Semester of Graduation

Fall 2017

Degree

Master of Science (MS)

Department

Biological Sciences

Document Type

Thesis

Abstract

Carbonic anhydrases (CAs) are zinc metalloenzymes which catalyze the interconversion of carbon dioxide and bicarbonate. It is hypothesized that CA activity facilitates CO2 flux in leaf mesophyll cells to maintain optimal rates of photosynthesis or to maintain a pool of inorganic carbon for other metabolic processes. There are three classes of CAs (α, β, and γ) in the model plant Arabidopsis thaliana. The βCAs and γCAs have been well studied, but the αCAs have received relatively little study. There are eight αCA genes in Arabidopsis. This study investigates which αCAs are expressed in shoots, their tissue-specific expression, their subcellular localization, and the effects of their mutation on growth.

RNAseq data show that only three of the eight αCA genes (αCA1, αCA2, and αCA3) are expressed in significant amounts in shoots. Therefore, our studies focused on αCA1, αCA2, and αCA3. The GUS reporter system was used to determine which specific tissues expressed αCA genes. The αCA1-GUS and αCA3-GUS constructs both show expression in petioles whereas the αCA2-GUS construct shows specific expression in trichomes. In addition, eGFP constructs were made to determine the subcellular localization of αCA proteins. Preliminary localization of αCA1-eGFP, αCA2-eGFP, and αCA3-eGFP in Nicotiana benthamiana leaf tissue suggests localization to the epidermal cell membrane. Stable expression of the αCA1-eGFP and αCA3-eGFP constructs in Arabidopsis appear to show localization at the epidermal cell membrane as well. Stably expressing Arabidopsis protoplasts specifies the localization as cell wall. Furthermore, T-DNA knockout mutants were obtained to assess above-ground biomass and projected rosette area under low (200 ppm) and high (1000 ppm) CO2 conditions. Growth studies were conducted on T-DNA knockout lines of single mutants αca1, αca2, βca4, and the double mutants αca1βca4 and αca2βca4. All mutant lines showed slight decreases in fresh weight, dry weight, and projected rosette area under low CO2 conditions. Exposure to high CO2 conditions restored the αca1 and αca2 single mutants to wild type (COL). However, the βca4 single mutant and the αca1βca4 and αca2βca4 double mutants continued to show an appreciably lower growth phenotype than COL.

Date

11-8-2017

Committee Chair

Moroney, James

DOI

10.31390/gradschool_theses.4333

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