ppGpp Research Articles

Metabolomic pathway regulation to achieve optimal control of inorganic carbon in anammox process

The significance of inorganic carbon (IC) for anaerobic ammonium oxidation (anammox) bacteria has been verified. However, the regulation of metabolic pathways under IC stress is not clear, limiting the optimization of IC supply. In this study, the regulatory pathways at IC concentration of 5–150 mg/L were explored to achieve optimal control of IC. The results show that the changes of metabolic pathway under IC stress determined anammox characteristics. At IC concentration of 5 mg/L, the anammox activity distinctly decreased due to the guanosine tetraphosphate (ppGpp) -mediated regulation under IC limitation. With less than 15 mg/L of IC, the decrease of carbon fixation limited the biosynthesis of gluconeogenesis and amino acids, causing the decline of extracellular polymeric substance synthesis. With more than 50 mg/L of IC, the improvement of purine and pyrimidine metabolism enhanced the electron transport capacity and growth potential of anammox bacteria. This study provides metabolic insights into IC influence on anammox consortia and a novel method of IC concentration optimization using metabolomics analysis.

Integrative biology of persister cell formation: molecular circuitry, phenotypic diversification and fitness effects.

Microbial populations often contain persister cells, which reduce the extinction risk upon sudden stresses. Persister cell formation is deeply intertwined with physiology. Due to this complexity, it cannot be satisfactorily understood by focusing only on mechanistic, physiological or evolutionary aspects. In this review, we take an integrative biology perspective to identify common principles of persister cell formation, which might be applicable across evolutionary-distinct microbes. Persister cells probably evolved to cope with a fundamental trade-off between cellular stress and growth tasks, as any biosynthetic resource investment in growth-supporting proteins is at the expense of stress tasks and vice versa. Natural selection probably favours persister cell subpopulation formation over a single-phenotype strategy, where each cell is prepared for growth and stress to a suboptimal extent, since persister cells can withstand harsher environments and their coexistence with growing cells leads to a higher fitness. The formation of coexisting phenotypes requires bistable molecular circuitry. Bistability probably emerges from growth-modulated, positive feedback loops in the cell's growth versus stress control network, involving interactions between sigma factors, guanosine pentaphosphate and toxin–antitoxin (TA) systems. We conclude that persister cell formation is most likely a response to a sudden reduction in growth rate, which can be achieved by antibiotic addition, nutrient starvation, sudden stresses, nutrient transitions or activation of a TA system.

An expanded whole-cell model of E. coli links cellular physiology with mechanisms of growth rate control

Growth and environmental responses are essential for living organisms to survive and adapt to constantly changing environments. In order to simulate new conditions and capture dynamic responses to environmental shifts in a developing whole-cell model of E. coli, we incorporated additional regulation, including dynamics of the global regulator guanosine tetraphosphate (ppGpp), along with dynamics of amino acid biosynthesis and translation. With the model, we show that under perturbed ppGpp conditions, small molecule feedback inhibition pathways, in addition to regulation of expression, play a role in ppGpp regulation of growth. We also found that simulations with dysregulated amino acid synthesis pathways provide average amino acid concentration predictions that are comparable to experimental results but on the single-cell level, concentrations unexpectedly show regular fluctuations. Additionally, during both an upshift and downshift in nutrient availability, the simulated cell responds similarly with a transient increase in the mRNA:rRNA ratio. This additional simulation functionality should support a variety of new applications and expansions of the E. coli Whole-Cell Modeling Project.

Guanosine tetraphosphate (ppGpp) accumulation inhibits chloroplast gene expression and promotes super grana formation in the moss Physcomitrium (Physcomitrella) patens.

The nucleotides guanosine tetraphosphate and pentaphosphate (or (p)ppGpp) are implicated in the regulation of chloroplast function in plants. (p)ppGpp signalling is best understood in the model vascular plant Arabidopsis thaliana in which it acts to regulate plastid gene expression to influence photosynthesis, plant development and immunity. However, little information is known about the conservation or diversity of (p)ppGpp signalling in other land plants. We studied the function of ppGpp in the moss Physcomitrium (previously Physcomitrella) patens using an inducible system for triggering ppGpp accumulation. We used this approach to investigate the effects of ppGpp on chloroplast function, photosynthesis and growth. We demonstrate that ppGpp accumulation causes a dramatic drop in photosynthetic capacity by inhibiting chloroplast gene expression. This was accompanied by the unexpected reorganisation of the thylakoid system into super grana. Surprisingly, these changes did not affect gametophore growth, suggesting that bryophytes and vascular plants may have different tolerances to defects in photosynthesis. Our findings point to the existence of both highly conserved and more specific targets of (p)ppGpp signalling in the land plants that may reflect different growth strategies.

Adenosine Awakens Metabolism to Enhance Growth-Independent Killing of Tolerant and Persister Bacteria across Multiple Classes of Antibiotics.

ABSTRACTMetabolic and growth arrest are primary drivers of antibiotic tolerance and persistence in clinically diverse bacterial pathogens. We recently showed that adenosine (ADO) suppresses bacterial growth under nutrient-limiting conditions. In the current study, we show that despite the growth-suppressive effect of ADO, extracellular ADO enhances antibiotic killing in both Gram-negative and Gram-positive bacteria by up to 5 orders of magnitude. The ADO-potentiated antibiotic activity is dependent on purine salvage and is paralleled with a suppression of guanosine tetraphosphate synthesis and the massive accumulation of ATP and GTP. These changes in nucleoside phosphates coincide with transient increases in rRNA transcription and proton motive force. The potentiation of antibiotic killing by ADO is manifested against bacteria grown under both aerobic and anaerobic conditions, and it is exhibited even in the absence of alternative electron acceptors such as nitrate. ADO potentiates antibiotic killing by generating proton motive force and can occur independently of an ATP synthase. Bacteria treated with an uncoupler of oxidative phosphorylation and NADH dehydrogenase-deficient bacteria are refractory to the ADO-potentiated killing, suggesting that the metabolic awakening induced by this nucleoside is intrinsically dependent on an energized membrane. In conclusion, ADO represents a novel example of metabolite-driven but growth-independent means to reverse antibiotic tolerance. Our investigations identify the purine salvage pathway as a potential target for the development of therapeutics that may improve infection clearance while reducing the emergence of antibiotic resistance.

Regulation of nitrogen starvation responses by the alarmone (p)ppGpp in rice

Nitrogen is an essential macronutrient for all living organisms and is critical for crop productivity and quality. In higher plants, inorganic nitrogen is absorbed through roots and then assimilated into amino acids by the highly conserved glutamine synthetase/glutamine:2-oxoglutarate aminotransferase (GS/GOGAT) cycle. How nitrogen metabolism and nitrogen starvation responses of plants are regulated remains largely unknown. Previous studies revealed that mutations in the rice ABNORMAL CYTOKININ RESPONSE1 (ABC1) gene encoding Fd-GOGAT cause a typical nitrogen deficiency syndrome. Here, we show that ARE2 (for ABC1 REPRESSOR2) is a key regulator of nitrogen starvation responses in rice. The are2 mutations partially rescue the nitrogen-deficient phenotype of abc1 and the are2 mutants show enhanced tolerance to nitrogen deficiency, suggesting that ARE2 genetically interacts with ABC1/Fd-GOGAT. ARE2 encodes a chloroplast-localized RelA/SpoT homolog protein that catalyzes the hydrolysis of guanosine pentaphosphate or tetraphosphate (p)ppGpp, an alarmone regulating the stringent response in bacteria under nutritional stress conditions. The are2 mutants accumulate excessive amounts of (p)ppGpp, which correlate with lower levels of photosynthetic proteins and higher amino acid levels. Collectively, these observations suggest that the alarmone (p)ppGpp mediates nitrogen stress responses and may constitute a highly conserved mechanism from bacteria to plants.

A guanosine tetraphosphate (ppGpp) mediated brake on photosynthesis is required for acclimation to nitrogen limitation in Arabidopsis.

Guanosine pentaphosphate and tetraphosphate (together referred to as ppGpp) are hyperphosphorylated nucleotides found in bacteria and the chloroplasts of plants and algae. In plants and algae artificial ppGpp accumulation can inhibit chloroplast gene expression, and influence photosynthesis, nutrient remobilization, growth, and immunity. However, it is so far unknown whether ppGpp is required for abiotic stress acclimation in plants. Here, we demonstrate that ppGpp biosynthesis is necessary for acclimation to nitrogen starvation in Arabidopsis. We show that ppGpp is required for remodeling the photosynthetic electron transport chain to downregulate photosynthetic activity and for protection against oxidative stress. Furthermore, we demonstrate that ppGpp is required for coupling chloroplastic and nuclear gene expression during nitrogen starvation. Altogether, our work indicates that ppGpp is a pivotal regulator of chloroplast activity for stress acclimation in plants.

Identification of a RelA/SpoT Homolog and Its Possible Role in the Accumulation of Astaxanthin in Haematococcus pluvialis.

A RelA/SpoT homolog, HpRSH, was identified in Haematococcus pluvialis. HpRSH was found to catalyze Mg2+-dependent guanosine tetraphosphate (ppGpp) synthesis and Mn2+-dependent ppGpp hydrolysis, respectively. The transcription of HpRSH was significantly upregulated by environmental stresses, such as darkness, high light, nitrogen limitation, and salinity stress. The intracellular ppGpp level was also increased when exposed to these stresses. In addition, the classical initiator of stringent response, serine hydroxamate (SHX), was found to upregulate the transcription of HpRSH and increase the level of ppGpp. Moreover, stringent response induced by SHX or environmental stresses was proven to induce the accumulation of astaxanthin. These results indicated that stringent response regulatory system involved in the regulation of astaxanthin biosynthesis in H. pluvialis. Furthermore, stringent response was unable to induce astaxanthin accumulation under dark condition. This result implied that stringent response may regulate astaxanthin biosynthesis in a light-dependent manner.

PpGpp is a bacterial cell size regulator

Growth and division are central to cell size. Bacteria achieve size homeostasis by dividing when growth has added a constant size since birth, termed the adder principle, by unknown mechanisms.1,2 Growth is well known to be regulated by guanosine tetraphosphate (ppGpp), which controls diverse processes from ribosome production to metabolic enzyme activity and replication initiation and whose absence or excess can induce stress, filamentation, and small growth-arrested cells.3-6 These observations raise unresolved questions about the relation between ppGpp and size homeostasis mechanisms during normal exponential growth. Here, to untangle effects of ppGpp and nutrients, we gained control of cellular ppGpp by inducing the synthesis and hydrolysis enzymes RelA and Mesh1. We found that ppGpp not only exerts control over the growth rate but also over cell division and thus the steady state cell size. In response to changes in ppGpp level, the added size already establishes its new constant value while the growth rate still adjusts, aided by accelerated or delayed divisions. Moreover, the magnitude of the added size and resulting steady-state birth size correlate consistently with the ppGpp level, rather than with the growth rate, which results in cells of different size that grow equally fast. Our findings suggest that ppGpp serves as a key regulator that coordinates cell size and growth control.

Pathophysiology of enteropathogenic Escherichia coli during a host infection.

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea in developing countries. However, sporadic outbreaks caused by this microorganism in developed countries are frequently reported recently. As an important zoonotic pathogen, EPEC is being monitored annually in several countries. Hallmark of EPEC infection is formation of attaching and effacing (A/E) lesions on the small intestine. To establish A/E lesions during a gastrointestinal tract (GIT) infeciton, EPEC must thrive in diverse GIT environments. A variety of stress responses by EPEC have been reported. These responses play significant roles in helping E. coli pass through GIT environments and establishing E. coli infection. Stringent response is one of those responses. It is mediated by guanosine tetraphosphate. Interestingly, previous studies have demonstrated that stringent response is a universal virulence regulatory mechanism present in many bacterial pathogens including EPEC. However, biological signficance of a bacterial stringent response in both EPEC and its interaction with the host during a GIT infection is unclear. It needs to be elucidated to broaden our insight to EPEC pathogenesis. In this review, diverse responses, including stringent response, of EPEC during a GIT infection are discussed to provide a new insight into EPEC pathophysiology in the GIT.

Diverse molecular mechanisms of transcription regulation by the bacterial alarmone ppGpp.

Bacteria must rapidly detect and respond to stressful environmental conditions. Guanosine tetraphosphate (ppGpp) is a universal stress signal that, in most bacteria, drives the reprograming of transcription at a global level. However, recent studies have revealed that the molecular mechanisms utilized by ppGpp to rewire bacterial transcriptomes are unexpectedly diverse. In Proteobacteria, ppGpp regulates the expression of hundreds of genes by directly binding to two sites on RNA polymerase (RNAP), one in combination with the transcription factor, DksA. Conversely, ppGpp indirectly regulates transcription in Firmicutes by controlling GTP levels. In this case, ppGpp inhibits enzymes that salvage and synthesize GTP, which indirectly represses transcription from rRNA and other promoters that use GTP for initiation. More recently, two different mechanisms of transcription regulation involving the direct binding of transcription factors by ppGpp have been described. First, in Francisella tularensis, ppGpp was shown to modulate the formation of a tripartite transcription factor complex that binds RNAP and activates virulence genes. Second, in Firmicutes, ppGpp allosterically regulates the transcription repressor, PurR, which controls purine biosynthesis genes. The diversity in bacterial ppGpp signaling revealed in these studies suggests the likelihood that additional paradigms in ppGpp‐mediated transcription regulation await discovery.

NirD curtails the stringent response by inhibiting RelA activity in Escherichia coli.

Bacteria regulate their metabolism to adapt and survive adverse conditions, in particular to stressful downshifts in nutrient availability. These shifts trigger the so-called stringent response, coordinated by the signaling molecules guanosine tetra and pentaphosphate collectively referred to as (p)ppGpp. In Escherichia coli, accumulation of theses alarmones depends on the (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthetase/hydrolase SpoT. A tight regulation of these intracellular activities is therefore crucial to rapidly adjust the (p)ppGpp levels in response to environmental stresses but also to avoid toxic consequences of (p)ppGpp over-accumulation. In this study, we show that the small protein NirD restrains RelA-dependent accumulation of (p)ppGpp and can inhibit the stringent response in E. coli. Mechanistically, our in vivo and in vitro studies reveal that NirD directly binds the catalytic domains of RelA to balance (p)ppGpp accumulation. Finally, we show that NirD can control RelA activity by directly inhibiting the rate of (p)ppGpp synthesis.

Enhancing recombinant Chaetomium thermophilium Formate Dehydrogenase Expression with CRISPR Technology.

Genetic manipulation of Escherichia coli influences the regulation of bacterial metabolism, which could be useful for the production of different targeted products. The RpoZ gene encodes for the ω subunit of the RNA polymerase (RNAP) and is involved in the regulation of the relA gene pathway. RelA is responsible for the production of guanosine pentaphosphate (ppGpp), which is a major alarmone in the stringent response. Expression of relA is reduced in the early hours of growth of RpoZ mutant E. coli. In the absence of the ω subunit, ppGpp affinity to RNAP is decreased; thus, rpoZ gene deleted E. coli strains show a modified stringent response. We used the E. coli K-12 MG1655 strain that lacks rpoZ (JEN202) to investigate the effect of the modified stringent response on recombinant protein production. However, the absence of the ω subunit results in diminished stability of the RNA polymerase at the promoter site. To avoid this, we used a deactivated CRISPR system that targets the ω subunit to upstream of the promoter site in the expression plasmid. The expression plasmid encodes for Chaetomium thermophilum formate dehydrogenase (CtFDH), a valuable enzyme for cofactor regeneration and CO2 reduction. A higher amount of CtFDH from the soluble fraction was purified from the JEN202 strain compared to the traditional BL21(DE3) method, thus offering a new strategy for batch-based recombinant enzyme production.

Emerging and divergent roles of pyrophosphorylated nucleotides in bacterial physiology and pathogenesis.

Bacteria inhabit diverse environmental niches and consequently must modulate their metabolism to adapt to stress. The nucleotide second messengers guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) (collectively referred to as (p)ppGpp) are essential for survival during nutrient starvation. (p)ppGpp is synthesized by the RelA-SpoT homologue (RSH) protein family and coordinates the control of cellular metabolism through its combined effect on over 50 proteins. While the role of (p)ppGpp has largely been associated with nutrient limitation, recent studies have shown that (p)ppGpp and related nucleotides have a previously underappreciated effect on different aspects of bacterial physiology, such as maintaining cellular homeostasis and regulating bacterial interactions with a host, other bacteria, or phages. (p)ppGpp produced by pathogenic bacteria facilitates the evasion of host defenses such as reactive nitrogen intermediates, acidic pH, and the complement system. Additionally, (p)ppGpp and pyrophosphorylated derivatives of canonical adenosine nucleotides called (p)ppApp are emerging as effectors of bacterial toxin proteins. Here, we review the RSH protein family with a focus on its unconventional roles during host infection and bacterial competition.

Enzymatic properties of Myxococcus xanthus exopolyphosphatases mxPpx1 and mxPpx2

Myxococcus xanthus possesses two exopolyphosphatases, mxPpx1 and mxPpx2, which belong to the family of Ppx/GppA phosphatases; however, their catalytic properties have not been described. mxPpx1 and mxPpx2 contain 311 and 505 amino acid residues, respectively; mxPpx2 has an additional C-terminal region, which corresponds to the metal-dependent HDc phosphohydrolase domain. mxPpx1 mainly hydrolyzed short-chain polyPs (polyP3 and polyP4), whereas mxPpx2 preferred long-chain polyP60–70 and polyP700–1000. mxPpx2 was activated by 25–50 mM KCl, but mxPpx1 did not significantly depend on K+. In addition, mxPpx1 and mxPpx2 showed weak hydrolysis of ATP and GTP in the absence of K+, and mxPpx2 could also hydrolyze guanosine pentaphosphate (pppGpp) in the presence of K+. The exopolyphosphatase activity of mxPpx1 toward polyP3 was inhibited by polyP700–1000 and that of mxPpx2 toward polyP60–70 and polyP700–1000, by pyrophosphate. To clarify the function of the mxPpx2 C-terminal domain, it was fused to mxPpx1 (mxPpx1-2C) and deleted from mxPpx2 (mxPpx2∆C). Compared to wild-type mxPpx2, mxPpx2∆C had significantly reduced exopolyphosphatase activity toward long-chain polyPs (by 90%), whereas that toward polyP3 and polyP4 was much less affected; furthermore, the phosphohydrolase activity toward pppGpp, ATP, and GTP was also decreased (by 30–75%). In contrast, mxPpx1-2C had increased hydrolytic activity compared to mxPpx1. Furthermore, mxPpx2∆C lost the requirement for K+ characteristic for the wild-type enzyme, whereas mxPpx1-2C acquired it. These results suggest that the C-terminal domain of mxPpx2 is necessary for its maximum hydrolytic activity, especially toward long-chain polyPs, and defines mxPpx2 dependency on K+ for activation.

(p)ppGpp-Dependent Regulation of the Nucleotide Hydrolase PpnN Confers Complement Resistance in Salmonella enterica Serovar Typhimurium.

The stringent response is an essential mechanism of metabolic reprogramming during environmental stress that is mediated by the nucleotide alarmones guanosine tetraphosphate and pentaphosphate [(p)ppGpp]. In addition to physiological adaptations, (p)ppGpp also regulates virulence programs in pathogenic bacteria, including Salmonella enterica serovar Typhimurium. S Typhimurium is a common cause of acute gastroenteritis, but it may also spread to systemic tissues, resulting in severe clinical outcomes. During infection, S Typhimurium encounters a broad repertoire of immune defenses that it must evade for successful host infection. Here, we examined the role of the stringent response in S Typhimurium resistance to complement-mediated killing and found that the (p)ppGpp synthetase-hydrolase, SpoT, is required for bacterial survival in human serum. We identified the nucleotide hydrolase, PpnN, as a target of the stringent response that is required to promote bacterial fitness in serum. Using chromatography and mass spectrometry, we show that PpnN hydrolyzes purine and pyrimidine monophosphates to generate free nucleobases and ribose 5'-phosphate, and that this metabolic activity is required for conferring resistance to complement killing. In addition to PpnN, we show that (p)ppGpp is required for the biosynthesis of the very long and long O-antigen in the outer membrane, known to be important for complement resistance. Our results provide new insights into the role of the stringent response in mediating evasion of the innate immune system by pathogenic bacteria.

Guanosine Tetraphosphate Has a Similar Affinity for Each of Its Two Binding Sites on Escherichia coli RNA Polymerase.

During nutrient deprivation, the bacterial cell undergoes a stress response known as the stringent response. This response is characterized by induction of the nucleotide derivative guanosine tetraphosphate (ppGpp) that dramatically modulates the cell’s transcriptome. In Escherichia coli, ppGpp regulates transcription of as many as 750 genes within 5 min of induction by binding directly to RNA polymerase (RNAP) at two sites ~60 Å apart. One proposal for the presence of two sites is that they have different affinities for ppGpp, expanding the dynamic range over which ppGpp acts. We show here, primarily using the Differential Radial Capillary Action of Ligand Assay (DRaCALA), that ppGpp has a similar affinity for each site, contradicting the proposal. Because the ppGpp binding sites are formed by interactions of the β’ subunit of RNAP with two small protein factors, the ω subunit of RNAP which contributes to Site 1 and the transcription factor DksA which contributes to Site 2, variation in the concentrations of ω or DksA potentially could differentially regulate ppGpp occupancy of the two sites. It was shown previously that DksA varies little at different growth rates or growth phases, but little is known about variation of the ω concentration. Therefore, we raised an anti-ω antibody and performed Western blots at different times in growth and during a stringent response. We show here that ω, like DksA, changes little with growth conditions. Together, our data suggest that the two ppGpp binding sites fill in parallel, and occupancy with changing nutritional conditions is determined by variation in the ppGpp concentration, not by variation in ω or DksA.

The stringent response and physiological roles of (pp)pGpp in bacteria.

The stringent response is a stress signalling system mediated by the alarmones guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) in response to nutrient deprivation. Recent research highlights the complexity and broad range of functions that these alarmones control. This Review provides an update on our current understanding of the enzymes involved in ppGpp, pppGpp and guanosine 5'-monophosphate 3'-diphosphate (pGpp) (collectively (pp)pGpp) turnover, including those shown to produce pGpp and its analogue (pp)pApp. We describe the well-known interactions with RNA polymerase as well as a broader range of cellular target pathways controlled by (pp)pGpp, including DNA replication, transcription, nucleotide synthesis, ribosome biogenesis and function, as well as lipid metabolism. Finally, we review the role of ppGpp and pppGpp in bacterial pathogenesis, providing examples of how these nucleotides are involved in regulating many aspects of virulence and chronic infection.

The nucleotide pGpp acts as a third alarmone in Bacillus, with functions distinct from those of (p)ppGpp

The alarmone nucleotides guanosine tetraphosphate and pentaphosphate, commonly referred to as (p)ppGpp, regulate bacterial responses to nutritional and other stresses. There is evidence for potential existence of a third alarmone, guanosine-5′-monophosphate-3′-diphosphate (pGpp), with less-clear functions. Here, we demonstrate the presence of pGpp in bacterial cells, and perform a comprehensive screening to identify proteins that interact respectively with pGpp, ppGpp and pppGpp in Bacillus species. Both ppGpp and pppGpp interact with proteins involved in inhibition of purine nucleotide biosynthesis and with GTPases that control ribosome assembly or activity. By contrast, pGpp interacts with purine biosynthesis proteins but not with the GTPases. In addition, we show that hydrolase NahA (also known as YvcI) efficiently produces pGpp by hydrolyzing (p)ppGpp, thus modulating alarmone composition and function. Deletion of nahA leads to reduction of pGpp levels, increased (p)ppGpp levels, slower growth recovery from nutrient downshift, and loss of competitive fitness. Our results support the existence and physiological relevance of pGpp as a third alarmone, with functions that can be distinct from those of (p)ppGpp.

Quantification of guanosine triphosphate and tetraphosphate in plants and algae using stable isotope-labelled internal standards

Guanosine tetraphosphate (G4P) and guanosine pentaphosphate (G5P) are signalling nucleotides found in bacteria and photosynthetic eukaryotes that are implicated in a wide-range of processes including stress acclimation, developmental transitions and growth control. Measurements of G4P/G5P levels are essential for studying the diverse roles of these nucleotides. However, G4P/G5P quantification is particularly challenging in plants and algae due to lower cellular concentrations, compartmentalization and high metabolic complexity. Despite recent advances the speed and accuracy of G4P quantification in plants and algae can still be improved. Here, we report a new approach for rapid and accurate G4P quantification which relies on the use of synthesized stable isotope-labelled as internal standards. We anticipate that this approach will accelerate research into the function of G4P signaling in plants, algae and other organisms.