Multiple Roles of Glycerate Kinase-From Photorespiration to Gluconeogenesis, C4 Metabolism, and Plant Immunity.

Plant glycerate kinase (GK) was previously considered an exclusively chloroplastic enzyme of the glycolate pathway (photorespiration), and its sole predicted role was to return most of the glycolate-derived carbon (as glycerate) to the Calvin cycle. However, recent discovery of cytosolic GK revealed metabolic links for glycerate to other processes. Although GK was initially proposed as being solely regulated by substrate availability, subsequent discoveries of its redox regulation and the light involvement in the production of chloroplastic and cytosolic GK isoforms have indicated a more refined regulation of the pathways of glycerate conversion. Here, we re-evaluate the importance of GK and emphasize its multifaceted role in plants. Thus, GK can be a major player in several branches of primary metabolism, including the glycolate pathway, gluconeogenesis, glycolysis, and C4 metabolism. In addition, recently, the chloroplastic (but not cytosolic) GK isoform was implicated as part of a light-dependent plant immune response to pathogen attack. The origins of glycerate are also discussed here; it is produced in several cell compartments and undergoes huge fluctuations depending on light/dark conditions. The recent discovery of the vacuolar glycerate transporter adds yet another layer to our understanding of glycerate transport/metabolism and that of other two- and three-carbon metabolites.

Exploring Redox Modulation of Plant UDP-Glucose Pyrophosphorylase.

UDP-glucose (UDPG) pyrophosphorylase (UGPase) catalyzes a reversible reaction, producing UDPG, which serves as an essential precursor for hundreds of glycosyltransferases in all organisms. In this study, activities of purified UGPases from sugarcane and barley were found to be reversibly redox modulated in vitro through oxidation by hydrogen peroxide or oxidized glutathione (GSSG) and through reduction by dithiothreitol or glutathione. Generally, while oxidative treatment decreased UGPase activity, a subsequent reduction restored the activity. The oxidized enzyme had increased Km values with substrates, especially pyrophosphate. The increased Km values were also observed, regardless of redox status, for UGPase cysteine mutants (Cys102Ser and Cys99Ser for sugarcane and barley UGPases, respectively). However, activities and substrate affinities (Kms) of sugarcane Cys102Ser mutant, but not barley Cys99Ser, were still prone to redox modulation. The data suggest that plant UGPase is subject to redox control primarily via changes in the redox status of a single cysteine. Other cysteines may also, to some extent, contribute to UGPase redox status, as seen for sugarcane enzymes. The results are discussed with respect to earlier reported details of redox modulation of eukaryotic UGPases and regarding the structure/function properties of these proteins.

Pyrophosphate as an alternative energy currency in plants.

In the conditions of [Mg2+] elevation that occur, in particular, under low oxygen stress and are the consequence of the decrease in [ATP] and increase in [ADP] and [AMP], pyrophosphate (PPi) can function as an alternative energy currency in plant cells. In addition to its production by various metabolic pathways, PPi can be synthesized in the combined reactions of pyruvate, phosphate dikinase (PPDK) and pyruvate kinase (PK) by so-called PK/PPDK substrate cycle, and in the reverse reaction of membrane-bound H+-pyrophosphatase, which uses the energy of electrochemical gradients generated on tonoplast and plasma membrane. The PPi can then be consumed in its active forms of MgPPi and Mg2PPi by PPi-utilizing enzymes, which require an elevated [Mg2+]. This ensures a continuous operation of glycolysis in the conditions of suppressed ATP synthesis, keeping metabolism energy efficient and less dependent on ATP.

Optimization of nucleotide sugar supply for polysaccharide formation via thermodynamic buffering.

Plant polysaccharides (cellulose, hemicellulose, pectin, starch) are either direct (i.e. leaf starch) or indirect products of photosynthesis, and they belong to the most abundant organic compounds in nature. Although each of these polymers is made by a specific enzymatic machinery, frequently in different cell locations, details of their synthesis share certain common features. Thus, the production of these polysaccharides is preceded by the formation of nucleotide sugars catalyzed by fully reversible reactions of various enzymes, mostly pyrophosphorylases. These 'buffering' enzymes are, generally, quite active and operate close to equilibrium. The nucleotide sugars are then used as substrates for irreversible reactions of various polysaccharide-synthesizing glycosyltransferases ('engine' enzymes), e.g. plastidial starch synthases, or plasma membrane-bound cellulose synthase and callose synthase, or ER/Golgi-located variety of glycosyltransferases forming hemicellulose and pectin backbones. Alternatively, the irreversible step might also be provided by a carrier transporting a given immediate precursor across a membrane. Here, we argue that local equilibria, established within metabolic pathways and cycles resulting in polysaccharide production, bring stability to the system via the arrangement of a flexible supply of nucleotide sugars. This metabolic system is itself under control of adenylate kinase and nucleoside-diphosphate kinase, which determine the availability of nucleotides (adenylates, uridylates, guanylates and cytidylates) and Mg2+, the latter serving as a feedback signal from the nucleotide metabolome. Under these conditions, the supply of nucleotide sugars to engine enzymes is stable and constant, and the metabolic process becomes optimized in its load and consumption, making the system steady and self-regulated.

Magnesium Signaling in Plants.

Free magnesium (Mg2+) is a signal of the adenylate (ATP+ADP+AMP) status in the cells. It results from the equilibrium of adenylate kinase (AK), which uses Mg-chelated and Mg-free adenylates as substrates in both directions of its reaction. The AK-mediated primary control of intracellular [Mg2+] is finely interwoven with the operation of membrane-bound adenylate- and Mg2+-translocators, which in a given compartment control the supply of free adenylates and Mg2+ for the AK-mediated equilibration. As a result, [Mg2+] itself varies both between and within the compartments, depending on their energetic status and environmental clues. Other key nucleotide-utilizing/producing enzymes (e.g., nucleoside diphosphate kinase) may also be involved in fine-tuning of the intracellular [Mg2+]. Changes in [Mg2+] regulate activities of myriads of Mg-utilizing/requiring enzymes, affecting metabolism under both normal and stress conditions, and impacting photosynthetic performance, respiration, phloem loading and other processes. In compartments controlled by AK equilibrium (cytosol, chloroplasts, mitochondria, nucleus), the intracellular [Mg2+] can be calculated from total adenylate contents, based on the dependence of the apparent equilibrium constant of AK on [Mg2+]. Magnesium signaling, reflecting cellular adenylate status, is likely widespread in all eukaryotic and prokaryotic organisms, due simply to the omnipresent nature of AK and to its involvement in adenylate equilibration.

Identification and characterization of inhibitors of UDP-glucose and UDP-sugar pyrophosphorylases for in vivo studies.

UDP-sugars serve as ultimate precursors in hundreds of glycosylation reactions (e.g. for protein and lipid glycosylation, synthesis of sucrose, cell wall polysaccharides, etc.), underlying an important role of UDP-sugar-producing enzymes in cellular metabolism. However, genetic studies on mechanisms of UDP-sugar formation were frequently hampered by reproductive impairment of the resulting mutants, making it difficult to assess an in vivo role of a given enzyme. Here, a chemical library containing 17 500 compounds was separately screened against purified UDP-glucose pyrophosphorylase (UGPase) and UDP-sugar pyrophosphorylase (USPase), both enzymes representing the primary mechanisms of UDP-sugar formation. Several compounds have been identified which, at 50 µm, exerted at least 50% inhibition of the pyrophosphorylase activity. In all cases, both UGPase and USPase activities were inhibited, probably reflecting common structural features of active sites of these enzymes. One of these compounds (cmp #6), a salicylamide derivative, was found as effective inhibitor of Arabidopsis pollen germination and Arabidopsis cell culture growth. Hit optimization on cmp #6 yielded two analogs (cmp #6D and cmp #6D2), which acted as uncompetitive inhibitors against both UGPase and USPase, and were strong inhibitors in the pollen test, with apparent inhibition constants of less than 1 µm. Their effects on pollen germination were relieved by addition of UDP-glucose and UDP-galactose, suggesting that the inhibitors targeted UDP-sugar formation. The results suggest that cmp #6 and its analogs may represent useful tools to study in vivo roles of the pyrophosphorylases, helping to overcome the limitations of genetic approaches.

Defense Responses in Aspen with Altered Pectin Methylesterase Activity Reveal the Hormonal Inducers of Tyloses.

Tyloses are ingrowths of parenchyma cells into the lumen of embolized xylem vessels, thereby protecting the remaining xylem from pathogens. They are found in heartwood, sapwood, and in abscission zones and can be induced by various stresses, but their molecular triggers are unknown. Here, we report that down-regulation of PECTIN METHYLESTERASE1 (PtxtPME1) in aspen (Populus tremula × tremuloides) triggers the formation of tyloses and activation of oxidative stress. We tested whether any of the oxidative stress-related hormones could induce tyloses in intact plantlets grown in sterile culture. Jasmonates, including jasmonic acid (JA) and methyl jasmonate, induced the formation of tyloses, whereas treatments with salicylic acid (SA) and 1-aminocyclopropane-1-carboxylic acid (ACC) were ineffective. SA abolished the induction of tyloses by JA, whereas ACC was synergistic with JA. The ability of ACC to stimulate tyloses formation when combined with JA depended on ethylene (ET) signaling, as shown by a decrease in the response in ET-insensitive plants. Measurements of internal ACC and JA concentrations in wild-type and ET-insensitive plants treated simultaneously with these two compounds indicated that ACC and JA regulate each other's concentration in an ET-dependent manner. The findings indicate that jasmonates acting synergistically with ethylene are the key molecular triggers of tyloses.

The redox control of photorespiration: from biochemical and physiological aspects to biotechnological considerations.

Photorespiration is a complex and tightly regulated process occurring in photosynthetic organisms. This process can alter the cellular redox balance, notably via the production and consumption of both reducing and oxidizing equivalents. Under certain circumstances, these equivalents, as well as reactive oxygen or nitrogen species, can become prominent in subcellular compartments involved in the photorespiratory process, eventually promoting oxidative post-translational modifications of proteins. Keeping these changes under tight control should therefore be of primary importance. In order to review the current state of knowledge about the redox control of photorespiration, we primarily performed a careful description of the known and potential redox-regulated or oxidation sensitive photorespiratory proteins, and examined in more details two interesting cases: the glycerate kinase and the glycine cleavage system. When possible, the potential impact and subsequent physiological regulations associated with these changes have been discussed. In the second part, we reviewed the extent to which photorespiration contributes to cellular redox homeostasis considering, in particular, the set of peripheral enzymes associated with the canonical photorespiratory pathway. Finally, some recent biotechnological strategies to circumvent photorespiration for future growth improvements are discussed in the light of these redox regulations.

Magnesium and cell energetics: At the junction of metabolism of adenylate and non-adenylate nucleotides.

Free magnesium (Mg2+) represents a powerful signal arising from interconversions of adenylates (ATP, ADP and AMP). This is a consequence of the involvement of adenylate kinase (AK) which equilibrates adenylates and uses defined species of Mg-complexed and Mg-free adenylates in both directions of its reaction. However, cells contain also other reversible Mg2+-dependent enzymes that equilibrate non-adenylate nucleotides (uridylates, cytidylates and guanylates), i.e. nucleoside monophosphate kinases (NMPKs) and nucleoside diphosphate kinase (NDPK). Here, we propose that AK activity is tightly coupled to activities of NMPK and NDPK, linking adenylate equilibrium to equilibria of other nucleotides, and with [Mg2+] controlling the ratios of Mg-chelated and Mg-free nucleotides. This coupling establishes main hubs for adenylate-driven equilibration of non-adenylate nucleotides, with [Mg2+] acting as signal arising from all nucleotides rather than adenylates only. Further consequences involve an overall adenylate control of UTP-, GTP- and CTP-dependent pathways and the availability of substrates for RNA and DNA synthesis.

Toward understanding the emergence of life: A dual function of the system of nucleotides in the metabolically closed autopoietic organization.

General structure of metabolism includes the reproduction of catalysts that govern metabolism. In this structure, the system becomes autopoietic in the sense of Maturana and Varela, and it is closed to efficient causation as defined by Robert Rosen. The autopoietic maintenance and operation of the catalysts takes place via the set of free nucleotides while the synthesis of catalysts occurs via the information encoded by the set of nucleotides arranged in polymers of RNA and DNA. Both energy charge and genetic information use the components of the same pool of nucleoside triphosphates, which is equilibrated by thermodynamic buffering enzymes such as nucleoside diphosphate kinase and adenylate kinase. This occurs in a way that the system becomes internally stable and metabolically closed, which initially could be realized at the level of ribozymes catalyzing basic metabolic reactions as well as own reproduction. The function of ATP, GTP, UTP, and CTP is dual, as these species participate both in the general metabolism as free nucleotides and in the transfer of genetic information via covalent polymerization to nucleic acids. The changes in their pools directly impact both bioenergetic pathways and nucleic acid turnover. Here we outline the concept of metabolic closure of biosystems grounded in the dual function of nucleotide coenzymes that serve both as energetic and informational molecules and through this duality generate the autopoietic performance and the ability for codepoietic evolutionary transformations of living systems starting from the emergence of prebiotic systems.

Magnesium and cell energetics in plants under anoxia.

Stress conditions (e.g. anoxia) frequently result in a decrease of [ATP] and in an increase of [ADP] and [AMP], with a concomitant increase of [Mg(2+)] and other cations, e.g. Ca(2+). The elevation of [Mg(2+)] is linked to the shift in the apparent equilibrium of adenylate kinase. As a result, enzymes that use Mg(2+) as a cofactor are activated, Ca(2+) activates calcium-dependent signalling pathways, and PP(i) can serve as an alternative energy source in its active form of MgPP(i) or Mg2PP(i). Under anoxic conditions in plants, an important source of PP(i) may come as a result of combined reactions of PK (pyruvate kinase) and PPDK (pyruvate, phosphate dikinase). The PP(i) formed in the PPDK/PK cycle ignites glycolysis in conditions of low [ATP] by involving PP(i)-dependent reactions. This saves ATP and makes metabolism under stress conditions more energy efficient.

A common structural blueprint for plant UDP-sugar-producing pyrophosphorylases.

Plant pyrophosphorylases that are capable of producing UDP-sugars, key precursors for glycosylation reactions, include UDP-glucose pyrophosphorylases (A- and B-type), UDP-sugar pyrophosphorylase and UDP-N-acetylglucosamine pyrophosphorylase. Although not sharing significant homology at the amino acid sequence level, the proteins share a common structural blueprint. Their structures are characterized by the presence of the Rossmann fold in the central (catalytic) domain linked to enzyme-specific N-terminal and C-terminal domains, which may play regulatory functions. Molecular mobility between these domains plays an important role in substrate binding and catalysis. Evolutionary relationships and the role of (de)oligomerization as a regulatory mechanism are discussed.

Effects of Magnesium, Pyrophosphate and Phosphonates on Pyrophosphorolytic Reaction of UDP-Glucose Pyrophosphorylase.

UDP-glucose pyrophosphorylase (UGPase) carries a freely reversible reaction, using glucose-1-P and UTP to produce UDP-glucose (UDPG) and pyrophosphate (PPi), with UDPG being essential for glycosylation reactions in all organisms including, e.g., synthesis of sucrose, cellulose and glycoproteins. In the present study, we found that free magnesium (Mg2+) had profound effects on the reverse reaction of purified barley UGPase, and was absolutely required for its activity, with an apparent Km of 0.13 mM. More detailed analyses with varied concentrations of MgPPi allowed us to conclude that it is the MgPPi complex which serves as true substrate for UGPase in its reverse reaction, with an apparent Km of 0.06 mM. Free PPi was an inhibitor in this reaction. Given the key role of PPi in the UGPase reaction, we have also tested possible effects of phosphonates, which are analogs of PPi and phosphate (Pi). Clodronate and etidronate (PPi analogs) had little or no effect on UGPase activity, whereas fosetyl-Al (Pi analog), a known fungicide, acted as effective near-competitive inhibitor versus PPi, with Ki of 0.15 mM. The data are discussed with respect to the role of magnesium in the UGPase reaction and elucidating the use of inhibitors in studies on cellular function of UGPase and related enzymes.

Metabolic systems maintain stable non-equilibrium via thermodynamic buffering.

Here, we analyze how the set of nucleotides in the cell is equilibrated and how this generates simple rules that help the cell to organize itself via maintenance of a stable non-equilibrium state. A major mechanism operating to achieve this state is thermodynamic buffering via high activities of equilibrating enzymes such as adenylate kinase. Under stable non-equilibrium, the ratios of free and Mg-bound adenylates, Mg(2+) and membrane potentials are interdependent and can be computed. The adenylate status is balanced with the levels of reduced and oxidized pyridine nucleotides through regulated uncoupling of the pyridine nucleotide pool from ATP production in mitochondria, and through oxidation of substrates non-coupled to NAD(+) reduction in peroxisomes. The set of adenylates and pyridine nucleotides constitutes a generalized cell energy status and determines rates of major metabolic fluxes. As the result, fluxes of energy and information become organized spatially and temporally, providing conditions for self-maintenance of metabolism.

Domain-specific determinants of catalysis/substrate binding and the oligomerization status of barley UDP-glucose pyrophosphorylase.

UDP-glucose (UDPG) pyrophosphorylase (UGPase) produces UDPG for sucrose and polysaccharide synthesis and glycosylation reactions. In this study, several barley UGPase mutants were produced, either single amino acid mutants or involving deletions of N- and C-terminal domains (Ncut and Ccut mutants, respectively) and of active site region ("NB loop"). The Del-NB mutant yielded no activity, whereas Ncut deletions and most of Ccut mutants, including short deletions at the so called "I-loop" region of C-terminal domain, as well as a single K260A mutant resulted in very low activity. For wt and the mutants, kinetics with UDPG were linear on reciprocal plots, whereas PPi at concentrations above 1 mM exerted strong substrate inhibition. Both K260A and most of the Ccut mutants had very high Km with PPi (up to 33 mM), whereas Ncut deletions had greatly increased Km with UDPG (up to 57 mM). Surprisingly, an 8 amino acid deletion from end of the C-terminus resulted in an enzyme (Ccut-8 mutant) with 44% higher activity when compared to wt, but with similar Km values. Whereas Ccut-8 existed solely as a monomer, other deletion mutants had a more oligomerized status, e.g. Ncut mutants existing primarily as dimers. Overall, the data confirmed the essential role of NB loop in catalysis, but also pointed out to the role of both N- and C-termini for activity, substrate binding and oligomerization. The importance of oligomerization status for enzymatic activity of UGPase is discussed.

Molecular and kinetic characterization of two UDP-glucose pyrophosphorylases, products of distinct genes, from Arabidopsis.

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme in the production (and conversions) of UDP-glucose, a key precursor for carbohydrate biosynthesis. cDNAs corresponding to two UGPase isozymes in Arabidopsis were overexpressed in Escherichia coli and, subsequently, the recombinant proteins were purified and characterized. Both proteins were highly conserved, sharing 93% identity. Based on crystal structure-derived images, the main amino acid differences mapped to N- and C-termini domains, but not to central active site region. The two proteins existed mainly as monomers, and they had similar molecular masses of ca. 53 kDa. However, comparison of molecular masses of UGPases from Arabidopsis root and leaf extracts revealed that the root protein was slightly larger, suggesting a post-translational modification. Specific activity of the purified UGPase-1 was ca. 10-30% lower than that of UGPase-2, depending on direction of the reaction, whereas its K(m) values with all substrates in both directions of the reaction were consistently ca. twice lower than those of UGPase-2 (0.03-0.14 mM vs. 0.07-0.36 mM, respectively). Both proteins were "true" UGPases, and had no activity with ADP-glucose/ATP or galactose-1-P. Equilibrium constant for both proteins was ca. 0.3, suggesting preference for the pyrophosphorolysis direction of the reaction. The data are discussed with respect to potential roles of UGPase in carbohydrate synthesis/metabolism in Arabidopsis.

UDP-glucose pyrophosphorylase is not rate limiting, but is essential in Arabidopsis.

UDP-glucose pyrophosphorylase (UGPase) produces UDP-glucose which is essential for sucrose and polysaccharide synthesis. Using Arabidopsis, we demonstrated that two UGPase genes (UGP1 and UGP2) are differentially expressed in a variety of organs, with UGP1 being pre-dominant. Co-expression analyses of UGP genes suggest that UGP1 is closely co-regulated with carbohydrate metabolism genes, late embryogenesis and seed loading, while UGP2 is co-regulated with stress response genes, fertilized flowers and photosynthetic genes. We have used Arabidopsis mutants for the UGP genes to characterize the role of both genes. The UGPase activity/protein was reduced by 70, 10 and 85% in ugp1, ugp2 and ugp1/ugp2 double mutant (DK) plants, respectively. A decrease in UGPase activity/protein was accompanied by an increase in expression of USP, a gene for UDP-sugar pyrophosphorylase, suggesting a compensatory mechanism. Generally, the mutants had no effects on soluble sugar/starch content (except in certain cases for DK plants), and there were no differences in cell wall composition/content between the wild type and the mutants. On the other hand, DK plants had greater hypocotyl and root lengths. When grown in the field, the mutants had as much as a 50% decrease in the number of seeds produced (consistent with a substantial decrease in field fitness), suggesting that they would be outcompeted in the field in a few generations. Overall, the data suggest that UGPase is not rate limiting for sucrose/starch and cell wall synthesis, but that it is essential in Arabidopsis.

Thermodynamic buffering, stable non-equilibrium and establishment of the computable structure of plant metabolism.

The equilibria of coenzyme nucleotides and substrates established in plant cells generate simple rules that govern the plant metabolome and provide optimal conditions for the non-equilibrium fluxes of major metabolic processes such as ATP synthesis, CO2 fixation, and mitochondrial respiration. Fast and abundant enzymes, such as adenylate kinase, carbonic anhydrase or malate dehydrogenase, provide constant substrate flux for these processes. These "buffering" enzymes follow the Michaelis-Menten (MM) kinetics and operate near equilibrium. The non-equilibrium "engine" enzymes, such as ATP synthase, Rubisco or the respiratory complexes, follow the modified version of MM kinetics due to their high concentration and low concentration of their substrates. The equilibrium reactions serve as control gates for the non-equilibrium flux through the engine enzymes establishing the balance of the fluxes of load and consumption of metabolic components. Under the coordinated operation of buffering and engine enzymes, the concentrations of free and Mg-bound adenylates and of free Mg2+ are set, serving as feedback signals from the adenylate metabolome. Those are linked to various cell energetics parameters, including membrane potentials. Also, internal levels of reduced and oxidized pyridine nucleotides are established in the coordinated operation of malate dehydrogenase and respiratory components, with proton concentration as a feedback from pyridine nucleotide pools. Non-coupled pathways of respiration serve to equilibrate the levels of pyridine nucleotides, adenylates, and as a pH stat. This stable non-equilibrium organizes the fluxes of energy spatially and temporally, controlling the rates of major metabolic fluxes that follow thermodynamically and kinetically defined computational principles.

The structure-activity relationship of the salicylimide derived inhibitors of UDP-sugar producing pyrophosphorylases.

UDP-sugars are key precursors for biomass production in nature (synthesis of cellulose, hemicellulose, etc.). They are produced de novo by distinct UDP-sugar producing pyrophosphorylases. Studies on the roles of these enzymes using genetic knockouts were hampered by sterility of the mutants and by functional-complementation from related enzyme(s), hindering clear interpretation of the results. In an attempt to override these difficulties, we turned to the reverse chemical genetics approaches to identify compounds which interfere with the activity of those enzymes in vivo. Hit expansion on one of such compounds, a salicylimide derivative, allowed us to identify several inhibitors with a range of activities. The present study provides a structure-activity relationship for these compounds.

Corrigendum: The Glycerate and Phosphorylated Pathways of Serine Synthesis in Plants: The Branches of Plant Glycolysis Linking Carbon and Nitrogen Metabolism.

[This corrects the article DOI: 10.3389/fpls.2018.00318.].