Carbon fixation and water use efficiency (WUE)
Our data clearly demonstrate that the constitutive overexpression ofFbβCA3 in the cytoplasm of the transgenics resulted in higher rates of photosynthetic carbon assimilation, per unit leaf area, than in the VC plants (Fig. 6a ); this corresponds to the higher electron transport rates of PSI and PSII that provided sufficient NADPH and ATP for increased CO2 assimilation. Higher protein content must have been the reason not only for the elevation of the rates of photosynthesis, but for the increased metabolic processes including Chl biosynthesis (Table1 ), and photosynthesis. In the transgenics, as compared to VC, the quantum yield of CO2assimilation measured in limiting light intensities increased by 17% to 20%. These observations suggest that the transgenics had inherently higher photosynthetic efficiency and that they utilized the absorbed light energy much more efficiently.
The FbβCA3 overexpressors had lower stomatal conductance, decreased transpiration rate per unit leaf area, and a better WUE (Figs. 6d,e,f ). Overexpression of βCA1 or βCA4 in guard cells of Arabidopsis thaliana has shown a reduced stomatal conductance and a substantial increase in the WUE (cf. Hu et al., 2010); however, there was no increase in the rate of photosynthesis in that work.
Owing to the overexpression of FbβCA3 , having a higher affinity for CO2 than that of endogenous CA in the cytosol, the production of HCO3- is definitely expected to be higher diminishing the cytoplasmic [CO2]. It would provide a higher concentration gradient for a better diffusion of CO2 from the intercellular space to the cytoplasm of the mesophyll cells to keep a balance between CO2 uptake and HCO3- assimilation by cytoplasmic endogenous PEP carboxylase of Arabidopsis thaliana . The overexpression of FbβCA3 could, indeed, produce enough HCO3- under ambient CO2 to partially close the stomata to reduce the stomatal conductance and transpiration rate and increase the WUE (Figs. 6d,e,f ). Conversely, Kolbe et al. (2018) found that theβca1βca2 maize mutants, with 3% of wild type CA activity, had higher stomatal conductance, a slower stomatal closure in response to an increase in CO2 concentration. Further, in transgenic lines of Setaria viridis (C4 grass), with less than 13% CA activity, Osborn et al. (2017) observed that the photosynthetic rates decreased at lower CO2concentrations. Further, in CA-deficient (less than 20% of the wild type) F. bidentis little effect was found on carbon assimilation, and on g m (Cousins et al., 2006; Ogee et al., 2018). However, a knockout of chloroplastic βCA1 in A. thalianahas been shown to have poor seedling establishment due to decreased photosynthetic performance under ambient CO2 (Ferreira et al., 2008). For efficient photosynthetic performance by plants, relatively high CA activity is one of the important requirements inA. thaliana . On the basis of the above results, we conclude that the CAx plants had higher photosynthetic efficiency with higher water use efficiency.