Features of Photosynthesis in Arabidopsis thaliana Plants with Knocked Out Gene of Alpha Carbonic Anhydrase 2.

The knockout of the At2g28210 gene encoding α-carbonic anhydrase 2 (α-CA2) in Arabidopsis thaliana (Columbia) led to alterations in photosynthetic processes. The effective quantum yields of both photosystem II (PSII) and photosystem I (PSI) were higher in α-carbonic anhydrase 2 knockout plants (α-CA2-KO), and the reduction state of plastoquinone pool was lower than in wild type (WT). The electron transport rate in the isolated thylakoids measured with methyl viologen was higher in α-CA2-KO plants. The amounts of reaction centers of PSII and PSI were similar in WT and α-CA2-KO plants. The non-photochemical quenching of chlorophyll a fluorescence in α-CA2-KO leaves was lower at the beginning of illumination, but became slightly higher than in WT leaves when the steady state was achieved. The degree of state transitions in the leaves was lower in α-CA2-KO than in WT plants. Measurements of the electrochromic carotenoid absorbance shift (ECS) revealed that the light-dependent pH gradient (ΔpH) across the thylakoid membrane was lower in the leaves of α-CA2-KO plants than in WT plants. The starch content in α-CA2-KO leaves was lower than in WT plants. The expression levels of the genes encoding chloroplast CAs in α-CA2-KO changed noticeably, whereas the expression levels of genes of cytoplasmic CAs remained almost the same. It is proposed that α-CA2 may be situated in the chloroplasts.

The Role of Carbonic Anhydrase αCA4 in Photosynthetic Reactions in Arabidopsis thaliana Studied, Using the Cas9 and T-DNA Induced Mutations in Its Gene.

An homozygous mutant line of Arabidopsis thaliana with a knocked out At4g20990 gene encoding thylakoid carbonic anhydrase αCA4 was created using a CRISPR/Cas9 genome editing system. The effects of the mutation were compared with those in two mutant lines obtained by the T-DNA insertion method. In αCA4 knockouts of all three lines, non-photochemical quenching of chlorophyll a fluorescence was lower than in the wild type (WT) plants due to a decrease in its energy-dependent component. The αCA4 knockout also affected the level of expression of the genes encoding all proteins of the PSII light harvesting antennae, the genes encoding cytoplasmic and thylakoid CAs and the genes induced by plant immune signals. The production level of starch synthesis during the light period, as well as the level of its utilization during the darkness, were significantly higher in these mutants than in WT plants. These data confirm that the previously observed differences between insertional mutants and WT plants were not the result of the negative effects of T-DNA insertion transgenesis but the results of αCA4 gene knockout. Overall, the data indicate the involvement of αCA4 in the photosynthetic reactions in the thylakoid membrane, in particular in processes associated with the protection of higher plants' photosynthetic apparatus from photoinhibition.

Effect of CO2 Content in Air on the Activity of Carbonic Anhydrases in Cytoplasm, Chloroplasts, and Mitochondria and the Expression Level of Carbonic Anhydrase Genes of the α- and ß-Families in Arabidopsis thaliana Leaves.

The carbonic anhydrase (CA) activities of the preparations of cytoplasm, mitochondria, chloroplast stroma, and chloroplast thylakoids, as well as the expression levels of genes encoding αCA1, αCA2, αCA4, ßCA1, ßCA2, ßCA3, ßCA4, ßCA5, and ßCA6, were measured in the leaves of Arabidopsis thaliana plants, acclimated to different CO2 content in the air: low (150 ppm, lCO2), normal (450 ppm, nCO2), and high (1200 ppm, hCO2). To evaluate the photosynthetic apparatus operation, the carbon assimilation and chlorophyll a fluorescence were measured under the same conditions. It was found that the CA activities of the preparations of cytoplasm, chloroplast stroma, and chloroplast thylakoids measured after two weeks of acclimation were higher, the lower CO2 concentration in the air. That was preceded by an increase in the expression levels of genes encoding the cytoplasmic form of ßCA1, and other cytoplasmic CAs, ßCA2, ßCA3, and ßCA4, as well as of the chloroplast CAs, ßCA5, and the stromal forms of ßCA1 in a short-term range 1-2 days after the beginning of the acclimation. The dependence on the CO2 content in the air was most noticeable for the CA activity of the preparations of the stroma; it was two orders higher in lCO2 plants than in hCO2 plants. The CA activity of thylakoid membranes from lCO2 plants was higher than that in nCO2 and hCO2 plants; however, in these plants, a significant increase in the expression levels of the genes encoding αCA2 and αCA4 located in thylakoid membranes was not observed. The CA activity of mitochondria and the expression level of the mitochondrial ßCA6 gene did not depend on the content of carbon dioxide. Taken together, the data implied that in the higher plants, the supply of inorganic carbon to carboxylation sites is carried out with the cooperative functioning of CAs located in the cytoplasm and CAs located in the chloroplasts.

Advances in understanding the physiological role and locations of carbonic anhydrases in C3 plant cells.

The review describes the structures of plant carbonic anhydrases (CAs), enzymes catalyzing the interconversion of inorganic carbon forms and belonging to different families, as well as the interaction of inhibitors and activators of CA activity with the active sites of CAs in representatives of these families. We outline the data that shed light on the location of CAs in green cells of C3 plants, algae and angiosperms, with the emphasis on the recently obtained data. The proven and proposed functions of CAs in these organisms are listed. The possibility of the involvement of several chloroplast CAs in acceleration of the conversion of bicarbonate to CO2 and in supply of CO2 for fixation by Rubisco is particularly considered. Special attention is paid to CAs in various parts of thylakoids and to discussion about current knowledge of their possible physiological roles. The review states that, despite the significant progress in application of the mutants with suppressed CAs synthesis, the approach based on the use of the inhibitors of CA activity in some cases remains quite effective. Combination of these two approaches, namely determining the effect of CA activity inhibitors in plants with certain knocked-out CA genes, turns out to be very useful for understanding the functions of other CAs.

Regulation of the size of photosystem II light harvesting antenna represents a universal mechanism of higher plant acclimation to stress conditions.

We investigated acclimatory responses of Arabidopsis plants to drought and salinity conditions before the appearance of obvious signs of damage caused by these factors. We detected changes indicating an increase in the reduction level of the chloroplast plastoquinone pool (PQ pool) 5-7 days after introduction of the stress factors. After 10-14 days, a decrease in the size of PSII light harvesting antenna was observed in plants under conditions of drought and salinity. This was confirmed by a decrease in content of PSII antenna proteins and by downregulation of gene expression levels of these proteins under the stress conditions. No changes in values of performance index and maximum quantum yield of PSII were detected. Under drought and salinity, the content of hydrogen peroxide in leaves was higher than in control leaves. Thus, we propose that reduction of the size of PSII antenna represents one of the universal mechanisms of acclimation of higher plants to stress factors and the downsizing already begins to manifest under mild stress conditions. Both the PQ pool reduction state and the hydrogen peroxide content are important factors needed for the observed rearrangement.

Influence of knockout of At4g20990 gene encoding α-CA4 on photosystem II light-harvesting antenna in plants grown under different light intensities and day lengths.

Effect of knockout of the At4g20990 gene encoding α-carbonic anhydrase 4 (α-CA4) in Arabidopsis thaliana in plants grown in low light (LL, 80 µmol photons m-2 s-1) or in high light (HL, 400 µmol photons m-2 s-1) under long (LD, 16 h) or short (SD, 8 h) day length was studied. In α-CA4 knockout plants, under all studied conditions, the non-photochemical quenching was lower; the decrease was more pronounced under HL. This pointed to α-CA4 implication in the processes leading to energy dissipation in PSII antenna. In this context the content of major antenna proteins Lhcb1 and Lhcb2 was lower in α-CA4 knockouts than in wild-type (WT) plants under all growth conditions. The expression level of lhcb2 gene was also lower in mutants grown under LD, LL and HL in comparison to WT. At the same time, this level was higher in mutants grown under SD, LL and it was the same under SD, HL. Overall, the data showed that the knockout of the At4g20990 gene affected both the contents of proteins of PSII light-harvesting complex and the expression level of genes encoding these proteins, with peculiarities dependent on day length. These data together with the fact of a decrease of non-photochemical quenching of leaf chlorophyll a fluorescence in α-CA4-mut as compared with that in WT plants implied that α-CA4 participates in acclimation of photosynthetic apparatus to light intensity, possibly playing important role in the photoprotection. The role of this CA can be especially important in plants growing under high illumination conditions.

Carbonic anhydrase activity in Arabidopsis thaliana thylakoid membrane and fragments enriched with PSI or PSII.

The procedure of isolating the thylakoids and the thylakoid membrane fragments enriched with either photosystem I or photosystem II (PSI- and PSII-membranes) from Arabidopsis thaliana leaves was developed. It differed from the one used with pea and spinach in durations of detergent treatment and centrifugation, and in concentrations of detergent and Mg(2+) in the media. Both the thylakoid and the fragments preserved carbonic anhydrase (CA) activities. Using nondenaturing electrophoresis followed by detection of CA activity in the gel stained with bromo thymol blue, one low molecular mass carrier of CA activity was found in the PSI-membranes, and two carriers, a low molecular mass one and a high molecular mass one, were found in the PSII-membranes. The proteins in the PSII-membranes differed in their sensitivity to acetazolamide (AA), a specific CA inhibitor. AA at 5 × 10(-7) M inhibited the CA activity of the high molecular mass protein but stimulated the activity of the low molecular mass carrier in the PSII-membranes. At the same concentration, AA moderately inhibited, by 30%, the CA activity of PSI-membranes. CA activity of the PSII-membranes was almost completely suppressed by the lipophilic CA inhibitor, ethoxyzolamide at 10(-9) M, whereas CA activity of the PSI-membranes was inhibited by this inhibitor even at 5 × 10(-7) M just the same as for AA. The observed distribution of CA activity in the thylakoid membranes from A. thaliana was close to the one found in the membranes of pea, evidencing the general pattern of CA activity in the thylakoid membranes of C3-plants.

Multiple sources of carbonic anhydrase activity in pea thylakoids: soluble and membrane-bound forms.

Carbonic anhydrase (CA) activity of pea thylakoids, thylakoid membranes enriched with photosystem I (PSI-membranes), or photosystem II (PSII-membranes) as well as both supernatant and pellet after precipitation of thylakoids treated with detergent Triton X-100 were studied. CA activity of thylakoids in the presence of varying concentrations of Triton X-100 had two maxima, at Triton/chlorophyll (triton/Chl) ratios of 0.3 and 1.0. CA activities of PSI-membranes and PSII-membranes had only one maximum each, at Triton/Chl ratio 0.3 or 1.0, respectively. Two CAs with characteristics of the membrane-bound proteins and one CA with characteristics of the soluble proteins were found in the medium after thylakoids were incubated with Triton. One of the first two CAs had mobility in PAAG after native electrophoresis the same as that of CA residing in PSI-membranes, and the other CA had mobility the same as the mobility of CA residing in PSII-membranes, but the latter was different from CA situated in PSII core-complex (Ignatova et al. 2006 Biochemistry (Moscow) 71:525-532). The properties of the "soluble" CA removed from thylakoids were different from the properties of the known soluble CAs of plant cell: apparent molecular mass was about 262 kD and it was three orders more sensitive to the specific CA inhibitor, ethoxyzolamide, than soluble stromal CA. The data are discussed as indicating the presence of, at least, four CAs in pea thylakoids.