Metabolic Fate of [8-14C]Adenine and [8-14C]Hypoxanthine in Higher Plants

Metabolic Fate of [8- 14 C]Adenine and [8- 14 C]Hypoxanthine in Higher Plants 1) HIROSHI ASHIHARA

and ERI NOBUSAWA

Department of Biology, Faculty of Science, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo, 112 Japan Received June 15, 1981 . Accepted August 7,1981

Summary Metabolism of [8- 14 C]adenine and [8- 14C]hypoxanthine was studied in several plant materials including cotyledons and embryonic axes of black gram seedlings, shoots of light- and darkgrown pea seedlings, young leaves and calluses of tobacco, phloem and xylem tissues of mature carrot roots, suspension culture cells of carrot, leaves of Ginkgo biloba and Acer buergerianum and shoot of wheat seedlings. In all plant materials examined, the «salvage» pathways of adenine and hypoxanthine for nucleotides and nucleic acids were present and the «adenine salvage» pathway was more active than the «hypoxanthine salvage» pathway. In addition to the «salvage» pathways, adenine and hypoxanthine were degraded to allantoin, allantoic acid and CO 2 , The rate of degradation of hypoxanthine was higher than that of adenine in all plant material examined. A large accumulation of allantoin and allantoic acid was observed in seedlings of black gram and peas, tobacco calluses, carrot suspension culture cells and leaves of Ginkgo and Acer. In contrast, little incorporation of radioactivity of labelled purines into allantoin and allantoic acid was found in xylem and phloem of carrot roots and tobacco leaves. A large quantity of 14C02 release from [8- 14C]hypoxanthine was observed in the leaves of tobacco, Ginkgo and Acer and wheat shoots. The data suggest that adenine and hypoxanthine are used for nucleotide and nucleic acid biosynthesis in all plant species. Adenine and hypoxanthine are also degraded in all plant species, but the end products of purine catabolism were different in each organ and tissue of plants. Accumulation of allantoin and allantoic acid may occur in leguminous plants and cultured tissues and cells. A strong degradation system of allantoin and allantoic acid may be present in leaf tissues.

Key words: Ginkgo biloba, Acer buergerianum, Phaseolus mungo, Pisum sativum, Nicotiana tao bacum, Daucus carota, Triticum aestivum, purine metabolism, nucleotide biosynthesis, purine degradation, adenine, hypoxanthine, allantoin.

Introduction

Purine nucleotides are essential precursors for nucleic acid synthesis as well as important compounds for energy metabolism. Considerable attention has been 1) Part I of the series, «Purine Metabolism and its Regulation in Higher Plants». Abbreviation: PCA, perchloric acid. Z. Pjlanzenphysiol. Bd. 104. S. 443-458. 1981.

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HIROSHI ASHIHARA and ERI NOBUSAWA

focused on purine metabolism in microorganisms and mammals (Henderson and Paterson, 1973), although little is known on this topic in higher plants (Brown, 1975; Suzuki and Takahashi, 1977). In many organisms, exogenously supplied adenine and hypoxanthine are metabolized in two ways: some are utilized for nucleotide and nucleic acid synthesis and others are degraded (Fig. 1). Studies on the purine degradation system in animals have been one of the most attractive topics in comparative biochemistry (Baldwin, 1964). Several studies on the adenine and hypoxanthine metabolism in plant cells and tissues have been carried out and the results were reviewed by Suzuki and Takahashi (1977). Nucleic ac ids

Nucleic acids

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Wide variation in the pattern of purine metabolism has been found in the plant kingdom; e.g., adenine was degraded into urea in the silver maple and horseshoe geranium (Barnes, 1959; Schlee and Reinbothe, 1965), however it was not metabolized in pea plants (Silver and Gilmore, 1%9), and adenine was salvaged for adenosine and adenine nucleotide biosynthesis in hazel plants (Bradbeer and Floyd, 1964). However, different methods used in different studies have rendered comparison of the metabolic patterns among the plant species difficult. In this investigation, as a first step in the study of purine metabolism and its regulation in higher plants, the overall metabolism of [8- 14 C]adenine and [8- 14 C]hypoxanthine in 12 different tissues and cells of 7 different plant species was investigated under the same experimental conditions.

Materials and Methods 1. Plant materials Embryonic axes and cotyledons of 24 h-old dark-grown black gram (Phaseolus mungo) seedlings, shoots of 10 day-old light- and dark-grown pea plants (Pisum sativum cv. Kelvedon

Z. Pjlanzenphysiol. Bd. 104. S. 443-458.1981.

Purine metabolism in plants

445

Wonder), young leaves of approx. one month-old tobacco plants (Nicotiana tabacum cv. Bright Consolation), 10 day-old green tobacco callus originating from leaves grown on solid agar containing Linsmaier and Skoog (1965) medium, phloem and xylem tissues of domestic carrot roots obtained according to Morohashi et al. (1967), suspension culture cells of carrot (the «D" cells) described in a previous paper Ashihara et al. Daucus carota (1981), light-grown shoots of 7 day-old seedlings of wheat (T..-iticum aestivum cv. Norin 26), green mature leaves of Acer buerge· rianum and Ginkgo biloba collected in July on the campus of Ochanomizu University were used as experimental materials.

2. Metabolism o/14C-labelled compounds Leaves of tobacco, Acer and Ginkgo and pea shoots were sterilized according to the methods of Colquhoun and Hillman (1972). Carrot roots were sterilized by the method of Komamine et al. (1969). Other plant materials grown under sterile conditions (Ashihara and Matsumura, 1977) were used without additional sterilization. Unless otherwise indicated, samples (approx. 250 mg) were incubated in 2.1 ml of 30 mM KH zP0 4 (pH 5.7) containing 10 mM sucrose and 0.2 ml (10 4 Bq) of [8- 14C]adenine (sp. act. 234 . 107 Bq mmol- 1) or [8- 14 C]hypoxanthine (sp. act. 188 . 10 7 Bq mmol- 1) solution in a 30 ml Erlenmeyer flask with a centre well (containing 0.2 ml of 20 % KOH). The flasks were incubated in an oscillating water bath at 27°C under room light (below 100 lux). After the incubation periods, the medium was removed, and the plant samples were washed with water, frozen in liquid nitrogen, and then ground finely in a chilled mortar. The frozen powder was extracted successively with (I) 6 % (w/v) perchloric acid (PCA) at 2°C for 10 min (twice), (II) an ethanol-ether (1 : 1 by vol) mixture at 50°C for 15 min (once) and at 2°C for 15 min (once), (III) 6 % PCA at 2 °C for 10 min, (IV) an ethanol-ether (1 : 1 by vol) mixture at 2 °C for 10 min, (V) 0.3 M KOH for 18 hat 37°C and (VI) 6 % PCA at 100°C for 15 min. The KOH-soluble and the last PCA-soluble fractions constituted the RNA and DNA fractions, respectively. The first PCA-soluble fraction was neutralized with KOH. After removal of precipitating perchlorate, a part of the fraction was concentrated in vacuo below 35°C and fractionated further by thin layer chromatography. Labelled nucleotides and related compounds were subjected to chromatography on Avicel cellulose plates (Asahi Kasei Kogyo, Tokyo) or PEl-cellulose F plates (E. Merck, Darmstadt) using five different solvent systems as shown in Table 1. To avoid decomposition of labelled compounds, the procedure mentioned up to this section was carried out within 48 h of the beginning of the experiments. Autoradiograms were made on medical X-ray film (Fuji Photo Film Co., Tokyo) exposed for approx. 1 month. Labelled compounds were identified by co-chromatography of standard unlabelled compounds.

Results and Discussion

1. Time course o/incorporation of[8- 14 C]adenine and [8_ 14 C]-hypoxanthine into nucleic acids and metabolites Time course studies for incorporation of radioactivity from labelled adenine and hypoxanthine were carried out using embryonic axes of 24 h-old black gram seedlings (Fig. 2 A and B). Incorporation of 14C from [8-14C]adenine into nucleotides increased during the initial 30 min after [8- 14 C]adenine was added, and then gradually decreased. Incorporation into nucleic acids remained almost constant within 4 h of incubation, although initial rapid increase was observed. Z. Pjlanzenphysiol. Bd. 104. S. 443-458.1981.

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HIROSHI ASHIHARA and ERI NOBUSAWA

Table 1: Rf values of purine nucleotides and related compounds. The developing solvents and thin layer used were as follows: I. Butanol-acetic acid-water, 4: 1 :2, v/v (cellulose). II. Butanol-methanol-water-ammonia, 60 : 20: 20: 1, v/v (cellulose). III. Propernolammonia-water, 100: 60 :40, v/v (cellulose). IV. 0.2 M LiCl2 min, 1.0 M LiCl6 min, 1.6 M LiCl 15 cm, (PEI-cellulose). V. Sodium formate buffer (pH 3.4), 0.5 M 30 sec, 2.0 M2 min, 4.0 M 15 cm, (PEI -cellulose). System

II

III

IV

V

Bases Adenine Guanine Hypoxanthine Xanthine

0.52 0.30 0.48 0.24

0.50 0.25 0.35 0.13

0.65 0.45 0.68 0.45

0.24 0.22 0.45

0.54 0.41 0.53 0.43

Nucleosides Adenosine Guanosine Inosine Xanthosine

0.50 0.38 0.42 0.37

0.43 0.25 0.30 0.20

0.69 0.62 0.72 0.66

0.43 0.48 0.56

0.60 0.57 0.71 0.61

Nucleotides AMP GMP IMP XMP

0.21 0.18 0.22 0.19

0.06 0.02 0.02 0.02

0.52 0.42 0.49 0.44

0.54 0.53 0.68 0.50

0.67 0.58 0.69 0.63

ADP GDP

0.12 0.13

0.03 0.02

0.49 0.41

0.49 0.46

0.54 0.34

ATP GTP

0.06 0.12

0.02 0.01

0.49 0.40

0.36 0.33

0.26 0.14

Degadation products Uric acid 0.12 Allantoin 0.34 Allantoic acid 0.31

0.03 0.20 0.10

0.31 0.57 0.59

0.51 0.72 0.75

0.44 0.72 0.71

The solvent systems used were made according to ASHIHARA (1977) (System I), RANDERATH and RANDERATH (1967) (System II, IV and V) and NGUYEN (1973) (System III).

On the other hand, the incorporation from [8- 14 C]adenine into allantoin and allantoic acid increased continuously during the incubation periods. Small amounts of radioactivity were found in adenine and adenosine throughout the incubation periods. The radioactivity of 14C02 released from [8- 14 C]adenine was observed, but the rate was only up to 0.2 % of the total uptake of [8- 14 C]adenine. The incorporation pattern of 14C from [8- 14 C]hypoxanthine into nucleotides was similar to that from [8- 14 C]adenine, but the radioactivity of the fraction was lower than that from [8- 14 C]adenine. Incorporation of [8- 14 C]hypoxanthine into nucleic acids increased for the first 30 min incubation period and decreased slightly for the next 30 min period and then remained constant throughout the experimental period after the initial 60 min. Z. Pjlanzenphysiol. Bd. 104. S. 443-458.1981.

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Fig. 2: Time course of metabolism of [8- 14C]adenine (A) and [8- 14C]hypoxanthine (B) in embryonic axes of 24 h-old black gram seedlings. Absorbed 14C-precursors (_, dashed lines) are shown as nmoles/g fresh weight, and incorporation of 14C into several cellular constituents (solid lines) is shown as percentage of 14C absorbed. 0, RNA; e, nucleotides; !;", hypoxanthine; A, adenine + adenosine; D, allantoin + allantoic acid.

A large amount of [8- 14 C]hypoxanthine was incorporated into allantoin and allantoic acid. These compounds were labelled rapidly without lag for the first 1 hr and then reached an almost constant level. A small amount of radioactivity from [8- 14 C]hypoxanthine was found in hypoxanthine, adenine, adenosine and other bases and nucleosides. Changes in the rate of incorporation into these compounds were not apparent during the incubation period. The data obtained here indicated that adenine and hypoxanthine were metabolized via both the «salvage» and «degradation» pathways shown in Fig. 1. Also the pattern of 14C distribution in cellular constituents from [8- 14 C]adenine and [8- 14 C]hypoxanthine changed during the incubation period. The metabolic pattern obtained from a 4 h incubation period may be used to comZ. Pjlanzenphysiol. Bd. 104. S. 443-458. 1981.

448

HIROSHI ASHIHARA

and ERI NOBUSAWA

pare each sample, since the incorporation rate into most compounds has already reached a steady level. Therefore, the incubation time for all experiments described below was fixed at 4 hrs. However, it is to be emphasized that the incorporation from [8- 14 C]adenine into the nucleotide and the allantoin and allantoic acid fractions may not remain constant throughout the 4 h incubation. 2. Effect ofconcentration ofexogenous precursors on the metabolism of[8- 14 C]adenine

and [8· 14 Cjhypoxanthine The effect of different concentrations of adenine and hypoxanthine on their metabolism was studied in the embryonic axes of 24 h-old black gram seedlings (Table 2). Table 2: Distribution of the radioactivity in the several intracellular compounds produced from exogenous [8- 14 C]adenine and [8- 14 C]hypoxanthine for different initial concentrations. [S-14C]Adenine

Precursors Initial Concentration

4.7[!M

Total Uptake (nmollg fro wt.!hr)

5.S3

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26.S 0.2 31.3 4.1 0.1 30.1 0.2 7.5

122.4 [!M 71.6 24.1 0.3 17.9 9.1 0.1 39.5 0.2 8.5

[S-14C]Hypoxanthine 5.9 [!M 7.29 14.2 0.1 2.7 5.4 4.2 63.0 O.S 9.8

123.5 [!M 43.2 14.0 0.1 4.1 6.2 4.4 59.4 0.9 11.1

The higher and lower initial concentrations used in this experiment were 4.7 and 122.4 11M for adenine, and 5.9 and 123.5 11M for hypoxanthine, respectively. The former concentrations of adenine and hypoxanthine were 21 times lower than the latter. The total uptake of adenine and hypoxanthine from the solutions at the higher initial concentrations were 12.3 and 5.9 times higher than those from the solutions at the lower concentration of these compounds, respectively. Raising the adenine concentration increased the incorporation of 14C into allantoin and allantoic acid, and decreased that into the nucleotide fraction. A slightly higher incorporation rate expressed as the percentage of total uptake into RNA fraction was observed when a lower initial concentration of adenine was used. The data suggest that the adenine anabolic enzyme system can be saturated with adenine at increased concentration, and subsequently, significant amounts of adenine overflowed to the catabolic pathway, resulting in incorporation of radioactivity of [8- 14 C]adenine into allantoin and allantoic acid. The observation might be explained by the fact that the Km value of adenine phosphoribosyltransferase for adenine is Z. Pjlanzenphysiol. Bd. 104. S. 443-458. 1981.

Purine metabolism in plants

449

considerably lower than that of adenine deaminase; the Km value obtained in the enzyme from soybean callus and Jerusalem artichoke shoots are 1.5 and 5.5 IlM, respectively (Nicholls and Murray, 1968; leFl6ch and Lafleuriel, 1978). The activity of adenine deaminase was detected in tobacco protoplasts (Barankiewicz and Paszkowski, 1980) and cultured cells of Vinca rosea (Ashihara, unpublished results), but the Km value for plant adenine deaminase has not been reported. The values for adenine obtained from Candida utilis and Azotobacter vinelandii are 30 and 10 IlM, respectively (Hartenstein and Fridovich, 1967; Heppel et aI., 1957). A higher concentration of hypoxanthine slightly increased the rate of incorporation of hypoxanthine into nucleotides and decreased that into allantoin and allantoic acid. In the case of hypoxanthine, the result might be due to the activity of the hypoxanthine degrading enzyme, xanthine dehydrogenase, and/or the affinity of hypoxanthine. This may be higher than that of the hypoxanthine salvage enzyme, hypoxanthine phosphoribosyltransferase. Although these two enzymes were detected in higher plants (Nguyen and Feierabend, 1978; Barankiewicz and Paszkowski, 1980) the detailed kinetics of these enzymes have not yet been reported in higher plants. If the above assumption is true, the hypoxanthine degradation system might be saturated with hypoxanthine at the higher concentration and the metabolic flow might be changed into an anabolic system.

3. Metabolism of[8· 14 Cjadenine and [8· 14 Cjhypoxan thine in several plant materials a) Embryonic axes and cotyledons of etiolated black gram seedlings The metabolic pattern of [8- 14 C]adenine and [8- 14 C]hypoxanthine in embryonic axes and cotyledons of black gram seedlings are shown in Fig. 3 A and B. A large amount (60 -70 %) of labelled adenine was incorporated into nucleic acids and nucleotides in both embryonic axes and cotyledons. The radioactivity of the nucleic acids of the embryonic axes was higher than that of the cotyledons. In contrast, incorporated of [8- 14 C]adenine into the nucleotide fraction was higher in the cotyledons than in the axes. Incorporation of [8- 14 C]adenine into A TP in cotyledons and embryonic axes comprised 84 % and 53 % of the total radioactivity of the nucleotide fractions, respectively. 30 and 16 % of radioactivity from [8- 14 C]adenine were found in the allantoin and allantoic acid fraction of embryonic axes and cotyledons, respectively. Not more than 0.2 % of the total radioactivity was released as 14eo2 from the embryonic axes and from the cotyledons of the seedlings. The results indicate that a very active adenine salvage system in cotyledons and embryonic axes of 24 h-old black gram seedlings contributes to the active nucleic acids and A TP synthesis for rapid growth of the seedlings. Guranowski and Barankicwicz (1979), using germinating lupin seeds, suggested that adenine is salvaged mainly by adenine phosphoribosyltransferase, because purine nucleoside phosphorylase activity was absent in the seeds throughout the germination. The adenine phosphoribosyltransferase activity was high in the early phase of lupin germination and then Z. Pjlanzenphysiol. Bd. 104. S. 443-458. 1981.

450

HIROSHI ASHIHARA

and ERI NOBUSAWA '/,

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Fig. 3: Incorporation of [8- H qadenine (A) and [8- 14 qhypoxanthine (B) into several fractions of cellular constituents of embryonic axes (0) and cotyledons (_) of dark-grown 24 h-old black gram seedlings. The samples were incubated with the labelled compounds for 4 hrs. Incorporation rates are expressed as percentage of total radioactivity absorbed.

decreased in cotyledons of the seedlings. Since the activity of adenine phosphoribosyltransferase was also detected in cotyledons and embryonic axes of black gram seedlings (Nobusawa and Ashihara, unpublished results), the enzyme may participate in the adenine salvage in the seedlings. On the other hand, about 60 % of radioactivity from [S-HC]hypoxanthine was incorporated into the allantoin and allantoic acid fraction of 24 h-old embryonic axes and cotyledons. Fujiwara and Yamaguchi (197S) reported that the main pathway of allantoin formation in germinating soybean seedlings was through purine decomposition, via xanthine and uric acid, and allantoin accumulation during an early stage of germination may be due to degradation of «storage RNA». The data obtained here using black gram seedlings seems to support the idea. The radioactivity in the nucleic acid fraction was 10-15 % of totalHC absorbed in the axes and cotyledons and only 2 - 3 % of the radioactivity was recovered in the nucleotide fraction. Almost all the radioactivity found in the nucleotide fraction was distributed in the guanine nucleotides. A higher incorporation of [S-HC]hypoxanthine into guanine nucleotide rather than into adenine nucleotides has been reported in tea shoot tips (Suzuki and Takahashi, 1975) and soybean axes (Anderson, 1979). The incorporation of [S-14C]hypoxanthine into the CO 2 fraction was extremely low (up to o.s %) in both embryonic axes and cotyledons of the seedlings. The results suggest that allantoin and allantoic acid were accumulated without further degradation and then probably transported as a nitrogen source to the other parts of the plants. These ureides have Z. Pjlanzenphysiol. Ed. 104. s. 443-458. 1981.

Purine metabolism in plants

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Fig. 4: Incorporation of [8- 14C]adenine (A) and [8- 14C]hypoxanthine (B) into several fractions of cellular constituents of shoots of light- (0) and dark-grown (_) 7 day-old pea seedlings. The samples were incubated with the labelled compounds for 4 hrs. Incorporation rates are expressed as percentages of total radioactivity absorbed.

been detected in both xylem and phloem saps of many higher plants including Phasealus vulgaris (Thomas and Schrader, 1981). b) Shoots of light-grown and dark-grown pea seedlings Silver and Gilmore (1969) studied the pattern of the metabolism of adenine and hypoxanthine infiltrated into pea seedlings and reported that hypoxanthine was converted to xanthine but adenine was not metabolized. In order to clarify whether the metabolism of these purines in pea plants show a unique pattern and is different from the black gram shown above, metabolism of [8- 14 C]adenine and [8-I4C]hypoxanthine was examined. As Silver and Gilmore (1969) did not mention the light conditions for the growth of pea plants in their paper, both light- and dark-grown pea seedlings were used in this work. Fig. 4 shows the metabolic pattern of [8- 14 C]adenine and [8- 14 C]hypoxanthine in shoots of light- and dark-grown pea seedlings. In contrast to the results of Silver and Gilmore (1969), adenine and hypoxanthine were converted to nucleic acids, nucleoti des and degradation products. Although incorporation of [8- 14 C]hypoxanthine into xanthine was observed, xanthine was not a major product of hypoxanthine degradation. Metabolic patterns of adenine and hypoxanthine found in pea seedlings were similar to those found in black grarp seedlings except that incorporation into allantoin and allantoic acid was lower in pea plants, and the percentages of un metabolized Z. Pjlanzenphysiol. Bd. 104. S. 443-458.1981.

452

HIROSHI ASHIHARA

and ERI NOBUSAWA

adenine and hypoxanthine observed in pea plants were higher than those in black gram. As Silver and Gilmore (1969) did not use radioisotopes in their experiments, they probably could not detect the details of the metabolism of these purines. Little difference in the metabolic patterns of [8- 14 C]adenine and [8- 14 C]hypoxanthine between light-grown and dark-grown pea shoots was observed: Incorporation of [8- 14 C]adenine into the nucleic acid, nucleotide and adenosine fractions was slightly higher in light-grown than in dark-grown seedlings. Incorporation of [8- 14 C]hypoxanthine into the nucleotide fraction was higher in the dark-grown seedlings, while the incorporation into allantoin and allantoic acid was slightly higher in light-grown seedlings. '/.

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25

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Fig. 5: Incorporation of [8- 14 C]adenine (A) and [8- H C]hypoxanthine (B) into several fractions of cellular constituents of tabacco leaves (0) and calluses (_). The samples were incubated with the labelled compounds for 4 h. Incorporation rates are expressed as percentages of total He absorbed.

c) Tobacco leaves and its callus Incorporation of labelled adenine and hypoxanthine into various fractions of young tobacco leaves and rapidly growing callus is shown in Fig. 5. In both leaf and callus tissues, adenine was easily salvaged for nucleotide and nucleic acid synthesis, while only a small amount of hypoxanthine was used for the synthesis. A similar observation was reported in protoplasts of tobacco leaves (Barankiewicz and Paszkowski, 1980). It is noteworthy that some distinct differences in purine catabolism were observed between leaves and calluses: Incorporation of [8- 14 C]adenine and [8- 14 C]hypoxanthine into allantoin and allantoic acid in the calluses was considerably higher than that in the leaves. In contrast, , the rate of release of Heo2from [8- 14C]hypoxanthine in the leaf tissue was 11 times higher than in the callus tissue. The results suggest that Z. P/lanzenphysiol. Ed. 104. S. 443-458. 1981.

Purine metabolism in plants

453

the leaves contain an allantoic acid degradation system (i.e., allantoicase and urease), but this capacity is partially depressed in the callus tissue. About 20 % of the radioactivity was recovered as xanthine when [8-14C]hypoxanthine was used in tobacco leaf discs. Barankiewicz and Paszkowski (1980) reported about the metabolism of purines in tobacco leaf protoplasts. The results are partially consistent with the results obtained here with tobacco leaves, but they did not mention the incorporation of [814C]adenine and [8- 14 C]hypoxanthine into the allantoin and allantoic acid and the CO 2 fractions. d) Phloem and xylem of carrot roots and carrot suspension culture cells The incorporation patterns of [8- 14 C]adenine and [8- 14 C]hypoxanthine in phloem and xylem tissues of mature carrot roots and suspension culture cells of carrot are shown in Fig. 6. More than 60 % of the absorbed 14C from [8- 14 C]adenine was salvaged and incorporated into the nucleotide and nucleic acid fractions in the carrot tissues and cells. In phloem tissue, 25.6 % of the total radioactivity absorbed was recovered in the adenosine fraction, while not more than 10 % of the radioactivity was observed in this fraction in xylem tissue and suspension culture cells. Incorporation of [8- 14 C]adenine into allantoin and allantoic acid was not detected either in phloem or xylem tissues of carrot roots. In contrast, 16.7 % of the total radioactivity was recovered in the fraction of the suspension culture cells. The incorporation of [8- 14 C]hypoxanthine into nucleotide and nucleic acid fractions of phloem, xylem and suspension culture cells was 46.6, 29.5 and 20.3 % of absorbed 14C, respectively. These rates were much higher as compared with other plant materials, although the incorporation rates into the nucleotide fraction were less than those of [8- 14 C]adenine. About 25 % of absorbed [8- 14 C]hypoxanthine was degraded in phloem and xylem tissues of the carrot roots, whereas about 60 % of [8- 14 C]hypoxanthine was degraded into allantoin, allantoic acid and CO 2 in the cultured cells. A considerable amount of unmetaholized [8- 14 C]hypoxanthine (28.9 and 43.2 % of radioactivity absorbed) was observed in phloem and xylem tissues. The results show that deamination activity of adenine is considerably repressed in carrot root tissues. Therefore allantoin and alantoic acid are probably not synthesized via an adenine deaminase step in carrot roots. e) Leaves of Ginkgo and Acer and shoots of wheat In order to examine whether the purine metabolism pattern of gymnosperms is different from that of dicotyledons and monocotyledons, the metabolism of [8_ 14 C]adenine and [8- 14 C]hypoxanthine in leaves of Ginkgo biloba, Acer buergerianum and wheat was determined and the results are shown in Fig. 7 A and B. In Ginkgo leaves, more than 75 % of the [8- 14 C]adenine absorbed was salvaged for

z. Pjlanzenphysiol. Bd.

104. S. 443-458. 1981.

454

HIROSHI ASHIHARA

and 'I,

ERI NOBUSAWA

of

Total

Radioactivity

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Nucleic acids

N uc leoti des

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Others

Adenosine Adenine

Fig. 6: Incorporation of [8- 14 C]adenine (A) and [8- 14C]hypoxanthine (B) into several fractions of cellular constituents of phloem (D) and xylem (0) tissues of carrot roots and suspension culture cells of carrot (_). The samples were incubated with the labelled compounds for 4 h. Incorporation rates are expressed as percentages of total He absorbed.

nucleotide and nucleic acid biosynthesis, and adenine degradation was extremely low. In contrast, at least 60 % of the absorbed [8- H C]adenine was degraded in wheat shoots. The incorporation pattern of [8- H C]adenine in Acer leaves was intermediate between that in Ginkgo leaves and that in wheat shoots. About 40 % of the [814C]adenine in Acer leaves remained unmetabolized. In Ginkgo leaves, little hypoxanthine salvage activity was observed and a large amount of hypoxanthine was degraded to allantoin, allantoic acid and CO 2 , Approximately 25 % of the total radioactivity was incorporated into the nucleotide and nucleic acid fraction of Acer leaves and a fairly large amount of [8- 14 C]hypoxanthine was recovered in allantoin, allantoic acid and CO 2 , In wheat shoots, a considerable amount of [8- 14 C]hypoxanthine was incorporated into the degradation products, mainly COz. A large amount (36.7 % of the total radioaktivity) of HC from [8- H C]hypoxanthine was observed in the adenine and adenosine fraction. Incorporation of [8- 14 C]hypoxanthine into adenine nucleotides in wheat shoots was also reported by Rybicka (1974). Her data suggest that labelled adenine and adenosine found in wheat shoots seem to originate from the following sequence of reactions: Hypoxanthine -+ IMP -+ AMP -+ Adenosine -+ Adenine. Accumulation of adenine and adenosine in wheat shoots is probably due to the high activity of nucleotidase(s), phosphatase(s) and nucleosidase(s). From the fact that HCOZ was released to a large extent from [8- 14 C]hypoxanthine, it is inferred that a higher degradation activity of allantoic acid occurred in all leaf Z. Pjlanzenphysiol. Bd. 104. S. 443-458. 1981.

Purine metabolism in plants '/,

Nucleic ac ids

o!

Total

50

75

A

455

Radioactivity

0 Nucleic acids

N ucle otides

Nucleotides

Adenosine

-+

Adenine

Hypoxan th ine

Allantoin • Allantoicacid

Allantoin • Allantoicac id

50

75

B

Adenosine Adenine

CO2

Others

Others

Fig. 7: Incorporation of [8- 14C]adenine (A) and [8- 14C]hypoxanthine (B) into several fractions of cellular constituents of leaves of Ginkgo biloba (0) and Acer buergerianum (0) and wheat shoots (_). The samples were incubated with the labelled compounds for 4 h. Incorporation rates are expressed as percentages of total 14C absorbed.

tissues examined in this study. The degradative capacity of allantoin and allantoic acid may be localized in the leaves of every plant.

4. Conclusions Although many workers have reported about purine metabolism in higher plants (Barnes, 1959 and 1961; Reinbothe, 1961; Bradbeer and Floyd, 1964; Butler et aI., 1961; Schelee and Reinbothe, 1965; Silver and Gilmore, 1969; Price and Murray, 1969; Doree et al., 1970; Doree, 1973; Ngnyen, 1977; Barankiewicz and Paszkowski, 1980), the data are fragmentary, and a general profile of the purine metabolism in plants has not yet been obtained. One of the reasons for the inconsistency of the results obtained is the difference in the incubation time in different studies. The data obtained here (Fig. 2) indicate that the rate of incorporation into several cell components changes during the incubation period. Another reason is the difference in the concentration of labelled precursors administered to the sample. The metabolic pattern of purine is influenced by the initial concentration of the precursors (Table 2). In addition, the different results obtained may be due to the different experimental conditions, i.e., buffer used, energy supply etc., and the procedures of extraction and identification of metabolites. In this investigation, the metabolism of [8- 14 C]adenine and [8- 14 C]hypoxanthine in several kinds of plant material was determined under identical experimental conditions. The results obtained revealed the general profiles of purine metabolism describZ. Pjlanzenphysiol. Bd. 104. S. 443-458. 1981.

456

HIROSHI ASHIHARA and ERI NOBUSAWA

ed below. The «adenine-» and «hypoxanthine salvage» pathways are present in all plant species and the activity of the former pathway is usually much higher than that of the latter. All plant species possess catabolic activity for both adenine and hypoxanthine, but hypoxanthine is usually more easily degraded than adenine. The end products of purine cata!}olism differ to some extent in different plant organs and in fresh and cultured tissues. However, the patterns of purine catabolism in leaves of angiosperms and gymnosperms resemble each other. «Adenine-» and «hypoxanthine salvage» pathways have also been found in microorganisms and animals (Hender.son and Paterson, 1973). Lalanne and Willemot (1980) measured the rate ~f incorporation of adeni~e and hypoxanthine into ribonucleotides in the intact erythrocytes of eight mammalian species. As is the case in higher plants, their data also indicated that the activity of adenine salvage for ribonucleotide biosynthesis was higher than that of hypoxanthine in all the intact erythrocytes examined. The catabolic route of hypoxanthine in higher plants may be the same as that found in animals, although the end products of the pathway in higher plants are different from those in man and higher primates, in which the end product is uric acid (Henderson and Paterson, 1973). The phylogenic distribution of various enzymes of purine catabolism in animals were generalized by Florkin and Duchateau (see Henderson and Paterson, 1973). In contrast, there is no evidence of the phylogenic distribution of the end product of purine catabolism in higher plants. Acknowledgements The authors are grateful to Prof. Seki Shimizu, Miss Akiko Kondo and Dr. Tatsuhito Fujimura for the supply of tobacco leaves, tobacco calluses and carrot suspension culture cells, respectively. They wish to thank Prof. Atsushi Komamine, Department of Botany, University of Tokyo, for his critical reading of this paper.

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