The removal of two cytoplasmic carbonic anhydrases, βCA2 and βCA4, from Arabidopsis causes reduced growth in plants grown in a low-CO2 environment.

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.

Phosphoenolpyruvate (PEP) carboxylase (PEPc) catalyzes the first committed step of C₄ photosynthesis generating oxaloacetate from bicarbonate (HCO₃⁻) and PEP. It is hypothesized that PEPc affinity for HCO₃⁻ has undergone selective pressure for a lower K_HCO3 (K_m for HCO₃⁻) to increase the carbon flux entering the C₄ cycle, particularly during conditions that limit CO₂ availability. However, the decrease in K_HCO3 has been hypothesized to cause an unavoidable increase in K_PEP (K_m for PEP). Therefore, the amino acid residue S774 in the C₄ enzyme, which has been shown to increase K_PEP, should lead to a decrease in K_HCO3. Several studies reported the effect S774 has on K_PEP; however, the influence of this amino acid substitution on K_HCO3 has not been tested. To test these hypotheses, membrane-inlet mass spectrometry (MIMS) was used to measure the K_HCO3 of the photosynthetic PEPc from the C₄Flaveria trinervia and the non-photosynthetic PEPc from the C₃F. pringlei. The cDNAs for these enzymes were overexpressed and purified from the PEPc-less PCR1 Escherichia coli strain. Our work in comparison with previous reports suggests that K_HCO3 and K_PEP are linked by specific amino acids, such as S774; however, these kinetic parameters respond differently to the tested allosteric regulators, malate and glucose-6-phosphate.