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Direct Observation of the Effects of 3,5-Diisopropylsalicylate and Light on Maize Coleoptiles using 31P NMR
Gustav Fischer Verlag Jena
Direct Observation of the Effects of 3,S-Diisopropylsalicylate and Light on Maize Coleoptiles using 31p NMR SNEZANA OBRENOVI( 1), R. GEORGE RATCLIFFE 2 ) and TIMOTHY E. SOUTHON 2 ) 1) Institute for Biological Research "Sinisa Stankovic", Belgrade, Yugoslavia; 2) Department of
Plant Sciences, University of Oxford, Oxford, U.K.
Key Term Index: 3,5-diisopropylsalicylic acid, maize coleoptiles, metabolic effects, nuclear magnetic resonance, phytochrome; Zea mays
Summary Metabolic effects caused by 3,5-diisopropylsalicylate (DIPS) and light were observed in etiolated maize coleoptiles using in vivo 31p NMR spectroscopy. A decrease in the nucleotide triphosphate (NTP) resonances and an increase in nucleotide diphosphate hexose (NDP-hexose) signals were induced by 200 11M DIPS, with an acidification of the cytoplasm occurring in a fraction of the cells. Increased hexose phosphate and NDP-hexose signals, accompanied by a decreased cytoplasmic Pi resonance, were observed under intermittent blue light, while in red light only the increase of the hexose-P and decrease in cytoplasmic Pi signals occurred. These results provide evidence for an analogy between the action of DIPS and light at the molecular level, while the similar effects of red and blue light are compatible with phytochrome as the principal photoreceptor responsible for the metabolic changes.
Introduction Various compounds, in addition to plant hormones, can mimic the regulatory action of light in plant development. In Amaranthus caudatus L. seedlings, the enhancement of betacyanin formation by lumiflavin showed a clear interaction with light (OBRENOVIC 1986), while another lipophilic compound, DIPS and its copper complex, simulated the effect of light in a manner similar to that of an allosteric activator (OBRENOVIC and SPASIC 1988). A more detailed study of lumiflavin (OBRENOVIC 1986) and Cu DIPS action in betacyanin formation revealed changes of the apparent Km and So.5 values that resembled the light-inactivation of PEPC from CAM plants in G-6-P (Wu and WEDDING 1985; OBRENOVIC 1990). Indeed, fusicoccin, which also mimicks the action of light (ELLIOTT 1979), increased the amount of G-6-P and malate in other plant systems (JOHNSON and RAYLE 1976; GUERN et al. 1982; TRocKNER and MARRE 1988). Abbreviations: B, blue light; CAM, crassulacean acid metabolism; DIPS, 3,5-diisopropylsalicylic acid; F-2,6-BP, fructose-2,6-bisphosphate; G-6-P, glucose-6-phosphate; hexose-P, hexose phosphate; Ka, activation constant; MES, 4-morpholinoethanesulfonic acid; NDP, nucleotide diphosphate; NMR, nuclear magnetic resonance; NTP, nucleotide triphosphate; Pi, inorganic phosphate; PEPC, phosphoenolpyruvate carboxylase; Pfr, far-red absorbing form of phytochrome; Pr, red absorbing form of phytochrome; R, red light; TEMDP, tetraethylester of methylene diphosphate.
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An increase in G-6-P has been observed in a unicellular alga upon blue light treatment, using in vivo 31p NMR spectroscopy (RUYTERS 1988), and we have explored the possibility of using the same approach to investigate the metabolic effects of light and a light-mimicking compound, DIPS, in higher plant tissues. The immediate problem is that in contrast to the situation in an algal cell suspension, the number of cells containing phytochrome in higher plant tissue is very limited (PRATT et al. 1976). However using maize coleoptiles, which respond to both R and B (BRIGGS 1960), we have observed small spectroscopic changes in response to illumination and we report these results and the spectroscopic effect of DIPS, in this paper.
Material and Methods Maize seeds (Zea mays L.) cv. ZP SC 46A, from Maize Research Institute (Zemun Polje, Belgrade, Yugoslavia) were washed with tap water for 2 h and germinated in the dark for 5 days at 25°C between sheets of absorbent paper soaked in 0.1 mM CaS04' The 5 mm coleoptile tips, with negligible contamination from leaf tissue, were excised under green safelight and vacuum infIltrated for 15 min in 10 mM MES/O.l mM CaS04, pH 6.0 (MES buffer). Vacuum infIltration was necessary to eliminate the line-broadening effects of the intercellular air spaces. Coleoptiles with air spaces floated and gave poor quality NMR spectra that were unsuitable for detailed interpretation, whereas coleoptiles that had been infIltrated until they sank gave well resolved spectra. Approximately 80 coleoptiles were transferred to a 10 mm diameter tube and oxygenated MES buffer was circulated through the tube at 4ml min-I. The tissue was allowed to stabilize for 15 min before starting the NMR recordings. The 31p NMR spectra were recorded at 121.49 MHz on a Bruker CXP 300 spectrometer, using procedures described elsewhere (KIME et al. 1982a; LEE and RATCLIFFE 1983; RATCLIFFE 1987). The plant tissue was maintained at 21-22 °C (in the air-conditioned dark room) and the spectra were usually accumulated with a 90° pulse and a recycle time of 0.8 s. The number of scans per 30 min interval was 2,250. Chemical shifts were measured relative to the signal from a capillary containing a 2% aqueous solution of TEMDP (KIME et al. 1982a) and are quoted relative to the resonance at oppm from 85% H3P04. The tissue was illuminated in the NMR tube, using a 150 W Schott cold light source (KL-1500T) and a fibre optic cable, 1 m long, inserted through the probehead to the NMR tube. This arrangement produced up to 6.500 Ilmol m- 2 S-I of light at the end of the light pipe, in the range 400-700 nm. Light filters were inserted between the light source and the fibre optical cable, and their characteristics, as well as the photoconversion of phytochrome, computed according to phytochemical parameters reported by KELLY and LAGARIAS (1985), are indicated in the figure captions. DIPS, obtained from Aldrich (U.K.), was dissolved immediately before use, recycled through the tube during treatments, and thoroughly rinsed from the circulating system with a detergent before the next experiment. The solubility of DIPS was enhanced by a concentrated NaOH solution in an initially small volume of MES buffer, and the pH readjusted to 6.0 after approximating the final volume.
Results and Discussion Figure 1a shows the 31p NMR spectrum of a sample of excised maize coleoptiles. The spectrum is similar to the 31p NMR spectra of other plant tissues and the signals can be assigned by analogy with previous work on the maize root tip spectrum (ROBERTS et al. 1980, KIME et al. 1982a, LEE and RATCLIFFE 1983, PFEFFER et al. 1988). There was very little change in the spectrum over a 6 h period, indicating that the tissue preparation was stable and that spectroscopic changes arising from experimental treatments could be BPP 188 (1992) 2
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Fig. 1. 31P NMR spectra of a maize coleoptile sample during a control experiment. The starting spectrum (a) was recorded 15 min after the sample had been transferred to the NMR tube and the second spectrum (b) was recorded 5 h later. Each spectrum took 1 h to accumulate with a 0.8 s recycling time . The resonance assignements are: 1, several phosphomonoesters, including G-6-P (1 a) and phosphorylcholine (1 c) ; 2, cytoplasmic Pi ; 3, vacuolar Pi with several underlying phosphodiesters; 4, the y-phosphate of nucleotide triphosphates (NTP, mainly ATP); 5, (X-NTP (ATP); 6, nucleoside diphospho-sugars (NDP-hexose), principally UDP-glucose; 7, NDP-hexose; 8, ~-NTP (ATP).
looked for with confidence. In contrast, attempts to use pea internodes in this work were less successful because of a pronounced drift in the vacuolar Pi signal during control experiments (data not shown). Exposure to 200 flM DIPS for 2 h caused the following changes in the maize coleoptile spectrum (Fig. 2): (i) an increase in the phosphomonoester region (peak 1): (ii) an increase in intensity upfield of peak 2, indicating the acidification of the cytoplasm in a fraction of the tissue ; (iii) a decrease in the NTP resonances; and (iv) an increase in the NDP-hexose resonances (peak 6 and 7). Preliminary experiments with pea internodes and 50 flM DIPS showed a similar shift in the cytoplasmic Pi resonance and a decrease in the NTP signals (data not shown). As a lipophilic weak acid, DIPS might be expected to reduce transmembrane proton gradients and thus uncouple oxidative phosphorylation. Two of the spectroscopic effects, the shift in the cytoplasmic Pi signal and the fall in the 138
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Fig. 2. 3 / P NMR spectra of a maize coleoptile sample recorded: (a) with MES buffer circulating through the NMR tube ; and (b) after switching the circulating medium to 200 tJ.M DIPS in MES buffer. Each spectrum took 2 h to accumulate. Increases (e) and decreases (0) occurred in the intensities of the labelled , cytoplasmic resonances .
NTP resonances, are consistent with this and have been observed previously in maize root tips exposed to the uncoupler 2,4-dinitrophenol (KIME et al. 1982a). However, the effect of DIPS differed from 2,4-dinitrophenol in that firstly, only a fraction of the tissue appeared to be affected, since a strong cytoplasmic signal remained at the original position (Fig. 2b), and secondly, the NDP-hexose signals increased whereas with 2,4dinitrophenol they disappeared. The increased NDP-hexose level, which can be attributed to an increase in UDP-glucose , would be expected to lead to an increase in G-6-P since the UTP:glucose-l-phosphate uridyltransferase reaction is near equilibrium in vivo (ROBERTS 1990), and this suggests that the increased intensity in phosphomonoester region could be due to increased G-6-P in cells with a reduced cytoplasmic pH. While DIPS, as a permeant weak acide, might affect the bulk of the coleoptile tissue, the effects of light are expected to occur only in cells containing phytochrome. These cells usually represent no more than 10 % of plant tissues (PRATT et al. 1976), and so irrespective of the uncertainty inherent in contrOlling the illumination of the tissue in the NMR tube , light-induced spectroscopic changes are likely to be smaller than those observed with DIPS . In keeping with this prediction, the changes induced by B and R were small and not easy to observe (Figs . 3 and 4). Pulses of B caused an increase in the G-6-P resonance (Perak 1 a), a decrease in the cytoplasmic Pi resonance (Peak 2) and a BPP 188 (1992) 2
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Fig. 3. 31p NMR spectra of a maize coleoptile sample recorded: (a) before illumination; and (b) while irradiated with intermittent blue light. Each spectrum took 2 h to accumulate and the intensity changes in the cytoplasmic resonances are indicated by the labels. The blue interference filter had a maximum transmission of 32 % at 451 nm and a half-band width of 7.5 nm. The 2 min of blue light, given each 10 min at a 2.5 cm distance from the sample in the tube, would be expected to maintain the Pfr fraction at 0.24. The corresponding fractions of phytochrome dimers are: 0.577 Prz, 0.365 PrPfr and 0.058 Pfrz.
small increase in the NDP-hexose signals (Fig. 3). Pulses of R caused a small increase in G-6-P and a small decrease in the cytoplasmic Pi (Fig. 4). The spectra in Figs. 3 and 4 are representative of at least three independent experiments. The changes in these spectra point to a similarity between the metabolic effects of light and DIPS. Thus, an increase in G-6-P was observed directly in B (Fig. 3) and R (Fig. 4), while the same effect could be inferred in the DIPS experiments from the increased NDP-hexose resonance (Fig. 2). This raises the possibility of an analogy at the molecular level between the effects of light and the light-mimicking action of DIPS (OBRENOVIC and SPASIC 1988). The decrease of NTP by DIPS could trigger the breakdown of carbohydrates, indicated by the increased NDP-hexose resonance, via the increased cytoplasmic Pi and/or decreased nucleotide energy charge (NEWSHOLME and START 1980). However, this possibility is not compatible with the decrease of NDP-hexose in 2,4-dinitrophenol (KIME et al. 1982a). On the other hand, the small 140
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Fig. 4. The effect of intermittent red light on phosphorus metabolites in maize coleoptiles. The spectra were recorded: (a) before illumination and (b) while irradiated with intermittent red light (20 s of red light applied at 10 min intervals). Each spectrum took 1 h to accumulate and the intensity changes in the cytoplasmic resonances are indicated by the label . The red interference filter, with maximum emission 40% at 651 nm and a half-band width of 7 nm, could maintain the fraction of Pfr at roughly 0.82 . The fractions of phytochrome dimers are : 0.032 Pr2 , 0.295 PrPfr and 0.672 Pfr2.
increase in G-6-P by light is accompanied by a decreased cytoplasmic Pi, suggesting a more specific mechanism that initiates carbohydrate breakdown. This would suggest an increase of F-2,6-BP, in accordance with current views on metabolic regulation (HUBER 1986). The changes induced by DIPS could also result from a similar mechanism, if allosteric activation of a more active PEPC (like the dark form in CAM plants) produces an excess of malate which favours a non-protonomotive electron flow, according to RUSTIN et al. (1984). The resulting decrease of the adenylate energy charge and increase of Pi, which could support a positive feedback in sucrolysis, closely resembles the effect offusicoccin in other plants (JOHNSON and RAYLE 1976; GUERN et aI. 1982; TROCKNER and MARRE 1988), in agreement with the light mimicking action of both compounds in betacyanin formation (ELLIOTT 1979; OBRENOVIC and SPASIC 1988). This possibility is compatible with the analogy between the kinetic parameters for CuDIPS action and allosteric activation of a CAM-like PEPC (OBRENOVIC 1991), supporting the view that PEPC plays a crucial role in light signal transduction. It is also consistent with the same BPP 188 (1992) 2
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effect of lower DIPS concentrations on pea internodes (data not shown), which can be attributed to lower Ka values of PEPC (WEDDING et al. 1989). The increase of G-6-P, induced by continuous B illumination in a chlorophyll-free mutant of Chiorella (RUYTERS 1988), was also ascribed to an enhanced breakdown of carbohydrates (KOWALLIK 1982), but it was considered in terms of a hypothetical photoreceptor "cryptochrome" (KOWALLIK 1982; RUYTERS 1988). However, the same effects of both Rand B on G-6-P and cytosolic Pi signals in maize coleoptiles (also a chlorophyll-free tissue) are consistent with phytochrome as the only identified photoreceptor in higher plants. The difference between Rand B, with respect to NDP-hexose resonance (Figs. 3 and 4 ), may be due to the presence of Pr2 dimers (see comments with Figs. 3 and 4), which support the more active (dark) form of PEPC and/or F-6-P 2kinase. The only possible source of carbohydrates in coleoptiles is sucrose supplied from cotyledons before excision, which seems to be sufficient to maintain a steady state level of phosphorus metabolites over the long period of acquisition (Fig. 1). In conclusion, 31p NMR spectroscopy provides some evidence for a common metabolic effect of light and a light-mimicking compound, although only a limited number of cells appear to undergo detectable metabolic changes, making the use of the technique difficult, but still more plausible than any extraction procedures, where sample variations can level off the small differences in crucial metabolites. Acknowledgements S. OBRENOVIC acknowledges the financial support of the British Council and the Cecil Pilkington Charitable Trust. This work was also supported by the Scientific Research Fund of SR Serbia to S.O. and by the U. K. Agricultural and Food Research Council to R.G.R. and T.E.S.
References BRIGGS, W. R.: Red light, auxin production and the phototropic responses of com and oat coleoptiles. Am. J. Bot. 50, 196-207 (1960). ELLIOTT, D. C.: Differential effects of fusicoccin- and cytokinin-stimulated betacyanin synthesis by ATP-ase inhibitors and uncouplers. Plant Sci. Lett. 15, 251-264 (1979). GUERN, J., KURKDJIAN, A., and MATHIEU, Y.: Hormonal regulation of intracellular pH: hypothesis versus facts. In: Plant Growth Substances 1982. (Ed. P. F. WAREING) pp. 427-437. Academic Press, London 1982. HUBER, S. c.: Fructose-2,6-bisphosphate as a regulatory metabolite in plants. Annu. Rev. Plant Physiol. 37, 233-246 (1986). JOHNSON, K. D., and RAYLE, D. L.: Enhancement of CO 2 uptake in Avena coleoptiles by fusicoccin. Plant Physiol. 57, 806-811 (1976). KELLY, J. M., and LAGARIAS, J. c.: Photochemistry of 124-kilodalton Avena phytochrome under constant illumination in vitro. Biochemistry 24, 6003-6010 (1985). KIME, M. J., LOUGHAM, B. c., and RATCLIFFE, R. G.: The application of 31p nuclear magnetic resonance to higher plant tissue. 2. Detection of intracellular changes. J. Exp. Bot. 33, 670-681 (l982a). KIME, M. J.. LOUGHAM. B. C., RATCLIFFE, R. G., and WILLIAMS, R. J. P.: The application of 31p nuclear magnetic resonance to higher plant tissue. 1. Detection of spectra. J. Exp. Bot. 33, 656-669 (1982b). 142
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KOWALLIK, W.: Blue light effects on respiration. Annu. Rev. Plant Physiol. 33, 51-72 (1982). LEE, R. B., and RATCLIFFE, R. G.: Development of an aeration system for use in plant tissue NMR experiments. J. Exp. Bot. 34, 1213-1221 (1983). NEWSHOLME, E. A., and START, c.: Regulation of Metabolism, John Wiley & Sons, ChichesterNew York-Brisbone-Toronto 1980. OBRENOVIC, S.: Effect of lumiflavin on light induction of betacyanin accumulation in Amaranthus caudatus L. seedlings. Photobiochem. Photobiophys. 13, 95-103 (1986). OBRENOVIC, S., and SPASIC, M.: Effects of copper chelates on betacyanin accumulation in Amaranthus seedlings. Plant Physiol. Biochem. 26, 597-601 (1988). OBRENOVIC, S.: Molecular basis for the wavelength, fluence rate and daylength sensing by plants. J. Photochem. Photobiol. 8, 237-239 (1991). PFEFFER, P. E., Tu, S. I., GERASIMOWICZ, W. V., and CAVANAUGH, J. R.: In vivo 31p NMR studies of corn tissue and its uptake of toxic metals. Plant Physiol. 80, 77-84 (1986). PRATT, L. H., COLEMAN, R. A., and MACKENZIE, J. M.: Immunological visualization of phytochrome. In: Light and Plant Development (Ed. H. SMITH) pp. 75-94. Butterworths, London-Boston 1976. RA TCLIFFE, R. G.: Applicaiton of nuclear magnetic resonance methods to plant tissues. Methods in Enzymology 148, 683-700 (1987). ROBERTS, J. K. M.: Observation of uridine triphosphate: glucose-I-phosphate uridyltransferase activity in maize root tips by saturation transfer 31p NMR. Estimation of cytoplasmic PPi. Biochem. Biophys. Acta 1051, 29-36 (1990). ROBERTS, J. K. M., RAY, P. M., WADE-JARDETZKY, N., and JARDETZKY, 0.: Estimation of cytoplasmic and vacuolar pH in higher plant cells using 31p NMR. Nature 283, 870-872 (1980). RUSTIN, P., DUPONT, J., and LANCE, c.: Involvement of lipid peroxy radicals in the cyanideresistant electron transport pathway. Physiol. Veg. 22, 643-663 (1984). RUYTERS, G.: Regulation of carbohydrate breakdown of Chiarella mutant No 20, studied by 31p NMR spectroscopy and enzymatic analysis. Plant & Cell Physiol. 29, 355- 363 (1988). TROCKNER, V., and MARRE, E.: Plasmamembrane redox chain and H+ extrusion. II. respiratory and metabolic changes associated with fusicoccin-induced and with ferricyanide-induced H+ extrusion. Plant Physiol. 87, 30- 35 (1988). WEDDING, R. T., BLACK, J. M., and MEYER, C. R.: Activation of higher plant phosphoenolpyruvate carboxylase by glucose-6-phosphate. Plant Physiol. 90, 648-652 (1989). Wu, M.-X., and WEDDING, R. T.: Diurnal regulation of phosphoenolpyruvate carboxylase from Crassula. Plant Physiol. 77, 667-675 (1985). Received May 6, 1991; revised form accepted September 23, 1991. Authors' address: SNEZANA OBRENOVIC, Institute for Biological Research "Sinisa Stankovic", 29. Novembra 142, 11060 Belgrade. Yugoslavia.
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Direct Observation of the Effects of 3,S-Diisopropylsalicylate and Light on Maize Coleoptiles using 31p NMR SNEZANA OBRENOVI( 1), R. GEORGE RATCLIFFE 2 ) and TIMOTHY E. SOUTHON 2 ) 1) Institute for Biological Research "Sinisa Stankovic", Belgrade, Yugoslavia; 2) Department of
Plant Sciences, University of Oxford, Oxford, U.K.
Key Term Index: 3,5-diisopropylsalicylic acid, maize coleoptiles, metabolic effects, nuclear magnetic resonance, phytochrome; Zea mays
Summary Metabolic effects caused by 3,5-diisopropylsalicylate (DIPS) and light were observed in etiolated maize coleoptiles using in vivo 31p NMR spectroscopy. A decrease in the nucleotide triphosphate (NTP) resonances and an increase in nucleotide diphosphate hexose (NDP-hexose) signals were induced by 200 11M DIPS, with an acidification of the cytoplasm occurring in a fraction of the cells. Increased hexose phosphate and NDP-hexose signals, accompanied by a decreased cytoplasmic Pi resonance, were observed under intermittent blue light, while in red light only the increase of the hexose-P and decrease in cytoplasmic Pi signals occurred. These results provide evidence for an analogy between the action of DIPS and light at the molecular level, while the similar effects of red and blue light are compatible with phytochrome as the principal photoreceptor responsible for the metabolic changes.
Introduction Various compounds, in addition to plant hormones, can mimic the regulatory action of light in plant development. In Amaranthus caudatus L. seedlings, the enhancement of betacyanin formation by lumiflavin showed a clear interaction with light (OBRENOVIC 1986), while another lipophilic compound, DIPS and its copper complex, simulated the effect of light in a manner similar to that of an allosteric activator (OBRENOVIC and SPASIC 1988). A more detailed study of lumiflavin (OBRENOVIC 1986) and Cu DIPS action in betacyanin formation revealed changes of the apparent Km and So.5 values that resembled the light-inactivation of PEPC from CAM plants in G-6-P (Wu and WEDDING 1985; OBRENOVIC 1990). Indeed, fusicoccin, which also mimicks the action of light (ELLIOTT 1979), increased the amount of G-6-P and malate in other plant systems (JOHNSON and RAYLE 1976; GUERN et al. 1982; TRocKNER and MARRE 1988). Abbreviations: B, blue light; CAM, crassulacean acid metabolism; DIPS, 3,5-diisopropylsalicylic acid; F-2,6-BP, fructose-2,6-bisphosphate; G-6-P, glucose-6-phosphate; hexose-P, hexose phosphate; Ka, activation constant; MES, 4-morpholinoethanesulfonic acid; NDP, nucleotide diphosphate; NMR, nuclear magnetic resonance; NTP, nucleotide triphosphate; Pi, inorganic phosphate; PEPC, phosphoenolpyruvate carboxylase; Pfr, far-red absorbing form of phytochrome; Pr, red absorbing form of phytochrome; R, red light; TEMDP, tetraethylester of methylene diphosphate.
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An increase in G-6-P has been observed in a unicellular alga upon blue light treatment, using in vivo 31p NMR spectroscopy (RUYTERS 1988), and we have explored the possibility of using the same approach to investigate the metabolic effects of light and a light-mimicking compound, DIPS, in higher plant tissues. The immediate problem is that in contrast to the situation in an algal cell suspension, the number of cells containing phytochrome in higher plant tissue is very limited (PRATT et al. 1976). However using maize coleoptiles, which respond to both R and B (BRIGGS 1960), we have observed small spectroscopic changes in response to illumination and we report these results and the spectroscopic effect of DIPS, in this paper.
Material and Methods Maize seeds (Zea mays L.) cv. ZP SC 46A, from Maize Research Institute (Zemun Polje, Belgrade, Yugoslavia) were washed with tap water for 2 h and germinated in the dark for 5 days at 25°C between sheets of absorbent paper soaked in 0.1 mM CaS04' The 5 mm coleoptile tips, with negligible contamination from leaf tissue, were excised under green safelight and vacuum infIltrated for 15 min in 10 mM MES/O.l mM CaS04, pH 6.0 (MES buffer). Vacuum infIltration was necessary to eliminate the line-broadening effects of the intercellular air spaces. Coleoptiles with air spaces floated and gave poor quality NMR spectra that were unsuitable for detailed interpretation, whereas coleoptiles that had been infIltrated until they sank gave well resolved spectra. Approximately 80 coleoptiles were transferred to a 10 mm diameter tube and oxygenated MES buffer was circulated through the tube at 4ml min-I. The tissue was allowed to stabilize for 15 min before starting the NMR recordings. The 31p NMR spectra were recorded at 121.49 MHz on a Bruker CXP 300 spectrometer, using procedures described elsewhere (KIME et al. 1982a; LEE and RATCLIFFE 1983; RATCLIFFE 1987). The plant tissue was maintained at 21-22 °C (in the air-conditioned dark room) and the spectra were usually accumulated with a 90° pulse and a recycle time of 0.8 s. The number of scans per 30 min interval was 2,250. Chemical shifts were measured relative to the signal from a capillary containing a 2% aqueous solution of TEMDP (KIME et al. 1982a) and are quoted relative to the resonance at oppm from 85% H3P04. The tissue was illuminated in the NMR tube, using a 150 W Schott cold light source (KL-1500T) and a fibre optic cable, 1 m long, inserted through the probehead to the NMR tube. This arrangement produced up to 6.500 Ilmol m- 2 S-I of light at the end of the light pipe, in the range 400-700 nm. Light filters were inserted between the light source and the fibre optical cable, and their characteristics, as well as the photoconversion of phytochrome, computed according to phytochemical parameters reported by KELLY and LAGARIAS (1985), are indicated in the figure captions. DIPS, obtained from Aldrich (U.K.), was dissolved immediately before use, recycled through the tube during treatments, and thoroughly rinsed from the circulating system with a detergent before the next experiment. The solubility of DIPS was enhanced by a concentrated NaOH solution in an initially small volume of MES buffer, and the pH readjusted to 6.0 after approximating the final volume.
Results and Discussion Figure 1a shows the 31p NMR spectrum of a sample of excised maize coleoptiles. The spectrum is similar to the 31p NMR spectra of other plant tissues and the signals can be assigned by analogy with previous work on the maize root tip spectrum (ROBERTS et al. 1980, KIME et al. 1982a, LEE and RATCLIFFE 1983, PFEFFER et al. 1988). There was very little change in the spectrum over a 6 h period, indicating that the tissue preparation was stable and that spectroscopic changes arising from experimental treatments could be BPP 188 (1992) 2
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Fig. 1. 31P NMR spectra of a maize coleoptile sample during a control experiment. The starting spectrum (a) was recorded 15 min after the sample had been transferred to the NMR tube and the second spectrum (b) was recorded 5 h later. Each spectrum took 1 h to accumulate with a 0.8 s recycling time . The resonance assignements are: 1, several phosphomonoesters, including G-6-P (1 a) and phosphorylcholine (1 c) ; 2, cytoplasmic Pi ; 3, vacuolar Pi with several underlying phosphodiesters; 4, the y-phosphate of nucleotide triphosphates (NTP, mainly ATP); 5, (X-NTP (ATP); 6, nucleoside diphospho-sugars (NDP-hexose), principally UDP-glucose; 7, NDP-hexose; 8, ~-NTP (ATP).
looked for with confidence. In contrast, attempts to use pea internodes in this work were less successful because of a pronounced drift in the vacuolar Pi signal during control experiments (data not shown). Exposure to 200 flM DIPS for 2 h caused the following changes in the maize coleoptile spectrum (Fig. 2): (i) an increase in the phosphomonoester region (peak 1): (ii) an increase in intensity upfield of peak 2, indicating the acidification of the cytoplasm in a fraction of the tissue ; (iii) a decrease in the NTP resonances; and (iv) an increase in the NDP-hexose resonances (peak 6 and 7). Preliminary experiments with pea internodes and 50 flM DIPS showed a similar shift in the cytoplasmic Pi resonance and a decrease in the NTP signals (data not shown). As a lipophilic weak acid, DIPS might be expected to reduce transmembrane proton gradients and thus uncouple oxidative phosphorylation. Two of the spectroscopic effects, the shift in the cytoplasmic Pi signal and the fall in the 138
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Fig. 2. 3 / P NMR spectra of a maize coleoptile sample recorded: (a) with MES buffer circulating through the NMR tube ; and (b) after switching the circulating medium to 200 tJ.M DIPS in MES buffer. Each spectrum took 2 h to accumulate. Increases (e) and decreases (0) occurred in the intensities of the labelled , cytoplasmic resonances .
NTP resonances, are consistent with this and have been observed previously in maize root tips exposed to the uncoupler 2,4-dinitrophenol (KIME et al. 1982a). However, the effect of DIPS differed from 2,4-dinitrophenol in that firstly, only a fraction of the tissue appeared to be affected, since a strong cytoplasmic signal remained at the original position (Fig. 2b), and secondly, the NDP-hexose signals increased whereas with 2,4dinitrophenol they disappeared. The increased NDP-hexose level, which can be attributed to an increase in UDP-glucose , would be expected to lead to an increase in G-6-P since the UTP:glucose-l-phosphate uridyltransferase reaction is near equilibrium in vivo (ROBERTS 1990), and this suggests that the increased intensity in phosphomonoester region could be due to increased G-6-P in cells with a reduced cytoplasmic pH. While DIPS, as a permeant weak acide, might affect the bulk of the coleoptile tissue, the effects of light are expected to occur only in cells containing phytochrome. These cells usually represent no more than 10 % of plant tissues (PRATT et al. 1976), and so irrespective of the uncertainty inherent in contrOlling the illumination of the tissue in the NMR tube , light-induced spectroscopic changes are likely to be smaller than those observed with DIPS . In keeping with this prediction, the changes induced by B and R were small and not easy to observe (Figs . 3 and 4). Pulses of B caused an increase in the G-6-P resonance (Perak 1 a), a decrease in the cytoplasmic Pi resonance (Peak 2) and a BPP 188 (1992) 2
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Fig. 3. 31p NMR spectra of a maize coleoptile sample recorded: (a) before illumination; and (b) while irradiated with intermittent blue light. Each spectrum took 2 h to accumulate and the intensity changes in the cytoplasmic resonances are indicated by the labels. The blue interference filter had a maximum transmission of 32 % at 451 nm and a half-band width of 7.5 nm. The 2 min of blue light, given each 10 min at a 2.5 cm distance from the sample in the tube, would be expected to maintain the Pfr fraction at 0.24. The corresponding fractions of phytochrome dimers are: 0.577 Prz, 0.365 PrPfr and 0.058 Pfrz.
small increase in the NDP-hexose signals (Fig. 3). Pulses of R caused a small increase in G-6-P and a small decrease in the cytoplasmic Pi (Fig. 4). The spectra in Figs. 3 and 4 are representative of at least three independent experiments. The changes in these spectra point to a similarity between the metabolic effects of light and DIPS. Thus, an increase in G-6-P was observed directly in B (Fig. 3) and R (Fig. 4), while the same effect could be inferred in the DIPS experiments from the increased NDP-hexose resonance (Fig. 2). This raises the possibility of an analogy at the molecular level between the effects of light and the light-mimicking action of DIPS (OBRENOVIC and SPASIC 1988). The decrease of NTP by DIPS could trigger the breakdown of carbohydrates, indicated by the increased NDP-hexose resonance, via the increased cytoplasmic Pi and/or decreased nucleotide energy charge (NEWSHOLME and START 1980). However, this possibility is not compatible with the decrease of NDP-hexose in 2,4-dinitrophenol (KIME et al. 1982a). On the other hand, the small 140
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-10
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Fig. 4. The effect of intermittent red light on phosphorus metabolites in maize coleoptiles. The spectra were recorded: (a) before illumination and (b) while irradiated with intermittent red light (20 s of red light applied at 10 min intervals). Each spectrum took 1 h to accumulate and the intensity changes in the cytoplasmic resonances are indicated by the label . The red interference filter, with maximum emission 40% at 651 nm and a half-band width of 7 nm, could maintain the fraction of Pfr at roughly 0.82 . The fractions of phytochrome dimers are : 0.032 Pr2 , 0.295 PrPfr and 0.672 Pfr2.
increase in G-6-P by light is accompanied by a decreased cytoplasmic Pi, suggesting a more specific mechanism that initiates carbohydrate breakdown. This would suggest an increase of F-2,6-BP, in accordance with current views on metabolic regulation (HUBER 1986). The changes induced by DIPS could also result from a similar mechanism, if allosteric activation of a more active PEPC (like the dark form in CAM plants) produces an excess of malate which favours a non-protonomotive electron flow, according to RUSTIN et al. (1984). The resulting decrease of the adenylate energy charge and increase of Pi, which could support a positive feedback in sucrolysis, closely resembles the effect offusicoccin in other plants (JOHNSON and RAYLE 1976; GUERN et aI. 1982; TROCKNER and MARRE 1988), in agreement with the light mimicking action of both compounds in betacyanin formation (ELLIOTT 1979; OBRENOVIC and SPASIC 1988). This possibility is compatible with the analogy between the kinetic parameters for CuDIPS action and allosteric activation of a CAM-like PEPC (OBRENOVIC 1991), supporting the view that PEPC plays a crucial role in light signal transduction. It is also consistent with the same BPP 188 (1992) 2
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effect of lower DIPS concentrations on pea internodes (data not shown), which can be attributed to lower Ka values of PEPC (WEDDING et al. 1989). The increase of G-6-P, induced by continuous B illumination in a chlorophyll-free mutant of Chiorella (RUYTERS 1988), was also ascribed to an enhanced breakdown of carbohydrates (KOWALLIK 1982), but it was considered in terms of a hypothetical photoreceptor "cryptochrome" (KOWALLIK 1982; RUYTERS 1988). However, the same effects of both Rand B on G-6-P and cytosolic Pi signals in maize coleoptiles (also a chlorophyll-free tissue) are consistent with phytochrome as the only identified photoreceptor in higher plants. The difference between Rand B, with respect to NDP-hexose resonance (Figs. 3 and 4 ), may be due to the presence of Pr2 dimers (see comments with Figs. 3 and 4), which support the more active (dark) form of PEPC and/or F-6-P 2kinase. The only possible source of carbohydrates in coleoptiles is sucrose supplied from cotyledons before excision, which seems to be sufficient to maintain a steady state level of phosphorus metabolites over the long period of acquisition (Fig. 1). In conclusion, 31p NMR spectroscopy provides some evidence for a common metabolic effect of light and a light-mimicking compound, although only a limited number of cells appear to undergo detectable metabolic changes, making the use of the technique difficult, but still more plausible than any extraction procedures, where sample variations can level off the small differences in crucial metabolites. Acknowledgements S. OBRENOVIC acknowledges the financial support of the British Council and the Cecil Pilkington Charitable Trust. This work was also supported by the Scientific Research Fund of SR Serbia to S.O. and by the U. K. Agricultural and Food Research Council to R.G.R. and T.E.S.
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KOWALLIK, W.: Blue light effects on respiration. Annu. Rev. Plant Physiol. 33, 51-72 (1982). LEE, R. B., and RATCLIFFE, R. G.: Development of an aeration system for use in plant tissue NMR experiments. J. Exp. Bot. 34, 1213-1221 (1983). NEWSHOLME, E. A., and START, c.: Regulation of Metabolism, John Wiley & Sons, ChichesterNew York-Brisbone-Toronto 1980. OBRENOVIC, S.: Effect of lumiflavin on light induction of betacyanin accumulation in Amaranthus caudatus L. seedlings. Photobiochem. Photobiophys. 13, 95-103 (1986). OBRENOVIC, S., and SPASIC, M.: Effects of copper chelates on betacyanin accumulation in Amaranthus seedlings. Plant Physiol. Biochem. 26, 597-601 (1988). OBRENOVIC, S.: Molecular basis for the wavelength, fluence rate and daylength sensing by plants. J. Photochem. Photobiol. 8, 237-239 (1991). PFEFFER, P. E., Tu, S. I., GERASIMOWICZ, W. V., and CAVANAUGH, J. R.: In vivo 31p NMR studies of corn tissue and its uptake of toxic metals. Plant Physiol. 80, 77-84 (1986). PRATT, L. H., COLEMAN, R. A., and MACKENZIE, J. M.: Immunological visualization of phytochrome. In: Light and Plant Development (Ed. H. SMITH) pp. 75-94. Butterworths, London-Boston 1976. RA TCLIFFE, R. G.: Applicaiton of nuclear magnetic resonance methods to plant tissues. Methods in Enzymology 148, 683-700 (1987). ROBERTS, J. K. M.: Observation of uridine triphosphate: glucose-I-phosphate uridyltransferase activity in maize root tips by saturation transfer 31p NMR. Estimation of cytoplasmic PPi. Biochem. Biophys. Acta 1051, 29-36 (1990). ROBERTS, J. K. M., RAY, P. M., WADE-JARDETZKY, N., and JARDETZKY, 0.: Estimation of cytoplasmic and vacuolar pH in higher plant cells using 31p NMR. Nature 283, 870-872 (1980). RUSTIN, P., DUPONT, J., and LANCE, c.: Involvement of lipid peroxy radicals in the cyanideresistant electron transport pathway. Physiol. Veg. 22, 643-663 (1984). RUYTERS, G.: Regulation of carbohydrate breakdown of Chiarella mutant No 20, studied by 31p NMR spectroscopy and enzymatic analysis. Plant & Cell Physiol. 29, 355- 363 (1988). TROCKNER, V., and MARRE, E.: Plasmamembrane redox chain and H+ extrusion. II. respiratory and metabolic changes associated with fusicoccin-induced and with ferricyanide-induced H+ extrusion. Plant Physiol. 87, 30- 35 (1988). WEDDING, R. T., BLACK, J. M., and MEYER, C. R.: Activation of higher plant phosphoenolpyruvate carboxylase by glucose-6-phosphate. Plant Physiol. 90, 648-652 (1989). Wu, M.-X., and WEDDING, R. T.: Diurnal regulation of phosphoenolpyruvate carboxylase from Crassula. Plant Physiol. 77, 667-675 (1985). Received May 6, 1991; revised form accepted September 23, 1991. Authors' address: SNEZANA OBRENOVIC, Institute for Biological Research "Sinisa Stankovic", 29. Novembra 142, 11060 Belgrade. Yugoslavia.
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