CO2 response curves
In general, response of the light-saturated CO2assimilation rate (A ) to leaf intercellular CO2mole fraction (C i) consists of three phases: (i) the first phase is when assimilation is limited by the amount of activerubisco (slope of the initial phase); (ii) the second phase is an inflection to a slower rise, whereA max is reached due to limitation by the supply of substrate (ribulose 1,5-bisphosphate, RuBP); and (iii) the third phase is where photosynthesis does not respond to increasing CO2, nor it is inhibited by increasing oxygen concentration; further, it is often limited by triose-phosphate utilization (von Caemmerer and Farquhar 1981; Sharkey, 1985;; Ainsworth and Long, 2005; Sharkey et al., 2007; Bernacchi et al., 2013). In 21% O2 the transgenics had higher photosynthetic capacity,CE and V cmax than VC. This was partly due to increased gm and higherC c in the transgenics, albeit to a smaller extent. Higher g m has potential to simultaneously improve photosynthetic efficiency and intrinsic water use efficiency (Flexas et al., 2013). The CO2 must diffuse to the cytoplasm and the chloroplasts either through the lipid bilayer or probably through the pores of certain aquaporins (Tyerman et al., 2002; Flexas et al., 2008; Groszmann et al., 2017). However, the diifusion is slow and dependent upon the concentration gradient and inversely proportional to the distance. Therefore, diffusion of CO2 has to be high enough to match the rate of carbon assimilation. The cytoplasmic reserve of HCO3- generated by overexpressedFbβCA , when required, could be converted back to CO2 by the reverse reaction of CA, and the CO2 thus produced could diffuse through the envelope membrane due to high CO2 gradient created by consumption of CO2 by rubisco in the stroma during the day time (Fig. 1 ). This would have resulted in a partially higher [CO2] in the stroma (C c) to enhance photosynthetic carbon assimilation (Fig. 7a ). In higher plants, no bicarbonate transporter has been found in their chloroplast envelope membranes (Poschenrieder et al., 2018). However, bicarbonate functions as a small molecule activator of SLAC1 anion channels in the guard cells and may partly contribute to bicarbonate transport into the stroma (Xue et al., 2011). Nonetheless, the possibility of HCO3- directly diffusing from the cytosol to stroma to act as a reservoir for CO2 to increased plastidic [CO2], is rather small.
Increase in CE and V cmax in the transgenics suggest that at the atmospheric [CO2], the higher rates of photosynthesis is due to enhancedC c although the impact of pleiotropic effect on the overexpressors is not ruled out. We have used theA/C i curves to obtain information on theCE and V cmax ; we found it to be clearly higher in the transgenics. We suggest that this was, in part, due to an increase in the rubisco content and in chloroplastic CO2 (C c). In several earlier studies, the V cmax/CE has been shown to be strongly correlated with the content of rubisco (von Caemmerer and Farquhar, 1981; Makino et al., 1994; Jacob et al., 1995, Manter et al., 2004). The transgenics, in our work, had 8%-10% higher concentration of C c than the VC, and, we suggest that it must have partially contributed to the enhanced CE . As the CO2 compensation point remained almost similar both in the VC and in the transgenic plants, the rate of photorespiration, probably, did not decrease in the CA overexpressors. Increase in photosynthetic capacity could be attributed to an increase in the rates of ETRII and ETRI. The RuBP regeneration was better in the transgenics probably due to a general increase in the protein content. Rubisco activation state and RuBP levels were shown to be higher in other transgenic plants (Miyagawa et al., 2001; also see von Caemmerer & Evans, 2010). It has been demonstrated in C3 plants that facilitating electron transport by overexpressing the components of electron transfer chain can result in higher assimilation rates (Simkin et al., 2017). The higher carbon fixation by transgenics could be partly due to recapture of photorespiratory CO2 by the efficient FbβCApresent in the cytoplasm. The increase in the rate of carbon assimilation in transgenics when photosynthesis becomes oxygen insensitive cannot be the result of better CO2 supply. It may be due to better RuBP regeneration capacity in the transgenics. The A /C i curves further suggest that transgenics may have limitation in triose phosphate utilization (Sharkey et al., 2007). The higher photosynthesis rate in transgenics is also due to higher g m. The relationship betweeng m and CA activity on photosynthesis rate is dependent on the environment and/or the photosynthetic pathway (e.g. Cano et al., 2019; also see a review by Momayyezi et al., 2020).
Under 2% O2, the rate of photorespiration is expected to have been very minimal. The backward extrapolation of theA/C i curve in 2% O2 shows that the CO2 compensation point was close to 7 µmol mol-1 in these experiments. Under 2% O2 the A/C i curves for the VC as well as the transgenics saturated almost at ambient CO2. In 2% O2, the CE of transgenics was higher than that in the VC plants which suggests that the increases in CE and in the maximum rates of photosynthesis, under ambient CO2, were not due to a reduction in photorespiration but due to an increase in the inherent efficiency of photosynthesis of the transgenics possibly due to pleiotropic effect of CAoverexpression. In 2% O2, the maximum rate of CO2 assimilation is the same whether measured in 20% or 2% oxygen; this indicates that starch and sucrose synthesis set a ceiling on the rate of photosynthesis. This ceiling is higher in the plants with extra CA.
Due to their higher photosynthetic efficiency, the starch content of the transgenic plants was substantially higher than in the VC plants. The additional starch produced in CA overexpressors was partly used in the dark period by respiration, most likely for increased growth of plants that had higher biomass. The increase in dark respiration rate in the transgenics supported the energy demand for higher plant growth. This finding is in complete agreement with data from several earlier reports (Lefebvre et al., 2005; Biswal et al., 2012; Kandoi et al., 2016; Ermakova et al., 2019), where enzymes, such as sedoheptulose-bisphosphatase, chlorophyllide a oxygenase, PEPC and Rieske FeS protein of the Cytochrome b6f complex, were overexpressed one at a time. In these experiments, PSII photosynthetic efficiency, carbon assimilation, starch content, and dry matter accumulation were shown to have consistently increased. It is obvious to us that the increases in overall biomass is very important towards the goal of obtaining high yielding bioenergy crops (see Ort et al., 2015). Our results, reported here, on overexpressing cytoplasmic C4 carbonic anhydrase fromFlaveria bidentis in Arabidopsis thaliana clearly demonstrates highly significant increases in photosynthesis which is in the right direction to meet the global needs ahead of us.