Enzymatic Characteristics of Higher Plant Carbonic Anhydrase and Its Role in Photosynthesis

Carbonic anhydrase, a metalloenzyme which catalyses the reversible interconversion of HCO3 and CO2, is a major protein component of higher plant tissues. It is now shown that DNA sequence and encoded proteins for the different , β, ɑ γ, δ, ε, and ζ forms of carbonic anhydrases are present in living organisms. While there are no sequence homologies between the different type of carbonic anhydrases, all the , β, and ɑ γ carbonic anhydrase gene family enzymes in higher plants catalyze the same chemical reaction. Its specific function is generally assumed to convert CO2 to HCO3 for the phosphoenolpyruvate carboxylase reaction and convert HCO3 to CO2 for the ribulose-1,5-bisphosphate carboxylase reaction in photosynthesis. Moreover, carbonic anhydrase activity in guard cells is required for CO2-mediated stomatal regulation and carbonic anhydrases may provide an approach for plant alternatively protection against stress conditions. Recent studies on carbonic anhydrases described in this manuscript include the characterization and gene family of carbonic anhydrases, physiological function of higher plant CAs, and gene engineerings about higher plant CAs.


Introduction
It is approved that yield potentials of cereal crops cannot further be increased by addition of chemical fertilizer, and photosynthesis increasing could be the major avenue to increase crop yield in next 50 years (Surridge, 2002;Reynolds et al., 2009).Plant photosynthetic organisms have developed methods for the acquisition of inorganic carbon (Ci) to aid ribulose-1,5-bisphosphate carboxylase (RuBisco) and to enhance CO 2 fixation.Carbonic anhydrases often play important roles in this process.Carbonic anhydrase (CA, EC: 4.2.1.1),a ubiquitous zinc metalloenzyme among living organisms, catalyses the reversible interconversion with very high catalysis rates reaching 10 6 s -1 : CO 2 + H 2 O ↔ HCO 3 − + H + (Raven, 1995).After CA was first discovered in red blood cells (Meldrum & Roughton, 1933), the enzyme has been found in animals, plants, archaebacteria and eubacteria.Besides carboxylase and decarboxylase rates are influenced by CAs, the processes of pH fluctuations, ion regulation, ion exchange and inorganic carbon diffusion are also influenced by CAs (Raven, 1995).
There is a high degree of structural homology among CAs from different higher plants.In plant leaves, CA concentration is 1-20% of total soluble protein and is next only to RuBisco in chloroplast (Fett & Coleman, 1994).Plant CAs facilitate CO 2 supply to phosphoenolpyruvate carboxylase (PEPC) in C 4 and CAM plants, and facilitate CO 2 supply to RuBisco in C 3 plants (Tiwari et al., 2005).The higher plants have developed their versions of photosynthetic CO 2 concentrating mechanisms (CCMs) to aid Rubisco in CO 2 capture.An important aspect of CCMs is the critical roles of CAs play in the overall process, participating in the interconversion of CO 2 and HCO 3 − both inside and outside the cell.The capacity to restrict the photorespiration loss of carbon could be one of important trait to develop plants with efficient photosynthetic capacity and could be a good option to enhance crop productivity.So, it would be one important research subject to enhance the CO 2 assimilation intensity of C 4 pathway in C 3 plants by the artificially regulation of CA expression.

General Enzymatic Characterization of Higher Plant CAs
There are distinct differences in primary protein structures of the various CAs, and their secondary and tertiary structures must be somewhat different.CAs shows a striking similarity in their metal-coordinating sites (Kimber & Pai, 2000).CA is a metalloenzyme requiring Zn 2+ for its activity, besides, some CAs containing Co and Cd were also published (Price & Morel, 1990;Morel et al., 1994).CA exhibits diverse compartmentalization among organs, tissues, and cellular organelles commensurate with different physiological roles.The CA activities in different plants are varies (Wu et al., 2006).The CA enzyme activity follows pattern in tobacco: leaves>stem>pods (Majeau, Arnoldo, & Coleman, 1994), and the enzyme has been found in plant roots and fruits (Diamantopoulos et al., 2013).
There are similarity between cytoplasm CA and chloroplast CA in higher plants including CA kinetic properties, affinity for CO 2 and sensitivity to inhibitors (Hatch & Burnell, 1990).It is shown from the purification of the Solanum cytosolic isoform that it is structurally and biochemically similar to the chloroplastic form, although its monomeric mass is larger (Rumeau et al., 1996).The difference of promoter region between cytoplasm and chloroplast CA induces the difference of expression between the two kinds of CAs (Badger & Price, 1994).The cytoplastic CAs, maintains HCO 3 − pools and compensates leakage of free CO 2 from the cytoplasm.Chloroplast CAs appear to be associated with other enzymes of the Calvin cycle in a large multienzyme complex (Jebanathirajah & Coleman, 1998).Although the bulk of the CA activity is associated with the chloroplast fraction in C 3 plants, there are at least 10 to 15 % of the total activity is cytoplastic (Rumeau et al., 1996).Chloroplast CA contents in higher plants are saturated and cytoplasm CA contents are inadequate (Badger, 2003), and it is important to study the function and application of cytoplasm CAs in higher plant resistance because the CA activity was up-regulated by salts and osmotic stresses (Yu et al., 2007).

Different Gene Family of CAs in Higher Plants
With regard to higher plants, the distribution of the number of CA genes in each gene family also varies.Arabidopsis has 19 CA genes (8ɑ-CA, 6β-CA, 5γ-CA) (Arabidopsis Genome Initiative, 2000), and rice (Yuan et al., 2005) have a similar number.Despite their structural difference, the ɑ-CA, β-CA and γ-CA isoforms in higher plants share the same general catalytic mechanism (Lindskog, 1997).
The ɑ-CAs were widely identified in vertebrates (Meldrum & Roughton, 1933), algae (Fujiwara, Ishida, & Tsuzuki, 1996), higher plants (Arabidopsis Genome Initiative, 2000), and eubacteria (Chirica, Elleby, & Lindskog, 2001).Most a-CAs is monomers of about 30 kDa with at least three histidines.The Arabidopsis ɑ-CA molecular structure is dominated by antiparallel β-sheets forming a spherical molecule with two halves and the active site is located in a funnel shaped crater with the zinc atom (Moroney, Bartlett, & Samuelsson, 2001).It is shown from EST sequence information in Arabidopsis that there are three ɑ-CA coding sequences are expressed.
The γ-CAs are discovered in the archaebacterium (Alber & Ferry, 1994), algae (Klodmann et al., 2010), eubacteria and plants (Newman, 1994).Recent work has indicated that γ-or γ-like CAs were part of Complex I of the mitochondrial electron transport chain in plants and algae, and plant γ-CA might play roles in CO 2 cycles of photorespiration, but chloroplastic γ-CA has not yet been reported (Martin et al., 2009).δ-CAs have only been described in some diatoms.The δ-CA families appears to be a case of convergent evolution with almost no sequence similarity with the ɑ-, β-, or γ-CA types (Roberts, Lane, & Morel, 1997).The ε-CA family is limited to bacteria containing a-type carboxysomes and has not been found in eukaryotes.ε-CAs is part of the carboxysome shell and has additional domains that serve the function in bacteria (Tanaka et al., 2008).ζ-CA is limited to maline protists and resembles the β-CA family, with other metals such as Cd or Co substitute for Zn (Lane et al., 2005).

Physiological Functions in Photosynthesis of Higher Plant CAs
As CA appears to be found in all organisms studied, the enzyme would seem to indicate important physiological functions.The connection between CA and photosynthesis is perhaps the most widely understood roles of plant CA.This role is most important because the uncatalyzed interconversion between CO 2 and HCO 3 − is 10 4 times slower compared with the flux of CO 2 in photosynthesis (Badger & Price, 1994).CA is the only enzyme of photosynthetic carbon metabolism, and any change in CA activity directly affects the rate of photosynthetic CO 2 fixation under CO 2 limiting conditions.
General physiological roles of plant CA is given below: One role is to supply CO 2 or HCO 3 − for some metabolic reactions, such as HCO 3 − for the PEPC reaction in C 4 photosynthesis and CO 2 for the Rubsico reaction in photosynthesis, requiring the intervention role of CA enzyme.Even within the C 3 mesophyll cell cytosol, CA may be required for the provision of HCO 3 -for PEPC which plays a compensating role.Besides Rubisco and PEPC, a number of other biological reactions require either CO 2 or HCO 3 − which can be supplied by CA.Take the acetyl CoA carboxylase for example, which catalyzes the initial step in fatty acid biosynthesis and uses only CO 2 (Vats, Kumar, & Ahuja, 2011).
At the same time, CA is the delivery of Ci to the correct location within the cell and is the reduction of CO 2 leakage (Braun & Zabaleta, 2007).CO 2 which enter into cells from the external gaseous environment can pass through biological membranes and quickly leak out of the cell.Plants may deal with this problem by converting CO 2 generated by the cell to HCO 3 − .The ectopic expression of CA activity in the cytosol of bundle-sheath cells of Flaveria bidentis leaded to an increase of CO 2 leakage in bundle-sheath (Ludwig et al., 1998).It showed that the absence of CA from the bundle sheath cells may be limit the conversion of accumulated CO 2 to HCO 3 − and this may limit the reduction of CO 2 leakage.
The next role of CA is in the CO 2 signaling pathway.It is suggest that low chloroplastic CA plants were compensating by increasing stomatal conductance to improve CO 2 entry into the leaf (Williams, Flanagan, & Coleman, 1996).Hu et al. (2010) suggested that guard cell expression of mammalian α CAII complemented the reduced sensitivity of ca1 ca4 mutant plants, showing that CA-mediated catalysis is an important mechanism for CO 2 induced stomatal closure.
Moreover, it has also been suggested that CA plays indirect roles in photosynthesis.CA activity has been hypothesized that it is possibly required for regulating chloroplast pH during rapid changes in light intensity and photosynthetic electron transport (Stemler, 1997).Ferreira et al. (2008) study demonstrated that βCA1 plays a significant role in seedling establishment in Arabidopsis.Besides, CA is involved in a variety of biological processes including pH regulation, ion exchange, respiration, biosynthesis, antioxidation and so on (Badger & Price, 1994;Moroney et al., 2001;Tetu et al., 2007).

The Application of Gene Engineering About Higher Plant CAs
Biosynthesis of CA may be regulated by some environmental factors such as light, salt content, osmetic stress, photon flux density, availability of Zn and CO 2 concentration (Karlsson et al., 1998).These results suggest that the expression of CA was related to environmental stresses.Transgenic Arabidopsis over-expressing OsCA1 had a greater salt tolerance at the seedling stage than wild-type plants and the CA activity was up-regulated by salts and osmotic stresses (Yu et al., 2007).The possible mechanism of over-expressing OsCA1 had a greater salt tolerance was that plants might rely on CA to catalyze HCO 3 -rapidly transformed to CO 2 in physiological drought condition caused by salt stress, to meet the physiology needs of the plants.Inhibition of CA activity in ethoxyzolamide-infiltrated C 3 plant leaf pieces resulted in an 80-90 % inhibition of photosynthesis at low CO 2 concentrations, and indicated an essential role for CA (Badger & Pfanz, 1995).Consequently, the regulation function of CA would play important role under stress conditions.
Over-expression of two specific isozymes, βCA1 or βCA4 considerably increased instantaneous water use efficiency in Arabidopsis thaliana plants indicating that βCA expression levels are not saturated in wild-type guard cells (Hu et al., 2010).All overexpression lines showed enhanced fresh weight from excised leaves compared with wild-type plants.Therefore, guard cell-targeted overexpression of βCA was sufficient to modulate CO 2 regulation of stomatal conductance and might provide an approach for improving the water use efficiency of C 3 plants.

Conclusions
In general, the studies achieve the efforts to reengineer crops to improve its photosynthetic efficiency under such conditions as CO 2 availability is limited.In most of the discussions on biotechnological approaches aiming to transfer C 4 -like features into C 3 plants, CA has received much less attention though CAs play important roles in photosynthesis process (Häusler et al., 2002).CAs take part in the adaptation to low CO 2 concentration in C 3 plants and it is one important research subject that how to enhance the CO 2 assimilation intensity of C 4 pathway in C 3 plants by artificially modifying the CA promoters.It is important that the study on the differ of CAs (especialy β-CA) between C 3 plants and C 4 plants, and the application of plant CA may contributed to enhance crop photosynthetic efficiency and increase crop yields.Some study data suggest that although increased CA levels occur, they alone are unable to maintain CO 2 concentrations within the chloroplast (Li et al., 2010;Vats et al., 2011).It is possible that an appropriate CA/Rubisco ratio would be more effective in maintaining chloroplast CO 2 levels.Moreover, identification of the molecular mechanisms of CA-participant CO 2 -mediated stomatal regulation is fundamental to understanding the regulation of gas exchange between plants and the atmosphere.Meanwhile, there is a striking correlation between stomatal conductance and CO 2 assimilation rate that differs between C 3 and C 4 plants.It would be interesting to determine if similar regulatory cis elements are present in genomic promoter sequences of both CA and C 4 PEPC isoforms.The mechanism behind these relationships is not yet understood.So, it is assumed that although we believe that plant CAs are important for biological functions, more work is required to resolve the specific field of CAs in photosynthesis improving and water use efficiency controlling.