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 Co-Chair (if applicable)

Perlin, Michael

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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