The non-photosynthetic phosphoenolpyruvate carboxylases of the C4 dicot Flaveria trinervia -- implications for the evolution of C4 photosynthesis

C4 phospho enolpyruvate carboxylases (PEPCase; EC 4.1.1.3) have evolved from ancestral non-photosynthetic (C3) isoforms during the evolution of angiosperms and thereby gained distinct kinetic and regulatory properties. In order to obtain insight into this evolutionary process we have studied the C3 isoforms, ppcB and ppcC, of the C4 dicot Flaveria trinervia (Spreng.) C. Mohr and compared them with the C4 enzyme of this species, ppcA, and its orthologue in the C3 species F. pringlei Gandoger. Phylogenetic analyses indicate that the ppcB PEPCase is the closest relative of the ppcA enzyme. In addition, the presence of ppcB also in the closely related C3 species F. pringlei suggests that this gene was present already in the ancestral C3 species and consequently that ppcA has evolved by gene duplication of ppcB. Investigation of the enzymatic properties of the ppcB and ppcC enzymes showed low and similar K(0.5)-PEP values and limited activation by glucose-6-phosphate, typical of non-photosynthetic PEPCases, at pH 8.0. However, at the more physiological pH of 7.6, the ppcC enzyme displayed a substantially higher K(0.5)-PEP than the ppcB counterpart, indicating their involvement in different metabolic pathways. This indication was strengthened by malate inhibition studies in which the ppcC enzyme showed 10 times higher tolerance to the inhibitor. The ppcA enzyme was, however, by far the most tolerant enzyme towards malate. Interestingly, the increased malate tolerance was correlated with a decrease in enzyme efficiency displayed by the turnover constant k(cat). We therefore suggest that the increased malate tolerance, which is imperative for an efficient C4 cycle, is connected with a decreased enzyme efficiency that in turn is compensated by increased enzyme expression.     

Evolution of c4 phosphoenolpyruvate carboxylase. Genes and proteins: a case study with the genus Flaveria

C4 photosynthesis is characterized by a division of labour between two different photosynthetic cell types, mesophyll and bundle-sheath cells. Relying on phosphoenolpyruvate carboxylase (PEPC) as the primary carboxylase in the mesophyll cells a CO2 pump is established in C4 plants that concentrates CO2 at the site of ribulose 1,5-bisphosphate carboxylase/oxygenase in the bundle-sheath cells. The C4 photosynthetic pathway evolved polyphyletically implying that the genes encoding the C4 PEPC originated from non-photosynthetic PEPC progenitor genes that were already present in the C3 ancestral species. The dicot genus Flaveria (Asteraceae) is a unique system in which to investigate the molcular changes that had to occur in order to adapt a C3 ancestral PEPC gene to the special conditions of C4 photosynthesis. Flaveria contains not only C3 and C4 species but also a large number of C3-C4 intermediates which vary to the degree in which C4 photosynthetic traits are expressed. The C4 PEPC gene of Flaveria trinervia, which is encoded by the ppcA gene class, is highly expressed but only in mesophyll cells. The encoded PEPC protein possesses the typical kinetic and regulatory features of a C4-type PEPC. The orthologous ppcA gene of the C3 species Flaveria pringlei encodes a typical non-photosynthetic, C3-type PEPC and is weakly expressed with no apparent cell or organ specificity. PEPCs of the ppcA type have been detected also in C3-C4 intermediate Flaveria species. These orthologous PEPCs have been used to determine the molecular basis for C4 enzyme characteristics and to understand their evolution. Comparative and functional analyses of the ppcA promoters from F. trinervia and F. pringlei make it possible to identity the cis-regulatory sequences for mesophyll-specific gene expression and to search for the corresponding trans-regulatory factors.     

Molecular evolution of C4 phospho<Emphasis Type="Italic">enol</Emphasis>pyruvate carboxylase in the genus<Emphasis Type="Italic"> Flaveria</Emphasis>—a gradual increase from C3 to C4 characteristics

In order to elucidate the discrete steps in phospho enolpyruvate carboxylase (PEPC) evolution concerning K(m)-PEP and malate tolerance a comparison was made between C3, C3-C4 and C4 species of the dicot genus Flaveria. The PEPCs of this genus are encoded by a gene family comprising three classes: ppcA, ppcB and ppcC [J. Hermans and P. Westhoff (1990) Mol Gen Genet 224:459-468, (1992) Mol Gen Genet 234:275-284]. The ppcA of F trinervia (C4) codes for the C4 PEPC isoform but other plants of the genus contain ppcA orthologues too. The C3 plant F. pringlei showed the lowest levels of ppcA PEPC mRNA followed by F. pubescens (C3-C4) while the C4-like plant F. brownii displayed RNA amounts close to the C4 species F. trinervia. In contrast to the similar expression profiles of F. brownii (C4-like) and F. trinervia (C4) the PEPC amino acid sequence of F. brownii was more similar to the C3 and C3-C4 ppcA PEPCs than to the C4 PEPC. Similarly, the C3, C3-C4 and C4-like ppcA PEPCs showed almost identical PEP saturation kinetics when activated by glucose-6-phosphate ( K(m)-PEP: 17-20 microM) while the K(m)-PEP for the C4 PEPC was determined to be 53 microM. However, without activation the ppcA PEPCs of F. pubescens and F. brownii displayed C3-C4 intermediate values. A similar picture was obtained when the malate sensitivities were compared. In the non-activated state the F. trinervia (C4) enzyme was 10 times more tolerant to malate than the F. pringlei counterpart. The ppcA enzymes of F. pubescens (C3-C4) and F. brownii (C4-like) displayed intermediate values. In contrast, the inclusion of 5 mM glucose-6-phosphate in the reaction mixture changed the order totally. Interestingly, the activation rendered the C4 enzyme about 50% less tolerant to malate than the C3 PEPC. The activation had a positive effect on malate tolerance of the F. pubescens (C3-C4) PEPC while the ppcA PEPC of F. brownii (C4-like) was almost unaffected.     

cis-Regulatory elements for mesophyll-specific gene expression in the C4 plant Flaveria trinervia, the promoter of the C4 phosphoenolpyruvate carboxylase gene

C(4) photosynthesis depends on the strict compartmentalization of CO(2) assimilatory enzymes. cis-regulatory mechanisms are described that ensure mesophyll-specific expression of the gene encoding the C(4) isoform of phosphoenolpyruvate carboxylase (ppcA1) of the C(4) dicot Flaveria trinervia. To elucidate and understand the anatomy of the C(4) ppcA1 promoter, detailed promoter/reporter gene studies were performed in the closely related C(4) species F. bidentis, revealing that the C(4) promoter contains two regions, a proximal segment up to -570 and a distal part from -1566 to -2141, which are necessary but also sufficient for high mesophyll-specific expression of the beta-glucuronidase reporter gene. The distal region behaves as an enhancer-like expression module that can direct mesophyll-specific expression when inserted into the ppcA1 promoter of the C(3) plant F. pringlei. Mesophyll expression determinants were restricted to a 41-bp segment, referred to as mesophyll expression module 1 (Mem1). Evolutionary and functional studies identified the tetranucleotide sequence CACT as a key component of Mem1.     

Evolution of C4 phosphoenolpyruvate carboxylase in Flaveria, a conserved serine residue in the carboxyl-terminal part of the enzyme is a major determinant for C4-specific characteristics

C4 phosphoenolpyruvate carboxylases have evolved from ancestral C3 isoforms during the evolution of angiosperms and gained distinct kinetic and regulatory properties compared with the C3 isozymes. To identify amino acid residues and/or domains responsible for these C4-specific properties the C4 phosphoenolpyruvate carboxylase of Flaveria trinervia (C4) was compared with its orthologue in the closely related C3 plant Flaveria pringlei. Reciprocal enzyme chimera were constructed and the kinetic constants, K(0.5) and k(cat), as well as the Hill coefficient, h, were determined for the substrate phosphoenolpyruvate both in the presence and absence of the activator glucose 6-phosphate. By this approach two regions were identified which determined most of the kinetic differences of the C4 and C3 ppcA phosphoenolpyruvate carboxylases with respect to the substrate PEP. In addition, the experiments suggest that the two regions do not act additively but interact with each other. The region between amino acids 296 and 437 is essential for activation by glucose 6-phosphate. The carboxyl-terminal segment between amino acids 645 and 966 contains a C4 conserved serine or a C3 invariant alanine at position 774 in the respective enzyme isoform. Site-directed mutagenesis shows that this position is a key determinant for the kinetic properties of the two isozymes.     

Evolution of C4 phosphoenolpyruvate carboxylase

C4 plants are known to be of polyphyletic origin and to have evolved independently several times during the evolution of angiosperms. This implies that the C4 isoform of phosphoenolpyruvate carboxylase (PEPC) originated from a nonphotosynthetic PEPC gene that was already present in the C3 ancestral species. To meet the special requirements of the C4 photosynthetic pathway the expression program of the C4 PEPC gene had to be changed to achieve a strong and selective expression in leaf mesophyll cells. In addition, the altered metabolite concentrations around C4 PEPC in the mesophyll cytoplasm necessitated changes in the enzyme's kinetic and regulatory properties. To obtain insight into the evolutionary steps involved in these altered enzyme characteristics, and even the order of these steps, the dicot genus Flaveria (Asteraceae) appears to be the experimental system of choice. Flaveria contains closely related C3, C3-C4, and C4 species that can be ordered by their gradual increase in C4 photosynthetic traits. The C4 PEPC of F. trinervia, which is encoded by the ppcA gene class, possesses typical kinetic and regulatory features of a C4-type PEPC. Its nearest neighbor is the orthologous ppcA gene of the C3 species F. pringlei. This latter gene encodes a typical nonphotosynthetic C3-type PEPC which is believed to be similar to the C3 ancestral PEPC. This pair of orthologous PEPCs has been used to map C4-specific molecular determinants for the kinetic and regulatory characteristics of C4 PEPCs. The most notable finding from these investigations was the identification of a C4 PEPC invariant site-specific mutation from alanine (C3) to serine (C4) at position 774 that was a necessary and late step in the evolution of C3 to C4 PEPC. The C3-C4 intermediate ppcA PEPCs are used to identify the sequence of events leading from a C3- to a C4-type PEPC.     

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

Phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.3) is a key enzyme of C₄ photosynthesis. It has evolved from ancestral non-photosynthetic (C₃) isoforms and thereby changed its kinetic and regulatory properties. We are interested in understanding the molecular changes, as the C₄ PEPCases were adapted to their new function in C₄ photosynthesis and have therefore analysed the PEPCase genes of various Alternanthera species. We isolated PEPCase cDNAs from the C₄ plant Alternanthera pungens H.B.K., the C₃/C₄ intermediate plant A. tenella Colla, and the C₃ 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 (C₄) as a C₄ PEPCase isoform. In contrast, both the PEPCases from A. sessilis (C₃) and A. tenella (C₃/C₄) were found to be typical C₃ PEPCase isozymes. The C₄ characteristics of the PEPCase of A. pungens were accompanied by the presence of the C₄-invariant serine residue at position 775 reinforcing that a serine at this position is essential for being a C₄ 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.     

Distinct But Conserved Functions for Two Chloroplastic NADP-Malic Enzyme Isoforms in C3 and C4 Flaveria Species

In the most common C4 pathway for carbon fixation, an NADP-malic enzyme (NADP-ME) decarboxylates malate in the chloroplasts of bundle sheath cells. Isoforms of plastidic NADP-ME are encoded by two genes in all species of Flaveria, including C3, C3-C4 intermediate, and C4 types. However, only one of these genes, ChlMe1, encodes the enzyme that functions in the C4 pathway. We compared the expression patterns of the ChlMe1 and ChlMe2 genes in developing leaves of Flaveria pringlei (C3) and Flaveria trinervia (C4) and in transgenic Flaveria bidentis (C4). ChlMe1 expression in C4 species increases in leaves with high C4 pathway activity. In the C3 species F. pringlei, ChlMe1 expression is transient and limited to early leaf development. In contrast, ChlMe2 is expressed in C3 and C4 species concurrent with stages in chloroplast biogenesis. Because previous studies suggest that NADP-ME activities generally reflect the level of its mRNA abundance, we discuss possible roles of ChlMe1 and ChlMe2 based on these expression patterns.     

Expression of a C3 plant Rubisco SSU gene in regenerated C4 Flaveria plants

We report the successful transformation, via Agrobacterium tumefaciens infection, and regeneration of two species of the genus Flaveria: F. brownii and F. palmeri. We document the expression of a C₃ plant gene, an abundantly expressed ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit gene isolated from petunia, in these C₄ plants. The organ-specific expression of this petunia gene in Flaveria brownii is qualitatively identical to its endogenous pattern of expression.     

Primary structure of NADP-dependent malic enzyme in the dicotyledonous C4 plant Flaveria trinervia

The primary structure of NADP-dependent malic enzyme (NADP-ME) of the dicotyledonous C4 plant Flaveria trinervia was determined from sequence analysis of a cDNA clone containing the complete coding region. Comparison of the mature F. trinervia NADP-ME with the maize enzyme reveals extensive sequence similarity. In contrast, no significant similarity can be detected between the putative transit peptides of the two enzymes. This suggests that the corresponding parts of the genes arose independently from each other during evolution of mono- and dicotyledonous C4 plants.     

Two genes encode highly similar chloroplastic NADP-malic enzymes in Flaveria. Implications for the evolution of C4 photosynthesis.

To gain an understanding of the molecular events underlying the evolution of C4 photosynthesis, we have undertaken as detailed study of the NADP-malic enzyme gene family in C4 and C3 species of Flaveria. Three genomic clones form the C4 species Flaveria bidentis were characterized and found to encode two highly similar chloroplastic forms of NADP-malic enzyme, termed ME1 and ME2. Genomic southern blotting with gene-specific probes showed that both Me1 and Me2 are found in Flaveria trinervia (C4) and Flaveria pringlei (C3) as well as in F. bidentis. Northern blots demonstrated that Me1 expression in leaves parallels the degree of C4 photosynthesis in seven Flaveria species. Furthermore, whereas Me2 was expressed at a low level in both roots and leaves of F. bidentis, Me1 expression was seen only in leaves and was light-regulated. We discuss these results in the context of the evolution of C4 photosynthesis in Flaveria.     

Molecular properties of phosphoenolpyruvate carboxylase from C3, C3−C4 intermediate,and C4 Flaveria species

Phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) from Flaveria trinervia Mohr (C₄), F. floridana Johnston (C₃–C₄), and F. cronquistii Powell (C₃) leaves were compared by electrotransfer blotting/enzyme-linked immunoassay (Western-blot analysis), mobility of the native enzyme in polyacrylamide gels and in isoelectric focusing (IEF) gels, peptide mapping, and in-vitro translation of RNA isolated from each plant. The PEPCases from the C₃ and C₃–C₄ plants were very similar to each other in terms of electrophoretic mobilities on gels and isoenzyme patterns on IEF gels, and identical in peptide mapping. Quantitative differences were noted, however, in that the C₃–C₄ intermediate plant contained more PEPCase overall and that the relative activity of individual isoenzymes shifted between the C₃ and C₃–C₄ intermediate PEPCases. The PEPCase from the C₄ plant had a different isoenzyme pattern, a different peptide map, and was far more abundant than the other two enzymes. Western blot analysis demonstrated the cross-reactivity of PEPCases from all three Flaveria species with antibody raised against maize PEPCase. The results provide evidence, at the molecular level, that supports the view of C₃–C₄ intermediate species as C₃-like plants with some C₄-like photosynthetic characteristics, but there are differences from the C₃ plant in the quantity and properties of the PEPCase from the C₃–C₄ intermediate plant.Abbreviations IEF isoelectric focusing - kDa kilodalton - PEPCase phosphoenolpyruvate carboxylase - Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase     

Differential regulation of transcripts encoding cytosolic NADP-malic enzyme in C3 and C4 Flaveria species.

A cytosolic NADP-malic enzyme (CYTME) has been described previously in several plants, all C3 species. CYTME is distinct from the chloroplastic NADP-malic enzyme (CHLME) that is highly active in C4 species. We show that at least one CytMe gene is present in all Flaveria spp., including C3, C4, and C3-C4 intermediate types. Based on the CytMe expression patterns in Flaveria pringlei (C3) and Flaveria trinervia (C4), we suggest CYTME has several distinct roles, including the supplying of NADPH for cytosolic metabolism, the supporting of wound response or repair, and the balancing of cellular pH in illuminated leaves. These three roles are likely correlated with CytMe mRNAs of apparent sizes 2.0, 2.2, and 2.4 kb, respectively, which differ in the length of the 5' untranslated regions. Various regulatory mechanisms involving RNA processing and translational efficiency are discussed.