Dale L. Shaner

Assay of acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, has received attention recently because of the finding that it is the site of action of several new herbicides. The most commonly used assay for detecting the enzyme is spectrophotometric involving an indirect detection of the product acetolactate. The assay involves the conversion of the end product acetolactate to acetoin and the detection of acetoin via the formation of a creatine and naphthol complex. There is considerable variability in the literature as to the details of this assay. We have investigated a number of factors involved in detecting AHAS in crude ammonium sulfate precipitates using this spectrophotometric method. Substrate and cofactor saturation levels, pH optimum, and temperature optimum have been determined. We have also optimized a number of factors involved in the generation and the detection of acetoin from acetolactate. The results of these experiments can serve as a reference for new investigators in the study of AHAS.

Biosynthesis of Branched Chain Amino Acids: From Test Tube to Field

The branched chain amino acids-valine, leucine, and isoleucine-are among the 10 essential amino acids that are not synthesized in mammals. Therefore, biosynthesis of branched chain amino acids in plants is of interest due to their importance in human and animal diets. In addition, in the early 198Os,

Mutations in corn (Zea mays L.) conferring resistance to imidazolinone herbicides

SummaryThree corn (Zea mays L.) lines resistant to imidazolinone herbicides were developed by in vitro selection and plant regeneration. For all three lines, resistance is inherited as a single semidominant allele. The resistance alleles from resistant lines XA17, XI12, and QJ22 have been crossed into the inbred line B73, and in each case homozygotes are tolerant of commercial use rates of imidazolinone herbicides. All resistant selections have herbicide-resistant forms of acetohydroxyacid synthase (AHAS), the known site of action of imidazolinone herbicides. The herbicide-resistant phenotypes displayed at the whole plant level correlate directly with herbicide insensitivity of the AHAS activities of the selections. The AHAS activities from all three selections have normal feedback regulation by valine and leucine, and plants containing the mutations display a normal phenotype.

Physiological responses of corn (Zea mays) to AC 243,997 in combination with valine, leucine, and isoleucine

Abstract Various physiological processes were measured in corn after treatment with AC 243,997. Neutral sugar levels in leaves increased 39% over the control 24 hr after application of AC 243,997. Protein synthesis, measured by [ 14 C]leucine and [ 14 C]cystine incorporation, and lipid synthesis were not inhibited 24 hr after application of 150 μ M of AC 243,997, while respiration and RNA synthesis were inhibited 32 and 15%, respectively. DNA synthesis was severely inhibited (70–90%) by 150 μ M of the herbicide 24 hr after application. The inhibition of DNA synthesis by AC 243,997 did not begin until 5 to 7 hr after application. Although protein synthesis rates were apparently unaffected by AC 243,997, the level of the soluble proteins decreased 40% while free amino acid levels increased 32% 24 hr after application of the herbicide. An exogenous supply of valine, leucine, and isoleucine to corn prevented the inhibition of growth and reversed the inhibition of DNA synthesis caused by AC 243,997. All three amino acids at a concentration of 1 m M were needed to provide maximum protection. The results support the hypothesis that AC 243,997 kills plants by interfering with the biosynthesis of valine, leucine, and isoleucine.

合衆国におけるアセト乳酸合成酵素(ALS)阻害剤に対する抵抗性: 研究史, 出現, 検定, および管理

Acetolactate synthase (ALS) inhibitors are highly potent herbicides that include the sulf onylureas, imidazolinones, triazolopyrimidine sulfonamides and pyrimidyl salicylates. Currently there are ALS-inhibiting herbicides for use in all major crops including maize, soybeans, rice, and cereals. These herbicides kill plants by inhibiting the first enzyme in the biosynthetic pathway of the branched chain amino acids. Resistance to ALS inhibitors can be conferred by single mutations at multiple sites within the ALS gene. The first case of resistance to ALS inhibitors was discovered in 1987 in a wild lettuce (Lactuca serriola) population in Idaho, U.S. Resistance to ALS inhibitors in the U.S. is now found in populations of 26 species in all of the major crops. In all the cases studied, the mechanism of resistance has been due to selection of an altered form of the ALS enzyme. Detection of resistance to ALS inhibitors can be done through whole plant screening, in vitro and in vivo enzyme assays. ALS resistance can be managed through the by using ALS inhibitors in an integrated system which utilizes other herbicides as well as mechanical, cultural and biological weed control methods.

MECHANISMS OF RESISTANCE TO ACETOLACTATE SYNTHASE/ACETOHYDROXYACID SYNTHASE INHIBITORS

Acetolactate synthase/acetohydroxyacid synthase (ALS) inhibitors can be very potent, broad spectrum herbicides. Two classes of inhibitors, the imidazolinones and the sulfonylureas, have been developed as herbicides and are becoming widely used. Resistant plant populations to these two chemical classes have been isolated through deliberate selection in the laboratory and through continuous use of the sulfonylureas in the field. Mutations have been selected in corn, soybeans, flax, tobacco, Chlamydomonas, Datura, Kochia, and Arabidopsis which are resistant to either the sulfonylureas, the imidazolinones or both. In fact, some mutations result in resistance to one subclass within sulfonylureas or imidazolinones but not other subclasses within the same chemical groups. Studies on the mechanism of resistance of these populations to these ALS inhibitors have shown that most of the resistant plant populations have a mutant ALS enzyme that is no longer sensitive to the herbicides. These studies have also demonstrated that although the imidazolinones and sulfonylureas inhibit the same enzyme, they do not appear to bind with the enzyme in exactly the same manner. The reason for these differences lies in the fact that multiple mutations can occur within the ALS gene that give rise to different levels of resistance. These different mutations must affect the way the imidazolinones and sulfonylureas bind to the ALS enzyme.

Separation and characterization of two forms of acetohydroxy acid synthase from black mexican sweet corn cells

Abstract Acetohydroxy acid synthase (EC 4.1.3.18), the first enzyme unique to the biosynthesis of the branched chain amino acids valine, leucine and isoleucine, is the site of action of the two different classes of herbicides, the imidazolinones and the sulfonylureas. Multiple forms of this enzyme with different sensitivity to feedback inhibition by amino acids or to inhibition by herbicides have been reproted in micro-organisms only, although the presence of similar forms of isozymes in plants has long been speculated. This is the first report of the isolation of two forms of acetohydroxy acid synthase from a plant source. The two forms were separated by both fast protein liquid chromatography and conventional chromatography. These forms exhibit significant differences in their physical and kientic properties as well as in their sensitivity to inhibition by amino acids and by different herbicides.

SAMOATM: one company's approach to herbicide-resistant weed management†‡

SAMOA (Second Active Mode of Action) is a program that was developed by American Cyanamid as a way to prevent/delay selection of resistance to the imidazolinones and to manage resistant weed populations if they are selected. The program consisted of three major components, all of which had to be implemented for the program to be successful. These components comprised educating sales persons, distributors and farmers on resistance management, developing efficacious and cost-effective tank mixtures or sequential programs and providing incentives for using this program.

Absorption and translocation of imazapyr in Imperata cylindrica (L.) Raeuschel and effects on growth and water usage

Abstract Imazapyr rapidly penetrates and translocates throughout Imperata cylindrica (L.) Raeuschel in the first four days after treatment. Leaf elongation ceases within one day after treatment and water usage, on both a per plant basis and a per leaf basis, decreases to approximately 20% of the untreated plant within 5 days after treatment.

Determination of 2-Keto Acids and Amino Acids in Plant Extracts

Abstract 2-keto acids and amino acids were extracted using liquid nitrogen and 0.25 N HCl. The keto acids and amino acids were seperated by cation exchange chromatography on AG50W-X8 resin. The cation exchange chromatography is vital for the determination of keto acids. The keto acids were derivatized with 1,2-diamino-4,5-methylenedioxybenzene (DMB), a specific derivatizing agent for 2-keto acids. The derivatized keto acids were quantified by reversed phase high performance liquid chromatography (HPLC). This assay is highly sensitive and can measure as low as 10 fmole of the keto acids per 10 μl injection. The amino acids were analyzed by an automatic amino acid analyzer. These methods were used to show that 2-ketobutyrate (2-KB) and 2-aminobutyrate (2-AB) accumulate in plants treated with an acetohydroxyacid synthase inhibiting herbicide.