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.