Assay of acetohydroxyacid synthase

Singh, Bijay; Stidham, Mark A.; Shaner, Dale L.

doi:10.1016/0003-2697(88)90139-x

Cited by 161 publications

(123 citation statements)

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“…AHAS activity was measured by estimation of the formation of product, acetolactate, after conversion by acid decarboxylation to acetoin (20). Standard reaction mixtures contained the enzyme in 50 mm potassium phosphate buffer (pH 7.0) containing 100 mm sodium pyruvate, 10 mM magnesium chloride, 1 mm thiamine pyrophosphate, and 10 gM FAD.…”

Section: Ahas Assaymentioning

confidence: 99%

Overexpression of Acetohydroxyacid Synthase from Arabidopsis as an Inducible Fusion Protein in Escherichia coli

et al. 1991

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Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) is the first enzyme unique to the biosynthesis of valine, leucine, and isoleucine. This enzyme is the target site of several classes of structurally unrelated herbicides. The conventional method of antibody production using purified protein has not been successful with this enzyme. Two separate fragments of a gene encoding a portion of the mature region of AHAS from Arabidopsis were fused with the trpE gene from Escherichia coli using the pATH1 vector. E. coli cells transformed with each respective plasmid expressed a fusion protein at levels greater than 10% of the total cell protein. The fusion protein was purified and used to immunize rabbits. Antisera obtained from the immunized rabbits immunoprecipitated AHAS activity from Arabidopsis cell free extracts. The anti-AHAS antisera reacted with a 65 kilodalton protein band in electrophoretically resolved extracts of Arabidopsis. In crossreactivity tests, this antibody was able to immunoprecipitate AHAS activity from various plant species. Furthermore, a protein band with a molecular mass of 65 kilodaltons was detected in the crude extracts of all plant species tested on a Westem blot. These results indicate that the 65 kilodalton protein represents AHAS in various plant species. The wide spectrum of crossreactivity for the antisera supports the view that the AHAS enzyme is highly conserved across all plant species.

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Section: Ahas Assaymentioning

confidence: 99%

Overexpression of Acetohydroxyacid Synthase from Arabidopsis as an Inducible Fusion Protein in Escherichia coli

et al. 1991

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“…The procedures used for the in vitro assays of AHAS activity have been published (18). Acetolactate produced by the enzyme was acid converted to acetoin, which was measured by the Westerfeld reaction (18).…”

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confidence: 99%

“…Acetolactate produced by the enzyme was acid converted to acetoin, which was measured by the Westerfeld reaction (18). Either leaf blades or lower portions of shoots were used as a source of plant tissue for the assay.…”

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confidence: 99%

Tolerance to Imidazolinone Herbicides in Wheat

Newhouse¹,

Smith²,

Starrett³

et al. 1992

Plant Physiol.

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An imidazolinone-tolerant wheat (Triticum aestivum L. em Thell) mutant in the winter wheat cultivar Fidel has been identified and characterized. The mutant was isolated from a population derived through seed mutagenesis of the variety with an aqueous solution containing sodium azide. Imidazolinone-tolerant wheat seedlings were selected from the M2 generation of the population in the presence of imazethapyr herbicide and identified as herbicideinsensitive individuals. The trait is inherited as a single semidominant gene and confers high levels of tolerance to imazethapyr. Acetohydroxyacid synthase activity in extracts from imidazolinonetolerant plants was less inhibited by imazethapyr than the enzyme from the wild type. The herbicide-tolerant plants have a completely normal phenotype and display no negative effects on growth and yield in either the absence or presence of imazethapyr.find imidazolinone-tolerant wheat (Triticum aestivum L. em Thell) through seed mutagenesis and selection with an M2 seedling screen. MATERIALS AND METHODS Seed MutagenesisFive thousand seeds of the French winter wheat (Triticum aestivum L. em Thell cv Fidel) were mutagenized by the procedure of Kueh and Bright (9). Wheat seeds were soaked in water for 18 h at 50C. Air was then bubbled through the seeds for 6 h at 200C. Following imbibition, seeds were soaked in 1 mm sodium azide (pH 3) for 2 h. The seeds were rinsed for 30 min, dried on paper towels, and planted in the field. The plants arising from these seeds were Ml generation plants. M2 seeds were harvested from these plants at maturity.The imidazolinone class of herbicides has been demonstrated to have a broad spectrum of weed control activity, flexibility in timing of application, low usage rates, and low mammalian toxicity. These herbicides inhibit the enzymic activity of AHAS2 (EC 4.1.3.18) (15), the first enzyme in the pathway for the synthesis of the branched chain amino acids valine, leucine, and isoleucine. This same enzyme has been shown to be the site of action for the sulfonylurea and triazolopyrimidine herbicides (4,19). The basis for crop selectivity of the imidazolinones has been shown to be the result of a difference in the nature or rate of metabolism of the herbicide (2,3,16 882Screening Procedure M2 seeds were surface disinfected in 70% ethanol for 30 s. This was followed by a disinfection in a solution of 2.6% sodium hypochlorite for 30 min under vacuum with gentle agitation. Two drops of the nonionic surfactant Tween 20 were used per 100 mL of the sodium hypochlorite solution to aid the disinfestation procedure. Subsequently, seeds were rinsed three times in sterile distilled water and placed into sterile plastic 10-x 1.5-cm Petri dishes at 250 seeds/dish. Twenty-five milliliters of 1 M imazethapyr was placed in each dish. After 3 d in the imazethapyr solution, the seeds were drained and blotted dry. The seeds were planted at a 1-to 2-cm depth in 15-x 20-cm peat flats containing Metromix 350 (W.R. Grace Co.) at the rate of 1000 seeds/flat. These flats...

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“…AHAS activity was assayed using the method of Singh et al [15] with some modifications. In brief, each chemical in the library was dissolved in dimethyl sulfoxide (DMSO).…”

Section: Microplate Assay For Ahasmentioning

confidence: 99%