Jun-ichi Ohnishi

Reducing Sludge Production and the Domination of Comamonadaceae by Reducing the Oxygen Supply in the Wastewater Treatment Procedure of a Food-Processing Factory

Sludge production was reduced remarkably by reducing the dissolved oxygen supply to less than 1 mg/l in the conventional wastewater treatment procedure of a food-processing factory that produced 180 m3 of wastewater of biochemical oxygen demand (BOD) of about 1,000 mg/l daily. DNA was extracted from the sludge and subjected to PCR amplification. The PCR product was cloned into a plasmid and sequenced. Estimation of the resident bacterial distribution by 16S rDNA sequences before and after improvement of the system suggested a remarkable gradual change in the major bacterial population from Anaerolinaeceae (15.6%) to Comamonadaceae (52.3%), members of denitrifying bacteria of Proteobacteria. Although we did not directly confirm the ability of denitrification of the resulting sludge, a change in the major final electron acceptors from oxygen to nitrate might explain the reduction in sludge production in a conventional activated sludge process when the oxygen supply was limitted.

Transcriptional profiles of organellar metabolite transporters during induction of crassulacean acid metabolism in Mesembryanthemum crystallinum

Metabolite transport across multiple organellar compartments is essential for the operation of crassulacean acid metabolism (CAM). To investigate potential circadian regulation of inter-organellar metabolite transport processes, we have identified eight full-length cDNAs encoding an organellar triose phosphate / Pi translocator (McTPT1), a phosphoenolpyruvate / Pi translocator (McPPT1), two glucose-6-phosphate / Pi translocators (McGPT1, 2), two plastidic Pi translocator-like proteins (McPTL1, 2), two adenylate transporters (McANT1, 2), a dicarboxylate transporter (McDCT2), and a partial cDNA encoding a second dicarboxylate transporter (McDCT1) in the model CAM plant, Mesembryanthemum crystallinum L. We next investigated day / night changes in steady-state transcript abundance of each of these transporters in plants performing either C3 photosynthesis or CAM induced by salinity or water-deficit stress. We observed that the expression of both isogenes of the glucose-6-phosphate / Pi translocator (McGPT1, 2) was enhanced by CAM induction, with McGPT2 transcripts exhibiting much more pronounced diurnal changes in transcript abundance than McGPT1. Transcripts for McTPT1, McPPT1, and McDCT1 also exhibited more pronounced diurnal changes in abundance in the CAM mode relative to the C3 mode. McGPT2 and McDCT1 transcripts exhibited sustained oscillations for at least 3 d under constant light and temperature conditions suggesting their expression is under circadian clock control. McTPT1 and McGPT2 transcripts were preferentially expressed in leaf tissues in either C3 or CAM modes. The leaf-specific and / or circadian controlled gene expression patterns are consistent with McTPT1, McGPT2 and McDCT1 playing CAM-specific metabolite transport roles.

Na+-induced uptake of pyruvate into mesophyll chloroplasts of a C4 plant, Panicum miliaceum

Uptake of [1‐14C]pyruvate into the sorbitol‐impermeable space of mesophyll chloroplasts of a C4 plant, Panicum miliaceum L., was investigated using a silicone oil filtering centrifugation method. An abrupt change of Na+ concentration in the suspending medium of the chloroplasts in the dark induced accumulation of pyruvate in the stroma, similar to the case of light‐driven active uptake. The effect was specific to Na+ among various mono‐ and divalent cations. The apparent K m for Na+ was in the range 2–5 mM. The K m for pyruvate was about 0.7 mM, which was similar to the value obtained in light‐driven uptake. The possible role of Na+ symport in active pyruvate uptake by C4 mesophyll chloroplasts is discussed.

Pyruvate uptake induced by a pH jump in mesophyll chloroplasts of maize and sorghum, NADP-malic enzyme type C4 species

A sudden pH decrease (pH jump) of the medium enhanced pyruvate uptake in the dark in mesophyll chloroplasts (MCp) of Zea mays and Sorghum bicolor, NADP‐malic enzyme type C4 plants, while it was reported that a Na+ jump enhanced pyruvate uptake in MCp of P. miliaceum, a NAD‐malic enzyme type [(1987) FEBS Lett. 219, 347]. The enhancement effect of the pH jump decayed completely in 5 min and the decay was accelerated by proton gradient‐collapsing reagents. The results suggest that active pyruvate uptake into MCp of NADP‐malic enzyme type C4 species is primarily driven by the proton gradient across the envelope.

Glycerate Uptake into Mesophyll and Bundle Sheath Chloroplasts of a C4 Plant, Panicum miliaceum

Summary Glycerate uptake into mesophyll and bundle sheath chloroplasts of a C 4 plant, Panicum miliaceum L. was compared. Although the glycerate metabolizing enzyme, glycerate kinase, was undetectable in bundle sheath chloroplasts, they showed a similar time course of light-enhanced glycerate uptake as mesophyll chloroplasts which contained significant kinase activity. The uptake in light was partially inhibited by glycolate and enhanced by preincubation of the chloroplasts with glycolate. These results suggest the existence of a glycolate/glycerate translocator in both types of C 4 chloroplasts similar to that reported for C 3 chloroplasts.

Characterization of the plastidic phosphate translocators in the inducible crassulacean acid metabolism plant Mesembryanthemum crystallinum.

In plant Mesembryanthemum crystallinum, which has the inducible crassulacean acid metabolism (CAM), isoforms of plastidic phosphate translocators (pPTs) are categorized into three subfamilies: the triose phosphate/phosphate translocator (McTPT1), the phosphoenolpyruvate/phosphate translocator (McPPT1), and the glucose 6-phosphate/phosphate translocator (McGPT1 and McGPT2). In order to elucidate the physiological roles of these pPTs in M. crystallinum, we determined the substrate specificity of each pPT isoform. The substrate specificities of McTPT1, McPPT1, and McGPT1 showed overall similarities to those of orthologs that have been characterized. In contrast, for glucose 6-phosphate, McGPT2 showed higher selectivity than McGPT1 and other GPT orthologs. Because the expression of McGTP2 is specific to CAM while that of McGTP1 is constitutively expressed in both the C3- and the CAM-state in M. crystallinum, we propose that McGPT2 functions as a CAM system-specific GPT in this plant.

Characterization of alkali-soluble polysaccharides in deep subsoil layers

Abstract Plant cell wall polysaccharides undergo a slower degradation process in deep subsoil layers than in topsoil. Through the identification of organic compounds in subsoil, we may gain an understanding of this degradation process. In the present study, we extracted alkali-soluble polysaccharides from subsoil bore samples at depths of 5, 18, 29, 35 and 40–43 m, and performed sugar composition and sugar linkage analyses. Based on the results, we suggest that cellulose, arabinoxylan, mannan and pectic polysaccharides derived from plant cell walls and β-1,3-glucan and/or β-1,3:1,6-glucan from fungal cell walls exist in deep subsoil layers.

Isolation and Characterization of a Polyubiquitin Gene and Its Promoter Region from Mesembryanthemum crystallinum

Transcript levels of the polyubiquitin gene McUBI1 had been reported to be constant during Crassulacean acid metabolism (CAM) induction in the facultative CAM plant, Mesembryanthemum crystallinum. Here, we report the sequences of the full-length cDNA of McUBI1 and its promoter, and validation of the McUBI1 promoter as an internal control driving constitutive expression in transient assays using the dual-luciferase system to investigate the regulation of CAM-related gene expression. The McUBI1 promoter drove strong, constitutive expression during CAM induction. We compared the activities of this promoter with those of the cauliflower mosaic virus (CaMV) 35S promoter in detached C3- and CAM-performing M. crystallinum and tobacco leaves. We confirmed stable expression of the genes controlled by the McUBI1 promoter with far less variability than under the CaMV 35S promoter in M. crystallinum, whereas both promoters worked well in tobacco. We found the McUBI1 promoter more suitable than the CaMV 35S promoter as an internal control for transient expression assays in M. crystallinum.

Light-enhanced uptake of metabolites in mesophyll protoplasts: evidence for a novel pyruvate transport system.

3) and sorghum (C4) leaves for the measurements of osmotic volume change and metabolite uptake. We first investigated whether the silicone oil layer filtering centrifugation method could be applied to the protoplasts. The density of the silicone oil was optimized (ρ =1.026) and 0.5M betaine was chosen as an osmoticum in the protoplast suspending medium. By using [14C] sorbitol and [14C] inulin as the marker of the medium carried over into the pellet, protoplast osmotic or internal volume was estimated to be 200–300 μl (mg Chl)−1, with the medium space in the pellet of 8–15 μl (mg Chl)−1. Lowering of the osmotic pressure of the medium induced protoplast swelling as expected. Light also induced swelling. Using this system, we could detect light-enhanced uptake of ascorbate, glutamate and pyruvate in both barley and sorghum protoplasts. Pyruvate uptake was far higher in barley than in sorghum and inhibited by various inhibitors, showed saturation kinetics and, therefore, seemed to be mediated by a translocator protein.