Document Type

Dissertation

Degree

Doctor of Philosophy

Major

Biology, Molecular and Cellular Biology

Date of Defense

4-18-2019

Graduate Advisor

Bethany K. Zolman

Committee

Bethany K. Zolman

Sam Wang

Wendy M. Olivas

Lon Chubiz

Abstract

Many peroxisomal proteins act in β-oxidation processes on a range of substrates. It is unclear how these proteins are coordinated to determine the flux of peroxisomal processes and meet the requirements for plant growth and development. Using mutant analysis and metabolic profiling, I examined proteins predicted to act in fatty acid and indole-butyric-acid (IBA) β-oxidation. ECH2 confers enoyl-CoA hydratase activity for the auxiliary β-oxidation of fatty acids with an even cis-unsaturated bond. ECH2 was suggested to function in IBA β-oxidation, as ech2 seedlings have altered IBA response. ech2 seedlings have reduced root length and cotyledon area. ech2 seedlings accumulate 3-hydroxyoctenoate (C8:1-OH) and 3-hydroxyoctanoate (C8:0-OH), putative hydrolysis products of catabolic intermediates for α-linolenic acid and linoleic acid, respectively. Wild-type seedlings treated with 3-hydroxyoctanoate have ech2-like growth defects and altered IBA responses. ech2 phenotypes are not rescued by sucrose or auxin, but are suppressed in combination with core β-oxidation mutants mfp2 or ped1.Consistently, ech2 mfp2 seedlings accumulate less C8:1-OH and C8:0-OH. These results indicate that ech2 phenotypes require efficient upstream core β-oxidation. Our findings suggest low ECH2 activity results in metabolic alterations through a toxic effect of the accumulating intermediates. These effects manifest in altered lipid metabolism, IBA responses, and gene expression pathways with resulting disruptions to seedling growth and development. Similarly, IBR3 and IBR1 are predicted to act at the oxidation step and dehydrogenation step of IBA β-oxidation, respectively. Mutant complementation reveals the requirement of the IBR3 FAD cofactor binding site and IBR1 catalytic triad for function. Transgenic plants co-overexpressing IBR1 and IBR3 display phenotypes indicative of IBA catabolism, suggesting direct activities in IBA β-oxidation. The importance of IBR10 as a Δ3, Δ2-enoyl-CoA isomerase in fatty acid catabolism is revealed by analysis of ibr10 higher-order mutants. IBR10 was predicted to have enoyl-CoA hydratase activity for IBA catabolism. However, this prediction is contradictory to the ability of IBR10 with an altered key glutamate residue to rescue ibr10. Involvement of multifunctional protein AIM1 in fatty acid and IBA β-oxidation was reported; AIM1RNAi lines display altered IBA responses. Future studies will determine the potential enoyl-CoA hydratase activity of AIM1 and/or IBR10 on IBA-CoA intermediates.

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