The bacterial-type phosphoenolpyruvate carboxylase isozyme from developing castor oil seeds is subject to in vivo regulatory phosphorylation at serine-451.

Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled anaplerotic enzyme situated at a pivotal branch point of plant carbohydrate-metabolism. In developing castor oil seeds (COS) a novel allosterically-densensitized 910-kDa Class-2 PEPC hetero-octameric complex arises from a tight interaction between 107-kDa plant-type PEPC and 118-kDa bacterial-type PEPC (BTPC) subunits. Mass spectrometry and immunoblotting with anti-phosphoSer451 specific antibodies established that COS BTPC is in vivo phosphorylated at Ser451, a highly conserved target residue that occurs within an intrinsically disordered region. This phosphorylation was enhanced during COS development or in response to depodding. Kinetic characterization of a phosphomimetic (S451D) mutant indicated that Ser451 phosphorylation inhibits the catalytic activity of BTPC subunits within the Class-2 PEPC complex.

Tissue-specific expression and post-translational modifications of plant- and bacterial-type phosphoenolpyruvate carboxylase isozymes of the castor oil plant, Ricinus communis L.

This study employs transcript profiling together with immunoblotting and co-immunopurification to assess the tissue-specific expression, protein:protein interactions, and post-translational modifications (PTMs) of plant- and bacterial-type phosphoenolpyruvate carboxylase (PEPC) isozymes (PTPC and BTPC, respectively) in the castor plant, Ricinus communis. Previous studies established that the Class-1 PEPC (PTPC homotetramer) of castor oil seeds (COS) is activated by phosphorylation at Ser-11 and inhibited by monoubiquitination at Lys-628 during endosperm development and germination, respectively. Elimination of photosynthate supply to developing COS by depodding caused the PTPC of the endosperm and cotyledon to be dephosphorylated, and then subsequently monoubiquitinated in vivo. PTPC monoubiquitination rather than phosphorylation is widespread throughout the castor plant and appears to be the predominant PTM of Class-1 PEPC that occurs in planta. The distinctive developmental patterns of PTPC phosphorylation versus monoubiquitination indicates that these two PTMs are mutually exclusive. By contrast, the BTPC: (i) is abundant in the inner integument, cotyledon, and endosperm of developing COS, but occurs at low levels in roots and cotyledons of germinated COS, (ii) shows a unique developmental pattern in leaves such that it is present in leaf buds and young expanding leaves, but undetectable in fully expanded leaves, and (iii) tightly interacts with co-expressed PTPC to form the novel and allosterically-desensitized Class-2 PEPC heteromeric complex. BTPC and thus Class-2 PEPC up-regulation appears to be a distinctive feature of rapidly growing and/or biosynthetically active tissues that require a large anaplerotic flux from phosphoenolpyruvate to replenish tricarboxylic acid cycle C-skeletons being withdrawn for anabolism.

Phosphorylation of bacterial-type phosphoenolpyruvate carboxylase at Ser425 provides a further tier of enzyme control in developing castor oil seeds.

PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightly controlled anaplerotic enzyme situated at a pivotal branch point of plant carbohydrate metabolism. Two distinct oligomeric PEPC classes were discovered in developing COS (castor oil seeds). Class-1 PEPC is a typical homotetramer of 107 kDa PTPC (plant-type PEPC) subunits, whereas the novel 910-kDa Class-2 PEPC hetero-octamer arises from a tight interaction between Class-1 PEPC and 118 kDa BTPC (bacterial-type PEPC) subunits. Mass spectrometric analysis of immunopurified COS BTPC indicated that it is subject to in vivo proline-directed phosphorylation at Ser425. We show that immunoblots probed with phosphorylation site-specific antibodies demonstrated that Ser425 phosphorylation is promoted during COS development, becoming maximal at stage IX (maturation phase) or in response to depodding. Kinetic analyses of a recombinant, chimaeric Class-2 PEPC containing phosphomimetic BTPC mutant subunits (S425D) indicated that Ser425 phosphorylation results in significant BTPC inhibition by: (i) increasing its Km(PEP) 3-fold, (ii) reducing its I50 (L-malate and L-aspartate) values by 4.5- and 2.5-fold respectively, while (iii) decreasing its activity within the physiological pH range. The developmental pattern and kinetic influence of Ser425 BTPC phosphorylation is very distinct from the in vivo phosphorylation/activation of COS Class-1 PEPC's PTPC subunits at Ser11. Collectively, the results establish that BTPC's phospho-Ser425 content depends upon COS developmental and physiological status and that Ser425 phosphorylation attenuates the catalytic activity of BTPC subunits within a Class-2 PEPC complex. To the best of our knowledge, this study provides the first evidence for protein phosphorylation as a mechanism for the in vivo control of vascular plant BTPC activity.

Bacterial-type phosphoenolpyruvate carboxylase (PEPC) functions as a catalytic and regulatory subunit of the novel class-2 PEPC complex of vascular plants.

Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated anaplerotic enzyme situated at a major branch point of the plant C metabolism. Two distinct oligomeric classes of PEPC occur in the triglyceride-rich endosperm of developing castor oil seeds (COS). Class-1 PEPC is a typical homotetramer composed of identical 107-kDa plant-type PEPC (PTPC) subunits (encoded by RcPpc3), whereas the novel Class-2 PEPC 910-kDa hetero-octameric complex arises from a tight interaction between Class-1 PEPC and distantly related 118-kDa bacterial-type PEPC (BTPC) polypeptides (encoded by RcPpc4). Here, COS BTPC was expressed from full-length RcPpc4 cDNA in Escherichia coli as an active PEPC that exhibited unusual properties relative to PTPCs, including a tendency to form large aggregates, enhanced thermal stability, a high K(m)((PEP)), and insensitivity to metabolite effectors. A chimeric 900-kDa Class-2 PEPC hetero-octamer having a 1:1 stoichiometry of BTPC:PTPC subunits was isolated from a mixture of clarified extracts containing recombinant RcPPC4 and an Arabidopsis thaliana Class-1 PEPC (the PTPC, AtPPC3). The purified Class-2 PEPC exhibited biphasic PEP saturation kinetics with high and low affinity sites attributed to its AtPPC3 and RcPPC4 subunits, respectively. The RcPPC4 subunits: (i) catalyzed the majority of the Class-2 PEPC V(max), particularly in the presence of the inhibitor l-malate, and (ii) also functioned as Class-2 PEPC regulatory subunits by modulating PEP binding and catalytic potential of its AtPPC3 subunits. BTPCs appear to associate with PTPCs to form stable Class-2 PEPC complexes in vivo that are hypothesized to maintain high flux from PEP under physiological conditions that would otherwise inhibit Class-1 PEPCs.

Characterization of the NADP malic enzyme gene family in the facultative, single-cell C4 monocot Hydrilla verticillata.

Hydrilla verticillata has a facultative single-cell system that changes from C3 to C4 photosynthesis. A NADP+-dependent malic enzyme (NADP-ME) provides a high [CO2] for Rubisco fixation in the C4 leaf chloroplasts. Of three NADP-ME genes identified, only hvme1 was up-regulated in the C4 leaf, during the light period, and it possessed a putative transit peptide. Unlike obligate C4 species, H. verticillata exhibited only one plastidic isoform that may perform housekeeping functions, but is up-regulated as the photosynthetic decarboxylase. Of the two cytosolic forms, hvme2 and hvme3, the latter exhibited the greatest expression, but was not light-regulated. The mature isoform of hvme1 had a pI of 6.0 and a molecular mass of 64 kD, as did the recombinant rHVME1m, and it formed a tetramer in the chloroplast. The recombinant photosynthetic isoform showed intermediate characteristics between isoforms in terrestrial C3 and C4 species. The catalytic efficiency of rHVME1m was four-fold higher than the cytosolic rHVME3 and two-fold higher than recombinant cytosolic isoforms of rice, but lower than plastidic forms of maize. The Km (malate) of 0.6 mM for rHVME1 was higher than maize plastid isoforms, but four-fold lower than found with rice. A comprehensive phylogenetic analysis of 25 taxa suggested that chloroplastic NADP-ME isoforms arose from four duplication events, and hvme1 was derived from cytosolic hvme3. The chloroplastic eudicot sequences were a monophyletic group derived from a cytosolic clade after the eudicot and monocot lineages separated, while the monocots formed a polyphyletic group. The findings support the hypothesis that a NADP-ME isoform with specific and unusual regulatory properties facilitates the functioning of the single-cell C4 system in H. verticillata.

Identification of C4 responsive genes in the facultative C4 plant Hydrilla verticillata.

The aquatic monocot Hydrilla verticillata (L.f.) Royle is a well-documented facultative C4 NADP-malic enzyme species in which the C4 and Calvin cycles operate in the same cell with the specific carboxylases confined to the cytosol and chloroplast, respectively. Several key components had already been characterized at the molecular level, thus the purpose of this study was to begin to identify other, less obvious, elements that may be necessary for a functional single-cell C4 system. Using differential display, mRNA populations from C3 and C4 H. verticillata leaves were screened and expression profiles compared. From this study, 65 clones were isolated and subjected to a customized macroarray analysis; 25 clones were found to be upregulated in C4 leaves. Northern and semi-quantitative RT-PCR analyses were used for confirmation. From these screenings, 13 C4 upregulated genes were identified. Among these one encoded a previously recognized C4 phosphoenolpyruvate carboxylase, and two encoded distinct pyruvate orthophosphate dikinase isoforms, new findings for H. verticillata. Genes that encode a transporter, an aminotransferase and two chaperonins were also upregulated. Twelve false positives, mostly housekeeping genes, were determined from the Northern/semi-quantitative RT-PCR analyses. Sequence data obtained in this study are listed in the dbEST database (DV216698 to DV216767). As a single-cell C4 system that lacks Kranz anatomy, a better understanding of how H. verticillata operates may facilitate the design of a transgenic C4 system in a C3 crop species.

Photosynthetic and other phosphoenolpyruvate carboxylase isoforms in the single-cell, facultative C(4) system of Hydrilla verticillata.

The submersed monocot Hydrilla verticillata (L.f.) Royle is a facultative C(4) plant. It typically exhibits C(3) photosynthetic characteristics, but exposure to low [CO(2)] induces a C(4) system in which the C(4) and Calvin cycles co-exist in the same cell and the initial fixation in the light is catalyzed by phosphoenolpyruvate carboxylase (PEPC). Three full-length cDNAs encoding PEPC were isolated from H. verticillata, two from leaves and one from root. The sequences were 95% to 99% identical and shared a 75% to 85% similarity with other plant PEPCs. Transcript studies revealed that one isoform, Hvpepc4, was exclusively expressed in leaves during C(4) induction. This and enzyme kinetic data were consistent with it being the C(4) photosynthesis isoform. However, the C(4) signature serine of terrestrial plant C(4) isoforms was absent in this and the other H. verticillata sequences. Instead, alanine, typical of C(3) sequences, was present. Western analyses of C(3) and C(4) leaf extracts after anion-exchange chromatography showed similar dominant PEPC-specific bands at 110 kD. In phylogenetic analyses, the sequences grouped with C(3), non-graminaceous C(4), and Crassulacean acid metabolism PEPCs but not with the graminaceous C(4), and formed a clade with a gymnosperm, which is consistent with H. verticillata PEPC predating that of other C(4) angiosperms.

C4 mechanisms in aquatic angiosperms: comparisons with terrestrial C4 systems.

Aquatic C4 photosynthesis probably arose in response to dissolved CO2 limitations, possibly before its advent in terrestrial plants. Of over 7600 C4 species, only about a dozen aquatic species are identified. Amphibious Eleocharis species (sedges) have C3-C4 photosynthesis and Kranz anatomy in aerial, but not submersed, leaves. Aquatic grasses have aerial and submersed leaves with C4 or C3-C4 photosynthesis and Kranz anatomy, but some lack Kranz anatomy in the submersed leaves. Two freshwater submersed monocots, Hydrilla verticillata and possibly Egeria densa, are C4 NADP-malic enzyme (NADP-ME) species. A marine macroalga, Udotea flabellum (Chlorophyta), and possibly a diatom, are C4, so it is not confined to angiosperms. Submersed C4 species differ from terrestrial in that ß-carboxylation is cytosolic with chloroplastic decarboxylation and Rubisco carboxylation, so the C4 and Calvin cycles operate in the same cell without Kranz anatomy. Unlike terrestrial plants, Hydrilla is a facultative C4 that shifts from C3 to C4 in low [CO2]. It is well documented, with C4 gas exchange and pulse-chase characteristics, enzyme kinetics and localization, high internal [CO2], relative growth rate, and quantum yield studies. It has multiple phosphoenolpyruvate carboxylase isoforms with C3-like sequences. Hvpepc4 appears to be the photosynthetic form induced in C4 leaves, but it differs from terrestrial C4 isoforms in lacking a C4 signature Serine. The molecular mass of NADP-ME (72 kDa) also resembles a C3 isoform. Hydrilla belongs to the ancient Hydrocharitaceae family, and gives insight to early C4 development. Hydrilla is an excellent 'minimalist' system to study C4 photosynthesis regulation without anatomical complexities.