Degree Type

Thesis

Date of Award

2019

Degree Name

Master of Science

Department

Biochemistry, Biophysics and Molecular Biology

Major

Molecular, Cellular and Developmental Biology

First Advisor

Basil J. Nikolau

Abstract

A number of economic and environmental imperatives have spurred an increased demand for biologically sourced, chemically reduced forms of carbon. Fatty acids, and closely related derivatives, represent an abundant form of such biological carbon. The biosynthesis of these molecules requires acetyl-CoA as a primer substrate, and malonyl-CoA as the extender substrate. Malonyl-CoA is generated by the carboxylation of acetyl-CoA, a reaction catalyzed by acetyl-CoA carboxylase (ACCase), which is thought to be an important determinant of the rate limiting reaction of fatty acid biosynthesis. In Arabidopsis, as in most dicotyledonous plants, two distinct isozymes catalyze this reaction, a cytosolic homomeric form (At.ACC1) and a plastidic heteromeric form (htACCase).

Here, we have implemented several different transgenic strategies to evaluate whether the supply of malonyl-CoA limits the generation of fatty acids based on the transgenic over-expression of the homomeric ACCase. The ectopic expression of a plastid-targeted homomeric ACCase (Pt.At.ACC1) resulted in an increased accumulation of epicuticular lipids and fatty acids in leaves, which is also associated with a morphological phenotype of wrinkled leaves. In other organs a different set of phenotypic alterations were noted, including bent stems, fused reproductive tissues and protrusions of the stem and in the most severe cases, vegetative death. These latter phenotypic impacts were associated with metabolic alterations in cuticular lipids and fatty acids. In parallel, we attempted to use this Pt.At.ACC1 transgene to replace the htACCase that was eliminated by a genetic mutation. We conclude that the plastid-targeted homomeric form (Pt.At.ACC1) cannot substitute for the htACCase enzyme in supplying the malonyl-CoA needed for the de novo biosynthesis of fatty acids but can significantly increase fatty acids and their derivatives within leaf tissue and alter the metabolic profile of the cuticular lipids and fatty acids in the vegetative plant structures.

An analogous experimental strategy was applied to assess if the ectopic constitutive expression of At.ACC1 could genetically complement mutations at the at.acc1 locus of Arabidopsis, and the sc.acc1 locus of S. cerevisiae. Both these mutations are known to be lethal, and our results indicate that ectopic expression of the At.ACC1 does not rescue these mutations, in spite of the fact that when At.ACC1 is expressed in the wild-type backgrounds the metabolic state of fatty acids is impacted in both organisms.

Copyright Owner

Troy Matthew Bunch

Language

en

File Format

application/pdf

File Size

44 pages

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