Year of Publication

2010

Degree Name

Doctor of Philosophy (PhD)

Document Type

Dissertation

College

Agriculture

Department

Plant Physiology

First Advisor

Dr. Sharyn E. Perry

Abstract

Nicotine, nornicotine, anabasine and anatabine are four major alkaloids in tobacco, of which nicotine is predominant. In many tobacco cultivars and also in other Nicotiana species, nicotine is converted to nornicotine, which in turn gives rise to potent carcinogen NNN. Nicotine to nornicotine conversion via nicotine-N-demethylation is mediated by the CYP82E family of P450 enzymes. Tobacco (Nicotiana tabacum) converts in senescing leaves, while its diploid progenitors N.tomentosiformis and N.sylvestris convert in both green and senescing and only in senescing leaves, respectively. Previously it has been shown that N.tomentosiformis has different active conversion loci in green and senescing leaves. The green leaf conversion enzyme CYP82E3 is inactivated in tobacco by a single amino acid substitution, while the senescing leaf converter enzyme CYP82E4 is active in tobacco, which gave tobacco a ‘senescing leaf converter’ phenotype. In nonconverter tobacco, CYP82E4 shows transcriptional silencing.

The nicotine-N-demethylase gene NsylCYP82E2 involved in nicotine to nornicotine conversion in senesced leaves of N. sylvestris was isolated. NsylCYP82E2 is active in N. sylvestris, but it has become inactivated in tobacco through mutations causing two amino acid substitutions. The conversion factor from N.sylvestris was characterized and a model for the alkaloid profile evolution in the amphidiploid N.tabacum from its diploid progenitors was proposed.

Regulation of conversion phenomenon was tested under different spatio-temporal conditions and various stresses. The promoter region for NtabCYP82E4 was isolated and promoter-reporter construct was used to determine that NtabCYP82E4 is specifically induced only during senescence. This pattern correlates with the nornicotine accumulation as measured by alkaloid profiling. Thus the regulatory regions of NtabCYP82E4 represent a senescence specific promoter.

In another project functional characterization of tobacco EIN2 (NtabEIN2) was undertaken. EIN2 from tobacco and N.sylvestris were cloned, their genomic structure was deduced and NtabEIN2 was silenced using RNAi approach. Silenced plants showed significant delay in petal senescence and abscission; as well as anther dehiscence, pod maturation, pod size, seed yield and defense against tobacco hornworm. Mechanism of delayed petal senescence phenotype, including possible cross-talk with Auxin Response Factor 2 and potential involvement of tasiRNA3 were also investigated.

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