Introduction
The current global research efforts are actively focusing on increasing crop yield for sustainable food and fuel production (Long et al., 2015). C3 photosynthesis is limited by available CO2. Therefore, it is important to pump more CO2 into the leaf cells to alleviate carbon limitation in C3 plants. We consider it to be an important option to enhance crop productivity by manipulating carbonic anhydrase (CA, EC 4.2.1.1), which is mostly a zinc-containing metalloenzyme that catalyses the inter-conversion of CO2 and HCO3 - and is widely distributed in eukaryotes and prokaryotes (Hewett-Emmett and Tashian, 1996; Liljas and Laurberg, 2000; Moroney et al., 2001; Bonacci et al., 2012). CAs belong to 6 independent gene families (Moroney et al., 2011): ɑ-CAs , β-CAs , γ-CAs , δ-CAs ,ε-CA and ζ-CA . In higher plants, only α-CA ,β-CA , and γ-CA exist. In spite of their structural differences, these 3 isoforms share the same general catalytic mechanism (Lindskog, 1997). In C3 plants, up to 2% of the total leaf protein is CA and 95% of the total CA activity is in chloroplast stroma (Okabe et al., 1984; Tsuzuki et al., 1985). Further, in C3 plants, chloroplastic CA is responsible for maintaining CO2 around r ibu losebis phosphate c arbo xylase/o xygenase (rubisco ) by facilitating its diffusion across the chloroplast envelope, after its formation from bicarbonate (Giordano et al., 2005). Without the CA , the hydration reaction of CO2 is usually very slow (Raven, 1997). CA also participates in respiration, pH regulation, inorganic carbon transport, ion transport, and water and electrolyte balance (Tashian, 1989; Henry, 1996; Smith and Ferry, 2000; Badger and Price, 2003). In Arabidopsis thaliana ,βCAs are responsible for the uptake of HCO3- from soil by the roots. Bicarbonate absorbed by roots is transported to stem and leaves and is assimilated by rubisco or phosphoenolpyruvate carboxylase,PEPC (Dąbrowska-Bronk et al., 2016). High light and long photoperiods favor rubisco activation leading to higher biomass. Conversely, low light and short photoperiods activate PEPC in C4 plants (Bailey et al., 2007). Further, it has been suggested that a thylakoid CA may be involved in Photosystem II reactions (Stemler, 1997; Shevela et al., 2012). In the cytoplasm of C4 mesophyll cells, CA is known to catalyse the very first reaction in its carbon fixation pathway (Hatch and Burnell, 1990), which is the hydration of CO2 producing HCO3 , the latter being the substrate for PEPC (also see Ludwig, 2012). Plants with C4 photosynthesis are efficient in carbon hydration and assimilation and have an advantage over C3photosynthesis. In the recent past, there have been several attempts to overexpress or regulate C4 pathway genes into C3 plants (for a review, see Miyao et al., 2011; Schuler et al., 2016; for attempts from our Lab, see: Kandoi et al., 2016, 2018; Borba et al., 2018; Lin et al., 2020). To enhance CO2uptake by C3 plants, carbonic anhydrase content needs to be substantially increased in the cytosol. Most studies have attempted to overexpress C3 CA in the cytosol or chloroplasts of C3 plants (Hu et al., 2010; Pal et al., 2015); however, this has not resulted in enhancing their rates of photosynthesis. Further, Arabidopsis double knockout mutants of the cytosolic β-CA2 and β-CA4 had even reduced growth rates and chlorosis of the younger leaves relative to that in the wild-type plants, when grown at low concentration of CO2(DiMario et al., 2016). Amino acid sequence alignment of cytoplasmicFlaveria bidentis (C4 plant) βCA3(FbβCA3 ), with different isoforms of Arabidopsis thaliana (C3 plant) βCAs (AtβCAs ), demonstrates that all the sequences have similar amino acid binding sites for Zn2+ as well as for the substrates (seeFig. S1 ).
A pertinent question that remains to be answered is whether overexpression of C4 carbonic anhydrase, having low Km for CO2 (Hatch and Burnell, 1990; Ignatova et al., 1998) and ability to efficiently hydrate CO2, in the cytosol of C3 plants, improves photosynthetic efficiency. To explore this question, we have, in this study, overexpressed carbonic anhydrase βCA3 from a dicot C4 plantFlaveria bidentis in the C3 plantArabidopsis thaliana (Fig. 1 ). Our results show that overexpression of FbβCA3 in the C3 plant results in improved photosynthesis, water use efficiency, higher starch content and biomass.