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 β-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, βCA1, βCA2, βCA3, βCA4, βCA5, and βCA6, 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 βCA1, and other cytoplasmic CAs, βCA2, βCA3, and βCA4, as well as of the chloroplast CAs, βCA5, and the stromal forms of βCA1 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 βCA6 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.


Introduction
In all living organisms, cells have several buffer systems, including those based on forms of Ci, CO 2 , and HCO 3 − . This is, apparently, one of the main reasons why the enzyme carbonic anhydrase (CA), which accelerates the interconversion of CO 2 and bicarbonate with the release and consumption of a proton, emerged at least eight times in the course of evolution in different groups of living organisms.
In autotrophs, which include inorganic carbon (Ci) into organic compounds, the interconversion of Ci forms is of particular importance. On the way from the medium to the carboxylation centers, where the fixation of inorganic carbon into organic compounds takes place, this interconversion must repeatedly occur to overcome the membranes and aqueous in these mutants. One of the promising ideas in solving the problem of the conversion of bicarbonate to CO 2 and the supply of the latter to Rubisco is the assumption that thylakoid CAs are involved in this process, additionally to the stromal CAs, αCA1, and/or βCA1 [23]. Most likely, this function can be carried out by αCA5, since this CA is situated on the stromal side of stromal thylakoid membranes, where the part of Rubisco associated with the thylakoid membrane is located [24,25].
The presence of Ci forms is important not only for the dark phase of photosynthesis but also for the optimal functioning of the electron transport chain. Bicarbonate is required for the electron transport on the acceptor side of PSII [26]. HCO 3 − interaction with non-heme iron ensures rapid electron transfer from Q A to Q B [27]. Bicarbonate is also required on the donor side of PSII, and it was suggested that CA located there could be involved in HCO 3 − delivery [27,28]. Studies on the functioning of αCA4 in Arabidopsis thylakoid implied that this CA is located close to light-harvesting PSII antennae [10,11,29]. The assumption about the role of αCA4 is that it directs the protons that arise from CO 2 hydration to the proteins (PsbS and/or violaxanthin de-epoxidase), which initiate the non-photochemical quenching of leaf chlorophyll a fluorescence (NPQ) [30][31][32]. These processes protect PSII from photoinhibition. Because of the CO 2 hydration bicarbonate molecules released in this reaction, they can bind with protons, which produce during water oxidation in the water-oxidizing PSII complex [33].
In recent years, more data on the involvement of CAs in higher plant cells for protection against stresses, in particular in transmitting a response signal to a negative environmental impact, have begun to appear [34][35][36][37]. Dabrowska-Bronk et al. [38] have shown that Arabidopsis plants with single mutations in all six βCAs genes had a weakened stress tolerance under high light conditions. At the same time, these authors emphasize that the regulation of βCAs' gene expression and the enzymatic activities of CAs are important for optimal plant growth and photosynthesis.
An understanding of the physiological roles of CAs in higher plants is still something elusive. Studies using mutants with the suppressed synthesis of CA genes usually turn out to be of little information, and we assume that the reason for this may be that CAs in plants cell function together, and some can replace the weakened function of one of them. The main aim of the present work was to determine the changes in the activity of CAs in different compartments of plant cells in parallel with the expression level of the genes encoding these CAs by exposing Arabidopsis plants to altered carbon dioxide content in the air, which is the main nutrition source for plants and the main substrate of CA reaction.

Photosynthetic and Stress State Characteristics of Arabidopsis Plants during Acclimation to Changed CO 2 Conditions
The acclimation of Arabidopsis plants grown under normal CO 2 levels in the air (450 ppm, nCO 2 ) to changed CO 2 levels in the air, low (150 ppm, lCO 2 ) and high (1200 ppm, hCO 2 ), was measured as changes of the induction of the chlorophyll a fluorescence before and 3, 9, and 16 days after exposure to changed CO 2 content. Plants grown at nCO 2 were used as a control for lCO 2 and hCO 2 plants of the same age.
In lCO 2 -and hCO 2 -acclimated plants versus nCO 2 plants, all parameters of OJIP kinetics commonly used for stress evaluation were changed. The photosynthetic performance index of PSII (PI abs ), which is estimated to absorption energy per the photochemically active reaction center of PSII, as well as the maximum quantum yield of PSII (Fv/Fm), decreased in the lCO 2 plants ( Figure 1A,B) after three days, whereas in hCO 2 plants, a decrease in these parameters was observed only after 9 and 16 days, respectively. These changes were in parallel with an increase in the dissipation of light energy per the active reaction center (DIo/RC), i.e., a parameter which characterizes the activation of the defense mechanisms of the photosynthetic apparatus in response to stress, also three days after exposure to lCO 2 and 16 days after the exposure to hCO 2 ( Figure 1C). The effect of the carbon dioxide level on the performance index of the total photosynthetic electron chain  (PI total ) was weaker than on PI abs , Fv/Fm, and DIo/RC ( Figure 1D). A decrease in the PI total in hCO 2 and lCO 2 plants compared to nCO 2 plants has appeared only by the ninth day.
Plants 2022, 11, x FOR PEER REVIEW 4 of reaction center (DIo/RC), i.e., a parameter which characterizes the activation of the defen mechanisms of the photosynthetic apparatus in response to stress, also three days aft exposure to lCO2 and 16 days after the exposure to hCO2 ( Figure 1C). The effect of t carbon dioxide level on the performance index of the total photosynthetic electron cha (PItotal) was weaker than on PIabs, Fv/Fm, and DIo/RC ( Figure 1D). A decrease in the PIt in hCO2 and lCO2 plants compared to nCO2 plants has appeared only by the ninth day. After 16 days of acclimation to low, normal, and high CO2 content in the air, we ha measured the expression level (expression intensity) of the genes, which are the marke of the induction of stress transcriptional cascades activated by the molecules of phytoho mones: abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA) (Figure 2). ABA an JA are synergists and both of them are antagonists of SA [39]. At1g29395 and At1g528 are both ABA-induced genes. At1g29395 encodes the integral membrane protein Co Regulated 414 Thylakoid Membrane 1 (COR414-TM1), located in the inner envelope chloroplasts, which provides tolerance to cold and water deprivation. At1g52890 encod NAC Domain Containing Protein19, one of the main transcription factors in plant abio stress responses. At1g17420 (lox3) and At5g42650 (aos) are induced by JA. At1g74710 e codes a lipoxygenase, which catalyzes the oxygenation of fatty acids. At5g42650 encod the allene oxide synthase that catalyzes the dehydration of the hydroperoxide to oxide the JA biosynthetic pathway. At1g74710 (icsi), At3g52430 (pad), and At1g64280 (npr1) a SA-induced genes. At1g74710 encodes a protein with isochorismate synthase activit After 16 days of acclimation to low, normal, and high CO 2 content in the air, we have measured the expression level (expression intensity) of the genes, which are the markers of the induction of stress transcriptional cascades activated by the molecules of phytohormones: abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA) (Figure 2). ABA and JA are synergists and both of them are antagonists of SA [39]. At1g29395 and At1g52890 are both ABA-induced genes. At1g29395 encodes the integral membrane protein Cold Regulated 414 Thylakoid Membrane 1 (COR414-TM1), located in the inner envelope of chloroplasts, which provides tolerance to cold and water deprivation. At1g52890 encodes NAC Domain Containing Protein19, one of the main transcription factors in plant abiotic stress responses. At1g17420 (lox3) and At5g42650 (aos) are induced by JA. At1g74710 encodes a lipoxygenase, which catalyzes the oxygenation of fatty acids. At5g42650 encodes the allene oxide synthase that catalyzes the dehydration of the hydroperoxide to oxide in the JA biosynthetic pathway. At1g74710 (icsi), At3g52430 (pad), and At1g64280 (npr1) are SA-induced genes. At1g74710 encodes a protein with isochorismate synthase activity, which is important for SA accumulation. At3g52430 encodes a lipase-like gene that is important for salicylic acid signaling. The nonexpressor of Pathogenesis-Related (NPR1) is a key regulator of the SA-mediated systemic acquired resistance (SAR) pathway.  In lCO2 plants, the contents of the transcripts of ABA-and JA-inducible ge At1g29395, At1g17420 (lox3), and At5g42650 (aos) were 16, 10, and 3 times higher than nCO2 plants, respectively (Figure 2A,B). The expression levels of SA-induced ge At3g52430 (pad) and At1g64280 (npr1) were 7 and 15 times lower in lCO2 plants than nCO2 plants ( Figure 2C).
In hCO2 plants, the response of stress marker genes was less expressed. Only a fo to five-fold increase in the content of the transcripts of the SA-induced At1g74710 (i gene versus nCO2 plants was observed ( Figure 2C). The other analyzed stress-mar genes had a tendency to be 10-50% higher in hCO2 plants than in nCO2 plants (Figure 2 C).

CO2 Assimilation and Carbon Levels in Leaves of Arabidopsis Plants Acclimated to Chang CO2 Content in the Air
The CO2 assimilation rate was measured in Arabidopsis plant leaves after 9 ( Fig  3A) and 14-16 ( Figure 3B) days of exposure to lCO2 and hCO2 with nCO2 plants of same age as a control. For this purpose, the measurement chamber of the LI-6800 Porta Photosynthesis System using the dynamic assimilation technique at CO2 concentratio varying from 0 to 1200 ppm has been used. By the ninth day of acclimation, the differen in CO2 assimilation rate between lCO2, nCO2, and hCO2 plants was imperceptible (Fig In lCO 2 plants, the contents of the transcripts of ABA-and JA-inducible genes At1g29395, At1g17420 (lox3), and At5g42650 (aos) were 16, 10, and 3 times higher than in nCO 2 plants, respectively (Figure 2A,B). The expression levels of SA-induced genes At3g52430 (pad) and At1g64280 (npr1) were 7 and 15 times lower in lCO 2 plants than in nCO 2 plants ( Figure 2C).
In hCO 2 plants, the response of stress marker genes was less expressed. Only a fourto five-fold increase in the content of the transcripts of the SA-induced At1g74710 (icsi) gene versus nCO 2 plants was observed ( Figure 2C). The other analyzed stress-marker genes had a tendency to be 10-50% higher in hCO 2 plants than in nCO 2 plants (Figure 2A-C).

CO 2 Assimilation and Carbon Levels in Leaves of Arabidopsis Plants Acclimated to Changed CO 2 Content in the Air
The CO 2 assimilation rate was measured in Arabidopsis plant leaves after 9 ( Figure 3A) and 14-16 ( Figure 3B) days of exposure to lCO 2 and hCO 2 with nCO 2 plants of the same age as a control. For this purpose, the measurement chamber of the LI-6800 Portable Photosynthesis System using the dynamic assimilation technique at CO 2 concentrations varying from 0 to 1200 ppm has been used. By the ninth day of acclimation, the difference in CO 2 assimilation rate between lCO 2 , nCO 2 , and hCO 2 plants was imperceptible ( Figure 3A). This difference became noticeable after about two weeks after the exposure to the changed CO 2 level ( Figure 3B). In lCO 2 plants, the CO 2 assimilation rate was lower than in nCO 2 plants. This difference was the highest in atmospheric CO 2 content in the measurement chamber.  . This difference became noticeable after about two weeks after the exposure to the changed CO2 level ( Figure 3B). In lCO2 plants, the CO2 assimilation rate was lower than in nCO2 plants. This difference was the highest in atmospheric CO2 content in the measurement chamber. The lowest CO 2 assimilation rate has been detected in the hCO 2 plants ( Figure 3B), which was the most expressed at 1200 ppm in the measurement chamber. Photosynthetic down-regulation in the hCO 2 plants ( Figure 1B), which has also been observed by Zheng et al. [40] at elevated CO 2 levels, could be the reason for the relatively low CO 2 assimilation rate in the hCO 2 plants against the nCO 2 and even the lCO 2 ones.
The carbon fixation products, starch and soluble carbohydrates, were determined in the Arabidopsis plants' leaves after 16 days of acclimation to the changed CO 2 level in the air. It was the lowest in the lCO 2 plants' leaves ( Table 1). The starch content in these plants was 70-80% lower, and the content of soluble carbohydrates was 20-30% lower than in the nCO 2 plants. In the hCO 2 plants, the content of starch was also lower than in the nCO 2 plants by about 30-40%, which correlates with the lower CO 2 assimilation rate in these plants ( Figure 3B). However, the content of soluble carbohydrates was slightly higher in the hCO 2 plants than in the nCO 2 ones. Thus, the presented results demonstrate that Arabidopsis plants grown at short day photoperiods and low photosynthetically active radiation (see Materials and Methods) require at least two weeks of acclimation to the changed CO 2 content in the air, both low and high.

CA Activity in Preparations of Cytoplasm, Mitochondria, and Chloroplasts Isolated from Leaves of Plants Acclimated to Low, Normal, and High CO 2 Level in the Air
We have found that CA activity in preparations of cytoplasm, where βCA4, βCA2, βCA1.4, and βCA3 are located, was about 30% higher in lCO 2 plants than in nCO 2 and hCO 2 ones ( Figure 4A). The CA activity of mitochondria preparations incubated with 1% Triton X-100 was independent of the CO 2 level in the air ( Figure 4B).
The CA activities of preparations isolated from chloroplasts were the most sensitive to the CO 2 levels in the air ( Figure 4C-E). We measured the CA activity of the stroma and thylakoids separately. The CA activity of the preparations of chloroplast stroma, where βCA1.1, βCA1.2, and αCA1 are located, increased three-fold in the lCO 2 plants compared to the nCO 2 plants. In the hCO 2 plants, the CA activity in these preparations was reduced to 3% of that in the nCO 2 plants. Thus, the activity of the CAs in the stroma of the lCO 2 plants was two orders higher than of those in the stroma of the hCO 2 plants ( Figure 4C). The CA activities of preparations isolated from chloroplasts were the most se to the CO2 levels in the air ( Figure 4C-E). We measured the CA activity of the strom thylakoids separately. The CA activity of the preparations of chloroplast stroma, βCA1.1, βCA1.2, and αCA1 are located, increased three-fold in the lCO2 plants com to the nCO2 plants. In the hCO2 plants, the CA activity in these preparations was re to 3% of that in the nCO2 plants. Thus, the activity of the CAs in the stroma of th plants was two orders higher than of those in the stroma of the hCO2 plants (Figur The thylakoids during the isolation process were thoroughly washed from ex lakoid CAs according to [7,41,42]. The CA activity of the thylakoids was determine incubation with the detergent Triton X-100. The addition of Triton X-100 at a Trit ratio of 0.3 (Thyl.0.3) exhibits the maximum activity of the CA located in the stroma of the thylakoid membranes, i.e., close to PSI [6,[42][43][44]. We have recently identifie αCA5 [13]. The CA activity of Thyl.0.3 increased by about six times after acclima 150 ppm of CO2 and by 2.5 times after acclimation to 1200 ppm of CO2, respectivel ure 4D), if compared with nCO2 plants.
The CA activity of CAs located in the granal thylakoid membranes, i.e., close t shows its maximum after the incubation of the thylakoids with Triton X-100 at ton/Chl ratio of 1.0 (Thyl.1.0) [6,[42][43][44]. This CA activity is apparently determined the presence of αCA4 [10,11,30-32] and αCA2 [11]. The CA activity of Thyl.1.0 wa The thylakoids during the isolation process were thoroughly washed from extrathylakoid CAs according to [7,41,42]. The CA activity of the thylakoids was determined after incubation with the detergent Triton X-100. The addition of Triton X-100 at a Triton/Chl ratio of 0.3 (Thyl.0.3) exhibits the maximum activity of the CA located in the stromal parts of the thylakoid membranes, i.e., close to PSI [6,[42][43][44]. We have recently identified it as αCA5 [13]. The CA activity of Thyl.0.3 increased by about six times after acclimation to 150 ppm of CO 2 and by 2.5 times after acclimation to 1200 ppm of CO 2 , respectively ( Figure 4D), if compared with nCO 2 plants.
The CA activity of CAs located in the granal thylakoid membranes, i.e., close to PSII, shows its maximum after the incubation of the thylakoids with Triton X-100 at a Triton/Chl ratio of 1.0 (Thyl.1.0) [6,[42][43][44]. This CA activity is apparently determined due to the presence of αCA4 [10,11,[30][31][32] and αCA2 [11]. The CA activity of Thyl.1.0 was twice higher in the lCO 2 plants than in the nCO 2 plants ( Figure 4E). In the hCO 2 plants, the CA activity of Thyl.1.0 was significantly, about 97%, lower than in the nCO 2 plants, i.e., the activity of the CAs from the granal thylakoid membranes of the plants acclimated to the lCO 2 level in the air was 30 times higher than of those from plants acclimated to the high CO 2 level.

The Effect of Acclimation of Adult nCO 2 Plants to Changed CO 2 Content in the Air on the Expression Level of the Genes Encoding CAs of α-and β-Families
We have measured the expression level, i.e., the content of transcripts, of the genes encoding αCA1, αCA2, αCA4, βCA1, βCA2, βCA3, βCA4, βCA5, and βCA6 in Arabidopsis leaves of different ages grown at nCO 2 ( Table 2). The content of the transcripts of the gene encoding thylakoid αCA5, located in stromal thylakoid membranes, i.e., close to PSI, has not been measured, since it is too low to be detected by the Real-Time RT PCR [13]. There are four alternative splicing forms of the βCA1 gene, with two pairs with the same sequences on 3 ends: βca1.1 + βca1.2 and βca1.3 + βca1. 4. We have determined the correspondent pairs together and denoted them as βca1.1+1.2 and βca1.3+1.4. βCA1.1 and βCA1.2 are situated in the chloroplasts [3], and the βca1.1+1.2 transcripts were related to the group of chloroplast CAs. The other form of βCA1, βCA1.3, was determined in the chloroplast envelope, whereas βCA1.4 was detected in the cytoplasm with a much stronger GFP fluorescence signal than for βCA1.3 [3]. Thereafter, we related the βca1.3+1.4 transcripts to the group of extrachloroplast CAs. Alternative splicing forms of the βCA4 gene, which encodes βCA4.2 located in the cytosol [2] and βCA4.1 located in the plasma membrane [2], also possess the same sequences on 3 ends of exons, and the correspondent transcripts could not be defined separately. The intensity of the expression of the CA genes in control plants under the constant CO 2 level at the normal growth conditions, i.e., at atmospheric CO 2 content, was the highest in the leaves of young, 26 days-old plants for most CA genes ( Table 2) and gradually decreased with age. In the nCO 2 plants, this expression level was rather stable from 44 to 52 days of age ( Figures 5 and 6, white columns) and decreased for all analyzed genes by the age of about 59-60 days. In addition, in 59-60 days (two months) old plants, the effects of CO 2 on the intensity of the CAs genes expression, are absent ( Figures 5 and 6). Plants 2022, 11, x FOR PEER REVIEW 10 of 24   Figure 5 shows the content of the same transcripts (Table 2) in the leaves of adult plants during the acclimation to different CO2 content in the air. In two-month-old plants, the intensity of the expression of βCA4 did not show a significant change under lCO2 ( Figure 5A). However, after 2 days of introduction to lCO2, it was about 30% higher than in the nCO2 plants of the same age. Its expression was not significantly changed in the lCO2 plants during CO2 acclimation.
Until recently, βCA2 was considered as the main CA in the cytoplasm of higher plants [45,46] due to its abundance [1,47] and high expression level [48,49]. The data in Table 2 also show that the expression level of βca2 was one of the highest. However, the (E)-βca5. Asterisks denote statistically significant differences between values for different CO 2 levels on the same experimental day, p < 0.01. Figure 5 shows the content of the same transcripts (Table 2) in the leaves of adult plants during the acclimation to different CO 2 content in the air. In two-month-old plants, the intensity of the expression of βCA4 did not show a significant change under lCO 2 ( Figure 5A). However, after 2 days of introduction to lCO 2 , it was about 30% higher than in the nCO 2 plants of the same age. Its expression was not significantly changed in the lCO 2 plants during CO 2 acclimation.
Until recently, βCA2 was considered as the main CA in the cytoplasm of higher plants [45,46] due to its abundance [1,47] and high expression level [48,49]. The data in Table 2 also show that the expression level of βca2 was one of the highest. However, the contents of the βca4 and βca1.3+1.4 transcripts are also high. The expression level of the genes encoding cytoplasmic Cas βca2, βca1.3+1.4, and βca3 increased by two times on the second day under lCO 2 ( Figure 5B,C) versus nCO 2 . During further acclimation to lCO 2 , the expression level of all four genes of the cytoplasmic Cas decreased, and by the ninth day, became lower than in nCO 2 ( Figure 5A-D).
The significant effect of hCO 2 has been observed only for the expression of βca2. On the first day of exposure to hCO 2 , it decreased to 12% from that in nCO 2 and increased to 212% by the third day of acclimation ( Figure 5B).
The expression level of the mitochondrial βCA6 was independent from CO 2 concentration in the air during all the acclimation ( Figure 5E). These data correspond with the constant CA activity in the preparations of mitochondria at any CO 2 content in the air ( Figure 4B).
The intensity of the expression of the genes encoding chloroplast CAs was fluctuating during acclimation ( Figure 6). However, in lCO 2 conditions versus nCO 2 , the reliable increase in the content of the βca1.1+1.2 transcripts ( Figure 6A) and the βca5 transcripts ( Figure 6E), eight and three times, respectively, have been observed. During further acclimation to lCO 2 , the expression levels of βca1.1+1.2 and βca5 decreased and became lower by the ninth day than in control plants, i.e., changed in the same way as the expression intensity of the genes of cytoplasmic CAs ( Figure 5A-D). The expression level of the genes, encoding stromal αCA1 and thylakoid CAs αCA2 and αCA4 were not significantly dependent on CO 2 content in the air ( Figure 6B-D).
Thus, the observed high-CA activity of cytoplasmic and chloroplast preparations in two-month-old lCO 2 plants if compared to nCO 2 plants and, to an even greater extent, to hCO 2 (Figure 4), was preceded by an increase in the expression levels of the genes encoding chloroplast βCA1 and βCA5 and cytoplasmic βCA2, βCA3, and βCA4 ( Figures 5 and 6) in a short-term range 1-2 days after the beginning of the acclimation.

The effect of Acclimation of Young Plants to low CO 2 Content in the Air on the Expression Level of the Genes Encoding CAs of α-and β-Families
A low concentration of carbon dioxide in the air is a significant stress factor for plants (Figures 1 and 2), leading to a considerable increase in the CA activity of most of the studied fractions (Figure 4), with a much less pronounced increase in gene expression intensity ( Figures 5 and 6). Therefore, the additional studies on changes in the levels of CA gene expression in Arabidopsis plants younger than two months of age after acclimation to lCO 2 are noted. In 26 days-age plants, a decrease in the CO 2 content for two weeks led to an increase in the expression levels of most genes of the cytoplasmic and chloroplast CAs ( Figure 7A). This expression level was 1.5-2 times higher for αca2 and βca5, 3-4 times higher for βca2 and βca3 genes, and 7-9 times higher for βca1.1+1.2 and βca1.3+1.4. The expression level of only αca4 was about 60% lower in lCO 2 plants than in nCO 2 plants.
In 50 days-age plants, 16 days of acclimation to lCO 2 led to an increase in the intensity of the expression of other CA genes to a much lesser extent than in 26 days-age plants. The expression level of βca4 and αca1 increased by two times; the αca4 and βca5 gene expressions were about three times higher ( Figure 7B). The expression levels of cytoplasmic βca2, βca3 and βca1.3+1.4 were about two to three times lower in lCO 2 plants than in nCO 2 plants.   In 50 days-age plants, 16 days of acclimation to lCO2 led to an increase in the intensity of the expression of other CA genes to a much lesser extent than in 26 days-age plants. The expression level of βca4 and αca1 increased by two times; the αca4 and βca5 gene expressions were about three times higher ( Figure 7B). The expression levels of cytoplasmic βca2, βca3 and βca1.3+1.4 were about two to three times lower in lCO2 plants than in nCO2 plants.

Discussion
In the present study, we have determined the changes in CA activities not in the total leave extract from C3 higher plant A. thaliana in response to CO2 content in the air, as in previous studies [50,51], but in different cell fractions isolated from leaves of C3 higher  Table 2, for 26 (A) and for 50 (B) days of age grown at 450 ppm. Data are shown as mean ± the SE. Asterisks denote statistically significant differences between values for different CO 2 levels on the same experimental day, p < 0.01.

Discussion
In the present study, we have determined the changes in CA activities not in the total leave extract from C3 higher plant A. thaliana in response to CO 2 content in the air, as in previous studies [50,51], but in different cell fractions isolated from leaves of C3 higher plant A. thaliana acclimated to the low and high CO 2 content in the air. The changes in the contents of transcripts of the correspondent CA genes during acclimation were analyzed in order to evaluate the response of their expression during such acclimations.
We have studied the above-listed changes when the negative consequences of acclimations had already taken place, manifesting primarily in a decrease in the maximum quantum yield of PSII (Fv/Fm) and performance indexes parameters (PI total and P abs ). In the plants exposed to lCO 2 , the changes in these photosynthetic parameters started in three days and were expectedly lower than in nCO 2 plants (Figure 1). However, the decline in the carbon dioxide assimilation rate in these plants, as well as in the content of starch and soluble carbohydrates (Table 1) and the lCO 2 plant size (not shown) has been observed not earlier than about two weeks after exposure to lCO 2 ( Figure 3B). These data are easily explained in terms of the shortage of the CO 2 content, i.e., of the basic material for photosynthesis. At the same time, the parameters of photosynthesis in plants exposed to hCO 2 also decreased versus those in nCO 2 ones, although later than in lCO 2 plants, only on the 9 th day for PI total and PI abs and on the 16 th day for Fv/Fm ( Figure 1A,B,D). Despite the larger size of the hCO 2 plants, the CO 2 assimilation rate in their leaves and the content of starch (mg/g of fresh weight) were lower than in the nCO 2 plants ( Figure 3B, Table 1). Photosynthetic down-regulation in the hCO 2 plants ( Figure 1B), which has also been observed by Zheng et al. [40] at elevated CO 2 levels, could be the reason for the relatively low CO 2 assimilation rate in hCO 2 plants against nCO 2 and even lCO 2 ones. This can be explained by the known effects of the changes in leaf configuration, i.e., the reduction in stomatal apertures and mesophyll tissue size at an elevated level of atmospheric CO 2 [40,52]. Another reason for the decrease in the assimilation rate may be the acidification of the stroma as a result of indirect proton transfer because of the absorption of excessive CO 2 [53]. Probably, in the conditions of the changed carbon dioxide level, some compensatory mechanisms in the total photosynthetic electron chain take place. Thus, in Arabidopsis plants, the maximum stress effect from the changed CO 2 level developed on the 16 th day.
One of the questions raised by investigations of the effect of CO 2 content changes is if a decrease in CO 2 content increases stress, or if the stress weakens the plants' response to changes in the CO 2 level [48]. Our results imply the first conclusion. The dissipation of light energy per active reaction center (DI 0 /RC) in both lCO 2 and hCO 2 plants was higher than in nCO 2 ones ( Figure 1C). Changes in the expression of stress-induced genes showed that both lCO 2 and hCO 2 conditions are stressful for plants, wherein, in lCO 2 plants and in hCO 2 plants, apparently, different stress responses are induced. In lCO 2 plants, the expression levels of the ABA-and JA-induced genes were higher than in nCO 2 and hCO 2 plants (Figure 2A,B), while the expression levels of the marker genes of SA pathways were lower. It is important that the expression of the npr1 gene, which is the key regulator of SA-mediated SAR, was about seven times lower in lCO 2 plants than in nCO 2 and hCO 2 ones ( Figure 2C). Since it is known that ABA and JA are synergists and both of them are antagonists of SA [39], these data mean that in plants at lCO 2 , the ABA-and JA-induced SAR pathways are activated, while the SA-mediated pathway is suppressed.
Our data indicate that for Arabidopsis plants, not only the conditions of low levels but also the conditions of high levels of carbon dioxide in the air, are stressful, although to a lesser extent. This is demonstrated both by the intensity of expression of stress genes and by photosynthetic parameters (Figures 1 and 2). The response of stress marker genes to the acclimation to hCO 2 was less expressed than in lCO 2 plants; for most genes analyzed, there was a small, only 10-50% increase in their expression in the hCO 2 plants versus the nCO 2 plants (Figure 2A-C).
The lipophilic CO 2 molecule should easily diffuse across lipid membranes [54,55]. However, this diffusion through cell and organelle membranes is slowed down by a series of resistances due to a high content of protein and sterol molecules in these membranes [55,56]. The diffusion to the site of carboxylation can also be slowed down due to diffusion resistance in the aqueous phases of the cytoplasm and chloroplast stroma [57]. It has long been hypothesized that the CAs of the plasma membrane, cytoplasm, mitochondria, and chloroplasts are involved in Ci transport, in particular, in the supply of CO 2 to carboxylation centers [8,16,47].
The CA activity of mitochondria is determined by the presence of the complex of γCAs-subunits attached to the inner mitochondrial membrane [58] and of the presence of βCA6 in the matrix [1]. Soto et al. [4] showed that the expression intensity of the encoding γCAs genes decreased under conditions of high CO 2 content, and Fabre et al. [1], using semi-quantitative PCR, demonstrated a higher intensity of the band of PCR products in plants grown at high CO 2 levels versus normal CO 2 . In our experiments, the CA activity of the mitochondria preparations incubated with 1% Triton X-100 and the contents of the βca6 transcripts were independent of the CO 2 level in the air (Figures 4B, 5E and 7A). One of the earlier hypotheses [59] about the role of mitochondrial CAs assumed their participation in Ci supply to chloroplasts under conditions of low CO 2 in the apoplast, for example, under conditions leading to stomatal closure. Our data do not support these assumptions. Participation in the processes of dark respiration in mitochondria seems to be more probable. This is indicated by a significant increase in the intensity of βca6 gene expression after 48 h in the dark [48].
The CA activities of the cytoplasm and all analyzed fractions of chloroplasts, i.e., preparations of stroma and thylakoids, were higher in 1CO 2 plants, than in nCO 2 plants ( Figure 4A,C-E). At that, the CA activity of the preparations of the stroma and Thyl.1.0 (CA activity of granal thylakoid membranes) in 1CO 2 plants turned out significantly higher than in hCO 2 plants. These data imply that CAs located in the cytoplasm of photosynthesizing cells and CAs located in the chloroplasts, both in stroma and in thylakoids, participate in Ci supply to carboxylation sites in higher plants. Participation in the conversion of bicarbonate into CO 2 to provide it to Rubisco in the stroma by several soluble CAs in parallel with thylakoid CAs seems to be even necessary. This would ensure the optimal rate of CO 2 supply to the carboxylation centers, which is the most important physiological process for plants. At that, the cooperative participation of stromal and thylakoid CAs in this process would improve plants' ability to adapt to changing environmental conditions. It seems most likely that of all the thylakoid CAs, it is αCA5, located on the stromal side of the stromal thylakoid membranes, which can be involved in this process [13]. The CA activity of the stromal thylakoids was the highest in lCO 2 ( Figure 4D). Herein, the activity of the stromal thylakoids was 2.5 times higher in hCO 2 plants versus nCO 2 plants. Under hCO 2 , the activity of the stromal thylakoids was even higher than the CA activity of the preparations of stroma ( Figure 4C). These data imply that under high CO 2 , the function of CO 2 supply to Rubisco is carried out, to a greater extent, by αCA5 than by the stromal CAs.
Under the lCO 2 level, i.e., in conditions of a deficiency of a Calvin-Benson cycle substrate, the value of NPQ increases. This is confirmed by the increment of the DI 0 /RC parameter in lCO 2 plants versus nCO 2 ones ( Figure 1B). The CA activity of the granal thylakoids under lCO 2 is more likely determined by the need for the participation of αCA4 located here [10,11] in the development of NPQ [30][31][32] than by the involvement of granal thylakoid CAs in Ci transport.
The expression level of CA-encoding genes in higher plants is daytime-dependent. That was shown for A. thaliana [48] and CAM plants, Sedum album, Ananas comosus, Kalanchoe fedtschenkoi, and Isoetes taiwanensis [60]. In our experiments, leaves were taken at the same time of the day for the measurements of the level of gene expression. The expression level of almost all CA genes in adult, 2-month-old plants, after 16 days of acclimation to the changed CO 2 concentrations in the air did not differ much from that in nCO 2 plants of the same age (Figures 5 and 6). The increase in the level of the βca1.3+1.4 ( Figure 5C), βca2 ( Figure 5B), βca3 (Figure 5D), and βca4 ( Figure 5A) transcripts encoding cytoplasmic CAs as well as of the βca1.1+1.2 ( Figure 6A) and βca5 ( Figure 6E) transcripts encoding chloroplast CAs in a short-term range 1-2 days after the beginning of the acclimation was preceded to the high CA activity (Figure 4). This result is surprising due to the convincing data of the role of βCA4 and βCA1 in CO 2 transport into cells and in CO 2 -dependent regulation of stomatal permeability [14,15].
βCA5, presumably located in the thylakoid lumen [8,9], was the only thylakoid CA of which the expression level responded to the CO 2 content in the air. The content of transcripts of the genes encoding thylakoid CAs, αca2 and αca4, was not increased in lCO 2 plants versus nCO 2 ones, whereas the CA activities of both preparations of thylakoids, Thyl.1.0 and Thyl.0.3, were the highest in lCO 2 plants ( Figure 4D,E).
From two stromal Cas, only βca1.1+1.2 transcripts showed an increase from exposure to lCO 2 ( Figure 6A). Thus, with a decrease in the concentration of carbon dioxide, it was intensified the synthesis of βCA1, i.e., of that CA, which participation in photosynthesis is constantly being questioned. The level of αca1 transcripts was independent of CO 2 content in the air ( Figure 6B). However, In plants with knocked-out genes encoding αCA1, the number of indicators of photosynthetic activity, as well as the ability to accumulate starch, was decreased [61]. These data indicate that both stromal CAs, βCA1 and αCA1, as well as the thylakoid αCA5 ( Figure 4D), play an important role in maintaining the Ci concentration close to carboxylation sites of Rubisco.
A significant decrease in the CA activity of the preparations of the chloroplast stroma and Thyl.1.0 in hCO 2 plants versus nCO 2 plants had no parallelism with the expression intensity of the corresponding genes. The expression levels of the genes encoding the stromal and thylakoid CAs in hCO 2 plants were about the same or even slightly higher, against nCO 2 plants ( Figure 6). These data mean that the changes in the intensities of the synthesis of these CAs at the stage of transcription of the genes encoding them is not the main way of CA activity regulation, at least in the growth conditions used and at age of about two months. Apparently, this mechanism is associated, first of all, with the regulation of CAs' activities, and to a lesser extent, with an increase in their biosynthesis at the level of the transcription of the genes encoding them. The mechanism of the regulation of CA activity was revealed for βCA1 by studying the action of high-temperature and water deficit stresses in the leaves of Helianthus annum [62] and Brassica napus [21]. The key regulation mechanisms of the CA activity in the chloroplasts in these plants were the nitration and phosphorylation of tyrosine residues in the active site of CA. The binding and dissociation of these nitrate and/or phosphate groups block and open, respectively, the passage of the substrate to the active site cavity according to plant needs. It is very likely that similar mechanisms of the regulation of CA activity exist for other CAs.
The additional studies on the effect of a decrease in the CO 2 content in the air on the expression levels of CAs genes in Arabidopsis plants younger than two months have shown that in 26-days-old seedlings, these conditions caused a significant intensification of the synthesis of CA gene transcripts ( Figure 7A). The expression levels of most cytoplasmic and chloroplast CAs genes were higher in 26-days-old plants after about two weeks of exposure to lCO 2 versus nCO 2 plants ( Figure 7A). In 50 days-old plants after 16 days of acclimation to lCO 2, an increase in the intensity of the transcription of CA genes was less pronounced ( Figure 7B). Thus, the younger the plants exposed to low CO 2 , the more the effect of an increase in the expression level of the genes encoding cytoplasmic and chloroplast CAs in them. Apparently, in young Arabidopsis plants, the increased intensity of the synthesis of these CAs at the stage of transcription of the genes encoding them makes a greater contribution to the increase in the content of CAs in the plant cell than that in mature plants.
Studies conducted using CA mutants, especially those with single mutations, most often demonstrate little pronounced effects on photosynthesis [15,22,63], except mutation in the βca5 gene, which leads to significant suppression of the growth of Arabidopsis plants [36]. In recent years, more data on the involvement of CAs in higher plant cells in protection against stress or in transmitting a response signal to a negative environmental impact have begun to appear. Initially, these data were obtained for only βCA1 [34,35,63]. The functioning of βCA1 in these processes was ascribed to the possibility of βCA1 participation in fatty acids (FA) biosynthesis [64] and/or to βCA1'ss ability to bind salicylic acid (SA) [34]. These two assumptions, in fact, not only do not exclude, but also complement each other, since SAs and FAs are the key molecules of the stress-induced regulation of metabolic pathways. Recently, Hines et al. [37] have found that leaves of ∆β-ca1ca5 tobacco double mutants developed abnormally, and their leaves were significantly damaged from necrosis even when supplied with sucrose. Apparently, all six βCAs participated in these processes. Medina-Puche et al. [36] have shown the association of βCAs 1-6 from Arabidopsis with NPR receptors, which are the main participants of SA-induced stress signals.
These authors concluded that βCAs are not involved in photosynthetic processes in higher plants. Wherein, our data show that under such stress as a low CO 2 concentration in the air, a significant increase in CA activity occurs (Figure 4), but the intensity of npr1 gene transcription, on the contrary, decreases ( Figure 2C). These data show that at least under these conditions, an increase in the activity of CAs is not associated with their participation in the stress signal transmission through binding with the NPR1 protein.
In fact, the involvement of CAs in stress signaling does not exclude the possibility of their participation in photosynthetic processes. Dabrowska-Bronk et al. [38] have shown that in Arabidopsis plants all six βCAs are involved in the uptake of HCO 3 − ions by roots, and their functioning is important for plant growth and cell homeostasis, especially under such stresses as lack of water and high light. The possible reason for the absence of dramatic effects of CAs mutations is that the CAs function together in plant cells, replacing each other in case of the suppression of the synthesis of any of them. In support of this hypothesis, it has been demonstrated that the addition of ethoxyzolamide, which is able to penetrate cell membranes and inhibit thus all cellular Cas, has led to a decrease in photosynthesis in the leaves of C3 and C4 plants at low CO 2 concentrations [65] and at the CO 2 -dependent O 2 release by pea leaf protoplasts both at low and optimal CO 2 concentrations [66,67].
The described changes in the intensities of CAs gene expression, depending on the time of exposure to changed CO 2 in the air and on the age of plants ( Figures 5-7), as well as the data of Hu et al. [15], DiMario et al. [2], Dabrowska-Bronk et al. [38], Medina-Puche et al. [36], and Hines et al. [37] indicate that the functioning of Cas in plants cells is carried out together, interdependently, and complexly. Our data show that this functioning depends significantly on the carbon dioxide content in the air, and this dependence is appreciably determined by the age of the plants.

Plant Material
Experiments were performed with Arabidopsis thaliana (L.) Heynh. ecotype Columbia-0 (Col). Three-week-old seedlings were planted into pots, one per pot, containing a commercially available soil mixture, and were grown in a growing chamber (CO 2 content of 450 ppm, temperature 19 • C, 8 h day/16 h night photoperiod, photosynthetically active radiation of 50-70 µmol quanta m −2 s −1 ) for 25 days and then exposed to conditions of the low and high CO 2 content, 150 and 1200 ppm under the same other conditions (Supplementary Materials Figure S1).
For the additional determination of the effects of age on the changes in the genes' expression level after adaptation to low CO 2 , young Arabidopsis plants were grown in the conditions described above. At the age of 10 days (Supplementary Materials Figure S2A) and 34 days (Supplementary Materials Figure S2B) the plants were exposed to conditions of the low CO 2 content (150 ppm). Redundant seedlings were removed from the pots of plants aged 10 days.

Measurement of Chlorophyll a Fluorescence
The maximum quantum yield of PSII (Fv/Fm), performance indexes (PI abs and PI total ), and the dissipation of light energy per active reaction center (DI 0 /RC) were calculated according to Kalaji et al. [68] after the measurement of OJIP chlorophyll a fluorescence kinetics. The OJIP chlorophyll a fluorescence transient was measured using a Handy-PEA (Hansatech) fluorometer, with the leaves illuminated with a 1 s flash of red light of 3000 µmol quanta m −2 s −1 . Before measurements, the plants were adapted to dark conditions for two hours.

Measurement of CO 2 Assimilation Rate
The CO 2 assimilation rate was measured in a leaf chamber using the LI-6800 Portable Photosynthesis System (Li-Cor, Lincoln, NE, USA) according to LI-6800 manual in the range of CO 2 concentration of 0-1200 ppm under a constant light intensity of 350 µmol quanta m −2 s −1 (90% red, 10% blue light), 23 • C, and 50% relative humidity. Before measurement, the plants were pre-adapted to the illumination of 350 µmol quanta m −2 s −1 for 2 h. The leaf areas were measured using the Petiole application (Petiole LTD).

Determination of Starch and Soluble Carbohydrates Content
The starch content was analyzed by measuring the absorbance at 620 nm of leaf aqueous extracts supplemented with KI after thorough washing from pigments [69]. Prior measurements of leaves were kept on a wet filter paper for an hour in order to normalize a turgor of leaves. The content of soluble carbohydrates was determined in hydroalcoholic extract using phenol-sulfuric acid reaction according to Du Bois et al. [70].
The supernatant "a" was centrifugated for 10 min at 8000× g, yielding precipitate (preparations of mitochondria) and supernatant "a ", enriched with the proteins of cytoplasm. The preparations of the mitochondria obtained were suspended in Medium 1 and were used for analysis after incubation for 20 min with 1% Triton X-100.
Supernatants "a' " and "b" were additionally centrifuged for 1 h at 175,000× g to remove the rest of the membranes yielding preparations of cytoplasm and stroma, respectively, which were used for analysis. The preparations of the stroma were enriched with Rubisco and the preparations of the thylakoids and mitochondria had no Rubisco that was checked by a Western blot assay, using antibodies against the large subunit of Rubisco (Agrisera) (not shown).
The thylakoids were washed three times by suspending the pellets in Medium 2 followed by centrifugation for 5 min at 2500× g. Part of the preparations of the thylakoids was used for analysis after incubation for 20 min with Triton X-100 at a Triton/Chl ratio of 0.3 (Thyl.0.3). The other part of the preparations of the thylakoids was used for analysis after incubation for 20 min with Triton X-100 at a Triton/Chl ratio of 1.0 (Thyl.1.0).

Determination of the Protein Content
The protein content in the supernatants was determined using a DC TM Protein Assay kit Bio-Rad according to Bio-Rad protocol.

Determination of the Chlorophyll Content
The chlorophyll content was determined in ethanol extracts according to Lichtenthaler [72].

Measurement of Carbonic Anhydrase Activity
Carbonic anhydrase activity was evaluated according to Khristin et al. [73] as the difference between the rates of pH decrease, measured with a pH electrode, from 8.3 to 7.8 in the course of CO 2 hydration at 2 • C in 13.6 mM Veronal buffer (pH 8.4) in the presence and in the absence of an aliquot of the preparation. The difference in the buffer capacities was taken into account to express the CA activity as the extent of the proton release. The CA activity was calculated as the difference between the rates of the pH decrease in the presence and the absence of the preparation and was expressed in µmol H+ per 1 mg of Chl or protein per 1 min.

Conclusions
In summary, this study demonstrated that in photosynthesizing cells, the conditions that require an increase in the intensity of inorganic carbon entry into cells lead to an increase in the activity of CAs located in the cytoplasm and CAs located in the chloroplasts, both stromal and thylakoid ones. Changes in the intensity of the expression of the genes encoding these CAs depended on the age of the plants. The increase in the level of CAs gene expression in lCO 2 plants versus hCO 2 plants was most pronounced in young 26-days-old plants. In 50 days-old plants, it was less noticeable, and in two-month-old plants, after 16 days of acclimation to the changed CO 2 concentrations in the air, it did not differ much from that in nCO 2 plants of the same age. The increase in the CA activities of the preparations of the cytoplasm, stroma, and thylakoids was preceded by an increase in the content of the transcripts of βca1.3+1.4, βca2, βca3, and βca4 encoding cytoplasmic forms of CAs as well as of βca1.1+1.2 and βca5, encoding chloroplast forms of CAs in a short-term range of 1-2 days after the beginning of the acclimation. The CA activity of the preparations of the mitochondria as well as the expression level of the gene encoding mitochondrial βCA6 was independent of the content of the carbon dioxide level in the air. These data do not support the assumptions of the participation of mitochondrial CAs in Ci supply to chloroplasts under conditions of low CO 2 .
Taken together, our data imply that CAs located in the cytoplasm and CAs located in the chloroplasts, both in stroma and in thylakoids, cooperatively participate in inorganic carbon supply to carboxylation sites in higher plants (Figure 8).
tions of the mitochondria as well as the expression level of the gene encoding mitochondrial βCA6 was independent of the content of the carbon dioxide level in the air. These data do not support the assumptions of the participation of mitochondrial CAs in Ci supply to chloroplasts under conditions of low CO2.
Taken together, our data imply that CAs located in the cytoplasm and CAs located in the chloroplasts, both in stroma and in thylakoids, cooperatively participate in inorganic carbon supply to carboxylation sites in higher plants (Figure 8). Supplementary Materials: The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Figure S1: Scheme of Arabidopsis plants cultivation during the experiment. Figure S2: Scheme of Arabidopsis plants cultivation during the experiment on the effect of the low CO2 level on the expression of CA genes. Figure S3: Scheme of isolation from Arabidopsis leaves of preparations of cytoplasm, mitochondria, stroma, and thylakoids.   Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants11162113/s1, Figure S1: Scheme of Arabidopsis plants cultivation during the experiment. Figure S2: Scheme of Arabidopsis plants cultivation during the experiment on the effect of the low CO2 level on the expression of CA genes. Figure S3: Scheme of isolation from Arabidopsis leaves of preparations of cytoplasm, mitochondria, stroma, and thylakoids.