SummaryTwo 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 nonproteolysed 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.

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 bacterialtype 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. Phosphoenolpyruvate (PEP)2 carboxylase (PEPC; EC 4.1.1.31) is an important enzyme of plant C metabolism that catalyzes the irreversible ␤-carboxylation of phosphoenolpyruvate to yield oxaloacetate and P i . This enzyme has been intensively studied with regards to its crucial role in catalyzing atmospheric CO 2 fixation in C 4 and crassulacean acid metabolism photosynthesis (1, 2). It also plays essential functions in bacteria and non-green plant cells, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates withdrawn for biosynthesis and N-assimilation (3, 4). Most vascular plant PEPCs exist as a homotetrameric "dimer of dimer" structure composed of four identical 100 -110-kDa subunits known as Class-1 PEPC. Class-1 PEPCs are subject to tight control by a combination of allosteric effectors and reversible phosphorylation at a conserved N-terminal seryl residue catalyzed by a dedicated Ca 2ϩ -independent PEPC protein kinase and protein phosphatase type 2A (1-3). Allosteric activation by hexose phosphates and inhibition by malate have been routinely observed, whereas phosphorylation activates the enzyme by reducing its sensitivity to malate inhibition and simultaneously enhancing activation by hexose phosphates (1-3). Our understanding of the post-translational control...

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 β-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.

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.

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.