Abstract
C4 photosynthesis enables the capture of atmospheric CO2 and its concentration at the site of RuBisCO, thus counteracting the negative effects of low atmospheric levels of CO2 and high atmospheric levels of O2 (21 %) on photosynthesis. The evolution of this complex syndrome was a multistep process. It did not occur by simply recruiting pre-exiting components of the pathway from C3 ancestors which were already optimized for C4 function. Rather it involved modifications in the kinetics and regulatory properties of pre-existing isoforms of non-photosynthetic enzymes in C3 plants. Thus, biochemical studies aimed at elucidating the functional adaptations of these enzymes are central to the development of an integrative view of the C4 mechanism. In the present review, the most important biochemical approaches that we currently use to understand the evolution of the C4 isoforms of malic enzyme are summarized. It is expected that this information will help in the rational design of the best decarboxylation processes to provide CO2 for RuBisCO in engineering C3 species to perform C4 photosynthesis.
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CSA, MFD, MS, MGW and MAT are members of the Researcher Career of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina) and CEA is a fellow of the same institution.
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11120_2013_9879_MOESM1_ESM.tiff
Supplemental Fig. 1 Protein design using DNA technology. a Site-directed mutagenesis. For an amino acid replacement, primers that harbour a mismatch and match in an internal region of cDNA template introduce the mutation in a PCR cycle generating a modified DNA fragment. Then, in a second PCR cycle the DNA generated is used as ‘large primer’ to obtain the modified full cDNA. b Chimeric protein synthesis. This strategy involves the interchange of regions or domains between two parental proteins. For this, it is necessary to identify the conserved recognition sites for restriction enzymes (REs) on the cDNAs that encode the parental proteins. Thus, both cDNA are digested with the REs, the products are purified and put together under ligation conditions. Usually, one of the cDNA lacks the recognition sequence for the RE so it is first introduced by site-directed mutagenesis as was described. c Protein co-expression. Western blot, using a mixture of antibodies against A. thaliana NADME1 and NAD-ME2, on fractions from purification steps from E. coli BL21 cells co-transformed with pET32-NAD-ME1 and pET29-NAD-ME2 (lanes 1–3) or E. coli BL21 cells transformed with pET29-NAD-ME2 (lanes 5–7). Lanes 1 and 5, E. coli crude extract after induction; lanes 2 and 6, last Ni-NTA acid column fraction wash; lanes 3 and 7, elute fraction. Purified NAD-ME1 fusion protein (80 kDa) was loaded in lane 4 (Adapted from Tronconi et al. 2010a). The estimated molecular weight of mature NAD-ME2 is 58 kDa. Supplementary material 1 (TIFF 1503 kb)
11120_2013_9879_MOESM2_ESM.tiff
Supplemental Fig. 2 Methodological approach for the study of C4 NADP-malic enzyme evolution. The biochemical characterization of an enzyme requires the coordinated combination of different analytical and methodological blocks: Recombinant proteins, Kinetics, Regulation and Structure. The scheme of the working flow for the study of ZmC4-NADP-ME is presented as an example. This summarizes the works described in Detarsio et al. (2007); Alvarez et al. (2012) and Saigo et al. (2013). Supplementary material 2 (TIFF 575 kb)
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Saigo, M., Tronconi, M.A., Gerrard Wheeler, M.C. et al. Biochemical approaches to C4 photosynthesis evolution studies: the case of malic enzymes decarboxylases. Photosynth Res 117, 177–187 (2013). https://doi.org/10.1007/s11120-013-9879-1
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DOI: https://doi.org/10.1007/s11120-013-9879-1