A rapid, noninvasive technique involving imaging of chlorophyll fluorescence parameters for detecting perturbations of leaf metabolism and growth in seedlings is described. Arabidopsis seedlings were grown in 96-well microtitre plates for 4 d and then treated with eight herbicides with differing modes of action to induce perturbations in a range of different metabolic processes. Imaging of chlorophyll fluorescence emissions from 96 seedlings growing on a microtitre plate enabled images of a number of fluorescence parameters to be rapidly and simultaneously produced for the plants in each well. Herbicideinduced perturbations in metabolism, even in metabolic reactions not directly associated with photosynthetic metabolism, were detected from the changes in the images of fluorescence parameters considerably before any visual effects on seedling growth were observed. Evaluations of seedling growth were made from measurements of the area of chlorophyll fluorescence emission in images of plants growing in the 96-well plates. Decreased seedling growth related directly to herbicideinduced changes in the imaged chlorophyll fluorescence parameters. The applicability of this rapid-screening technique for metabolic perturbations in monocotyledonous species was demonstrated by treating Agrostis tenuis seedlings with Imazapyr, an inhibitor of branched-chain amino acid synthesis.In many areas of plant biology and agrochemical research, there is an increasing requirement for more rapid-screening techniques to identify plants with impaired metabolism and growth. The rapid expansion of Arabidopsis genomics research programs has highlighted the need for new technologies to facilitate the identification of potentially interesting mutants with genetic modifications that impair specific metabolic reactions and growth (e.g. Borevitz et al., 2000; Nakazawa et al., 2001). Also, within the agrochemical industry, more efficient screening processes for crop protection chemical discovery are needed to deal effectively with the increases in the numbers of molecules entering the initial cascade of screening. For many years, conventional screening methods involved screening 5,000 to 20,000 new chemical entities per year in greenhouse screens, which for herbicides involved pot-grown plants in greenhouses (Evans, 1999). Combinatorial chemistry can provide at least a 10-fold increase in the numbers of molecules for screening, however, quantities of each molecule are normally less than 1 to 2 mg, which is insufficient for conventional greenhouse screens. In parallel to the introduction of combinatorial chemistry has been the introduction of new microscreening or high-throughput screening, which, for herbicide screening, involves growing plants in 96-well microtitre plates (Berg et al., 1999; Evans, 1999). In the past, conventional greenhouse screening has involved detailed visual assessment of herbicidal activity over a 2-to 3-week period following treatment. This is not possible using microscreening or highthroughput screening, and less time-con...

This paper reviews the discovery of isoxa¯utole (IFT), focusing on the chemical and physicochemical properties which contribute to the herbicidal behaviour of this new herbicide. IFT (5-cyclopropyl-1,2-isoxazol-4-yl aaa-tri¯uoro-2-mesyl-p-tolyl ketone) is a novel herbicide for pre-emergence control of a wide range of important broadleaf and grass weeds in corn and sugarcane. The ®rst benzoyl isoxazole lead was synthesised in 1989 and IFT in 1990, and the herbicidal potential of the latter was identi®ed in 1991. The decision to develop the molecule was taken after two years of ®eld testing in North America.The biochemical target of IFT is 4-hydroxyphenylpyruvate dioxygenase (HPPD), inhibition of which leads to a characteristic bleaching of susceptible species. The inhibitor of HPPD is the diketonitrile derivative of IFT formed from opening of the isoxazole ring. The diketonitrile (DKN) is formed rapidly in plants following root and shoot uptake. The DKN is both xylem and phloem mobile leading to high systemicity. IFT also undergoes conversion to the DKN in the soil. The soil half-life of IFT ranges from 12 h to 3 days under laboratory conditions and is dependent on several factors such as soil type, pH and moisture. The log P of IFT is 2.19 and the water solubility is 6.2 mg litre À1 , whereas the corresponding values for the DKN are 0.4 and 326 mg litre À1 , respectively. These properties restrict the mobility of IFT, which is retained at the soil surface where it can be taken up by surface-germinating weed seeds. The DKN, which has a laboratory soil half-life of 20±30 days, is more mobile and is taken up by the roots. In addition to in¯uencing the soil behaviour of IFT and DKN, the greater lipophilicity of IFT leads to greater uptake by seed, shoot and root tissues. In both plants and soil, the DKN is converted to the herbicidally inactive benzoic acid. This degradation is more rapid in maize than in susceptible weed species and this contributes to the mechanism of selectivity, together with the greater sowing depth of the crop.

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The steady-state kinetics of two multifunctional isoforms of acetyl-CoA carboxylase (ACCase) from maize leaves (a major isoform, ACCase1 and a minor isoform, ACCase2) have been investigated with respect to reaction mechanism, inhibition by two graminicides of the aryloxyphenoxypropionate class (quizalofop and fluazifop) and some cellular metabolites. Substrate interaction and product inhibition patterns indicated that ADP and P(i) products from the first partial reaction were not released before acetyl-CoA bound to the enzymes. Product inhibition patterns did not match exactly those predicted for an ordered Ter Ter or a random Ter Ter mechanism, but were close to those postulated for an ordered mechanism. ACCase2 was about 1/2000 as sensitive as ACCase1 to quizalofop but only about 1/150 as sensitive to fluazifop. Fitting inhibition data to the Hill equation indicated that binding of quizalofop or fluazifop to ACCase1 was non-cooperative, as shown by the Hill constant (n(app)) values of 0.86 and 1.16 for quizalofop and fluazifop respectively. Apparent inhibition constant values (K' from the Hill equation) for ACCase1 were 0.054 microM for quizalofop and 21.8 microM for fluazifop. On the other hand, binding of quizalofop or fluazifop to ACCase2 exhibited positive co-operativity, as shown by the (napp) values of 1.85 and 1.59 for quizalofop and fluazifop respectively. K' values for ACCase2 were 1.7 mM for quizalofop and 140 mM for fluazifop. Kinetic parameters for the co-operative binding of quizalofop to maize ACCase2 were close to those of another multifunctional ACCase of limited sensitivity to graminicide, ACC220 from pea. Inhibition of ACCase1 by quizalofop was mixed-type with respect to acetyl-CoA or ATP, but the concentration of acetyl-CoA had the greater effect on the level of inhibition. Neither ACCase1 nor ACCase2 was appreciably sensitive to CoA esters of palmitic acid (16:0) or oleic acid (18:1). Approximate IC50 values were 10 microM (ACCase2) and 50 microM (ACCase1) for both CoA esters. Citrate concentrations up to 1 mM had no effect on ACCase1 activity. Above this concentration, citrate was inhibitory. ACCase2 activity was slightly stimulated by citrate over a broad concentration range (0.25-10 mM). The significance of possible effects of acyl-CoAs or citrate in vivo is discussed.