Date of Award

12-2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry

Committee Chair

Natalia Dudareva

Committee Member 1

Clint Chapple

Committee Member 2

Ann Kirchmaier

Committee Member 3

Joshua Widhalm

Abstract

Sessile organisms, such as plants, have developed intricate means of responding and interacting with their environment in order to grow, reproduce, and survive environmental stresses such as attacks by other organisms and competition for resources with neighboring organisms. Plant volatile organic compounds (VOCs) play vital roles in resolving these evolutionary constraints associated with the sedentary nature of plants by attracting pollinators and seed dispersers necessary for reproduction. VOCs also mediate plant-plant interactions and provide defense against biotic stresses (pathogens, predators, and herbivores) and abiotic stresses (oxidative stress, high temperature stress, drought). Beyond the importance of VOCs to plants, humans have used VOCs for centuries as perfumes, therapeutics, food additives, and contribute to the flavor and aroma of fruits and vegetables. Chapter 1 of this dissertation offers a brief overview of plant VOCs, their functions, and biosynthetic pathways. The factors influencing their emission and the long-standing diffusion model of volatile release from plant cells are also discussed. Chapter 2 challenges the diffusion model and provides evidence for the involvement of active transport in the passage of VOCs across the plasma membrane. Downregulation of an ATP-binding cassette (ABC) transporter, PhABCG1, by RNA interference (RNAi) in Petunia hybrida flowers led to a decrease in volatile emission and accumulating of the internal pools of volatiles to toxic levels in the plasma membrane. In addition, PhABCG1 was shown to directly transport benzenoid volatiles using Nicotiana tabacum BY2 cells overexpressing PhABCG1. Together, these results alter the default assumption that VOCs simply diffuse out of cells.
Chapter 3 completes the identification of the biosynthetic genes in the peroxisomal β-oxidative pathway of benzoic acid (BA) biosynthesis by the identification of a petunia cinnamoyl-CoA hydratase-dehydrogenase (PhCHD). Kinetic analysis of recombinant PhCHD shows that it converts cinnamoyl-CoA to 3-oxo-3-phenylpropanoyl-CoA in vitro. Furthermore, downregulation of PhCHD in petunia flowers, using an RNAi approach, led to a decrease in benzoyl-CoA (BA-CoA), BA and other benzenoid derived volatiles further demonstrating the involvement of this gene to the peroxisomal β-oxidative pathway of BA biosynthesis. Lastly, Chapter 4 investigates the possible mechanisms of transport of the final product BACoA, of β-oxidative BA metabolism out of peroxisomes. Since the CoA moiety is membrane impermeable and BA-CoA thioesterase activity is enriched in purified peroxisomes, we hypothesized that BA-CoA is converted to BA by a thioesterase prior to transport or diffusion across the membrane and then reconverted to BA by a CoA ligase such as Ph4CL1 (petunia 4- coumarate: CoA ligase 1) or BZL1 (benzoate: CoA ligase) or by an unknown membrane associated ligase. Characterization of recombinant PhTE1 shows that it efficiently converts several hydroxycinnamoyl-CoA thioesters, including BA-CoA, to their free acids. Also, downregulation of PhTE1 led to an increase in the levels of BA-CoA and its derived volatiles, suggesting that BA-CoA is most likely transported out of peroxisomes. Furthermore, the levels of volatile phenylpropenes, anthocyanin and lignin were also altered suggesting cross-talk between the β-oxidative and the general phenylpropanoid pathways. Together, these results suggest the auxiliary roles thioesterases play in β-oxidative metabolism.

Download

Share

COinS