Date on Master's Thesis/Doctoral Dissertation

8-2019

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biology

Degree Program

Biology, PhD

Committee Chair

Running, Mark

Committee Member

Perlin, Michael

Committee Member

Schultz, David

Committee Member

Davis, Keith

Committee Member

Himes, Paul

Committee Member

Harnett, Cindy

Author's Keywords

Prenylation; arabidopsis; abscisic acid; moss; protein

Abstract

Lipid post-translational modifications enhance a protein’s interaction with membranes and other proteins. In this dissertation, I studied a recently identified Arabidopsis gene, PROTEIN PRENYLTRANSFERASE ALPHA SUBUNIT-LIKE (PPAL), by mapping a second-site mutation that rescues the original mutation phenotype and could be an additional factor involved in sugar homeostasis/sensing. I found that mutant phenotype associated with suppressor line was too weak and variable to consistently score, which resulted in not finding any linked markers. I also generated crosses between ppal-1 and ABA biosynthesis and signal transduction mutants to find any possible connection between these pathways and PPAL. By using several physiological screens, I concluded that PPAL’s response is dependent on ABA. A cell wall composition analysis of ppal-1xaba3-1 showed a severe drop in the lignin content of these plants, which shows promise for biofuels and biomaterial applications and indicates possible crosstalk among ABA biosynthesis, sugar homeostasis, and lignin biosynthesis. I found that PPAL functions in the ABA signal-transduction pathway. To investigate the possible cause for distorted segregation of ppal-1 in this study, reciprocal crosses were generated between ppal-1 and Col-0, and F1 genotyping again revealed only Col-0 band and no ppal-1 insertion was found, indicating a possible gametophytic lethality. I also used a mutant of the moss Phycomitrella patens to establish a novel heterologous protein expression system. This mutant, ggb, reverts the plants into undifferentiated, unicellular plants resembling green algae. ggb was used successfully to express three fungal lignolytic enzymes: aryl alcohol dehydrogenase, glyoxal oxidase and laccase. Western blots of total protein from transgenic lines showed the expected protein bands. I found the laccase activity in extracellular fluid from transgenic lines by development of blue-green color with ABTS; however, similar color was also found in an untransformed ggb line. In this dissertation, a bioreactor was developed to test the activity of recombinant proteins. I showed the advantages of ggb as a heterologous protein expression system, including its amenability to easy protein isolation compared to other plant-based protein expression systems.

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