Nitrite Transport in Chloroplast Inner Envelope Vesicles (I. Direct Measurement of Proton-Linked Transport)

Chloroplast inner envelope membrane vesicles that are loaded with the pH-sensitive fluorophore, pyranine, show rapid internal acidification when nitrite is added. Acidification is dependent upon [delta]pH, with the inside of vesicles being alkaline with respect to the outside. The rate of vesicle acidification was directly proportional to the concentration of nitrite that was added and the imposed pH difference across the membrane. In contrast, added nitrate had no effect on vesicle acidification. Nitrite also caused acidification of asolectin vesicles. The extent of vesicle acidification is dependent on the internal volume of vesicles. Inner envelope and asolectin vesicles that were prepared by extrusion were approximately the same size, allowing them to be compared when the final extent of acidification, measured after the pH gradient had collapsed, was similar. The rate of nitrite-dependent acidification was similar in these two preparations at any single nitrite concentration. These results indicate that nitrite movement occurs by rapid diffusion across membranes as nitrous acid, and this movement is dependent on a proton gradient across the lipid bilayer. Under conditions approximating those in vivo, the rate of diffusion of nitrous acid far exceeds that of nitrite reduction within chloroplasts.     

Altered photosynthesis,flowering, and fruiting in transgenic tomato plants that have an increased capacity for sucrose synthesis

Photosynthesis, leaf assimilate partitioning, flowering, and fruiting were examined in two lines of Lycopersicon esculentum Mill. transformed with a gene coding for sucrose-phosphate synthase (SPS) (EC 2.3.1.14) from Zea mays L. expressed from a tobacco ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit promoter. Plants were grown at either 35 or 65 Pa CO₂ and high light (1000 mol photons·m^–2·s^–1). Limiting and maximum SPS activities were significantly greater (up to 12 times) in the leaves of SPS-transformed lines for all treatments. Partitioning of carbon into sucrose increased 50% for the SPS transformants. Intact leaves of the control lines exhibited CO₂-insensitivity of photosynthesis at high CO₂ levels, whereas the SPS transformants did not exhibit CO₂-insensitivity. The O₂-sensitivity of photosynthesis was also greater for the SPS-transformed lines compared to the untransformed control when measured at 65 Pa CO₂. These data indicate that the SPS transformants had a reduced limitation on photosynthesis imposed by end-product synthesis. Growth at 65 Pa CO₂ resulted in reduced photosynthetic capacity for control lines but not for SPS-transformed lines. When grown at 65 Pa CO₂, SPS transformed lines had a 20% greater photosynthetic rate than controls when measured at 65 Pa CO₂ and a 35% greater rate when measured at 105 Pa CO₂. Photosynthetic rates were not different between lines when grown at 35 Pa CO₂. The time to 50% blossoming was reduced and the total number of inflorescences was significantly greater for the SPS transformants when grown at either 35 or 65 Pa CO₂. At 35 Pa CO₂, the total fruit number of the SPS transformants was up to 1.5 times that of the controls, the fruit matured earlier, and there was up to a 32% increase in total fruit dry weight. Fruit yield was not significantly different between the lines when grown at 65 Pa CO₂. Therefore, there was not a strict relationship between yield and leaf photosynthesis rate. Flowering and fruit development of the SPS-transformed lines grown at 35 Pa CO₂ showed similar trends to the controls grown at 65 Pa CO₂. Incidences of blossom-end rot were also reduced in the SPS-transformed lines. These data indicate that altering starch/sucrose partitioning by increasing the capacity for sucrose synthesis can affect acclimation to elevated CO₂ partial pressure and flowering and fruiting in tomato.Abbreviations DAS days after seeding - nptII neomycin phos-photransferase - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SPS sucrose-phosphate synthase - SSU Rubisco small subunit This research was supported by U.S. Department of Energy grant FG02-87ER13785. B.J.M. thanks the Natural Sciences and Engineering Research Council of Canada for financial support. We are grateful to Toni A. Voelker (Calgene Inc.) for supplying tomato seeds and valuable advice.     

Decrease in carbamylation of rubisco by high CO2 concentration is due to decrease of rubisco activase in kidney bean

Decrease in rubisco activation at high CO₂ concentration was caused by decrease in carbamylation of rubisco (Rohet al., 1996). However, it is unclear whether decrease in carbamylation rate at high CO₂ concentration is due to decrease in activity itself or content of rubisco activase. To clarify this ambiguity, investigation was performed to determine effects of CO₂ concentration on rubisco activase with kidney bean (Phaseolus vulgaris L.) leaves grown at normal CO₂ (350 ppm) and high CO₂ (650 ppm) concentration. The analysis of Western blotting showed that the 50 and 14.5 kl) polypeptides were identified immunochemically as the large and small subunits of rubisco in the preparation, respectively. For the 14.5 kD small subunit, the degree of intensity at high CO₂ concentration was similar to that at normal CO₂ concentration. For the 50 kD large sububit, however, the intensity of a band at high CO, concentration was significantly higher than that at normal CO₂ concentration, indicating that only the large subunit is affected by high CO₂ concentration. The analysis of Western immunoblotting showed two major polypeptides at 46 and 42 kD which were identified as rubisco activase subunits. The intensities of two bands were shown to be higher at normal CO₂ than high CO₂ concentration. These data indicate that decrease of carbamylation resulting from increase of CO₂ concentration was caused by rubisco activase. Finally, by employing ATP hydrolysis assay and ELISA, we also observed a significant decrease in both activity and content of rubisco activase as CO₂ concentration was raised from normal to high CO₂ concentration. These results suggest that decrease in rubisco carbamylation at high CO₂ concentration is caused by activity itself and/or content of rubisco activase.     

Soil pH and electrical conductivity are key edaphic factors shaping bacterial communities of greenhouse soils in Korea

Soil microorganisms play an essential role in soil ecosystem processes such as organic matter decomposition, nutrient cycling, and plant nutrient availability. The land use for greenhouse cultivation has been increasing continuously, which involves an intensive input of agricultural materials to enhance productivity; however, relatively little is known about bacterial communities in greenhouse soils. To assess the effects of environmental factors on the soil bacterial diversity and community composition, a total of 187 greenhouse soil samples collected across Korea were subjected to bacterial 16S rRNA gene pyrosequencing analysis. A total of 11,865 operational taxonomic units at a 97% similarity cutoff level were detected from 847,560 sequences. Among nine soil factors evaluated; pH, electrical conductivity (EC), exchangeable cations (Ca²⁺, Mg²⁺, Na⁺, and K⁺), available P₂O₅, organic matter, and NO₃-N, soil pH was most strongly correlated with bacterial richness (polynomial regression, pH: R² = 0.1683, P < 0.001) and diversity (pH: R² = 0.1765, P < 0.001). Community dissimilarities (Bray-Curtis distance) were positively correlated with Euclidean distance for pH and EC (Mantel test, pH: r = 0.2672, P < 0.001; EC: r = 0.1473, P < 0.001). Among dominant phyla (> 1%), the relative abundances of Proteobacteria, Gemmatimonadetes, Acidobacteria, Bacteroidetes, Chloroflexi, and Planctomycetes were also more strongly correlated with pH and EC values, compared with other soil cation contents, such as Ca²⁺, Mg²⁺, Na⁺, and K⁺. Our results suggest that, despite the heterogeneity of various environmental variables, the bacterial communities of the intensively cultivated greenhouse soils were particularly influenced by soil pH and EC. These findings therefore shed light on the soil microbial ecology of greenhouse cultivation, which should be helpful for devising effective management strategies to enhance soil microbial diversity and improving crop productivity.     

Metabolic engineering to produce γ-linolenic acid in <Emphasis Type="Italic">Brassica napus</Emphasis> using a Δ6-desaturase from pike eel

We generated γ-linolenic acid (GLA, C18:3^Δ6,9,12)-producing transgenic Brassica napus transformed with McD6DES, the Δ6-desaturase gene identified from pike eel (Muraenesox cinereus) under the control of the seed-specific vicilin promoter. Seed-specific expression of McD6DES in B. napus produced up to 8.4% of GLA by creating a double bond at the sixth position from the carboxyl end of linoleic acid (LA, C18:2^Δ9,12) in seeds. These results demonstrate that McD6DES expression enables to reconstitute in polyunsaturated fatty acid biosynthetic pathways, highlighting the potential of GLA biosynthesis as a target for metabolic engineering of oilseed crops.     

Carcinoembryonic antigen gene and carcinoembryonic antigen expression in the liver metastasis of colorectal carcinoma.

The presence of the carcinoembryonic antigen (CEA) gene and CEA expression in the liver was tested to identify their possible roles in the liver metastasis of colorectal carcinoma. The CEA gene in the liver was identified by amplifying the CEA-specific N-terminal domain exon with digoxigenin-dUTP labeling in 16 colorectal carcinomas with liver metastases. Next, CEA expression was tested by immunostaining using the anti-CEA monoclonal antibody (T84.66, ATCC). Liver tissues from 13 stomach cancer patients and 12 colorectal cancer patients without liver metastasis were also tested as control groups. Three grades (<25%, 25-50%, and 50%< or =) were given according to the proportion of positive cells. The CEA gene was amplified in the metastatic tumor cells of the liver (2.6 +/- 0.2, mean grade +/- SEM) and their surrounding hepatocytes (1.5 +/- 0.2) in all cases. CEA expression was found in all metastatic tumor cells and 14 cases of the surrounding hepatocytes. Among the control groups, the CEA gene of the hepatocytes was found in 9 cases each of the colorectal and the stomach cancers that did not exhibit CEA expression. The level of serum CEA was related with the numbers and volume of liver metastases, but not with CEA expression in tumor cells and surrounding hepatocytes. The CEA gene in the metastatic tumor cells, not in the hepatocytes, was closely associated with CEA expression in the surrounding hepatocytes (p<0.01). Although the precise mechanism of CEA gene regulation in hepatocytes remains to be proven, the CEA gene in the metastatic tumor of the liver seems to affect CEA expression in the surrounding hepatocytes facilitating liver metastasis in colorectal carcinoma.     

Rat liver 10-formyltetrahydrofolate dehydrogenase, carbamoyl phosphate synthetase 1 and betaine homocysteine S-methytransferase were co-purified on Kunitz-type soybean trypsin inhibitor-coupled sepharose CL-4B

An Asp/His catalytic site of 10-formyltetrahydrofolate dehydrogenase (FDH) was suggested to have a similar catalytic topology with the Asp/His catalytic site of serine proteases. Many studies supported the hypothesis that serine protease inhibitors can bind and modulate the activity of serine proteases by binding to the catalytic site of serine proteases. To explore the possibility that soybean trypsin inhibitor (SBTI) can recognize catalytic sites of FDH and can make a stable complex, we carried out an SBTI-affinity column by using rat liver homogenate. Surprisingly, the Rat FDH molecule with two typical liver proteins, carbamoyl-phosphate synthetase 1 (CPS1) and betaine homocysteine S-methyltransferase (BHMT) were co-purified to homogeneity on SBTI-coupled Sepharose and Sephacryl S-200 followed by Superdex 200 FPLC columns. These three liver-specific proteins make a protein complex with 300 kDa molecular mass on the gel-filtration column chromatography in vitro. Immuno-precipitation experiments by using anti-FDH and anti-SBTI antibodies also supported the fact that FDH binds to SBTI in vitro and in vivo. These results demonstrate that the catalytic site of rat FDH has a similar structure with those of serine proteases. Also, the SBTI-affinity column will be useful for the purification of rat liver proteins such as FDH, CPS1 and BHMT.     

Propagation of Bombyx mori Nucleopolyhedrovirus in nonpermissive insect cell lines

This study addresses the susceptibility of Spodoptera frugiperda (Sf9 and Sf21), Trichoplusia ni (Hi5), and S. exigua (Se301) cells to the Bombyx mori nucleopolyhedrovirus (BmNPV). Although these cells have classically been considered nonpermissive to BmNPV, the cytopathic effect, an increase in viral yield, and viral DNA synthesis by BmNPV were observed in Sf9, Sf21, and Hi5 cells, but not in Se301 cells. Very late gene expression by BmNPV in these cell lines was also detected via beta-galactosidase expression under the control of the polyhedrin promoter. Sf9 cells were most susceptible to BmNPV in all respects, followed by Sf21 and Hi5 cells in decreasing order, while the Se301 cells evidenced no distinct viral replication. This particular difference in viral susceptibility in each of the cell lines can be utilized for our understanding of the mechanisms underlying the host specificity of NPVs.     

Enumeration, isolation and identification of diazotrophs from Korean wetland rice varieties grown with long-term application of N and compost and their short-term inoculation effect on rice plants

AIM: This study has been aimed (i) to isolate and identify diazotrophs from Korean rice varieties; (ii) to examine the long-term effect of N and compost on the population dynamics of diazotrophs and (iii) to realize the shot-term inoculation effect of these diazotrophs on rice seedlings. METHODS AND RESULTS: Diazotrophic and heterotrophic bacterial numbers were enumerated by most probable number method and the isolates were identified based on morphological, physiological, biochemical and 16s rDNA sequence analysis. Long-term application of fertilizer N with compost enhanced both these numbers in rice plants and its environment. Bacteria were high in numbers when malate and azelaic acids were used as carbon source, but less when sucrose was used as a carbon substrate. The combined application promoted the association of diazotrophic bacteria like Azospirillum spp., Herbaspirillum spp., Burkholderia spp., Gluconacetobacter diazotrophicus and Pseudomonas spp. in wetland rice plants. Detection of nifD genes from different diazotrophic isolates indicated their nitrogen fixing ability. Inoculation of a representative isolate from each group onto rice seedlings of the variety IR 36 grown in test tubes indicated the positive effect of these diazotrophs on the growth of rice seedlings though the percentage of N present in the plants did not differ much. CONCLUSIONS: Application of compost with fertilizer N promoted the diazotrophic and heterotrophic bacterial numbers and their association with wetland rice and its environment. Compost application in high N fertilized fields would avert the reduction of N(2)-fixing bacterial numbers and their association was beneficial to the growth of rice plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibitory effect of high N fertilization on diazotrophic bacterial numbers could be reduced by the application of compost and this observation would encourage more usage of organic manure. This study has also thrown light on the wider geographic distribution of G. diazotrophicus with wetland rice in temperate region where sugarcane (from which this bacterium was first reported to be associating and thereon from other plant species) is not cultivated.