Balazs Siminszky

Plant cytochrome P450-mediated herbicide metabolism

In the last two decades it has become apparent that enzymes of the P450 monooxygenase (P450) superfamily are responsible for the Phase I metabolism of numerous herbicides representing several classes of organic compounds. The majority of experimental evidence for P450 involvement in herbicide metabolism has been derived from in vitro studies in which the catalytic activity of plant microsomes towards herbicidal substrates was measured in the presence of various P450 inhibitors and activators. While the studies with microsomes elicited much appreciation for the pivotal roles of plant P450s in herbicide metabolism, detailed characterization of these enzymes only became possible after the isolation of genes encoding specific isoforms responsible for herbicide conversion. Several lines of evidence suggest that the development of herbicide resistance in weeds by enhanced detoxification is frequently associated with elevated levels of P450 activity. Enhanced detoxification-based herbicide resistance is particularly difficult to control, because it can involve resistance to multiple, chemically unrelated classes of herbicides. Continued research efforts are aimed at elucidating the role of P450s in the metabolic fates of herbicides in plants and the development of herbicide resistance in weeds. Recent advances made in the isolation and genetic manipulation of P450 enzymes have created new opportunities for their application in engineering herbicide tolerance and bioremediation.

Functional Analysis of Nicotine Demethylase Genes Reveals Insights into the Evolution of Modern Tobacco

Tobacco (Nicotiana tabacum L.) is a natural allotetraploid derived from the interspecific hybridization between ancestral Nicotiana sylvestris and Nicotiana tomentosiformis. The majority of cultivated tobacco differs from both of its progenitor species in that tobacco typically contains nicotine as the primary alkaloid, in contrast to its two progenitors that accumulate nornicotine in the senescing leaves. However, most, if not all, tobacco cultivars possess an unstable mutation, commonly referred to as the conversion locus, that when activated mediates the conversion of a large percentage of nicotine to nornicotine in the senescing leaf. We have recently identified CYP82E4, a tobacco nicotine N-demethylase gene whose expression was highly induced during senescence in plants that have converted, and CYP82E3, a closely related homolog that exhibited no nicotine N-demethylase activity. In this study, domain swapping and site-directed mutagenesis studies identified a single amino acid change that fully restored nicotine N-demethylase activity to CYP82E3. An examination of the N. tomentosiformis orthologs of CYP82E3 and CYP82E4 revealed that both are functional nicotine N-demethylase genes in N. tomentosiformis. Collectively, our results suggest that a single base pair mutation in CYP82E3 and transcriptional suppression of CYP82E4 played important roles in the evolution of the alkaloid profile characteristic of modern tobacco.

A cytochrome P450 monooxygenase cDNA (CYP71A10) confers resistance to linuron in transgenic Nicotiana tabacum

Abstract The isolation of a Glycine max cytochrome P450 monooxygenase (P450) cDNA designated CYP71A10 that conferred linuron resistance to laboratory-grown, transgenic Nicotiana tabacum seedlings was previously reported. A nonsegregating transgenic N. tabacum line has been established that possesses two independent copies of the G. max CYP71A10 transgene. Five-week-old progeny plants of this selected line were grown in a controlled environmental chamber and treated with linuron using either pretransplant incorporated (PTI) or postemergence (POST) applications. CYP71A10-transformed N. tabacum was more tolerant to linuron than the wild type for both application methods. The transgenic N. tabacum line tolerated an approximately 16-fold and 12-fold higher rate of linuron than wild-type N. tabacum when the herbicide was applied PTI or POST, respectively. These results provide evidence that plant-derived P450 genes can be employed effectively to confer herbicide resistance to transgenic plants. Nomenclature: Cytochrome P450; linuron; Glycine max L. Merr. ‘Dare’, soybean; Nicotiana tabacum L. ‘SR1’, tobacco.

Enantioselective Demethylation of Nicotine as a Mechanism for Variable Nornicotine Composition in Tobacco Leaf

Background: Nornicotine composition in tobacco leaf has been a puzzle for more than half a century. Results: Three recombinant nicotine demethylases selectively use (R)-nicotine in vitro and in planta. Conclusion: Nornicotine composition in tobacco leaf can be reasonably explained by the combination of three nicotine demethylases. Significance: This knowledge lays the base for the optimization of nornicotine composition in tobacco leaf in the future. Nicotine and its N-demethylation product nornicotine are two important alkaloids in Nicotiana tabacum L. (tobacco). Both nicotine and nornicotine have two stereoisomers that differ from each other at 2′-C position on the pyrrolidine ring. (S)-Nicotine is the predominant form in the tobacco leaf, whereas the (R)-enantiomer only accounts for ∼0.2% of the total nicotine pool. Despite considerable past efforts, a comprehensive understanding of the factors responsible for generating an elevated and variable enantiomer fraction of nornicotine (EFnnic of 0.04 to 0.75) from the consistently low EF observed for nicotine has been lacking. The objective of this study was to determine potential roles of enantioselective demethylation in the formation of the nornicotine EF. Recombinant CYP82E4, CYP82E5v2, and CYP82E10, three known tobacco nicotine demethylases, were expressed in yeast and assayed for their enantioselectivities in vitro. Recombinant CYP82E4, CYP82E5v2, and CYP82E10 demethylated (R)-nicotine 3-, 10-, and 10-fold faster than (S)-nicotine, respectively. The combined enantioselective properties of the three nicotine demethylases can reasonably account for the nornicotine composition observed in tobacco leaves, which was confirmed in planta. Collectively, our studies suggest that an enantioselective mechanism facilitates the maintenance of a reduced (R)-nicotine pool and, depending on the relative abundances of the three nicotine demethylase enzymes, can confer a high (R)-enantiomer percentage within the nornicotine fraction of the leaf.

Denaturing high-performance liquid chromatography efficiently detects mutations of the acetolactate synthase gene

Abstract Acetolactate synthase (ALS), a common enzyme in the biosynthesis of branched-chain amino acids, is a target for the sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidinylthiobenzoate, and the sulfonylaminocarbonyltriazolinone classes of herbicides. Widespread resistance to the ALS-inhibiting herbicides has been attributed to single-base mutations in the ALS gene. The objective of this study was to investigate the feasibility of using denaturing high-performance liquid chromatography (DHPLC), a recently developed method of mutation analysis, for the detection of three ALS mutations, Ala122Thr, Leu574Trp, and Ser653Thr, which confer herbicide resistance. The mutated variants of the ALS gene were isolated from herbicide-resistant biotypes of smooth pigweed and Powell amaranth using polymerase chain reaction (PCR). The PCR products were hybridized with a wild-type sample and subjected to DHPLC analysis. All three mutations could be detected using a modified High performance liquid chromatography system; however, the sensitivity of the method was strongly dependent on the melting temperature profile of the analyzed DNA fragment. Once the primers and the DHPLC conditions are optimized, the procedure is economical, rapid, and requires little sample preparation. Because of these favorable features, DHPLC can be used as an alternative to other commonly used mutation detection methods. Nomenclature: Imidazolinone; pyrimidinylthiobenzoate; sulfonylaminocarbonyltriazolinone; sulfonylurea; triazolopyrimidine; Powell amaranth, Amaranthus powellii S. Wats. AMAPO; smooth pigweed, Amaranthus hybridus L. AMACH.