L. K. Ignatova

Carbonic Anhydrase Activities in Pea Thylakoids

Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 µmol H+ (mg Chl)−1 h−1 at pH 7.0) were prepared. Western blot analysis using antibodies raised against the soluble stromal β-CA from spinach clearly showed that this activity is not a result of contamination of the thylakoids with the stromal CA but is derived from a thylakoid membrane-associated CA. Increase of the CA activity after partial membrane disintegration by detergent treatment, freezing or sonication implies the location of the CA in the thylakoid interior. Salt treatment of thylakoids demonstrated that while one part of the initial enzyme activity is easily soluble, the rest of it appears to be tightly associated with the membrane. CA activity being measured as HCO3− dehydration (dehydrase activity) in Photosystem II particles (BBY) was variable and usually low. The highest and most reproducible activities (approximately 2000 µmol H+ (mg Chl)−1 h−1) were observed in the presence of detergents (Triton X-100 or n-octyl-β-D-glucopyranoside) in low concentrations. The dehydrase CA activity of BBY particles was more sensitive to the lipophilic CA inhibitor, ethoxyzolamide, than to the hydrophilic CA inhibitor, acetazolamide. CA activity was detected in PS II core complexes with average rate of 13,000 µmol H+ (mg Chl)−1 h−1 which was comparable to CA activity in BBY particles normalized on a PS II reaction center basis.

Light-induced stimulation of carbonic anhydrase activity in pea thylakoids

Stimulation of the bicarbonate dehydration reaction in thylakoid suspension under conditions of saturating light at pH 7.6–8.0 was discovered. This effect was inhibited by nigericin or the lipophilic carbonic anhydrase (CA) inhibitor ethoxyzolamide (EZ), but not by the hydrophilic CA inhibitor, acetazolamide. It was shown that the action of EZ is not caused by an uncoupling effect. It was concluded that thylakoid CA is the enzyme utilizing the light‐generated proton gradient across the thylakoid membrane thus facilitating the production of CO2 from HCO3 − and that this enzyme is covered from the stroma side of thylakoids by a lipid barrier.

Diversity in forms and functions of carbonic anhydrase in terrestrial higher plants

The review summarizes current data on the existence in terrestrial higher plants of several carbonic anhydrase forms differing in their properties, molecular structure, and intracellular localization. Possible functions of these carbonic anhydrases are discussed as well as specific features of carbon-concentrating mechanisms in phototrophic tissues of plants with C3 and C4 pathways of photosynthesis.

Photosystem II associated carbonic anhydrase activity in higher plants is situated in core complex

The thylakoid membrane containing photosystem II (PSII membranes) from pea and wheat leaves catalyzed the reaction of CO2 hydration with low rate, which increased after their incubation either with Triton X‐100, up to Triton/chlorophyll ratio 1:1, or 1 M CaCl2. The presence of the inhibitor of CAs, p‐aminomethylbenzensulfonamide (mafenide), at the start line in the course of electrophoresis of PSII membranes solubilized by n‐dodecyl‐β‐maltoside (DM) decreased the amount of PSII core complex in the gel. The elution of PSII core complex from the column with immobilized mafenide occurred only either by mafenide or another inhibitor of CAs, ethoxyzolamide. The above results led to a conclusion that membrane‐bound CA activity associated with PSII is situated in the core complex.

Carbonic anhydrases in photosynthetic cells of higher plants

This review presents information about carbonic anhydrases, enzymes catalyzing the reversible hydration of carbon dioxide in aqueous solutions. The families of carbonic anhydrases are described, and data concerning the presence of their representatives in organisms of different classes, and especially in the higher plants, are considered. Proven and hypothetical functions of carbonic anhydrases in living organisms are listed. Particular attention is given to those functions of the enzyme that are relevant to photosynthetic reactions. These functions in algae are briefly described. Data about probable functions of carbonic anhydrases in plasma membrane, mitochondria, and chloroplast stroma of higher plants are discussed. Update concerning carbonic anhydrases in chloroplast thylakoids of higher plants, i.e. their quantity and possible participation in photosynthetic reactions, is given in detail.

Heterogeneous origin of carbonic anhydrase activity of thylakoid membranes

Carbonic anhydrase activities of pea thylakoids as well as thylakoid fragments enriched either in Photosystem 1 (PS1-membranes) or Photosystem 2 (PS2-membranes) were studied. The activity of PS1-membranes if calculated on chlorophyll basis was much higher than the activity of PS2-membranes. Acetazolamide, a non-permeable inhibitor of carbonic anhydrases, increased carbonic anhydrase activity of PS2-membranes at concentrations lower than 10−6 M and suppressed this activity only at higher concentrations. A lipophilic inhibitor of carbonic anhydrases, ethoxyzolamide, effectively suppressed the carbonic anhydrase activity of PS2-membranes (I50 = 10−9 M). Carbonic anhydrase activity of PS1-membranes was suppressed alike by both inhibitors (I50 = 10−6 M). In the course of the electrophoresis of PS2-membranes treated with n-dodecyl-β-maltoside “high-molecular-mass” carbonic anhydrase activity was revealed in the region corresponding to core-complex of this photosystem. Besides, carbonic anhydrase activity in the region of low-molecular-mass proteins was discovered in the course of such an electrophoresis of both PS2-and PS1-membranes. These low-molecular-mass carbonic anhydrases eluted from corresponding gels differed in sensitivity to specific carbonic anhydrase inhibitors just the same as PS1-membranes versus PS2-membranes. The results are considered as evidence for the presence in the thylakoid membranes of three carriers of carbonic anhydrase activity.

Growth, photosynthesis, and metabolism of sugar beet at an early stage of exposure to elevated CO2

The effects of CO2 concentration (Ca) on growth, photosynthesis, and the activity of enzymes associated with the translocation and assimilation of CO2 were studied in sugar beet (Beta vulgaris L. subsp. saccharifera, cv. Ramonskaya) plants. The plants were grown in controlled-climate chamber to the stage of 3–4 leaves and then used in experiments. Experimental plants were exposed in boxes to doubled Ca (700 µl/l, 2C plants), whereas control plants were kept in a chamber with ambient atmosphere (350 µl/l, 1C plants). As compared with 1C plants, in 3 and 8 days, the leaf area of 2C plants increased by 14 and 26%, respectively. The rate of their photosynthesis (Pn) measured in 3, 6, and 8 days increased by 85, 47, and 52%, respectively, whereas in normal air, the values of Pn in 2C plants were by 12, 19, and 15% lower than in 1C plants. After 8-day growth, the content of soluble carbohydrates in the leaves of 2C plants attained 7.2%, being by 80% greater than in 1C plants; the content of starch did not exceed 3%. The total content of chlorophylls a and b in the leaves of 2C plants was by 14% greater than in 1C plants, but their ratio was essentially the same. The level of protein in 2C plants was by 13.4% lower than in 1C plants. The activity and content of Rubisco in 1C and 2C plants were similar. As compared with 1C plants, in 2C plants the activity of soluble carbonic anhydrase (sCA) was lower by 34% in 3 days and by 18% in 8 days; the activity of carbonic anhydrase of membrane preparations (mCA), was lower by 24 and 77%, respectively. Catalase activity in 2C plants became by 8% lower than in 1C plants only after 8 days. A reduction in the photosynthetic ability of 2C plants in ambient atmosphere, a decrease in activity of sCA and, especially, of mCA observed together with invariable activity and content of Rubisco in the leaf extracts are interpreted as early symptoms of acclimation of young plants of sugar beet to elevated CO2.

Effect of knockout of α-carbonic anhydrase 4 gene on photosynthetic characteristics and starch accumulation in leaves of Arabidopsis thaliana

Effects of knockout of the gene encoding α-carbonic anhydrase 4 (α-CA4) on growth and photosynthesis of Thale cress (Arabidopsis thaliana (L.) Heynh., var. Columbia) were investigated. The shoot weight of mutant plants was found to be higher than in wild-type plants, and the leaves of mutants were enriched in starch content. The electron microscopy study revealed a considerable increase in the number and size of starch grains in chloroplasts of mutant plants. Comparison of wild-type and mutant plant leaves in terms of chlorophyll a fluorescence, coefficient of photochemical fluorescence quenching, effective quantum yield of photosystem II reaction, and nonphotochemical fluorescence quenching under steady-state illumination and saturating CO2 content in the air led to the proposal that α-CA4 participates in the development of nonphotochemical energy dissipation by accelerating the supply of protons for activation of violaxanthin deepoxidase and for structural changes in the light-harvesting complexes.

Participation of two carbonic anhydrases of the alpha family in photosynthetic reactions in Arabidopsis thaliana

The expression of genes of two carbonic anhydrases (CA) belonging to the a-family, α-CA2 and α-CA4 (according to the nomenclature in N. Fabre et al. (2007) Plant Cell Environ., 30, 617-629), was studied in arabidopsis (Arabidopsis thaliana, var. Columbia) leaves. The expression of the At2g28210 gene coding α-CA2 decreased under increase in plant illumination, while the expression of the At4g20990 gene coding α-CA4 increased. Under conditions close to optimal for photosynthesis, in plants with gene At2g28210 knockout, the effective quantum yield of photosystem 2 and the light-induced accumulation of hydrogen peroxide in leaves were lower than in wild type plants, while the coefficient of non-photochemical quenching of leaf chlorophyll a fluorescence and the rate of CO2 assimilation in leaves were higher. In plants with At4g20990 gene knockout, the same characteristics changed in opposite ways relative to wild type. Possible mechanisms of the participation of αa-CA2 and α-CA4 in photosynthetic reactions are discussed, taking into account that protons can be either consumed or released in the reactions they catalyze.

The presence of soluble carbonic anhydrase in the thylakoid lumen of chloroplasts from Arabidopsis leaves

Supernatant obtained after high-speed centrifugation of disrupted thylakoids that had been washed free from extrathylakoid carbonic anhydrases demonstrated carbonic anhydrase activity that was inhibited by the specific inhibitors acetazolamide and ethoxyzolamide. A distinctive feature of the effect of Triton X-100 on this activity also suggested that the source of the activity is a soluble protein. Native electrophoresis of a preparation obtained using chromatography with agarose/mafenide as an affinity sorbent revealed one protein band with carbonic anhydrase activity. The same protein was revealed in a mutant deficient in soluble stromal carbonic anhydrase β-CA1, and this indicated that the newly revealed carbonic anhydrase is not a product of the At3g01500 gene. These data imply the presence of soluble carbonic anhydrase in the thylakoid lumen of higher plants.