Herbicide Resistance in Datura innoxia

Abstract

Acetolactate synthase (ALS, EC 4.1.3.18), the first enzyme in the biosynthesis of branched-chain amino acids, was isolated from wild-type and sulfonylurea-resistant Datura innoxia cell variants and characterized. Apparent Km values of the ALS for pyruvate from three sulfonylurea-resistant variants (CSR2, CSR6, and CSR10) were manyfold greater than that of the wild type. The inhibition of wild-type and herbicide-resistant ALS activity by chlorsulfuron (CS), a sulfonylurea herbicide, and L-leucine (LLeu), one of the feedback inhibitors of the enzyme, was examined. ALS from two CS-resistant variants exhibited severalfold greater resistance to CS than did the wild-type enzyme. Inhibition of ALS by L-Leu fitted a partially competitive pattern most closely.It is proposed that the herbicide resistance mutation accentuated the partial inhibition characteristics of ALS by L-Leu. ALS from one of the two CS-resistant variants (CSR6) had a Ki for L-Leu an order of magnitude greater than that of the wild-type enzyme. The alterations in kinetic properties observed in the ALS from sulfonylurea-resistant variants are discussed in relation to the possible evolutionary significance of the herbicide binding site of this enzyme, the physiological effects of such biochemical alterations, and their practical utility in genetic studies.ALS3 (EC 4.1.3.18, also known as acetohydroxy acid synthase) is the first enzyme in the biosynthesis of Val, Leu, and Ile. This enzyme catalyzes the condensation of an acetaldehyde moiety derived from pyruvate either with another molecule of pyruvate to form 2-acetolactate or with 2-ketobutyrate to form 2-aceto-2-hydroxybutyrate.ALS is feedback inhibited by the end products Val and Leu in both micro-organisms and in plants (5,11,26). Recently, it has been discovered that ALS is the target of several structurally diverse herbicidal compounds such as SUs, IZs, and TPs (9,16,22,24). The In our laboratory, a number of SU-resistant and IZ-resistant variants of Datura innoxia have been isolated, and ALS from these variants exhibits different degrees of cross-resistance to SUs and IZs. The lack of cross-resistance in some of the variants has been used to support the hypothesis that there are two separable binding domains for SUs and IZs on the ALS molecule (18,19). ALS from several of these variants has also been found to be altered in feedback sensitivity to Val, Leu, and Ile (14). In this report, the properties of ALS from SU-resistant variants are compared with that ofthe wild type. We report the mechanism of inhibition of ALS from D. innoxia by the feedback inhibitor, Leu. The implications of the altered kinetic constants of the ALS from SU-resistant variants are discussed in relation to our current understanding of the herbicide binding site of ALS and the regulation of branched-chain amino acid biosynthesis in higher plants. MATERIALS AND METHODS Cell Suspension CulturesMaintenance and isolation of SU-resistant cell variants of Datura innoxia from a predominantly haploid (>90%) cell culture we...

Mutagenesis of Escherichia coli acetohydroxyacid synthase isoenzyme II and characterization of three herbicide-insensitive forms

“…They found that most substitutions cause a change in the K m for the substrate pyruvate and the activation constants (K c ) for the cofactors thiamin diphosphate (ThDP) and FAD, the largest being a 53-fold increase in the K c for ThDP. Similarly, Rathinasabapathi and King [17] found increases of 3-to 16-fold in the K m for substrate in five herbicide-insensitive mutants of Datura innoxia. Subramanian et al [18] observed no change in the K m for pyruvate of one tobacco and one cotton mutant but a second cotton mutant displayed a 4-to 9-fold increase ; there was no change in the K c for ThDP of these three variants.…”

Effect of four classes of herbicides on growth and acetolactate-synthase activity in several variants of Arabidopsis thaliana

“…It will also be possible to investigate the influence that specific base substitutions in the ALS gene have on the substrate-and amino-acid-binding sites on the protein product of the gene. Other studies indicate that the induction of resistance to herbicides can have a substantial influence on the feedback inhibition (Rathinasabapathi et al 1990) and the kinetic characteristics of ALS (Rathinasabapathi and King 1991) as well as on the physiological responses of plant cells to branched-chain amino acids (Rathinasabapathi and King 1992).…”

“…More recently, our investigations have included mutants resistant to herbicides that have an altered acetolactate synthase (ALS; see Rathinasabapathi and King 1991).…”

The use of mutants and transgenic plants to study amino acid metabolism

“…The I50 value for chlorsulphuron for the wild-type AHAS enzyme was 10"m ol m"^, and for the resistant line CSR2, the I50 value was 2mmolm~^, whilst for CSR6, 70% of the AHAS activity remained uninhibited at 50 mmol m~^. The chlorsulphuron resistant AHAS enzymes were shown to be less sensitive to feedback inhibition by leucine (Rathinasabapathi & King 1991). AHAS inhibitor resistant mutant lines of important crop plants have recently been isolated; for example, soybean (Sebastian et al 1989), canola (Swanson et al 1989), wheat (Newhouse et al 1992) and maize (Bright 1992).…”