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Adenylate Metabolism in Phosphate-depleted Isolated Soybean Leaf Cells and Wheat Leaf Fragments
Adenylate Metabolism in Phosphate-depleted Isolated Soybean Leaf Cells and Wheat Leaf Fragments M. MIGINIAC-MASLOW, J. NGUYEN*} and A. HOARAU Laboratoire de Photosynthese et Metabolisme (UA CNRS 1128), Bat. 430, Universite de Paris-Sud, 91405 ORSAY Cedex and *) Institut de Physiologie Vegetale, CNRS, 91190 GIF sur Yvette, France
Received July 17, 1985 . Accepted September 27, 1985
Summary The influence of phosphate deficiency on adenylate degradation and on adenine incorporation into adenylates was examined in isolated soybean leaf cells and in wheat leaf fragments. Phosphate deficiency was obtained either by sequestration with non-metabolizable sugars (glucosamine or mannose) or by cultivating the plants on phosphate-deprived media. Adenylate catabolism was studied on adenine)4C prelabelled plant material submitted to phosphate sequestrating treatments. Adenine incorporation into adenylates was studied by measuring adenylate)4C labelling rates in the presence of adenine- 14C on already phosphate-deficient plant material. Phosphate sequestration resulted in enhanced adenylate degradation rates with an accumulation of purine oxidation products. Phosphate-deficient plant material showed lower adenine14C incorporation rates into adenylates. Then, the lower adenylate levels observed in phosphate-deficient plants are very likely due to the combined effect of lower adenylate synthesis rates and higher degradation rates.
Key words: Glycine max., Triticum aestivum, adenylate metabolism, inorganic phosphate.
Introduction In the course of a study on the influence of phosphate depletion on the energetic status of either isolated soybean cells, or intact wheat seedlings and wheat leaf fragments, we observed that a drop in the intracellular phosphate content resulted in all cases in a slight decrease in energy charge values and in a marked decrease in the total adenylate content (Miginiac-Maslow et al., 1981; Miginiac-Maslow and Hoarau, 1982; Miginiac-Maslowet al., 1983). This observation suggested that inorganic phosphate regulated the intracellular adenylate content thus preventing wide variations in the energy charge, in a similar way as it does in animal tissues or cells (Chapman and Atkinson, 1973; Matsumoto et al., 1979) and yeasts (Erecinska et al., 1977; Yoshino and Murakami, 1981). This point was further studied in the present work where the influence of phosphate depletion was examined in relation to adenylate metabolism. It was found that under conditions of phosphate deficiency, adenylate synthesis was slowed down and adenylate degradation was stimulated. j. Plant Physiol. Vol. 123. pp. 69-77 (1986)
70
M. MIGINIAc-MAsLOW, J. NGUYEN and A. HOARAU
Material and Methods Plant material The soybean (Glycine max. cv. Amsoy) and wheat (Triticum aestivum cv. Capitole) plants were grown in a greenhouse under natural daylight supplemented with fluorescent bulbs providing 60W'm- 2 for 16h a day, on vermiculite watered daily, alternatively with deionized water or with complete nutrient solution. Phosphorous-deficient plants were obtained as already described (Miginiac-Maslow et al., 1983) by growing the seedlings on aerated liquid nutrient from which phosphate was omitted. Isolated soybean cells were obtained by mechanical grinding, as described previously (Miginiac-Maslow and Hoarau, 1982). Their chlorophyll content was determined on ethanol extracts according to Wintermans and De Moots (1965). Wheat leaf fragments were prepared by cutting the third mature wheat leaves with scissors into about 2 mm wide fragments. The leaves were previously washed with distilled water containing a few drops of T eepol and surface-sterilized by dipping into 0.6 % Ca hypochlorite solution.
Adenylate catabolism studies In order to examine the effect of phosphorous deficiency on adenylate degradation rates, the adenylate pool was first labelled for 1 hr with radioactive adenine under the conditions described below: at this time, a steady-state labelling level of the adenylates and of their degradation products was reached. Then, the plant material was washed and resuspended in a medium without adenine, either in the presence or in the absence of a phosphate-sequestrating compound (glucosamine or mannose: Herold et al., 1976; Miginiac-Maslow and Hoarau, 1982). Soybean cells were suspended in 50 mM HEPES buffer, pH 7.8, 0.3 M mannitol, 12.5 mM K2S04, and 2 mM dithiothreitol, at a concentration of 150 /Lg chlorophyll/ ml (corresponding to about 50 mg fresh weight), in the presence of 10 to 50!Lhl adenine 8_ 14C (CEA, France. Specific activity: 185.107 Bq/mMole). After 1 h incubation, they were washed by centrifugation and resuspended in the same buffer without adenine, but containing either 50 mM glucosamine or 25 mM mannose in the phosphate sequestration assay. Wheat leaf fragments were suspended in 50 mM HEPES buffer pH 7, at about 100 mg fresh weight per ml in the presence of 50!Lhl adenine 14c. After 1 h incubation, they were washed on a nylon net with distilled water and resuspended in the same buffer containing 25 or 50 mM glucosamine or mannose in the phosphate sequestration assay. The antibiotic cephaloridine (300 /Lg! ml) was routinely included in all the incubation media in order to avoid bacterial development (Paul and Bassham, 1977). The incubations were carried out at 27°C in the light (90 W . m -2). At the end of the experiment, the plant material ,was rapidly washed by centrifugation (soybean cells) or filtration on nylon net and blotting with filter paper (wheat leaf fragments), frozen in liquid nitrogen, and extracted after thawing with trichloroacetic acid as previously described (Miginiac-Maslow and Hoarau, 1979). The neutralized extracts were chromatographed by two-dimensional thin-layer chromatography on cellulose, as described by Nguyen (1973). The adenylate degradation compounds were localized by autoradiography. The spots were scraped off, eluted with water and their radioactivity counted by scintillation counting in ACS (Aqueous Counting Scintillant, Amersham).
Adenylate synthesis and degradation studies The short-time kinetics of adenine-'4C incorporation into adenylates and intermediary products of purine catabolism were investigated on phosphate deprived plant cells and leaf fragments, compared to control plant material.
J. Plant Pbysiol. Vol.
123. pp. 69-77 (1986)
Adenylate metabolism in P-depleted leaf cells
71
Phosphate deficiency was obtained in two ways: either by using plants grown on a medium without phosphate, or by submitting the isolated cells or leaf fragments to a phosphate sequestration treatment for 2 hrs before adenine incorporation. The analytical procedures were the same as for adenylate catabolism studies, but higher (237/LM) adenine)4C concentrations were used, in order to obtain maximal adenine incorporation into adenylates (Hirose and Ashihara, 1984). All the assays were duplicated, and each experiment was repeated independently 2 or 3 times and provided similar results.
Results
1) Adenylate catabolism studies In order to elucidate the mechanism of the variation in adenylate contents during phosphate depletion, it seemed appropriate to start with samples containing identical amounts of labelled nucleotides and to examine the variations of these amounts and the appearance of degradation products in samples where the inorganic phosphate content was gradually decreased by chemical sequestration. Adenine- 14C was used for this purpose as AMP, as well as adenosine, are not taken up by plant tissues (Nguyen, 1979). Table 1: Labelling of adenylates and purine degradation products in wheat leaf fragments before and after a 2 h incubation either in buffer, or in the presence of a phosphate sequestrating agent. Each assay contained 100 mg fresh weight. The samples were prelabelled with 30/LM adenine for 1 h and then either extracted or submitted to a 2 h incubation, as indicated. The values are the means of two independent samples. Adenylates = ATP+ADP+AMP. Deamination products = IMP + inosine + hypoxanthine. Oxidation products = xanthine + uric acid + allanto"in + allanto"ic acid. Treatment
2 h incubation in buffer
2 h 25 mM glucosamine 2 h 25 mM mannose
cpm recovered per assay Adenylates
Deamination products
Oxidation products
195115 98372 27347 18306
13323 22928 13444 9420
67719 111645 214299 180596
In Table 1 are listed the groups of the main compounds which appeared in wheat leaf fragments after 1 h of adenine- 14C labelling and after incubation of the prelabelled samples for 2 h either in buffer or in the presence of a phosphate-sequestrating agent. The adenylate group is the sum of labelled ATP + ADP + AMP. Adenylate deamination products represent IMP, inosine, and hypoxanthine, and oxidation products are xanthine, uric acid, allantoin, allantoic acid, and urea. Both ureides were heavily labelled while the radioactivity of urea was weak, c.a. 5 % of that of ureides. The adenylate labelling confirmed the results previously obtained by the means of adenine nucleotide determinations by the luciferase method (Miginiac-Maslow and
J. Plant Physiol. Vol.
123. pp. 69-77 (1986)
M. MIGINIAc-MAsww, J. NGUYEN and A.
72
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Fig. 1: Percent distribution of radioactivity between adenylates and purine degradation products in isolated soybean cells prelabelled with 14C-adenine, as a function of the duration of incubation in the presence or absence of 50 mM glucosamine. Adenine concentration in the prelabelling (1 h) period: 30 JLM. Total radioactivity recovered in adenylates + degradation products at 0 time (= 100 %): 25000 cpm. Total radioactivity after 120 min treatment: control sample: ·22 500 cpm Glucosamine-treated: 20000 cpm - Ad: adenylates: (. - - . ) control; (0- - - - -0) glucosamine; Oxid: oxidized products (.6.--.6.) control; (~- - - - -~) glucosamine; Deam: deaminated products (____) control; (0- - - - -0) glucosamine.
Hoarau, 1982): compared to the controls incubated in buffer, the glucosamine or mannose-treated samples contained much less labelled adenylate. The deaminated products (IMP, IR, and Hx) were also less labelled in phosphate-depleted samples while the label recovered in terminal degradation products (oxidation products) was doubled. This indicates that the degradation of adenylates was markedly stimulated under conditions of phosphate depletion. Then the lower labelling of deamination products-which never accumulate in high amounts (Nguyen, 1979) may be considered to be due to an increase in oxidative purine breakdown. Similar, although less marked, results were obtained with soybean leaf cells. A kinetic study provided more precise information: the label of the adenylate pool decreased more steeply in glucosamine-treated cells than in control cells, while an increase in the label of the oxidation products was observed (Fig. 1).
2) Adenylate synthesis and degradation studies While it clearly appearted that in the case of phosphate sequestration, the degradation of adenylates was stimulated, the lower adenylate levels observed in phosphatedepleted plants might also be due to a lower rate of adenylate synthesis. This was tested by a time-course study of adenine- 14C incorporation into normal or phosphate-depleted plant material. The phosphate depletion was realized either by phosphate sequestration, or by cultivating the plants on a phosphate-deprived medium. Fig. 2 shows adenine incorporation by control or glucosamine-preteated wheat leaf fragments. The label recovered in adenylates was much higher in control plants than in preteated plants. The labellilng of oxidized products was also higher but, when expressed as a percent of adenylate label, the phosphate-depleted fragments had a label
J. Plant Physiol. Vol.
123. pp. 69-77 (1986)
Adenylate metabolism in P-depleted leaf cells
73
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Fig. 2: Time-course of adenine incorporation into adenylates and of the appearance of labelled oxidation products in wheat leaf fragments pretreated with 25 mM glucosamine. Comparison with untreated control samples. Adenine concentration: 30IlM. Adenine was given after a 2 hr preincubation of samples either in buffer or in buffer supplemented with 25 mM glucosamine. Adenyl: adenylate (e--e) control; (0- - - - -0) pretreated with glucosamine. Oxid: oxidation products (.A--.A) control; (6- - - - -6), glucosamine pretreated. Oxidation products are given as the percentage of the label recovered in adenylates.
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Fig. 3: Time-course of adenine incorporation into adenylates and of the appearance of labelled oxidation products in soybean cells isolated either from control plants or from plants cultivated on a phosphate-deprived medium. Adenine concentration: 237 11M. Adenyl: adenylates (e--e) control; (0- - - - -0) phosphate-deficient cells. Ox: Oxidation products (.A--.A) control; (6- - - - -6) phosphate-deficient.
J Plant Physiol.
Vol. 123. pp. 69-77 (1986)
74
M.
MIGINIAC-MASLOW,
J. NGUYEN and A. HOARAU
nearly twice as high, while the deaminated products represented a quite constant percentage of adenylate label in all cases (around 30 %, result not shown). Similar results were obtained with wheat leaf fragments from phosphorous-deficient plants (data not shown), and with soybean leaf cells either pretreated with glucosamine (data not shown) or isolated from phosphorous-deficient plants compared to control plants (Fig. 3). In all cases, phosphorous deficiency resulted in lower rates of adenine incorporation into adenylates and in a relatively faster appearance of their oxidation products. In these rather long time-course experiments, no demonstrative changes could be noticed in the labelling of deaminated products. Therefore, we checked whether in short-time experiments some differences could be found to provide some information about the deamination sequence and a putative phosphorous-sensitive deamination reaction. The first deaminated product appearing in short-time labelling expriments was hypoxanthine (Table 2) followed by inosine and finally IMP. In phosphorous-deficient plants IMP appearance was delayed, while a relatively high percentage of oxidation products (compared to adenylate labelling) was observed. The appearance of hypoxanthine labelling before adenylate labelling suggests that part of the adenine was transformed directly into hypoxanthine via adenine aminohydrolase. Discussion Our results clearly show that in all cases of phosphate depletion the rate of degradation of the adenylates was enhanced. Degradation of adenylate molecules led not only to the formation of deamination products, but also, and mainly, to an accumulation of oxidation products of purine catabolism. Higher levels of oxidation products were found in phosphate-depleted plant material than in control plant material while no increase in labelling of deaminated compounds was observed. In most cases (Table 1) the labelling of deaminated products was even decreased. This observation suggests that the oxidation rates were enhanced comparative to the deamination rates: otherwise, higher labelling of deamination products should be observed in phosphate-depleted plants. Even in short-time 14C adenine incorporation experiments (Table 2) no transient increase in deamination products was observed in phosphate-depleted plants compared to control plants. The early appearance of hypoxanthine prior to IMP and even of adenylates is remarkable in phosphate-deficient plants in which adenine incorporation was slowed down (Table2). This indicates that a direct deamination of adenine into hypoxanthine can occur in wheat leaves via adenine aminohydrolase. Such an activity was found in tobacco protoplasts (Barankiewicz and Paszkowski, 1980) and cotyledons of aseptically-cultured Pharbitis nil plants (Nguyen, unpublished). This does not exclude the possibility of a further deamination at the nucleotide level: an AMP
J Plant Physiol. Vol. 123. pp. 69-77 (1986)
Adenylate metabolism in P-depleted leaf cells
75
aminohydrolase activity has been found in pea seeds (Turner and Turner, 1961), spinach leaves (Yoshino and Murakami, 1980) and Jerusalem artichoke tubers (Le Floc'h et al., 1982). On the other hand, the labelling of inosine was observed after that of hypoxanthine but prior to IMP labelling. As radiolabelling of inosine is concomitant with adenyl ate labelling, two possible ways of inosine production might be considered: either through a nucleoside phosphorylase acting towards nucleoside synthesis or through adenylate catabolism reactions involving a nucleotidase or a less specific phosphatase and an adenosine aminohydrolase. It should be noted that adenosine was strongly labelled (Table 2). Table 2: Short time incorporation of labelled adenine)4C into normal or phosphate-depleted wheat leaf fragments. Phosphate depletion was obtained by cultivating plants on a medium without phosphate. Adenine)4C concentration: 237 JLM. Plant material
Incubation time (min)
Products formed (cpm per sample) Adenosine
Adenylate
Hx
IR
IMP
Oxidized products
Control plants
0.5 2 5 10
920 3124 11368 25221
416 2038 9549 25036
66 78 87 279
22 116 303 1022
0 19 43 279
o (0) 97 (4.8) 281 (2.9) 790 (3.2)
Phosphatedepleted plants
0.5 2 5 10
0 843 4283 10101
0 422 2089 4413
64 37 105 53
0 18 148 585
0 0 0 0
42 73 232 744
() (17.3) (11.1) (16.9)
The numbers in the brackets are the percentage of label compared to the label recovered in adenylates (100 %). Labelled IMP appeared last: in phosphate-depleted plants it was detectable only after a 30 min incubation. Despite the fact that spinach leaf AMP aminohydrolase has been shown to be inhibited by inorganic phosphate (Yoshino and Murakami, 1980), in our experiments, the hypothesis of a specific stimulation of AMP aminohydrolase by phosphate removal must be ruled out: such a stimulation should result in early IMP appearance, and in higher IMP levels in phosphate-depleted plants. In addition this enzyme is not systematically present in all types of plants: it has not been detected in tobacco leaf protoplasts (Barankiewicz and Paszkowski, 1980). In view of our results, it seems that AMP aminohydrolase does not play a regulatory role in phosphate-dependent changes of adenylate degradation rates in our plant material. The lower adenyl ate content of phosphate-deficient plants is probably not exclusively accounted for by the enhancement of the adenylate degradation rates. When adenine- 14C incorporation rate into adenylates was examined, a lower adenylate labelling was observed in phosphate-depleted plant material. This indicates
J. Plant Pbysiol. Vol.
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M. MIGINIAC-MASLOW, J. NGUYEN and A. HOARAU
that the «salvage pathway» of adenylate biosynthesis i.e. AMP synthesis from adenine and phosphoribosyl pyrophosphate was slowed down under conditions of phosphate deficiency. Whatever the reaction directly affected by intracellular phosphate concentrations, it may be considered that low adenylate levels occurring under conditions of phosphate deficiency are the combined effect of a lower adenylate biosynthesis rate and a higher purine catabolism rate. Acknowledgements The authors wish to thank Dr. M. L. Champigny for critical reading of the manuscript. References BARANKlEWICZ, J and P. PASZKOWSKI: Purine metabolism in mesophyll protoplasts of tobacco (Nicotiana tabaccum) leaves. Biochem. J 186, 343 -350 (1980). CHAPMAN, A. G. and D. E. ATKINSON: Stabilization of adenylate energy charge by the adenylate deaminase reaction. J BioI. Chern. 248,8309-8312 (1973). EKECINSKA, M., M. STUBBS, Y. MrYATA, C. DITKE, and D. F. WILSON: Regulation of cellular metabolism by intracellular phosphate. Biochim. Biophys. Acta 462,20-35 (1977). HIROSE, F. and H. ASHIHAKA: Fine control of purine nucleotide biosynthesis in intact cells of Catharanthus roseus. J Plant Physiol. 116, 417-423 (1984). HEROLD, A., D. H. LEVIS, and D. A. WALKER: Sequestration of cytoplasmic orthophosphate by mannose and its differential effect on photosynthetic starch synthetis in C 3 and C4 species. New Phytol. 76, 397 -407 (1976). LE FLOC'H, F., J. LAFLEURIEL, and A. GUILLOT: Interconversion of purine nucleotides in Jerusalem artichoke shoots. Plant Sci. Lett. 27, 309-316 (1982). MATSUMOTO, S. S., K. O. RAMo, and J E. SEEGMILLER: Adenine nucleotide degradation during energy depletion in human lymphoblasts. Adenosine accumulation and adenylate energy charge correlation. J BioI. Chern. 254, 8956-8962 (1979). MIGINIAC-MAsLOW, M. and A. HOARAU: The adenirie nucleotide levels and the adenylate energy charge values of different Triticum and Aegilops species. Z. Pflanzenphysiol. 193, 387 -394 (1979).
MIGINIAC-MAsLOW, A. and A. HOARAU: Variations in the adenylate levels during phosphate depletion in isolated soybean cells and wheat leaf fragments. Z. Pflanzenphysiol. 107, 427 -436 (1982).
MIGINIAC-MASLOW, M., Y. MATHIEU, A. NATO, and A. HOARAU: Contribution of photosynthesis to the growth and differentiation of cultured tobacco cells. The regulatory role of inorganic phosphate. Photosynthesis V 977:-984. Balaban Int. Sci. Services, Philadelphia, 1981.
.
MIGINIAC-MAsLOW, M.,J VIDAL, E. BISMUTH, A/HoARAU; and M. L. CHAMPIGNY: Effets de la carenee et de la nfalimentation en phosphate sur l'equilibre energetique et l'activite phosphoenol pyruvate carboxylase de jeunes plants de Ble. Physiol. veg. 21,325-336 (1983). NGUYEN, J: Influence de l'eclairement sur Ie transport et Ie metabolisme de l'adenine 8)4C chez Ie Pharbitis nil Chois. Physiol. veg. 11, 593-613 (1973). NGUYEN, J: Effect of light on deamination and oxidation of adenylic compounds in cotyledons of Pharbitis nil. Physiol. Plant. 46, 255-259 (1979). PAUL, J S. and J A. BASSHAM: Maintenance of high photosynthetic rates in mesophyll cells isolated from Papaver somniferum. Plant Physiol. 60, 775 -778 (1977). TURNER, D. H. and J. F. TURNER: Adenylic deaminase in pea seeds. Biochem. J. 79, 143-147 (1961).
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Adenylate metabolism in P-depleted leaf cells
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WINTERMANS, J. F. G. M. and A. DE MOOTS: Spectrophotometric characteristics of chlorophyll and their pheophytins in ethanol. Biochim. Biophys. Acta 109, 448-453 (1965). YOSHINO, M. and K. MURAKAMI: AMP deaminase from spinach leaves. Purification and some regulatory properties. Z. Pflanzenphysiol. 99, 331- 338 (1980). YOSHINO, M. and K. MURAKAMI: In situ studies on AMP deaminase as a control system of the adenylate energy charge in yeasts. Biochim. Biophys. Acta 672, 16-20 (1981).
J. Plant Physiol. Vol.
123. pp. 69-77 (1986)
Received July 17, 1985 . Accepted September 27, 1985
Summary The influence of phosphate deficiency on adenylate degradation and on adenine incorporation into adenylates was examined in isolated soybean leaf cells and in wheat leaf fragments. Phosphate deficiency was obtained either by sequestration with non-metabolizable sugars (glucosamine or mannose) or by cultivating the plants on phosphate-deprived media. Adenylate catabolism was studied on adenine)4C prelabelled plant material submitted to phosphate sequestrating treatments. Adenine incorporation into adenylates was studied by measuring adenylate)4C labelling rates in the presence of adenine- 14C on already phosphate-deficient plant material. Phosphate sequestration resulted in enhanced adenylate degradation rates with an accumulation of purine oxidation products. Phosphate-deficient plant material showed lower adenine14C incorporation rates into adenylates. Then, the lower adenylate levels observed in phosphate-deficient plants are very likely due to the combined effect of lower adenylate synthesis rates and higher degradation rates.
Key words: Glycine max., Triticum aestivum, adenylate metabolism, inorganic phosphate.
Introduction In the course of a study on the influence of phosphate depletion on the energetic status of either isolated soybean cells, or intact wheat seedlings and wheat leaf fragments, we observed that a drop in the intracellular phosphate content resulted in all cases in a slight decrease in energy charge values and in a marked decrease in the total adenylate content (Miginiac-Maslow et al., 1981; Miginiac-Maslow and Hoarau, 1982; Miginiac-Maslowet al., 1983). This observation suggested that inorganic phosphate regulated the intracellular adenylate content thus preventing wide variations in the energy charge, in a similar way as it does in animal tissues or cells (Chapman and Atkinson, 1973; Matsumoto et al., 1979) and yeasts (Erecinska et al., 1977; Yoshino and Murakami, 1981). This point was further studied in the present work where the influence of phosphate depletion was examined in relation to adenylate metabolism. It was found that under conditions of phosphate deficiency, adenylate synthesis was slowed down and adenylate degradation was stimulated. j. Plant Physiol. Vol. 123. pp. 69-77 (1986)
70
M. MIGINIAc-MAsLOW, J. NGUYEN and A. HOARAU
Material and Methods Plant material The soybean (Glycine max. cv. Amsoy) and wheat (Triticum aestivum cv. Capitole) plants were grown in a greenhouse under natural daylight supplemented with fluorescent bulbs providing 60W'm- 2 for 16h a day, on vermiculite watered daily, alternatively with deionized water or with complete nutrient solution. Phosphorous-deficient plants were obtained as already described (Miginiac-Maslow et al., 1983) by growing the seedlings on aerated liquid nutrient from which phosphate was omitted. Isolated soybean cells were obtained by mechanical grinding, as described previously (Miginiac-Maslow and Hoarau, 1982). Their chlorophyll content was determined on ethanol extracts according to Wintermans and De Moots (1965). Wheat leaf fragments were prepared by cutting the third mature wheat leaves with scissors into about 2 mm wide fragments. The leaves were previously washed with distilled water containing a few drops of T eepol and surface-sterilized by dipping into 0.6 % Ca hypochlorite solution.
Adenylate catabolism studies In order to examine the effect of phosphorous deficiency on adenylate degradation rates, the adenylate pool was first labelled for 1 hr with radioactive adenine under the conditions described below: at this time, a steady-state labelling level of the adenylates and of their degradation products was reached. Then, the plant material was washed and resuspended in a medium without adenine, either in the presence or in the absence of a phosphate-sequestrating compound (glucosamine or mannose: Herold et al., 1976; Miginiac-Maslow and Hoarau, 1982). Soybean cells were suspended in 50 mM HEPES buffer, pH 7.8, 0.3 M mannitol, 12.5 mM K2S04, and 2 mM dithiothreitol, at a concentration of 150 /Lg chlorophyll/ ml (corresponding to about 50 mg fresh weight), in the presence of 10 to 50!Lhl adenine 8_ 14C (CEA, France. Specific activity: 185.107 Bq/mMole). After 1 h incubation, they were washed by centrifugation and resuspended in the same buffer without adenine, but containing either 50 mM glucosamine or 25 mM mannose in the phosphate sequestration assay. Wheat leaf fragments were suspended in 50 mM HEPES buffer pH 7, at about 100 mg fresh weight per ml in the presence of 50!Lhl adenine 14c. After 1 h incubation, they were washed on a nylon net with distilled water and resuspended in the same buffer containing 25 or 50 mM glucosamine or mannose in the phosphate sequestration assay. The antibiotic cephaloridine (300 /Lg! ml) was routinely included in all the incubation media in order to avoid bacterial development (Paul and Bassham, 1977). The incubations were carried out at 27°C in the light (90 W . m -2). At the end of the experiment, the plant material ,was rapidly washed by centrifugation (soybean cells) or filtration on nylon net and blotting with filter paper (wheat leaf fragments), frozen in liquid nitrogen, and extracted after thawing with trichloroacetic acid as previously described (Miginiac-Maslow and Hoarau, 1979). The neutralized extracts were chromatographed by two-dimensional thin-layer chromatography on cellulose, as described by Nguyen (1973). The adenylate degradation compounds were localized by autoradiography. The spots were scraped off, eluted with water and their radioactivity counted by scintillation counting in ACS (Aqueous Counting Scintillant, Amersham).
Adenylate synthesis and degradation studies The short-time kinetics of adenine-'4C incorporation into adenylates and intermediary products of purine catabolism were investigated on phosphate deprived plant cells and leaf fragments, compared to control plant material.
J. Plant Pbysiol. Vol.
123. pp. 69-77 (1986)
Adenylate metabolism in P-depleted leaf cells
71
Phosphate deficiency was obtained in two ways: either by using plants grown on a medium without phosphate, or by submitting the isolated cells or leaf fragments to a phosphate sequestration treatment for 2 hrs before adenine incorporation. The analytical procedures were the same as for adenylate catabolism studies, but higher (237/LM) adenine)4C concentrations were used, in order to obtain maximal adenine incorporation into adenylates (Hirose and Ashihara, 1984). All the assays were duplicated, and each experiment was repeated independently 2 or 3 times and provided similar results.
Results
1) Adenylate catabolism studies In order to elucidate the mechanism of the variation in adenylate contents during phosphate depletion, it seemed appropriate to start with samples containing identical amounts of labelled nucleotides and to examine the variations of these amounts and the appearance of degradation products in samples where the inorganic phosphate content was gradually decreased by chemical sequestration. Adenine- 14C was used for this purpose as AMP, as well as adenosine, are not taken up by plant tissues (Nguyen, 1979). Table 1: Labelling of adenylates and purine degradation products in wheat leaf fragments before and after a 2 h incubation either in buffer, or in the presence of a phosphate sequestrating agent. Each assay contained 100 mg fresh weight. The samples were prelabelled with 30/LM adenine for 1 h and then either extracted or submitted to a 2 h incubation, as indicated. The values are the means of two independent samples. Adenylates = ATP+ADP+AMP. Deamination products = IMP + inosine + hypoxanthine. Oxidation products = xanthine + uric acid + allanto"in + allanto"ic acid. Treatment
2 h incubation in buffer
2 h 25 mM glucosamine 2 h 25 mM mannose
cpm recovered per assay Adenylates
Deamination products
Oxidation products
195115 98372 27347 18306
13323 22928 13444 9420
67719 111645 214299 180596
In Table 1 are listed the groups of the main compounds which appeared in wheat leaf fragments after 1 h of adenine- 14C labelling and after incubation of the prelabelled samples for 2 h either in buffer or in the presence of a phosphate-sequestrating agent. The adenylate group is the sum of labelled ATP + ADP + AMP. Adenylate deamination products represent IMP, inosine, and hypoxanthine, and oxidation products are xanthine, uric acid, allantoin, allantoic acid, and urea. Both ureides were heavily labelled while the radioactivity of urea was weak, c.a. 5 % of that of ureides. The adenylate labelling confirmed the results previously obtained by the means of adenine nucleotide determinations by the luciferase method (Miginiac-Maslow and
J. Plant Physiol. Vol.
123. pp. 69-77 (1986)
M. MIGINIAc-MAsww, J. NGUYEN and A.
72
HOARAU
100
:::-::-::-g, "-
u
g
"
50
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Fig. 1: Percent distribution of radioactivity between adenylates and purine degradation products in isolated soybean cells prelabelled with 14C-adenine, as a function of the duration of incubation in the presence or absence of 50 mM glucosamine. Adenine concentration in the prelabelling (1 h) period: 30 JLM. Total radioactivity recovered in adenylates + degradation products at 0 time (= 100 %): 25000 cpm. Total radioactivity after 120 min treatment: control sample: ·22 500 cpm Glucosamine-treated: 20000 cpm - Ad: adenylates: (. - - . ) control; (0- - - - -0) glucosamine; Oxid: oxidized products (.6.--.6.) control; (~- - - - -~) glucosamine; Deam: deaminated products (____) control; (0- - - - -0) glucosamine.
Hoarau, 1982): compared to the controls incubated in buffer, the glucosamine or mannose-treated samples contained much less labelled adenylate. The deaminated products (IMP, IR, and Hx) were also less labelled in phosphate-depleted samples while the label recovered in terminal degradation products (oxidation products) was doubled. This indicates that the degradation of adenylates was markedly stimulated under conditions of phosphate depletion. Then the lower labelling of deamination products-which never accumulate in high amounts (Nguyen, 1979) may be considered to be due to an increase in oxidative purine breakdown. Similar, although less marked, results were obtained with soybean leaf cells. A kinetic study provided more precise information: the label of the adenylate pool decreased more steeply in glucosamine-treated cells than in control cells, while an increase in the label of the oxidation products was observed (Fig. 1).
2) Adenylate synthesis and degradation studies While it clearly appearted that in the case of phosphate sequestration, the degradation of adenylates was stimulated, the lower adenylate levels observed in phosphatedepleted plants might also be due to a lower rate of adenylate synthesis. This was tested by a time-course study of adenine- 14C incorporation into normal or phosphate-depleted plant material. The phosphate depletion was realized either by phosphate sequestration, or by cultivating the plants on a phosphate-deprived medium. Fig. 2 shows adenine incorporation by control or glucosamine-preteated wheat leaf fragments. The label recovered in adenylates was much higher in control plants than in preteated plants. The labellilng of oxidized products was also higher but, when expressed as a percent of adenylate label, the phosphate-depleted fragments had a label
J. Plant Physiol. Vol.
123. pp. 69-77 (1986)
Adenylate metabolism in P-depleted leaf cells
73
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Fig. 2: Time-course of adenine incorporation into adenylates and of the appearance of labelled oxidation products in wheat leaf fragments pretreated with 25 mM glucosamine. Comparison with untreated control samples. Adenine concentration: 30IlM. Adenine was given after a 2 hr preincubation of samples either in buffer or in buffer supplemented with 25 mM glucosamine. Adenyl: adenylate (e--e) control; (0- - - - -0) pretreated with glucosamine. Oxid: oxidation products (.A--.A) control; (6- - - - -6), glucosamine pretreated. Oxidation products are given as the percentage of the label recovered in adenylates.
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Fig. 3: Time-course of adenine incorporation into adenylates and of the appearance of labelled oxidation products in soybean cells isolated either from control plants or from plants cultivated on a phosphate-deprived medium. Adenine concentration: 237 11M. Adenyl: adenylates (e--e) control; (0- - - - -0) phosphate-deficient cells. Ox: Oxidation products (.A--.A) control; (6- - - - -6) phosphate-deficient.
J Plant Physiol.
Vol. 123. pp. 69-77 (1986)
74
M.
MIGINIAC-MASLOW,
J. NGUYEN and A. HOARAU
nearly twice as high, while the deaminated products represented a quite constant percentage of adenylate label in all cases (around 30 %, result not shown). Similar results were obtained with wheat leaf fragments from phosphorous-deficient plants (data not shown), and with soybean leaf cells either pretreated with glucosamine (data not shown) or isolated from phosphorous-deficient plants compared to control plants (Fig. 3). In all cases, phosphorous deficiency resulted in lower rates of adenine incorporation into adenylates and in a relatively faster appearance of their oxidation products. In these rather long time-course experiments, no demonstrative changes could be noticed in the labelling of deaminated products. Therefore, we checked whether in short-time experiments some differences could be found to provide some information about the deamination sequence and a putative phosphorous-sensitive deamination reaction. The first deaminated product appearing in short-time labelling expriments was hypoxanthine (Table 2) followed by inosine and finally IMP. In phosphorous-deficient plants IMP appearance was delayed, while a relatively high percentage of oxidation products (compared to adenylate labelling) was observed. The appearance of hypoxanthine labelling before adenylate labelling suggests that part of the adenine was transformed directly into hypoxanthine via adenine aminohydrolase. Discussion Our results clearly show that in all cases of phosphate depletion the rate of degradation of the adenylates was enhanced. Degradation of adenylate molecules led not only to the formation of deamination products, but also, and mainly, to an accumulation of oxidation products of purine catabolism. Higher levels of oxidation products were found in phosphate-depleted plant material than in control plant material while no increase in labelling of deaminated compounds was observed. In most cases (Table 1) the labelling of deaminated products was even decreased. This observation suggests that the oxidation rates were enhanced comparative to the deamination rates: otherwise, higher labelling of deamination products should be observed in phosphate-depleted plants. Even in short-time 14C adenine incorporation experiments (Table 2) no transient increase in deamination products was observed in phosphate-depleted plants compared to control plants. The early appearance of hypoxanthine prior to IMP and even of adenylates is remarkable in phosphate-deficient plants in which adenine incorporation was slowed down (Table2). This indicates that a direct deamination of adenine into hypoxanthine can occur in wheat leaves via adenine aminohydrolase. Such an activity was found in tobacco protoplasts (Barankiewicz and Paszkowski, 1980) and cotyledons of aseptically-cultured Pharbitis nil plants (Nguyen, unpublished). This does not exclude the possibility of a further deamination at the nucleotide level: an AMP
J Plant Physiol. Vol. 123. pp. 69-77 (1986)
Adenylate metabolism in P-depleted leaf cells
75
aminohydrolase activity has been found in pea seeds (Turner and Turner, 1961), spinach leaves (Yoshino and Murakami, 1980) and Jerusalem artichoke tubers (Le Floc'h et al., 1982). On the other hand, the labelling of inosine was observed after that of hypoxanthine but prior to IMP labelling. As radiolabelling of inosine is concomitant with adenyl ate labelling, two possible ways of inosine production might be considered: either through a nucleoside phosphorylase acting towards nucleoside synthesis or through adenylate catabolism reactions involving a nucleotidase or a less specific phosphatase and an adenosine aminohydrolase. It should be noted that adenosine was strongly labelled (Table 2). Table 2: Short time incorporation of labelled adenine)4C into normal or phosphate-depleted wheat leaf fragments. Phosphate depletion was obtained by cultivating plants on a medium without phosphate. Adenine)4C concentration: 237 JLM. Plant material
Incubation time (min)
Products formed (cpm per sample) Adenosine
Adenylate
Hx
IR
IMP
Oxidized products
Control plants
0.5 2 5 10
920 3124 11368 25221
416 2038 9549 25036
66 78 87 279
22 116 303 1022
0 19 43 279
o (0) 97 (4.8) 281 (2.9) 790 (3.2)
Phosphatedepleted plants
0.5 2 5 10
0 843 4283 10101
0 422 2089 4413
64 37 105 53
0 18 148 585
0 0 0 0
42 73 232 744
() (17.3) (11.1) (16.9)
The numbers in the brackets are the percentage of label compared to the label recovered in adenylates (100 %). Labelled IMP appeared last: in phosphate-depleted plants it was detectable only after a 30 min incubation. Despite the fact that spinach leaf AMP aminohydrolase has been shown to be inhibited by inorganic phosphate (Yoshino and Murakami, 1980), in our experiments, the hypothesis of a specific stimulation of AMP aminohydrolase by phosphate removal must be ruled out: such a stimulation should result in early IMP appearance, and in higher IMP levels in phosphate-depleted plants. In addition this enzyme is not systematically present in all types of plants: it has not been detected in tobacco leaf protoplasts (Barankiewicz and Paszkowski, 1980). In view of our results, it seems that AMP aminohydrolase does not play a regulatory role in phosphate-dependent changes of adenylate degradation rates in our plant material. The lower adenyl ate content of phosphate-deficient plants is probably not exclusively accounted for by the enhancement of the adenylate degradation rates. When adenine- 14C incorporation rate into adenylates was examined, a lower adenylate labelling was observed in phosphate-depleted plant material. This indicates
J. Plant Pbysiol. Vol.
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M. MIGINIAC-MASLOW, J. NGUYEN and A. HOARAU
that the «salvage pathway» of adenylate biosynthesis i.e. AMP synthesis from adenine and phosphoribosyl pyrophosphate was slowed down under conditions of phosphate deficiency. Whatever the reaction directly affected by intracellular phosphate concentrations, it may be considered that low adenylate levels occurring under conditions of phosphate deficiency are the combined effect of a lower adenylate biosynthesis rate and a higher purine catabolism rate. Acknowledgements The authors wish to thank Dr. M. L. Champigny for critical reading of the manuscript. References BARANKlEWICZ, J and P. PASZKOWSKI: Purine metabolism in mesophyll protoplasts of tobacco (Nicotiana tabaccum) leaves. Biochem. J 186, 343 -350 (1980). CHAPMAN, A. G. and D. E. ATKINSON: Stabilization of adenylate energy charge by the adenylate deaminase reaction. J BioI. Chern. 248,8309-8312 (1973). EKECINSKA, M., M. STUBBS, Y. MrYATA, C. DITKE, and D. F. WILSON: Regulation of cellular metabolism by intracellular phosphate. Biochim. Biophys. Acta 462,20-35 (1977). HIROSE, F. and H. ASHIHAKA: Fine control of purine nucleotide biosynthesis in intact cells of Catharanthus roseus. J Plant Physiol. 116, 417-423 (1984). HEROLD, A., D. H. LEVIS, and D. A. WALKER: Sequestration of cytoplasmic orthophosphate by mannose and its differential effect on photosynthetic starch synthetis in C 3 and C4 species. New Phytol. 76, 397 -407 (1976). LE FLOC'H, F., J. LAFLEURIEL, and A. GUILLOT: Interconversion of purine nucleotides in Jerusalem artichoke shoots. Plant Sci. Lett. 27, 309-316 (1982). MATSUMOTO, S. S., K. O. RAMo, and J E. SEEGMILLER: Adenine nucleotide degradation during energy depletion in human lymphoblasts. Adenosine accumulation and adenylate energy charge correlation. J BioI. Chern. 254, 8956-8962 (1979). MIGINIAC-MAsLOW, M. and A. HOARAU: The adenirie nucleotide levels and the adenylate energy charge values of different Triticum and Aegilops species. Z. Pflanzenphysiol. 193, 387 -394 (1979).
MIGINIAC-MAsLOW, A. and A. HOARAU: Variations in the adenylate levels during phosphate depletion in isolated soybean cells and wheat leaf fragments. Z. Pflanzenphysiol. 107, 427 -436 (1982).
MIGINIAC-MASLOW, M., Y. MATHIEU, A. NATO, and A. HOARAU: Contribution of photosynthesis to the growth and differentiation of cultured tobacco cells. The regulatory role of inorganic phosphate. Photosynthesis V 977:-984. Balaban Int. Sci. Services, Philadelphia, 1981.
.
MIGINIAC-MAsLOW, M.,J VIDAL, E. BISMUTH, A/HoARAU; and M. L. CHAMPIGNY: Effets de la carenee et de la nfalimentation en phosphate sur l'equilibre energetique et l'activite phosphoenol pyruvate carboxylase de jeunes plants de Ble. Physiol. veg. 21,325-336 (1983). NGUYEN, J: Influence de l'eclairement sur Ie transport et Ie metabolisme de l'adenine 8)4C chez Ie Pharbitis nil Chois. Physiol. veg. 11, 593-613 (1973). NGUYEN, J: Effect of light on deamination and oxidation of adenylic compounds in cotyledons of Pharbitis nil. Physiol. Plant. 46, 255-259 (1979). PAUL, J S. and J A. BASSHAM: Maintenance of high photosynthetic rates in mesophyll cells isolated from Papaver somniferum. Plant Physiol. 60, 775 -778 (1977). TURNER, D. H. and J. F. TURNER: Adenylic deaminase in pea seeds. Biochem. J. 79, 143-147 (1961).
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WINTERMANS, J. F. G. M. and A. DE MOOTS: Spectrophotometric characteristics of chlorophyll and their pheophytins in ethanol. Biochim. Biophys. Acta 109, 448-453 (1965). YOSHINO, M. and K. MURAKAMI: AMP deaminase from spinach leaves. Purification and some regulatory properties. Z. Pflanzenphysiol. 99, 331- 338 (1980). YOSHINO, M. and K. MURAKAMI: In situ studies on AMP deaminase as a control system of the adenylate energy charge in yeasts. Biochim. Biophys. Acta 672, 16-20 (1981).
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