe adaptation of cyanobacteria to salt shock. (Received June 11, 1997; Accepted September 24, 1997)     

The Expression Pattern of NAD(P)H Oxidases and the Cyclic Electron Transport Pathway around Photosystem I of Synechocystis sp. PCC6803 Depend on Growth Conditions

Cyclic electron transport and NADH and/or NADPH (NAD(P)H)-oxidizing activities were investigated in Synechocystis sp. PCC6803 grown under various stressed conditions and in ndhB-less (M55) and ycf33-deletion mutants. Activity staining and inhibitor data suggested that the ferredoxin-quinone reductase (FQR) route is the main pathway in ycf33-deletion and high-light (300 μE m^?2 s^?1)-grown cells as well as in M55 cells. The FQR route was highly sensitive to HgCl₂, but not to diphenyleneiodonium (DPI). On the other hand, cells grown under low CO₂ (0.03%) or normal (100 μE m^?2 s^?1, 3% CO₂) conditions were found perhaps to use the complex I-type NAD(P)H dehydrogenase route, which was found to be highly sensitive to DPI but not to HgCl₂. In high-salt (0.55 M NaCl)-grown cells, the amount of ferredoxin-NADP⁺ oxidoreductase (FNR) increased, and the main cyclic electron flow was perhaps the FNR route. Both DPI and HgCl₂ were strong inhibitors of the FNR route.     

集胞蓝藻PCC6803含疏水亚基的NAD（P）H脱氢酶亚复合体的分离

利用离子交换与凝胶过滤层析 ,从n dodecylβ D maltoside(DM)处理的集胞蓝藻SynechocystisPCC6 80 3细胞粗提液中 ,首次分离到两个包含NDH疏水亚基NdhA的亚复合体。酶活性分析表明 ,分离到的NDH亚复合体具有NADPH 氮蓝四唑 (NBT)氧化还原酶活性 ,以NADPH为电子供体可以还原铁氰化钾、二溴百里香醌 (DBMIB)、二氯酚靛酚 (DCPIP)、duroquinone以及UQ 0等质醌类电子受体。     

Properties of the Respiratory NAD(P)H Dehydrogenase Isolated from the Cyanobacterium Synechocystis PCC6803

Activity staining with NADPH-nitroblue tetrazolium after native-PAGEof membrane proteins of Synechocystis PCC6803, solubilized with3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),revealed four NAD(P)H dehydrogenase (NDH) activities; an NDHcomplex of the respiratory chain, a ferredoxin NADP⁺ reductase(FNR), a drgA product which oxidized both NADH and NADPH, andan uncharacterized NADH-specific enzyme. The NDH complex waspurified with anion exchange and gel filtration chromatographies.The purified complex had a molecular mass of 376 kDa and wascomposed of 9 subunits. Western analysis showed that the complexcontained the NDH-H subunit, but not NDH-A or B. The enzymereduced ferricyanide much faster than plastoquinone and usedNADPH as its prefered electron donor rather than NADH. The enzymaticactivity was inhibited by diphenyleneiodonium chloride and salicylhydroxamicacid, but not by rotenone, p-chloromercuribenzoate, N-ethylmaleimide,flavon, dicumarol, or antimycin A. These results suggest thatthe purified complex is a hydrophilic subcomplex which containsan NADPH binding site and flavin, and is dissociated from ahydrophobic subcomplex, which contains quinone binding site. ¹Present address: Division of Applied Life Sciences, GraduateSchool of Agriculture, Kyoto University, Sakyo, Kyoto, 606-8502Japan ³Present address: Department of Biotechnology, Faculty of Engineering,Fukuyama University, 1 Gakuencho, Fukuyama, Hiroshima, 729-0292Japan     

Reduction of Photosystem I Reaction Center in DrgA Mutant of the Cyanobacterium Synechocystis sp. PCC 6803 Lacking Soluble NAD(P)H:Quinone Oxidoreductase

Photoautotrophically grown cells of the cyanobacterium Synechocystis sp. PCC 6803 wild type and the Ins2 mutant carrying an insertion in the drgA gene encoding soluble NAD(P)H:quinone oxidoreductase (NQR) did not differ in the rate of light-induced oxygen evolution and Photosystem I reaction center (P700+) reduction after its oxidation with a white light pulse. In the presence of DCMU, the rate of P700+ reduction was lower in mutant cells than in wild type cells. Depletion of respiratory substrates after 24 h dark-starvation caused more potent decrease in the rate of P700+ reduction in DrgA mutant cells than in wild type cells. The reduction of P700+ by electrons derived from exogenous glucose was slower in photoautotrophically grown DrgA mutant than in wild type cells. The mutation in the drgA gene did not impair the ability of Synechocystis sp. PCC 6803 cells to oxidize glucose under heterotrophic conditions and did not impair the NDH-1-dependent, rotenone-inhibited electron transfer from NADPH to P700+ in thylakoid membranes of the cyanobacterium. Under photoautotrophic growth conditions, NADPH-dehydrogenase activity in DrgA mutant cells was less than 30% from the level observed in wild type cells. The results suggest that NQR, encoded by the drgA gene, might participate in the regulation of cytoplasmic NADPH oxidation, supplying NADP+ for glucose oxidation in the pentose phosphate cycle of cyanobacteria.     

Isolation of a Novel NAD(P)H-Quinone Oxidoreductase from the Cyanobacterium Synechocystis PCC6803

A novel NAD(P)H-quinone oxidoreductase (NQR) was isolated fromthe cyanobacterium Synechocystis PCC6803 by ion-exchange, affinityand gel-filtration chro-matographies. Isolated NQR was foundto be a drgA gene product that was a homodimer composed of 23-kDasub-units. It showed NAD(P)H-plastoquinone oxidoreductase activitywith K_m values for NADPH and NADH of 12 and 48 µM respectively.The activity was inhibited by thiol-modifying reagents, butnot by rotenone, amobarbital, salicylhydroxamic acid, dicumarol,flavone, or diphenylene-iodonium chloride. Therefore, the Cys-147residue is probably involved in the catalytic reaction. Theamino acid sequence of the purified NQR had some homology withthose of NADH oxidase, NAD(P)H-flavin oxidoreductase, and nitroreductasebut did not contain either an adenine-bind-ing motif or a phosphate-bindingmotif, thus, it is a new type of NQR. ¹Present address: Division of Applied Life Sciences, GraduateSchool of Agriculture, Kyoto University, Sakyo, Kyoto, 606-8502Japan. ³Present address: Department of Biotechnology, Faculty of Engineering,Fukuyama University, 1 Gakuencho, Fukuyama, 729- 0251 Japan.     

PsaE Is Required for in Vivo Cyclic Electron Flow around Photosystem I in the Cyanobacterium Synechococcus sp. PCC 7002

Electron transfer rates to P700+ have been determined in wild-type and three interposon mutants (psaE-, ndhF-, and psaE- ndhF-) of Synechococcus sp. PCC 7002. All three mutants grew significantly more slowly than wild type at low light intensities, and each failed to grow photoheterotrophically in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and a metabolizable carbon source. The kinetics of P700+ reduction were similar in the wild-type and mutant whole cells in the absence of DCMU. In the presence of DCMU, the P700+ reduction rate in the psaE mutant was significantly slower than in the wild type. In the presence of DCMU and potassium cyanide, added to inhibit the outflow of electrons through cytochrome oxidase, P700+ reduction rates increased for both the psaE- and ndhF- strains. The reduction rates for these two mutants were nonetheless slower than that observed for the wild-type strain. The further addition of methyl viologen caused the rate of P700+ reduction in the wild type to become as slow as that for the psaE mutant in the absence of methyl viologen. Given the ability of methyl viologen to intercept electrons from the acceptor side of photosystem I, this response reveals a lesion in cyclic electron flow in the psaE mutant. In the presence of DCMU, the rate of P700+ reduction in the psaE ndhF double mutant was very slow and nearly identical with that for the wild-type strain in the presence of 2,4-dibromo-3-methyl-6-isopropyl-p-benzoquinone, a condition under which physiological electron donation to P700+ should be completely inhibited. These results suggest that NdhF- and PsaE-dependent electron donation to P700+ occurs only via plastoquinone and/or cytochrome b6/f and indicate that there are three major electron sources for P700+ reduction in this cyanobacterium. We conclude that, although PsaE is not required for linear electron flow to NADP+, it is an essential component in the cyclic electron transport pathway around photosystem I.     

Role of NAD(P)H:Quinone Oxidoreductase Encoded by drgA Gene in Reduction of Exogenous Quinones in Cyanobacterium Synechocystis sp. PCC 6803 Cells

Insertion mutant Ins2 of the cyanobacterium Synechocystis sp. PCC 6803, lacking NAD(P)H:quinone oxidoreductase (NQR) encoded by drgA gene, was characterized by higher sensitivity to quinone-type inhibitors (menadione and plumbagin) than wild type (WT) cells. In photoautotrophically grown cyanobacterial cells more than 60% of NADPH:quinone-reductase activity, as well as all NADPH:dinoseb-reductase activity, was associated with the function of NQR. NQR activity was observed only in soluble fraction of cyanobacterial cells, but not in membrane fraction. The effects of menadione and menadiol on the reduction of Photosystem I reaction center (P700(+)) after its photooxidation in the presence of DCMU were studied using the EPR spectroscopy. The addition of menadione increased the rate of P700(+) reduction in WT cells, whereas in Ins2 mutant the reduction of P700(+) was strongly inhibited. In the presence of menadiol the reduction of P700(+) was accelerated both in WT and Ins2 mutant cells. These data suggest that NQR protects the cyanobacterial cells from the toxic effect of exogenous quinones by their reduction to hydroquinones. These data may also indicate the probable functional homology of Synechocystis sp. PCC 6803 NQR with mammalian and plant NAD(P)H:quinone oxidoreductases (DT-diaphorases).     

Localization of NAD(P)H dehydrogenase in the cyanobacterium Synechocystis sp. strain PCC 6803

Antibodies raised against NdhH and NdhB detected these proteins in the thylakoid membrane of Synechocystis sp. strain PCC 6803, but not in a purified cytoplasmic membrane. We conclude that NAD(P)H dehydrogenase is largely, if not exclusively, confined to the thylakoid membrane.     

Thylakoid Membrane Reduction Affects the Photosystem Stoichiometry in the Cyanobacterium Synechocystis sp. PCC 6803

Biogenesis of thylakoid membranes in both chloroplasts and cyanobacteria is largely not understood today. The vesicle-inducing protein in plastids 1 (Vipp1) has been suggested to be essential for thylakoid membrane formation in Arabidopsis (Arabidopsis thaliana), as well as in the cyanobacterium Synechocystis sp. PCC 6803, although its exact physiological function remains elusive so far. Here, we report that, upon depletion of Vipp1 in Synechocystis cells, the number of thylakoid layers in individual Synechocystis cells decreased, and that, in particular, the content of photosystem I (PSI) complexes was highly diminished in thylakoids. Furthermore, separation of native photosynthetic complexes indicated that PSI trimers are destabilized and the monomeric species is enriched. Therefore, depletion of thylakoid membranes specifically affects biogenesis and/or stabilization of PSI in cyanobacteria.In chloroplasts and cyanobacteria the energy transfer between PSI and PSII is regulated in a light-dependent manner (for a recent review, see Kramer et al., 2004). The two photosystems are connected by the cytochrome b₆f complex, and electron transfer from PSII via the cytochrome b₆f complex to PSI is believed to be regulated by the redox state of the plastoquinol pool potentially also involving the cytochrome b₆f complex (Fujita et al., 1987; Murakami and Fujita, 1993; Schneider et al., 2001, 2004; Pfannschmidt, 2003; Volkmer et al., 2007). Transfer of light energy to the two photosystems is mediated by light-harvesting complexes, and in cyanobacteria light is harvested by the soluble extramembranous phycobilisomes. The efficient energy transfer to PSI and PSII has to be balanced to synchronize the function of the two photosystems. In response to changing light intensities and qualities, energy coupling between the phycobilisomes and the photosystems changes, which allows a rapid adjustment of light absorbance by the individual photosystems. Furthermore, besides this short-term adaptation mechanism, it has been shown in many studies that on a longer term in cyanobacteria the ratio of the two photosystems changes depending on the light conditions (Manodori and Melis, 1986; Murakami and Fujita, 1993; Murakami et al., 1997). Upon shifting cyanobacterial cells from low-light to high-light growth conditions, the PSI-to-PSII ratio decreases due to selective suppression of the amount of functional PSI. In recent years, some genes have already been identified that are involved in this regulation of the photosystem stoichiometry (Hihara et al., 1998; Sonoike et al., 2001; Fujimori et al., 2005; Ozaki et al., 2007).Whereas in chloroplasts of higher plants and green algae the amounts of the two photosystems change in response to changing light conditions (Melis, 1984; Chow et al., 1990; Smith et al., 1990; Kim et al., 1993), it has already been noted a long time ago that the chloroplast ultrastructure also adapts to high-light and low-light conditions (Melis, 1984). Chloroplasts of plants grown under low light or far-red light have more thylakoid membranes than chloroplasts of plants grown under high light or blue light (Anderson et al., 1973; Lichtenthaler et al., 1981; Melis and Harvey, 1981). There appears to be a direct correlation between the chlorophyll content and the amount of thylakoids per chloroplast because light harvesting is increased by enhanced chlorophyll and thylakoid membrane content per chloroplast. Thus, chloroplasts adapt to high light both by a reduction of thylakoid membranes and by a decrease in the PSI-to-PSII ratio.Thylakoid membranes are exclusive features of both cyanobacteria and chloroplasts, and it still remains mysterious how formation of thylakoid membranes is organized. Many cellular processes, like lipid biosynthesis, membrane formation, protein synthesis in the cytoplasm and/or at a membrane, protein transport, protein translocation, and protein folding have to be organized and aligned for formation of internal thylakoid membranes. The recent observation that deletion of the vipp1 gene in Arabidopsis (Arabidopsis thaliana) results in complete loss of thylakoid membranes has indicated that Vipp1 is involved in biogenesis of thylakoid membranes. Further analysis has suggested that Vipp1 could be involved in vesicle trafficking between the inner envelope and the thylakoid membrane of chloroplasts (Kroll et al., 2001). Because of this, the protein was named Vipp1, for vesicle-inducing protein in plastids 1. Depletion of Vipp1 strongly affected the ability of cyanobacterial cells to form proper thylakoid membranes (Westphal et al., 2001) and, consequently, also in cyanobacteria Vipp1 appears to be involved in formation of thylakoid membranes. A Vipp1 depletion strain of Arabidopsis is deficient in photosynthesis, although the defect could not be assigned to a deficiency of a single photosynthetic complex, but appeared to be caused by dysfunction of the entire photosynthetic electron transfer chain (Kroll et al., 2001). Therefore, depletion of Vipp1 in Arabidopsis seems to affect thylakoid membrane formation rather than the assembly of thylakoid membrane protein complexes (Aseeva et al., 2007). However, for cyanobacteria, it is not clear yet how diminishing the amount of thylakoid membrane layers would affect the amount and stoichiometry of the two photosystems.Here, we present the generation and characterization of a Vipp1 depletion strain of the cyanobacterium Synechocystis sp. PCC 6803. Upon depletion of Vipp1, a decrease in thylakoid membrane pairs in the generated mutant strain and, furthermore, a significant decrease in active PSI centers was observed. Moreover, trimerization of PSI also appeared to be impaired in the mutant strain. These results suggest that thylakoid membrane perturbations caused by the Vipp1 depletion directly affects PSI assembly and stability in cyanobacterial thylakoid membranes.     

Genetic Manipulation of the Cyanobacterium Synechocystis sp. PCC 6803 (Development of Strains Lacking Photosystem I for the Analysis of Mutations in Photosystem II)

We have taken a genetic approach to eliminating the presence of photosystem I (PSI) in site-directed mutants of photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803. By selecting under light-activated heterotrophic conditions, we have inactivated the psaA-psaB operon encoding the PSI reaction center proteins in cells containing deletions of the three psbA genes. We have also introduced deletions into both copies of psbD in a strain containing a mutation that inactivates psaA (ADK9). These strains, designated D1-/PSI- and D2-/PSI-, may serve as recipient strains for the incorporation of site-directed mutations in either psbA2 or psbD1. The characterization of these cells, which lack both PSI and PSII, is described.     

Two types of functionally distinct NAD(P)H dehydrogenases in Synechocystis sp. strain PCC6803

The ndhD gene encodes a membrane protein component of NAD(P)H dehydrogenase. The genome of Synechocystis sp. PCC6803 contains 6 ndhD genes. Three mutants were constructed by disrupting highly homologous ndhD genes in pairs. Only the DeltandhD1/DeltandhD2 (DeltandhD1/D2) mutant was unable to grow under photoheterotrophic conditions and exhibited low respiration rate, although the mutant grew normally under photoautotrophic conditions in air. The DeltandhD3/DeltandhD4 (DeltandhD3/D4) mutant grew very slowly in air and did not take up CO(2). The results demonstrated the presence of two types of functionally distinct NAD(P)H dehydrogenases in Synechocystis PCC6803 cells. TheDeltandhD5/DeltandhD6 (DeltandhD5/D6) mutant grew like the wild-type strain. Under far-red light (>710 nm), the level of P700(+) was high in DeltandhD1/D2 and M55 (ndhB-less mutant) at low intensities. The capacity of Q(A) (tightly bound plastoquinone) reduction by plastoquinone pool, as measured by the fluorescence increase in darkness upon addition of KCN, was much less in DeltandhD1/D2 and M55 than in DeltandhD3/D4 and DeltandhD5/D6. We conclude that electrons from NADPH are transferred to the plastoquinone pool mainly by the NdhD1.NdhD2 type of NAD(P)H dehydrogenases.     

Thylakoid Membrane-Bound, NADPH-Specific Pyridine Nucleotide Dehydrogenase Complex Mediates Cyclic Electron Transport in the Cyanobacterium Synechocystis sp. PCC 6803

The donation of electrons from NADPH to the intersystem chain,as monitored by an increase in Chl fluorescence, occurred inthe isolated thylakoid membranes of Synechocystis PCC 6803.The stimulation by NADPH of the methyl viologen-dependent photoreductionof dioxygen and of the reduction of P700⁺ after photooxidationin the presence of DCMU also confirmed the donation of electronsfrom NADPH to the electron carriers in the intersystem. Thesereactions were sensitive to rotenone, capsaicin, l-(2-thenoyl)-3,3,3-trifluoroacetoneand HgCl₂ but not to antimycin A or flavone. In contrast tothe thylakoid membranes from the wild type, those from a mutant,designated M55, in which a gene of a subunit of the pyridinenucleotide dehydrogenase complex (NDH) had been inactivated,did not show evidence of such reactions. These results supportour previous hypothesis that the transport of electrons fromNADPH to the intersystem chain is mediated by NDH [Mi et al.(1994) Plant Cell Physiol. 35: 163] and indicate the bindingof an NADPH-specific NDH to the thylakoid membranes. The Chlfluorescence was quenched transiently by addition of ferredoxinand NADP₊ to the thylakoid membranes but showed a subsequentincrease. This result suggests the reduction of plastoquinoneby the photoreduced NADP₊ and initiation of the NADPH-mediatedcyclic flow of electrons around PSI. Furthermore, a similarresponse of Chl fluorescence was observed upon the additionof ferredoxin only, demonstrating the ferredoxin-dependent cyclicflow of electrons. Both pathways of cyclic electron transportwere inhibited by rotenone, and were not detected in the NDH-defectedthylakoid membranes from M55, indicating the participation ofthe NDH complex. These results confirm that, in Synechocystis,the thylakoid-bound NDH complex mediates the ferredoxin-dependentcyclic electron flow, as well as the NADPH-dependent cyclicelectron flow. (Received November 24, 1994; Accepted March 16, 1995)     

Response of NAD(P)H dehydrogenase complex to the alteration of CO2 concentration in the cyanobacterium Synechocystis PCC6803

An NADPH-specific NDH-1 sub-complex was separated by native-polyacrylamide gel electrophoresis and detected by activity staining from the whole cell extracts of Synechocystis PCC6803. Low CO2 caused an increase in the activity of this sub-complex quickly, accompanied by an evident increase in the expression of NdhK and PSI-driven NADPH oxidation activity that can reflect the activity of NDH-1-mediated cyclic electron transport. During incubation with high CO2, the activities of NDH-1 sub-complex and PSI-driven NADPH oxidation as well as the protein level of NdhK slightly increased at the beginning, but decreased evidently in various degrees along with incubation time. These results suggest that CO2 concentration in vitro as a signal can control the activity of NDH-1 complex, and NDH-1 complex may in turn function in the regulation of CO2 uptake.     

NAD(P)H Dehydrogenase-Dependent, Antimycin A-Sensitive Electron Donation to Plastoquinone in Tobacco Chloroplasts

Cyclic electron transport around PSI through the NAD(P)H dehydrogenasecomplex (NDH) in tobacco leaf disks, measured as an increasein the dark level of Chl fluorescence after the onset of darkness,was inhibited by antimycin A, an inhibitor of ferredoxin quinonereductase (FQR), suggesting that antimycin A inhibits not onlythe FQR-mediated cyclic flow but also the NDH-dependent flow.This electron flow was inhibited also by amytal, an inhibitorof mitochondrial NDH and by nigericin. The reduction of plastoquinonewas detected when NADPH and ferredoxin were added to the suspensionof the osmotically ruptured chloroplasts of the wild type andNDH-defective mutant. Because the addition of NADPH alone didnot induce the reduction, membrane-bound ferredoxin NADP⁺reductase(FNR) was supposed to reduce ferredoxin, which may be a moredirect electron donor for the plastoquinone reduction. The presenceof two types of reducing enzymes was suggested from the bi-phasicinhibition of plastoquinone reduction by antimycin A in thewild type. It is proposed that the reducing activity inhibitedby antimycin A at a low concentration is attributed to FQR andthe less sensitive activity to NDH. (Received June 29, 1998; Accepted September 7, 1998)     

Electron Transport Controls Glutamine Synthetase Activity in the Facultative Heterotrophic Cyanobacterium Synechocystis sp. PCC 6803

Glutamine synthetase (GS) from Synechocystis sp. PCC 6803 was inactivated in vivo by transferring cells from light to darkness or by incubation with the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea but not with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. Addition of glucose prevented both dark and 3-(3,4-dichlorophenyl)-1,1-dimethylurea GS inactivation. In a Synechocystis psbE-psbF mutant (T1297) lacking photosystem II, glucose was required to maintain active GS, even in the light. However, in nitrogen-starved T1297 cells the removal of glucose did not affect GS activity. The fact that dark-inactivated GS was reactivated in vitro by the same treatments that reactivate the ammonium-inactivated GS points out that both nitrogen metabolism and redox state of the cells lead to the same molecular regulatory mechanism in the control of GS activity. Using GS antibodies we detected that dark-inactivated GS displayed a different electrophoretic migration with respect to the active form in nondenaturing polyacrylamide gel electrophoresis but not in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The possible pathway to modulate GS activity by the electron transport flow in Synechocystis cells is discussed.