Title page for ETD etd-11202009-051306

Type of Document Dissertation
Author Hildreth, Sherry Boston
Author's Email Address shildret@vt.edu
URN etd-11202009-051306
Title Investigation of Protein – Protein Interactions among Nicotine Biosynthetic Enzymes and Characterization of a Nicotine Transporter
Degree PhD
Department Plant Pathology, Physiology, and Weed Science
Advisory Committee
Advisor Name Title
John Jelesko Committee Chair
Dorothea Tholl Committee Member
Elizabeth Grabau Committee Member
Vinatzer, Boris A. Committee Member
Winkel, Brenda S. J. Committee Member
  • NUP1
  • Nicotiana tabacum
  • putrescine methyltransferase
  • SAHH
  • S-adenosylhomocysteine hydrolase
  • nicotine
  • PMT
  • nicotine uptake permease
  • MPO
  • N-methylputrescine oxidase
Date of Defense 2009-09-29
Availability unrestricted
Alkaloids are a class of plant secondary metabolites produced in about 20% of plant families. Domesticated tobacco, Nicotiana tabacum produces nicotine as the predominant alkaloid. The biosynthesis of nicotine occurs exclusively in the roots of tobacco, yet accumulates in the leaves of tobacco where it is acts as a defense compound to deter insect herbivory. The research detailed in this dissertation addresses two aspects of nicotine physiology in tobacco: 1) an investigation of hypothesized protein-protein interactions among nicotine biosynthetic enzymes and 2) the characterization of a novel nicotine transporter.

A hypothesized metabolic channel including the two nicotine biosynthetic enzymes putrescine N-methyltransferase (PMT), N-methylputrescine Oxidase (MPO) and the S-adenosylmethionine (SAM) recycling enzyme S-adenosylhomocysteine hydrolase (SAHH) has been proposed. To further explore this hypothesis, protein-protein interactions among nicotine biosynthetic enzymes PMT, MPO and SAHH were investigated using yeast two-hybrid assays and co-immunoprecipitation experiments. The yeast two-hybrid was conducted as both a directed screen to detect interactions between the hypothesized metabolic channel members and as a library screen to detect interactions between hypothesized metabolic channel members and proteins from a tobacco root cDNA library.

Co-immunoprecipitation experiments were conducted using proteins produced in an in vitro transcription/ translation system and using native proteins from a tobacco root extract. The outcome of these experiments provided no further evidence of a nicotine metabolic channel and a discussion of the methods and outcomes of the experiments conducted is presented.

The nicotine uptake permease, NUP1, was identified in tobacco roots and was shown to preferentially transport nicotine when expressed in Schizosaccharomyces pombe. This report presents the characterization of tobacco plants and hairy roots with diminished NUP1 transcripts created by using RNAi. The NUP1-RNAi hairy roots and plants showed a decreased level of nicotine and the hairy root cultures displayed an altered distribution of nicotine from the root to the culture medium. Additionally NUP1-GFP was used to determine that NUP1 localized to the plasma membrane of tobacco BY-2 protoplasts. Potential models for the role of NUP1 in nicotine physiology will be discussed.

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