Toshio Iwaki

A Bacterial Transgene for Catalase Protects Translation of D1 Protein during Exposure of Salt-Stressed Tobacco Leaves to Strong Light

During photoinhibition of photosystem II (PSII) in cyanobacteria, salt stress inhibits the repair of photodamaged PSII and, in particular, the synthesis of the D1 protein (D1). We investigated the effects of salt stress on the repair of PSII and the synthesis of D1 in wild-type tobacco (Nicotiana tabacum ‘Xanthi’) and in transformed plants that harbored the katE gene for catalase from Escherichia coli. Salt stress due to NaCl enhanced the photoinhibition of PSII in leaf discs from both wild-type and katE-transformed plants, but the effect of salt stress was less significant in the transformed plants than in wild-type plants. In the presence of lincomycin, which inhibits protein synthesis in chloroplasts, the activity of PSII decreased rapidly and at similar rates in both types of leaf disc during photoinhibition, and the observation suggests that repair of PSII was protected by the transgene-coded enzyme. Incorporation of [35S]methionine into D1 during photoinhibition was inhibited by salt stress, and the transformation mitigated this inhibitory effect. Northern blotting revealed that the level of psbA transcripts was not significantly affected by salt stress or by the transformation. Our results suggest that salt stress enhanced photoinhibition by inhibiting repair of PSII and that the katE transgene increased the resistance of the chloroplasts translational machinery to salt stress by scavenging hydrogen peroxide.

Change of Ascorbic Acid Level after Grafting of Tomato Seedlings

Grafting is an easy way to produce a new seedling, which can tolerate against various stresses. During the acclimation after grafting, however, the seedlings still suffer a severe water stress. It is well known that water stress produces active oxygen to oxidize ascorbic acid. The concentration of ascorbic acid in the leaves was analyzed by HPLC equipped with an electrochemical detector. The column used was SP-120-5-ODS-BP (DAISO, JAPAN) and elution was performed with 0.1 ᴍ phosphate buffer, pH 3.0. After grafting the seedlings were acclimated under a 6-hr light/dark regimen. The content of ascorbic acid increased gradually during 2 days compared with control. The ascorbate peroxidase showed about constant activity, so the increase of ascorbic acid may be due to its requirement to cure the grafting

REACTION MECHANISM OF PHOSPHORIBULOKINASE FROM A CYANOBACTERIUM, SYNECHOCOCCUS PCC7942

The dependence of the activity of phosphoribulokinase isolated from a cyanobacterium, Synechococcus PCC7942, on Mg2+ showed that its real substrates were Mg-ATP and free D-ribulose 5-phosphate. On the basis of results of kinetic inhibition studies and previously reported result of affinity chromatography, an ordered bi bi mechanism in which Mg-ATP binds before ribulose 5-phosphate is proposed. The Km values for ATP and D-ribulose 5-phosphate were 0.09 and 0.27 mM, respectively. Ki values of ADP and D-ribulose 1,5-bisphosphate were 0.32 and 10.0 mM, respectively. Inhibition constants Ki1 and Ki2 for 6-phosphogluconate were 9.3 and 0.49 mM. Kia was 0.13 mM. New kinetics on PRK gave higher control coefficient than the kinetics on Spinach PRK did in the model with PRK activity from 175 to 1000 µmol min−1 mg−1 chl.

Regulation of the expression of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) in a cyanobacterium, Synechococcus PCC7942

When cyanobacterium cells are grown under extremely low CO2 concentration, the number of carboxysomes, structures containing ribulose-bisphosphate carboxylase (Rubisco; EC 4.1.1.39), is known to increase. This suggests that Rubisco helps to regulate photosynthesis in cyanobacteria. However, no studies have been done on the changes of Rubisco content and activity in response to the extracellular CO2 concentration, and no information is available on its effect on photosynthesis. To elucidate the relationship between the expression responses of Rubisco and extracellular CO2, wild-type cells (Synechococcus PCC7942) and carboxysome-lacking cells were grown under various CO2 concentrations, and Rubisco activity was determined. In both strains, Rubisco activity increased when the cells were grown under a CO2 concentration around, or less than, K1/2(CO2) of photosynthesis. In carboxysome-lacking cells, Rubisco activity increased five to six times at most, and a simultaneous increase in the rate of photosynthesis was observed. These results suggest that stimulation of expression of Rubisco occurs to compensate for the decrease in the rate of photosynthesis under CO2-limited conditions.

Inhibition of RuBisCO cloned from Thermosynechococcus vulcanus and expressed in Escherichia coli with compounds predicted by Molecular Operation Environment (MOE).

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) of a thermophilic cyanobacterium, Thermosynechococcus vulcanus, was cloned and expressed in Escherichia coli. The purified enzyme had higher thermostability than RuBisCOs isolated from mesophilic cyanobacteria. Prediction of the tertiary structure was performed using the software Molecular Operating Environment (MOE). The predicted structure did not give any clue about the basis of thermostability. Then, the molecular docking of substrates and inhibitors in the catalytic site were carried out to test analogs for consistency of ribulose 1,5-bisphosphate, a RuBisCO substrate. The analogs were searched in the Kyoto Encyclopedia of Genes and Genomes (KEGG), and 99 compounds were selected for the docking. The mol files from LIGAND Database in KEGG were changed to a three dimensional (3D) structure for use in docking simulation. The docking simulation was performed on ASEDock of MOE, and the SiteFinder command suggested about 20 candidates for the docking site of the compounds. Based on the homology of these candidate sites with the xylulose 1,5-bisphosphate (XBP)-binding site of RuBisCO isolated from Synechococcus PCC 6301, one site was selected for the docking simulation. The 40 compounds with the highest docking energies included synthetic organic substances that had never been demonstrated to be inhibitors of RuBisCO. The total docking energies were -102 kcal/mol, -104 kcal/mol, -94.0 kcal/mol, and -57.7 kcal/mol for ribulose 1,5-bisphosphate (RuBP), etidronate, risedronate, and citrate respectively. Kinetic analysis of RuBisCO revealed a K(m) value of 315 microM for RuBP, and K(i) values of 1.70, 0.93, and 2.04 mM for etidronate, risedronate, and citrate respectively. From these values, the binding energies were estimated to be -4.85, -3.84, -4.20, and -3.73 kcal/mol for RuBP, etidronate, risedronate, and citrate respectively. The differences between the values estimated from experimental data and by simulation may mainly depend on the dissimilarity of the environment for the protein and ligands between the experiments and the simulation. The results obtained here suggested a few new inhibitors, which might be useful as tools for studying the relationship between the structure and the function of RuBisCO.

Modification of CO2 fixation of photosynthetic prokaryote.

Resemblances and differences on photosynthesis of C3 plant and cyanobacterium Synechococcus PCC7942 were discussed with a simplified model of photosynthesis and photorespiration. Then the transformation of the cyanobacterium was shown with Chromatium vinosum (Cv) RuBisCO. Total RuBisCO activity and photosynthesis were compared with wild type S. 7942 and RuBisCO high expression mutant. Results were explained with RuBisCO environment of S. 7942 mutant. For excluding the influence of inorganic carbon concentrating mechanism (CCM) on the effect of RuBisCO, carboxysome less mutant was created with homologous recombination of ccm operon to be replaced with kanamycine resistant gene. And the deletion of the operon was confirmed by genomic PCR on the transformant. The confirmation was performed with three primers. Electron micrograms of S. 7942 show the existence of carboxysome in wild type and its absence in the mutant. Growth rate of CL mutant on different CO2 concentration shows that there is a possibility to increase a photosynthetic ability of a cyanobacterium with increases the RuBisCO activity.

Expression of Chromatium vinosum rubisco in the carboxysome deficient mutant of cyanobacterium Synechococcus PCC7942

The control coefficient is an important index to show how an enzyme regulates a metabolic pathway. On higher plants, antisence technique decrease RuBisCO protein for the elucidation of a regulatory role of the enzyme in photosynthetic inorganic carbon anabolism (1). The technique can not be applicable to cyanobacteria because of their high possibility of homologous recombination. On the other hand, the control coefficient could be estimated dependent on the increase of the enzyme concerned. As a trial for the estimation, RuBisCO protein of purple sulfur photosynthetic bacterium Chromatium vinosum was expressed in the cyanobacterium Synechococcus PCC7942 by the transformation with bidirectional expression vector. In this transformant MT1, we confirmed that introduced expression vector replicated stably And C. vinosum RuBisCO was expressed in the cytosol in active L8S8 form. But the expression level of C. vinosum RuBisCO was around 30% of total RuBisCO activity. The low expression level makes it difficult to analyze the effect of the foreign enzyme on the photosynthetic physiology of the transformant. Thus, in this paper, we try to increase its expression level after checking promoters derived from cyanobacterium and E. coli with luciferase as a reporter.