The search for new active molecules with novel modes of action and desirable physical properties is an ongoing endeavour. This publication describes the follow-up chemistry of a biological hit discovered in the screening system of Novartis Crop Protection, the legacy agrochemical parent of Syngenta Crop Protection. This chemistry was optimized through classical synthetic methods and automated parallel synthesis with coverage of important physical properties such as lipophilicity or Clog P and solubility. Preliminary biological activity from the greenhouse and field data with symptomology is presented.

Developing cotton (Gossypium hirsutum) fibers, cultured in vitro with their associated ovules, were used to compare the effects of two herbicides that inhibit cellulose synthesis: 2,6-dichlorobenzonitrile (DCB) and an experimental thiatriazine-based herbicide, CGA 325'615. CGA 325'615 in nanomolar concentrations or DCB in micromolar concentrations causes inhibition of synthesis of crystalline cellulose. Unlike DCB, CGA 325'615 also causes concomitant accumulation of noncrystalline beta -1,4-glucan that can be at least partially solubilized from fiber walls with ammonium oxalate. The unusual solubility of this accumulated glucan may be explained by its strong association with protein. Treatment of the glucan fraction with protease changes its size distribution and leads to precipitation of the glucan. Treatment of the glucan fraction with cellulase digests the glucan and also releases protein that has been characterized as GhCesA-1 and GhCesA-2-proteins that are believed to represent the catalytic subunit of cellulose synthase. The fact that cellulase treatment is required to release this protein indicates an extremely tight association of the glucan with the CesA proteins. In addition, CGA 325'615, but not DCB, also causes accumulation of CesA protein and a membrane-associated cellulase in the membrane fraction of fibers. In addition to the effects of CGA 325'615 on levels of both of these proteins, the level of both also shows coordinate regulation during fiber development, further suggesting they are both important for cellulose synthesis. The accumulation of non-crystalline glucan caused by CGA 325'615 mimics the phenotype of the cellulose-deficient rsw1 mutant of Arabi- dopsis that also accumulates an apparently similar glucan (T. Arioli, L. Peng, A.S. Betzner, J. Burn, W. Wittke, W. Herth, C. Camilleri, H. Hofte, J. Plazinski, R. Birch et al. [1998] Science 279: 717).

The induction of glutathione S-transferase (GST, EC 2.5.1.18) with 2,3,5-triiodobenzoic acid (TIBA) in soybean (Glycine max) hypocotyls was diminished by the natural antioxidants ascorbate and glutathione but not with 1,4-dithio-threito (DTT). DTT, 1,2-benzenediol and 1,6-benzenediol induced GST activity in soybean hypocotyls. Furthermore, in the presence of luminol TIBA-treated soybean suspension cells exhibited strong biphasic chemiluminescence. The induced chemiluminescence was inhibited by added catalase (EC 1.11.1.6) and superoxide dismutase (EC 1.15.1.1) but not by the peroxidase inhibitor azide. These findings indicate that as a response to TIBA-treatment, soybean cells produce H2O2 and O2- without involvement of peroxidase (EC 1.11.1.7). The time course of active oxygen species production was nearly identical to that described for cells treated with an incompatible pathogen. (C) 1998 Elsevier Science Ltd. All rights reserved.

Higher plants are equipped with a remarkably versatile system that protects them from the potentially phytotoxic actions of xenobiotics, i.e. synthetic chemicals present in the plant's environment. Particularly striking is the natural tolerance of certain plants toward herbicides that profoundly affect closely related species. This phenomenon of herbicide selectivity is widely exploited in agriculture to control competing weeds in a field of crop plants that are tolerant to the particular herbicide. Herbicide selectivity is, in most cases, based primarily on the differential ability of plant species to metabolically detoxify the herbicide (Lamoureux et al., 1991; Cole, 1994). Metabolic herbicide inactivation has been employed in the genetic engineering of crops for herbicide tolerance (Hinchee et al., 1993). Finally, enhanced herbicide detoxification is one of the mechanisms of herbicide resistance, in addition to altered target site susceptibility and yet-unknown mechanisms, that may emerge within formerly susceptible weed species upon continuous use of the same herbicide or herbicide class (Holt et al., 1993). It is well documented that plants are able to metabolize and detoxify herbicides by a variety of enzymatic reactions and with extraordinary diversity among species. Furthermore, recent research has revealed that transporters in the vacuolar membrane mediate the energydependent export of herbicide metabolites into the large central vacuole (Martinoia et al., 1993).

Glutathione S-transferases (GSTs) with additional activities as fatty acid hydroperoxidases were investigated in soybean (Glycine max L.) hypocotyls. Aside from the GSTs present in total soluble tissue extracts, enzyme activities and distinct immunoreactive GST polypeptides were also detected in the intercellular washing fluid. Whereas the intracellular isoenzymes were both constitutive and inducible, apoplastic GST and glutathione peroxidase was detectable only in tissues treated with the known GST inducer 2,3,5-triiodobenzoic acid. Monensin inhibited the induced accumulation of apoplastic GST but did not affect the intracellular isoforms. The discovery of apoplastic inducible GST will be discussed in light of the putative function of these enzymes in plants.

Glycosylation of endogenous secondary plant products and abiotic substances such as herbicides increases their water solubility and enables vacuolar deposition of these potentially toxic substances. We characterized and compared the transport mechanisms of two glucosides, isovitexin, a native barley flavonoid C-glucoside and hydroxyprimisulfuron-glucoside, a herbicide glucoside, into barley vacuoles. Uptake of isovitexin is saturable (Km = 82 μM) and stimulated by MgATP 1.3-1.5-fold. ATP-dependent uptake was inhibited by bafilomycin A1, a specific inhibitor of vacuolar H+-ATPase, but not by vanadate. Transport of isovitexin is strongly inhibited after dissipation of the ΔpH or the ΔΨ across the vacuolar membrane. Uptake experiments with the heterologue flavonoid orientin and competition experiments with other phenolic compounds suggest that transport of flavonoid glucosides into barley vacuoles is specific for apigenin derivatives. In contrast, transport of hydroxyprimisulfuron-glucoside is strongly stimulated by MgATP (2.5-3 fold), not sensitive toward bafilomycin, and much less sensitive to dissipation of the ΔpH, but strongly inhibited by vanadate. Uptake of hydroxyprimisulfuron-glucoside is also stimulated by MgGTP or MgUTP by about 2-fold. Transport of both substrates is not stimulated by ATP or Mg2+ alone, ADP, or the nonhydrolyzable ATP analogue 5′-adenylyl-β,γ-imidodiphosphate. Our results suggest that different uptake mechanisms exist in the vacuolar membrane, a ΔpH-dependent uptake mechanism for specific endogenous flavonoid-glucosides, and a directly energized mechanism for abiotic glucosides, which appears to be the main transport system for these substrates. The herbicide glucoside may therefore be transported by an additional member of the ABC transporters.

Glutathione S‐transferases (GSTs; EC 2.5.1.18) have recently been proposed to form one large group among the auxin‐induced proteins. However. the properties and regulation of such auxin‐responsive GSTs in the plant still await detailed investigation. In this study, a 2,4‐dichloro‐phenoxyacetic acid (2,4‐D)‐inducible GST isozyme from soybean (Glycine max [L.] Merr. cv. Williams) was purified to near homogeneity by anion‐exchange and affinity chromatography on S‐hexylglutathione agarose. The native enzyme had a molecular mass of 49 kDa, as determined by gel filtration, and consisted of 26‐kDa subunits. The purified GST conjugated glutathione to 1‐chloro‐2,4‐dinitrobenzene and to the herbicide metolachlor, but not to the other GST substrates atrazine. fluorodifen or trans‐cinnamic acid. The N‐termmal amino acid sequence shared significant homology with the deduced polypeptide sequences of two 2,4‐D‐inducible genes from tobacco, par A and CNT107. The levels of the 26‐kDa GST subunit protein in soybean hypocotyls were analysed by immunoblotting. At micromolar concentrations, 2,4‐D induced a transient increase in net accumulation of GST, whereas indole‐3‐acetic acid or I‐naphthaleneacetic acid did not increase the GST levels. Known inhibitors of polar auxin transport, including 2.3.5‐tri‐iodobenzoic acid. N‐I‐naphthylphthalamic acid and analogues thereof, differed widely in their ability to elicit GST protein accumulation. It is concluded that the induction of soybean GST by 2,4‐D and by some of the auxin transport inhibitors is not related to auxin activity or to changes in the endogenous auxin levels.

Ethanol treatment of etiolated maize seedlings led to a marked enhancement in metabolism of the herbicides metolachlor and prosulfuron. cDNA clones representing eight genes that encode putative cytochrome P450 enzymes were isolated from maize. They fall into three families and are designated CYP71C5, CYP73A6, A7, and A8, and CYP81A1, A2, A3, and A4. Ethanol treatment induced the CYP81A subfamily at the mRNA level in both roots and shoots of etiolated seedlings.

In plants potentially toxic compounds are ultimately deposited in the large central vacuole. In this report we show that isolated barley mesophyll vacuoles take up the glucoside conjugate of the herbicide derivate [5-hydroxyphenyl]primisulfuron. Transport is stimulated by Mg-ATP and is distinct from that previously described for glutathione conjugates. Treatment of barley with different herbicide antidotes (safeners) revealed that the safener cloquintocet-mexyl doubles the vacuolar transport activities for both the glutathione and glucoside conjugates. Stimulation of the uptake of the metolachlor—glutathione conjugate was the result of an increased uptake velocity whereas the Km remained unaltered, suggesting that the higher activity was due to a higher expression of the transporter. These results indicate that modulation of vacuolar transport activities are an integral part of the detoxification mechanism of plants.

Herbicide safeners are chemicals which protect crop plants from injury by certain herbicides, without affecting weed control efficacy of the herbicides. The protective mechanism of herbicide safeners has not yet been fully elucidated, but there is increasing evidence that safeners act by selectively enhancing herbicide detoxification in crop plants. To date, two main detoxification pathways have been related to the mode of action of herbicide safeners. The first includes oxidation and subsequent glucose conjugation, mediated by cytochrome P450 ‐dependent monooxygenases and UDP‐glucosyltransferases, respectively. This pathway appears to be important predominantly in safener protection to aryloxyphenoxypropionate and sulfonylurea herbicides. The second pathway represents the conjugation of thiocarbamate sulfoxides and chloroacetanilide herbicides with glutathione. This mechanism is accomplished by either elevating the levels of reduced glutathione or the activity of glutathione S‐transferase, or both. Since glutathione has been reported to be involved in several stress situations of plants its function associated with safener‐induced herbicide tolerance will be discussed in more detail in this review.

The effect of decreased glutathione levels on the action of the herbicide metolachlor was studied in shoots of maize seedlings. Metolachlor inhibited growth by impairing the length, the dry weight, and the fresh weight of maize shoots. Conversely, buthionine-S,R-sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase (EC 6.3.2.2.), when applied in concentrations of up to 2 mM had no effect on shoot growth, but it decreased the glutathione content up to 22-fold compared to untreated plants. When 100 μM metolachlor and various BSO levels were applied together, the growth of the shoots was inhibited with increasing concentrations of BSO. This growth inhibition significantly correlated with the decreased glutathione content. As BSO increased the metolachlordependent activity of glutathione transferase, our results suggest that decreased intracellular glutathione levels obtained with BSO are one of the limiting factors for the tolerance of maize plants to metolachlor.

PLANTS are exposed to many potentially phytotoxic foreign compounds, such as microbial toxins and agrochemicals (xenobiotics). Detoxification and elimination of these compounds within or from the cell is a prerequisite for their survival. Metabolism and detoxification of xenobiotics are remarkably similar in plants and animals and can generally be divided into three phases1,2. In the first phase, a foreign compound may be oxidized, reduced or hydrolysed to introduce or reveal a functional group. In a second step, the activated xenobiotic is conjugated to either glutathione, glucuronate (animals), or malonyl or glucosyl moieties (plants) by the respective transferases. In animals the third step, excretion of conjugated xenobiotics to the extracellular medium, is mediated by a specific ATPase1,3-5. In plants, instead of excretion, conjugates of xenobiotics appear to be stored in the large central vacuole6, but it is not known how they are transported into this organelle. We show here that glutathione S-conjugate uptake into the vacuole is mediated by a specific ATPase which is remarkably similar to the glutathione S-conjugate export pumps in the canalicular membrane of mammalian liver.

Tolerance of maize to sulfonylurea herbicides such as primisulfuron has recently been reported to be impaired by the use of some organophosphorus insecticides. In an effort to elucidate the mechanism of this interaction, the effect of the insecticide, malathion, on the metabolism of primisulfuron was studied in whole plants, in excised leaves, and in a microsomal in vitro system from maize. Foliar application of malathion to 7-day-old plants had no influence on leaf uptake and translocation of primisulfuron, but caused a decrease in the rate of herbicide metabolism. In excised leaves, malathion increased the metabolic half-life of primisulfuron. In microsomal preparations, malathion inhibited cytochrome P450-dependent primisulfuron phenyl- and pyrimidinering hydroxylation. Loss of primisulfuron phenyl-ring hydroxylase activity was time-dependent, saturable with respect to malathion concentration, and attenuated in the absence of NADPH. The kinetic data suggest a mechanism-based cytochrome P450 inactivation by malathion. The oxoanalogue of malathion, malaoxon, did not influence the metabolic half-life of primisulfuron in excised leaves and was a poor inhibitor of microsomal primisulfuron hydroxylation. Neither insecticide had any effect in vitro on total microsomal cytochrome P450 content. From the present results it may be concluded that malathion affects primisulfuron tolerance of maize due to the inhibition of cytochrome P450 monooxygenases involved in herbicide metabolism.

The influence of the safener, CGA 185072 (5-chloro-8-quinolinoxy-acetic acid-1-methyl-hexyl-ester), on the metabolism of the aryloxyphenoxypropanoate herbicide, CGA 184927 (2-propynyl-R-2-[4-(5-chloro-3-fluoro-2-pyridinyloxy)-phenoxy]-propionate), was studied in excised leaves of wheat, barley, and maize. In wheat and barley, CGA 184927 readily underwent ester hydrolysis followed by hydroxylation at the pyridinyl moiety as well as ether cleavage between the pyridinyl and the phenyl ring. Ether cleavage constituted the minor pathway in both species. All metabolites were subject to glycosyl conjugation. Tetcyclacis strongly inhibited pyridinyl-ring hydroxylation in wheat. Metabolism by hydroxylation and ether cleavage was more rapid in wheat than in barley, and was found to be accelerated in the presence of the safener CGA 185072 in both wheat and, to a lesser degree, in barley. Moreover, the safener increased the capacity for O-glycoside formation in wheat as suggested from studies using the C-14-labelled pyridinyl-ring hydroxylated metabolite as a precursor. In maize, which is highly susceptible to CGA 184927, rapid ester hydrolysis of CGA 184927 and partial conversion of the corresponding carboxylic acid to glycosyl ester conjugate(s) occurred. However, no further transformation of the herbicide was found in maize, both in the absence or presence of CGA 185072. It is concluded that the ability of CGA 185072 to protect wheat from injury by the herbicide, CGA 184927, and to confer partial protection to barley, is related to the ability of the safener to stimulate herbicide metabolism in these crop species.