Alkaloid; cytochrome P450; gene silencing; nicotine N-demethylase; N'-nitrosonornicotine; plant genetic engineering; metabolic engineering; Nicotiana tabacum L.; real-time PCR; RNA interference; tobacco-specific nitrosamines.
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.
Nicotine to nornicotine conversion in tobacco (Nicotiana tabacum L.) is regulated by an unstable converter locus which in its activated state gives rise to a high nornicotine, low nicotine phenotype in the senescing leaves. In plants that carry the high nornicotine trait, nicotine conversion is primarily catalyzed by a cytochrome P450 protein, designated CYP82E4 whose transcription is strongly upregulated during leaf senescence. To further investigate the regulation of CYP82E4 expression, we examined the spatiotemporal distribution and the stress- and signaling molecule-elicited expression patterns of CYP82E4 using alkaloid analysis and a fusion construct between the 2.2 kb upstream regulatory region of CYP82E4 and the beta-glucurodinase (GUS) gene. Histochemical and fluorometric analyses of GUS expression revealed that the CYP82E4 promoter confers high levels of expression in the senescing leaves and flowers, and in the green stems of young and mature plants, but only very low activity was detected in the roots. In the leaves, GUS activity was strongly correlated with the progression of senescence. Treatments of leaf tissue with various signaling molecules including abscisic acid, ethylene, jasmonic acid, salicylic acid and yeast extract; and stresses, such as drought, wounding and tobacco mosaic virus infection did not enhance nicotine conversion or GUS activity in the green leaves, but an increase in CYP82E4 expression was observed in response to ethylene- or tobacco mosaic virus-induced senescence. These results suggest that the expression of CYP82E4 is senescence-specific in the leaves and the use of the CYP82E4 promoter could provide a valuable tool for regulating gene expression in the senescing leaves.
Right click to open a feedback form in a new tab to let us know how this document benefits you.ABSTRACT. Biologically catalyzed reactions often produce enantiomers of the product; however, only one configuration is desired. Reaction conditions are known to affect enantiomer ratios and reaction kinetics, but little is known regarding the effect of processing conditions on whole-cell biocatalysis. Saccharomyces cerevisiae cells were grown in batch on glucose at pH = 4, 5, and 7, and then immobilized on Celite beads or in calcium alginate beads and used as the biocatalyst for the conversion of acetophenone in hexane to (S)-1-phenylethanol at water activities of 0.37, 0.61, and 0.80. S. cerevisiae was used as a model microorganism for the whole-cell catalyzed reaction. The initial reaction rate (IRR) and the final (S)-1-phenylethanol concentration were quantified for each treatment. The highest IRR value (94.9 mmol/h) and the highest final concentration of (S)-1-phenylethanol (17.8 mM) were observed on Celite-immobilized cells grown at pH 5 or 7, with the main effect of growth medium pH highly statistically significant. The main effect of water activity and the interactions of the two were not statistically significant (a = 0.05).The cells immobilized in calcium alginate beads favored a water activity of 0.61, resulting in an IRR of 916.2 mmol/h/g dcw, averaged over pH. The highest final concentration of (S)-1-phenylethanol (4.8 mM) was achieved with cells grown at pH 5 or 7.Calcium alginate beads gave the highest initial reaction rate with a growth pH of 7 and a water activity of 0.61. However, pH of 5 and water activity of 0.61 resulted in the highest final concentration of (S)-1-phenylethanol.
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