Characterization of acetolactate synthase from sulfonylurea herbicide‐resistant Schoenoplectus juncoides

Ten accessions of sulfonylurea‐resistant Schoenoplectus juncoides were collected from paddy fields in Japan. In order to characterize acetolactate synthase from sulfonylurea‐resistant S. juncoides, acetolactate synthase amino acid substitutions, whole‐plant growth inhibition and acetolactate synthase enzyme inhibition were examined. Schoenoplectus juncoides has two acetolactate synthase genes (ALS1 and ALS2). The sulfonylurea‐resistant accessions harbored amino acid substitutions at Pro197 or Trp574 in either ALS1 or ALS2 (the amino acid number is standardized to the Arabidopsis thaliana sequence). The whole plants of all the sulfonylurea‐resistant accessions showed resistance to imazosulfuron. The resistance level depended on the altered amino acid residues in acetolactate synthase. The acetolactate synthase enzyme that was partially purified from all the sulfonylurea‐resistant accessions was less sensitive to imazosulfuron, compared to the susceptible accession, suggesting that the resistance is related to the altered acetolactate synthase enzyme. In addition, the concentration–response inhibition of acetolactate synthase activity by imazosulfuron in the sulfonylurea‐resistant accessions was remarkably different with the presence of an amino acid substitution in either ALS1 or ALS2. Furthermore, the concentration–response inhibition of acetolactate synthase activity in the sulfonylurea‐resistant accessions with a P197S, P197T or W574L mutation showed a double‐sigmoid curve. The regression analysis of enzyme inhibition suggested that the abundance ratio of ALS1 to ALS2 enzymes was approximately 70:30%, with a range of ±15%. Taken together, these results suggest that the resistance of sulfonylurea‐resistant accessions of S. juncoides is related to altered acetolactate synthase in either ALS1 or ALS2, although the abundance of the altered acetolactate synthase in the plants is different among the sulfonylurea‐resistant accessions.     

Effect of activation treatments of recipient oocytes on subsequent development of bovine nuclear transfer embryos

Effects of recipient oocyte activation methods on the development of nuclear transfer (NT) embryos were investigated. In Exp. 1, cell-cycle phase of serum-starved bovine cumulus cells was examined by flow cytometry. Majority (95.5%) of medium-sized (16-20 microm) cells that made up 56% of total cells was at the G0/G1 phase. NT embryos were constructed by electric fusion with the medium-sized serum-starved cumulus cells and bovine oocytes of 3 different preparations: enucleated oocytes treated with calcium ionophore A 23187 for 5 min and cycloheximide for 5 hr (A 23187/CHX), those treated with ethanol for 7 min and cycloheximide for 2 hr(ethanol/CHX) and those without treatment. In Exp. 2 and 3, developmental competence of NT embryos constructed with A 23187/CHX- and ethanol/CHX-treated oocytes was compared to that of NT embryos constructed with non-treated oocytes, respectively. Further, nuclear behavior in 3 different NT embryos was examined in Exp. 4. Within 1 hr after fusion, majority of the NT embryos constructed with non-treated oocytes showed condensed chromosome. Three hours after fusion, about 50% of NT embryos constructed with non-treated or ethanol/CHX-treated oocytes showed a single pronucleus-like structure. NT embryos constructed with ethanol/CHX-treated oocytes showed similar rates of fusion, cleavage and blastocyst formation to those of the non-treated oocytes. In contrast, NT embryos constructed with A 23187/CHX-treated oocytes did not show any pronucleus-like structure and showed lower cleavage rate and no development to blastocysts. The results indicate that ethanol/CHX-treated oocytes could support development of somatic cell NT embryos to the blastocyst stage at a similar rate to that of non-treated oocytes.     

In vitro culture of mouse preantral follicles using membrane inserts and developmental competence of in vitro ovulated oocytes

To develop a reliable follicle culture system, mouse preantral follicles 150-200 microm in diameter were cultured individually for 5 or 6 days in membrane inserts or in droplets, and then induced to ovulate with hCG (Experiment 1). The nuclear maturation and developmental competence of the oocytes that ovulated from the follicles cultured in inserts were determined (Experiment 2). There was no significant difference between the two culture systems in the survival rate (83 and 77%). However, follicles cultured in inserts showed a higher ovulation rate (63%) than those cultured in droplets (39%, P<0.05). About 80% of the oocytes that ovulated from the follicles cultured in inserts were at the metaphase II stage. After in vitro fertilization, 75 and 48% of in vitro ovulated oocytes cleaved and developed into blastocysts, respectively. These results demonstrate that the insert culture system is superior to the droplet culture system in terms of follicular growth and ovulation, and can be used to investigate the growth and ovulation of follicles in vitro.     

Expression of <Emphasis Type="Italic">Xanthomonas oryzae </Emphasis>pv. <Emphasis Type="Italic">oryzae hrp</Emphasis> Genes in XOM2, a Novel Synthetic Medium

To analyze the regulation of hrp expression and to detect and identify hrp-dependent secretion proteins of plant-pathogenic bacteria, an appropriate hrp-inducing medium is indispensable. In this study, two efficient hrp-inducing media for Xanthomonas oryzae pv. oryzae were designed by assaying the expression of a hrcU (the first gene of the hrpC operon) and a gus (β-glucuronidase) fusion gene. We modified XVM2, which is a hrp-inducing medium for X. campestris pv. vesicatoria, by adding 0.01% xylose in place of fructose and sucrose (0.18 and 0.34%, respectively) as a sugar source. The resulting medium induced approximately 15-fold more GUS activity from transformants containing a hrcU::gus gene than did XVM2. Moreover, a methionine-containing synthetic medium with 0.18% xylose as a sugar source was able to induce much stronger expression of HrcU::GUS, with GUS activity approximately 100-fold greater than that in XVM2. Induction depended on a regulator, HrpXo, and the PIP (plant-inducible-promoter) box, suggesting that HrcU::GUS was expressed in a hrp-dependent manner. The induction of operons hrpA to hrpF in XOM2 was also confirmed. These results suggest that both media, especially XOM2, are highly efficient hrp-inducing media for X. oryzae pv. oryzae. Received 7 October 2002/ Accepted in revised form 22 November 2002     

Seasonal foraging patterns of forest‐grazing Japanese Black heifers with increased plasma total antioxidant capacity

Forest‐grazing enables the intake of high total antioxidant capacity (TAC) plants that might be beneficial for the TAC status of cattle. This study evaluated the relation between the seasonal foraging patterns of forest‐grazing Japanese Black (JB) heifers or the TAC levels in shrubs and trees and the changes of plasma TAC. We examined 12 JB heifers, four each of which were allocated to forest‐grazing (F), pasture‐grazing, and pen‐housed groups. The plasma TAC level in F heifers on July 26, August 13, 30 and September 17 were significantly higher than those on April 27 and June 4 (P < 0.05). In F group, the mean rates of foraging frequency (FF) of shrubs and trees during July 5–8 and September 13–16 were much higher than that during May 31–June 3 (P < 0.05). The rate of FF of grass significantly decreased later in the season (P < 0.05). The mean TAC levels in these shrubs and trees were higher than those in grasses, concentrates, and timothy hay. Results suggest that an important factor in the increase of plasma TAC in forest‐grazing cattle might be the increased foraging of TAC‐rich shrubs and trees during summer–fall.     

Effect of increased concentrate allotment before evening grazing on herbage intake,nitrogen utilization and rumen fermentation in dairy cows grazed on perennial ryegrass pasture

Two experiments were conducted to elucidate the effect of increased concentrate allotment before evening grazing on herbage intake, nitrogen utilization and rumen fermentation in dairy cows. In experiment 1, nine lactating cows were grazed in the morning and evening sessions (2.5 h each). The cows were allocated to treatments of three concentrate allotment levels before the evening grazing session by altering proportions to daily total offered; 25%, 50% and 75%. Daily herbage dry matter intake quadratically decreased with increasing the concentrate allotment levels (P < 0.05). Nitrogen utilization was similar among treatments. To investigate diurnal changes in rumen fermentation, a second experiment was conducted where six ruminally cannulated non‐lactating dairy cows grazed in the morning and evening sessions (3 h each) were subjected to the same treatments as experiment 1. Total volatile fatty acid concentration in the rumen linearly increased with increasing the concentrate allotment levels throughout the pre‐evening grazing session to the post‐morning grazing session (P < 0.01). The results indicate that dairy cows reduce daily herbage intake but do not alter nitrogen utilization with increasing concentrate allotment before evening grazing. © 2016 Japanese Society of Animal Science     

Evaluation of serum amyloid A and surfactant protein D in sera for identification of the clinical condition of horses with bacterial pneumonia

In the present study, the concentrations of serum amyloid A and surfactant protein D in sera were measured to evaluate them for identification of the clinical condition of horses with bacterial pneumonia. The study utilized 185 clinically healthy control thoroughbreds and 9 thoroughbreds for experimental infectious study with S. zooepidemicus. Blood samples were collected from the 185 healthy control thoroughbreds. The 9 thoroughbreds were experimentally infected S. zooepidemicus using an endoscopic injection to a lung lobe and were then observed of clinical conditions. Blood samples were collected before inoculation and on the 1-15th, 22nd, and 29th days after inoculation (follow-up group). The levels of SAA and SP-D in the healthy control thoroughbreds were very low. In the follow-up group, the levels of SAA and SP-D changed in parallel with the horses' clinical condition. The pyrexia observed after bacterial inoculation faded by the 11th day, and the changes in SAA and SP-D occurred simultaneous to disappearance of the clinical signs. Measurement of SAA and SP-D proved useful for monitoring the clinical condition of the horses with bacterial pneumonia. Changes in the SP-D value were preceded by changes in the SAA value. Since the changes in SP-D occurred approximately simultaneous to the changes in the horses' clinical signs, we believe that they reflect the condition of the alveolar membranes. We conclude that measurement of SAA and SP-D in sera is useful for identification of the clinical condition of horses with bacterial pneumonia.     

Differences in CO2 and N2O emission rates following crop residue incorporation with or without field burning: A case study of adzuki bean residue and wheat straw

In the context of sustainable soil-quality management and mitigating global warming, the impacts of incorporating raw or field-burned adzuki bean (Vigna angularis (Willd.) Ohwi & Ohashi) and wheat (Triticum aestivum L.) straw residues on carbon dioxide (CO₂) and nitrous oxide (N₂O) emission rates from soil were assessed in an Andosol field in northern Japan. Losses of carbon (C) and nitrogen (N) in residue biomass during field burning were much greater from adzuki bean residue (98.6% of C and 98.1% of N) than from wheat straw (85.3% and 75.3%, respectively). Although we noted considerable inputs of carbon (499 ± 119 kg C ha^–1) and nitrogen (5.97 ± 0.76 kg N ha^–1) from burned wheat straw into the soil, neither CO₂ nor N₂O emission rates from soil (over 210 d) increased significantly after the incorporation of field-burned wheat straw. Thus, the field-burned wheat straw contained organic carbon fractions that were more resistant to decomposition in soil in comparison with the unburned wheat straw. Our results and previously reported rates of CO₂, methane (CH₄) and N₂O emission during wheat straw burning showed that CO₂-equivalent greenhouse gas emissions under raw residue incorporation were similar to or slightly higher than those under burned residue incorporation when emission rates were assessed during residue burning and after subsequent soil incorporation.     

Incorporation of fallow weed increases phosphorus availability in a farmer’s organic rice fields on allophanic Andosol in eastern Japan

ABSTRACT

We investigated the amount of soil phosphorus (P) in a farmer’s paddy fields under organic farming (OF) for various periods from 0 to 22 years as well as other farmers’ fields under conventional farming. All the fields are located in allophanic Andosol with long history of P fertilizer application, and some of them have been converted to OF across years. After conversion to OF, P was supplied only with winter fallow weeds mainly Foxtail (Alopecurus aequalis), rice residues (rice bran and straw), and guano. We determined total-P (Tot-P) and plant-available P (Av-P), which consists of Truog-P (Tru-P) and Bray-2-P under reducing condition with ascorbic acid (Asc-P), in soils of each field. For both Av-Ps, the ratio to Tot-P increased across years under OF following quadratic functions with both linear and quadratic terms being statistically significant. The ratios showed little changes for the initial 15 (Tru-P) or 10 (Asc-P) years and increased rapidly thereafter. These temporal changes in Av-P were consistent with the rapid increase of the amount of P accumulated in the winter fallow weeds and incorporated in the fields after beginning of OF. These results led us to the hypothesis that the incorporation of winter weeds has contributed to the increase of Av-P in the organic fields across years. We tested this hypothesis by investigating temporal changes of Av-P after suspending the weed incorporation for 2 consecutive years in plots of the organic fields. Both Av-Ps were significantly greater in plots with continued weed incorporation (CWI) than those in plots with its suspension. We further found that the increase of Asc-P in plots with CWI was 4.9-fold the input of total P in the incorporated weeds. This suggests that the incorporation of winter fallow weeds enhanced soil-P availability beyond the supply of P accumulated in the weeds.