NMR and Plant Metabolism Richard Bligny and Roland Douce*

191

NMR and plant metabolism Richard Bligny and Roland Douce*

Recent advances in NMR methodology offer a way to acquire (Figure 1). NMR experiments using 13C-labelled com- a comprehensive profile of a wide range of metabolites from pounds to decipher a metabolic pathway are usually various plant tissues or cells. NMR is a powerful approach for hampered by the low sensitivity of the 13C nucleus (which plant metabolite profiling and provides a capacity for the is four-fold lower than that of the 31P nucleus). This dynamic exploration of plant metabolism that is virtually problem can be overcome by using the cyclic j-cross unmatched by any other analytical technique. polarisation technique, which allows the indirect detection of 13C nuclei coupled to 1H nuclei by exploiting the high Current Opinion in Plant Biology 2001, 4:191–196 NMR sensitivity of protons [5]. The signal provided by the sensitive protons theoretically increases the signal/noise 1369-5266/01/$ — see front matter ratio by a factor of 64 over that of the 13C signal. It is also © 2001 Elsevier Science Ltd. All rights reserved. possible to enhance the 13C-signal using the nuclear Addresses Overhauser effect (i.e. a change in the intensity of a spec- Département de Biologie Moléculaire et Structurale, Laboratoire de troscopic signal caused by the irradiation of another Physiologie Cellulaire Végétale, CEA, CNRS et Université Joseph [ •] Fourier, 17 rue des martyrs, F 38054 Grenoble, Cedex 9, France nucleus during the NMR experiment) 6 . *e-mail: [email protected] NMR signal intensities are important analytically because at Abbreviation NMR nuclear magnetic resonance a given pulse sequence (signals are elicited by pulses of radiofrequency irradiation) they can be related to the con- tent within the tissue of the molecules that produce the Introduction signals. On the other hand, the chemical-shift effect ensures Nuclear magnetic resonance (NMR) spectroscopy can be that many metabolites, such as glucose, glucose-6-P and used to analyse the composition of tissues both in vivo and ATP, can be identified on the basis of their characteristic in various extracts. NMR has its origin in the net magnetic NMR signal patterns observed either in vivo (in a non-inva- moment or spin of an atomic nucleus that has an odd atom- sive way) or in vitro (in a perchloric extract). Moreover, the ic mass and/or an odd atomic number [1]. Common nuclei strength of NMR for tackling metabolism lies in the fact that that exhibit such magnetic properties are the highly abun- isotopic labels in different positions of different compounds dant isotopes 1H (99.98% in nature) and 31P (100% in can be simultaneously measured. nature) or the low abundance isotopes 13C (1.1% in nature) and 15N (0.37% in nature). The widespread use of NMR to In vivo NMR methods are frequently used to provide analyse plant metabolism and its compartmentation has information on the absolute concentrations of the more been reviewed recently [1,2,3••]. We have chosen to abundant metabolites, including sucrose, glucose 6-P and focus on restricted recent examples to convince the reader inorganic phosphate, and on how these concentrations that NMR is a powerful approach for plant metabolite change during biochemical transformation. These methods profiling [4••]. eliminate the need for extractions and sample preparation procedures. Several devices exist that are used to maintain NMR spectra a living system in a physiologically viable and controllable NMR signals (i.e. resonance) are observed when a sample state [1,2,3••]. These include the use of small leaf pieces is irradiated with pulses of radiofrequency electromagnetic that are infiltrated with perfusion medium, cell suspen- radiation in a strong magnetic field. Each nucleus within a sions at high cell density and root tips. These systems molecule experiences a slightly different magnetic field entail maintaining the supply of oxygen and various because of its distinct chemical environment and absorbs nutrients, including sugars, and the removal of waste energy at a slightly different frequency. The separation of products such as ethanol. By modifying the composition of these resonance frequencies from an arbitrarily chosen the circulating medium (e.g. by introducing phosphate or reference is called the chemical shift (δ), which is calculated amino acids, sucrose starvation or removing oxygen) it is on the basis of the relationship: possible to perturb the metabolism of the living system and to monitor the spectral changes caused by these δ = (resonance frequency of the sample [νs] – resonance changes simultaneously, thereby obtaining several succes- frequency of the reference [νr]) / (νr.106) sive spectra from the same sample. For example, aerobic and anaerobic conditions could be alternated at intervals of δ, which is independent of the field strength of the magnet, as short as 2 min. Examination of intact cells or tissues by is a unitless number that is expressed in parts per million NMR, however, gives relatively poor separation of signals (ppm). The result of an NMR analysis of a tissue is a spec- (i.e. allows more overlapping signals), loss of coupling multi- trum, that is, a plot of intensity (area) against chemical shift plicities (i.e. loss of J-coupling) and relatively poor in which each signal occurs at a characteristic energy signal-to-noise ratios. Hence, the detection and quantification 192 Physiology and metabolism

Figure 1

vac-His 12 h (Incubation with 100 µ M histidine) C3 His C2 cyt-His His His C5 C3 12 h

His His His C1 C4 C6

9 h

C 2 3 Sucrose HO 1 CHCH 2 4 5 6 h

NH2 N N H Histidine 6

3 h cit mal cit 0 h (Reference spectrum) Glu Glu 0 h

180 160 140 120 100 80 60 40 20 34 32 30 28 26 Chemical shift (δ), ppm Current Opinion in Plant Biology

13 A pulse-chase experiment utilising in vivo C-NMR techniques. Series incubation medium contained 1 mM KCl, 5 mM KNO3, 0.5 mM of NMR spectra registered during the incorporation of histidine by MgSO4, 0.5 mM Ca(NO3)2, 0.1 mM KH2PO4, and 4 mM glucose. The heterotrophic culture sycamore cells. The spectra were recorded at pH of the external medium was regulated to 6.0. Histidine (100 µM) 20 °C on a Bruker AMX 400, WB, spectrometer, which was equipped was added at time 1 h; a chase was started at time 16 h (see Figure 2) with a 25-mm probe tuned at 161.9 MHz. They are the result of after the rinsing of perfused cells with a nutrient medium devoid of 900 scans (1 h). The signal-to-noise ratio was maximised using a histidine. The total height of the 12 sucrose peaks is not shown. specific perfusion arrangement [13•], which optimised the Citrate (cit), malate (mal) and glutamate (Glu) are also identified. The homogeneity of the analysed samples. Cells (10 g wet weight) were profile of soluble metabolites present in the reference spectrum of placed on a porous plate near the bottom of a 25-mm NMR tube. The sycamore cells shows that sucrose is the most abundant compound. porous plate was crossed by a central output glass tube; an inlet tube Quantification assays show that its concentration was circa 80 µmol/g and a safety output tube were positioned 2 cm above the surface of cell wet weight. The incorporated histidine appears as six pairs of the sedimented cells. The nutrient medium was circulated though the peaks corresponding to the six carbon atoms of the molecule. Except cells via a peristaltic pump attached to these tubes and recycled in a for carbon C5, the right peak in each pair corresponds to histidine well-oxygenated external reservoir. The conditions for 13C-NMR present in an acidic compartment (i.e. the vacuole at pH 5.7) and the acquisition utilised 70-µs pulses (90°) at 5.6-s intervals and a sweep left one to histidine present in an alkaline compartment (i.e. the width of 20.73 kHz. Broad-band decoupling at 4W during acquisition cytoplasm at pH 7.5). Carbon C5 splits in the opposite direction and 0.5 W during delay were applied using the Waltz sequence. according to pH shifts. cyt-His, cytoplasmic histidine; vac-His, Spectra were referenced to hexamethyldisiloxane at 2.7 ppm. The cells vacuolar histidine.

of signals, especially of less intense signals, are typically from different elements is considerably reduced and inferior to those achieved using extracted tissue samples. coupling multiplicities are seen. In addition to one-dimen- For this reason, spectroscopy in vivo is usually supported sional NMR spectra from plant extracts, a wide range of by spectra taken from extracts of the cells or tissues being NMR-spectroscopic methods that generate two-dimen- investigated (i.e. from perchloric extracts that are devoid of sional spectra of different nuclei have now been paramagnetic cations such as manganese). Such extract developed. Indeed, a typical metabolite contains more spectra have sharp lines and hence overlapping of signals than one element that is detectable by NMR. NMR and plant metabolism Bligny and Douce 193

Furthermore, atoms of the same element within molecules Figure 2 are chemically non-equivalent and therefore worth distin- guishing from one another by NMR. The type of 20 additional information provided by 2D spectra and how it is interpreted is carefully described by Fan [7].

The various NMR spectroscopy techniques offer, there- Total (cyt+vac) fore, some unique ways to decipher metabolic networks in plants, to probe the metabolic response of tissues or cells to physiological [2,8–10,11•–13•] and chemical [14•] perturba- vac-His tions, to follow the fate of a stable-isotope-labelled molecule along a metabolic pathway [15–24,25•,26•], to visualise the unpredictable changes in metabolism reported mol/g cell wet wt) mol/g cell wet in genetically engineered plants [3••], and to analyse the µ 10 metabolic dialogue between plants and their microbial symbionts ([27]; see also Update).

NMR is also a useful technique for the determination of intracellular pH in a variety of tissues or cells. pH determination by NMR is based on the dependence of the Incorporated histidine ( chemical shift of various endogenous molecules (i.e. inor- cyt-His ganic phosphate, organic acids and amino acids) on intracellular pH [3••,13•] (Figures 1,2). 31P-NMR, for example, can discriminate vacuolar Pi (pH~5.5) from cytoplasmic Pi (pH~7.5), and this technique allows accurate 0 0 10 20 30 and non-invasive studies of trans-vacuolar proton move- Time (h) 31 ments. The usefulness of Pi as a P-NMR probe for the Current Opinion in Plant Biology measurement of pH in cells is, however, hampered by its sensitivity to ionic strength, its low and varying concen- Kinetics of incorporation of histidine in cells and compartmentation tration during metabolism (e.g. during Pi starvation) and the between the cytoplasm and the vacuole during the phase of large line widths of cellular peaks. (The line widths of incorporation and during the chase. Histidine accumulated first in the intracellular peaks originated from the natural heterogeneity cytoplasm up to circa 6 µmol/g cell wet weight. Then it accumulated steadily in the vacuole. During the first hours of chase (which begins at of cytoplasmic and vacuolar pHs within plant cells. The the arrows), this amino-acid decreased in the cytoplasm and increased broader the signal, the more difficult it is to define its symmetrically in the vacuole. As the vacuole-to-cytoplasm volume ratio shape and extent.) Consequently, it is almost impossible to is circa 7 in this material [2], one can estimate that the concentration characterise distinct pools of Pi within the cytoplasm. For of histidine was 20–24 mM in the cytoplasm and 12–14 mM in the vacuole after 14 h of chase. Note the slow decrease of the total pool 31 example, P-NMR cannot be utilised to study trans- of histidine, cytoplasmic plus vacuolar, corresponding to the mitochondrial or trans-plastidial proton movements (which metabolism of this amino acid (E Gout, R Bligny, R Douce, involve a ∆pH of less than 0.2 units). To solve these problems, unpublished data). Pietri et al. [28•] have designed a new series of non-toxic uncharged α- and β-aminophosphonates in which alkoxyl groups are linked to the phosphorus atom. This new class enzymatic reaction involving the translocation of a nucleus of highly sensitive 31P-NMR pH indicators, which are per- with a spin, transfer of magnetism directly reflects transfer meable to cell membranes and exhibit a low sensitivity to of mass. Metabolites of interest are labelled by orienting ionic strength, allow the investigation of a large range of their magnetic nuclei using radiowaves. Two-dimensional pKa values and offer the maximum NMR sensitivity at a NMR exchange spectroscopy has been used in an elegant given pKa value. way to study unidirectional fluxes through several of the enzymes of central metabolism (e.g. the activities of Two-dimensional phosphorus NMR exchange ATPases, phosphoglyceromutase, enolase, phosphogluco- spectroscopy mutase and UDP-glucose pyrophosphorylase) in hypoxic There are numerous reactions in plant cells in which maize root tips [29]. This method can potentially allow metabolites such as ATP or glucose 6-P turnover rapidly; the simultaneous monitoring of several unidirectional their rate of turnover can be quantified by the saturation enzymatic reactions at steady states in intact cells. This is transfer NMR technique [3••,8]. This is the technique of a distinct advantage over previous one-dimensional satu- choice for measuring the forward and backward rates of a ration transfer NMR experiments in which only single reaction driven in situ by an enzyme and, therefore, for exchange reactions were observed. Using this strategy, measuring unidirectional fluxes through the enzyme Roscher et al. [29] have shown that ATP turnover and gly- in situ in cells or tissues at steady states. Indeed, for any colytic flux increase with temperature up to the point at 194 Physiology and metabolism

Figure 3 which oxygen availability limits respiratory rate (i.e. until hypoxia). During the course of hypoxia they observed a net flux through phosphoglucomutase and UDP-glucose Time of sucrose starvation: pyrophosphorylase toward carbohydrate synthesis. Such a 120 h situation leads to a net production of pyrophosphate, P-Cho Pi which appears to be of the same order of magnitude as the flux needed for PPi-dependent phosphofructokinase to operate in the glycolytic direction.

P-EA Conclusions Non-destructive and non-invasive NMR can be used to investigate the metabolism of plants. This method allows the identification of molecules and ions in tissues or 48 h cells as well as in various extracts, the determination GPC of the absolute concentrations of the more abundant GPI mobile metabolites, the measurement of the change in concentration of key molecules during biochemical trans- formations, and the measurement of unidirectional 24 h Glycerol-3-P fluxes in intact cells or tissues at steady state. In addi- GPE tion, NMR can reveal unexpected information, including the discovery of novel compounds (Figure 3) that would escape detection by other analytical methods. 12 h Undoubtedly, this method will continue to decipher metabolic networks under changing physiological conditions in an increasingly sophisticated way. Research in GPG plant biology has been revolutionised over the past α decade by the creation of transgenic plants with the Glc-6-P intention of causing a specific perturbation along a metabolic pathway. There would seem to be considerable β Reference spectrum scope for using in vivo NMR to visualise, in a single snap-shot, the metabolites induced by the introduction Man-6-P Phytate Gluconate-6-P Fru-6-P of a foreign gene into a plant genome. Finally, rapid developments are occurring at the interface between NMR spectroscopy and NMR imaging [30]. Indeed PGA NMR imaging, which allows repeated imaging of the same specimen (e.g. a hypocotyl or a root), has a great 54 20 potential for the study of various physiological processes, Chemical shift (δ), ppm growth and development, water flow and environmental Current Opinion in Plant Biology effects.

Acknowledgements Representative in vitro 31P-NMR spectra for perchloric acid extracts of We would like to thank Dr Elizabeth Gout for her unflagging collaboration. sucrose-supplied sycamore cells (bottom spectrum) and cells starved of We would also like to thank Dr Claude Roby for his kind interest. sucrose for various periods (12–120 h). Cells harvested from the culture medium (9 g) were rinsed three times by successive resuspension in fresh culture medium devoid of sucrose and incubated at zero time in References and recommended reading sucrose-free culture medium. The spectra recorded at 20 °C are the Papers of particular interest, published within the annual period of review, results of 1024 transients (1h). Note the disappearance of hexoses-P have been highlighted as: and the steady accumulation of phosphorylcholine during the course of • of special interest sucrose starvation. The accumulation of phosphorylcholine, which •• of outstanding interest exhibits a remarkable metabolic inertness, is attributable to a massive intracellular membrane degradation (i.e. autophagy). In fact, this 1. Ratcliffe RG: In vivo NMR spectroscopy: biochemical and phosphodiester is derived from phosphatidylcholine degradation via the physiological applications to plants. In Nuclear Magnetic Resonance in Plant Biology. Edited by Shachar-Hill Y, Pfeffer PE. transient accumulation of glycerylphosphorylcholine. Fatty acids released Rockville: American Society of Plant Physiology; 1996:1-32. during the course of polar lipid degradation are utilised to fuel the remaining mitochondria with respiratory substrates (for more details 2. Aubert S, Bligny R, Douce R: NMR studies of metabolism in cell see [31]). Fru-6-P, fructose 6-P; Glc-6-P, glucose 6-P; GPC, suspensions and tissue culture. In Nuclear Magnetic Resonance in Plant Biology. Edited by Shachar-Hill Y, Pfeffer PE. Rockville: glycerylphosphorylcholine; GPE, glycerylphosphorylethanolamine; GPG, American Society of Plant Physiology; 1996:109-154. glycerylphosphorylglycerol; GPI, glycerylphosphorylinositol; Man-6-P, mannose 6-P; P-Cho, phosphorylcholine; P-EA, phosphorylethanolamine; 3. Roberts JKM: NMR adventures in the metabolic labyrinth within •• plants. 5 PGA, 3-phosphoglycerate. Trends Plant Sci 2000, :30-34. Roberts explains in an elegant way in which NMR has been used to obtain quantitative information about fluxes in metabolic networks. NMR and plant metabolism Bligny and Douce 195

••4. Fien O, Kopka J, Dörmann P, Altmann T, Trethewey RN, Willmitzer L: 15. Aubert S, Gout E, Bligny R, Douce R: Multiple effects of glycerol Metabolite profiling for plant functional genomics. Nature on plant cell metabolism. Phosphorus-31 nuclear magnetic Biotechnol 2000, 18:1157-1161. resonance studies. J Biol Chem 1994, 269:21420-21427. Gas chromatography coupled to electron-impact quadrupole mass spectrometry is a powerful technology for establishing rapidly, and in a reliable and sensitive way, 16. Dieuaide-Noubhani M, Raffard G, Canioni P, Pradet A, Raymond P: the metabolite profile of a single plant extract. Metabolite profiling provides a direct Quantification of compartmented metabolic fluxes in maize root link between a gene sequence and the function of the metabolic network in plants. tips using isotope distribution from 13C- or 14C-labeled glucose. J Biol Chem 1995, 270:13147-13159. 5. Heidenreich M, Köckenberger W, Kimmich R, Chandrakumar N, Bowtell R: Investigation of carbohydrate metabolism and transport 17. Ford YY, Ratcliffe RG, Robins RJ: Phytohormone-induced GABA in castor bean seedlings by cyclic j cross polarization imaging and production in transformed root cultures of Datura spectroscopy. J Magn Reson 1998, 132:109-124. stramonium: an in vivo 15N NMR study. J Exp Botany 1996, 47:811-818. 6. Kalusche B, Versht J, Gebauer G, Komor E, Haase A: Sucrose • unloading in the hypocotyl of the Ricinus communis L. seedling 18. Prabbhu V, Chatson KB, Abrams GD, King J: 13C nuclear magnetic measured by 13C-nuclear magnetic resonance spectroscopy resonance detection of interactions of serine in vivo. Planta 1999, 208:358-364. hydroxymethyltransferase with C1-tetrahydrofolate synthase and Nuclear-Overhauser-enhanced 13C-spectroscopy is developed to study the glycine decarboxylase complex activities in Arabidopsis. 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Roberts JKM, Aubert S, Gout E, Bligny R, Douce R: Cooperation and the involvement of putrescine in phytoformone-induced competition between adenylate kinase, nucleoside diphosphate de-differentiation. 205 kinase, electron transport, and ATP synthase in plant Planta 1998, :205-213. mitochondria studied by 31P-nuclear magnetic resonance. Plant 22. Krook J, Vreugdenhil D, Dijkema C, van der Plas LHW: Sucrose and Physiol 1997, 113:191-199. starch metabolism in carrot (Daucus carota L.) cell suspensions 13 11. Aubert S, Hennion F, Bouchereau A, Gout E, Bligny R, Dorne AJ: analysed by C-labelling: indications for a cytosol and a plastid- • Subcellular compartmentation of proline in the leaves of the localized oxidative pentose phosphate pathway. J Exp Botany subantarctic Kerguelen cabbage Pringlea antiscorbutica R. Br. 1998, 49:1917-1924. 13 In vivo C-NMR study. Plant Cell Environ 1999, 22:255-259. 23. Prabhu V, Chatson KB, Lui H, Abrams GD, King J: Effect of 13 Using in vivo C-NMR techniques in Pringlea leaves, it was possible to sulfanilamide and methotrexate on 13C fluxes through the glycine visualise the subcellular compartmentation of proline between cytoplasmic decarboxylase/serine hydroxymethyltransferase enzyme system and vacuolar compartments for the first time. This osmolyte accumulated at in Arabidopsis. Plant Physiol 1998, 116:137-144. a 2–3 times greater concentration in the cytoplasm than in the vacuole. 24. Schleucher J, Vanderveer PJ, Sharkey TD: Export of carbon from Carbon uptake 12. Pfeffer PE, Douds DD Jr, Bécard G, Shachar-Hill Y: chloroplasts at night. Plant Physiol 1998, 118:1439-1445. • and the metabolism and transport of lipids in an arbuscular mycorrhiza. Plant Physiol 1999, 120:587-598. 25. Mouillon JM, Aubert S, Bourguignon J, Douce R, Rébeillé F: Glycine Arbuscular mycorrhizal fungi are obligate symbionts that colonise the roots of • and serine catabolism in non-photosynthetic higher plant cells: the majority of crop plants. 13C-NMR is an ideal technique for following the their role in C1 metabolism. Plant J 1999, 20:197-205. fate of 13C-glucose through the fungus metabolic network. In this article, the Using the 13C-NMR technique and various 13C-labelled substrates (e.g. authors propose an elegant model for major fluxes of carbon in arbuscular [2-13C]serine and [2-13C]glycine), Mouillon et al. have shown that serine mycorrhizal fungi in the mycorrhizal state. According to this model, lipids are catabolism in plants (a cytosolic affair) is essentially connected to C1 metab- actively synthesised by the fungus within the roots (i.e. intraradically) from olism and that the glycine formed during this process is rapidly oxidised by 13C-glucose and are stored or exported to the extraradical mycelium. There, the mitochondrial glycine decarboxylase-serinehydroxymethyltransferase they are stored or metabolised to produce glucose the precursor of trehalose, enzymatic system. This serine–glycine cycle therefore involves reactions that a major storage disaccharide commonly found in fungi. take place in both the cytosolic and mitochondrial compartments. Indeed in Origin of the most organisms, the single carbon involved in folate-dependent processes is 13. Gout E, Boisson AM, Aubert S, Douce R, Bligny R: β • cytoplasmic pH changes during anaerobic stress in higher plants. derived from the -carbon of serine with the concomitant formation of Carbon-13 and phosphorous-31 nuclear magnetic resonance glycine. Assuming that for each utilisation of one C1 unit there is the pro- studies. Plant Physiol 2001, 125:1-14. duction of one glycine, it is clear that glycine catabolism via mitochondrial 31P NMR is a useful technique for determining the intracellular pH of a vari- GDC–SHMT coupled reactions is necessary. ety of tissues or cells. pH is determined on the basis of the dependence of 26. Gout E, Aubert S, Bligny R, Rébeillé F, Nonomura AR, Benson AA, the endogenous inorganic phosphate (Pi) chemical shift on intracellular pH. • Douce R: Metabolism of methanol in plant cells. Carbon-13 The intracellular pHs of Pi-deprived cells were measured using methylphos- nuclear magnetic resonance studies. Plant Physiol 2000, phonate as a non-metabolisable pH probe. The combined use of 13C- and 123:287-296. 31P-NMR to analyse changes during anaerobic stress in higher plant cells Using 13C-NMR, Gout et al. demonstrated that [13C]methanol admini- led to the following major conclusions: first, the proton-releasing metabolism [ 13 ] [13 ] stered to plant cells is metabolised to 3- C serine, CH3 methionine of ATP was at the origin of the cytoplasmic acidisis established in cells [13 ] and CH3 phosphatidylcholine. These authors concluded that the immediately after the imposition of anoxia, and second, the proton pump of [13 ] [13 ] the plasmalemma did not operate under anaerobic conditions. assimilation of C methanol occurs through the formation of CH3 tetrahydrofolate and S-adenosyl-methionine. Unexpectedly, during the 14. Aubert S, Pallett KE: Combined use of 13C and 19F-NMR to analyse course of this investigation the metabolism of [13C]methanol in plant cells • the mode of action and the metabolism of the herbicide also revealed the assimilation of label into a new cellular product, which [13 ] β isoxaflutole. Plant Physiol Biochem 2000, 38:517-523. was identified as CH3 methyl- -D-glucopyranoside. A specific methyl- The authors of this paper describe the use of 19F-NMR (many herbicides transferase using S-adenosyl-methionine is probably not involved in the including isoxaflutole are fluorinated molecules) to follow the metabolic fate synthesis of this compound. Instead, it could be synthesised via a non- of isoxaflutole in plant cells. 13C-NMR was used to characterise the effects specific transglycosylation process that is catalysed by an unknown of isoxaflutole on plant metabolism. hydrolase. 196 Physiology and metabolism

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