The intracellular distribution of inorganic carbon fixing enzymes does not support the presence of a C4 pathway in the diatom Phaeodactylum tricornutum.

Diatoms are unicellular algae and important primary producers. The process of carbon fixation in diatoms is very efficient even though the availability of dissolved CO2 in sea water is very low. The operation of a carbon concentrating mechanism (CCM) also makes the more abundant bicarbonate accessible for photosynthetic carbon fixation. Diatoms possess carbonic anhydrases as well as metabolic enzymes potentially involved in C4 pathways; however, the question as to whether a C4 pathway plays a general role in diatoms is not yet solved. While genome analyses indicate that the diatom Phaeodactylum tricornutum possesses all the enzymes required to operate a C4 pathway, silencing of the pyruvate orthophosphate dikinase (PPDK) in a genetically transformed cell line does not lead to reduced photosynthetic carbon fixation. In this study, we have determined the intracellular location of all enzymes potentially involved in C4-like carbon fixing pathways in P. tricornutum by expression of the respective proteins fused to green fluorescent protein (GFP), followed by fluorescence microscopy. Furthermore, we compared the results to known pathways and locations of enzymes in higher plants performing C3 or C4 photosynthesis. This approach revealed that the intracellular distribution of the investigated enzymes is quite different from the one observed in higher plants. In particular, the apparent lack of a plastidic decarboxylase in P. tricornutum indicates that this diatom does not perform a C4-like CCM.

Phylogenic and phosphorylation regulation difference of phosphoenolpyruvate carboxykinase of C3 and C4 plants.

In C4 plants, phosphoenolpyruvate carboxykinase (PEPCK) plays a key role in the C4 cycle. PEPCK is also involved in gluconeogenesis and is conserved in both lower and higher organisms, including in animals and plants. A phylogenic tree constructed from PEPCK sequences from bacteria to higher plants indicates that the C4 Poaceae PEPCKs are conserved and have diverged from the PEPCKs of C3 plants. The maximum enzymatic activities of wild-type and phosphorylation mimic PEPCK proteins indicate that there is a significant difference between C3 and C4 plant PEPCKs. The conserved PEPCK phosphorylation sites are regulated differently in C3 and C4 plants. These results suggest that the functions of PEPCK have been conserved, but that sequences have diverged and regulation of PEPCK is important in C4 plants, but not in herbaceous and, in particular, woody C3 plants.

Identification and phylogeny of putative PEPC genes in three toxin-producing Karenia (Dinophyta) species.

Dense blooms of toxin-producing Karenia brevis increase local surface ocean pH through CO2 uptake. To identify genes that may contribute to bloom-related environmental pH and pCO2 changes, transcriptomes with RNA from K. brevis Wilson cultures that had been acclimated to low CO2 (250 ppm) or recent CO2 (350 ppm) pCO2 levels were assembled. Among the annotated transcripts were PEPC, PPDK, and PEPCK enzymes found in the model C4 carbon fixation pathway. Previous studies have demonstrated that the enzymatic activity of PEPC, PPDK, and/or PEPCK in some algae species, including marine diatoms, is influenced by variations in dissolved inorganic carbon. We found significantly similar PEPC, PPDK, and PEPCK enzymes in the transcriptomes of K. brevis and two sister species Karenia papilionacea, and Karenia mikimotoi. One or more isoforms of PEPC were also identified in the transcriptomes of thirty additional photosynthetic phytoplankton species from nine phyla. Phylogenetic trees were constructed with neighbor joining and maximum likelihood techniques to characterize the evolutionary relationship among phytoplankton, terrestrial plant C4, and terrestrial plant C3 PEPC sequences. Based on the nucleotide trees constructed during this study, the Karenia PEPC transcripts were more closely related to the terrestrial C4 genes than the terrestrial C3 genes. Furthermore, PEPC phylogeny among phytoplankton closely resembles phylogenetic trees constructed with ribosomal RNA. This study confirmed that the toxin-producing dinoflagellates K. brevis, K. mikimotoi, and K. papilionacea express putative PEPC, PEPCK, and PPDK transcripts.

Bacterial- and plant-type phosphoenolpyruvate carboxylase polypeptides interact in the hetero-oligomeric Class-2 PEPC complex of developing castor oil seeds.

Two classes of phosphoenolpyruvate carboxylase (PEPC) sharing the same 107-kDa catalytic subunit (p107) were previously purified from developing castor oil seed (COS) endosperm. The association of p107 with an immunologically unrelated 64-kDa polypeptide (p64) causes pronounced physical and kinetic differences between the Class-1 PEPC p107 homotetramer and Class-2 PEPC p107/p64 hetero-octamer. Tryptic peptide sequencing matched p64 to the deduced C-terminal half of several bacterial-type PEPCs (BTPCs) of vascular plants. Immunoblots probed with anti-(COS p64 peptide or p107)-IgG established that: (i) BTPC exists in vivo as an approximately 118-kDa polypeptide (p118) that is rapidly truncated to p64 by an endogenous cysteine endopeptidase during incubation of COS extracts on ice, and (ii) mature and germinated COS contain Class-1 PEPC and p107, but no detectable Class-2 PEPC nor p118. Non-denaturing PAGE, in-gel PEPC activity staining and immunoblotting of developing COS extracts demonstrated that p118 and p107 are subunits of the non-proteolysed approximately 910-kDa Class-2 PEPC complex. As total PEPC activity of clarified COS extracts was unaffected following p118 truncation to p64, the BTPC p118 may function as a regulatory rather than catalytic subunit of the Class-2 PEPC. Moreover, recombinant AtPPC3 and AtPPC4 (Arabidopsis orthologs of COS p107 and p118) expressed as active and inactive PEPCs, respectively. Cloning of cDNAs encoding p118 (RcPpc4) and p107 (RcPpc3) confirmed their respective designation as bacterial- and plant-type PEPCs. Levels of RcPpc3 and RcPpc4 transcripts generally mirrored the respective amounts of p107 and p118. The collective findings provide insights into the molecular features and functional significance of vascular plant BTPCs.

Evolution of C(4) phosphoenolpyruvate carboxylase in the genus Alternanthera: gene families and the enzymatic characteristics of the C(4) isozyme and its orthologues in C(3) and C(3)/C(4) Alternantheras.

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.

Isolation of a cDNA for a phosphoenolpyruvate carboxylase from a monocot CAM-plant, Aloe arborescens: structure and its gene expression.

A phosphoenolpyruvate carboxylase (PEPCase) cDNA was isolated from Aloe arborescens, a monocot CAM plant. Northern analysis of the PEPCase transcript indicated that it is specifically expressed in green leaves, strongly suggesting its involvement in CAM photosynthesis. No diurnal change in expression level was evident. Western blot analysis also showed no alteration of the amount of the PEPCase protein. These results suggest that circadian rhythm in PEPCase activity may be regulated post-translationally. The representative cDNA clone contained an ORF encoding 964 amino acid residues. Deduced amino acid sequence of the aloe PEPCase is highly conserved as compared with other PEPCases. The phosphorylation site which may be modified by PEPC-kinase was conserved. An evolutional map with known PEPCases suggested that CAM-type PEPCases were located between C4 and housekeeping PEPCases.