Analysis of promoter activity for the gene encoding pyruvate orthophosphate dikinase in stably transformed C4 flaveria species

The C4 enzyme pyruvate orthophosphate dikinase is encoded by a single gene, Pdk, in the C4 plant Flaveria trinervia. This gene also encodes enzyme isoforms located in the chloroplast and in the cytosol that do not have a function in C4 photosynthesis. Our goal is to identify cis-acting DNA sequences that regulate the expression of the gene that is active in the C4 cycle. We fused 1.5 kb of a 5' flanking region from the Pdk gene, including the entire 5' untranslated region, to the uidA reporter gene and stably transformed the closely related C4 species Flaveria bidentis. beta-Glucuronidase (GUS) activity was detected at high levels in leaf mesophyll cells. GUS activity was detected at lower levels in bundle-sheath cells and stems and at very low levels in roots. This lower-level GUS expression was similar to the distribution of mRNA encoding the nonphotosynthetic form of the enzyme. We conclude that cis-acting DNA sequences controlling the expression of the C4 form in mesophyll cells and the chloroplast form in other cells and organs are co-located within the same 5' region of the Pdk gene.

Genomic structure and expression of the pyruvate, orthophosphate dikinase gene of the dicotyledonous C4 plant Flaveria trinervia (Asteraceae).

Pyruvate orthophosphate dikinase (PPDK) is a key enzyme of C4 photosynthesis providing the acceptor molecule for the primary CO2 fixation in the mesophyll cells. Here we present the isolation and characterisation of the corresponding gene (termed pdk) from the C4 plant Flaveria trinervia (Asteraceae). Southern analysis indicates that in contrast to maize pdk sequences in F. trinervia are present as single copy. Sequence analysis of the entire gene reveals that its coding sequence is identical to the previous isolated PPDK-cDNA from this species. The gene spans about 13 kb and consists of 21 exons, it thus contains two additional exons compared to the maize gene. As in maize, a long intervening sequence of 6.1 kb is positioned at the boundary of the transit peptide segment and the mature protein region. Pdk transcripts accumulate abundantly in leaves, but are also detectable in stems and roots. While the leaf and stem transcripts are 3.4 kb in size and encode the chloroplastic PPDK isoform, a 3.0 kb transcript lacking the region encoding the plastidic transit peptide accumulates in roots. Thus two different transcripts can be produced from a single pdk gene most likely by use of alternative promoters and not by alternative splicing. The accumulation of the 3.4 kb transcript is under light control. Darkening leads to a drastic depletion of this transcript in both leaves and stems. Instead, the 3.0 kb transit peptide-lacking pdk transcript accumulates, but only in stems and roots, not in leaves.

The C3 plant Flaveria pringlei contains a plastidic NADP-malic enzyme which is orthologous to the C4 isoform of the C4 plant F. trinervia.

To study the molecular evolution of NADP-dependent malic enzyme (NADP-ME) in the genus Flaveria a leaf-specific cDNA library of the C3 plant F. pringlei was screened for the presence of sequences homologous to the C4 isoform gene (named modA) of the C4 plant F. trinervia. The cDNAs isolated contained varying numbers of identical restriction fragments suggesting that they were derived from a single gene. This was supported by Southern hybridisation experiments with genomic DNA from F. trinervia and F. pringlei. Nucleotide sequence analysis of a full-size clone identified the presence of a typical plastidic transit peptide and revealed that the mature modA proteins of F. trinervia (C4) and F. pringlei (C3) are 90% similar. These findings indicate that C3 plants, like C4 species, possess a plastidic isoform of NADP-ME and that the modA genes of the two species represent orthologous genes. Northern analyses showed that modA transcripts accumulate to similar levels in leaves, stems and roots of F. pringlei. The expression of this gene in F. pringlei thus appears to be rather constitutive. In contrast, the modA gene of F. trinervia is abundantly expressed in leaves, but maintains its expression in stems and roots. It has to be concluded from these data that the leaf-specific increase in the expression level was a key step which was taken during the evolution of the C4 isoform modA gene starting from a C3 ancestral gene.

Primary structure of the photosynthetic pyruvate orthophosphate dikinase of the C3 plant Flaveria pringlei and expression analysis of pyruvate orthophosphate dikinase sequences in C3, C3-C4 and C4 Flaveria species.

We have isolated full-size cDNA sequences encoding the photosynthetic isoform of pyruvate orthophosphate dikinase (PPDK) of the C3 plant Flaveria pringlei. The encoded protein shares 96% identical amino acid residues with the C4 isoform of PPDK in the C4 species F. trinervia. The differing amino acid residues are evenly distributed along the polypeptide chain. Genomic Southern analysis of photosynthetic PPDK sequences in F. pringlei (C3), F. chloraefolia (C3-C4), F. linearis (C3-C4), F. floridana (C3-C4), F. brownii (C4-like) and F. trinervia (C4) reveals a simple hybridization pattern which is suggestive of a single gene. Northern hybridization experiments show that the abundance of PPDK transcripts in leaves correlates with the degree of C4 characteristics expressed in the various photosynthetic types analysed. This finding demonstrates that the increase in expression levels must have played a crucial role in evolving the C4-PPDK gene in the genus Flaveria.

Primary structure of pyruvate, orthophosphate dikinase in the dicotyledonous C4 plant Flaveria trinervia.

We have isolated and characterized cDNA clones encoding the entire precursor for the leafspecific isoform of pyruvate, orthophosphate dikinase (PPDK) from the dicotyledonous C4 plant Flaveria trinervia. The deduced amino acid sequence reveals a high degree of similarity to the corresponding maize protein indicating a common evolutionary basis. However, no significant similarities are apparent upon comparison of the putative transit peptides. The implications of this divergence are discussed with respect to the evolution of PPDK genes.