The knockout of either At3g01500 or At3g52720 gene encoding Arabidopsis thaliana βCA1 and αCA1 carbonic anhydrase (CA), respectively, led to a lower CA activity of the chloroplast stroma preparations from the knockout mutant plants (αCA1-KO and βCA1-KO) compared to such preparations from the wild-type (WT) plants. To identify the differences in the photosynthetic characteristics of mutant and WT plants, they were grown in low light (LL; 50-70 µmol quanta∙m⁻²∙s⁻¹, natural conditions) and high light (HL; 400 µmol quanta∙m⁻²∙s⁻¹, stressful conditions). The rate of CO₂ assimilation measured at 400 µmol quanta∙m⁻²∙s⁻¹ in plants grown under LL was lower in αCA1-KO and βCA1-KO mutants compared to WT plants. The rate of photosynthetic electron transport was lower in αCA1-KO plants and higher in βCA1-KO plants than in WT plants; the content of CO₂ in chloroplasts was lower in βCA1-KO plants than in both WT and αCA1-KO plants, where it differed little. The value of the proton-motive force was higher in βCA1-KO plants and lower in αCA1-KO plants than in WT plants due to changes in ΔpH value. The obtained results suggest that βCA1 facilitates the intake of inorganic carbon into chloroplasts, while αCA1 ensures the conversion of bicarbonate into CO₂ in the chloroplast stroma for its use in the reaction catalyzed by Ribulose bisphosphate carboxylase/oxygenase (RuBisCO). In both αCA1-KO and βCA1-KO mutants, the expression levels of genes encoding other chloroplast CAs differed markedly from those in WT plants; the pattern of the changes in the genes expression depended on the light intensity during cultivation. The content of hydrogen peroxide in the leaves of both αCA1-KO and βCA1-KO mutants was higher in LL and lower in HL than in WT plants. The expression levels of stress marker genes changed similarly in both types of mutant plants. A possible involvement of the chloroplast stroma CAs in the transmission of stress signals in higher plants is discussed.

I. According to the World Resources Institute, the global population is expected to reach 10 billion by 2050 (Searchinger et al. 2019). The Green Revolution of the 1960s increased crop yields throu...

Arabidopsis thaliana plants of two mutant lines with a knockout of 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 55% higher in αCA1-KO plant leaves than in Col. Under LCO₂, the content of H₂O₂ 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 34% compared to that in NCO₂ plants. Under NCO₂ conditions, the contents of the stromal, cytosolic, 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 4 mM for Col. Our data demonstrate the involvement of the stromal chloroplast αCA1 in providing A. thaliana cells with CO₂, and imply the significance of αCA1 in supplying inorganic carbon for starch and 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.

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.