Isolation and expression of genes for acetolactate synthase and acetyl-CoA carboxylase in Echinochloa phyllopogon, a polyploid weed species

BACKGROUND: Target‐site resistance is the major cause of herbicide resistance to acetolactate synthase (ALS)‐ and acetyl‐CoA carboxylase (ACCase)‐inhibiting herbicides in arable weeds, whereas non‐target‐site resistance is rarely reported. In the Echinochloa phyllopogon biotypes resistant to these herbicides, target‐site resistance has not been reported, and non‐target‐site resistance is assumed to be the basis for resistance. To explore why target‐site resistance had not occurred, the target‐site genes for these herbicides were isolated from E. phyllopogon, and their expression levels in a resistant biotype were determined. RESULTS: Two complete ALS genes and the carboxyltransferase domain of four ACCase genes were isolated. The expression levels of ALS and ACCase genes were higher in organs containing metabolically active meristems, except for ACC4, which was not expressed in any organ. The differential expression among examined organs was more prominent for ALS2 and ACC2 and less evident for ALS1, ACC1 and ACC3. CONCLUSION: E. phyllopogon has multiple copies of the ALS and ACCase genes, and different expression patterns were observed among the copies. The existence of three active ACCase genes and the difference in their relative expression levels could influence the occurrence of target‐site resistance to ACCase inhibitors in E. phyllopogon. Copyright © 2012 Society of Chemical Industry     

Bolting resistant breeding of Chinese cabbage. 1. Flower induction of late bolting variety without chilling treatment

Summary When a local slow bolting variety Osaka Shirona Bansei (Brassica rapa L. ssp. pekinensis, syn. B. campestris L. ssp. pekinensis) was grown in a phytotron (25°C, 16 hours day length without chilling treatment), one third of the plants bolted and flowered. In order to clarify the different flowering responses in the variety, a progeny line (FNC) of the flowering plants was chilled for 4 different periods (0, 22, 36 and 53 days) in a chamber of 2 7°C, then transplanted to three different conditions, i.e. PHY: 25–20°C day and night temperatures, 16 hours day length, GHL: 10 25°C, 16 hours day length with supplementary light and GHN: 10 25°C, natural day length (10 15 hours). In PHY, FNC bolted and flowered with almost the same leaf numbers in all 4 different chilling treatments. This means that FNC has very low sensitivity and no requirement to low temperature for its reproductive growth. In GHN (short day length), FNC bolted very slowly. Then the bolting and flowering of FNC were promoted by both long day length and high temperature. The newly found bolting characteristics of Osaka Shirona Bansei could be applied to breed unique slow bolting Chinese cabbage (B. rapa L. ssp. pekinensis) which might be non-sensitive to low temperature and its bolting and flowering would be induced with the combination of long day length and high temperature. Using the unique variety, it might be also possible to establish a new cropping type of Chinese cabbage (late autumn sowing, spring harvest).Abbreviations PHY Phytotron, long day and high temperature condition - GHL Greenhouse, long day and medium temperature condition - GHN Greenhouse, natural (short) day and medium temperature condition - FNC A progeny line of Osaka Shirona Bansei which flowered with no chilling treatment - FC A progeny line of Osaka Shirona Bansei which flowered with chilling treatment     

Amiprophosmethyl-induced efficient in vitro production of polyploids in raphanobrassica with the aid of aminoethoxyvinylglycine (AVG) in the culture medium

Optimum conditions for obtaining tetraploid were investigated in raphanobrassica, the intergeneric hybrid between radish (Raphanus sativus) and kale (Brassica oleracea var. acephala) by treating in vitro plants with an anti-mitotic agent, amiprophosmethyl (APM). Initially, no tetraploids but hexaploids and octaploids were induced by the treatments. Although the leaves of these polyploids of raphanobrassica showed chlorosis during subcultures in in vitro conditions, the chlorosis could be successfully prevented by the ethylene inhibitors, both AVG and AgNO₃. Based on this result, AVG was added into medium used for the culture after the chromosome doubling treatment, which subsequently resulted in increased survival rates of the treated plant materials as well as increased production rates of polyploids including tetraploid. These polyploid plants showed obviously different characters from the original diploid plant. The tetraploid plant had bigger sizes in shoot, flower and leaf, and more number of leaves than the diploid. On the other hand, the hexaploid and octaploid plants had smaller sizes in shoots and leaves, and less number of leaves than the diploid. Concentration of glucosinolates, functional substances of Brassicaceae crops, did not significantly differ between diploid and tetraploid of raphanobrassica, but reduced in hexaploid and octaploid.     

Biochemical properties of the cell wall in the Arabidopsis mutant bor1‐1 in relation to boron nutrition

We examined concentrations of boron (B) and dimerization of rhamnogalacturonan II (RG‐II), a B‐binding polysaccharide, in the cell wall of a low‐B sensitive mutant of Arabidopsis thaliana, bor1‐1, to investigate possible effects of the bor1‐1 mutation on the biochemical form of pectins in the cell wall. In the bor1‐1 mutant, B concentrations in the cell wall from shoots were lower than those in the wild type at low B supply, whereas they were similar at sufficient B supply. The amount of B present as borate ester of the RG‐II dimer (dRG‐II‐B) in the bor1‐1 mutant was lower than that in the wild type at low B supply. In the wild type, about 90 % of RG‐II was present as dRG‐II‐B, both, at low and sufficient B supply. In the bor1‐1 mutant, about 60 % of RG‐II was in its monomeric form (mRG‐II) at low B supply, whereas more than 85 % of it was present as dRG‐II‐B at sufficient B supply. However, similar as the wild type, mRG‐II derived from the bor1‐1 mutant was able to form dRG‐II‐B in vitro in the presence of borate and lead. Sugar composition of cell wall fractions was similar in both genotypes. These results suggest that the polysaccharide composition in the cell wall was not strongly affected by the bor1‐1 mutation. The observed difference in dimerization of RG‐II at low B supply is most likely due to a reduced B concentration in the shoots of the bor1‐1 mutant.     

The Function of a Maize-Derived Phosphoenol pyruvate Carboxylase (PEPC) in Phosphorus-Deficient Transgenic Rice

Transgenic rice ( Oryza sativa L., a C₃ plant) lines carrying a complete phospho enol pyruvate carboxylase (PEPC) gene from maize (a C₄ plant) were tested for their performance in terms of organic acid synthesis and organic acid exudation into the rhizosphere under phosphorus (P)-deficient conditions. High PEPC activity increased the fraction of photosynthetically fixed carbon allocated to the organic acid pool, and P deficiency enhanced oxalate exudation from the roots of the transgenic plants. There was no evidence that the transformed PEPC was involved in internal P recycling in the plant. However, the root PEPC activity was positively correlated with the oxalate exudation and negatively correlated with the root P concentration, and a higher root PEPC activity led to a higher oxalate exudation. Thus, it is suggested that C₄-PEPC transgenic rice plants had acquired the ability to exude oxalate, which enhanced their capacity to adapt to low P soil conditions.     

Structural characterization and biological activities of an exopolysaccharide kefiran produced by Lactobacillus kefiranofaciens WT-2B(T)

Lactobacillus kefiranofaciens, isolated from kefir grains, produces an extracellular polysaccharide when cultured, not only in PYG10 medium but also in a liquid medium containing a rice hydrolysate that had been previously degraded by treatment with a glucoamylase. The maximum yield of the polysaccharide, using the rice hydrolysate as the medium, was 2.5 g/L after a 7-day culture period at pH 5.0 and 33 degrees C. Compositional analysis, methylation analysis, specific rotation, and (1)H and (13)C NMR spectroscopy revealed that the structures of polysaccharides obtained from these two different culture media are essentially identical. The polysaccharide is composed of a hexasaccharide repeating unit and, thus, is known as kefiran. The weight-average molecular weight and the z-average radius of gyration of a sample, purified from the rice hydrolysate medium, were determined to be 7.6 x 10(5) g/mol and 39.9 nm, respectively, by gel permeation chromatography equipped with a multiangle laser-light-scattering photometer. Changes in blood pressure and serum components were examined in SHRSP/Hos rats, using doses of 100 and 300 mg of kefiran/kg of rat. A suppression in the increase in blood pressure was observed in these rats after 30 days. This activity is discussed in terms of the concentration of serum components of the rat, with emphasis on lipid components such as cholesterols, triglycerides, and free fatty acids.     

Host status of 10 fungal isolates for two nematode species, Filenchus misellus and Aphelenchus avenae

The classification of nematodes in the family Tylenchidae into plant parasites, plant associates or fungal-feeders for community analyses, have been much discussed by nematode ecologists. For an appropriate classification, fungal-feeding habits in the family need to be studied. To evaluate the host status of 10 fungal isolates for Filenchus misellus (Tylenchidae) and Aphelenchus avenae (Aphelenchida, Aphelenchidae), population growth rates, body length and width and sex ratios of the nematodes were measured after 40-day culture on fungal colonies at 25 °C. For F. misellus, the fungi determined as good hosts were two Basidiomycota fungi (Agaricus bisporus, Coprinus cinereus), three Ascomycota fungi (Chaetomium cochlioides, Chaetomium funicola, Chaetomium globosum) and a plant-pathogenic fungus (Rhizoctonia solani) on the basis of nematode population growth rate and female body length. Interestingly Pleurotus ostreatus, known as a predaceous fungus for the other nematodes, was also a good host for F. misellus. While, for A. avenae, good hosts were four plant-pathogenic fungi (Fusarium oxysporum f. sp. conglutinans, F. oxysporum f. sp. cucumerinum, Pythium ultimum, R. solani) and A. bisporus. A. avenae was trapped and preyed upon by Pleurotus hyphae. In F. misellus, males were 7-21% of adults, but the ratio did not correlate significantly with the population growth rate. In A. avenae, no male occurred. Differences in habitat preference between Filenchus and Aphelenchus were explained on the basis of the host status and habitat preferences of the tested fungi.     

New approach to immunochemical determinations for triclopyr and 3,5,6-trichloro-2-pyridinol by using a bifunctional hapten,and evaluation of polyclonal antiserum

The present work describes the design and synthesis of the structurally unique hapten, "bifunctional hapten", to produce a group-specific polyclonal antiserum to triclopyr and 3,5,6-trichloro-2-pyridinol. A bifunctional hapten was designed and synthesized by conjugating commercially available Nepsilon-2,4-dinitrophenyl (DNP)-L-lysine to triclopyr, and then coupling this to carrier proteins such as bovine serum albumin (BSA). The synthesized bifunctional hapten greatly raised the antiserum titer in comparison with that of the conventional hapten, triclopyr. Antiserum with a sufficiently high titer to provide the determinations of targeted compounds was obtained only 63 days after the primary immunization. The obtained antiserum showed the highest affinity to triclopyr (IC(50) = 3.5 nM) and 3,5,6-trichloro-2-pyridinol (IC(50) = 5.1 nM) in homologous ELISA. The cross-reactivities to various agrochemicals and some chlorinated phenolic compounds were determined. Significant cross-reactivity was found to the herbicide 2,4,5-T. The antiserum reacted to both triclopyr and its metabolite. Assay sensitivity was evaluated for effects of various assay conditions, including pH value and concentrations of organic solvents and detergents. Under optimized assay conditions, the quantitative working range of triclopyr ELISA was from 0.1 to 5.2 ng/mL with a limit of detection (LOD) of 0.037 ng/mL, and an IC(50) of 0.72 ng/mL. On the other hand, the quantitative working range of 3,5,6-trichloro-2-pyridinol ELISA was from 0.13 to 6.0 ng/mL with a LOD of 0.052 ng/mL, and an IC(50) of 0.95 ng/mL. Water samples fortified with triclopyr or its metabolite at 1, 5, and 10 ng/mL were directly analyzed without extraction and cleanup by the proposed ELISA. The mean recovery was 101.6%, and the mean coefficient of variation (CV) was 7.1% in the case of the triclopyr ELISA. In the case of the 3,5,6-trichloro-2-pyridinol ELISA, the mean recovery was 99.8%, and the mean CV was 9.5%. The proposed ELISA turned out to be a powerful tool for monitoring of residual triclopyr or 3,5,6-trichloro-2-pyridinol in water samples at trace level.