Pyrophosphate inhibits gluconeogenesis by restricting UDP-glucose formation in vivo

Ferjani, Ali; Kawade, Kensuke; Asaoka, Mariko; Oikawa, Akira; Okada, Takashi; Mochizuki, Atsushi; Maeshima, Masayoshi; Hirai, Masami Yokota; Saito, Kazuki; Tsukaya, Hirokazu

doi:10.1038/s41598-018-32894-1

Download PDF

Article
Open access
Published: 02 October 2018

Pyrophosphate inhibits gluconeogenesis by restricting UDP-glucose formation in vivo

Scientific Reports volume 8, Article number: 14696 (2018) Cite this article

3716 Accesses
22 Citations
Metrics details

Subjects

An Author Correction to this article was published on 21 May 2019

This article has been updated

Abstract

Pyrophosphate (PPi) is produced by anabolic reactions and serves as an energy donor in the cytosol of plant cells; however, its accumulation to toxic levels disrupts several common biosynthetic pathways and is lethal. Before acquiring photosynthetic capacity, young seedlings must endure a short but critical heterotrophic period, during which they are nourished solely by sugar produced from seed reserves by the anabolic process of gluconeogenesis. Previously, we reported that excess PPi in H⁺-PPase-knockout fugu5 mutants of Arabidopsis thaliana severely compromised gluconeogenesis. However, the precise metabolic target of PPi inhibition in vivo remained elusive. Here, CE-TOF MS analyses of major metabolites characteristic of gluconeogenesis from seed lipids showed that the Glc6P;Fru6P level significantly increased and that Glc1P level was consistently somewhat higher in fugu5 compared to wild type. In contrast, the UDP-Glc level decreased significantly in the mutants. Importantly, specific removal of PPi in fugu5, and thus in AVP1_pro:IPP1 transgenic lines, restored the Glc1P and the Glc6P;Fru6P levels, increased the UDP-Glc level ~2.0-fold, and subsequently increased sucrose synthesis. Given the reversible nature of the Glc1P/UDP-Glc reaction, our results indicate that UGP-Glc pyrophosphorylase is the major target when excess PPi exerts inhibitory effects in vivo. To validate our findings, we analyzed metabolite responses using a mathematical theory called structural sensitivity analysis (SSA), in which the responses of concentrations in reaction systems to perturbations in enzyme activity are determined from the structure of the network alone. A comparison of our experimental data with the results of pure structural theory predicted the existence of unknown reactions as the necessary condition for the above metabolic profiles, and confirmed the above results. Our data support the notion that H⁺-PPase plays a pivotal role in cytosolic PPi homeostasis in plant cells. We propose that the combination of metabolomics and SSA is powerful when seeking to identify and predict metabolic targets in living cells.

Metabolite profiling of Arabidopsis mutants of lower glycolysis

Article Open access 11 October 2022

A novel variant of the Calvin–Benson cycle bypassing fructose bisphosphate

Article Open access 16 March 2022

Transport efficiency of AtGTR1 dependents on the hydrophobicity of transported glucosinolates

Article Open access 24 March 2022

Introduction

Inorganic pyrophosphate (PPi) was discovered in the 19^th century and, in 1941, was found to accumulate in rat livers; this was the first report on PPi accumulation in a biological system¹. Later, Kornberg described the first PPi-producing biological reaction² and proposed that pyrophosphorylases acted in the direction of PPi production favoring the formation of biochemical compounds³. Further, it was suggested that inorganic pyrophosphatase (PPase)-mediated PPi hydrolysis rendered the above reactions practically irreversible⁴, a hypothesis that is now widely accepted. PPi hydrolysis has a ∆G^′° of −33.4 kJ/mol and can therefore drive reactions that are otherwise energetically unfavorable, including many biosynthetic steps⁵. Almost 200 different reactions produce PPi^6,7,8,9. The loss of PPase activity arrested growth in bacteria¹⁰ and yeast¹¹ and triggered developmental blockage at an early larval stage in worms¹², supporting a vital role for PPi homeostasis in living cells. In the model plant Arabidopsis thaliana (hereinafter, Arabidopsis), we previously reported that vacuolar proton pyrophosphatase (H⁺-PPase) is essential for maintaining adequate PPi levels¹³, and that cytosolic PPa isozymes that exhibit non-overlapping subcellular localization patterns¹⁴, particularly PPa1, act cooperatively with H⁺-PPase to prevent an increase in PPi concentrations to toxic levels¹⁵. The PPi concentration in the cytosol of plant cells was 0.2–0.3 mM¹⁶. Moreover, the constitutive expression of vacuolar proton pyrophosphatase (H⁺-PPase) increases plant growth under a variety of abiotic stresses, rendering the encoding gene of critical importance to crop breeders^17,18. However, the actual target of excess PPi in vivo and the physiological roles of PPases remain enigmatic in all living organisms, and little is known about the master regulator of cytosolic PPi homeostasis in plants⁵.

Against this background, we isolated¹⁹ and characterized vacuolar H⁺-PPase loss-of-function fugu5 mutants of Arabidopsis; these are viable but exhibit defects in cotyledon development and hypocotyl elongation¹³. The postgerminative growth defects recover when sucrose (Suc) is supplied or when PPi is removed by the yeast cytosolic PPase IPP1 in the AVP1_pro:IPP1 lines¹³. This indicated that H⁺-PPase played a major role in the hydrolysis of inhibitory PPi^9,13,20,21. The PPi level was ~2.5-fold higher, and the Suc level 50% lower, in fugu5 etiolated seedlings compared to those of wild-type (WT)¹³. Thus, excess PPi likely inhibits gluconeogenesis from seed storage lipids (triacylglycerols; TAG), but the precise metabolic target remained unclear^9,13. Thus, we examined how excess PPi inhibited gluconeogenesis in vivo.

Here, CE-TOF MS analyses of major metabolites produced during TAG mobilization showed that relatively few metabolites were significantly affected in three fugu5 alleles compared to WT (Fig. 1a). In fact, only 8 anions and 16 cations were commonly up- or down-regulated (Fig. 1b,c). The levels of Fru6P;Glc6P were significantly higher (~2.0-fold) in the mutants, and the Glc1P level was consistently somewhat higher (Fig. 1d). Also, the citrate, Gly3P, GlcNAc6P, and S7P levels increased significantly in the mutants (Fig. 1c,d; Table S1). Interestingly, the UDP-Glc level was significantly reduced (up to ~0.6-fold) in all three fugu5 strains (Fig. 1d; Table S1). Principal component analysis (PCA) of the above metabolic changes indicated that the WT and fugu5 strains clearly differed (Fig. 1e). On the other hand, the levels of several amino acids were significantly reduced in the fugu5 strains (Fig. 1c; Table S1). Of the enzymes active on the above metabolites, only UDP-Glc pyrophosphorylase (UGPase) produces PPi. Given that gluconeogenesis is compromised in fugu5 mutants¹³, the results suggest that UGPase was the likely target of inhibition by excess cytosolic PPi in the fugu5 background.

To confirm this hypothesis, two AVP1_pro:IPP1 transgenic lines¹³, in which cytosolic PPi is specifically hydrolyzed in the fugu5 background, were subjected to CE-TOF MS analysis along with WT and a representative fugu5-1 strain (Fig. 2). The fugu5 metabolic defects were reversed in AVP1_pro:IPP1 (Fig. 2a), where Glc1P and the Glc6P;Fru6P levels equaled or were only slightly lower than those in WT, respectively (Fig. 2b; Table S2). Additionally, the UDP-Glc levels in AVP1_pro:IPP1#8-3 and AVP1_pro:IPP1#17-3 were 1.74- and 2.25-fold higher, respectively, than in the WT (Fig. 2b; Table S2). In other words, simply removing PPi from the fugu5 background increased the levels of UDP-Glc by 3.4- and 4.5-fold in AVP1_pro:IPP1#8-3 and AVP1_pro:IPP1#17-3 respectively (Fig. 2b; Table S2). Finally, to assess the impact of PPi removal on gluconeogenesis, we measured Suc levels. Consistently, the Suc level was 45% higher in AVP1_pro:IPP1#8-3 and 31% higher in AVP1_pro:IPP1#17-3 (Fig. 2c). Again, provided that the UGPase-catalyzed reaction is readily reversible^16,22, our data strongly indicate that UGPase is a major metabolic target of excess PPi in vivo.

TAG mobilization is a multistep process that has been extensively investigated; several key enzymes have been identified²³. The mutants icl-2, mls-2, and pck1-2 are defective in isocitrate lyase (ICL), malate synthase (MS), and phopshoenolpyruvate carboxykinase (PEPCK), respectively (Fig. 2b)^24,25,26. All exhibit significant reductions in Suc synthesis from TAG and mimic the fugu5 developmental defects²⁷. Therefore, comparative analyses of the metabolic profiles of these three mutants would confirm whether UGPase was a specific target of excess PPi, as the icl-2, mls-2, and pck1-2 mutants express the functional H⁺-PPase. Interestingly, CE-TOF MS metabolic profiling revealed that key metabolites, such as Glc6P;Fru6P, Glc1P, and UDP-Glc, were differentially affected in the icl-2, mls-2, and pck1-2 strains compared to the fugu5 strain (Fig. 3a,b). For example, although the UDP-Glc level was significantly reduced in the fugu5 strain, the levels were almost unaffected in the icl-2, mls-2, and pck1-2 strains (Fig. 3b; Table S2). The Glc1P and Glc6P;Fru6P levels were elevated in the fugu5 strain, but reduced in the icl-2, mls-2, and pck1-2 strains (Fig. 3b; Table S2). Additionally, the levels of malate, succinate, and citrate were 3.5-, 2.3-, and 5.4-fold higher, respectively, in the pck1-2 strain than in WT, indicating that metabolic flow was severely suppressed (Fig. 3b; Table S2). On the other hand, the succinate and malate levels were severely reduced (by up to ~40% and ~10%, respectively, compared to the WT strain) in both the icl-2 and mls-2 mutants (Fig. 3b; Table S2). Finally, PCA of the metabolic changes in the three mutants indicated that they all clearly differed (Fig. 3c). Taken together, the results clearly indicated that UDP-Glc production was disrupted by excess PPi in planta.

Figure 3d shows the metabolic pathway responsible for Suc production from Fru1,6P₂. The circuit contains two reversible reactions in which PPi is released as a byproduct. At first glance, any defect in H⁺-PPase would be expected to result in excess PPi in plant cells, reducing the forward rate of reaction, eventually reducing Suc production. We used structural sensitivity analysis (SSA)^28,29 to analyze the effect of the fugu5 mutation on metabolic dynamics. The qualitative responses of the steady state metabolite concentrations and fluxes induced by perturbations in enzyme levels can be determined from the network structure alone; it is unnecessary to assume reaction rate functions or parameter values. When applied to the metabolic pathway responsible for Suc production from Fru1,6P₂ (Fig. 3d), SSA (surprisingly) predicted that excess PPi should not influence the concentration of Suc if the network structure of Fig. 3d was, in fact, correct. This indicated that the network structure had to be revised to explain the experimentally measured metabolite profile of the fugu5 strains. After analysis of all possible modifications (Fig. S1), we found that addition of at least one of four reactions (involving Fru1,6P₂, Fru6P, Glc6P, and/or Glc1P) was required to explain the empirical results (please see the Methods for details). Figure 3e shows the structure of one network that successfully explains the observed decrease in Suc levels and also predicts changes in the concentrations of other metabolites caused by excess PPi. Thus, SSA not only allows information on known networks to be validated but also predicts the existence of unsuspected reactions that explain the observed responses of various systems.

PPi is universally produced in large amounts by a variety of vital biosynthetic reactions^5,7. The UGPase-catalyzed reaction is readily reversible; the in vivo equilibrium point depends on the PPi concentration^5,16,22. Plant cells contain substantial levels of PPi in the cytosol; these remain remarkably constant under a variety of conditions. It has been suggested that PPi-dependent phosphofructokinase (PFP) acts to control PPi levels, but the cytosolic PPi level of transgenic plants with very low levels of this enzyme are barely affected, suggesting that other mechanisms are involved³⁰. H⁺-PPase has been proposed as a potential key player in PPi metabolism. However, no H⁺-PPase mutants were available, and any possible role for the enzyme thus remained unresolved²². The widespread belief that proton pumping is the major role of H⁺-PPase is partially supported by the increased tolerance to abiotic stress of many crops engineered to overexpress the enzyme to date¹⁷, but any possible contribution made by PPi hydrolysis has been overlooked^9,13,18. Characterization of fugu5 mutants revealed the pivotal role played by H⁺-PPase in PPi homeostasis and suggested that gluconeogenesis might be compromised by excess PPi¹³. Here, we used a comparative metabolomics approach to confirm that gluconeogenesis is, indeed, affected and provide robust evidence that UGPase is a major target of excess PPi in vivo.

UGPase is an important enzyme for the metabolism of UDP-Glc, a key precursor in the synthesis of Suc, cellulose, and callose^15,31,32, and is thought to be regulated by substrate availability alone at the enzyme level³¹. UGPase has been purified from a wide variety of organisms, including yeast, plants, animals, the slime mold Dictyostelium discoidium, and several bacterial species³³. Although UGPase has been suggested to be classified structurally into both prokaryotic and eukaryotic groups, they have almost identical catalytic properties³⁴. The Arabidopsis genome contains three genes encoding UGPase: AtUGP1, AtUGP2, and AtUGP3^35,36. We have previously demonstrated that recombinant AtUGP3 catalyzed the formation of UDP-Glc from Glc1P and UTP³⁶. Moreover, we examined the effect of PPi on the UGPase activity of recombinant UGP3 and found that the addition of various PPi concentrations (0–10 mM) strongly inhibited the UGPase activity of the recombinant UGP3³⁶, as has been reported for other UGPases, via product inhibition (see ref.³⁶, and the citations therein for details). Thus, the enhanced stress tolerance of crops constitutively expressing H⁺-PPase is, in part, attributable to increased photosynthetic efficiency, and UGPase is a novel useful target in efforts to genetically engineer crops with increased yields^17,18.

Methods

Plant material and growth conditions

Arabidopsis Col-0 was the WT strain, and all the mutants and transgenics were in the Col-0 background. The fugu5 mutants and the AVP1_pro:IPP1#8-3 and AVP1_pro:IPP1#17-3 strains have been previously described¹³. Seeds of the icl-2, mls-2, and pck1-2 strains were the kind gift of Professor Ian Graham (the University of York) and have been described previously^24,25,26. The seeds were sterilized and sown in plates containing Murashige and Skoog Plant Salt Mixture (Wako Pure Chemical Industries), 0.1% (w/v) 2-(N-morpholino) ethanesulfonic acid (MES) (pH = 5.8 as adjusted with KOH), and 0.2% (w/v) gellun gum³⁷. The seeds were left in the dark for 3 days at 4 °C, exposed to light (50 µmol m⁻² s⁻¹) to facilitate germination at 22 °C for 6 h, and then maintained at 22 °C in the dark for 66 h. Three sets of experiments were conducted in parallel for each genotype. The first set featured the WT, fugu5-1, fugu5-2, and fugu5-3 strains. The second set included the WT, fugu5-1, AVP1_pro: IPP1#8-3, AVP1_pro: IPP1#17-3, icl-2, mls-2, and pck1-2 strains. Three-day-old etiolated seedlings were immediately frozen in liquid nitrogen after sampling and stored at −80 °C prior to CE-TOF MS analysis.

Metabolite profiling with CE-TOF MS

About 50-mg quantities of frozen seedlings were homogenized using a Zirconia bead in a Safe-seal micro tube (2 mL; PP; Sarsted) with the aid of a Mixer Mill (Retsch). Then, 500 µL methanol containing internal standards (each 8 µM; methionine sulfone and camphor 10-sulfonic acid for cation and anion analysis, respectively) was added, followed by repeat homogenization and centrifugation at 20,400 g for 3 min at 4 °C. Next, 500 µL chloroform and 200 µL water were added. The mixture was vortexed for 3 min and centrifuged at 20,400 g for 3 min at 4 °C. Methanol in the mixture was evaporated in a centrifugal concentrator for 30 min at 45 °C. The resulting upper layer (100–200 µL water associated fraction) was centrifugally filtered through a Millipore 5-kDa-cutoff filter (Merck Millipore) at 9,100 g for 120 min. The filtrate was evaporated to dryness in a centrifugal concentrator for 120 min. The residue was dissolved in 20 µL water and other internal standards (200 µM of each of 3-aminopyrrolidine and trimesic acid for cation and anion analysis, respectively) added before CE-TOF MS analyses. All compounds and solvents were purchased from Sigma-Aldrich (St. Louis, MO) and Wako Pure Chemical Industries (Osaka, Japan), respectively.

CE-TOF MS experiments were performed using an Agilent G7100A CE Instrument (Agilent Technologies), an Agilent G6224A TOF LC/MS, an Agilent G1311C 1260 Infinity Quat Pump VL, a G1603A Agilent CE-MS adapter, an Agilent G1607-60002 CE ESI Sprayer II (Agilent Technologies), and G1601BA Agilent ChemStation Ver. B.04.03 software. Separations were performed with the aid of a capillary filled with fused silica (50 mm internal diameter × 100 cm total length) filled with 1 M formic acid (FA) or 20 mM ammonium formate (pH 10.0) as the electrolyte for cation or anion analyses, respectively. The capillary temperature was maintained at 20 °C. Fifteen nanoliters of sample solution was injected under 50 mbar for 15 s. The sample tray was held at <10 °C. Prior to each run, the capillary was flushed with the electrolyte for 5 min. The voltage used for separation was 30 kV. Methanol/water (50%, v/v) containing 0.5 µM reserpine served as the sheath liquid, delivered at 10 µL/min. ESI-TOF MS was conducted in the positive-ion mode for cation analyses and in the negative-ion mode for anion analyses, and the capillary voltage was set to 4 kV. The flow rate of hot (300 °C) dry nitrogen was 10 psig. The fragmentor, skimmer, and Oct1 RF Vpp values were set to 105, 65, and 750 V, respectively, for cation analysis and to 100, 60, and 750 V, respectively, for anion analysis. Automatic recalibration of each spectrum was performed using the masses of reference standards. The methanol dimer ion ([2 M + H]⁺, m/z = 65.0597) and reserpine ion ([M + H]⁺, m/z = 609.2806) (cation analyses) or the FA dimer ion ([2M–H]^-, m/z = 91.0037) and reserpine ion ([M–H]^-, m/z = 607.2661) (anion analyses) afforded the lock masses allowing precise mass measurements. Mass data were acquired at 1.5 cycles/s over the 50–1,000 m/z range. Further experimental information has been presented elsewhere^38,39. The raw CE-TOF MS data were converted, and the peaks were automatically identified, aligned, and annotated, using our in-house software (“Masterhands”)⁴⁰. Suc levels in 3-day-old etiolated seedlings were measured as described previously¹³. The two-tailed Student’s t-test was used for statistical evaluations. Principle component analysis (PCA) was performed as described previously⁴¹.

Structural sensitivity analysis

Structural sensitivity analysis is a mathematical method to determine responses of steady state concentrations and fluxes in chemical reaction networks to the perturbation of each of reaction rate parameters from structure of networks alone^28,29. In the following, we label chemicals by m (m = 1, …, M) and reactions by i (i = 1, …, R). In general, the state of a chemical reaction system is specified by the concentrations x_m (t), which obey the following differential equations

$$\frac{d{x}_{m}}{dt}=\sum _{i=1}^{R}{\nu }_{mi}{W}_{i}({k}_{i};x)$$

Here, the matrix v is called a stoichiometric matrix. W_i is called a flux, which depends on metabolite concentrations and also on a reaction rate parameter k_i, which corresponds to amount/activity of enzyme mediating the reaction. We do not assume specific forms for the flux functions, but assume that each W_i is an increasing function of its substrate concentration:

$\frac{\partial {W}_{i}}{\partial {x}_{m}} > 0$ if x_m is a substrate of reaction i,

$\frac{\partial {W}_{i}}{\partial {x}_{m}}=0$ otherwise.

Below, we abbreviate and emphasize nonzero $\frac{\partial {W}_{i}}{\partial {x}_{m}}$ as r_im.

In this framework, enzyme knockdown of the jth reaction corresponds to changing the reaction coefficient as ${k}_{j}\to {k}_{j}+\delta {k}_{j}$. We assume steady state of this system both before knockdown and after knockdown leading the following condition:

$$\sum _{i=1}^{R}{\nu }_{mi}{\delta }_{j}{W}_{i}=\sum _{i=1}^{R}{\nu }_{mi}(\frac{\partial {W}_{i}}{\partial {k}_{j}}+\sum _{m^{\prime} =1}^{M}\frac{\partial {W}_{i}}{\partial {x}_{m^{\prime} }}\frac{\partial {x}_{m^{\prime} }}{\partial {k}_{j}})\delta {k}_{j}=0$$

Here ${\delta }_{j}\overrightarrow{W}$ is the flux change induced by the parameter change, which is also written as

$${\delta }_{j}\overrightarrow{W}=\sum _{n=1}^{{N}_{k}}{\overrightarrow{c}}^{n}{\delta }_{j}{\mu }^{n}\cdot $$

Here, $\{{\vec{c}}^{1},\ldots ,{\vec{c}}^{{N}_{k}}\}$ is a basis of the right-kernel space of the stoichiometric matrix v.

As shown in^28,29, the metabolite concentration change ${\delta }_{j}\overrightarrow{x}=\frac{\partial \overrightarrow{x}}{\partial {k}_{j}}\delta {k}_{j}$ and flux change ${\delta }_{j}\overrightarrow{W}$ at a steady state under the perturbation ${k}_{j}\to {k}_{j}+\delta {k}_{j}$ are given from network structure only. From a linear algebra derivation, we have a systematic method to determine response of each chemical to perturbation of each reaction rate in a system at the same time:

$$(\frac{{\delta }_{1}\overrightarrow{x}\cdots {\delta }_{R}\overrightarrow{x}}{{\delta }_{1}\overrightarrow{\mu }\cdots {\delta }_{R}\overrightarrow{\mu }})=-\,{{\bf{A}}}^{-1}diag.(\frac{\partial {W}_{1}}{\partial {k}_{1}}\delta {k}_{1},\cdots ,\frac{\partial {W}_{R}}{\partial {k}_{R}}\delta {k}_{R})\equiv {\bf{S}}\,diag.(\frac{\partial {W}_{1}}{\partial {k}_{1}}\delta {k}_{1},\cdots ,\frac{\partial {W}_{R}}{\partial {k}_{R}}\delta {k}_{R}),$$

where the square matrix A is given as

$${\bf{A}}=(\frac{\partial {W}_{i}}{\partial {x}_{m}}|-\,{\overrightarrow{c}}^{1},\cdots ,-{\overrightarrow{c}}^{{N}_{k}})\cdot $$

The matrix ${\bf{S}}\equiv -\,{{\bf{A}}}^{-1}\,\,$is called the sensitivity matrix. The flux change is obtained through the following equation

$$({\delta }_{1}\overrightarrow{W}\cdots {\delta }_{R}\overrightarrow{W})=({\overrightarrow{c}}^{1}\cdots {\overrightarrow{c}}^{{N}_{k}})({\delta }_{1}\,\overrightarrow{\eta }\cdots {\delta }_{R}\,\overrightarrow{\eta })$$

Note that distribution of nonzero entries in the matrix A reflects structure of reaction network. We determine qualitative response of each chemical and flux from distribution of nonzero entries in the matrix A only. This implies that our theory depends only on the structure of reaction network.

The metabolite pathway for Suc production in plant (Fig. 3d) consists of the following 15 reactions:

1.
F1,6P → F6P + PPi
2.
F6P + PPi → F1,6P
3.
F6P → G6P
4.
G6P → F6P
5.
G6P → G1P
6.
G1P → G6P
7.
G1P → PPi + UDPG
8.
PPi + UDPG → G1P
9.
UDPG + F6P → S6P
10.
S6P → UDPG + F6P
11.
S6P → Suc
12.
Suc → S6P
13.
PPi → ϕ (degradation)
14.
Suc → ϕ (outflow)
15.
ϕ → F1,6P (inflow)

The stoichiometry matrix v is given by

$$\nu =(\begin{array}{ccccccccccccccc}-\,1 & 1 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 1\\ 1 & -\,1 & -\,1 & 1 & 0 & 0 & 0 & 0 & -\,1 & 1 & 0 & 0 & 0 & 0 & 0\\ 1 & -\,1 & 0 & 0 & 0 & 0 & 1 & -\,1 & 0 & 0 & 0 & 0 & -\,1 & 0 & 0\\ 0 & 0 & 1 & -\,1 & -\,1 & 1 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0\\ 0 & 0 & 0 & 0 & 1 & -\,1 & -\,1 & 1 & 0 & 0 & 0 & 0 & 0 & 0 & 0\\ 0 & 0 & 0 & 0 & 0 & 0 & 1 & -\,1 & -\,1 & 1 & 0 & 0 & 0 & 0 & 0\\ 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 1 & -\,1 & -\,1 & 1 & 0 & 0 & 0\\ 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 1 & -\,1 & 0 & -\,1 & 0\end{array}),$$

where the raw indices correspond to {F1,6P; F6P; PPi; G6P; G1P; UDPG; S6P; Suc}.

The matrix A is computed as

$${\bf{A}}=(\begin{array}{ccccccccccccccc}{r}_{1,{\rm{F}}1,6{\rm{P}}} & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 2 & 0 & 0 & 0 & 0 & 0 & 1\\ 0 & {r}_{{\rm{2}},{\rm{F6P}}} & {r}_{2,{\rm{PPi}}} & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 1\\ 0 & {r}_{3,{\rm{F6P}}} & 0 & 0 & 0 & 0 & 0 & 0 & 1 & 0 & 0 & 0 & 0 & 1 & 0\\ 0 & 0 & 0 & {r}_{4,{\rm{G}}6{\rm{P}}} & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 1 & 0\\ 0 & 0 & 0 & {r}_{5,{\rm{G}}6{\rm{P}}} & 0 & 0 & 0 & 0 & 1 & 0 & 0 & 0 & 1 & 0 & 0\\ 0 & 0 & 0 & 0 & {r}_{6,{\rm{G}}1{\rm{P}}} & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 1 & 0 & 0\\ 0 & 0 & 0 & 0 & {r}_{7,{\rm{G}}1{\rm{P}}} & 0 & 0 & 0 & 1 & 0 & 0 & 1 & 0 & 0 & 0\\ 0 & 0 & {r}_{8,{\rm{PPi}}} & 0 & 0 & {r}_{8,{\rm{UDPG}}} & 0 & 0 & 0 & 0 & 0 & 1 & 0 & 0 & 0\\ 0 & {r}_{9,{\rm{F6P}}} & 0 & 0 & 0 & {r}_{9,{\rm{UDPG}}} & 0 & 0 & 1 & 0 & 1 & 0 & 0 & 0 & 0\\ 0 & 0 & 0 & 0 & 0 & 0 & {r}_{10,{\rm{S}}6{\rm{P}}} & 0 & 0 & 0 & 1 & 0 & 0 & 0 & 0\\ 0 & 0 & 0 & 0 & 0 & 0 & {r}_{11,{\rm{S}}6{\rm{P}}} & 0 & 1 & 1 & 0 & 0 & 0 & 0 & 0\\ 0 & 0 & 0 & 0 & 0 & 0 & 0 & {r}_{12,{\rm{Suc}}} & 0 & 1 & 0 & 0 & 0 & 0 & 0\\ 0 & 0 & {r}_{13,{\rm{PPi}}} & 0 & 0 & 0 & 0 & 0 & 3 & 0 & 0 & 0 & 0 & 0 & 0\\ 0 & 0 & 0 & 0 & 0 & 0 & 0 & {r}_{14,{\rm{Suc}}} & 1 & 0 & 0 & 0 & 0 & 0 & 0\\ 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 2 & 0 & 0 & 0 & 0 & 0 & 0\end{array})$$

By inverting the matrix A, the signs of the entries of S are determined as

$${\bf{S}}=(\begin{array}{ccccccccccccccc}- & + & - & + & - & + & - & + & - & + & - & + & - & - & +\\ 0 & 0 & - & + & - & + & - & + & - & + & - & + & - & - & +\\ 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & - & 0 & +\\ 0 & 0 & + & - & - & + & - & + & - & + & - & + & - & - & \pm \\ 0 & 0 & + & - & + & - & - & + & - & + & - & + & - & - & \pm \\ 0 & 0 & + & - & + & - & + & - & - & + & - & + & + & - & \pm \\ 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & - & + & 0 & - & +\\ 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & - & +\\ 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & -\\ 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & - & 0 & + & -\\ 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & - & + & - & 0 & + & -\\ 0 & 0 & - & + & - & + & - & - & + & - & + & - & + & + & \pm \\ 0 & 0 & - & + & - & - & + & - & + & - & + & - & + & + & \pm \\ 0 & 0 & - & - & + & - & + & - & + & - & + & - & + & + & \pm \\ 0 & - & + & - & + & - & + & - & + & - & + & - & + & + & -\end{array}),$$

where +, − represent qualitative responses to perturbations. The symbol ± means that the sign depends on quantitative values of r_im. The disruption of H⁺-PPase corresponds to the perturbation ${k}_{13}\to {k}_{13}+\delta {k}_{13}$ with $\,\delta {k}_{13} < 0$. The response presented in Fig. 3d is obtained by reversing the signs of the 13th column of the matrix S.

Data Availability

All data generated or analysed during this study are included in this published article (and its Supplementary Information files).

Change history

21 May 2019
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

References

Cori, G. T., Ochoa, S., Slein, M. W. & Cori, C. F. The metabolism of fructose in liver; isolation of fructose-1-phosphate and inorganic pyrophosphate. Biochim. Biophys. Acta 7, 304–317 (1951).
Article CAS Google Scholar
Kornberg, A. The participation of inorganic pyrophosphate in the reversible enzymatic synthesis of diphosphopyridine nucleotide. J. Biol. Chem. 176, 1475–1476 (1948).
CAS PubMed Google Scholar
Kornberg, A. Pyrophosphorylases and phosphorylases in biosynthetic reactions. Adv. Enzymol. 18, 191–240 (1957).
CAS Google Scholar
Kornberg, A. On the metabolic significance of phosphorolytic and pyrophosphorolytic reactions. In: Kasha, H., Pullman, B. (eds) Horizons in biochemistry. Academic Press, New York, pp 251–264 (1962).
Biochemistry and molecular biology of plants. 1st Edition (eds Buchanan, B. B., Gruissem, W. & Jones, R. L., 2000).
Maeshima, M. Vacuolar H⁺-pyrophosphatase. Biochim. Biophys. Acta 1465, 37–51 (2000).
Article CAS Google Scholar
Heinonen, J. K. Biological role of inorganic pyrophosphate. Kluwer Academic Publishers, Boston Dordrecht London (2001).
Book Google Scholar
Martinoia, E., Maeshima, M. & Neuhaus, H. E. Vacuolar transporters and their essential role in plant metabolism. J. Exp. Bot. 58, 83–102 (2007).
Article CAS Google Scholar
Ferjani, A., Segami, S., Asaoka, M. & Maeshima, M. Regulation of PPi levels through the vacuolar membrane H⁺-pyrophosphatase. Lüttge, U. et al. (eds), Springer-Verlag Berlin Heidelberg. Progress in Botany 75, 145–165 (2014).
Chen., J. et al. Pyrophosphatase is essential for growth of Escherichia coli. J. Bacteriol. 172, 5686–5689 (1990).
Article CAS Google Scholar
Lundin, M., Baltscheffsky, H. & Ronne, H. Yeast PPA2 gene encodes a mitochondrial inorganic pyrophosphatase that is essential for mitochondrial function. J. Biol. Chem. 266, 12168–12172 (1991).
CAS PubMed Google Scholar
Ko, K. M., Lee, W., Yu, J. R. & Ahnn, J. PYP-1, inorganic pyrophosphatase, is required for larval development and intestinal function in C. elegans. FEBS Lett. 581, 5445–5453 (2007).
Article CAS Google Scholar
Ferjani, A. et al. Keep an eye on PPi: the vacuolar-type H⁺-pyrophosphatase regulates postgerminative development in Arabidopsis. Plant Cell 23, 2895–2908 (2011).
Article CAS Google Scholar
Gutiérrez-Luna, F. M., Navarro de la Sancha, E., Valencia-Turcotte, L. G., Vázquez-Santana, S. & Rodríguez-Sotres, R. Evidence for a non-overlapping subcellular localization of the family I isoforms of soluble inorganic pyrophosphatase in Arabidopsis thaliana. Plant Sci. 253, 229–242 (2016).
Article Google Scholar
Segami, S. et al. Vacuolar H⁺-pyrophosphatase and cytosolic soluble pyrophosphatases cooperatively regulate pyrophosphate levels in Arabidopsis thaliana. Plant Cell 30, 1040–1061 (2018).
Article CAS Google Scholar
Weiner, H., Stitt, M. & Heldt, H. W. Subcellular compartmentation of pyrophosphate and alkaline pyrophosphatase in leaves. Biochim. Biophys. Acta 893, 13–21 (1987).
Article CAS Google Scholar
Wang, X. et al. Genetic variation in ZmVPP1 contributes to drought tolerance in maize seedlings. Nat. Genet. 48, 1233–1241 (2016).
Article CAS Google Scholar
Schilling, R. K., Tester, M., Marschner, P., Plett, D. C. & Roy, S. J. AVP1: one protein, many roles. Trends Plant Sci. 22, 154–162 (2017).
Article CAS Google Scholar
Ferjani, A., Horiguchi, G., Yano, S. & Tsukaya, H. Analysis of leaf development in fugu mutants of Arabidopsis reveals three compensation modes that modulate cell expansion in determinate organs. Plant Physiol. 144, 988–999 (2007).
Article CAS Google Scholar
Fukuda, M. et al. Lack of H⁺-pyrophosphatase prompts developmental damage in Arabidopsis leaves on ammonia-free culture medium. Front. Plant Sci. 7, 819 (2016).
Article Google Scholar
Asaoka, M., Segami, S., Ferjani, A. & Maeshima, M. Contribution of PPi-hydrolyzing function of vacuolar H⁺-pyrophosphatase in vegetative growth of Arabidopsis: Evidenced by expression of uncoupling mutated enzymes. Front. Plant Sci. 7, 415 (2016).
Article Google Scholar
Quick, P., Neuhaus, H. E., Feil, R. & Stitt, M. Fluoride leads to an increase of inorganic pyrophosphate and an inhibition of photosynthetic sucrose synthesis in spinach leaves. Biochim. Biophys. Acta 973, 263–271 (1989).
Article CAS Google Scholar
Graham, I. A. Seed storage oil mobilization. Annu. Rev. Plant Biol. 59, 115–142 (2008).
Article CAS Google Scholar
Eastmond, P. J. et al. Postgerminative growth and lipid catabolism in oilseeds lacking the glyoxylate cycle. Proc. Natl. Acad. Sci. USA 97, 5669–5674 (2000).
Article ADS CAS Google Scholar
Cornah, J. E., Germain, V., Ward, J. L., Beale, M. H. & Smith, S. M. Lipid utilization, gluconeogenesis, and seedling growth in Arabidopsis mutants lacking the glyoxylate cycle enzyme malate synthase. J. Biol. Chem. 279, 42916–42923 (2004).
Article CAS Google Scholar
Penfield, S. et al. Reserve mobilization in the Arabidopsis endosperm fuels hypocotyl elongation in the dark, is independent of abscisic acid, and requires PHOSPHOENOLPYRUVATE CARBOXYKINASE1. Plant Cell 16, 2705–2718 (2004).
Article CAS Google Scholar
Takahashi, K. et al. Compensated cell enlargement in fugu5 is specifically triggered by lowered sucrose production from seed storage lipids. Plant Cell Physiol. 58, 668–678 (2017).
Article CAS Google Scholar
Mochizuki, A. & Fiedler, B. Sensitivity of chemical reaction networks: a structural approach. 1. Examples and the carbon metabolic network. J. Theor. Biol. 367, 189–202 (2015).
Article MathSciNet CAS Google Scholar
Okada, T. & Mochizuki, A. Law of localization in chemical reaction networks. Phys. Rev. Lett. 117, 048101 (2016).
Article ADS Google Scholar
Hajirezaei, M. et al. Transgenic potato plants with strongly decreased expression of pyrophosphate:fructose-6-phosphate phosphotransferase show no visible phenotype and only minor changes in metabolic fluxes in their tubers. Planta 192, 16–30 (1994).
CAS Google Scholar
Kleczkowski, L. A. Glucose activation and metabolism through UDP-glucose pyrophosphorylase in plants. Phytochem. 37, 1507–1515 (1994).
Article CAS Google Scholar
Chen, R. et al. Rice UDP-glucose pyrophosphorylase1 is essential for pollen callose deposition and its cosuppression results in a new type of thermosensitive genic male sterility. Plant Cell 19, 847–861 (2007).
Article CAS Google Scholar
Turnquist, R. L. & Hansen, R. G. In “The enzymes”, eds. Boyer, P. D., Academic Press, Inc., New York and London, 8A, pp. 51–71 (1973).
Koo, H. M., Yim, S. W., Lee, C. S., Pyun, Y. R. & Kim, Y. S. Cloning, sequencing, and expression of UDP-glucose pyrophosphorylase gene from Acetobacter xylinum BRC5. Biosci. Biotechnol. Biochem. 64, 523–529 (2000).
Article CAS Google Scholar
Park, J. I. et al. UDP-glucose pyrophosphorylase is rate limiting in vegetative and reproductive phases in Arabidopsis thaliana. Plant Cell Physiol. 51, 981–996 (2010).
Article CAS Google Scholar
Okazaki, Y. et al. A chloroplastic UDP-glucose pyrophosphorylase from Arabidopsis is the committed enzyme for the first step of sulfolipid biosynthesis. Plant Cell 21, 892–909 (2009).
Article CAS Google Scholar
Murashige, T. & Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497 (1962).
Article CAS Google Scholar
Oikawa, A. et al. Effects of freeze-drying of samples on metabolite levels in metabolome analyses. J. Sep. Sci. 34, 3561–3567 (2011).
Article CAS Google Scholar
Oikawa, A., Fujita, N., Horie, R., Saito, K. & Tawaraya, K. Solid-phase extraction for metabolomics analysis of high-salinity samples by capillary electrophoresis-mass spectrometry. J. Sep. Sci. 34, 1063–1068 (2011).
Article CAS Google Scholar
Sugimoto, M., Wong, D. T., Hirayama, A., Soga, T. & Tomita, M. Capillary electrophoresis mass spectrometry-based saliva metabolomics identified oral, breast and pancreatic cancer-specific profiles. Metabolomics 6, 78–95 (2010).
Article CAS Google Scholar
Xia, J., Sinelnikov, I., Han, B. & Wishart, D. S. MetaboAnalyst 3.0 - making metabolomics more meaningful. Nucl. Acids Res. 43, W251–257 (2015).
Article CAS Google Scholar

Download references

Acknowledgements

We thank Mr. Ryosuke Sasaki for technical support with the CE-TOF MS analyses. This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan [Grant-in-Aid for Encouragement of Young Scientists (B) (21770036 to A.F.); Grant-in-Aid for Scientific Research (B) (16H04803 to A.F.); Grant-in-Aid for Scientific Research on Innovative Areas (25113002 to A.F. and H.T.; 26113506 to M.M.; 25113010 to M.Y.H., A.M, and A.O.)]; Grant-in-Aid for Scientific Research (A) (26252011 to M.M.); BIO-NEXT project from Okazaki Institute for Integrative Bioscience (K.K. and H.T); CREST program (JPMJCR13W6 to A.M.) of the Japan Science and Technology Agency (JST); and The Naito Foundation.

Author information

Mariko Asaoka & Hirokazu Tsukaya
Present address: Exploratory Research Center on Life and Living Systems (ExCELLS), Okazaki, Aichi, 444-8787, Japan
Kensuke Kawade
Present address: Department of Biology, Tokyo Gakugei University, Nukui-Kita 4-1-1, Koganei, Tokyo, 184-8501, Japan

Authors and Affiliations

Department of Biology, Tokyo Gakugei University, Koganei, Tokyo, 184-8501, Japan
Ali Ferjani
Okazaki Institute for Integrative Bioscience, Okazaki, Aichi, 444-8787, Japan
Kensuke Kawade & Hirokazu Tsukaya
National Institute for Basic Biology, Okazaki, Aichi, 444-8585, Japan
Kensuke Kawade
Department of Basic Biology, School of Life Science, Graduate University for Advanced Studies, Okazaki, Aichi, 444-8585, Japan
Kensuke Kawade
RIKEN Center for Sustainable Resource Science, Yokohama, 230-0045, Japan
Kensuke Kawade, Akira Oikawa, Masami Yokota Hirai & Kazuki Saito
Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601, Japan
Mariko Asaoka & Masayoshi Maeshima
Faculty of Agriculture, Yamagata University, Tsuruoka, 997-8555, Japan
Akira Oikawa
Theoretical Biology Laboratory, RIKEN, Wako, 351-0198, Japan
Takashi Okada & Atsushi Mochizuki
Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, 263-8522, Japan
Kazuki Saito
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, 113-0033, Japan
Hirokazu Tsukaya
Exploratory Research Center on Life and Living Systems (ExCELLS), Okazaki, Aichi, 444-8787, Japan
Kensuke Kawade
Department of Biology, Tokyo Gakugei University, Nukui-Kita 4-1-1, Koganei, Tokyo, 184-8501, Japan
Mariko Asaoka

Authors

Ali Ferjani
View author publications
You can also search for this author in PubMed Google Scholar
Kensuke Kawade
View author publications
You can also search for this author in PubMed Google Scholar
Mariko Asaoka
View author publications
You can also search for this author in PubMed Google Scholar
Akira Oikawa
View author publications
You can also search for this author in PubMed Google Scholar
Takashi Okada
View author publications
You can also search for this author in PubMed Google Scholar
Atsushi Mochizuki
View author publications
You can also search for this author in PubMed Google Scholar
Masayoshi Maeshima
View author publications
You can also search for this author in PubMed Google Scholar
Masami Yokota Hirai
View author publications
You can also search for this author in PubMed Google Scholar
Kazuki Saito
View author publications
You can also search for this author in PubMed Google Scholar
Hirokazu Tsukaya
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

A.F. conceived the project, designed the study, performed the experiments and analyzed the data. K.K. and A.O. performed experiments and CE-TOF MS analyses. T.O. and A.M. performed the structural localization analyses. M.Y.H., M.M., K.S. and H.T. directed the study. A.F., A.M., T.O. and M.A. wrote the paper with input from co-authors. Ali Ferjani is responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions.

Corresponding authors

Correspondence to Ali Ferjani or Atsushi Mochizuki.

Ethics declarations

Competing Interests

The authors declare no competing interests.

Additional information

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Supplementary Information

Supplementary Table 1

Supplementary Table 2

Supplementary Table 3

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Ferjani, A., Kawade, K., Asaoka, M. et al. Pyrophosphate inhibits gluconeogenesis by restricting UDP-glucose formation in vivo. Sci Rep 8, 14696 (2018). https://doi.org/10.1038/s41598-018-32894-1

Download citation

Received: 26 March 2018
Accepted: 18 September 2018
Published: 02 October 2018
DOI: https://doi.org/10.1038/s41598-018-32894-1

Keywords

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.