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.