Discussion
In our study, we have generated several lines of Arabidopsis thaliana overexpressing C4 Flaveria bidentis βCA3 that, in general, has a low Km for CO2. Overexpression of C4 FbβCA3 under the control of constitutive 35S promoter resulted in higher gene expression, higher protein abundance and higher enzymatic activity of CA , in the transgenic lines than in the VC (Fig. 2f, g ). In the CAoverexpressors, higher activity of CA resulted in hydration of extra CO2 and its conversion into bicarbonate, the substrate for PEPC (Hatch and Burnell, 1990) (see Fig. 1 ). However, in vitro activity of PEPC was similar in the transgenics and in the VC plants, as expressed on equal protein basis, in the presence of equal concentration of the substrate (Fig. 3a) . This implies that the protein abundance of PEPC did not substantially increase in response to CA overexpression.PEPC has a much higher (25 mM) Km of HCO3- (Bauwe 1986) than the concentration of HCO3- in the cytoplasm of C3 plants (Mukerji and Yang, 1974; Sato et al., 1988). Due to the higher concentration of HCO3- in the cytosol of CAoverexpressors, their endogenous PEPC activity must have increased generating more oxaloacetic acid (OAA) (see Fig. 1 ). The OAA generated by the increased carboxylation activity of endogenous cytoplasmic PEPC may lead to an increase in the carboxylic acid pool of the tricarboxylic acid (TCA) cycle. The anaplerotic role played by FbβCA in the cytoplasm by providing the pool of carboxylic acids as carbon skeleton to TCA cycle for amino acid and protein synthesis must have, we suggest, increased their content in the transgenics (see Fig. 1 ).
Although Chl and proteins are synthesized via two different metabolic processes, their biosynthetic pathways may be co-regulated by the nitrogen status of the system (Garai and Tripathy, 2018). Higher protein content in the transgenics would have resulted in increased Chl synthesis that is regulated by several genes. We propose that higher Chl content must have been due to increased expression of several Chl biosynthetic genes, such as PBGS , UROD , PPOX ,CHLI and PORC (Fig. 5a ). Similarly, overexpression of one of the Chl biosynthesis genes is known to up-regulate the abundance of message of several other genes involved in the greening process, suggested to be due to the presence of regulatory network among Chl biosynthesis genes (Alawady and Grimm, 2005; Pattanayak and Tripathy, 2011; Biswal et al., 2012). Our results demonstrate the pleiotropic effect of FbβCA3 overexpression on transcript abundance of genes involved in Chl biosynthesis.