Degree Type

Dissertation

Date of Award

2010

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Basil J. Nikolau

Abstract

Acetyl–CoA is metabolized via one of three mechanisms: carboxylation, acetylation and condensation. Acetoacetyl–CoA thiolase (AACT) catalyzes the condensation of two acetyl–CoA molecules to form acetoacetyl–CoA. The metabolic fate of acetoacetyl–CoA depends on the biological context in which it is generated. In microbes, such as Rhodospirillum rubrum, acetoacetyl–CoA is the precursor of the storage polymer polyhydroxyalkanoate (PHA). In the cytosol of plant cells, it is the precursor of mevalonate–derived isoprenoids.

In R. rubrum, the AACT enzyme is encoded within the phaABC1 operon, which is responsible for PHA biosynthesis, in addition there are two phaC1–like genes in the genome, called phaC2 and phaC3. Furthermore, R. rubrum contains one phaJ gene, encoding (R)–specific 2–enoyl–CoA hydratase. To characterize the roles of these genes in PHA biosynthesis, I generated over–expressing and deletion mutants of PHA genes. Characterization of these mutants show that PhaB is the key enzyme of the pha–operon pathway for PHA biosynthesis, and that PhaC2 is the major PHA polymerase for synthesizing PHA in vivo. These studies further demonstrated that PhaC2 is responsible for integrating 3–hydroxybutyrate, 3–hydroxyvalerate and 3–hydroxyhexanoate monomers into the PHA–polymer, whereas PhaC1 and PhaC3 are specific for integrating 3–hydroxybutyrate. These data also indicate that PhaC1 and PhaC3 may interact with each other. We also demonstrated that PhaJ is responsible for converting trans–2,3–enoylacyl–CoA to (R)–3–hydroxyacyl–CoA in vivo. Moreover, monitoring the growth and analyzing the PHA content of R. rubrum mutants indicate that eliminating PHA affects R. rubrum growth.

Genomic analyses revealed two AACT genes in the Arabidopsis genome, At5g47720 (AACT1) and At5g48230 (AACT2). These two genes code for proteins that share 78.4% sequence identity. Complementation of yeast AACT knock–out mutant Δerg10 shows that both Arabidopsis AACTs are functional. To study the physiological function of each AACT–coding genes, two T–DNA insertion alleles at AACT1 and one T–DNA insertion allele at AACT2 gene have been characterized. These characterizations indicate that although both genes are expressed (as evidenced by western analysis); mutation in AACT2 is embryo lethal whereas null alleles of AACT1 are viable and show no apparent growth phenotypes. Furthermore, segregation analysis and genetic complementation demonstrate that mutations in AACT2 affect male transmission, and in vivo pollen germination and elongation. Promoter::GUS fusion experiments indicate that AACT1 is primarily expressed in the vascular system and AACT2 is highly expressed in root tips, young leaves, top stems and anthers. AACT2–RNAi lines show pleiotropic phenotypes, including elongated life–span and flowering duration, sterility, dwarfing, reduced seed yield and shorter root length. Microscopic analysis reveals that dwarfing is caused by smaller cell size and reduced cell numbers and loss of pollen coat resulted in male–sterility, probably due to the faster degeneration of tapetum cells during pollen development. These phenotypes were rescued when mutant plants were grown in the presence of mevalonate. Phytosterol analysis of AACT2–RNAi plants shows reduced sterol content and altered composition in the seedling roots. The accumulation of these sterols was restored to wild type levels when the plants were feed with mevalonate. In contrast, no significant phytosterol changes were detected in the aact1 mutant. These results indicate that AACT2 is essential in plant growth and development and cannot be replaced by AACT1.

In combination, the product of acetyl–CoA condensation, acetoacetyl–CoA, is used for producing different biomolecules in different organisms. Correspondingly, knock–out of this pathway results in different consequences in different organisms. Deleting acetyl–CoA condensation pathway affects growth in microbes, whereas it leads to lethality in plants.

DOI

https://doi.org/10.31274/etd-180810-1683

Copyright Owner

Huanan Jin

Language

en

Date Available

2012-04-30

File Format

application/pdf

File Size

120 pages

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