GHB614 × T304-40 × GHB119 × COT102 Cotton: Protein Expression Analyses of Field-Grown Samples.

Food and feed safety assessment is not enhanced by performing protein expression analysis on stacked trait products. The expression levels of six proteins in cotton matrices from four single cotton events and three conventionally stacked trait cotton products are reported. Three proteins were for insect control; two proteins confer herbicide tolerance; and one protein was a transformation-selectable marker. The cotton matrices were produced at three U.S., five Brazil, and two Argentina field trials. Similar protein expression was observed for all six proteins in the stacked trait products and the single events. However, when two copies of the bar gene were present in the stacked trait products, the expression level of phosphinothricin acetyl transferase herbicide tolerance was additive. Conventional breeding of genetically engineered traits does not alter the level or pattern of expression of the newly introduced proteins, except when multiple copies of the same transgene are present.

Isoxaflutole: the background to its discovery and the basis of its herbicidal properties.

This paper reviews the discovery of isoxaflutole (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 alpha alpha alpha-trifluoro-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 first benzoyl isoxazole lead was synthesised in 1989 and IFT in 1990, and the herbicidal potential of the latter was identified in 1991. The decision to develop the molecule was taken after two years of field 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-1, whereas the corresponding values for the DKN are 0.4 and 326 mg litre-1, 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 influencing 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.

Increasing expression of P450 and P450-reductase proteins from monocots in heterologous systems.

Monocotyledonous crop plants are usually more resistant to herbicides than grass weeds and most dicots. Their resistance to herbicides is mediated in many cases by P450 oxygenases. Monocots thus constitute an appealing source of P450 enzymes for manipulating herbicide resistance and recombinant forms of the major xenobiotic metabolizing mooxygenases are potential tools for the optimization of new active molecules. We report here the isolation and functional characterization of the first P450 and P450 reductase coding sequences from wheat. The first attempts at expressing these cDNAs in yeast and tobacco led to levels of protein, which were extremely low, often not even detectable. The wheat P450 cDNAs were efficiently transcribed, but no protein or activity was found. Wheat coding sequences, like those of other monocots, are characterized by a high GC content and by a related strong bias of codon usage, different from that observed in yeast or dicots. Complete recoding of genes being costly, the reengineering their 5'-end using a single PCR megaprimer designed to comply with codon usage of the host was attempted. It was sufficient to relieve translation inhibition and to obtain good levels of protein expression. The same strategy also resulted in a dramatic increase in protein expression in tobacco. A basis for the success of such a partial recoding strategy, much easier and cheaper than complete recoding of the cDNA, is proposed.

Cloning and functional expression in yeast of a cDNA coding for an obtusifoliol 14alpha-demethylase (CYP51) in wheat.

Screening of a wheat cDNA library with an heterologous CYP81B1 probe from Helianthus tuberosus led to the isolation of a partial cDNA coding a protein with all the characteristics of a typical P450 with high homology (32-39% identity) to the fungal and mammalian CYP51s. Extensive screening of several wheat cDNA libraries isolated a longer cDNA (W516) coding a peptide of 453 amino acids. Alignment of W516 with other P450 sequences revealed that it was missing a segment corresponding to the N-terminal membrane anchor of the protein. The corresponding segment from the yeast lanosterol 14alpha-demethylase was linked to the partial wheat cDNA and the chimera expressed in Saccharomyces cerevisiae. Compared to microsomes from control yeasts, membranes of yeast expressing the chimera catalysed 14alpha-demethylation of obtusifoliol with an increased efficiency relative to lanosterol demethylase activity. W516 is thus a plant member of the most ancient and conserved P450 family, CYP51.

Kinetic studies on two isoforms of acetyl-CoA carboxylase from maize leaves.

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.

Tissue-Specific Expression of Germin-Like Oxalate Oxidase during Development and Fungal Infection of Barley Seedlings.

Oxalate oxidase activity was detected in situ during the development of barley seedlings. The presence of germin-like oxalate oxidase was confirmed by immunoblotting using an antibody directed against wheat germin produced in Escherichia coli, which is shown to cross-react with barley (Hordeum vulgare) oxalate oxidase and by enzymatic assay after electrophoresis of the protein extracts on polyacrylamide gels. In 3-d-old barley seedlings, oxalate oxidase is localized in the epidermal cells of the mature region of primary roots and in the coleorhiza. After 10 d of growth, the activity is detectable only in the coleorhiza. Moreover, we show that oxalate oxidase is induced in barley leaves during infection by the fungus Erysiphe graminis f. sp. hordei but not by wounding. Thus, oxalate oxidase is a new class of proteins that responds to pathogen attack. We propose that oxalate oxidase could have a role in plant defense through the production of H2O2.

Identification of barley oxalate oxidase as a germin-like protein.

A barley oxalate oxidase was purified to homogeneity and the N-terminal sequences of the protein and of two peptides generated by CNBr cleavage of this protein were determined. Searches for similarities in data bank revealed that the sequences are highly homologous to the amino-acid sequence of a wheat protein, germin, which is synthesized de novo during germination. The similarity of the two proteins was confirmed by showing that anti-oxalate oxidase antibodies strongly recognize germin produced in Escherichia coli. We show that like germin, oxalate oxidase is glycosylated, resistant to SDS denaturation, heat stable, and protease resistant. Moreover, oxalate oxidase activity is strongly induced during germination of barley embryos resulting from an accumulation of the protein. Thus, we conclude that barley oxalate oxidase is a germin-like protein.