PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightly controlled enzyme located at the core of plant C-metabolism that catalyses the irreversible β-carboxylation of PEP to form oxaloacetate and Pi. The critical role of PEPC in assimilating atmospheric CO(2) during C(4) and Crassulacean acid metabolism photosynthesis has been studied extensively. PEPC also fulfils a broad spectrum of non-photosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates consumed during biosynthesis and nitrogen assimilation. An impressive array of strategies has evolved to co-ordinate in vivo PEPC activity with cellular demands for C(4)-C(6) carboxylic acids. To achieve its diverse roles and complex regulation, PEPC belongs to a small multigene family encoding several closely related PTPCs (plant-type PEPCs), along with a distantly related BTPC (bacterial-type PEPC). PTPC genes encode ~110-kDa polypeptides containing conserved serine-phosphorylation and lysine-mono-ubiquitination sites, and typically exist as homotetrameric Class-1 PEPCs. In contrast, BTPC genes encode larger ~117-kDa polypeptides owing to a unique intrinsically disordered domain that mediates BTPC's tight interaction with co-expressed PTPC subunits. This association results in the formation of unusual ~900-kDa Class-2 PEPC hetero-octameric complexes that are desensitized to allosteric effectors. BTPC is a catalytic and regulatory subunit of Class-2 PEPC that is subject to multi-site regulatory phosphorylation in vivo. The interaction between divergent PEPC polypeptides within Class-2 PEPCs adds another layer of complexity to the evolution, physiological functions and metabolic control of this essential CO(2)-fixing plant enzyme. The present review summarizes exciting developments concerning the functions, post-translational controls and subcellular location of plant PTPC and BTPC isoenzymes.

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.

Contents Summary670I.Introduction671II.Principle 1 – Plant respiration performs three distinct functions673III.Principle 2 – Metabolic pathway flexibility underlies plant respiratory performance676IV.Principle 3 – Supply and demand interact over time to set plant respiration rate677V.Principle 4 – Plant respiratory acclimation involves adjustments in enzyme capacities679VI.Principle 5 – Respiration is a complex trait that helps to define, and is impacted by, plant lifestyle strategies680VII.Future directions680Acknowledgements682References682 Summary Respiration is a core biological process that has important implications for the biochemistry, physiology, and ecology of plants. The study of plant respiration is thus conducted from several different perspectives by a range of scientific disciplines with dissimilar objectives, such as metabolic engineering, crop breeding, and climate‐change modelling. One aspect in common among the different objectives is a need to understand and quantify the variation in respiration across scales of biological organization. The central tenet of this review is that different perspectives on respiration can complement each other when connected. To better accommodate interdisciplinary thinking, we identify distinct mechanisms which encompass the variation in respiratory rates and functions across biological scales. The relevance of these mechanisms towards variation in plant respiration are explained in the context of five core principles: (1) respiration performs three distinct functions; (2) metabolic pathway flexibility underlies respiratory performance; (3) supply and demand interact over time to set respiration rates; (4) acclimation involves adjustments in enzyme capacities; and (5) respiration is a complex trait that helps to define, and is impacted by, plant lifestyle strategies. We argue that each perspective on respiration rests on these principles to varying degrees and that broader appreciation of how respiratory variation occurs can unite research across scales.

The apoplast is the arena in which endophytic pathogens such as Pseudomonas syringae grow and interact with plant cells. Using metabolomic and ion analysis techniques, this study shows how the composition of Phaseolus vulgaris leaf apoplastic fluid changes during the first six hours of compatible and incompatible interactions with two strains of P. syringae pv. phaseolicola (Pph) that differ in the presence of the genomic island PPHGI‐1. Leaf inoculation with the avirulent island‐carrying strain Pph 1302A elicited effector‐triggered immunity (ETI) and resulted in specific changes in apoplast composition, including increases in conductivity, pH, citrate, γ‐aminobutyrate (GABA) and K+, that are linked to the onset of plant defence responses. Other apoplastic changes, including increases in Ca2+, Fe2/3+ Mg2+, sucrose, β‐cyanoalanine and several amino acids, occurred to a relatively similar extent in interactions with both Pph 1302A and the virulent, island‐less strain Pph RJ3. Metabolic footprinting experiments established that Pph preferentially metabolizes malate, glucose and glutamate, but excludes certain other abundant apoplastic metabolites, including citrate and GABA, until preferred metabolites are depleted. These results demonstrate that Pph is well‐adapted to the leaf apoplast metabolic environment and that loss of PPHGI‐1 enables Pph to avoid changes in apoplast composition linked to plant defences.

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

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...

Respiration rate measurements provide an important readout of energy expenditure and mitochondrial activity in plant cells during the night. As plants inhabit a changing environment, regulatory mechanisms must ensure that respiratory metabolism rapidly and effectively adjusts to the metabolic and environmental conditions of the cell. Using a high-throughput approach, we have directly identified specific metabolites that exert transcriptional, translational, and posttranslational control over the nighttime O 2 consumption rate (R N) in mature leaves of Arabidopsis (Arabidopsis thaliana). Multi-hour R N measurements following leaf disc exposure to a wide array of primary carbon metabolites (carbohydrates, amino acids, and organic acids) identified phosphoenolpyruvate (PEP), Pro, and Ala as the most potent stimulators of plant leaf R N. Using metabolite combinations, we discovered metabolite-metabolite regulatory interactions controlling R N. Many amino acids, as well as Glc analogs, were found to potently inhibit the R N stimulation by Pro and Ala but not PEP. The inhibitory effects of amino acids on Pro-and Ala-stimulated R N were mitigated by inhibition of the Target of Rapamycin (TOR) kinase signaling pathway. Supporting the involvement of TOR, these inhibitory amino acids were also shown to be activators of TOR kinase. This work provides direct evidence that the TOR signaling pathway in plants responds to amino acid levels by eliciting regulatory effects on respiratory energy metabolism at night, uniting a hallmark mechanism of TOR regulation across eukaryotes.

The phosphoenolpyruvate carboxylase (PEPC) interactome of developing castor oil seed (COS; Ricinus communis) endosperm was assessed using coimmunopurification (co-IP) followed by proteomic analysis. Earlier studies suggested that immunologically unrelated 107-kD plant-type PEPCs (p107/PTPC) and 118-kD bacterial-type PEPCs (p118/BTPC) are subunits of an unusual 910-kD hetero-octameric class 2 PEPC complex of developing COS. The current results confirm that a tight physical interaction occurs between p118 and p107 because p118 quantitatively coimmunopurified with p107 following elution of COS extracts through an anti-p107-IgG immunoaffinity column. No PEPC activity or immunoreactive PEPC polypeptides were detected in the corresponding flow-through fractions. Although BTPCs lack the N-terminal phosphorylation motif characteristic of PTPCs, Pro-Q Diamond phosphoprotein staining, immunoblotting with phospho-serine (Ser)/threonine Akt substrate IgG, and phosphate-affinity PAGE established that coimmunopurified p118 was multiphosphorylated at unique Ser and/or threonine residues. Tandem mass spectrometric analysis of an endoproteinase Lys-C p118 peptide digest demonstrated that Ser-425 is subject to in vivo proline-directed phosphorylation. The co-IP of p118 with p107 did not appear to be influenced by their phosphorylation status. Because p118 phosphorylation was unchanged 48 h following elimination of photosynthate supply due to COS depodding, the signaling mechanisms responsible for photosynthate-dependent p107 phosphorylation differ from those controlling p118's in vivo phosphorylation. A 110-kD PTPC coimmunopurified with p118 and p107 when depodded COS was used. The plastidial pyruvate dehydrogenase complex (PDC pl ) was identified as a novel PEPC interactor. Thus, a putative metabolon involving PEPC and PDC pl could function to channel carbon from phosphoenolpyruvate to acetyl-coenzyme A and/or to recycle CO 2 from PDC pl to PEPC.