OsαCA1 Affects Photosynthesis, Yield Potential, and Water Use Efficiency in Rice

Plant growth and crop yield are essentially determined by photosynthesis when considering carbon dioxide (CO2) availability. CO2 diffusion inside a leaf is one of the factors that dictate the CO2 concentrations in chloroplasts. Carbonic anhydrases (CAs) are zinc-containing enzymes that interconvert CO2 and bicarbonate ions (HCO3−), which, consequently, affect CO2 diffusion and thus play a fundamental role in all photosynthetic organisms. Recently, the great progress in the research in this field has immensely contributed to our understanding of the function of the β-type CAs; however, the analysis of α-type CAs in plants is still in its infancy. In this study, we identified and characterized the OsαCA1 gene in rice via the analysis of OsαCAs expression in flag leaves and the subcellular localization of its encoding protein. OsαCA1 encodes an α-type CA, whose protein is located in chloroplasts with a high abundance in photosynthetic tissues, including flag leaves, mature leaves, and panicles. OsαCA1 deficiency caused a significant reduction in assimilation rate, biomass accumulation, and grain yield. The growth and photosynthetic defects of the OsαCA1 mutant were attributable to the restricted CO2 supply at the chloroplast carboxylation sites, which could be partially rescued by the application of an elevated concentration of CO2 but not that of HCO3−. Furthermore, we have provided evidence that OsαCA1 positively regulates water use efficiency (WUE) in rice. In summary, our results reveal that the function of OsαCA1 is integral to rice photosynthesis and yield potential, underscoring the importance of α-type CAs in determining plant physiology and crop yield and providing genetic resources and new ideas for breeding high-yielding rice varieties.


Introduction
The ongoing population increase and climate change are increasingly threatening global food security [1,2]. Therefore, there is an urgent need to enhance plant productivity and boost food production to feed the world sustainably [3][4][5][6][7]. Plant growth and crop yield primarily rely on photosynthesis [8,9], which can be divided into two stages: one of which comprises the light-dependent reactions that harness solar energy to produce adenosine triphosphate (ATP) and nicotinomide-adenine dinucleotide phosphate (NADPH), and the other comprises the so-called dark reactions, which are often referred to as the Calvin cycle in reference to the reactions' discoverer, Melvin Calvin. Dark reactions are a group of reactions that take place within the stroma of a chloroplast, wherein ATP and NADPH are used to drive the biosynthesis of glucose [10]. The availability of carbon dioxide (CO 2 ) is expression levels of 11 OsαCA genes in flag leaves were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that the transcripts of OsαCA1 and OsαCA2 accumulate to a high level in flag leaves ( Figure S1). Given that protein localization in a cell is tightly controlled and strongly associated with its function, we sought to determine the subcellular localization of OsαCAs based on the green fluorescent protein (GFP) fusion protein strategy by transiently expressing OsαCAs-GFP in protoplasts. As demonstrated by the data presented in Figure S2, distinct from OsαCA2, which is located in the plasma membrane, OsαCA1 displays a characteristic location in the chloroplast. Therefore, in this study, we concentrated our efforts on OsαCA1.
Next, we explored the tissue expression of OsαCA1 in greater detail. As shown in Figure 1A, OsαCA1 was mainly expressed in the photosynthesis-conducting tissues, including the flag leaves, mature leaves, and panicles. Further examination concerning protein subcellular localization showed that the fluorescence signal of OsαCA1-GFP was specifically detected in chloroplasts ( Figure 1C), indicating that OsαCA1 is a chloroplastlocalized protein. Given that OsαCA1 consists of an N-terminal signal peptide (SP) and a C-terminal CA domain ( Figure 1B), we also wanted to know which component(s) of the OsαCA1 protein governs its subcellular localization. To this end, two additional constructs were generated in parallel with OsαCA1-GFP, one of which expressed the fusion protein of GFP plus the SP of OsαCA1 (SP-GFP), while the other one expressed the protein of GFP fused with the CA domain of OsαCA1 (CA-GFP). From the transfected protoplasts, no fluorescence of either of the two fusion constructs was observed in the chloroplasts under laser confocal microscopy in contrast to that of OsαCA1-GFP, suggesting that neither SP-GFP nor CA-GFP could be delivered to the chloroplasts and that OsαCA1 targeting to chloroplasts relies on both its SP and the CA domain ( Figure 1C).

Spatiotemporal Expression of OsαCA1 Gene in Rice
Photosynthesis mainly transpires in the chloroplasts of leaf mesophyll cells [46,47]. To identify whether or which OsαCAs are involved in the photosynthesis process in rice, the expression levels of 11 OsαCA genes in flag leaves were analyzed by quantitative realtime polymerase chain reaction (qRT-PCR). The results showed that the transcripts of OsαCA1 and OsαCA2 accumulate to a high level in flag leaves ( Figure S1). Given that protein localization in a cell is tightly controlled and strongly associated with its function, we sought to determine the subcellular localization of OsαCAs based on the green fluorescent protein (GFP) fusion protein strategy by transiently expressing OsαCAs-GFP in protoplasts. As demonstrated by the data presented in Figure S2, distinct from OsαCA2, which is located in the plasma membrane, OsαCA1 displays a characteristic location in the chloroplast. Therefore, in this study, we concentrated our efforts on OsαCA1.
Next, we explored the tissue expression of OsαCA1 in greater detail. As shown in Figure 1A, OsαCA1 was mainly expressed in the photosynthesis-conducting tissues, including the flag leaves, mature leaves, and panicles. Further examination concerning protein subcellular localization showed that the fluorescence signal of OsαCA1-GFP was specifically detected in chloroplasts ( Figure 1C), indicating that OsαCA1 is a chloroplast-localized protein. Given that OsαCA1 consists of an N-terminal signal peptide (SP) and a Cterminal CA domain ( Figure 1B), we also wanted to know which component(s) of the OsαCA1 protein governs its subcellular localization. To this end, two additional constructs were generated in parallel with OsαCA1-GFP, one of which expressed the fusion protein of GFP plus the SP of OsαCA1 (SP-GFP), while the other one expressed the protein of GFP fused with the CA domain of OsαCA1 (CA-GFP). From the transfected protoplasts, no fluorescence of either of the two fusion constructs was observed in the chloroplasts under laser confocal microscopy in contrast to that of OsαCA1-GFP, suggesting that neither SP-GFP nor CA-GFP could be delivered to the chloroplasts and that OsαCA1 targeting to chloroplasts relies on both its SP and the CA domain ( Figure 1C).  The tissue expression of OsαCA1 in rice. The total RNA of various tissues, including root, leaf sheath, young leaf, mature leaf, flag leaf, stem, panicle, and seed, was extracted to analyze the expression of OsαCA1 via qRT-PCR, with Ubiquitin incorporated as the control. Values are shown as means ± SEM, where n = 3. (B) The signal peptide and the conserved domain of OsαCA1 and diagram of vector construction. The green rectangle represents the signal peptide termed SP, and the blue rectangle represents the CA domain termed CA. (C) The subcellular localization of GFP-fused proteins. The expression vectors of SP-GFP and CA-GFP were constructed; then, the protoplasts of the rice leaf sheath were transfected. GFP-the view via GFP fluorescence; BF-the view via bright-field microscopy; chlorophyll-the view of chloroplast autofluorescence; and Merge-the merged view of GFP and chlorophyll. Scale bars: 5 µm.

OsαCA1 Gene Mutants Displayed a Significant Reduction in Photosynthesis Rates
To investigate the functions of the OsαCA1 gene, we generated two independent mutant lines of OsαCA1 (αca1-1 and αca1-2) by the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-mediated editing method. Both alleles had a 1 bp insertion of nucleotide A and T at +370 from position 0 in the third exon of OsαCA1, each introducing a premature termination codon ( Figure S3). The carbon assimilation rate (also known as photosynthesis rate) of αca1 was an average of 20% lower than that of the wild type (WT, ZH11) at both the vegetative growth stage and the reproductive growth stage (Figure 2A,D). Importantly, compared with the WT, the αca1 mutants exhibited increased transpiration rates and reduced WUE ( Figure 2B,C,E,F). These results indicate that the mutation of OsαCA1 led to the inhibition of photosynthesis. Since both αca1 mutants displayed the same phenotypes, our further investigation only focused on one of them: the αca1-2 line.

OsαCA1 Gene Mutants Displayed a Significant Reduction in Photosynthesis Rates
To investigate the functions of the OsαCA1 gene, we generated two independent mutant lines of OsαCA1 (αca1-1 and αca1-2) by the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-mediated editing method. Both alleles had a 1 bp insertion of nucleotide A and T at +370 from position 0 in the third exon of OsαCA1, each introducing a premature termination codon ( Figure S3). The carbon assimilation rate (also known as photosynthesis rate) of αca1 was an average of 20% lower than that of the wild type (WT, ZH11) at both the vegetative growth stage and the reproductive growth stage (Figure 2A,D). Importantly, compared with the WT, the αca1 mutants exhibited increased transpiration rates and reduced WUE ( Figure 2B,C,E,F). These results indicate that the mutation of OsαCA1 led to the inhibition of photosynthesis. Since both αca1 mutants displayed the same phenotypes, our further investigation only focused on one of them: the αca1-2 line. Values are shown as means ± SEM determined via one-way ANOVA, where different letters indicate a significant difference. p < 0.05; n ≥ 9.

OsαCA1 Mutation Caused a Severe Reduction in Biomass Production and Grain Yield in Rice
Photosynthesis contributes to most of the accumulation of dry matter for plant growth and yield [48]. Therefore, the phenotypes of plant growth and yield potential were analyzed in the WT and αca1-2. The results suggest that the mutation of OsαCA1 triggered significant growth defects as αca1-2 had shorter plant height, shorter root length, lower dry weight, and lower fresh weight values ( Figure 3A-G). The rice yield was determined by the effective tiller number, grain number per panicle, seed-setting rate, and 1000-grain weight [49][50][51]. There was no statistical difference in the effective tiller number between Values are shown as means ± SEM determined via one-way ANOVA, where different letters indicate a significant difference. p < 0.05; n ≥ 9.

OsαCA1 Mutation Caused a Severe Reduction in Biomass Production and Grain Yield in Rice
Photosynthesis contributes to most of the accumulation of dry matter for plant growth and yield [48]. Therefore, the phenotypes of plant growth and yield potential were analyzed in the WT and αca1-2. The results suggest that the mutation of OsαCA1 triggered significant growth defects as αca1-2 had shorter plant height, shorter root length, lower dry weight, and lower fresh weight values ( Figure 3A-G). The rice yield was determined by the effective tiller number, grain number per panicle, seed-setting rate, and 1000-grain weight [49][50][51]. There was no statistical difference in the effective tiller number between the WT and αca1-2 ( Figure 3L), whereas the panicle length of αca1-2 was significantly shorter than that of the WT due to the reduced primary branch number; as a result, a 40% reduction in grain number per panicle was consistently observed ( Figure 3H-K). In addition, the seed-setting rate and 1000-grain weight of αca1-2 were comparable to those of the WT ( Figure 3M,N). These results indicate that the inhibition of photosynthesis by the OsαCA1 mutation resulted in a significant decrease in biomass and yield. the WT and αca1-2 ( Figure 3L), whereas the panicle length of αca1-2 was significantly shorter than that of the WT due to the reduced primary branch number; as a result, a 40% reduction in grain number per panicle was consistently observed ( Figure 3H-K). In addition, the seed-setting rate and 1000-grain weight of αca1-2 were comparable to those of the WT ( Figure 3M,N). These results indicate that the inhibition of photosynthesis by the OsαCA1 mutation resulted in a significant decrease in biomass and yield. (M) seed-setting rate; (N) thousand-seed weight. All values are shown as means ± SEM determined via Student's t-test, where * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns indicates no significant difference, and n ≥ 20.

The CO2 Concentration in Chloroplasts Was Reduced Markedly by OsαCA1 Mutation
Light reactions and dark reactions are two stages of the photosynthesis process [10]. Several studies have suggested that αCAs in higher plants participate in the stage in which light reactions transpire [39][40][41]. In order to understand how OsαCA1 is involved in photosynthesis, the activity of photosystem II (PSII) was assessed in both the WT and αca1-2. As in the data presented in Figure S4, the maximum photochemical quantum efficiency (M) seed-setting rate; (N) thousand-seed weight. All values are shown as means ± SEM determined via Student's t-test, where * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns indicates no significant difference, and n ≥ 20.

The CO 2 Concentration in Chloroplasts Was Reduced Markedly by OsαCA1 Mutation
Light reactions and dark reactions are two stages of the photosynthesis process [10]. Several studies have suggested that αCAs in higher plants participate in the stage in which light reactions transpire [39][40][41]. In order to understand how OsαCA1 is involved in photosynthesis, the activity of photosystem II (PSII) was assessed in both the WT and αca1-2. As in the data presented in Figure S4, the maximum photochemical quantum efficiency (Fv/Fm), the actual photochemical quantum efficiency (Y(II)), and the electron transport rate (ETR(II)) of PSII in αca1-2 were comparable to those of the WT ( Figure S4). These results implied that the mutation of OsαCA1 might not have affected the light reactions, so we speculated that the compromised photosynthesis of the αca1-2 mutant might be a consequence of the inhibition of the dark reaction.
To evaluate the possible deleterious effects of the αca1-2 mutant on the dark reactions of photosynthesis, the expression of several genes that encode key enzymes of carbon assimilation and the accumulation of starch were examined. Our data suggest that the transcription of these genes was greatly down-regulated in αca1-2 ( Figure 4A). Consistent with this observation, the starch content was significantly reduced in αca1-2 relative to the WT ( Figure 4B). These results clearly indicate that the carbon assimilation in αca1-2 was impaired. Furthermore, the C c of αca1-2 was much lower than that of the WT ( Figure 5A), indicating that the reduction in the photosynthesis rate was likely caused by a decline in the C c at the dark reaction stage.
(Fv/Fm), the actual photochemical quantum efficiency (Y(II)), and the electron transport rate (ETR(II)) of PSII in αca1-2 were comparable to those of the WT ( Figure S4). These results implied that the mutation of OsαCA1 might not have affected the light reactions, so we speculated that the compromised photosynthesis of the αca1-2 mutant might be a consequence of the inhibition of the dark reaction.
To evaluate the possible deleterious effects of the αca1-2 mutant on the dark reactions of photosynthesis, the expression of several genes that encode key enzymes of carbon assimilation and the accumulation of starch were examined. Our data suggest that the transcription of these genes was greatly down-regulated in αca1-2 ( Figure 4A). Consistent with this observation, the starch content was significantly reduced in αca1-2 relative to the WT ( Figure 4B). These results clearly indicate that the carbon assimilation in αca1-2 was impaired. Furthermore, the Cc of αca1-2 was much lower than that of the WT ( Figure 5A), indicating that the reduction in the photosynthesis rate was likely caused by a decline in the Cc at the dark reaction stage.   rate (ETR(II)) of PSII in αca1-2 were comparable to those of the WT ( Figure S4). These results implied that the mutation of OsαCA1 might not have affected the light reactions, so we speculated that the compromised photosynthesis of the αca1-2 mutant might be a consequence of the inhibition of the dark reaction.
To evaluate the possible deleterious effects of the αca1-2 mutant on the dark reactions of photosynthesis, the expression of several genes that encode key enzymes of carbon assimilation and the accumulation of starch were examined. Our data suggest that the transcription of these genes was greatly down-regulated in αca1-2 ( Figure 4A). Consistent with this observation, the starch content was significantly reduced in αca1-2 relative to the WT ( Figure 4B). These results clearly indicate that the carbon assimilation in αca1-2 was impaired. Furthermore, the Cc of αca1-2 was much lower than that of the WT (Figure 5A), indicating that the reduction in the photosynthesis rate was likely caused by a decline in the Cc at the dark reaction stage.   To ascertain the possible reasons for the C c decrease in αca1-2, we measured g s (reflecting the resistance of stomata) and mesophyll conductance (g m , reflecting the resistance of mesophyll cells) and calculated the stomatal limitation value (L s ). g s increased while L s and g m decreased significantly in αca1-2 with reference to the WT ( Figure 5B-E), indicating that stomata were not the reason for the decline in photosynthesis in αca1-2. The reduction in C c might have resulted from increased resistance to CO 2 diffusion in the mesophyll cells.

OsαCA1 has Carbonic Anhydrase Activity Both In Vitro and In Vivo
The CA concentration in chloroplasts is one of the vital determinants of g m [25]. Given that OsαCA1 proteins are located in chloroplasts ( Figure 1C), the decrease in the Cc in αca1-2 might be a result of the declined CA concentration in the chloroplasts. To test this hypothesis, GST-fused OsαCA1 proteins were produced and purified from E. coli ( Figure S5). An enzyme activity assay was then performed, which showed that OsαCA1 has CA activity (Data S1 and Figure 6A). A comparative study on the CA activities in the whole leaves and chloroplasts of the WT and αca1-2 was also conducted, and our results suggest that the CA activities of αca1-2 were mildly yet consistently lower than their counterparts in the WT (Data S1 and Figure 6B,C).
To ascertain the possible reasons for the Cc decrease in αca1-2, we measured gs (reflecting the resistance of stomata) and mesophyll conductance (gm, reflecting the resistance of mesophyll cells) and calculated the stomatal limitation value (Ls). gs increased while Ls and gm decreased significantly in αca1-2 with reference to the WT ( Figure 5B-E), indicating that stomata were not the reason for the decline in photosynthesis in αca1-2. The reduction in Cc might have resulted from increased resistance to CO2 diffusion in the mesophyll cells.

OsαCA1 has Carbonic Anhydrase Activity Both In Vitro and In Vivo
The CA concentration in chloroplasts is one of the vital determinants of gm [25]. Given that OsαCA1 proteins are located in chloroplasts ( Figure 1C), the decrease in the Cc in αca1-2 might be a result of the declined CA concentration in the chloroplasts. To test this hypothesis, GST-fused OsαCA1 proteins were produced and purified from E. coli ( Figure  S5). An enzyme activity assay was then performed, which showed that OsαCA1 has CA activity (Data S1 and Figure 6A). A comparative study on the CA activities in the whole leaves and chloroplasts of the WT and αca1-2 was also conducted, and our results suggest that the CA activities of αca1-2 were mildly yet consistently lower than their counterparts in the WT (Data S1 and Figure 6B,C).

The Impaired Growth of αca1-2 Mutant Could Only be Partially Rescued by Application of Elevated CO2 Concentration but Not HCO3 − Treatment
The observations that the undersupply of CO2 in the αca1-2 chloroplasts caused the inhibition of photosynthesis and growth arrest ( Figure 5A) and that the expression of OsαCA1 could be induced either by the elevated CO2 or by 100 mM NaHCO3 ( Figure S6) prompted us to evaluate whether an elevated CO2 concentration or an extra HCO3 − treatment could restore the phenotypic defects in αca1-2. As shown in Figure 7A, the CO2 response curve suggested that the assimilation rate of αca1-2 was consistently lower than The chloroplast proteins of flag leaves were extracted to detect CA activity. All values are shown as means ± SEM determined via Student's t-test, where ** p < 0.01, ns indicates no significant difference, and n ≥ 8.

The Impaired Growth of αca1-2 Mutant Could Only be Partially Rescued by Application of Elevated CO 2 Concentration but Not HCO 3 − Treatment
The observations that the undersupply of CO 2 in the αca1-2 chloroplasts caused the inhibition of photosynthesis and growth arrest ( Figure 5A) and that the expression of OsαCA1 could be induced either by the elevated CO 2 or by 100 mM NaHCO 3 ( Figure  S6) prompted us to evaluate whether an elevated CO 2 concentration or an extra HCO 3 − treatment could restore the phenotypic defects in αca1-2. As shown in Figure 7A, the CO 2 response curve suggested that the assimilation rate of αca1-2 was consistently lower than that of the WT, while the additional input of CO 2 promoted carbon assimilation in αca1-2. Interestingly, around 800 ppm of CO 2 , a decrease in the photosynthesis rate could be observed in the WT but not in αca1-2 ( Figure 7A). Taken together, the results indicate that the αca1-2 mutant heavily restricted the supply of CO 2 for carbon fixation. In line with this observation, under 1000 ppm of CO 2 , the plant heights, root lengths, and dry weights of the WT and αca1-2 were all increased, while the αca1-2 seedlings benefited less from the growth-promoting effects of high CO 2 ( Figure 7B-F). These data indicate that the elevated concentration of CO 2 could only partially rescue the growth defects in αca1-2. Of note, the plant height, root length, and dry weight could be increased in the WT through the treatment of 100 µM of NaHCO 3 , whereas there was no detectable enhancement in αca1-2 ( Figure 7G-J), indicating that the HCO 3 − application did not effectively mitigate growth inhibition in αca1-2. These results evidently suggest that OsαCA1 deficiency compromised the conversion from HCO 3 − to CO 2 , and restricted the molecular diffusion of CO 2 , which, in turn, caused C c decline and, consequently, led to a CO 2 undersupply for photosynthesis. the WT and αca1-2 were all increased, while the αca1-2 seedlings benefited less from the growth-promoting effects of high CO2 (Figure 7B-F). These data indicate that the elevated concentration of CO2 could only partially rescue the growth defects in αca1-2. Of note, the plant height, root length, and dry weight could be increased in the WT through the treatment of 100 µM of NaHCO3, whereas there was no detectable enhancement in αca1-2 (Figure 7G-J), indicating that the HCO3 − application did not effectively mitigate growth inhibition in αca1-2. These results evidently suggest that OsαCA1 deficiency compromised the conversion from HCO3 − to CO2, and restricted the molecular diffusion of CO2, which, in turn, caused Cc decline and, consequently, led to a CO2 undersupply for photosynthesis.

Discussion
Photosynthesis is the basic process underlying plant growth and food production. CAs modulate photosynthesis through carbon-concentrating processes or other mechanisms, which influence photosynthetic carbon assimilation and, consequently, plant (J) dry weight of root. One-week-old seedlings were treated with or without 100 µM of NaHCO 3 for two weeks. Values are shown as means ± SEM in (C-J) determined via two-way ANOVA, where different letters indicate a significant difference, p < 0.05, and n ≥ 20.

Discussion
Photosynthesis is the basic process underlying plant growth and food production. CAs modulate photosynthesis through carbon-concentrating processes or other mechanisms, which influence photosynthetic carbon assimilation and, consequently, plant productivity [10,52,53]. However, genetic evidence of specific CA isoforms, particularly with respect to αCA, which plays various roles in dark reactions, is lacking. In this study, we demonstrated that OsαCA1 deficiency is a limiting factor of photosynthesis in rice (Figure 2). OsαCA1 influences CO 2 availability to ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) at the chloroplast carboxylation sites and is required to enhance the dark reactions of photosynthesis (Figures 4 and 5A). As a result, reduced biomass and yield were observed in the αca1-2 mutant (Figure 3). These results underscore the importance of CAs in determining plant physiology and crop productivity.

OsαCA1 Functions in Photosynthesis via Regulating CO 2 Availability
The CO 2 supply to Rubisco was affected by the resistance of stomata and mesophyll cells [11,12,14]. The function of CAs in stomata biology has been well docu-mented [34,35,45]. The knockout mutants βca1βca4 in Arabidopsis and βca1 in rice showed lower sensitivity to CO 2 -induced stomatal closure [34,45], and AtβCA1 and AtβCA4 mediated stomatal development regulated by CO 2 [35]. The higher g s observed in αca1-2 than that of the WT ( Figure 5C) corresponds well with the phenotypes of βca1βca4 in Arabidopsis and βca1 in rice [34,45]. However, the lower L s suggests that the stomata were not the reason for the reduced C c in αca1-2 ( Figure 5D). Despite its enhanced g s , the αca1-2 mutant had a decreased assimilation rate, resulting in a greatly decreased WUE (Figures 2 and 5C), suggesting that OsαCA1 may be a positive regulator of WUE in rice. The increased g s observed in the αca1-2 mutant was likely due to the feedback regulation to increase CO 2 uptake and compensate for the decreased CO 2 availability in chloroplasts to minimize the reduction in carbon assimilation. Surprisingly, we also observed an obvious reduction in g m in αca1-2, implying that CO 2 diffusion suffered from a larger degree of resistance from the intercellular space to the carboxylation sites in αca1-2 ( Figure 5E). Multiple studies have suggested that CAs play important roles in CO 2 diffusion based on their catalytic activity [13,53,54]. In one such study, plasma-membrane-located AtβCA4 interacted with AtPIP2;1 and was critical for the CO 2 permeability of the plasma membrane [55]. In this study, given that OsαCA1 was located in the chloroplasts ( Figure 1C), OsαCA1 might play a role in CO 2 diffusion in chloroplasts. Tholen and Zhu demonstrated that once entering the chloroplast, CO 2 is partially converted into HCO 3 − to promote its diffusion in the chloroplast stroma, while around the carboxylation sites, HCO 3 − is converted into CO 2 for the carboxylation reaction of Rubisco. This process is dependent on the CA concentrations in chloroplasts [25]. OsαCA1 presented CA activity ( Figure 6A). However, we failed to detect a significant difference in the CA activities in either the leaves or chloroplasts between the WT and αca1-2 ( Figure 6B,C). There were several explanations for this discrepancy. First, it could have been due to methodological problems. Considering the high abundance of chloroplast-located OsβCA1, which contributes 80% of the CA activity [45], it is conceivable that the relatively small decrease in the total CA activity caused by the OsαCA1 knockdown was beyond the relatively low measurement resolution of the method we used. To address this issue in the future, a more accurate tool for detecting subtle changes in CA activity would be required. The second possibility is that OsαCA1 might regulate CO 2 diffusion independent of its CA activity. The transformation of the active and inactive forms of OsαCA1 into OsαCA1-deficient mutants and a subsequent examination of CO 2 -supply-related phenotypes would be useful for evaluating this possibility in the future. The observation that growth inhibition in αca1-2 could only be partially complemented by the elevated CO 2 concentration but not by the HCO 3 − treatment (Figure 7) indicates that the conversion of HCO 3 − to CO 2 in the chloroplasts was impaired, which, in turn, resulted in an inadequate supply of CO 2 to Rubisco. If this is the case, OsαCA1 must have a distinct substrate preference mechanism from other CAs during the interconversion between CO 2 and HCO 3 − . This is, of course, speculation, but it is a topic that can be explored in the future.

OsαCA1 Is Conserved in Arabidopsis
Regarding CO 2 -induced stomatal closure, the loss of function of βCA1βCA4 in Arabidopsis or βCA1 in rice caused reduced sensitivity, indicating that AtβCA1, AtβCA4, and OsβCA1 had conserved roles in CO 2 -regulated stomatal movement [34,45]. We have explored whether the function of αCA1 is conserved in different plants. The sequence alignment and the collinearity analysis showed that AtCAH1 was the homologous protein of OsαCA1 in Arabidopsis ( Figure S7). It has been reported that the chloroplast localization of AtCAH1 depends on its N-terminal SP and glycosylation modification [42]. In this study, we found that the SP of OsαCA1 is vital for its chloroplast localization ( Figure 1C). The growth defects of the T-DNA insertion mutants of the AtCAH1 gene indicated the possibility that AtCAH1 also participates in plant growth regulation ( Figure S8). Determining whether AtCAH1 modulates photosynthesis and growth through influencing CO 2 availability and investigating the functional conservation between OsαCA1 and its close homologs in other species would be worth further investigation.

OsαCA1 may Be Beneficial to Environmental Adaptation of Rice
Accumulating evidence is suggesting that chloroplasts not only carry out photosynthesis but also produce various metabolites and participate in plant responses to adverse conditions [56,57]. Our observations that OsαCA1 is located in chloroplasts ( Figure 1C) and that OsαCA1 deficiency significantly restricted plant growth and reduced WUE ( Figure 2C,F and Figure 3A-G) suggest the importance of OsαCA1 functions in plants' adaptation to adverse conditions, especially drought, which has been a prime challenge for plant life since it moved onto land and one that will likely worsen if climate change continues [58]. Thus, we are currently generating transgenic lines that overexpress the OsαCA1 gene to further explore how OsαCA1 affects yield potential and plant responses to water limitation. A better understanding of the connection between the expression of OsαCA1 and rice photosynthesis and WUE will be informative with regard to the breeding of high-yielding and stress-tolerant crops against the backdrop of climate change and population rise.

Plant Material, Growth Conditions, and Treatments
The rice (Oryza sativa L.) used in this study was of the background of Japonica cv.  [59]. For the elevated CO 2 treatments, germinated seeds were cultured in an incubator with 1000 ppm CO 2 concentration for two weeks. For HCO 3 − treatment, one-week-old seedlings were cultured in a nutrient solution supplemented with 100 µM of NaHCO 3 . The plants in the soil pot were cultured in a greenhouse with 11 h light (28 • C)/13 h dark (26 • C) cycle.

RNA Extraction and qRT-PCR
Total RNA was extracted from rice tissues with Plant Total RNA Kit (ZOMANBIO, Beijing, China). PrimeScript TM RT reagent Kit (TaKaRa, Shiga, Japan) was used to perform reverse transcription. qRT-PCR was performed using 2× HQ SYBR qPCR Mix (without ROX) (ZOMANBIO). The qRT-PCR primers used are listed in Table S1.

Subcellular Localization Analysis
CDS of OsαCAs and the sequence encoding the SP and CA domains of OsαCA1 were cloned to the expression vector (PCUN 1300-GFP) and then transiently transformed to the protoplasts of rice leaf sheath for 12 h. The primers used are listed in Table S2. The fluorescence was observed using a confocal laser scanning microscope (TCS SP8, Leica Microsystems, Wetzlar, Germany) according to the process described by Wang et al. [60]. The excitation wavelengths of GFP and chlorophyll were 488 nm and 552 nm, respectively, and the emission wavelengths were from 498 nm to 540 nm and from 660 nm to 710 nm, respectively.

Measurement of Gas Exchange
The gas exchange parameters were measured by Li-6800 portable photosynthetic apparatus (Li-6800, LI-COR, Lincoln, NV, USA) with environment parameters set as 1000 µmol m −2 s −1 light intensity, 400 ppm CO 2 concentration, a temperature of 28 • C, and 60% relative humidity. The first fully expanded leaf of 2-week-old seedlings was used for measurement at the vegetative growth stage, and the flag leaves were used for measurement at the reproductive growth stage. The WUE is the ratio of the photosynthesis rate to the transpiration rate, and the L s was calculated according to the following formula: L s = (1 − C i /C a ) * 100%. C i -intercellular CO 2 concentration; C a -ambient CO 2 concentration [61].

Determination of Starch Content
A total of 0.25 g of dried leaf powder was added to 10 mL of 50% ethanol and stirred for 10 min. Then, 7.5 mL of 60% perchloric acid was added and stirred for 10 min. The samples diluted to 100 mL were filtered and analyzed via SAN ++ automatic wet chemical analyzer (SAN ++ , Skalar, Delft, Netherlands).

Determination of g m and C c
The g m was determined by the curve-fitting method [62] and the Variable J method [63]. For the curve-fitting method, the measured CO 2 response curve was fitted by a nonrectangular hyperbola version of the model [64]. For the latter method, g m and C c were calculated by the following formula. For Γ * (the CO 2 compensation point without respiration) and R d (the photorespiration rate), we used the empirical values: 40 µmol mol −1 and 1 µmol m −2 s −1 , respectively [65]. A N : net photosynthesis rate.

Protein Extraction and Purification
The production of protein via Escherichia coli was essentially performed as described below [66]. Briefly, CDS of OsαCA1 was cloned to the expression vector (pET-4T) and then transformed to Rosetta (DE3) to obtain GST-OsαCA1 fusion proteins. The primers used are listed in Table S2. BeyoGold TM GST-tag Purification Resin (Beyotime, Shanghai, China) was used for protein purification. A lysis buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , and pH 7.3) was added to the pellet. With high-pressure crushing, the bacterial lysate was centrifuged at 4 • C, 10,000 g for 30 min. The mixture of the supernatant and the resin (1:50) was gently shaken for 1 h and added into the empty column tubes of the affinity chromatography. The mixture was washed with lysis buffer 6 times, using 1 mL each time. The target protein was eluted with elution buffer (50 mM Tris-HCl, 10 mM GSH, and pH 8.0) 6 times, using 1 mL each time, and the purified protein was obtained.
The extraction of total proteins from rice leaves was performed according to the method reported by Chen et al. [67]. Briefly, 0.5 g sample was added into 300 µL of protein extract buffer (50 mM pH7.5 Tris-HCl, 0.5% Triton X-100, 150 mM NaCl, and protease inhibitor), mixed, and centrifuged at 12,000 g for 20 min at 4 • C. The supernatant was crude protein.
The extraction of chloroplast proteins from rice leaves was performed according to the method described by Du et al. [68]. A total of 3 g sample was added into 15 mL 1× CIB (2.5× CIB: 2.5 mM EDTA, 125 mM Tricine, 2.5 mM DTT, 2.5 mM MgCl 2 , 1.25 M Sorbitol, 2.5% BSA, and diluted to 1× CIB before use). After gentle shaking, the reaction was filtered and centrifuged at 4 • C and 200 g for 3 min. The supernatant was centrifuged at 1000 g for 7 min. A total of 1 mL 1× CIB was added to scatter the precipitate, and the chloroplast suspension was obtained. Then, 40% Percoll solution (Percoll: ddH 2 O: 2.5× CIB = 2:1:2) was transferred into 2 mL centrifuge tubes, applying 1.5 mL to each tube, and then 0.5 mL chloroplast suspension was carefully and slowly layered onto the Percoll solution and centrifuged at 4 • C and 1,700 g for 6 min. The complete chloroplastwas at the bottom. This was washed with 1× CIB (without BSA); then, lysis buffer was added (2 mM EDTA, 2 mM DTT, 10% glycerol, 10 mM Tricine, and 0.0025% PMSF). After being left on ice for 30 min, chloroplast proteins were obtained.

CA Activity Assay
The proteins were quantified with Quick Start™ Bradford Reagent (Bio-Rad, Hercules, California, USA) and diluted to the same concentration. CA activity assay was performed according to the steps described by Sun et al. [43]. CO 2 was continuously fed into 200 mL of ice water for 30 min to obtain CO 2 -saturated water. A total of 3 mL of 0.2 M, pH 8.3 Tris-HCl was added to 2 mL of CO 2 -saturated water and the pH was lowered, which was determined by pH meter (Orion Star TM A211, Thermo Fisher Scientific, Waltham, MA, USA). The time required for the pH to be reduced from 8.3 to 6.3 was recorded as T 0 . A total of 10 µL of enzyme was added to the mixture of 2 mL CO 2 -saturated water and 3 mL of Tris-HCl, and the time required for the pH to be reduced from 8.3 to 6.3 was recorded as T. The CA activity was calculated using the following formula: units = 2 * (T 0 − T)/T.

Statistical Analysis
All experiments were repeated at least three times, and similar results were obtained. GraphPad Prism 8 was used to analyze the data and create the figures. All data conformed to normal distribution. When there was only one variable in the experiment, Student's t-test was used to compare the differences between two sets of data with small sample size (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001), and one-way ANOVA was used for multiple sets of data. Two-way ANOVA was used to determine the significant differences between the two variables used in the experiment. When one-way ANOVA and two-way ANOVA were used, different letters were used to indicate a significant difference, which was determined at p < 0.05.

Conclusions
We identified OsαCA1 as a chloroplast-located CA. The distribution and abundance of OsαCA1 proteins correlated well with their proposed biological roles in plant photosynthesis reactions and productivity; thus, OsαCA1 is a beneficial gene for the improvement of the yield potential and environmental adaptation of crops against the backdrop of climate change and population rise. To the best of our knowledge, this is the first functional description of an α-type carbonic anhydrase in rice.