Partial purification and characterisation of a 2,4,5-trichlorophenol detoxifying O-glucosyltransferase from wheat

An enzyme preparation with UDP-glucose-dependent O-glucosyltransferase (OGT; EC 2.4.1.-) activity toward 2,4,5-trichlorophenol has been purified 215-fold from wheat shoots. The OGT co-purified with the major extractable glucosylating activity toward the flavonol quercetin and was characterised as a monomeric 53 kDa protein. Among the xenobiotic phenols tested, the purified enzyme preparation showed at least a 10-fold preference for 2,4,5-trichlorophenol. When assayed with flavonoids, the OGT was active toward flavonols and coumestrol, showing a clear preference for 3-hydroxy flavone when incubated with a range of monohydroxylated flavonoids. It was concluded that the major 2,4,5-trichlorophenol-detoxifying OGT in wheat shoots is most probably a flavonol-3-O-glucosytransferase.     

Selective disruption of wheat secondary metabolism by herbicide safeners

In wheat (Triticum aestivum L.), treatment with herbicide safeners enhances the expression of enzymes involved in pesticide detoxification and reduces crop sensitivity to herbicides. Since these same enzymes are involved in plant secondary metabolism, it was of interest to determine whether or not the safener cloquintocet mexyl perturbed phenolic metabolism in wheat seedlings. LC/ESI/MS analysis identified 14 phenolic substrates in the shoots of young wheat plants. Fragmentation imposed by collision induced dissociation identified specific C-glycosidic conjugates of 4′,5,7-trihydroxflavone (apigenin), 3′,4′,5,7-tetrahydroxyflavone (luteolin) and 3′-O-methylluteolin. Treatment of 7-day-old wheat shoots with cloquintocet mexyl resulted in an accelerated depletion of the conjugates of all three flavones, most notably with the glycosides of luteolin. In contrast, safener treatment caused the selective accumulation of 4′,5,7-trihydroxy-3′,5′-dimethoxyflavone (tricin) and the phenylpropanoid ferulic acid. Changes in phenolic content were associated with an increase in O-methyltransferase and C-glucosyltransferase activity toward flavonoid substrates as well as the classic enhancement of detoxifying glutathione transferases. Our results suggest that in addition to altering the capacity of wheat to metabolise herbicides and other xenobiotics, safeners can also cause a selective shift in the metabolism of endogenous phenolics.     

Carboxylesterase activities toward pesticide esters in crops and weeds

Proteins were extracted from maize, rice, sorghum, soybean, flax and lucerne; the weeds Abutilon theophrasti, Echinochloa crus-galli, Phalaris canariensis, Setaria faberii, Setaria viridis, Sorghum halepense and the model plant Arabidopsis thaliana and assayed for carboxylesterase activity toward a range of xenobiotics. These included the pro-herbicidal esters clodinafop-propargyl, fenoxaprop-ethyl, fenthioprop-ethyl, methyl-2,4-dichlorophenoxyacetic acid (2,4-d-methyl), bromoxynil-octanoate, the herbicide-safener cloquintocet-mexyl and the pyrethroid insecticide permethrin. Highest activities were recorded with alpha-naphthyl acetate and methylumbelliferyl acetate. Esters of p-nitrophenol were also readily hydrolysed, with turnover declining as the chain length of the acyl component increased. Activities determined with model substrates were much higher than those observed with pesticide esters and were of limited value in predicting the relative rates of hydrolysis of the crop protection agents. Substrate preferences with the herbicides were typically 2,4-d-methyl>clodinafop-propargyl>fenthioprop-ethyl, fenoxaprop-ethyl and bromoxynil-octanoate. Isoelectric focussing in conjunction with staining for esterase activity using alpha-naphthyl acetate as substrate confirmed the presence of multiple carboxylesterase isoenzymes in each plant, with major qualitative differences observed between species. The presence of serine hydrolases among the resolved isoenzymes was confirmed through their selective inhibition by the organophosphate insecticide paraoxon. Our studies identify potentially exploitable differences between crops and weeds in their ability to bioactivate herbicides by enzymic hydrolysis and also highlight the usefulness of Arabidopsis as a plant model to study xenobiotic biotransformation.     

Jacalin domain in wheat jasmonate-regulated protein Ta-JA1 confers agglutinating activity and pathogen resistance

Ta-JA1 is a jacalin-like lectin from wheat (Triticum aestivum) plants. To date, its homologs are only observed in the Gramineae family. Our previous experiments have demonstrated that Ta-JA1 contains a modular structure consisting of an N-terminal dirigent domain and a C-terminal jacalin-related lectin domain (JRL) and this protein exhibits a mannose-specific lectin activity. The over-expression of Ta-JA1 gene provides transgenic plants a broad-spectrum resistance to diseases. Here, we report the differential activities of the dirigent and JRL domains of Ta-JA1. In vitro assay showed that the recombinant JRL domain could effectively agglutinate rabbit erythrocytes and pathogen bacteria Pseudomonas syringe pv tabaci. These hemagglutination activities could be inhibited by mannose but not by galactose. In contrast, the recombinant dirigent domain did not show agglutination activity. Corresponding to these differentiations of activities, similar to full-length of Ta-JA1, the over-expression of JRL domain in transgenic plants also increased resistance to the infection of P. syringe. Unlike JRL, the over-expression of dirigent domain in transgenic plants led to alteration of the seedling sensitivity to salts. In addition, a d_N/d_S ratio analysis of Ta-JA1 and its related proteins showed that this protein family functionally limited to a few crop plants, such as maize, rice and wheat.     

Genomic regions for yield and yield parameters in Chinese winter wheat (Triticum aestivum L.) genotypes tested under varying environments correspond to QTL in widely different wheat materials

Field trials with a population of 108 doubled haploid (DH) lines of bread wheat (Triticum aestivum L.) derived from a cross between the Chinese winter wheat cultivars CA9613 and H1488 were carried out at Beijing (China) in 2000/2001 and 2001/2002. In addition, a field trial and a pot experiment were carried out at the experimental field stations of Giessen University (Germany) in the vegetation periods 2004/2005 and 2006/2007. Phenotypic data for major agronomic yield-related traits, i.e. grain weight per ear (GWE), grain number per ear (GNE), plant height and thousand-grain weight (TGW), were recorded in all experiments. In addition, biomass weight per tiller and ear weight were evaluated in the two field trials at Beijing. Based on the phenotypic data and a genetic map comprising 168 SSR markers, an analysis of quantitative trait loci (QTL) was carried out for yield and yield parameters using the composite interval mapping (CIM) approach. A total of 30 QTL were detected for these traits across four environments. Five of these QTL located on chromosomes 1A, 1B, 2B, 2D and 7D exhibited pleiotropic effects. Such pleiotropic gene loci will be very useful for understanding the homologous/homeologous relationships among QTL and designing an appropriate marker-assisted breeding programme including multi-trait selection in order to accumulate (“pyramide”) favorable alleles at different genetic loci.     

Evidence of two forms of poly(A) polymerase in germinated wheat embryos and their regulation by a novel protein kinase

Two forms of poly(A) polymerase (PAPI and PAPII) from germinated wheat embryos have been resolved on DEAE-cellulose ion-exchange chromatography by a linear gradient of 0-500 mM (NH(4))(2)SO(4). Further purification shows that both forms are monomeric in nature with an identical molecular weight, approximately 65 kDa. The phosphoprotein nature of PAPI and PAPII has been established by in vivo labelling with (32)P-orthophosphate. Acid hydrolysis of both (32)P-labelled purified PAPI and PAPII has revealed that phosphorylations generally take place in serine and threonine residues. PAPI and PAPII have also been characterised with respect to V(max) and K(m) for poly(A). The V(max) and K(m) values of PAPI are 28.57 and 11.37 microg, respectively, whereas 34.48 and 7.04 microg of PAPII. In vitro dephosphorylation of the purified enzyme by alkaline phosphatase leads to a significant loss of the enzyme activity, which is regained upon phosphorylation by a 65 kDa protein kinase (PK) purified from wheat embryos. The extent of phosphorylation by protein kinase shows that PK has similar affinity towards both PAPI and PAPII, whereas the phosphate incorporation in PAPII is twofold higher than PAPI suggesting their distinct chemical nature.