Sulfonylurea herbicide sensitivity

A triple-quadrupole mass spectrometer with an electrospray interface is recommended for achieving the best sensitivity and selectivity in the quantitative determination of sulfonylurea herbicides. Ion trap mass spectrometers may also be used, but reduced sensitivity may be observed, in addition to more severe matrix suppression due to the increased need for sample concentration or to the space charge effect. Also, we have observed that two parent to daughter transitions cannot be obtained for some of the sulfonylurea compounds when ion traps are used in the MS/MS mode. Most electrospray LC/MS and LC/MS/MS analyses of sulfonylureas have been done in the positive ion mode with acidic HPLC mobile phases. The formation of (M - - H)+ ions in solution and in the gas phase under these conditions is favorable, and fragmentation or formation of undesirable adducts can easily be minimized. Owing to the acid-base nature of these molecules, negative ionization can also be used, with the formation of (M - H) ions at mobile phase pH values of approximately 5-7, but the sensitivity is often reduced as compared with the positive ion mode. 

Thermospray LC/MS has been extensively used for the study of sulfonylurea herbicides (1-2). These compounds are thermally labile and can not be successfully analyzed by conventional GC/MS. Early applications of thermospray LC/MS included metabolite identification and product chemistry studies. We have recently evaluated the use of thermospray LC/MS for multi-sulfonylurea residue analysis in crops and have found the technique to meet the criteria for multiresidue methods. LC/MS offers both chromatographic separation and universal mass selectivity. Our study included optimization of the thermospray ionization and LC conditions to eliminate interferences and maximize sensitivity for trace level analysis. The target detection levels were SO ppb in crops. Selectivity of the LC/MS technique simplified sample extraction and minimized sample clean up, which saved time and optimized recovery. Average recovery for these compounds in crop was above 85%. 

For the six sulfonylurea herbicides included in Figure 1, we monitored the protonated molecular ion fen each herbicide in addition to one or two major fragment ions. Table 1 shows the ions selected for each of the sulfonylurea and Figure 4 shows the ion traces for each compound. The table shows two ions which are common for some of these sulfonylurea herbicides. HARMONY, ALLY and GLEAN contain the same triazine urea ion at m/z 184 while ALLY, OUST and EXPRESS contain the same sulfonamide ions at m/z 233. Selecting these common ions for quantitation will increase the overall sensitivity for multiresidue analysis. 

The sulfonylureas, an extremely potent class of herbicides, act by inhibiting acetolactate synthase (ALS), which is the first common enzyme in the biosynthetic pathways leading to the branched chain amino acids. Two other unrelated classes of herbicides also act by interfering with this enzyme. We have cloned and characterized the genes encoding ALS from several higher plants. The ALS genes isolated from herbicide sensitive and herbicide resistant plants have been compared, and several mutations which confer the herbicide resistant phenotype have been identified. 

It appears that resistance to the aryloxyphenoxypropionate herbicides, the cyclohexanedione herbicides and to the sulfonylurea herbicides is unlikely to be due to reductions in the sensitivity or increases in the amounts of their respective target enzymes (Figures 2 3). Studies have not yet been performed to examine if die resistance to the dinitroaniline herbicide trifluralin is associated with any change at the tubulin polymerization site. 

Any major differences in the sensitivity of the enzymic target sites of the aryloxyphenoxypropionate, cyclohexanedione or sulfonylurea herbicides. 

Site-directed mutagenesis was used to make additional amino acid substitutions at these sites in yeast ALS. At some of the sites, e.g. alall7, prol92, or trp586, nearly any substitution for the wild type amino acid that was tested resulted in a herbicide-resistant enzyme (Table I). Each of the mutant enzymes was characterized by enzyme assays to compare its activity, and its sensitivity to the sulfonylurea herbicide chlorimuron ethyl, to the wild type enzyme. These analyses have indicated that some of the mutations have little adverse effect on the activity of the enzyme, while decreasing sensitivity to the herbicide from three to greater than one thousandfold. The characteristics of these mutant enzymes were further evaluated in vivo in order to investigate the utility of particular herbicide/mutant enzyme combinations (Falco et al., manuscript in preparation). 

In the second approach, herbicide-resistance mutations in the Arabidopsis ALS gene were studied in E. coli. To do this, wild type and mutant Arabidopsis genes were functionally expressed in E. coli, such that the plant genes complemented a branched chain amino acid auxotrophy in the bacteria (Smith et al. 1989, PNAS in press). ALS enzyme assays on extracts prepared from E. coli expressing the mutant Arabidopsis gene indicated that the mutant enzyme is resistant to sulfonylurea herbicides but is sensitive to the imidazolinone herbicide imazaquin. This selective... 

Acetolactate synthase inhibition by imidazolinones and triazolopyrimidines, 460 sensitivity to sulfonylurea herbicides, 460 Acetolactate synthase gene activity and inheritance of resistance in tobacco, 461... 

The systemic morpholine fungicide Corbel with fenpropimorph as its active substance is frequently applied to cereal cropping. It is rapidly metabolized in soil to fenpropimorphic acid. For the assay of fenpropimorphic acid in soil, GC-MS coupling109 has been proposed. The GC technique was used to assure the best separation for the fenpropimorphic acid from the complex soil matrix. The MS technique is very sensitive and assures the best reliability of the analytical information. The same reliability is also achieved when the HPLC-MS tandem system is used for sulfonylurea herbicide assay in soil.110... 

A number of possible crop selectivity mechanisms have been investigated (see Brown, H. M. Pestic. Sci. 1990 (in press)). Differential uptake and/or translocation of the selective herbicide between the tolerant crop and sensitive weeds has been ruled out as the basis for crop selectivities in several specific cases. For example, Sweetser et al. ( found no ccnrelation between chlorsulfuron uptake or translocation and sensitivity to this herbicide in a study of 7 plant species, and similar conclusions were drawn in studies of thifensulfuron methyl tolerance in soybeans (28). Lichtner (29) has shown that sulfonylurea herbicide uptake and translocation in plants is not carrier-mediated, but instead depends on the physical properties of the herbicide (pKa, log P) and proceeds through an acid-trapping mechanism common to higher plants. Given this information, we conclude that differential uptake and/or translocation is unlikely to account for any of the sulfonylurea crop selectivities discovered to date. 

Differential herbicide sensitivity at the site of action is a second possible selectivity mechanism, with good precedence in the aryloxyphenoxy and cyclo-hexanedione grass herbicides (2Q, 51). However, several studies now clearly indicate that the inherent sulfonylurea crop selectivities listed in Table 1 are not based on differential sensitivity at the site of action. ALS preparations isolated fix>m crops and weeds are equally sensitive to several selective sulfonylurea herbicides (28.32.33). Although data have not appeared for all of the compounds shown in Table 1, we conclude that differential active site sensitivity is not a general sulfonylurea selectivity mechanism. The clear exceptions to this generalization are the cases of genetically-altered plants (crops and we s) which have acquired through mutation or deliberate transformation a resistant form of the ALS enzyme (see below). 

Triazine herbicides and quinoclamine, having the mode of action in inhibition of PS II (Photosynthesis at photosystem II) had low variability on sensitivities in different algal taxa. On tiie other hand. Amide herbicide such as Pretilachlor and Cafenstrole as well as sulfonylurea herbicides of bensulfiironmethyl and imazosulfuron had great variability on sensitivities in different algal taxa. These herbicide have in other the mode of action in inhibition of cell division or in inhibition of acetolactate synthase rather tiian in inhibition of PS II. Carbamate herbicide showed relatively low toxicity on algae. Daimuron and bentazone exhibit low toxicity on all tire tested species (Figure 3). 

Herbicides also inhibit 5- (9/-pymvylshikiniate synthase, a susceptible en2yme in the pathway to the aromatic amino acids, phenylalanine, tyrosine and tryptophan, and to the phenylpropanes. Acetolactate synthase, or acetohydroxy acid synthase, a key en2yme in the synthesis of the branched-chain amino acids isoleucine and valine, is also sensitive to some herbicides. Glyphosate (26), the sulfonylureas (136), and the imida2oles (137) all inhibit specific en2ymes in amino acid synthesis pathways. 

The selective toxicity of sulfonylureas to certain weeds without damage to the cereal crop arises from their rapid metabolism in the crop plant to inactive compounds, whereas in sensitive weeds the metabolism is much slower. The very high herbicidal activity suggests a specific biochemical mode of action, which is concluded to be the inhibition of plant cell division. Sulfonylureas block the enzyme acetolacetate synthase (ALS), which catalyses the biosynthesis of the essential branched chain amino acids valine, leucine and isoleucine. 

LC/MS has emeiged as a sensitive and selective residue methodology for the trace organic analysis of crop protection chemicals. This technology is especially applicable to low application rate herbicides such as sulfonylureas because it requires minimal sample processing and clean-up prior to chromatographic and spectroscopic quantitation. 

Acetolactate synthase (ALS) is the target enzyme for three unrelated classes of herbicides, the sulfonylureas, the imidazolinones, and the triazolopyrimidines. We have cloned the genes which specify acetolactate synthase from a variety of wild type plants, as well as from plants which are resistant to these herbicides. The molecular basis of herbicide resistance in these plants has been deduced by comparing the nucleotide sequences of the cloned sensitive and resistant ALS genes. By further comparing these sequences to ALS sequences obtained from herbicide-resistant yeast mutants, two patterns have become clear. First, the ALS sequences that can be mutated to cause resistance are in domains that are conserved between plants, yeast and bacteria. Second, identical molecular substitutions in ALS can confer herbicide resistance in both yeast and plants. 

Nonetheless unanswered questions remain. What are the relative contributions of branched chain amino acid deficiency and AKB overabundance to the cytotoxic effects of the sulfonylurea and Imldazollnone herbicides Will the delineation of the cytotoxicity of AKB towards typhimurium, the only system in which it has been approached, provide relevant information towards the roles of this molecule in the Inhibition of plant growth Can herbicide-resistant alleles of ALS structural genes be used as dominant selectable markers In the transformation of a wide variety of sensitive cell lines What are the structural details of the interaction of ALS with the sulfonylurea and imldazollnone herbicides Are eukaryotic ALS Isozymes composed of nonldentlcal subunits How does SM cause cessation of DNA synthesis in plants In the next few years answers to some of these questions may emerge. 

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