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.