Expression and functional roles of the two distinct NDH-1 complexes and the carbon acquisition complex NdhD3/NdhF3/CupA/Sll1735 in Synechocystis sp PCC 6803

To investigate the (co)expression, interaction, and membrane location of multifunctional NAD(P)H dehydrogenase type 1 (NDH-1) complexes and their involvement in carbon acquisition, cyclic photosystem I, and respiration, we grew the wild type and specific ndh gene knockout mutants of Synechocystis sp PCC 6803 under different CO2 and pH conditions, followed by a proteome analysis of their membrane protein complexes. Typical NDH-1 complexes were represented by NDH-1L (large) and NDH-1M (medium size), located in the thylakoid membrane. The NDH-1L complex, missing from the DeltaNdhD1/D2 mutant, was a prerequisite for photoheterotrophic growth and thus apparently involved in cellular respiration. The amount of NDH-1M and the rate of P700+ rereduction in darkness in the DeltaNdhD1/D2 mutant grown at low CO2 were similar to those in the wild type, whereas in the M55 mutant (DeltaNdhB), lacking both NDH-1L and NDH-1M, the rate of P700+ rereduction was very slow. The NDH-1S (small) complex, localized to the thylakoid membrane and composed of only NdhD3, NdhF3, CupA, and Sll1735, was strongly induced at low CO2 in the wild type as well as in DeltaNdhD1/D2 and M55. In contrast with the wild type and DeltaNdhD1/D2, which show normal CO2 uptake, M55 is unable to take up CO2 even when the NDH-1S complex is present. Conversely, the DeltaNdhD3/D4 mutant, also unable to take up CO2, lacked NDH-1S but exhibited wild-type levels of NDH-1M at low CO2. These results demonstrate that both NDH-1S and NDH-1M are essential for CO2 uptake and that NDH-1M is a functional complex. We also show that the Na+/HCO3- transporter (SbtA complex) is located in the plasma membrane and is strongly induced in the wild type and mutants at low CO2.     

Novel gene products associated with NdhD3/D4-containing NDH-1 complexes are involved in photosynthetic CO2 hydration in the cyanobacterium, Synechococcus sp. PCC7942

Cyanobacteria possess light-dependent CO2 uptake activity that results in the net hydration of CO2 to HCO3- and may involve a protein-mediated carbonic anhydrase (CA)-like activity. This process is vital for the survival of cyanobacteria and may be a contributing factor in the ecological success of this group of organisms. Here, via isolation of mutants of Synechococcus sp. PCC7942 that cannot grow under low-CO2 conditions, we have identified two novel genes, chpX and chpY, that are involved in light-dependent CO2 hydration and CO2 uptake reactions; co-inactivation of both these genes abolished both activities. The function and mechanism of the CO2 uptake systems supported by each chp gene product differs, with each associated with functionally distinct NAD(P)H dehydrogenase (NDH-1) complexes. The ChpX system has a low affinity for CO2 and is dependent on photosystem I cyclic electron transport, whereas the inducible ChpY system has a high affinity for CO2 and is dependent on linear electron transport. We believe that ChpX and ChpY are involved in a unique, net hydration of CO2 to HCO3-, that is coupled electron flow within the NDH-1 complex on the thylakoid membrane.     

CpcM posttranslationally methylates asparagine-71/72 of phycobiliprotein beta subunits in Synechococcus sp. strain PCC 7002 and Synechocystis sp. strain PCC 6803

Cyanobacteria produce phycobilisomes, which are macromolecular light-harvesting complexes mostly assembled from phycobiliproteins. Phycobiliprotein beta subunits contain a highly conserved gamma-N-methylasparagine residue, which results from the posttranslational modification of Asn71/72. Through comparative genomic analyses, we identified a gene, denoted cpcM, that (i) encodes a protein with sequence similarity to other S-adenosylmethionine-dependent methyltransferases, (ii) is found in all sequenced cyanobacterial genomes, and (iii) often occurs near genes encoding phycobiliproteins in cyanobacterial genomes. The cpcM genes of Synechococcus sp. strain PCC 7002 and Synechocystis sp. strain PCC 6803 were insertionally inactivated. Mass spectrometric analyses of phycobiliproteins isolated from the mutants confirmed that the CpcB, ApcB, and ApcF were 14 Da lighter than their wild-type counterparts. Trypsin digestion and mass analyses of phycobiliproteins isolated from the mutants showed that tryptic peptides from phycocyanin that included Asn72 were also 14 Da lighter than the equivalent peptides from wild-type strains. Thus, CpcM is the methyltransferase that modifies the amide nitrogen of Asn71/72 of CpcB, ApcB, and ApcF. When cells were grown at low light intensity, the cpcM mutants were phenotypically similar to the wild-type strains. However, the mutants were sensitive to high-light stress, and the cpcM mutant of Synechocystis sp. strain PCC 6803 was unable to grow at moderately high light intensities. Fluorescence emission measurements showed that the ability to perform state transitions was impaired in the cpcM mutants and suggested that energy transfer from phycobiliproteins to the photosystems was also less efficient. The possible functions of asparagine N methylation of phycobiliproteins are discussed.     

Identification and characterization of NuhA, a novel Nudix hydrolase specific for ADP-ribose in the cyanobacterium Synechococcus sp. PCC 7002

We cloned the gene for a novel Nudix hydrolase in the cyanobacterium Synechococcus sp. PCC 7002 and termed it nuhA. The deduced amino acid sequence of NuhA included the Nudix motif, GX(5)EX(7)RELXEEXGV, which is common to Nudix hydrolases, and in addition, a proline at the 15th amino acid from the C-terminus of the Nudix motif, which is characteristic of the subfamily of ADP-ribose pyrophosphatases. The recombinant NuhA with a hexahistidine tag was overexpressed in Escherichia coli and purified. The recombinant NuhA hydrolyzed ADP-ribose specifically among various nucleoside diphosphate derivatives. The hydrolytic activity for ADP-ribose required Mg(2+) and was optimal at pH 9.5. The V(max) and K(m) values of hydrolysis were 23.6 units mg(-1) and 0.094 mM, respectively. NuhA contained an uncharacterized domain in the C-terminal region, termed Pfam-B-3116, which is conserved in several hypothetical proteins. The mutated NuhA deficient in the Pfam-B-3116 domain failed to form the hexamers that are characteristic of NuhA, and exhibited a significantly higher K(m) value for ADP-ribose, suggesting that the Pfam-B-3116 domain might be responsible for oligomerization of NuhA and full binding affinity for ADP-ribose. These unique features suggest that NuhA is a novel type of ADP-ribose pyrophosphatase.     

1H, 15N, and 13C resonance assignments of the 2/2 hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002 in the ferric bis-histidine state

The hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002 is a monomeric 123-residue Group I 2/2 hemoglobin. Here, we report ¹H, ¹⁵N, and ¹³C assignments for the ferric (low-spin, S = ½) protein with a b heme cofactor and after post-translational modification leading to a c-like heme.     

Distinct roles of multiple NDH-1 complexes in the cyanobacterial electron transport network as revealed by kinetic analysis of P700+ reduction in various Ndh-deficient mutants of Synechocystis sp. strain PCC6803

While methyl viologen had only a small effect on P700(+) rereduction kinetics after far-red pulses in KCN-treated wild-type Synechocystis sp. strain PCC6803 and an NdhF3/NdhF4 (NdhF3/F4)-defective mutant, it involved a rather slow P700(+) rereduction in an NdhF1-defective mutant. This strongly indicates that (i) active electron flow from metabolites to plastoquinone is suppressed upon deletion of ndhF1 and (ii) photosystem 1-mediated cyclic electron transport is dependent on NdhF3/F4-type NDH-1 complexes.     

Regulation of the induction of bicarbonate uptake by dissolved CO2 in the marine diatom, Phaeodactylum tricornutum

Physiological properties of photosynthesis were determined in the marine diatom, Phaeodactylum tricornutum UTEX640, during acclimation from 5% CO₂ to air and related to H₂CO₃ dissociation kinetics and equilibria in artificial seawater. The concentration of dissolved inorganic carbon at half maximum rate of photosynthesis (K_0·5[DIC]) value in high CO₂‐grown cells was 1009 mmol m ^{? 3} but was reduced three‐fold by the addition of bovine carbonic anhydrase (CA), whereas in air‐grown cells K_0·5[DIC] was 71 mmol m ^{? 3}, irrespective of the presence of CA. The maximum rate of photosynthesis (P_max) values varied between 300 and 500 μ mol O₂ mg Chl ^{? 1} h ^{? 1} regardless of growth pCO₂. Bicarbonate dehydration kinetics in artificial seawater were re‐examined to evaluate the direct HCO₃ ^? uptake as a substrate for photosynthesis. The uncatalysed CO₂ formation rate in artificial seawater of 31·65°/_oo of salinity at pH 8·2 and 25 °C was found to be 0·6 mmol m ^{? 3} min ^{? 1} at 100 mmol m ^{? 3} DIC, which is 53·5 and 7·3 times slower than the rates of photosynthesis exhibited in air‐ and high CO₂‐grown cells, respectively. These data indicate that even high CO₂‐grown cells of P. tricornutum can take up both CO₂ and HCO₃ ^? as substrates for photosynthesis and HCO₃ ^? use improves dramatically when the cells are grown in air. Detailed time courses were obtained of changes in affinity for DIC during the acclimation of high CO₂‐grown cells to air. The development of high‐affinity photosynthesis started after a 2–5 h lag period, followed by a steady increase over the next 15 h. This acclimation time course is the slowest to be described so far. High CO₂‐grown cells were transferred to controlled DIC conditions, at which the concentrations of each DIC species could be defined, and were allowed to acclimate for more than 36 h. The K_0·5[DIC] values in acclimated cells appeared to be correlated only with [CO_2(aq)] in the medium but not to HCO₃ ^? , CO₃^{2 ?} , total [DIC] or the pH of the medium and indicate that the critical signal regulating the affinity of cells for DIC in the marine diatom, P. tricornutum, is [CO_2(aq)] in the medium.     

Photosynthesis, immediate export and carbon partitioning in source leaves of C3, C3-C4 intermediate, and C4Panicum and Flaveria species at ambient and elevated CO2 levels

Immediate export in leaves of C₃‐C₄ intermediates were compared with their C₃ and C₄ relatives within the Panicum and Flaveria genera. At 35 Pa CO₂, photosynthesis and export were highest in C₄ species in each genera. Within the Panicum, photosynthesis and export in ‘type I’ C₃‐C₄ intermediates were greater than those in C₃ species. However, ‘type I’ C₃‐C₄ intermediates exported a similar proportion of newly fixed ¹⁴C as did C₄ species. Within the Flaveria, ‘type II’ C₃‐C₄ intermediate species had the lowest export rather than the C₃ species. At ambient CO₂, immediate export was strongly correlated with photosynthesis. However, at 90 Pa CO₂, when photosynthesis and immediate export increased in all C₃ and C₃‐C₄ intermediate species, proportionally less C was exported in all photosynthetic types than that at ambient CO₂. All species accumulated starch and sugars at both CO₂ levels. There was no correlation between immediate export and the pattern of ¹⁴C‐labelling into sugars and starch among the photosynthetic types within each genus. However, during CO₂ enrichment, C₄Panicum species accumulated sugars above the level of sugars and starch normally made at ambient CO₂, whereas the C₄Flaveria species accumulated only additional starch.     

Differential responses of root uptake kinetics of NH4+ and NO3− to enriched atmospheric CO2 concentration in field-grown loblolly pine

The nitrogen requirement of plants is predominantly supplied by NH₄⁺ and/or NO₃^? from the soil solution, but the energetic cost of uptake and assimilation is generally higher for NO₃^? than for NH₄⁺. We found that CO₂ enrichment of the atmosphere enhanced the root uptake capacity for NO₃^?, but not for NH₄⁺, in field-grown loblolly pine saplings. Increased preference for NO₃^? at the elevated CO₂ concentration was accompanied by increased carbohydrate levels in roots. The results have important implications for the potential consequences of global climate change on plant-and ecosystem-level processes in many temperate forest ecosystems.     

Effects of elevated O3, alone and in combination with elevated CO2, on tree leaf chemistry and insect herbivore performance: a meta-analysis

We reviewed the effects of elevated ozone (O₃), alone and in combination with elevated carbon dioxide (CO₂) on primary and secondary metabolites of trees and performance of insect herbivores by means of meta‐analysis. Our database consisted of 63 studies conducted on 22 species of trees and published between 1990 and 2005. Ozone alone had no overall effect on concentrations of carbohydrates or nutrients, whereas in combination with CO₂, elevated O₃ reduced nutrient concentrations and increased carbohydrate concentrations. In contrast to primary metabolites, concentrations of phenolics and terpenes were significantly increased by 16% and 8%, respectively, in response to elevated O₃. Effects of ozone in combination with elevated CO₂ were weaker than those of ozone alone on phenolics, but stronger than those of ozone alone on terpenes. The magnitude of secondary metabolite responses depended on the type of ozone exposure facility and increased in the following order: indoor growth chamber 3 than gymnosperms, as shifts in concentrations of carbohydrate and phenolics were observed in the former, but not in the latter. Elevated O₃ had positive effects on some indices of insect performance: pupal mass increased and larval development time shortened, but these effects were counteracted by elevated CO₂. Therefore, despite the observed increase in secondary metabolites, elevated O₃ tends to increase tree foliage quality for herbivores, but elevated CO₂ may alleviate these effects. Our meta‐analysis clearly demonstrated that effects of elevated O₃ alone on leaf chemistry and some indices of insect performance differed from those of O₃+CO₂, and therefore, it is important to study effects of several factors of global climate change simultaneously.     

Winter hardening and glutathione status in the bilberry (Vaccinium myrtillus) in response to trace gases (CO2, O3) and nitrogen fertilization

Bilberry plants (Vaccinium myrtillus L.) at a field site in northern Finland (65°N) were subjected to nitrogen fertilization [6.5 mmol m^?2 NH₄NO₃× Ca(OH)₂] at the beginning of 3 growing seasons in late May and to trace gas fumigation (CO₂ and O₃) for 5 months (May–September) in 1993–1995 in order to investigate frost resistance and glutathione concentrations during the winter hardening period, and to assess the correlation between these variables. Harvesting was performed twice in the autumn of both 1994 and 1995, and the two-year data for each harvest were pooled. The frost resistance of the bilberry stems increased by about 10°C during the hardening period between the two harvests. Nitrogen fertilization increased the frost resistance towards late autumn. The fumigation treatments had no marked effect on it. The combination of elevated CO₂ and nitrogen fertilization induced a decrease in frost resistance. Increases in total glutathione concentrations and the proportion of reduced glutathione (GSH) in the stems were evident during hardening. Nitrogen fertilization positively affected the total glutathione concentration and the proportion of GSH at the beginning of the hardening period but the effect disappeared during the hardening process. Trace gas fumigation as such had no marked effect on glutathione concentration. Increases in glutathione concentrations during hardening did not correlate with frost resistance, possibly due to different timing of the appearence of the response to fertilization treatment, i.e., glutathione responded in the beginning of hardening while frost resistance at the end. The lack of correlation with frost resistance, and especially the different responses to nitrogen fertilization, may reflect the indirect role of glutathione in the development of winter hardening, as a transport and storage form of reduced nitrogen and sulphur. In conclusion, winter hardening and glutathione status in the bilberry seems to be sensitive to nitrogen fertilization, and not affected by elevated CO₂ and O₃.     

Polymorphic glutamate dehydrogenase in lilac vitroplants as revealed by combined preparative IEF and native PAGE: Effect of ammonium deprivation, darkness and atmospheric CO2 enrichment upon isomerization

The activity and polymorphism of glutamate dehydrogenase (GDH) were studied in basal callus of lilac ( Syringa vulgaris L.) vitroplants. Native PAGE alone revealed seven bands staggered at regular intervals. Preparative liquid-vein IEF allowed the separation of six to ten GDH fractions with charges ranging between 5.18 and 7.08. Analysis of these GDH fractions in native PAGE indicated that up to seven GDH bands can be detected for each fraction. This suggests the existence of seven isoforms of the enzyme with subunits presenting different isoelectric points. Dark- and ammonium-controlled forms were found to be the more acidic and faster migrating ones in native PAGE. The results support for the first time that atmospheric CO₂ enrichment increases GDH activity dramatically and modifies isomerization of the enzyme.     

Effects of PI3-k/Akt short hairpin RNA on proliferation, fibronectin production and synthesis of thrombospondin-1 and transforming growth factor-β1 in glomerular mesangial cells induced by sublytic C5b-9 complexes

Objectives: To explore proliferation of glomerular mesangial cells (GMC) and secretion of extracelluar matrix (fibronectin induced by sublytic C5b-9 complexes), and then ascertain the role of phosphatidylinositol 3-kinase (PI3-k)/Akt signal pathway in these processes, by using small hairpin RNAs.
Material and methods: The expression of cyclin D₂, ³H-thymidine into DNA and production of fibronectin including thrombospondin-1 and transforming growth factor-β₁ in the GMCs stimulated by sublytic C5b-9 or transfected with expression vectors of PI3-k and Akt short hairpin RNA or LY294002 (PI3-k inhibitor) were measured by Real-time quantitative polymerase chain reaction (PCR), Western blot, enzyme-linked immunosorbent assay (ELISA) and ³H-thymidine incorporation (³H-TdR), respectively.
Results: The expression of cyclin D₂, ³H-thymidine into DNA and fibronectin in the GMCs stimulated by sublytic C5b-9 could all be increased, and the elevations of these parameters mentioned above were also markedly reduced in the GMCs transfected with vectors of PI3-k and Akt short hairpin RNA or LY294002, respectively.
Conclusions: These data indicate that sublytic C5b-9 can promote proliferation of GMCs and secretion of fibronectin as well as synthesis of thrombospondin-1 and transforming growth factor-β₁. The PI3-k/Akt signal pathway in these reactions, mediated by sublytic C5b-9 complexes, may play at least a partial role.