Carbonic anhydrases in the cell wall and plasma membrane of Arabidopsis thaliana are required for optimal plant growth on low CO2

Introduction: Plants have many genes encoding both alpha and beta type carbonic anhydrases. Arabidopsis has eight alpha type and six beta type carbonic anhydrase genes. Individual carbonic anhydrases are localized to specific compartments within the plant cell. In this study, we investigate the roles of αCA2 and βCA4.1 in the growth of the plant Arabidopsis thaliana under different CO2 regimes.Methods: Here, we identified the intracellular location of αCA2 and βCA4.1 by linking the coding region of each gene to a fluorescent tag. Tissue expression was determined by investigating GUS expression driven by the αCA2 and βCA4.1 promoters. Finally, the role of these proteins in plant growth and photosynthesis was tested in plants with T-DNA insertions in the αCA2 and βCA4 genes.Results: Fluorescently tagged proteins showed that αCA2 is localized to the cell wall and βCA4.1 to the plasma membrane in plant leaves. Both proteins were expressed in roots and shoots. Plants missing either αCA2 or βCA4 did not show any growth defects under the conditions tested in this study. However, if both αCA2 and βCA4 were disrupted, plants had a significantly smaller above- ground fresh weight and rosette area than Wild Type (WT) plants when grown at 200 μL L−1 CO2 but not at 400 and 1,000 μL L−1 CO2. Growth of the double mutant plants at 200 μL L−1 CO2 was restoredif either αCA2 or βCA4.1 was transformed back into the double mutant plants.Discussion: Both the cell wall and plasma membrane CAs, αCA2 and βCA4.1 had to be knocked down to produce an effect on Arabidopsis growth and only when grown in a CO2 concentration that was significantly below ambient. This indicates that αCA2 and βCA4.1 have overlapping functions since the growth of lines where only one of these CAs was knocked down was indistinguishable from WT growth. The growth results and cellular locations of the two CAs suggest that together, αCA2 and βCA4.1 play an important role in the delivery of CO2 and HCO3− to the plant cell

DataSheet1_Carbonic anhydrases in the cell wall and plasma membrane of Arabidopsis thaliana are required for optimal plant growth on low CO2.PDF

Introduction: Plants have many genes encoding both alpha and beta type carbonic anhydrases. Arabidopsis has eight alpha type and six beta type carbonic anhydrase genes. Individual carbonic anhydrases are localized to specific compartments within the plant cell. In this study, we investigate the roles of αCA2 and βCA4.1 in the growth of the plant Arabidopsis thaliana under different CO2 regimes.Methods: Here, we identified the intracellular location of αCA2 and βCA4.1 by linking the coding region of each gene to a fluorescent tag. Tissue expression was determined by investigating GUS expression driven by the αCA2 and βCA4.1 promoters. Finally, the role of these proteins in plant growth and photosynthesis was tested in plants with T-DNA insertions in the αCA2 and βCA4 genes.Results: Fluorescently tagged proteins showed that αCA2 is localized to the cell wall and βCA4.1 to the plasma membrane in plant leaves. Both proteins were expressed in roots and shoots. Plants missing either αCA2 or βCA4 did not show any growth defects under the conditions tested in this study. However, if both αCA2 and βCA4 were disrupted, plants had a significantly smaller above- ground fresh weight and rosette area than Wild Type (WT) plants when grown at 200 μL L−1 CO2 but not at 400 and 1,000 μL L−1 CO2. Growth of the double mutant plants at 200 μL L−1 CO2 was restoredif either αCA2 or βCA4.1 was transformed back into the double mutant plants.Discussion: Both the cell wall and plasma membrane CAs, αCA2 and βCA4.1 had to be knocked down to produce an effect on Arabidopsis growth and only when grown in a CO2 concentration that was significantly below ambient. This indicates that αCA2 and βCA4.1 have overlapping functions since the growth of lines where only one of these CAs was knocked down was indistinguishable from WT growth. The growth results and cellular locations of the two CAs suggest that together, αCA2 and βCA4.1 play an important role in the delivery of CO2 and HCO3− to the plant cell.</p

Arabidopsis plastid carbonic anhydrase βCA5 is important for normal plant growth

Carbonic anhydrases (CAs) are zinc-metalloenzymes that catalyze the interconversion of CO2 and HCO3-. In heterotrophic organisms, CAs provide HCO3- for metabolic pathways requiring a carboxylation step. Arabidopsis (Arabidopsis thaliana) has 14 α- and β-type CAs, two of which are plastid CAs designated as βCA1 and βCA5. To study their physiological properties, we obtained knock-out (KO) lines for βCA1 (SALK_106570) and βCA5 (SALK_121932). These mutant lines were confirmed by genomic PCR, RT-PCR, and immunoblotting. While βca1 KO plants grew normally, growth of βca5 KO plants was stunted under ambient CO2 conditions of 400 µL L-1; high CO2 conditions (30,000 µL L-1) partially rescued their growth. These results were surprising, as βCA1 is more abundant than βCA5 in leaves. However, tissue expression patterns of these genes indicated that βCA1 is expressed only in shoot tissue, while βCA5 is expressed throughout the plant. We hypothesize that βCA5 compensates for loss of βCA1 but, owing to its expression being limited to leaves, βCA1 cannot compensate for loss of βCA5. We also demonstrate that βCA5 supplies HCO3- required for anaplerotic pathways that take place in plastids, such as fatty acid biosynthesis