Degree

Doctor of Philosophy (PhD)

Department

Department of Biological Sciences

Document Type

Dissertation

Abstract

Carbonic anhydrases (CAs) are zinc-metalloenzymes that interconvert two inorganic carbon (Ci) species, CO2 and HCO3-. In Arabidopsis thaliana, there are eight αCA genes, six βCA genes, three γCA genes, and two γCA-like genes. The majority of CA research in plants has focused on finding a link between CA activity and photosynthesis rates. Since the CA genes are expressed in different plant tissues and multiple CA isoforms are distributed among various organelles of the plant cell, I hypothesize that CAs facilitate CO2 diffusion among cell compartments and maintain Ci pools for carbon-requiring reactions by interconverting CO2 and HCO3-. This thesis focuses on the αCAs and βCAs of Arabidopsis and how they may affect various reactions throughout the plant. CA T-DNA insertion lines were used to determine if removing one or more CAs from Arabidopsis affects plant growth. As my first aim, the physiological roles of two plastid CAs, βCA1, and βCA5 were studied. The growth of the βca5 single mutant was severely stunted in ambient CO2 conditions, and high CO2 partially rescued wild-type growth in the βca5 plants. However, βca1 mutant plants grew like wild-type plants in ambient CO2. This is notable because βCA1 comprises nearly 1% of all soluble protein in the leaf. Also, βCA1 is expressed at much higher levels than βCA5 in leaves. Despite such high levels of βCA1 in the leaf, it was the βca5 mutant that showed a drastic growth reduction in ambient air. We hypothesize that it is the differences in tissue expression of βCA1 and βCA5 that explain these results. The lack of expression of βCA1 in roots suggests that root plastids have no CA activity when we eliminate βCA5 expression using gene disruption. This would then cause defects in important anaplerotic pathways requiring a carboxylase step that take place within plastids, like fatty acid biosynthesis. The lack of a plastid CA in the roots, therefore, leads to the very poor growth-phenotype of βca5 plants on ambient CO2. Here I present data demonstrating that βCA5 is supplying HCO3- required for anaplerotic pathways that take place in plastids, such as fatty acid synthesis. My second aim was to study the importance of cell wall and plasma membrane CAs, αCA2 and βCA4. For that, I used the cell wall and plasma membrane associated CA mutant line αca2βca4. The double mutant plants showed a significant reduction in growth when compared to wild-type plants when grown under low CO2 conditions. Interestingly, it seems the reduced growth of the αca2βca4 double mutant plants was linked to deficiencies in photosynthesis rates. These results suggest that CAs are playing more complex roles in plants than once thought and that the various isoforms are affecting different carbon-requiring pathways.

Date

4-16-2023

Committee Chair

Moroney, James

DOI

10.31390/gradschool_dissertations.6101

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