Synechococcus Vulcanus Research Articles

Cloning of a C-phycocyanin Alpha Subunit from Thermosynechococcus sp. TUBT-T01 and Prediction of Its Properties

C-Phycocyanin, a blue-colored and water soluble protein, is a class of phycobiliproteins that are the major light-harvesting pigments of a photosynthetic system in cyanobacteria. C-phycocyanins are utilized in many industries, including as natural colorants in food and cosmetics and as antioxidant compounds. However, the uses of C-phycocyanins have been limited due to their vulnerability to high temperatures. Therefore, the objective of this study was to identify and analyze the C-phycocyanin gene isolated from Thermosynechococcus sp. TUBT-T01, living in a hot spring in Surat Thani province, in the hope that this C-phycocyanin exhibited thermostable properties and that their applications could be expanded over a wide range of industries. In the present study, the polymerase chain reaction of the gene encoding alpha subunits of C-phycocyanin (cpcA) was performed, using primers designed based upon the sequence alignments of cpcA from Thermosynechococcus sp. available in the GenBank database. The putative cpcA, with an approximate size of 500 base pairs, was detected on an agarose gel. The DNA sequencing analysis indicated that the cpcA was 489 base pairs in length, and its nucleotide sequence was 94 % identical to those of thermophilic Thermosynechococcus sp. NK55, T. elongatus BP-1, and Synechococcus vulcanus. The deduced amino acid sequence was very similar to those of Thermosynechococcus sp. NK55, T. elongatus BP-1, and S. vulcanus. The data derived from the homologous model revealed that the presence of Asp28, Lys32, and Ser72 in the alpha subunit of C-phycocyanin from Thermosynechococcus sp. TUBT-T01 could provide the high thermostability property of this protein.

Characterization of Nicotiana tabacum plants expressing hybrid genes of cyanobacterial Δ9 or Δ12 acyl-lipid desaturases and thermostable lichenase

We established transgenic lines of Nicotiana tabacum expressing hybrid genes of Synechocystis sp. PCC 6803 Δ12 (desA) acyllipid desaturase and Synechococcus vulcanus Δ9 (desC) acyllipid desaturase with or without sequence coding for transit peptide of Rubisco small subunit of Arabidopsis thaliana under control of a constitutive promoter. Reliable increase of linoleic acid portion (C18:2; Δ9,12) accompanied with decrease of α-linolenic acid (C18:3; Δ9,12,15) relative amount was detected for plants expressing hybrid desA::licBM3 gene. No reliable changes were detected in fatty acid profiles and unsaturation index of plants transformed with Δ9 desaturase gene desC::licBM3 lacking signals of intracellular targeting while expression of this gene with Arabidopsis thaliana Rubisco small subunit transit peptide sequence caused growth of C18:3 α-linolenic acid part simultaneously with reduction of C18:2 linoleic acid part, as well as increase of unsat-uration index. No changes in relative amount of Δ9-monounsaturated fatty acids were observed in any of studied lines. All plants expressing desaturase genes exhibited enhanced levels of superoxide dismutase (SOD) activity after cold treatment in contrast to control lines with suppressed SOD activity after cold treatment.

Superoxide dismutase activity in transgenic canola

Superoxide dismutase (SOD) activity was investigated in leaves of transgenic canola plants which expressed heterologous genes of different origin, namely 1) herbicide resistance genes (bar and simultaneously bar and epsps); 2) DesC desaturase gene (desC) of cyanobacterium Synechococcus vulcanus; 3) human interferon alpha2b gene (huLFN-alpha2b); 4) esxA::fbpB(deltaTMD) fused gene, encoding ESAT-6 and Ag85b Mycobacterium tuberculosis proteins, inducing immune response against tuberculosis; 5) cyp11A1 gene of cytochrome P450(scc) from bovine adrenal cortex mitochondria. Introduction of herbicide resistance genes as well as desaturase gene of cyanobacterium and mycobacterium's genes did not change leaf SOD activity. At the same time it was shown that cyp11A1 and huIFN-alpha2b canola have increased leaf SOD activity up 58 and 33%, respectively, compared with control ones in non-stress conditions. It may be a prerequisite for improved resistance of these plants to the stressors of different origin.

Surface Display of Bacterial Metallothioneins and a Chitin Binding Domain on Escherichia coli Increase Cadmium Adsorption and Cell Immobilization

To increase the level of adsorption of cadmium ions to the surface of Escherichia coli, we fused cyanobacterial metallothioneins, SmtA (from Synechococcus elongatus PCC 3601) and MtnA (from Synechococcus vulcanus) to the E. coli cell surface using a Lpp'-OmpA-based display system. E. coli strains expressing Lpp'-OmpA-SmtA-linker-ChBD (chitin-binding domain from Bacillus pumillus SG2 chitinase S; chiS) and Lpp'-OmpA-MtnA-linker-ChBD on their surface adsorbed more cadmium compared to the E. coli cells expressing only the Lpp'-OmpA-linker-ChBD hybrid. These constructs also were bound to chitin through their chitin-binding domain, allowing them to be immobilized on a chitin matrix. We assessed surface presentation of Lpp'-OmpA-SmtA-linker-ChBD, Lpp'-OmpA-MtnA-linker-ChBD, and Lpp'-OmpA-linker-ChBD using immunostaining. The Lpp'-OmpA-SmtA-linker-ChBD chimera adsorbed metal and was bound to chitin with the highest efficiency compared to the other chimeras, suggesting that it is an effective bioadsorbent. This is the first example of coupling metal adsorption with cell immobilization using a whole-cell bioadsorbent.

Multiplex PCR assay for detection of recombinant genes encoding fatty acid desaturases fused with lichenase reporter protein in GM plants

Thermostable lichenase encoded by licB gene of Clostridium thermocellum can be used as a reporter protein in plant, bacterial, yeast, and mammalian cells. It has important advantages of high sensitivity and specificity in qualitative and quantitative assays. Deletion variants of LicB (e.g., LicBM3) retain its enzymatic activity and thermostability and can be expressed in translational fusion with target proteins without compromising with their properties. Fusion with the lichenase reporter is especially convenient for the heterologous expression of proteins whose analysis is difficult or compromised by host enzyme activities, as it is in case of fatty acid desaturases occurring in all groups of organisms. Recombinant desaturase-lichenase genes can be used for creating genetically modified (GM) plants with improved chill tolerance. Development of an analytical method for detection of fused desaturase-lichenase transgenes is necessary both for production of GM plants and for their certification. Here, we report a multiplex polymerase chain reaction method for detection of desA and desC desaturase genes of cyanobacteria Synechocystis sp. PCC6803 and Synechococcus vulcanus, respectively, fused to licBM3 reporter in GM plants.

Chloroplast ultrastructure in leaves of tobacco plants with the introduced gene for the acyl-lipid Δ9-desaturase from Synechococcus vulcanus at normal and low temperature

Ultrastructural changes in chloroplasts of tobacco plants (Nicotiana tabacum L.) with the introduced desC gene for the acyl-lipid Δ9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus were investigated during plant acclimation to cold. Control plants were transformed with an empty pGA482 binary vector. At optimum growth temperature, a decreased number of grana and thylakoids and an increased number of plastoglobules and their larger area were observed in transgenic plants when compared to control ones. In control plants, acclimation to cold (6 days at 10°C) resulted in the larger areas of chloroplasts and grana. These changes indicated starting cold-induced injuries manifested in swelling of the stroma and a slight decrease in the total number of thylakoids in the chloroplast. In contrast, transgenic plants responded to cold by reducing the chloroplast, granal, and plastoglobule areas. Meantime, the number of thylakoids per granum increased noticeably. The total number of thylakoids in the chloroplast increased from 123 to 203. It was concluded that expression of the acyl-lipid Δ9-desaturase gene in tobacco plants provided for the formation of the cell ultrastructure similar to one characteristic of cold-tolerant plants.

Deletion of PsbM in Tobacco Alters the QB Site Properties and the Electron Flow within Photosystem II

Photosystem II, the oxygen-evolving complex of photosynthetic organisms, includes an intriguingly large number of low molecular weight polypeptides, including PsbM. Here we describe the first knock-out of psbM using a transplastomic, reverse genetics approach in a higher plant. Homoplastomic Delta psbM plants exhibit photoautotrophic growth. Biochemical, biophysical, and immunological analyses demonstrate that PsbM is not required for biogenesis of higher order photosystem II complexes. However, photosystem II is highly light-sensitive, and its activity is significantly decreased in Delta psbM, whereas kinetics of plastid protein synthesis, reassembly of photosystem II, and recovery of its activity are comparable with the wild type. Unlike wild type, phosphorylation of the reaction center proteins D1 and D2 is severely reduced, whereas the redox-controlled phosphorylation of photosystem II light-harvesting complex is reversely regulated in Delta psbM plants because of accumulation of reduced plastoquinone in the dark and a limited photosystem II-mediated electron transport in the light. Charge recombination in Delta psbM measured by thermoluminescence oscillations significantly differs from the 2/6 patterns in the wild type. A simulation program of thermoluminescence oscillations indicates a higher Q(B)/Q(-)(B) ratio in dark-adapted mutant thylakoids relative to the wild type. The interaction of the Q(A)/Q(B) sites estimated by shifts in the maximal thermoluminescence emission temperature of the Q band, induced by binding of different herbicides to the Q(B) site, is changed indicating alteration of the activation energy for back electron flow. We conclude that PsbM is primarily involved in the interaction of the redox components important for the electron flow within, outward, and backward to photosystem II.

Specific features of oxidative stress in the chilled tobacco plants following transformation with the desC gene for acyl-lipid Δ9-desaturase from Synechococcus vulcanus

Tobacco plants (Nicotiana tabacum L.) transformed with the desC gene for acyl-lipid Δ9-desaturase from a thermophilic cyanobacterium Synechococcus vulcanus were cultivated on the agarized Murashige and Skoog medium at 22°C and a 16-h photoperiod. Tobacco plants transformed with an empty binary vector pGA482 served as the control. The investigations showed that, in contrast to the control, transgenic plants maintained a higher activity of antioxidant enzymes during 2-h incubation at 2°C; as a result, these plants resisted more efficiently the accumulation of reactive oxygen species and reduced the rate of the lipid peroxidation. The activity of antioxidant enzymes in the transformed plants is apparently related to the operation of the introduced desC gene for acyl-lipid Δ9-desaturase because the enhanced activity of the latter increased the relative content of polyunsaturated FAs in membrane lipids and in this way promoted the liquid state of membranes during the chilling period. These changes helped preserve the cellular homeostasis and thereby maintain the steady synthesis of antioxidant enzymes at hypothermic conditions; as a result, cold resistance of transformed tobacco plants increased.

The Effect of Tobacco Plant Transformation with a Gene for Acyl-Lipid Δ9-Desaturase from Synechococcus vulcanus on Plant Chilling Tolerance

Tobacco plants with the introduced desC gene for acyl-lipid Δ9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus were cultivated on agar-solidified Murashige and Skoog nutrient medium supplemented with ferulic acid and antibiotics at 22°C and a 16-h photoperiod. Control plants were transformed with an empty pGA482 vector. The analysis of fatty acids (FAs) showed that, in transgenic plants, the level of 16:0 and 18:0 FAs decreased substantially, whereas the levels of di- and trienoic FAs increased. Transformed plants were more cold-tolerant. The tolerance to chilling was evaluated from electrolyte leakage from tissues damaged by cold treatments and from the accumulation of a product of lipid peroxidation, malondialdehyde. It was concluded that acyl-lipid Δ9-desaturase was actively expressed in transgenic tobacco plants and converted stearic acid into oleic acid, thus producing a substrate for further synthesis of di- and trienoic FAs. An increased proportion of polyunsaturated FAs in membrane lipids resulted in improved tobacco plant tolerance to chilling.

Post-transcriptional control of the cyanobacterial hspA heat-shock induction

The hspA gene encodes a small heat-shock protein in the thermophilic cyanobacterium Synechococcus vulcanus. To gain insight into the post-transcriptional regulation, hspA was expressed in Escherichia coli. HspA was induced upon a temperature upshift from 30 to 42 °C, although the hspA transcription in E. coli occurred constitutively at both 30 and 42 °C. Neither replacement of the native hspA promoter with the lacZ promoter nor the addition of rifampin abolished the heat induction. Thus, the primary form of regulation of the heat induction is at the post-transcriptional level. Analyses of expression of a series of the transcriptional and translational hspA–lacZ fusions confirmed the constitutive transcription, and demonstrated that the heat induction occurred only in the translational fusions. They further indicated the presence of regulatory elements involved in the translational regulation. An element in the 5′-untranslated region of the hspA mRNA suppressed the translation, while that in the hspA coding region was required for the de-repression of the translation and for thermal regulation.

Transformation of tobacco with a gene for the thermophilic acyl-lipid desaturase enhances the chilling tolerance of plants.

The desC gene for the acyl-lipid Delta9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus was introduced into Nicotiana tabacum under control of the 35S promoter. Expression of the desaturase was confirmed by Western blotting. Lipid analysis revealed that lipid content and the extent of fatty acid unsaturation significantly increased in leaves of transgenic plants. Chilling tolerance of those plants also increased, as estimated by the electrolyte leakage from the tissues damaged by cold treatments. Seeds of plants that expressed the desC gene imbibed at low temperatures demonstrated higher chilling tolerance than those of the control plants. The results demonstrate that the cyanobacterial thermophilic acyl-lipid desaturase was efficiently expressed in tobacco at ambient temperatures, and its expression resulted in the enhanced chilling tolerance of the transgenic plants.

Near-IR irradiation of the S2 state of the water oxidizing complex of photosystem II at liquid helium temperatures produces the metalloradical intermediate attributed to S1Y(Z*).

Near-IR (NIR) excitation at liquid He temperatures of photosystem II (PSII) membranes from the cyanobacterium Synechococcus vulcanus or from spinach poised in the S2 state results in the production of a g = 2.035 EPR resonance, reminiscent of metalloradical signals. The signal is smaller in the spinach preparations, but it is significantly enhanced by the addition of exogenous quinones. Ethanol (2-3%, v/v) eliminates the ability to trap the signal. The g = 2.035 signal is identical to the one recently obtained by Nugent et al. by visible-light illumination of the S1 state, and preferably assigned to S1Y(Z*) [Nugent, J. H. A., Muhiuddin, I. P., and Evans, M. C. W. (2002) Biochemistry 41, 4117-4126]. The production of the g = 2.035 signal by liquid He temperature NIR excitation of the S2 state is paralleled by a significant reduction (typically 40-45% in S. vulcanus) of the S2 state multiline signal. This is in part due to the conversion of the Mn cluster to higher spin states, an effect documented by Boussac et al. [Boussac, A., Un, S., Horner, O., and Rutherford, A. W. (1998) Biochemistry 37, 4001-4007], and in part due to the conversion to the g = 2.035 configuration. Following the decay of the g = 2.035 signal at liquid helium temperatures (decay halftimes in the time range of a few to tens of minutes depending on the preparation), annealing at elevated temperatures (-80 degrees C) results in only partial restoration of the S2 state multiline signal. The full size of the signal can be restored by visible-light illumination at -80 degrees C, implying that during the near-IR excitation and subsequent storage at liquid helium temperatures recombination with Q(A-) (and therefore decay of the S2 state to the S1 state) occurred in a fraction of centers. In support of this conclusion, the g = 2.035 signal remains stable for several hours (at 11 K) in centers poised in the S2...Q(A) configuration before the NIR excitation. The extended stability of the signal under these conditions has allowed the measurement of the microwave power saturation and the temperature dependence in the temperature range of 3.8-11 K. The signal intensity follows Curie law temperature dependence, which suggests that it arises from a ground spin state, or a very low-lying excited spin state. The P1/2 (microwave power at half-saturation) value is 1.7 mW at 3.8 K and increases to 96 mW at 11 K. The large width of the g = 2.035 signal and its relatively fast relaxation support the assignment to a radical species in the proximity of the Mn cluster. The whole phenomenology of the g = 2.035 signal production is analogous to the effects of NIR excitation on the S3 state [Ioannidis, N., Nugent, J. H. A., and Petrouleas, V. (2002) Biochemistry 41, 9589-9600] producing an S2'Y(Z*) intermediate. In the present case, the intermediate is assigned to S1Y(Z*). The NIR-induced increase in the oxidative capability of the Mn cluster is discussed in relation to the photochemical properties of a Mn(III) ion that exists in both S2 and S3 states. The EPR properties of the S1Y(Z*) intermediate cannot be reconciled easily with our current understanding of the magnetic properties of the S1 state. It is suggested that oxidation of tyr Z alters the magnetic properties of the Mn cluster via exchange of a proton.

Low-molecular-mass polypeptide components of a photosystem II preparation from the thermophilic cyanobacterium Thermosynechococcus vulcanus.

Using a recently introduced electrophoresis system [Kashino et al. (2001) Electrophoresis 22: 1004], components of low-molecular-mass polypeptides were analyzed in detail in photosystem II (PSII) complexes isolated from a thermophilic cyanobacterium, Thermosynechococcus vulcanus (formerly, Synechococcus vulcanus). PsbE, the large subunit polypeptide of cytochrome b(559), showed an apparent molecular mass much lower than the expected one. The unusually large mobility could be attributed to the large intrinsic net electronic charge. All other Coomassie-stained polypeptides were identified by N-terminal sequencing. In addition to the well-known cyanobacterial PSII polypeptides, such as PsbE, F, H, I, L, M, U, V and X, the presence of PsbY, PsbZ and Psb27 was also confirmed in the isolated PSII complexes. Furthermore, the whole amino acid sequence was determined for the polypeptide which was known as PsbN. The whole amino acid sequence revealed that this polypeptide was identical to PsbTc which has been found in higher plants and green algae. These results strongly suggest that PsbN is not a member of the PSII complex. It is also shown that cyanobacteria have cytochrome b(559) in the high potential form as in higher plants.

Refined structure of c-phycocyanin from the cyanobacterium Synechococcus vulcanus at 1.6 Å: insights into the role of solvent molecules in thermal stability and co-factor structure

The crystal structure of the light-harvesting phycobiliprotein, c-phycocyanin from the thermophilic cyanobacterium Synechococcus vulcanus has been refined to 1.6 Å resolution based on the previously determined lower resolution structure (PDB entry 1I7Y). The improved data was collected using synchrotron radiation at 100 K. The significantly improved crystallographic data has lead to improved calculated electron density maps, allowing the unambiguous positioning of all protein and co-factor atoms and the positioning of 377 solvent molecules. The positions of solvent molecules at specific sites important for stabilization of different levels of self-assembly of the phycobilisome structure were identified and the bonding network is described. The presence of solvent molecules in the vicinity of the co-factors and in intermolecular spaces is identified and their possible roles are suggested. All three of the phycocyanobilin co-factors bind water molecules at specific sites between the propionic acid side chains. Molecular dynamic (MD) simulations support that these special waters have a role in stabilization of this conformation. On the basis of the crystal packing reported here and in comparison to other phycobiliprotein crystal forms, we have analyzed the roles of specific sites on the formation of the phycobilisome complex.

Specific binding of a protein to a novel DNA element in the cyanobacterial small heat-shock protein gene

Previously, it was shown that transcription of the small heat-shock protein gene, hspA, from the thermophilic cyanobacterium Synechococcus vulcanus is transiently heat-inducible at a vegetative promoter that lacks any known regulatory DNA elements. A novel regulatory mechanism that suppresses the expression of hspA under non-heat-shock condition has been postulated. In this study, it is demonstrated that a protein(s) in the extract of unstressed cells of the thermophilic cyanobacterium Thermosynechococcus elongatus, a cyanobacterium closely related to S. vulcanus, specifically binds to a 5 ′-untranslated region of the hspA gene. An AT-rich imperfect inverted-repeat sequence (ACAAgcAAA-TTTagTTGT) as a target for a putative DNA-binding protein has been identified. The DNA-binding activity in the cell as well as in the cell extract was lost much more quickly at a heat-shock temperature than a normal growth temperature. In a cell, the activity was restored within 45 min after a heat-shock by the heat-induced synthesis and stabilization of a DNA-binding protein. It is proposed that the inverted repeat is a specific target for a DNA-binding protein and that it plays a role in the regulation of the cyanobacterial hspA gene expression.

Molecular Characterization of a Phosphoenolpyruvate Carboxylase from a Thermophilic Cyanobacterium, Synechococcus vulcanus with Unusual Allosteric Properties

A gene for phosphoenolpyruvate carboxylase (PEPC) was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus, by screening a genomic DNA library using the coding region of Anacystis nidulans 6301 PEPC as a probe. The S. vulcanus PEPC gene (SvPEPC) had an open reading frame for a polypeptide of 1,011 amino acid residues with a calculated molecular mass of 116.4 kDa. SvPEPC was expressed in E. coli BL21 Codonplus (DE3), using pET32a as a vector. The purified recombinant SvPEPC protein with a tag showed a single band of 120 kDa on SDS-PAGE. The enzyme forms homotetramer as judged by gel filtration. SvPEPC retained full activity even after incubation at 50 degrees C for 60 min or exposure to 0.5 M guanidine-HCl at 30 degrees C for 20 h, being more stable than C4-form PEPC from Zea mays (ZmPEPC(C4)). SvPEPC activity showed a sharp optimum temperature of 42 degrees C at pH 7.5 and an optimum pH of 9.0 at 30 degrees C. The enzyme, unlike most plant PEPCs, was predominantly activated by fructose 1,6-bisphosphate (Fruc-1,6-P(2)), and slightly stimulated by 3-phosphoglycerate (3-PGA), glucose 6-phosphate (Gluc-6-P), glucose 1-phosphate, Glu and Gln. Acetyl-CoA known as a strong activator of most bacterial PEPCs but not of plant PEPCs, showed no effect on the enzyme activity. SvPEPC was more sensitive to the inhibition by Asp at higher pH (9.0) than lower pH (7.0), contrary to Coccochloris peniocystis PEPC and plant PEPCs. I(0.5) for Asp was increased about 2-fold by Gluc-6-P while markedly decreased by Fruc-1,6-P(2), Glu and Gln about 3- to 4-fold. The regulation mechanism of SvPEPC is not readily interpretable by conventional allosteric models.

Structure of c-phycocyanin from the thermophilic cyanobacterium Synechococcus vulcanus at 2.5 Å: structural implications for thermal stability in phycobilisome assembly

The crystal structure of the light-harvesting phycobiliprotein, c-phycocyanin from the thermophilic cyanobacterium Synechochoccus vulcanus has been determined by molecular replacement to 2.5 Å resolution. The crystal belongs to space group R32 with cell parameters a = b = 188.43 Å, c = 61.28 Å, α = β = 90 °, γ = 120 °, with one (αβ) monomer in the asymmetric unit. The structure has been refined to a crystallographic R factor of 20.2 % (R-free factor is 24.4 %), for all data to 2.5 Å. The crystals were grown from phycocyanin (αβ)3 trimers that form (αβ)6 hexamers in the crystals, in a fashion similar to other phycocyanins. Comparison of the primary, tertiary and quaternary structures of the S. vulcanus phycocyanin structure with phycocyanins from both the mesophilic Fremyella diplsiphon and the thermophilic Mastigocladus laminosus were performed. We show that each level of assembly of oligomeric phycocyanin, which leads to the formation of the phycobilisome structure, can be stabilized in thermophilic organisms by amino acid residue substitutions. Each substitution can form additional ionic interactions at critical positions of each association interface. In addition, a significant shift in the position of ring D of the B155 phycocyanobilin cofactor in the S. vulcanus phycocyanin, enables the formation of important polar interactions at both the (αβ) monomer and (αβ)6 hexamer association interfaces.

Phophoenolpyruvate carboxylase (PEPC) from a thermophilic caynobacterium, Synechococcus vulcanus

A gene for phosphoenolpyruvate carboxylase (PEPC) was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus and expressed in E.coli BL21 (DE3) under the control of T7 promoter. The purified fusion protein (recombinant SvPEPC) showed very unusual allosteric regulatory properties. The enzyme activity was strongly activated by Fruc-1,6-P2 and slightly stimulated by Gluc-6-P, Glu and Gln. SvPEPC was more sensitive to the inhibition by Asp at higher pH (9.0) than lower pH (7.0), contrary to Coccochloris peniocystis PEPC and plant PEPCs. I0.5 for Asp was increased about 2-fold by Gluc-6-P while markedly decreased about 4-fold by Fruc-1,6-P2. These allosteric properties are not readily explainable by conventional allosteric models. It seems that Fruc-1,6-P2 and Gluc-6-P do not share the same binding site on SvPEPC protein. When SvPEPC was introduced into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated in planta transformatiom under the control of the cauliflower mosaic virus 35S promoter, growth hindrance occurred on the transformants plants grown on agar plate with 1% sucrose under white light. In the early stage of growth, about 10% of plants showed bleaching in leaves or cotyledons in a gradient manner. A part of them could grow on soil but decreased apical dominance was observed in the adult plants. Leaf or cotyledon bleaching may be resulted from the exhaust of PEP due to the over-expression of SvPEPC in Arabidopsis thaliana.

Reconstitution of the extrinsic 23 and 17 kDa proteins with spinach PSII which had been exchanged for the 33 kDa protein from different plant species

The 33 kDa protein of photosystem II (PSII) plays an important role in maintaining the function of the Mn cluster, and is common to all of the organisms and totally exchangeable in binding to PSII from various different organisms (Enami et al., 2000, Plant Cell Physiol. 41, 1354-1364). The amino acid sequence of the 33 kDa protein showed a relatively high homology above 40% from cyanobacteria to higher plants. Thus, the structure of the 33 kDa protein has been considered to be conserved during evolution from cyanobacteria to higher plants. In this study, reconstitution experiments were performed for the extrinsic 23 and 17 kDa proteins from spinach with spinach PSII which had been exchanged for the 33 kDa protein from different sources. Spinach PSII was treated with 2.6 M urea/0.2 M NaCl to remove all of the extrinsic proteins, and then reconstituted with the 33 kDa protein from a cyanobacterium (Synechococcus vulcanus), a red alga (Cyanidium caldarium) or a higher plant (Spinach). The results showed that, while spinach PSII bearing the higher plant 33kDa protein retained the full ability of binidng the 23 kDa and 17 kDa proteins, PSII of which the 33 kDa protein had been replaced with the red algal or cyanobacterial 33kDa protein partially rebound the 23kDa protein but totally failed to bind the 17kDa protein. This suggests that the structure of the 33kDa protein interacting with other extrinsic proteins is different between higher plants and red algae or cyanobacteria.

Cross-reconstitution of extrinsic cytochrome c-550 and 12 kDa protein between cyanobacterial and red algal PSII

Red algal and cyanobacterial PSII contain two similar extrinsic proteins, namely, cytochrome c-550 and a 12 kDa protein. Their amino acid sequences showed a relatively high homology of about 40% between red algae and cyanobacteria. In this study, cross-reconstitution experiments of the extrinsic proteins with red algal (Cyanidium caldarium) or cyanobacterial (Synechococcus vulcanus) PSII were performed in order to compare the binding and functional properties of the extrinsic proteins between red algae and cyanobacteria. Red algal cytochrome c-550, as well as cyanobacterial cytochrome c-550, bound directly to cyanobacterial PSII. However, none of them bound directly to red algal PSII. This indicates that direct binding of cytochrome c-550 to PSII depends upon the structure of intrinsic proteins of PSII but not cytochrome c-550 itself. Red algal 12 kDa protein functionally bound to cyanobacterial PSII, while cyanobacterial 12 kDa protein did not bind to red algal PSII. The antibodies raised against cyanobacterial or red alga 12 kDa protein did not cross-react with red algal or cyanobacterial 12 kDa protein, respectively. These results imply that, while the structure and function of cytochrome c-550 were rather conserved, those of the 12 kDa protein have been changed during evolution from cyanobacteria to red algae.