Plastid carbonic anhydrases are essential for growth of Arabidopsis on ambient air but not for photosynthesis

The physiological role of chloroplast carbonic anhydrases (CAs) has long been debated, particularly in the context of photosynthesis. While early hypotheses proposed that CAs enhance CO₂ assimilation by rapidly accessing the HCO3- pool, direct evidence has been lacking. In this study, we examined Arabidopsis (Arabidopsis thaliana) mutants lacking both chloroplast-localized βCA1 (AT3G01500) and βCA5 (AT4G33580) to assess their impact on plant growth and photosynthetic performance. Our results show that plants deficient in chloroplast CA activity are unable to grow under ambient CO₂ conditions (400 μL·L⁻¹) but can complete their life cycle under elevated CO₂ levels (≥12,000 μL·L⁻¹). However, CO₂ assimilation rates and ΦII measurements in CA-deficient plants were comparable to those in wild-type plants under 0.04% (400 μL·L⁻¹), 0.4% (4,000 μL·L⁻¹), and 4% CO₂ (40,000 μL·L⁻¹) concentrations, indicating that chloroplast CAs are not essential for photosynthetic CO₂ fixation. Instead, our findings suggest that chloroplast CA activity is critical for supporting other metabolic pathways, namely amino acid, nucleic acid, and fatty acid biosynthesis. Expression of the Chlamydomonas (Chlamydomonas reinhardtii) bicarbonate transporter LCIA in chloroplast CA mutants partially rescued the growth phenotype under near-ambient CO₂ conditions. These LCIA-complemented lines showed no difference in photosynthesis, further supporting the role of CAs in non-photosynthetic reactions. This work provides direct evidence that while chloroplast CAs are dispensable for photosynthesis, they are essential for plant growth and development under ambient CO₂ due to their role in increasing the bicarbonate concentration for specific anaplerotic pathways.