A plastidial sodium-dependent pyruvate transporter.

Pyruvate serves as a metabolic precursor for many plastid-localized biosynthetic pathways, such as those for fatty acids, terpenoids and branched-chain amino acids. In spite of the importance of pyruvate uptake into plastids (organelles within cells of plants and algae), the molecular mechanisms of this uptake have not yet been explored. This is mainly because pyruvate is a relatively small compound that is able to passively permeate lipid bilayers, which precludes accurate measurement of pyruvate transport activity in reconstituted liposomes. Using differential transcriptome analyses of C(3) and C(4) plants of the genera Flaveria and Cleome, here we have identified a novel gene that is abundant in C(4) species, named BASS2 (BILE ACID:SODIUM SYMPORTER FAMILY PROTEIN 2). The BASS2 protein is localized at the chloroplast envelope membrane, and is highly abundant in C(4) plants that have the sodium-dependent pyruvate transporter. Recombinant BASS2 shows sodium-dependent pyruvate uptake activity. Sodium influx is balanced by a sodium:proton antiporter (NHD1), which was mimicked in recombinant Escherichia coli cells expressing both BASS2 and NHD1. Arabidopsis thaliana bass2 mutants lack pyruvate uptake into chloroplasts, which affects plastid-localized isopentenyl diphosphate synthesis, as evidenced by increased sensitivity of such mutants to mevastatin, an inhibitor of cytosolic isopentenyl diphosphate biosynthesis. We thus provide molecular evidence for a sodium-coupled metabolite transporter in plastid envelopes. Orthologues of BASS2 can be detected in all the genomes of land plants that have been characterized so far, thus indicating the widespread importance of sodium-coupled pyruvate import into plastids.

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.

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.

Structure and immunocytochemical localization of photosynthetic enzymes in the lamina joint and sheath pulvinus of the C4 grass Arundinella hirta.

The C(4) grass Arundinella hirta exhibits a unique C(4) anatomy, with isolated Kranz cells (distinctive cells) and C(4)-type expression of photosynthetic enzymes in the leaf sheath and stem as well as in the leaf blade. The border zones between these organs are pale green. Those between the leaf blade and sheath and between the sheath and stem are called the lamina joint and sheath pulvinus, respectively, and are involved in gravity sensing. We investigated the structure and localization of C(3) and C(4) photosynthetic enzymes in these tissues. In both zones the epidermis lacked stomata. The inner tissue was composed of parenchyma cells and vascular bundles. The parenchyma cells were densely packed with small intercellular spaces and contained granal chloroplasts with large starch grains. No C(4)-type cellular differentiation was recognized. Western blot analysis showed that the lamina joint and pulvinus accumulated substantial amounts of phosphoenolpyruvate carboxylase (PEPC), pyruvate,Pi dikinase (PPDK), and ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco). Immunogold electron microscopy revealed PEPC in the cytosol and both PPDK and rubisco in the chloroplasts of parenchyma cells, suggesting the occurrence of C(3) and C(4) enzymes within a single type of chlorenchyma cell. These data indicate that the lamina joint and pulvinus have unique expression patterns of C(3) and C(4) enzymes, unlike those in C(4)-type anatomy.

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 m(3) 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.

Structure and enzyme expression in photosynthetic organs of the atypical C4 grass Arundinella hirta.

In its leaf blade, Arundinella hirta has unusual Kranz cells that lie distant from the veins (distinctive cells; DCs), in addition to the usual Kranz units composed of concentric layers of mesophyll cells (MCs) and bundle sheath cells (BSCs; usual Kranz cells) surrounding the veins. We examined whether chlorophyllous organs other than leaf blades--namely, the leaf sheath, stem, scale leaf, and constituents of the spike--also have this unique anatomy and the C4 pattern of expression of photosynthetic enzymes. All the organs developed DCs to varying degrees, as well as BSCs. The stem, rachilla, and pedicel had C4-type anatomy with frequent occurrence of DCs, as in the leaf blade. The leaf sheath, glume, and scale leaf had a modified C4 anatomy with MCs more than two cells distant from the Kranz cells; DCs were relatively rare. An immunocytochemical study of C3 and C4 enzymes revealed that all the organs exhibited essentially the same C4 pattern of expression as in the leaf blade. In the scale leaf, however, intense expression of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) occurred in the MCs as well as in the BSCs and DCs. In the leaf sheath, the distant MCs also expressed Rubisco. In Arundinella hirta, it seems that the ratio of MC to Kranz cell volumes, and the distance from the Kranz cells, but not from the veins, affects the cellular expression of photosynthetic enzymes. We suggest that the main role of DCs is to keep a constant quantitative balance between the MCs and Kranz cells, which is a prerequisite for effective C4 pathway operation.

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.

Photosynthetic enzyme accumulation during leaf development of Arundinella hirta, a C4 grass having Kranz cells not associated with veins.

The leaf of the NADP-malic enzyme type C(4) grass, Arundinella hirta, has not only mesophyll cells (MCs) and bundle sheath cells (BSCs, usual Kranz cells) but also another type of Kranz cells (distinctive cells; DCs) that are not associated with vascular bundles. We investigated photosynthetic enzyme accumulation along the base-to-tip maturation gradient of developing leaves by immunogold electron microscopy. In mature leaves, phosphoenolpyruvate carboxylase (PEPC) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) were detected in the MC cytosol and in the BSC and DC chloroplasts, respectively. Pyruvate, P(i) dikinase (PPDK) was present in the chloroplasts of all photosynthetic cells but with higher levels in the MCs. Rubisco was first detected in the basal region of emerging leaf blades where the BSCs and DCs became discernable. Subsequently, the accumulation of PEPC and PPDK was initiated in the region where the granal proliferation in the chloroplasts was conspicuous; and, suberized lamellae were formed in the cell walls of the Kranz cells. There was no difference in the patterns of cellular development and enzyme accumulation between the BSCs and DCs or between the MCs adjacent to each type of Kranz cells. These results demonstrate that, although the DCs are not associated with veins, they behaved like BSCs with respect to enzyme induction and cellular differentiation.

Development of the Simulation Model InPest for Prediction of the Indoor Behavior of Pesticides.

The objective was to develop a computer software package (to be registered as InPest) that runs under Microsoft Excel on a personal computer to help in the risk assessment of indoor-use pesticides for both applicators and indoor occupants for various methods of application including space spraying, electric vaporizing, broadcast spraying, and residual spraying. For space spraying, the movement of the pesticide in a sprayed room including droplet settlement, permeation into the floor, degradation, transference, and discharge by ventilation were described as precisely as possible by various physicochemi-cal equations. The equations thus obtained were then incorporated into the Fugacity model (Level IV). When pesticide information regarding molecular weight, vapor pressure, water solubility, and octanol/water partition coefficient is available, InPest is able to simulate the time-dependent concentrations of the pesticide in the air and residual amounts on floor, wall, and ceiling materials under various conditions. Simulation data indicate that the predicted behavior of pesticides fully agrees with the measured data. Based on the predicted concentrations in the air and amounts of residue on the floor, the levels of exposure to room occupants via inhalation, dermal, or oral intake can be computed and compared with the mammalian toxicological data. Thus, InPest is a powerful tool for evaluating the safety of indoor-use pesticides with regard to human health.