α Carbonic Anhydrase 1 Affects Photosynthetic CO₂ Assimilation and Stomatal Conductance in Arabidopsis thaliana Plants

Arabidopsis thaliana plants of two mutant lines with knocked out the At3g52720 gene, encoding stromal α-carbonic anhydrase 1, αCA1 (αCA1-KO), and wild-type plants, of the Columbia ecotype (Col), were grown under 450 ppm (NCO₂) and 150 ppm (LCO₂) CO₂ in the air. The CO₂ assimilation rate (ACO₂) in plants grown under NCO₂ was lower in the αCA1-KO plants than in Col plants at CO₂ concentrations from 600 to 1200 ppm in the measurement chamber. In LCO₂-grown plants, ACO₂ values were lower than in NCO₂-grown plants both in Col and mutants, with the same values in mutants and Col. Water use efficiency (WUE) in NCO₂ grown mutant plants was lower than in Col at a CO₂ concentration of 600 ppm and above, and in LCO₂-grown mutants WUE became lower than in Col at 1200 ppm. The starch content was lower in leaves of mutants than in Col under both NCO₂ and LCO₂ conditions due to its reduced synthesis during the daytime photosynthesis. Under NCO₂, the content of hydrogen peroxide was twice higher in αCA1‑KO plant leaves than in Col. Under LCO₂, the content of Н₂О₂ in Col increased to more than three times its level under NCO₂, and became 2.5 times higher than in αCA1‑KO, in which it decreased by 1.5 times compared to that in NCO₂ plants. Under NCO₂ conditions, the contents of the stromal, cytoplasmic and peroxisomal forms of ascorbate peroxidases (APXs) were about twice higher in αCA1-KO than in Col plants. Contrariwise, in LCO₂-grown plants the contents of APXs were lower in mutants than in Col. The total protein amount, as well as the level of ribulose-1,5-bisphosphate carboxylase/oxygenase, were the same in Col and mutants under NCO₂ conditions, but were 35% and 45-50%, respectively, lower in αCA1-KO than in Col under LCO₂ conditions. In the leaves of NCO₂-grown plants, the stomatal conductance was higher in αCA1-KO than in Col with slower stomatal opening and delayed closure in response to decreases and increases in CO₂ level, respectively. At the same time, in the absence of αCA1, both the total number of stomata per unit leaf surface area, and the relative number of open stomata were higher than in Col leaves. In the protoplasts isolated from NCO₂-grown αCA1-KO plants, the steady-state rates of oxygen evolution were 30 % lower in mutants than in Col and were reached at a lower HCO₃¯ concentration (5 mM) than in protoplasts from Col (8 mM). The apparent half-saturating bicarbonate concentration was approximately 2 mM for αCA1-KO and 3 mM for Col. Our data demonstrate the involvement of the stromal chloroplast αCA1 in providing A. thaliana cells with CO₂, the importance of this enzyme for starch synthesis, as well as the significance of αCA1 for protein synthesis in plants. The increase in the total number of stomata per unit of leaf surface area and in the relative proportion of open stomata, as well as the reduced rate of stomatal responses in plants lacking αCA1, suggests the possible involvement of this enzyme in signaling processes regulating both stomatal movement and development.