Decoding Biosynthetic Pathways in Plants by Pulse-Chase ... - MDPI

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Decoding Biosynthetic Pathways in Plants by Pulse-Chase Strategies Using 13CO2 as a Universal Tracer † Adelbert Bacher, Fan Chen and Wolfgang Eisenreich * Lehrstuhl für Biochemie, Technische Universität München, 85748 Garching, Germany; [email protected] (A.B.); [email protected] (F.C.) * Correspondence: [email protected]; Tel.: +49-89-28913336; Fax: +49-89-28913363 † Dedicated to Dr. Karl-Heinz Michl on the occasion of his 90th birthday. Academic Editor: Peter Meikle Received: 24 May 2016; Accepted: 4 July 2016; Published: 14 July 2016

Abstract: 13 CO2 pulse-chase experiments monitored by high-resolution NMR spectroscopy and mass spectrometry can provide 13 C-isotopologue compositions in biosynthetic products. Experiments with a variety of plant species have documented that the isotopologue profiles generated with 13 CO2 pulse-chase labeling are directly comparable to those that can be generated by the application of [U-13 C6 ]glucose to aseptically growing plants. However, the application of the 13 CO2 labeling technology is not subject to the experimental limitations that one has to take into account for experiments with [U-13 C6 ]glucose and can be applied to plants growing under physiological conditions, even in the field. In practical terms, the results of biosynthetic studies with 13 CO2 consist of the detection of pairs, triples and occasionally quadruples of 13 C atoms that have been jointly contributed to the target metabolite, at an abundance that is well above the stochastic occurrence of such multiples. Notably, the connectivities of jointly transferred 13 C multiples can have undergone modification by skeletal rearrangements that can be diagnosed from the isotopologue data. As shown by the examples presented in this review article, the approach turns out to be powerful in decoding the carbon topology of even complex biosynthetic pathways. Keywords: biosynthesis; metabolism; isotopologue profiling; 13 CO2 ; flux analysis

1. Introduction Isotope incorporation analysis is a time-honored, major tool for the dissection of biosynthetic and metabolic processes. Labeling experiments using 2 H-isotopes were introduced in the 1930s when heavy water, 2 H2 O, became available [1]. Until about 1960, isotope use in biochemistry was then mostly dominated by the radioisotopes 3 H and 14 C [2,3]. Later, a variety of factors have been conducive to a progressive shift to the use of stable isotopes. Notably, stable isotopes involve no health hazards and are exempt from safety regulations. Moreover, dramatic progress in NMR and mass spectrometry instrumentation has greatly enhanced the sensitivity of stable isotope detection [4,5]. The major elemental components of organic matter, i.e., hydrogen, carbon, nitrogen and oxygen, are all present in nature as mixtures of stable isotopes. In natural organic matter, these isotopes show an almost random distribution. Small deviations from randomness arise by isotope fractionation inherent in chemical (including biochemical) reactions [6], but the non-random effects are small, and sensitive techniques are required for their assessment; these aspects are not addressed in the present study. Isotope tracer techniques designed to analyze biosynthetic and other metabolic processes invariably start with an artificially-induced perturbation of natural isotope distribution. In principle, a perturbation can be caused by the introduction of any chemical with an isotope distribution

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at variance from the average isotope composition of natural organic matter. More specifically, the perturbing agent could be universally isotope enriched or enriched at single or multiple discrete sites. Crucially, however, it needs to be able to access the metabolic network of the study organism. Selecting an appropriate tracer, with regard to the structure and labeling pattern of isotope-labeled tracers, depends on numerous boundary conditions, including, but not limited to (i) commercial or synthetic availability; (ii) uptake by the target organism and (iii) the availability (or absence) of a strong working hypothesis suggesting a biosynthetic relationship of the tracer and target product. A brief discussion of the network theory of intermediary metabolism is required in order to gain a rational basis for the choice of tracers under a wide variety of conditions. 2. Metabolic Networks The numbers of open reading frames specified by different genomes are now known to range from a few hundred (in the case of certain microorganisms) to tens of thousands [7]. Although not all translation products serve as enzymes, the numbers of enzymes and, hence, putative enzyme products are likewise in the range of hundreds to many thousands. By interpreting metabolites as nodes that can be connected by enzymes catalyzing the conversion of certain metabolites into other metabolites, the metabolome of any given organism can be described as a network whose complexity depends on the number of enzymes specified by the organism’s genome and by the specific connectivities of each respective node. Unsurprisingly, certain parts of metabolic networks are more closely interconnected than other parts. Notably, central intermediary metabolism (cf. the central metabolome in Figure 1), e.g., dealing with the interconversion of carbohydrates or carboxylic acids, is typically characterized by dense connectivity [8,9]. On the other hand, biosynthetic pathways conducive to structurally complex primary metabolites (such as aliphatic and aromatic amino acids, vitamins) and secondary metabolites (e.g., antibiotics, alkaloids) are more in the nature of “one-way roads” where nodes frequently have only two connections (to the direct precursor and the consecutive intermediary product, respectively). The introduction of a node compound, in isotope-labeled form, into the metabolome of a given organism converts that node into a source for perturbation of the near-equilibrium distribution of isotopes in natural matter. Depending on the number of connections of the given node, the potential division of the global network into modular sections [10,11] and the interconnections between these sections, the perturbation can spread into many or few directions where other nodes could serve as novel sources of the tracer isotope. As a simple case, consider the feeding of 13 C-labeled anthranilic acid to a microorganism that is capable of tryptophan biosynthesis [12] (Figure 1). The labeled tracer will be incorporated, via 3-indolylglycerol phosphate and indole, into the benzenoid ring of tryptophan, into proteins and potentially into all secondary products downstream from tryptophan, such as certain alkaloids; however, the central metabolome can only be accessed after catabolic degradation of the proffered precursor or one of its downstream products. As an example for a more general, but still limited mode of spreading, let us consider the introduction of an isotope-labeled purine base such as hypoxanthine (Figure 1). The perturbant will be able to access the complex network of purine nucleotides by the transfer of a ribose phosphate moiety, thus converting it into IMP, which will then serve as a secondary perturbation source [13]. From that secondary source, the label (and, hence, the perturbation) will be able to spread to a variety of sinks, including the entire purine nucleotide and deoxynucleotide network, and the realm of nucleic acids, but also the realm of purine-derived primary and secondary metabolites such as coenzymes (e.g., flavocoenzymes, folic acid derivatives, the molybdopterin cofactor family), some special tRNA bases, purine-derived antibiotics, to name only the most important [14–16]. Nevertheless, the spread of the perturbation will be far from universal, since the label will need a relatively long time to access the central carbohydrate pools (in fact, this will only be possible after catabolic disruption of the purine ring system that would be conducive to very simple molecules, such as formate and CO2 ).


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3 of 24 of the purine ring system that would be conducive to very simple molecules, such as formate and CO2). A totally totally different different situation situation arises arises when when aa major major nutrient, nutrient, such such as as glucose, glucose, is is proffered proffered in in A isotope-labeled form (Figure 1). After penetration into the cytoplasm, the tracer will immediately isotope-labeled form (Figure 1). After penetration into the cytoplasm, the tracer will immediately start start be diverted all metabolic compartments, andisotope the isotope spread out throughout to be to diverted to allto metabolic compartments, and the labellabel will will spread out throughout the the metabolic network (with intracellular glucose or its phosphoric acid ester, respectively, serving metabolic network (with intracellular glucose or its phosphoric acid ester, respectively, serving as a as a source, virtually all nodes will operate as sinks within a short time period). Hence, the net source, virtually all nodes will operate as sinks within a short time period). Hence, the net transfer of transfer of isotope to all nodes in the network fails to provide meaningful information about isotope to all nodes in the network fails to provide meaningful information about metabolic processes, metabolic processes, unless we make use of the fact that isotope atoms do not travel the metabolic unless we make use of the fact that isotope atoms do not travel the metabolic network alone, but as network alone, but as tightly (covalently) linked associations of atoms. In other words, the carbon tightly (covalently) linked associations of atoms. In other words, the carbon skeletons of metabolites skeletons of metabolites are pieced together as a patchwork assembled from precursor metabolites are pieced together as a patchwork assembled from precursor metabolites or their respective fragments or their respective fragments by the catalytic action of enzymes. by the catalytic action of enzymes.

Figure Principal strategies strategies for for in in vivo vivo labeling labeling experiments. experiments. A:A:Incorporation Incorporationexperiment experimentwith witha Figure 1. 1. Principal ametabolite metabolite(i.e., (i.e.,anthranilate anthranilateininthe theexample) example)biogenetically biogeneticallyclose close to to the the target target metabolite metabolite (i.e., indole (i.e., indole alkaloids alkaloids or or benzoxazinones benzoxazinones in in the the example). example). The The label label can can be be distributed distributed over over aa limited limited part part of of the the metabolic network shown by red arrows. B: Incorporation experiment with a metabolic precursor metabolic network shown by red arrows. B: Incorporation experiment with a metabolic precursor (i.e., hypoxanthine in the example) for a wider range of products (folates, flavines, pterins, DNA/RNA) (i.e., hypoxanthine in the example) for a wider range of products (folates, flavines, pterins, shown by blue arrows. C: Incorporation experiment with a general precursor (i.e., glucose in the DNA/RNA) shown by blue arrows. C: Incorporation experiment with a general precursor (i.e., example) where the label is distributed via the central metabolome of the organism shown by green glucose in the example) where the label is distributed via the central metabolome of the organism arrows. PRPP, 5-phosphoribosyl diphosphate. shown by green arrows. PRPP, 5-phosphoribosyl diphosphate.

As aa specific specific (and (and fairly fairly typical) typical) example, example, consider consider the theapplication application of ofuniversally universally1313C-labeled C-labeled 13 glucose ([UC66]glucose) ]glucose) to to aa biological biological target. Following Following its its incorporation, incorporation, itit may access one or ([U-13C more more catabolic catabolic pathways pathways resulting resulting in in fragmentation, fragmentation, affording compounds compounds carrying one (in the case case 13 13 13 13 of catabolite two or or more C C atoms, atoms, which which are are contributed contributed to the inventory of central catabolite CO22),), two central intermediary metabolites. metabolites. Henceforth, Henceforth,anabolic anabolic processes will start from mixtures of unlabeled processes will start from mixtures of unlabeled and and labeled intermediates of central intermediary metabolism (unlabeled intermediates from labeled intermediates of central intermediary metabolism (unlabeled intermediates from the the 13C-labeled tracer, labeled metabolic inventory inventory that that had had been been established established prior prior to to the the addition addition of of the the 13 labeled intermediates C-labeled their reactions with components of intermediates obtained obtainedby bycatabolism catabolismofofthe the1313 C-labeledtracer tracerand and their reactions with components the unlabeled metabolic inventory). The products resulting from anabolic processes will then appear of the unlabeled metabolic inventory). The products resulting from anabolic processes will then as mosaics with contributions by 13 C-labeled, as well unlabeled building blocks. Following the appear as mosaics with contributions by 13C-labeled, asas well as unlabeled building blocks. Following 13 C atoms13in isolation of individual metabolites from the experimental system, contiguous groups of the isolation of individual metabolites from the experimental system, contiguous groups of C the metabolites under study can study be diagnosed by NMR spectroscopy or by the combined atoms in the metabolites under can be diagnosed by NMR spectroscopy or by theapplication combined of NMR andofMS techniques. application NMR and MS techniques. The interpretation interpretation of of tracer tracer studies studies using using multiply-labeled multiply-labeled central central metabolic metabolic intermediates intermediates typically involves the comparison of the labeling labeling patterns of numerous numerous metabolites metabolites from a given given experiment. Importantly, Importantly, the the available available information information on on the the biosynthetic biosynthetic pathways of primary primary metabolites can be be used usedfor forthe thereconstruction reconstruction labeling patterns of central metabolic pools. ofof thethe labeling patterns of central metabolic pools. For For example, since tryptophan is exclusively biosynthesized via the shikimate pathway, the labeling

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example, since tryptophan is exclusively biosynthesized via the shikimate pathway, the labeling pattern pattern of of tryptophan tryptophan from from aa given given labeling labeling experiment experiment allows allows the the unequivocal unequivocal reconstruction reconstruction (retrodiction) of the labeling pattern of erythrose 4-phosphate (Figure 2). Moreover, (retrodiction) of the labeling pattern of erythrose 4-phosphate (Figure 2). Moreover, the the labeling labeling pattern also informs about the labeling of carbon atoms 1atoms and 2 of phosphate patternofoftryptophan tryptophan also informs about the labeling of carbon 1 the andpentose 2 of the pentose pool and ofpool carbon 2 andatoms 3 of phosphoenol pyruvate. pyruvate. phosphate andatoms of carbon 2 and 3 of phosphoenol

Figure 2. 2. Retrobiosynthetic Retrobiosynthetic dissection dissection of of tryptophan. tryptophan. The is formed formed via via the the Figure The aromatic aromatic amino amino acid acid is shikimate pathway using erythrose 4-phosphate (red labels), phosphoenol pyruvate (green labels), shikimate pathway using erythrose 4-phosphate (red labels), phosphoenol pyruvate (green labels), serine (blue (bluelabels) labels)and andphosphoribosyl phosphoribosyldiphosphate diphosphate(orange (orange labels) building blocks. basis serine labels) asas building blocks. OnOn thethe basis of of detected the detected labeling of tryptophan, the labeling patterns of the precursors are the labeling patternpattern of tryptophan, the labeling patterns of the precursors are reconstructed as reconstructed as shown “retro” arrows. Thebiosynthetically-equivalent colors represent biosynthetically-equivalent modules. shown by “retro” arrows.byThe colors represent modules.

The synopsis of retrodictive information from amino acids affords a detailed description of the The synopsis of retrodictive information from amino acids affords a detailed description of the labeling patterns of central metabolic intermediates (Table 1) that can then also be used as the basis labeling patterns of central metabolic intermediates (Table 1) that can then also be used as the basis for a pattern recognition approach with the aim to identify the building blocks in the biosynthesis of for a pattern recognition approach with the aim to identify the building blocks in the biosynthesis of a metabolite under study. a metabolite under study. Table 1. Reconstruction of the labeling patterns of biosynthetic precursorsa from the 13C-patterns of Table 1. Reconstruction of the labeling patterns of biosynthetic precursorsa from the 13 C-patterns of key amino acids. key amino acids. Amino Amino Acid Acid Ala Ala Arg Arg Arg Arg Asp Asp Glu Glu His His His His IleIle Ile

Precursora Precursora Pyruvate Pyruvate Glu Glu 2 CO CO2 Oxaloacetate Oxaloacetate α-Ketoglutarate α-Ketoglutarate 5-Phosphoribosyl 5-Phosphoribosyl diphosphate diphosphate ATP ATP Thr Thr Pyruvate

Related Upstream Related Upstream Intermediate Intermediate Glyceraldehyde 3-Phosphate, Malate Glyceraldehyde 3-Phosphate, Malate α-Ketoglutarate α-Ketoglutarate Phosphoenolpyruvate, Phosphoenolpyruvate, CO2 CO2 Oxaloacetate, Oxaloacetate, Acetyl-CoA Acetyl-CoA Ribulose Ribulose5-Phosphate 5-Phosphate 2 CO CO 2 Asp Asp Glyceraldehyde 3-Phosphate, Malate

Pathway Pathway Glycolysis, C4-dicarboxylic acid cycle, Non-mevalonate pathway Glycolysis, C4-dicarboxylic acid cycle, Non-mevalonate pathway Citrate cycle Citrate cycle C1-metabolism C1-metabolism Citrate cycle, C1-metabolism Citrate cycle, C1-metabolism Citrate cycle, Mevalonate pathway Citrate cycle, Mevalonate pathway Pentose phosphate pathway, Calvin cycle, Pentose phosphate pathway, C1-metabolism Calvin cycle, C1-metabolism Purinemetabolism, metabolism,C1-metabolism C1-metabolism Purine Citratecycle cycle Citrate Glycolysis, C4-dicarboxylic acid cycle, Non-mevalonate pathway


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Table 1. Cont. Related Upstream Intermediate

Amino Acid

Precursora

Ile

Pyruvate

Leu

Pyruvate

Leu

Acetyl-CoA

Pyruvate, Fatty acids

Lys

Asp

Phe

Erythrose 4-phosphate

Phe

Phosphoenol-pyruvate

Pro Thr

Glu Asp

Trp


Trp

Phosphoenol pyruvate

Oxaloacetate Fructose 6-Phosphate, Ribulose 5-Phosphate 3-Phosphoglycerate, Oxaloacetate α-Ketoglutarate Oxaloacetate Fructose 6-Phosphate, Ribulose 5-Phosphate 3-Phosphoglycerate, Oxaloacetate

Trp

Ser

3-Phosphoglycerate, Gly

Trp

5-Phosphoribosyl diphosphate

Ribulose 5-Phosphate

Tyr


Tyr

Phosphoenol pyruvate

Val

Pyruvate

Glyceraldehyde 3-Phosphate, Malate Glyceraldehyde 3-Phosphate, Malate

Fructose 6-Phosphate, Ribulose 5-Phosphate 3-Phosphoglycerate, Oxaloacetate Glyceraldehyde 3-Phosphate, Malate

Pathway Glycolysis, C4-dicarboxylic acid cycle, Non-mevalonate pathway Glycolysis, C4-dicarboxylic acid cycle, Non-mevalonate pathway Mevalonate pathway, Polyketide metabolism, Fatty acid metabolism Citrate cycle Chorismate pathway, Pentose phosphate pathway, Calvin cycle Chorismate pathway, Glycolysis, Calvin cycle Citrate cycle Citrate cycle Chorismate pathway, Pentose phosphate pathway, Calvin cycle Chorismate pathway, Glycolysis, Calvin cycle Chorismate pathway, Glycolysis, Calvin cycle, C1-metabolism Pentose phosphate pathway, Calvin cycle Chorismate pathway, Pentose phosphate pathway, Calvin cycle Chorismate pathway, Glycolysis, Calvin cycle Glycolysis, C4-dicarboxylic acid cycle, Non-mevalonate pathway

a

These precursors also serve as key intermediates in central metabolic pathways, and, as simple building units, in the biosynthetic pathways of complex natural products. For example, acetyl-CoA or pyruvate/glyceraldehyde phosphate (GAP) are basic precursors in terpene biosynthesis, acetyl-CoA serves in the formation of polyketides, phenylalanine in the biosynthesis of phenylpropanoids, and erythrose 4-phosphate and ribose 5-phosphate in the generation of glycosides).

3. Technical Hurdles for Tracer Studies in Plants Whereas the presented arguments apply, in principle, to studies in all biological kingdoms, specific problems arise with the application of isotope-labeled tracers to plants. With the exception of saprophytic and parasitic species, plants generate their biomass exclusively from CO2 . Pilot experiments with aseptically grown tobacco plants have provided proof of principle for feeding of 13 C-labeled glucose via the root system [17]. However, the availability of aseptically-growing plant specimens is the exception rather than the norm. Thus, conducting tracer experiments with unperturbed plants is not easily feasible. Work-arounds are possible and have been successfully used in numerous cases. Plant cell cultures or plant organ cultures (e.g., embryos or root cultures) growing in vitro enable experiments that are technically similar to work with microorganism [18–20]. Again, the availability of these systems for a given species will be the exception rather than the norm. Moreover, plant cells or organs growing in vitro may or may not be producing specific plant metabolites in quantities that are sufficient for analysis. Nevertheless, important problems in plant biochemistry and physiology could be solved with that approach. Experiments can also be conducted with cut plant segments that are immersed into nutrient solutions containing a tracer compound. However, cut segments are less metabolically prolific than whole plants; moreover, their condition is far from being physiological, is subject to progressive decline with time and, finally, may reflect metabolic pathways and fluxes triggered by the wounding of the


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plant and being absent in the intact plant under standard conditions. In addition, as with cultured plant cells, the metabolic productivity of cut segments may be less than that of intact plants. The single direct carbon source for most plants growing under natural conditions (with the exception of saprophytes and parasites) is of course CO2 . This review article describes examples for tracer experiments with 13 CO2 directed at the elucidation of biosynthetic pathways of plant secondary metabolites. The extensive use of 13 CO2 for a variety of research purposes in general plant physiology (for recent examples, see [21–27]) is not covered in this review. It is important to note that work on biosynthesis depends technically on high resolution NMR of metabolites that have been isolated in pure form. Due to the limited resolution and sensitivity of NMR spectroscopy, biosynthetic studies using 13 CO2 as a tracer have only been rarely used in the past century [28]. In contrast to 13 CO2 -based biosynthetic studies at high NMR resolution, general plant physiology analyses with 13 CO2 can also use solid state NMR techniques enabling the analysis of intact plant tissue, as initiated by work from the research group of Schaefer and others (for specific examples, cf. [29–32]). 4. Biochemistry of 13 CO2 Pulse-Chase Experiments Under conditions of optimal lighting, plants make adjustments for maximum utilization of the incoming light for photosynthesis. When proffered exclusively with 13 CO2 as the carbon source, photosynthesis will then generate 13 C-labeled carbohydrates that can be partially stored away as starch. In an ideal (but hypothetical) experiment, the photosynthetic products would approach universal labeling at a level equivalent to the 13 C abundance of the feeding CO2 , above 99%. However, since growing plants are endowed with an inventory of unlabeled metabolites and sugars (already present in the plants prior to the labeling experiment), the 13 C enrichments of these products will be lower (typically 0.2%–5% 13 C excess in the isolated target compounds) than the enrichment of the proffered 13 CO (i.e., 99% 13 C excess) during the short pulse period of the labeling experiment (typically 1–10 h). 2 When the plants are returned to normal atmosphere for a predetermined chase phase (typically 1–10 days), two different processes will play out: (i) during light periods, the plants under study will now produce new carbohydrates from CO2 with natural 13 C abundance of about 1.1%; (ii) during dark periods, the plants will use stored metabolites as sources of energy and metabolism and will thus mobilize 13 C-labeled metabolites from the pulse period; moreover, they will utilize unlabeled storage material that had been deposited prior the pulse period (quasi-“fossil” storage material), as well as low molecular weight and high molecular weight material generated during the chase period from CO2 with natural (i.e., low) 13 C abundance. The first committed step of carbon fixation in plants is catalyzed by ribulose bisphosphate carboxylase (RUBISCO), which generates two equivalents of 3-phosphoglycerate from one equivalent each of ribulose bisphosphate and CO2 [33]. 3-Phosphoglycerate can be converted into ribulose bisphosphate via the Calvin cycle under regeneration of 3-phosphoglycerate, which serves as the primary CO2 acceptor in the RUBISCO reaction. When a plant is grown in a synthetic atmosphere containing 13 CO2 , the carbon isotope will therefore access the inventory of Calvin cycle intermediates, resulting in a progressive wash-in of 13 C (and concomitant wash-out of 12 C) affecting the 3-phosphoglyerate pool and the whole metabolic network downstream from 3-phosphoglycerate. Thus, the average 13 C abundance of the soluble carbohydrate inventory should asymptotically approach the 13 C abundance of the proffered CO by way of monotonous progression. 2 In a typical pulse/chase experiment, the 13 CO2 -treated plant is subsequently returned to normal atmosphere (with different terminology, the 13 CO2 phase can be interpreted as the perturbation phase and the consecutive phase in normal atmosphere as the relaxation period in a perturbation/relaxation experiment). In the chase period (relaxation period), the plant will now produce additional photosynthate derived from natural CO2 comprising predominantly 12 C; moreover, unlabeled carbohydrates may also become available by the catabolism of storage polymers that had already been present prior to the 13 CO2 pulse phase. For the biosynthesis of primary and secondary metabolites, the plant can now retrieve material from all currently available pools of photosynthate


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(i.e., from unlabeled pre-pulse storage material, from labeled pulse-phase material and from unlabeled chase-phase material). Notably, carbohydrate molecules that had been formed in different periods can jointly undergo metabolic recycling. As a first approximation, the plant will be operating under conditions that are similar to a plant that is growing aseptically with an exogenous supply of [U-13 C6 ]glucose. In fact, there is detailed experimental evidence for this hypothesis [17,34] that will be presented below. However, in order to present that evidence, it is essential to introduce some technical details concerning the spectroscopic analysis of the metabolites. Readers who are familiar with the spectroscopic analysis of isotopologue mixtures are advised to skip the following paragraphs. 5. Terminology of 13 C Isotopologue Mixtures According to the rules of the International Union of Pure and Applied Chemistry (IUPAC), isomers having the same number of each isotopic atom, but differing in their positions are termed isotopomers. In contrast, molecular entities that differ more generally in their isotopic compositions are noted isotopologues. Since the latter term involves the full range of molecules that are subjected to NMR or MS analysis in the 13 C-labeling experiments, we prefer the isotopologue notation. IUPAC rules for the accurate description of isotopologues use square brackets to indicate the position, type and number of isotopic nuclei in a given isotopologue. For example, [1-13 C1 ]glucose carries a single 13 C label in position 1; [2,3-13 C ]glucose carries 13 C labels in positions 2 and 3; and all carbon atoms 2 are 13 C in [U-13 C6 ]glucose. Working with 13 CO2 as the tracer, one has to deal with complex sets of multiple isotopologues. Hence, we found it necessary to introduce a shorthand notation that is easily accessible to being handled by set theory. Briefly, the isotopologue status of a compound comprising n carbon atoms is rendered as a binary number with n positions, where each digit describes the status of a specific carbon atom. 12 C is rendered as 0 (zero); 13 C is rendered as 1 (one); and x serves as a wild card designating a molecular position with unknown isotope status. For example, [1-13 C1 ]-, [2,3-13 C2 ]and [U-13 C6 ]glucose are rendered as {100000}, {011000} and {111111}, respectively. Isotope mixtures are written as sets of binary numbers; hence {100000, 011000, 111111} designates the mixture of the three isotopologues mentioned above. {11xxxx}Glucose designates a mixture of isotopologues with 13 C in positions 1 and 2, but with the carbon isotope status of the remaining carbon atoms being not declared. 6. A Short Primer to 13 C-NMR Spectra of Complex Isotopologues and Isotopologue Mixtures Generally, the detection and quantification of 13 C isotopologue mixtures requires high-resolution NMR instruments (>400 MHz 1 H-frequency), preferably equipped with a cryo-probe head enabling 13 C-detection at high sensitivity (i.e., with the inner coil tuned to 13 C). In 13 C-NMR spectra of organic matter with natural 13 C abundance (i.e., about 1.1% 13 C), signals appear as singlets (since the 13 C-13 C coupling satellites typically disappear in the noise at 1.1% natural 13 C abundance). The probability for directly adjacent 13 C pairs is in the range of 100 ppm, and the probability of directly connected 13 C triples is in the range of 1 ppm; thus, the general probability of directly-connected 13 C pairs is low, and the probability of larger directly =connected 13 C ensembles is even lower. However, the abundance of molecules carrying ensembles of covalently connected ensembles of 13 C atoms can be dramatically enhanced by pulse labeling of intact plants with 13 CO2 . A consecutive chase period in air with natural 13 CO abundance results in the formation of metabolites containing multiply-labeled molecular species 2 in the single-digit % range, i.e., with an abundance of multiply 13 C-labeled molecular species that is orders of magnitude above their occurrence in natural organic material. 13 C spectra of samples derived from that type of experiment described in more detail below typically show much higher complexity. When ensembles of two or more 13 C atoms are connected by one to four covalent bonds, their signals appear split into multiplets that can be rather complex. The 13 C-13 C coupling constants relating directly-connected carbon atoms are in the range of 30–70 Hz. The connectivity of 13 C atoms via 2–4 bonds can show up with coupling constants up to 10 Hz. The subcomponents of a given multiplet can be directly related to sets of certain isotopologues (Figure 3).

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(Figure 3). The binary notation introduced was specifically designed for the quantitative analysis of The binary notation introduced was specifically designed for the quantitative analysis of isotopologue mixtures by1313C-NMR spectroscopy. The information gleaned from specific multiplets isotopologue mixtures by C-NMR spectroscopy. The information gleaned from specific multiplets can be expressed in the format of x-groups (sets of isotopologues where the isotopologue status of can be expressed in the format of x-groups (sets of isotopologues where the isotopologue status of certain molecular positions is unknown and is rendered as x). The comprehensive analysis of a given certain molecular positions is unknown and is rendered as x). The comprehensive analysis of a given spectrum affords quantitative information on certain x-group sets. From these, the abundance of spectrum affords quantitative information on certain x-group sets. From these, the abundance of individual isotopologues can be extracted by numerical deconvolution. The technical details have individual isotopologues can be extracted by numerical deconvolution. The technical details have been described earlier, and readers are directed to those papers for specifics [17,34,35]. been described earlier, and readers are directed to those papers for specifics [17,34,35].

Figure 3. 1313C-NMR-based isotopologue profiling of complex isotopologue mixtures. (A) 1313C-NMR Figure 3. C-NMR-based isotopologue profiling of complex isotopologue mixtures. (A) C-NMR signature of C-1 in glucose isolated from starch of maize kernels grown on agar containing signature of C-1 in glucose isolated from starch of maize kernels grown on agar containing [U-13 C6 ]glucose and unlabeled glucose [36]. The same 13 C-NMR signal when processed by [U-13C6]glucose and unlabeled glucose [36]. The same 13C-NMR signal when processed by multiplication with an exponential function (bottom) or a Gaussian function (top). Signals due to scalar multiplication with an exponential function (bottom) or a Gaussian function (top). Signals due to couplings between 13 C-atoms in the same molecule are indicated. The coupling partners are indicated scalar couplings between 13C-atoms in the same molecule are indicated. The coupling partners are by italic numbers. Colors indicate the corresponding x-groups with 1 = 13 C, 0 = 12 C and X = unknown. indicated by italic numbers. Colors indicate the corresponding x-groups with 1 = 13C, 0 = 12C and X = Filled or open circles in the structures indicate 13 C- or 12 C-atoms, respectively. (B) 13 C-Isotopologue unknown. Filled or open circles in the structures indicate 13C- or 12C-atoms, respectively. (B) abundances mol% in the 13 C-enriched glucose) by numerical deconvolution of the detected x-group 13C-Isotopologue abundances mol% in the 13C-enriched glucose) by numerical deconvolution of the abundances. One of the major isotopologues in the biosynthetic mixture, i.e., [1,2,3-13 C3 ]glucose due to detected x-group abundances. One of the major isotopologues in the biosynthetic mixture, i.e., glycolytic cycling of the proffered [U-13 C6 ]glucose, is displayed [36]. [1,2,3-13C3]glucose due to glycolytic cycling of the proffered [U-13C6]glucose, is displayed [36].

7. Similar Labeling Patterns of Metabolic Glucose in Tobacco Plants Exposed to [U-13 C ]glucose 7. Similar Labeling Patterns of Metabolic Glucose in Tobacco Plants Exposed to [U-13C66]glucose or 1313CO2 or CO2 Figure 4 compares the x-group profiles of glucose harvested from tobacco plants (i) after Figure 4 compares the x-group profiles of glucose harvested from tobacco plants (i) after pulse-chase with 13 CO2 and (ii) after growth with [U-13 C6 ]glucose supplied via the root system pulse-chase with 13CO2 and (ii) after growth with [U-13C6]glucose supplied via the root system of a of a plant growing on a matrix of sterile agar [34]. The patterns show remarkable similarity, which is plant growing on a matrix of sterile agar [34]. The patterns show remarkable similarity, which is not not surprising in light of the foregoing considerations. With this in mind, it appears reasonable to use surprising in light of the foregoing considerations. With this in mind, it appears reasonable to use the interpretational approach that has been developed in work with [U-13 C ]glucose as tool for the the interpretational approach that has been developed in work with [U-13C66]glucose as tool for the interpretation of 1313CO2 labeling work. interpretation of CO2 labeling work.

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13 C-enriched 4. Comparison of x-group profiles leafglucose glucose (relative (relative fractions samples) FigureFigure 4. Comparison of x-group profiles ofofleaf fractionsinin13C-enriched samples) 13CO2 (in green) and (ii) after growth with 13 harvested from tobacco plants (i) after pulse-chase with harvested from tobacco plants (i) after pulse-chase with CO2 (in green) and (ii) after growth with 13C6]glucose supplied via the root system of a plant growing on a matrix of sterile agar (in red) [U-13 C[U6 ]glucose supplied via the root system of a plant growing on a matrix of sterile agar (in red) [34].

[34].

The established technique for biosynthetic metabolomics data The established technique forthe the interpretation interpretation ofof biosynthetic and and metabolomics data from thefrom 13 13 the relatively bodyofofevidence evidence generated C6 ]glucose has been reviewed in some relativelylarge large body generated withwith [U- [UC6]glucose has been reviewed in some detail ofofprimary and secondary metabolites are are mosaics that that are are detail[37]. [37].Briefly, Briefly,the thecarbon carbonskeletons skeletons primary and secondary metabolites mosaics assembled building blocks derivedfrom fromthe theinventory inventory of and carbonic acids of of assembled fromfrom building blocks derived ofcarbohydrates carbohydrates and carbonic acids 13C central intermediary metabolites. The initial metabolization of proffered [U6]glucose in plants 13 central intermediary metabolites. The initial metabolization of proffered [U- C6 ]glucose in plants can proceed via glycolysis and the pentose phosphate cycle (which are both participants in the more can proceed via glycolysis and the pentose phosphate cycle (which are both participants in the more complex Calvin cycle). These processes involve the breaking of 1313C-1313 C bonds and generate novel complex Calvin cycle). These processes involve the breaking of C- C bonds and generate novel molecular entities comprising fewer than six1313C atoms. When these molecular entities are used as molecular entities comprising fewer than C atoms.ofWhen thesemolecules molecular entities are that used as building blocks in an environment withsix an abundance unlabeled (from biomass building blocks in an environment with an abundance of unlabeled molecules (from biomass 13 was present prior to the introduction of the C-labeled glucose), novel carbon-carbon bonds will be that 13 C-labeled glucose), novel carbon-carbon was present the introduction of thebetween bonds formed,prior whichto implicate covalent linkage fragments derived from 13C-labeled material, as will be formed, implicatematerial. covalent linkage between fragments derived from 13with C-labeled well aswhich from unlabeled Thus, the newly-generated molecules are mosaics labeledmaterial, and domains, depending the specific origin of each respective building block.with labeled and as wellunlabeled as from unlabeled material. on Thus, the newly-generated molecules are mosaics The biosynthetic pathways conducive to certain primary metabolites in plants are known in unlabeled domains, depending on the specific origin of each respective building block. detail. Biosynthetic amino acids can be isolated conveniently after hydrolysis of biomass, andknown their in The biosynthetic pathways conducive to certain primary metabolites in plants are isotopologue patterns can be determined by NMR. Mass spectroscopy can be used for further detail. Biosynthetic amino acids can be isolated conveniently after hydrolysis of biomass, and confirmation when required. On this basis, the labeling patterns of many central intermediary their isotopologue patterns can be determined by NMR. Mass spectroscopy can be used for further metabolites can be reconstructed on the basis of the labeling profiles of amino acids [37]. For confirmation required. Onare this basis, the labeling patterns of many central intermediary example, when aromatic amino acids exclusively formed via the shikimate pathway. Specifically, the metabolites be reconstructed on arises the basis of erythrose the labeling profiles of amino acids [37]. Forribose example, carboncan skeleton of tryptophan from phosphate, phosphoenol pyruvate, aromatic aminoand acids are(cf. exclusively formed viapattern the shikimate pathway. Specifically, the carbon phosphate serine Figure 2). The labeling of erythrose 4-phosphate and serine used as the source material for biosynthetic tryptophan can thus bephosphoenol easily deducedpyruvate, in their entirety the skeleton of tryptophan arises from erythrose phosphate, ribosefrom phosphate labeling Onpattern the other hand, since only two respective carbon and serine (cf.pattern Figure of 2).tryptophan. The labeling of erythrose 4-phosphate and serine usedatoms as theare source contributed to the amino acid from phosphoenol pyruvate and ribulose phosphate, the information material for biosynthetic tryptophan can thus be easily deduced in their entirety from the labeling accessible from the On tryptophan labeling is restricted to the label distribution carbon atoms to pattern of tryptophan. the other hand,pattern since only two respective carbon atomsinare contributed 1 and 2 of ribose phosphate or, more generally speaking, of the pentose phosphate pool, and carbon the amino acid from phosphoenol pyruvate and ribulose phosphate, the information accessible from atoms 2 and 3 of phosphoenol pyruvate; still, this is useful information in the context of additional the tryptophan labeling pattern is restricted to the label distribution in carbon atoms 1 and 2 of ribose information gleaned from the labeling patterns of other amino acids and nucleic acid building blocks phosphate or, more speaking, oftothe pentose and carbon atoms 2 and 3 of (the latter beinggenerally of course ideally suited diagnose thephosphate labeling of pool, the pentose phosphate pool). phosphoenol pyruvate; still, this is useful information in the context of additional information The labeling patterns of secondary metabolites can also be determined by NMR andgleaned by from the labeling patterns of other amino acids acid building blocks (the latter being supplementary MS analysis as required. Sinceand the nucleic carbon skeletons of secondary metabolites are of from participants central intermediary metabolites (e.g., carbohydrates and carbonic courseassembled ideally suited to diagnoseofthe labeling of the pentose phosphate pool). The labeling patterns of secondary metabolites can also be determined by NMR and by supplementary MS analysis as required. Since the carbon skeletons of secondary metabolites are assembled from participants of central intermediary metabolites (e.g., carbohydrates and carbonic


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6, 21 10 building of 24 acids)Metabolites and/or2016, from more complex primary metabolites, such as amino acids and nucleic acid blocks, it stands to reason that the labeling patterns of the primary building blocks should be reflected acids) and/or from more complex primary metabolites, such as amino acids and nucleic acid in the labeling patterns of more complex metabolites (a special problem arises when the mosaic pattern building blocks, it stands to reason that the labeling patterns of the primary building blocks should of a complex metabolite is somehow by onemetabolites or several (a structural rearrangements; be reflected in the labeling patternsscrambled of more complex special problem arises whenhowever, the the reconstruction of the primary building blocks is still possible and will be illustrated below in mosaic pattern of a complex metabolite is somehow scrambled by one or several structural connection with isoprenoid biosynthesis). rearrangements; however, the reconstruction of the primary building blocks is still possible and will

be illustrated below in connection with isoprenoid biosynthesis).

8. Comparison of Labeling Patterns of Plant Secondary Products as Observed in Experiments 13 C ]glucose with [Uor 13 CO2Patterns of Plant Secondary Products as Observed in Experiments 8. Comparison of Labeling 6 with [U-13C6]glucose or 13CO2

8.1. Nicotine 8.1. Nicotine

Studies on the biosynthesis of nicotine in tobacco plants have been conducted in parallel using 13 C ]glucose on the biosynthesis in tobacco plants have been the conducted in parallel using either [U-Studies or 13 CO2 asofa nicotine tracer and can serve to illustrate similarities, as well as the 6 either [U-13C6]glucose or 13CO2 as a tracer and can serve to illustrate the similarities, as well as the differences of the technologies [17,34]. Figure 5 shows labeling patterns obtained in independent differences of the technologies [17,34]. Figure 5 shows labeling patterns obtained in independent experiments with (i) a tobacco plant grown aseptically on a sterile agarose matrix containing a mixture experiments with (i) a tobacco plant grown aseptically on a sterile agarose matrix containing a of unlabeled glucose and [U-13 C6 ]glucose as tracer and (ii) a tobacco plant that was subjected to mixture of unlabeled glucose and [U-13C6]glucose as tracer and (ii) a tobacco plant that was subjected 13 CO . Specifically, the latter plant was grown for 5 h in synthetic a pulse-chase experiment with 2 2. Specifically, the latter plant was grown for 5 h in synthetic to a pulse-chase experiment with 13CO 13 13 atmosphere containing 600600 ppm keptininthe thegreenhouse greenhouse 10 days. In both atmosphere containing ppm CO CO and was was then then kept forfor 10 days. In both 2 2and experiments, nicotine waswas isolated, and patternwas wasdetermined determined NMR. In and this and experiments, nicotine isolated, anditsits1313CC labeling labeling pattern by by NMR. In this 13blocks subsequent Figures, ensembles two morecontiguously-labeled contiguously-labeled 13C are are indicated by bars. subsequent Figures, ensembles of of two orormore C blocks indicated by bars. 13CO 13 Most notably, the labeling patterns of nicotine from the pulse-chase experiment with 2 and with with Most notably, the labeling patterns of nicotine from the pulse-chase experiment with CO2 and 13C6]glucose, respectively, are closely similar. This adds additional support to the concept that [U-]glucose, [U-13 C respectively, are closely similar. This adds additional support to the concept that 6 13CO2 pulse labeling is conducive to a 13C distribution of the central intermediary carbohydrate pools 13 CO pulse labeling is conducive to a 13 C distribution of the central intermediary carbohydrate pools 2 that resembles that obtained by labeling with universally 13C-labeled glucose. that resembles that obtained by labeling with universally 13 C-labeled glucose. The isotopologue compositions of nicotine from both experiments supported its well-known The isotopologue compositions nicotine from both experiments supported its well-known formation via nicotinic acid that is of biosynthesized from aspartate and glyceraldehyde phosphate. formation via nicotinic that isfrom biosynthesized from aspartate and glyceraldehyde The pyrrolidine ring acid is obtained glutamate as reflected by the presence of two 13C2 unitsphosphate. in the 13 C units in the The pyrrolidine ring is obtained from glutamate as reflected by the presence of In two five-membered ring. For details, the reader is directed to the original paper [34]. conclusion, the 2 five-membered ring. For reader is directed the original paper [34].validity In conclusion, the2 pilot pilot experiment on details, nicotine the biosynthesis providedtostrong evidence for the of the 13CO approach. experiment on nicotine biosynthesis provided strong evidence for the validity of the 13 CO2 approach.

Figure 5. Experimental settings experiments analysis of nicotine biosynthesis Figure 5. Experimental settingsfor for labeling labeling experiments forfor thethe analysis of nicotine biosynthesis in tobaccoplants. plants. (A) (A) Tobacco Tobacco plantlets plantlets were conditions on on agaragar containing in tobacco weregrown grownunder understerile sterile conditions containing 13C6]glucose and unlabeled glucose. (B) 1313 CO 2 pulse/chase experiments with whole plants of [U[U-13 C CO 6 ]glucose and unlabeled glucose. (B) 2 pulse/chase experiments with whole plants tobacco[34]. [34]. The The resulting resulting isotopologue profiles (mixtures) of nicotine are indicated on top of of tobacco isotopologue profiles (mixtures) of nicotine are indicated onthe top of respective experiments. The bars indicate multiple 13C-isotopologues detected by NMR in the 13 the respective experiments. The bars indicate multiple C-isotopologues detected by NMR in the isotopologue mixtures. Filled circles indicate single 13C-labeled isotopologues. The numbers indicate isotopologue mixtures. Filled circles indicate single 13 C-labeled isotopologues. The numbers indicate the mol% fractions of the respective isotopologues. the mol% fractions of the respective isotopologues.


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8.2. Hermidin Hermidin 8.2. 13CO2 as the tracer have also been conducted Parallel studies studies using using either either [U[U-1313C C66]glucose Parallel ]glucose or or 13 CO2 as the tracer have also been conducted with hermidin, hermidin, an byby thethe wide-spread weed, Mercurialis annua [38]. [38]. The with an alkaloid alkaloidthat thatisisproduced produced wide-spread weed, Mercurialis annua similar labeling patterns obtained by the different methods are illustrated in Figure 6. Notably, the The similar labeling patterns obtained by the different methods are illustrated in Figure 6. Notably, 13CO 13C 13 13 2 experiment provides higher levels of labeling, and, as a consequence thereof, reveals the CO2 experiment provides higher levels of C labeling, and, as a consequence thereof, reveals substantially more more labeling labeling detail. detail. As As in in the the case case of of nicotine, nicotine, the the data data document document the the incorporation incorporation of of substantially a three-carbon motif into the heterocyclic ring of hermidin. In parallel to the biosynthesis of nicotine, a three-carbon motif into the heterocyclic ring of hermidin. In parallel to the biosynthesis of nicotine, this suggests suggests aa biosynthetic biosynthetic pathway pathway using using dihydroxyacetone dihydroxyacetone phosphate phosphate and and aspartate aspartate as as starting starting this units. Furthermore, units. Furthermore, in in parallel parallel with with the thestudies studieson onnicotine nicotinebiosynthesis, biosynthesis,nicotinic nicotinicacid acidappears appearsasasa likely intermediate in the biosynthesis of hermidin (Figure 6). a likely intermediate in the biosynthesis of hermidin (Figure 6).

Figure Comparisonofofthe theisotopologue isotopologue profiles hermidin from Mercurialis annua. (A) Data Figure 6. Comparison profiles in in hermidin from Mercurialis annua. (A) Data from 13 CO experiment. 1313C ]glucose. 13 from the (B) Data from a labeling experiment using [UCO2 experiment. (B) Data from a labeling experiment [U- C66 the 2 Experimentally-observed Experimentally-observed labeling labeling patterns patterns are are shown shown inin boxes. boxes. The bars indicate indicate multiple multiple 13 13C-isotopologues C-isotopologues detected detected by NMR in in the the isotopologue isotopologue mixtures. mixtures. The colors colors indicate indicate molecular molecular segments numbers indicate thethe molar amounts (mol%) of the segments that thatare arebiosynthetically biosyntheticallyequivalent. equivalent.The The numbers indicate molar amounts (mol%) of respective isotopologues. the respective isotopologues.

8.3. Nudicaulin Nudicaulin 8.3. Recent work work has has also also established established aa closely closely similar similar labeling labeling pattern pattern for for the the flower flower pigment pigment Recent 1313C nudicaulin in in parallel parallel work work with with[U[UC66]glucose ]glucose and and 1313CO CO22 (Figure 7) 7) [39,40]. [39,40]. The The biosynthetic biosynthetic nudicaulin implications of of these these data data are are discussed discussed in in the thesubsequent subsequentSection Section11. 11. implications


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Figure 7. Comparison of isotopologue profiles in nudicaulin from Papaver nudicaule [26,27]. (A) From Figure 7. Comparison of isotopologue profiles in nudicaulin from Papaver nudicaule [26,27]. (A) From 13CO2. (B) From a labeling experiment with [U-13C6]glucose. For more a labeling experiment with a labeling experiment with 13 CO2 . (B) From a labeling experiment with [U-13 C6 ]glucose. For more details, see the legend to Figure 6. details, see the legend to Figure 6.

9. Biosynthesis of Terpenes Studied by 13CO2 Experiments

9. Biosynthesis of Terpenes Studied by 13 CO2 Experiments

Terpenes are the largest family of known natural products, with at least 30,000 documented

members. They participate in an almost unlimited variety of physiological [41].documented To give Terpenes are the largest family of known natural products, with at processes least 30,000 just a few examples, terpenes act as components of biomembranes, pollinator attractants, members. They participate in an almost unlimited variety of physiological processes [41]. Todyes, give just repellents and Numerous pollinator terpenes are major drugs serving as a fewhormones, examples,vitamins, terpenespredator act as components oftoxins. biomembranes, attractants, dyes, hormones, vitamins, hormones or cytostatic agents. On the other hand, enzymes involved in terpenoid vitamins, predator repellents and toxins. Numerous terpenes are major drugs serving as vitamins, biosynthesis and metabolism are very important drug targets in humans, most notably for the hormones or cytostatic agents. On the other hand, enzymes involved in terpenoid biosynthesis and control of cholesterol biosynthesis. The remarkably large number of Nobel prizes that have been metabolism are very important drug targets in humans, most notably for the control of cholesterol awarded for work related to terpene chemistry and biochemistry provides ample testimony of the biosynthesis. remarkably large number of Nobel prizes that have been awarded for work related relevanceThe of terpene research. to terpeneIrrespective chemistry and biochemistry provides complexity ample testimony the relevance of terpenes terpene research. of the enormous structural of the of terpene family, all are Irrespective of the enormous structural complexity of the terpene family, terpenes are biosynthesized from only two simple five-carbon compounds, namely isopentenyl all diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (for a review, namely see [42]).isopentenyl Quite often,diphosphate the assembly (IPP) biosynthesized from only two simple five-carbon compounds, pattern, even ofdiphosphate structurally-complex terpenes, from branched five-carbon motifs be guessed and dimethylallyl (DMAPP) (for a review, see [42]). Quite often, thecan assembly pattern, using the “isoprenoid rule” that had been formulated by Ruziczka around the 1940s (in a the even of structurally-complex terpenes, from branched five-carbon motifs can be guessed using comprehensive article published in 1953 in Experientia [43], Ruzicka singlehandedly presented the “isoprenoid rule” that had been formulated by Ruziczka around the 1940s (in a comprehensive article isoprenoid dissections for a wide variety of terpenes). published in 1953 in Experientia [43], Ruzicka singlehandedly presented the isoprenoid dissections for Pioneering work by Lynen, Bloch and coworkers elucidated the biosynthesis of the universal a wide variety of terpenes). isoprenoid precursor, IPP and DMAPP, from acetyl-CoA via mevalonate (reviewed in [44]). A Pioneering by Lynen, Bloch and coworkers elucidated biosynthesis of the universal spinoff fromwork this work was the introduction of blockbuster drugsthe inhibiting the biosynthesis of isoprenoid precursor, IPP and DMAPP, from acetyl-CoA via mevalonate (reviewed in A spinoff cholesterol by inhibition of a mevalonate pathway enzyme, hydroxymethyl-glutaryl-CoA[44]). reductase. from Despite this work was the introduction of blockbuster inhibiting the biosynthesis cholesterol by some inconvenient anomalies, the questiondrugs of isoprenoid biosynthesis appearedof settled. the 1990s brought the discovery of a second major pathway affordingDespite IPP andsome inhibitionUnexpectedly, of a mevalonate pathway enzyme, hydroxymethyl-glutaryl-CoA reductase. DMAPP from glyceraldehyde phosphate and pyruvate via 1-deoxyxylulose 5-phosphate (DXP) and inconvenient anomalies, the question of isoprenoid biosynthesis appeared settled. 2C-methylerythritol 4-phosphate (MEP) [45,46]. Importantly, the biosynthetic pathways of different Unexpectedly, the 1990s brought the discovery of a second major pathway affording IPP and plant terpenes take their respective isoprenoid precursors either from the mevalonate or the DMAPP from glyceraldehyde phosphate and pyruvate via 1-deoxyxylulose 5-phosphate (DXP) non-mevalonate pathway (but crosstalk between the pathways is also possible; reviewed in [42,47]). and 2C-methylerythritol 4-phosphate (MEP) [45,46]. Importantly, the biosynthetic pathways of In order to determine the biosynthetic origin of the isoprenoid building blocks of a given plant different plantit terpenes take their precursors either from the mevalonate terpene, is essential that the respective mevalonateisoprenoid pathway uses three acetyl-CoA precursor molecules,or the non-mevalonate pathway (but crosstalk between the pathways is also possible; reviewed in [42,47]). whereas the non-mevalonate pathway uses only two precursor molecules, i.e., pyruvate and In order to determine the biosynthetic origin of the isoprenoid buildingThat blocks of athat given glyceraldehyde 3-phosphate, to assemble an isoprenoid precursor molecule. implies theplant 13C three 13C triples, terpene, it is essential that the acetyl-CoA molecules, mevalonate pathway canmevalonate contribute pathway pairs of uses atoms, but not precursor whereas whereas the 13C triples (from the glyceraldehyde 3-phosphate precursor), non-mevalonate pathway contributes the non-mevalonate pathway uses only two precursor molecules, i.e., pyruvate and glyceraldehyde 13C pairs (Figure 8). It turns out that the 13CO2 pulse-chase experiment is perfectly suited to as well asto 3-phosphate, assemble an isoprenoid precursor molecule. That implies that the mevalonate pathway distinguish between alternatives, asCwill become obvious asnon-mevalonate we proceed to specific examples. can contribute pairs of 13these C atoms, but not 13 triples, whereas the pathway contributes 13 C triples (from the glyceraldehyde 3-phosphate precursor), as well as 13 C pairs (Figure 8). It turns out that the 13 CO2 pulse-chase experiment is perfectly suited to distinguish between these alternatives, as will become obvious as we proceed to specific examples.


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The two different isoprenoid biosynthesis pathways are spatially separated inside plant cells, withThe the two mevalonate in the cytoplasmic compartment and the non-mevalonate different pathway isoprenoidoperating biosynthesis pathways are spatially separated inside plant cells, with pathway inside chloroplasts (Figure 8). To make matters more complicated, however, the isoprenoid the mevalonate pathway operating in the cytoplasmic compartment and the non-mevalonate pathway precursors for different steps in the biosynthesis of complex terpenes can be contributed from inside chloroplasts (Figure 8). To make matters more complicated, however, the isoprenoid precursors different sources. Hence, it is of interest to specifically assess the origin of each five-carbon unit of a for different steps in the biosynthesis of complex terpenes can be contributed from different sources. complex terpene. For that purpose, it is of course advantageous to be able to work at the level of Hence, it is of interest to specifically assess the origin of each five-carbon unit of a complex terpene. intact, rather than any to cellular which might unperturbed be prone to For thatunperturbed purpose, it isplants, of course advantageous be ableintovitro worksystem, at the level of intact, 13CO2 are perfectly artifacts. As shown by the subsequent examples, pulse-chase experiments with plants, rather than any cellular in vitro system, which might be prone to artifacts. As shown by the suited to distinguish between the {2+2+1} pattern origin and the {2+3} pattern of subsequent examples, pulse-chase experiments with 13of COmevalonate 2 are perfectly suited to distinguish between 13 non-mevalonate origin. Indeed, short-term CO2{2+3} labeling experiments with Arabidopsis thaliana the {2+2+1} pattern of mevalonate origin and the pattern of non-mevalonate origin. Indeed, 13CO2 is rapidly (within minutes) assimilated into intermediates of the showed that the label from 13 13 short-term CO2 labeling experiments with Arabidopsis thaliana showed that the label from CO2 is non-mevalonate pathwayassimilated [48]. rapidly (within minutes) into intermediates of the non-mevalonate pathway [48]. Figure 8 illustrates possible outcomes of of aa thought thought experiment experiment where where aa single single molecule molecule of of Figure 8 illustrates possible outcomes 13C3]glyceraldehyde phosphate is contributed to an isoprenoid diphosphate unit via the shortest [U[U-13 C3 ]glyceraldehyde phosphate is contributed to an isoprenoid diphosphate unit via the shortest possible path path (i.e., (i.e., without without reshuffling reshuffling in in any any metabolic metabolic cycles) cycles) and and in in the the presence presence of of unlabeled unlabeled possible 13C-labeled metabolites that can be used as cosubstrates. By way of the non-mevalonate pathway, the 13 metabolites that can be used as cosubstrates. By way of the non-mevalonate pathway, the C-labeled glyceraldehyde phosphate phosphate could could afford afford an an isoprenoid isoprenoid containing containing aa pair pair of of directly-connected directly-connected carbon carbon glyceraldehyde 13 13 atoms, plus a C-labeled methyl group in DMAPP or a C-labeled methylene group in IPP, that are atoms, plus a 13 C-labeled methyl group in DMAPP or a 13 C-labeled methylene group in IPP, that are not directly connected to the pair, as a consequence of the skeletal rearrangement that is engineered not directly connected to the pair, as a consequence of the skeletal rearrangement that is engineered by by methylerythritol phosphate synthase. Alternatively, [U-13C3]glyceraldehyde 3-phosphate could methylerythritol phosphate synthase. Alternatively, [U-13 C3 ]glyceraldehyde 3-phosphate could be be converted to [U-13C3]pyruvate, whose utilization for isoprenoid formation would afford a product converted to [U-13 C3 ]pyruvate, whose utilization for isoprenoid formation would afford a product carrying a directly-linked pair of 13C atoms. Notably, C-1 of pyruvate is lost as CO2 during the carrying a directly-linked pair of 13 C atoms. Notably, C-1 of pyruvate is lost as CO2 during the biosynthetic process (cf. Figure 8). biosynthetic process (cf. Figure 8).

Figure Figure 8.8. Biosynthesis Biosynthesis of of the the terpene terpene precursors, precursors, isopentenyl isopentenyl diphosphate diphosphate (IPP) (IPP) and and dimethylallyl dimethylallyl diphosphate (DMAPP), via the non-mevalonate (MEP) pathway in the plastidic compartment diphosphate (DMAPP), via the non-mevalonate (MEP) pathway in the plastidic compartment (in (in the the box) of a plant cell or via the mevalonate pathway in the cytosolic compartment. Biosynthetic motifs box) of a plant cell or via the mevalonate pathway in the cytosolic compartment. Biosynthetic motifs derived derived from from pyruvate pyruvate and and glyceraldehyde glyceraldehyde phosphate phosphate (GAP) (GAP) are are indicated indicated by by red red and and green green colors, colors, respectively. The green arrows indicate that the three-carbon connectivity of the original GAP precursor respectively. The green arrows indicate that the three-carbon connectivity of the original GAP becomes a skeletal rearrangement the biosynthetic Biosynthetic units precursormodified becomesby modified by a skeletal during rearrangement during process. the biosynthetic process. deriving from acetyl-CoA in the mevalonate route are indicated by blue colors. Biosynthetic units deriving from acetyl-CoA in the mevalonate route are indicated by blue colors.


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On the other hand, for utilization via the mevalonate pathway, the [U-13C3]glyceraldehyde phosphate molecule would have to be converted, via [U-13C3]pyruvate, into 13 [1,2-13C2]acetyl-CoA. On the other hand, for utilization via the mevalonate pathway, the [UC3 ]glyceraldehyde 13 Since three molecules of acetyl-CoA fuel the mevalonate pathway, a single C-labeled acetyl-CoA phosphate molecule would have to be converted, via [U-13 C3 ]pyruvate, into [1,2-13 C2 ]acetyl-CoA. molecule could afford two different isoprenoid isotopologues carrying directly-linked pairs of 13C Since three molecules of acetyl-CoA fuel the mevalonate pathway, a single 13 C-labeled acetyl-CoA atoms. Moreover, a double-labeled acetate unit could yield an isoprenoid isotopologue carrying a molecule could afford two different isoprenoid isotopologues carrying directly-linked pairs of 13 C 13C-labeled methyl or methylene group. atoms. Moreover, a double-labeled acetate unit could yield an isoprenoid isotopologue carrying Remarkably, the overall isotopologue patterns obtained via the two different pathways in our a 13 C-labeled methyl or methylene group. thought experiment look remarkably similar insofar as the 13C pairs have the same locations. Most Remarkably, the overall isotopologue patterns obtained via the two different pathways in our importantly, however, only the non-mevalonate pathway 13can contribute three 13C atoms from a thought experiment look remarkably similar insofar as the C pairs have the same locations. Most single precursor molecule, whereas the mevalonate pathway can at most contribute pairs of 13C importantly, however, only the non-mevalonate pathway can contribute three 13 C atoms from a single atoms. This is the single, crucial criterion to be used for the differentiation of mevalonate versus precursor molecule, whereas the mevalonate pathway can at most contribute pairs of 13 C atoms. This is non-mevalonate origin to be used in tracer experiments starting either with exogenously-labeled the 13single, crucial criterion to be used for the differentiation of mevalonate versus non-mevalonate [U- C6]glucose or ensembles of multiply-labeled carbohydrates generated in situ by photosynthesis origin13to be used in tracer experiments starting either with exogenously-labeled [U-13 C6 ]glucose from CO2. The following sections illustrate the practical application of this cutting edge criterion. or ensembles of multiply-labeled carbohydrates generated in situ by photosynthesis from 13 CO2 . The following sections illustrate the practical application of this cutting edge criterion. 9.1. Thymol 9.1. Thymol Thymol is a simple monoterpene. Anyone who is marginally familiar with the “isoprene rule” of terpene can easily execute the is dissection offamiliar thymolwith intothe the two precursor Thymolbiosynthesis is a simple monoterpene. Anyone who marginally “isoprene rule” of isoprenoids that yield carbon skeleton (Figure 9). The question however, whether these terpene biosynthesis canthe easily execute the dissection of thymol into theis,two precursor isoprenoids monomers arise by the {2+3} or the {2+2+1} scheme via the non-mevalonate or mevalonate pathway, that yield the carbon skeleton (Figure 9). The question is, however, whether these monomers arise by respectively. the {2+3} or the {2+2+1} scheme via the non-mevalonate or mevalonate pathway, respectively.

Figure Figure 9. 9. Biosynthesis Biosynthesis of thymol from geranyl diphosphate that is formed from DMAPP (red motif) and and IPP IPP (green (green motif). motif).

Evidence for for aa {2+3} {2+3} origin any isoprenoid isoprenoid by by NMR NMR should should be be easy easy enough, enough, except except that that the the Evidence origin of of any three-carbonunit unitis disrupted is disrupted byskeletal the skeletal rearrangement engineered by methylerythritol three-carbon by the rearrangement engineered by methylerythritol phosphate phosphate synthase (IspC) catalyzing the first committed step of the non-mevalonate pathway. synthase (IspC) catalyzing the first committed step of the non-mevalonate pathway. 13CO2 pulse-chase experiment with Thymus transcaucasicus, biosynthetic thymol was In aa13 CO In 2 pulse-chase experiment with Thymus transcaucasicus, biosynthetic thymol was analyzed analyzed exhaustively 1D and2D advanced 2D NMR techniques, as well as mass spectrometry [49]. exhaustively by 1D and by advanced NMR techniques, as well as mass spectrometry [49]. A battery of 13 A battery of simple, as well as advanced NMR tools uniformly confirmed the presence of simple, as well as advanced NMR tools uniformly confirmed the presence of fragmented C triples 13 fragmented C triples in technical the monoterpene. technical challenge is thethe necessity to diagnose the in the monoterpene. The challengeThe is the necessity to diagnose three-carbon fragment three-carbon long-range coupling with its 13characteristically small 13C-13To C coupling via long-rangefragment couplingvia with its characteristically small C-13 C coupling constants. this aim, constants. To this aim, harnessing advanced 2D-NMR techniques used in [36]. However, as harnessing advanced 2D-NMR techniques were used [36]. However,were as shown Figure 10, even 13 13 in 1D Figure 10, even acan humble 1D C by experiment can simultaneous add evidencecoupling by documenting ashown humble C experiment add evidence documenting of C-1 to simultaneous coupling of C-1 to the directly-connected carbon atom C-6 (J, 66.6 Hz) and to the directly-connected carbon atom C-6 (J, 66.6 Hz) and to the more remote carbon C-4 (J,the 8.1more Hz). 13C-triple could also be detected for C-3/C-2/C-11 in the 13 remote carbon C-4 (J, 8.1 Hz). Similarly, a Similarly, a C-triple could also be detected for C-3/C-2/C-11 in the other isoprenoid unit (Figure 10). other isoprenoid unit (Figure 10).profiles In conclusion, the isotopologue profiles for the first In conclusion, the isotopologue demonstrated for the first timedemonstrated the {2+3} origin of both time the {2+3} origin of both C 5 -precursors in the monoterpene under physiological conditions. C5 -precursors in the monoterpene under physiological conditions.

Metabolites 2016, 6, 21 Metabolites 2016, 6, 21 Metabolites 2016, 6, 21

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13C-NMR Figure 10. 13 C-NMR signals from ofThymus Thymustranscaucasicus transcaucasicus grown under an Figure 10. signalsof thymol isolated isolated from grown under an an 13 Figure 10. C-NMR signals ofofthymol thymol isolated from ofof Thymus transcaucasicus grown under 13CO13 13C-Couplings via one-bond or multiple bonds are indicated with the 13CO atmosphere containing atmosphere containing 2. 2. C-Couplings via one-bond or multiple bonds are indicated with the 13 13 atmosphere containing CO2 . C-Couplings via one-bond or multiple bonds are indicated with corresponding couplingconstants. constants. Carbon Carbon atoms atoms derived from the GAP precursor via the corresponding coupling derived from the the corresponding coupling constants. Carbon atoms derived fromthe theGAP GAP precursor precursor via via the non-mevalonate pathway are highlightedin in red. red. ** indicates signals due to impurities. non-mevalonate pathway are highlighted indicates signals due to impurities. non-mevalonate pathway are highlighted in red. * indicates signals due to impurities.

9.2. Artemisinin

9.2. Artemisinin 9.2. Artemisinin Artemisinin and its derivatives are at present the most important drugs for the treatment and Artemisinin and its derivatives are at present the most important drugs for the treatment and prevention of and malaria, which is rampant large parts of theimportant world and drugs claims for more than 600,000 and Artemisinin its derivatives are at in present the most the treatment prevention of malaria, which is rampant in large parts of the world and claims more than 600,000 lives per Although artemisinin available, the fight malaria lives prevention of year. malaria, whichsynthetic is rampant in large analogs parts ofare thenow world and claims moreagainst than 600,000 lives depends, per year.more Although synthetic artemisinin analogs are now available, theNotably, fight against malaria than anything else, on natural products from Artemisia plants. the Nobel per year. Although synthetic artemisinin analogs are now available, the fight against malaria depends, depends, than anything else,toon natural from Notably, to thetheNobel price more has been awarded in 2015 Youyou Tu,products at the age of 85Artemisia years, forplants. her contribution more than anything else, on natural products from Artemisia plants. Notably, the Nobel price has been the drug [50]. pricediscovery has beenofawarded in 2015 to Youyou Tu, at the age of 85 years, for her contribution to the awarded in 2015 to Youyou Tu, at the age of 85 years, for her contribution to the discovery of the a sesquiterpene assembled from three isoprenoid precursor moieties via farnesyl discoveryArtemisinin of the drugis[50]. drug diphosphate. [50]. The connectivity of the isoprenoid moiety shown in green precursor in Figure 11moieties is disrupted a Artemisinin is a sesquiterpene assembled from three isoprenoid via at farnesyl Artemisinin is a stage sesquiterpene assembled from three isoprenoid precursor via farnesyl later biosynthetic by oxidative cleavage of a carbon-carbon bond of a bicyclic moieties intermediate. diphosphate. The connectivity of the isoprenoid moiety shown in green in Figure 11 is disrupted at a diphosphate. The connectivity of the isoprenoid moiety shown in green in Figure 11 is disrupted at later biosynthetic stage by oxidative cleavage of a carbon-carbon bond of a bicyclic intermediate. a later biosynthetic stage by oxidative cleavage of a carbon-carbon bond of a bicyclic intermediate.

Figure 11. Studies on the biosynthesis of the sesquiterpene artemisinin. (A) Structure and carbon numbering of artemisinin. (B) Isoprene dissections of artemisinin indicated by the different colors. (C) 13C isotopologue composition of artemisinin from the experiment with 13CO2. Multiple Figure 11. Studies Studies biosynthesis the of the sesquiterpene artemisinin. (A) Structure and 13C-labeled Figure 11. onon thethe biosynthesis sesquiterpene artemisinin. (A) Structure carbon isotopologues are indicated of by colored bars, single-labeled isotopologues by filled and circles. carbon numbering of artemisinin. (B) Isoprene dissections of artemisinin indicated by the 13 13 13 detecteddissections by long-range C– C couplings in the biosynthetic sample. The arrow indicates a C-triple numbering of artemisinin. (B) Isoprene of artemisinin indicated by the different colors. 13 C isotopologue 13 CO . 13 different colors. (C) composition of artemisinin from the experiment with 2 The indicatecomposition C enrichments mol%. C numbers isotopologue of inartemisinin from the experiment with 13CO2. Multiple (C) 13 13 Multiple isotopologues are indicated by colored bars, single-labeled isotopologues by filled 13C-labeledC-labeled isotopologues are indicated by colored bars, single-labeled isotopologues by filled circles. circles. The arrow indicates a 13 C-triple detected by long-range 13 C–13 C couplings in the biosynthetic The arrow indicates a 13C-triple detected by long-range 13C–13C couplings in the biosynthetic sample. 13 C enrichments in mol%. sample. The numbers indicate The numbers indicate 13C enrichments in mol%.


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Improving Artemisia cultivars by breeding and/or genetic engineering could help to guarantee a sufficient supplyArtemisia of artemisinin-based antimalarials. the biosynthetic origin of eacha Improving cultivars by breeding and/orUnderstanding genetic engineering could help to guarantee of the three isoprenoid moieties of theantimalarials. farnesyl diphosphate precursor be of practical sufficient supply of artemisinin-based Understanding the could biosynthetic origin ofinterest each of in that context. the three isoprenoid moieties of the farnesyl diphosphate precursor could be of practical interest in Surprisingly, the retrodiction of the building blocks in a 13CO2 pulse-chase experiment that context. 13 CO documented the inclusion of a three-carbon for blocks the middle moiety (Figure 11C), whereas Surprisingly, the retrodiction of the moiety building in aIPP 2 pulse-chase experiment the DMAPP starter molecule and the terminal IPP moiety of the geranyl diphosphate precursor documented the inclusion of a three-carbon moiety for the middle IPP moiety (Figure 11C), whereas the 13C triples [51]. Hence, the middle isoprenoid precursor must have been showed no evidence of DMAPP starter molecule and the terminal IPP moiety of the geranyl diphosphate precursor showed 13 Cnon-mevalonate generated byofthe pathway inside chloroplasts, whereas both flanking precursors no evidence triples [51]. Hence, the middle isoprenoid precursor must have been generated by could arise via the mevalonate pathway inside the cytoplasmic compartment. Such a scenario would the non-mevalonate pathway inside chloroplasts, whereas both flanking precursors could arise via necessitate the transfer of inside DMAPP the cytoplasmic compartment to the plastid stroma, wherethe it the mevalonate pathway thefrom cytoplasmic compartment. Such a scenario would necessitate could react with an IPP moiety derived via the non-mevalonate pathway. In a consecutive step, the transfer of DMAPP from the cytoplasmic compartment to the plastid stroma, where it could react with resulting geranyl diphosphate would have to pathway. be re-exported to the cytoplasmic for an IPP moiety derived via the non-mevalonate In a consecutive step, the compartment resulting geranyl conversion farnesyl diphosphate by the addition of an IPP unit of mevalonate origin. Whereas this diphosphatetowould have to be re-exported to the cytoplasmic compartment for conversion to farnesyl hypothesis may need additional confirmation, the findings illustrate how the pulse-chase diphosphate by the addition of an IPP unit of mevalonate origin. Whereas this hypothesis may need experiment with 13CO2 can provide insights transportexperiment of intermediates between the additional confirmation, the also findings illustrate howinto the the pulse-chase with 13 CO 2 can also compartments ofinto a plant cell. provide insights the transport of intermediates between the compartments of a plant cell. 9.3. Ginsenosides Ginsenosides A plethora of putative health benefits has been attributed to the ginseng plant, and ginseng products are estimated estimated to have have a market market value value of of about about two two billion billion US$ US$ per per year year worldwide worldwide [52]. [52]. Among the numerous secondary products in ginseng extracts, triterpene type ginsenosides ginsenosides are dominant and are believed to be the main contributors to the claimed health benefits. benefits. The world-wide ginseng supply supply is is based based on on harvesting harvesting plants plants aged aged 4–8 4–8 years. years. Advantageously, Advantageously, the 1313CO CO22 pulse-chase pulse-chasemethod method be applied field conditions evenofto plants of cancan be applied underunder field conditions and even and to plants considerable considerable size; thus, a six-year old ginseng plant could be used for the study of triterpene-type size; thus, a six-year old ginseng plant could be used for the study of triterpene-type ginsenosides ginsenosides (Figure 12) [53]. (Figure 12) [53].

13 CO Figure 12. Transportable 2. The specific example shows an Transportable facility facilityto tolabel labelplants plantsininthe thefield fieldwith with13CO specific example shows 2 . The experiment with Panax ginseng plants aimed to elucidate thethe biosynthesis of ginsenosides. an experiment with Panax ginseng plants aimed to elucidate biosynthesis of ginsenosides.

Specifically, a pulse pulse period period of 7h h followed followed by by aa chase chase period period of of eight eight days days was was applied applied under Specifically, a of 7 under field conditions. The ginsenosides Rg1 and Rb1 were isolated from the roots. The but field conditions. The ginsenosides Rg1 and Rb1 were isolated from the roots. The small, but small, significant 13 13 significant couplingdetected satellites in thespectrum C-NMRgave spectrum gavefor evidence for inCthe 2-units in the 13 C -units coupling satellites indetected the 13 C-NMR evidence molecules 2 molecules indicating the mevalonate origin of the triterpenes under study. No evidence whatsoever indicating the mevalonate origin of the triterpenes under study. No evidence whatsoever was obtained 13C triples into ginsenosides. Hence, a {2+3} pattern appears was obtained for the incorporation of 13 for the incorporation of C triples into ginsenosides. Hence, a {2+3} pattern appears most unlikely, and most unlikely, and ginsenosides can be attributed to the mevalonate pathway. Moreover, the


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observed labeling patterns were perfect agreement with Moreover, a chair-chair-chair-boat conformation of ginsenosides can be attributed toin the mevalonate pathway. the observed labeling patterns the (S)-2,3-oxidosqualene precursor entering the cyclization process via the protosteryl cation were in perfect agreement with a chair-chair-chair-boat conformation of the (S)-2,3-oxidosqualene (Figure 13).entering the cyclization process via the protosteryl cation (Figure 13). precursor

13 13 -labeled plants of P. ginseng. The bold bonds Figure 13. Biosynthesis Rg1 from Figure Biosynthesisofofthe theginsenoside ginsenoside Rg1 fromCO CO 2 2-labeled plants of P. ginseng. The bold 13 13 13C-atoms 13C2]acetyl-CoA indicate adjacent C-atoms from [1,2(colored in blue) thevia mevalonate (MVA) bonds indicate adjacent from C[1,2(colored in via blue) the mevalonate 2 ]acetyl-CoA 13 C ]pyruvate 13 13C3]GAP pathway or fromor [U(colored in red)inand [U(colored in green) via the 3[U-13C3]pyruvate 3 ]GAP (MVA) pathway from (colored red) andC[U(colored in green) viaMEP the pathway. The observed labeling profile of Rg1 (in the middle) was perfectly in line with the MVA MEP pathway. The observed labeling profile of Rg1 (in the middle) was perfectly in line with the prediction, but at but odds MEP MVA prediction, at with oddsthe with theprediction. MEP prediction.

9.4. Lupeol Lupeol 9.4. Lupeol is aa triterpene been obtained from numerous plants, withwith occasionally high Lupeol triterpenealcohol alcoholthat thathas has been obtained from numerous plants, occasionally abundance. Whereas the later its biosynthesis have been elucidated in some detail, the origin its high abundance. Whereas thestages later of stages of its biosynthesis have been elucidated in some detail,ofthe basic isoprenoid building blocks,building i.e., IPP and DMAPP, established. In many conjugates origin of its basic isoprenoid blocks, i.e., had IPP not andbeen DMAPP, had not beencases, established. In 1 -R-hydroxy)-stearate of lupeol and fatty acids can foundand in plants. an example, lupeol-3-(3 is a many cases, conjugates of be lupeol fatty As acids can be found in plants. As an example, major compound in the Mexican medicinal Pentalinon andrieuxii. The assumed plant, polyketide origin lupeol-3-(3′-R-hydroxy)-stearate is a majorplant, compound in the Mexican medicinal Pentalinon of the long-chain β-hydroxycarboxylic acidof from moieties via acetyl-CoA provides an acetate elegant andrieuxii. The assumed polyketide origin the acetate long-chain β-hydroxycarboxylic acid from test for the of the 13 CO2 pulse-chase experiment Perfectly line2 with the prediction, each moieties viapower acetyl-CoA provides an elegant test for the[54]. power of thein13CO pulse-chase experiment 13 13 observed carboninatom the acyl is exclusively related by C- C coupling to aacyl single adjacent [54]. Perfectly lineofwith the moiety prediction, each observed carbon atom of the moiety is carbon atom,related thus unequivocally identifyingtothea biosynthetic acetate building blocks (Figure 14). exclusively by 13C-13C coupling single adjacent carbon atom, thus unequivocally identifying the biosynthetic acetate building blocks (Figure 14).

Metabolites 2016, 6, 21 Metabolites 2016, 6, 21 Metabolites 2016, 6, 21

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13C-NMR analysis of lupeol-3-(3′-R-hydroxy)-stearate 13 C-NMR Figure Figure 14.14. analysis of lupeol-3-(31 -R-hydroxy)-stearatefrom fromPentalinon Pentalinonandrieuxii andrieuxiilabeled labeled 13CO2. Many 13C-satellites. The corresponding 1313 13C-NMR 13 13 with of the NMR signals display pairs of 2-units in Figure 14. analysis of lupeol-3-(3′-R-hydroxy)-stearate from Pentalinon andrieuxii with CO2 . Many of the NMR signals display pairs of C-satellites. The corresponding Clabeled C2 -units in 13The 13CO2. Many 13C2-units biosynthetic compound areindicated indicated byblue blue bars. A pair pair of of 13 C-atoms that disrupted with of the NMR signals display pairs of 13C-satellites. corresponding in thethe biosynthetic compound are by bars. A C-atoms thatbecomes becomes disrupted 13C-atoms that becomes disrupted the biosynthetic compound are indicated by blue bars.(cf. A pair of during the cyclization process is shown by blue circles also Figure 15). during the cyclization process is shown by blue circles (cf. also Figure 15).

during the cyclization process is shown by blue circles (cf. also Figure 15).

Likewise, the pentacyclic isoprenoid moiety shows 13CO2 pairs, but no 13C triples whatsoever. 13C 13 Likewise, the the pentacyclic moiety shows CO no Cthe triples whatsoever. pentacyclic moiety shows 1313CO 2 pairs, butbut noone triples whatsoever. 13 2 pairs, Hence, Likewise, the isoprenoid moiety isoprenoid is isoprenoid exclusively of mevalonate origin. Notably, of C2-moieties is 13C213 Hence, the isoprenoid moiety is exclusively of mevalonate origin. Notably, one of the -moieties Hence, the isoprenoid moiety is exclusively of mevalonate origin. Notably, one of the C is 2 fractured by skeletal rearrangement in the later course of the biosynthetic pathway (Figure-moieties 15). is fractured by skeletal rearrangement thelater latercourse course of pathway (Figure 15). 15). fractured by skeletal rearrangement ininthe of the thebiosynthetic biosynthetic pathway (Figure

13CC2 units Figure 15. Biosynthetic steps formationofoflupeol. lupeol. 13 via the Figure 15. Biosynthetic steps in in thethe formation from13C132-acetyl-CoA C2 -acetyl-CoA via the 2 unitsfrom 13C2 units from 13C2-acetyl-CoA via the Figure 15. Biosynthetic steps in the formation of lupeol. 13 13 mevalonate pathway indicated bluebars. bars.One One pair pair of becomes disrupted during the the mevalonate pathway areare indicated bybyblue of CCatoms atoms becomes disrupted during mevalonate pathway are affords indicated by blue bars. Oneinpair of 13C atoms becomes disrupted during the 1313C cyclization process single C atoms atoms shown by by blue circles. cyclization process andand affords thethe single in the thestructure structure shown blue circles. 13 cyclization process and affords the single C atoms in the structure shown by blue circles.


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10. Polyacetylenes from Panax ginseng 10. Polyacetylenes from ginsengpattern of the fatty acid residue of the lupeol derivative, a In close similarity toPanax the labeling 13 strict In polyketide pattern also observed 2-labeled panaxynol and panaxydol from the close similarity to was the labeling pattern for of theCO fatty acid residue of the lupeol derivative, a strict 13 plethora of secondary products formed by Panax ginseng [55]. Specifically, the number atoms 13 polyketide pattern was also observed for CO2 -labeled panaxynol and panaxydol fromof the C plethora that had beenproducts importedformed jointlyby does notginseng exceed[55]. twoSpecifically, in any case.the Notably, the terminal of secondary Panax numberhowever, of 13 C atoms that had methyl carbon atoms of panaxynol or panaxydol did not show any co-transfer at all, thus suggesting been imported jointly does not exceed two in any case. Notably, however, the terminal methyl carbon that one moiety in the polyketide precursor has been shortened the loss of a one-carbon atoms of acetate panaxynol or panaxydol did not show any co-transfer at all, thusby suggesting that one acetate unit (Figure moiety in the16). polyketide precursor has been shortened by the loss of a one-carbon unit (Figure 16).

13 C atoms detected Figure 16. 16. Proposed Proposed biosynthetic biosynthetic pathway pathway of panaxynol and panaxydol. Adjacent 13 Figure C atoms detected 13 13 13 13 in experiments with CO or [UC ]glucose are indicated by blue bars. 6 in experiments with CO22or [U- C6]glucose are indicated by blue bars.

11. 11. Tracking TrackingShikimate ShikimateDerivatives Derivativesas asBuilding BuildingBlocks Blocksfor forSecondary SecondaryMetabolites Metabolites 13CO Figure Figure 17 17 summarizes summarizes data data from a 13 CO22 pulse-chase pulse-chase experiment experimentwith with Papaver Papavernudicaule nudicaule[39]. [39]. One thecompounds compoundsinin Figure nudicaulin, is responsible foryellow the yellow color of the flowers. plant’s One of the Figure 17,17, nudicaulin, is responsible for the color of the plant’s flowers. The other, kaempferol, is a whose flavonoid whose occurrence is shared with a variety of plant The other, kaempferol, is a flavonoid occurrence is shared with a variety of plant species. species.


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13 CO Figure Isotopologuecompositions compositions of of nudicaulin nudicaulin and 2 pulse/chase Figure 17. 17. Isotopologue and kaempferol kaempferolfrom from CO 2 pulse/chase 13 experiments with Papavernudicaule nudicaule [26]. bars indicate multiple C-labeled isotopologues. The 13 C-labeled experiments with Papaver [26]. The The bars indicate multiple isotopologues. numbers indicate mol% of the respective isotopologues. The colors indicate related precursor units The numbers indicate mol% of the respective isotopologues. The colors indicate related precursor units (for details, see the text). (for details, see the text). 13

The similarity of the rings f and B with, regard to the structure and labeling pattern are immediately obvious. Thus, these ringsBofwith, both compounds contiguous blocks ofpattern up to are The similarity of the rings f and regard to can the acquire structure and labeling 13C atoms, notably with overlapping topology. This is easily explained if the phenol rings arise three immediately obvious. Thus, these rings of both compounds can acquire contiguous blocks of up 13 Ca atoms, from four-carbon precursor that can topology. be endowed different labeling more arise to three notably with overlapping Thiswith is easily explained if thepatterns; phenol rings specifically, the four-carbon element is easily tracked to erythrose 4-phosphate, a precursor in the from a four-carbon precursor that can be endowed with different labeling patterns; more specifically, shikimate pathway that is the sole source for aromatic amino acid biosynthesis (see above). In both the four-carbon element is easily tracked to erythrose 4-phosphate, a precursor in the shikimate compounds shown in Figure 17, the rings b/e and C display a linker consisting of three carbon atoms pathway that is the sole source for aromatic amino acid biosynthesis (see above). In both compounds shown in green. Importantly, that linker can simultaneously import up to three 13C atoms, thus shown in Figure 17,itthe rings b/e and chain C display a linker consisting ofasthree carbon atoms shown in suggesting that represents the side of a tyrosine precursor, such coumarate. 13 13C13C suggesting green. Importantly, linker simultaneously importsurprising up to three C sight. atoms, thus The labelingthat pattern of can rings a and A may appear at first coupling is that it represents the of a tyrosine as coumarate. displayed forside eachchain ring carbon to eachprecursor, of the two such adjacent ring atoms; however, no ring carbon is 13 C-is 13 C ever labeling simultaneously carbon surprising atoms. This at seeming paradox incoupling fact a The patterncoupled of ringstoaboth and adjacent A may appear first sight. is well-known feature of ring systems of polyketide origin, as shown by the reaction sequence displayed for each ring carbon to each of the two adjacent ring atoms; however, no ring carbon is ever proposed incoupled Figure 18. the tyrosine precursor is extended by is a sequence of ester simultaneously toBriefly, both adjacent carbon atoms.coumarate This seeming paradox in fact a well-known condensations using malonyl CoA as the substrate. Ring closure affords a trihydroxyphenyl moiety feature of ring systems of polyketide origin, as shown by the reaction sequence proposed in Figure 18. that is initially free to rotate, but is subsequently immobilized by the introduction of an ether bridge Briefly, the tyrosine precursor coumarate is extended by a sequence of ester condensations using that results in the fixation of two discrete labeling patterns for ring a. malonyl CoA as the substrate. Ring closure affords a trihydroxyphenyl moiety that is initially free Unsurprisingly, the benzenoid ring of the indole motif in nudicaulin repeats in every detail the to rotate, but islabeling subsequently the in introduction an ether bridge that results shikimate pattern immobilized that we haveby seen the phenolicof ring of both compounds fromin the fixation of two discrete labeling patterns for ring a. P. nudicaule. The yellow pigment could arise by condensation of the tryptophan precursor, indole, Unsurprisingly, the benzenoid ring of the indole motif in repeats in every with the shikimate/polyketide-type intermediate pelargonidin vianudicaulin naringenin chalcone (Figure 18)detail [40]. the shikimate labeling pattern that we have seen in the phenolic ring of both compounds from P. nudicaule. The yellow pigment could arise by condensation of the tryptophan precursor, indole, with 12. The Role of the Time Coordinate in Work with 13CO2 the shikimate/polyketide-type intermediate pelargonidin via naringenin chalcone (Figure 18) [40]. Numerous plant physiology studies have used 13CO2 to analyze the kinetics of the transfer of 13 CO 12. The Rolephotosynthate of the Time Coordinate Work with primary into differentinmetabolic compartments. Not surprisingly, time is the most 2 important experimental parameter in work of this type13that is not discussed in detail in the present Numerous plant physiology studies have used CO2 to analyze the kinetics of the transfer review. of primary different metabolic pathways compartments. Not time isis the On photosynthate the other hand, into the work on biosynthesis discussed in surprisingly, the present review mostentirely important experimental parameter in work of this type that is not discussed in detail focused on the identification of biosynthetic building blocks, based on the concept that thein the present review. carbon skeletons of secondary metabolites are mosaic structures that are assembled from “Lego©” blocks fromtheprimary metabolite. Technically, that implicates the present detectionreview of On the extracted other hand, work on biosynthesis pathways discussed in the is 13C entities inside the target molecule structures by 13C-13C coupling via NMR and covalently-linked entirely focused on the identification of biosynthetic building blocks, based on the concept that 13C-labeled isotopologues. In other words, the required by massskeletons spectrometric of multiply the carbon of detection secondary metabolites are mosaic structures that are assembled from information resides in isotopologue space, as opposed to ime space. that implicates the detection of © “Lego ” blocks extracted from primary metabolite. Technically,

covalently-linked 13 C entities inside the target molecule structures by 13 C-13 C coupling via NMR and by mass spectrometric detection of multiply 13 C-labeled isotopologues. In other words, the required information resides in isotopologue space, as opposed to ime space.


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Figure 18. Proposed biosynthetic pathway for nudicaulin biosynthesis [40]. Figure 18. Proposed biosynthetic pathway for nudicaulin biosynthesis [40].

Closer analysis, however, reveals easily that the time coordinate is also relevant in biosynthetic 13C-labeled Closer however, reveals that time coordinate is also biosynthetic studies.analysis, In fact, the entire concept is easily based on thethe assembly of an inventory of relevant multiply in 13 studies. In fact, the concept is based the assembly an inventory multiply 13 C-labeled metabolites by aentire dynamic process, namelyon the formation of of highly C-labeledofphotosynthate in a 13 13CO2-containing atmosphere and its subsequent dispersal, by mixing with pools photosynthate of unlabeled in metabolites by a dynamic process, namely the formation of highly C-labeled photosynthate. Whereas the isotopologue distribution of biomatter is normally very close to a 13 CO 2 -containing atmosphere and its subsequent dispersal, by mixing with pools of unlabeled 13CO2 stochastic, a massive disequilibrium be generated by a dynamic process,very namely photosynthate. Whereas the isotopologuecan distribution of biomatter is normally closethe to stochastic, labeling pulse. The subsequent relaxation period is conducive to mixing of metabolites from highly a massive disequilibrium can be generated by a dynamic process, namely the 13 CO2 labeling different compartments of isotopologue space, namely the photosynthate from 13CO2 with biomass pulse. The subsequent relaxation period is conducive to mixing of metabolites from highly different that had been deposited in the prelabeling period and with biomass that is assembled de novo 13 compartments isotopologue during theof chase period. space, namely the photosynthate from CO2 with biomass that had been deposited The in the prelabeling period and with biomass that is assembled the chaseby period. chase (relaxation) period is conducive to the generationde ofnovo novelduring isotopologues 13 The chase (relaxation) is conducive to partially the generation of novel isotopologues recombination of building period blocks from unlabeled and or completely C-labeled precursor by molecules. of However, chase from period does not and havepartially a tendency reestablish13aC-labeled truly stochastic recombination buildingthe blocks unlabeled or to completely precursor isotopeHowever, distribution timescale of realistic Rather, the a return a stochastic molecules. thewithin chase the period does not have a experiments. tendency to reestablish trulyto stochastic isotope distribution within the timescale of realistic experiments. Rather, the return to a stochastic distribution, inside a closed system, would require very long periods of metabolization and would typically have to involve multiple species, including predators of plant biomass. In terms of isotopologue entropy,


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the chase period develops in the direction of a metastable state, rather than a new quasi-stochastic isotopologue distribution. A systematic optimization of pulse and chase duration has not been performed for the type of biosynthetic studies described in the first part of the article. In fact, the conditions used, with a pulse of a typical sunlight day and a chase period of several days, were essentially established serendipitously. In retrospect, it appears that the lengths of the time periods are not really critical in light of the fact that any state with a lowered isotope entropy that has been generated by the pulse period will coast toward a metastable state of isotopologue entropy during the chase period, rather than to a novel equilibrium state with an enhanced average abundance. In any case, the presently available data on biosynthetic studies using 13 CO2 as a universal precursor provide ample evidence that the approach is conducive to generating information on carbon metabolism that cannot easily be obtained by other techniques. Acknowledgments: We acknowledge the financial support of the Hans-Fischer Gesellschaft e.V., Munich, and the Deutsche Forschungsgemeinschaft (EI 384/8). We thank Bernd Schneider and Evangelos Tatsis for providing original data from the nudicaulin project and help with Figures 7, 17 and 18. Author Contributions: Adelbert Bacher and Wolfgang Eisenreich wrote the manuscript. Fan Chen helped to design the figures. Conflicts of Interest: The authors declare no conflict of interest.

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