Evolution of C4 phosphoenolpyruvate carboxylase in the genus Alternanthera: gene families and the enzymatic characteristics of the C4 isozyme and its orthologues in C3 and C3/C4 Alternantheras

Abstract

Phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.3) is a key enzyme of C(4) photosynthesis. It has evolved from ancestral non-photosynthetic (C(3)) isoforms and thereby changed its kinetic and regulatory properties. We are interested in understanding the molecular changes, as the C(4) PEPCases were adapted to their new function in C(4) photosynthesis and have therefore analysed the PEPCase genes of various Alternanthera species. We isolated PEPCase cDNAs from the C(4) plant Alternanthera pungens H.B.K., the C(3)/C(4) intermediate plant A. tenella Colla, and the C(3) plant A. sessilis (L.) R.Br. and investigated the kinetic properties of the corresponding recombinant PEPCase proteins and their phylogenetic relationships. The three PEPCases are most likely derived from orthologous gene classes named ppcA. The affinity constant for the substrate phosphoenolpyruvate (K (0.5) PEP) and the degree of activation by glucose-6-phosphate classified the enzyme from A. pungens (C(4)) as a C(4) PEPCase isoform. In contrast, both the PEPCases from A. sessilis (C(3)) and A. tenella (C(3)/C(4)) were found to be typical C(3) PEPCase isozymes. The C(4) characteristics of the PEPCase of A. pungens were accompanied by the presence of the C(4)-invariant serine residue at position 775 reinforcing that a serine at this position is essential for being a C(4) PEPCase (Svensson et al. 2003). Genomic Southern blot experiments and sequence analysis of the 3' untranslated regions of these genes indicated the existence of PEPCase multigene family in all three plants which can be grouped into three classes named ppcA, ppcB and ppcC.

C4 Cycles: Past, Present, and Future Research on C4 Photosynthesis

“…During this transition, 21 amino acids evolved under positive selection and converged to similar or identical amino acids. In some amino acid positions, identical changes have also been recorded in non-grass C 4 species (Blä sing et al, 2000; Gowik et al, 2006;Christin et al, 2007Christin et al, , 2011. At some sites, such convergence appears to reflect the need for a specific amino acid for C 4 function, whereas at other sites, there appears to be a requirement for loss of the C 3 -associated amino acid.…”

Species Having C4 Single-Cell-Type Photosynthesis in the Chenopodiaceae Family Evolved a Photosynthetic Phosphoenolpyruvate Carboxylase Like That of Kranz-Type C4 Species

“…Sequence comparison of plant PEPCs revealed that C 4 enzymes investigated to date, of both monocot and dicot origin, harbor a Ser residue at a position corresponding to 774 of F. trinervia PEPC, 775 of Alternanthera pungens, or 780 in maize C 4 -PEPC (Blässing et al, 2000;Gowik et al, 2006), whereas this position is occupied by an Ala in all nonphotosynthetic and CAM PEPCs. As expected for C 4 -type PEPC, the deduced amino acid sequence obtained from S. eltonica displays a Ser residue in the equivalent position, whereas the equivalent position in C 3 S. linifolia PEPC has an Ala.…”

“…Although more than one isoform may be present in leaves of the species under study, as previously seen in Arabidopsis (Arabidopsis thaliana), Flaveria spp., Alternanthera spp., and maize, among others (Westhoff et al, 1997;Dong et al, 1999;Sánchez and Cejudo, 2003;Gowik et al, 2006), activity on native IEF gels shows only one activity band for each species (Fig. 2).…”

Reversible Burst of Transcriptional Changes during Induction of Crassulacean Acid Metabolism in Talinum triangulare

“…During the evolution of C 4 , the change of a single amino acid residue in the N-terminal region of PEPC, namely from Ala to Ser, was shown to increase the enzyme's kinetic efficiency compared to C 3 sister plants (Svensson et al, 1997;Bläsing et al, 2000;Svensson et al, 2003;Gowik et al, 2006). It was recently shown that in C 4 and CAM species within the Caryophyllales, to which the Talinaceae belong, the isogenes of a recurrently recruited gene lineage encoding PEPC1, namely ppc1-E1c, almost exclusively encodes the C 4 -like Ser-780 (Christin et al, 2014).…”