The Metabolism of N6-(Δ2-Isopentenyl) [3H] adenine by Isolated Organs of Pisum sativum

The Metabolism of N 6.(A 2.Isopentenyl) [lH]adenine by Isolated Organs of Pisum sativum R. A.

KING

and].

VAN STADEN

UN / CSIR Research Unit for Plant Growth and Development, Department of Botany, Uni versity of Nat al, Pictcrmaritzburg 3200, South Africa

Received February II , 1987 . Accepted April 6, 1987

Summary Isolated roots, stems, leaves and nodal explants of Pisum sativum L. cv. Onward were incubated in a med ium containing N 6 {.12-isopentenyl) eH]adenine. Radioactive derivatives of N 6-{A2-isopentenyl)adcnine were separated using HPLC. Comparison of the uptake and metabol ism of this cytokinin by the different organs indicated that different types of cytokinin metabolites were associated with each organ. These results are discussed in relation to the regulation of cyrokini n acti vity in plant organs.

Key words: Pisum sa civum, f'.t-{/12·isopenten yl)adenine, cytokinin metabolism, cytokinin syn·

thesis.

Introduction

The production of free cytokinins from applied adenine (Burrows, 1978; Chen and Petschow, 1978) has promoted study of possible metabolic pathways and the nature and activity of precursors and intermediates in these pathways. From studies on the cell-free biosynthesis of cytokinins it would appear that adenosine (Ado) accepts the isopentenyl group directly to form N'-{<1.'-isopentenyl)adenosine (i'Ado) which is converted to trans-zeatin (tZ) via N6~<1.'-isopentenyl)adenine (i'Ade) by several enzymes (Miura and Miller, 1969; Miura and Hall, 1973; Nishinari and Syono, 1980). The metabolism of j6Ade has been studied in several plant systems (Laloue et a1., 1977) and the trans-hydroxylation of the terminal methyl group in the side chain resulting in the formation of cytokinins with increased activity has been demonstrated (Leonard et aI., 1969). This reaction may be an important controlling mechanism of endogenous cytokinin levels, in which inhibition or promotion of the terminal hydroxylation step could dictate the availability of active cytokinins necessary for cellular functions. Recent evidence has indicated that cytokinin biosynthesis may not be limited to the roots of plants (Chen and Petschow, 1978; Einset, 1984; Chen et al., 1985), but as yet the precise sites of cytokinin biosynthesis and the metabolism of applied cytokinins in the shoot system remains to be elucidated. This paper repons on [he uptake and differential metabolism of ['Hli'Ade by detached pea roOlS, stems, leaves and inhibited lateral bud explants. Abbt-cVJdtwns: Ade, aden inc; Ado, adcnosine; cZ, cis-zeat in; DHZ, dihydrozeatin; DHZR, dih ydroribosylzeatin ; il'i Ade. N6-{.6,1-isopemenyl)adenine; i6Ado. Nb-{/1 2-isopentenyl)adenosine; tZ, tr4m-zeatin; tZR , tr4ns-ribosylzeatin; ZOG, glu cosylzeatin .

!

Plant Phys.ol. Vol. 131. /'P. 181-190 (1987)

182

R. A.

KING

and J.

VAN STADEN

Materials and Methods Cultivation ofpea plants and isolation of organs Seeds of Pisum sativum L. cv. Onward were grown in a greenhouse for 2 weeks or until the seedlings had reached the four-node stage. At this stage the seedlings were divided into leaves, stems, roots and nodal explants bearing a stipule and inhibited lateral bud. Leaves were derived from the upper (fourth) node of the plants; stems from internodal segments between the basal (first) and second node, and the second and third node. Nodal explants were taken from the second node. Root tip sections 200 p.m in length were removed from the lateral roots,

Incubation and harvesting ofplants organs Replicates of 1 gram of fresh tissue were incubated in lOOmi of distilled water containing 60 x 106 Bq of eH]i 6Ade (Amersham). The cultures were incubated at 25°C on a shaker in the light, with the exception of the root cultures which were placed in the dark. The tissues were removed after 3 hand 6 h, washed in running distilled water and then frozen with liquid N 2 ,

Tentative identification offH]i6Ade metabolites To extract metabolites, the tissues were homogenised in cold 80% (v/v) ethanol and filtered through What man paper No. 1. The ethanol extract was taken to dryness in vacuo at 40°C, redissolved in 3 mt of 80 % ethanol and passed through a millipore filter (0.22 pm). Samples of 100 pi were then analysed using HPLC. A Hypersil 5 ODS column (5 p.m, C 18 bonded, 250 x 4mm i,d" flow rate 1 mt min - I) fitted to a Varian 5000 liquid chromatograph was used, Separation conditions were as described by Lee et aI., (1985). Absorbance was recorded with a Varian variable wavelength monitor at 26Snm, fitted with an 8,d flow-through cell. Fractions of 1 mt were collected, Only lOO,d of each 1 mt (1 min) fraction was used for determination of radioactivity, with the remainder stored for subsequent analysis. Individual radioactive peaks determined in this way were funher investigated by chemical treatment with /3-glucosidase

(Sigma) (Lee et a!., 1985) or Hel (Dyson et a!., 1972) and then rechromatographed against

authentic cytokinin standards using HPLC as described. Funher separation of radioactive peaks co-eluting with i6 Ade and i 6 Ado was achieved using a Supelcosil LC 18 DB Column (250 x 4.6 mm i.d., flow rate 1.0 mt min - I) in the manner described. This column yielded a superior separation of i6 Ade from i6 Ado. All fractions were counted in Ready Solv EP Scintillation fluid (Beckman) with a Beckman LS 3800 Scintillation counter. Separation of i6 Ade and j6 Ado was also achieved using TLC. The samples were spot loaded onto TLC plates (Merck, Silica gel PF 254) and the plates developed in n-butanol : 14M ammonium hydroxide: water (6: 1: 2; upper phase). Spots co-eluting with authentic markers were then scraped off and reeluted with 100% methanol. The samples were counted in POPOP scintillation cocktail (lOg

2,5-diphenyloxazol; 0,5 g dimethyl-POPOP; 2,51 toluene) with a Beckman LS 3800 Scintilla-

tion counter.

Results

Metabolism off'Hli6Ade by the roots Five major radioactive peaks were recovered from extracts of root tissue after 3 h

and 6 h incubation (Fig. 1). Four peaks were found to co-elute with Ade, Ado, tramribosylzeatin (tZR) and i'Adeli'Ado. The amount of radioactivity associated with these peaks remained constant for 3 hand 6 h. An unidentified peak, designated a

(56 - 60) was recovered after 3 h. After 6 h, this peak had increased and was designated b (56-60). Metabolites co-eluting with Ade for both time treatments were hydrolysed with acid and rechromatographed on HPLC. Both subsequently co-eluted

J. Plant Plrysiol. VoL 131. pp. 181-190(1987)

Metabolism of N 6{.:).1·isopentenyl) of Pisum sativum

@Ade Ado

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Fig. l; The distribution of [lH]i6Ade derived radioactivity from root extracts ( ... - -) and the UV trace of authentic cytokinin standards (--) after separation by HPLC: A, total extract after 6 h; B, peaks (49- 53) for 3 hand 6 h after treatment with HCL; and C, peak (75 - 90) for 6 h after further separation using a supclcosil column and TLC (vertical bars).

with Adc. Metabolites co-eluting with Ado were si milarly treated aher which the radioactivity coincided with the elution position of Ade. Treatment of possible tZR metabolites with acid resulted in the recovery of radioactive peaks co-eluting with tz (Fig, 1), Metabolites a and b failed to shift elution position following acid treatment. The radioactive peaks co-eluting with i6Ade/j6Ado were re-chromatographed using a Supelcosil column which yielded separation of the base and riboside for both time treatments (Fig. 1). Separation on TLC co nfirmed these results (Fig. 1). Metabolism o/f'Hli'Ade by the stems Stem tissue showed the most extensive metabolic activity with six major radioactive peaks being recovered after 3 h and nine peaks recovered after 6 h (Fig. 2). At 3 h. radioactive peaks co-eluting with Ade, Ado, cis-zeatin (cZ) and i6Ade/j6Ado were reco vered. Unidentified metabolites were designated C (55-59) and d (59 - 61). The metabolire co-el uting with Ade was hydrolysed with acid and re-chromarographed on HPLC, co-eluting with Ade agai n. The metabolite co-elu ting with Ado was similarly treated, after which the radioactivity coincided with the elution position of Ade_ j. PL.ntPhysiol. Vol.131.pp.181-190(1987)

184

R.

A.

KING

@Ade Ado

and 1. VAN STADEN

IZR

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Fig. 2: The distribution of [lH]j6 Ade derived radioactivity from stem extracts (- - - - -) and the UV trace of authentic cytokinin standards (--) after separation by HPLC ; a, total extract after 6 h; b, peak (44-49) for 6 h after treatment with HCL; c, peak (J4 - 39) for 6 h after treat ment with II-glucosidase; d, peak fat 6 h after treatment with HCL; and e, peak (73 - 90) for 6 h after further separation using a supelcosil column and TLC (vertical bars).

Acid treatment of the radioactive peak co-eluting with cZ failed to shift the peak, suggesting that this metabolite was cZ. Metabolite c when treated with acid shifted towards the elution positions of Ade and Ado. Metabolite d failed to shift position after treatment with acid. The radioactive peak co-eluting with i6Ade/i 6 Ado was treated as described for the root extract and separation of the base and riboside was achieved. After 6 h incubation, nine major radioactive peaks were recovered from extracts of stem tissue (Fig. 2). Of these. four were similar in position to those found at the elution positions of Ade, Ado, cZ and i 6Ade/ i6Ado, but had increased in radioactivity. Further, a radioactive peak co-eluting with the elution position of glucosylzeatin (ZOG). not found at 3 h. was recovered. Unidentified metabolites were designated e (24-26). f (26-29). g (55-58) and h (59-63). Metabolites g and h. similar to metabolites c and d respectively showed an increase in radioactivity from 3 h to 6 h. Radioactive peaks co-eluting with Ade and Ado were treated with acid which caused them to respond in a similar manner to the corresponding peaks at 3 h. Acid treatment again failed to shift the radioactive peak co-eluting with cZ (Fig. 2). The radioac-

J. Plant P/rysiol.

VoL 131. pp. 181 - 190 (1987)

Metabolism of N6-{.<3. 2-isopemenyl) of Pisum sativum

185

tive peak co-eluting with ZOG was treated with ,8-glucosidase which yielded a radioactive peak co-eluting with tZ when re-chromatographed (Fig. 2). Treatment of metabolite e with acid failed to shift this peak, while similar treatment of metabolite f produced two radioactive peaks, one of which had shifted from the original position (Fig. 2). Metabolites g and h, similar in elution position to metabolites c and d of the 3 h treatment, responded to acid treatment in the same way. Metabolite g shifted towards the elution positions of Ade and Ado, while metabolite h failed to shift position. The radioactive peak co-eluting with i6Ade/i 6Ado was treated as described for the root extract and separation of the base and riboside was achieved (Fig. 2).

Metabolism offHf;'Ade by the buds After 3 h, three major radioactive peaks were recovered in extracts from bud explants. One of the peaks co-eluted with Ade. one with i6Ade/ i6 Ado and the unidentified peak was designated as i (58-63). After 6h, four major radioactive peaks were recovered, three having elution positions identical to those of Ade, i6 Adeli 6 Ado and metabolite i found after 3 h (Fig. 3). No increase in radioactivity was noted for these

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Fig. 3: The distribution of CH]i Ade derived radioactivity from bud explant extracts (- - - --) and the UV tnee of :;I.llthentic cytokinin standards (--) after separation by HPLCj A, to(al extract after b h j B, peak i for 3 hafter treatment with HCL; C, peak j for 6 h after treatment 6

with iJ·glucosida.~~; and D, peak (73 - 90) for 6 h after further separatinn using a Supelcosil col. umn and TLC (vertical bars).

]. Plant Piry>iol, Vol, 131, pp, 181-190 (1981)

R. A.

186

KlNG

and J. VAN STADEN

three peaks. The unidentified peak co-eluting with the position of metabolite i was designated j (58 - 63). The fourth metabolite was designated k (65-68). Metabolites co-eluting with Ade for both time intervals were treated with acid and re-chromato-

graphed on HPLC. Both co-eluted with Ade. Metabolites i and j were treated with acid and is-glucosidase respectively which yielded radioactive peaks near the elution

positions of i'Ade and i'Ado (Fig. 3). The metabolite k, not found at 3 h was treated with acid but did not shift in position when re-chromatographed on HPLC. The radioa(:tive peaks co-eluting with j6 Ade/i 6Ado were treated as described for the root ex-

tract and separation of the base and riboside achieved (Fig. 3). Metabolism offffji'Ade by the leaves Three major peaks were recovered from extracts of leaf tissue after 3 h and 6 h

(Fig. 4). These peaks co-eluted with Ade, dihydroribosylzeatin (DHZR) and i'Adel i'Ado. A slight dedine in radioactivity associated with the Ade and DHZR peaks was noted after 6 h. Metabolites co-eluting with Ade for both treatments were treated

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Fig. 4: The distribution derived radioactivity from leaf extracts (- - - - -) and the UV trace of authentic cytokinin standards (- -) after separation by HPLC; A, total extract after 6 h; B, peak (51 - 56) for 3 h after treatment with acid; C, peak (51-56) for 6 h after treatment of eH]i 6Ade

with ,8-glucosidase; and E, peak (74 -90) for 6 h after further separation using a Supelcosil col· urnn and TLC (venical bars). ]. Plant P/rysiol. Vol. 131. pp. 181 - 190 (1987)

Metabolism of N 6-{A2-isopentenyl) of Pisum sativum

187

Table 1: Distribution of tentatively identified radioactive cytokinins recovered in extracts of pea organs after 3 and 6 hours. Results are expressed as the percentage of total radioactivity supplied at the commencement of the experiment. Radioactivity co-eluting with:

ROOTS 1h 6h

STEMS 1h 6h

BUDS 1h

6h

LEAVES 1h 6h

Ade

1.0 0.6

0.6 0.6

I.l

1.5

1.1

0.7

1.6 1.0 3.9

1.0 2.3 0.66

1.0

0.9 0.7 0.66

4.5

4.1

3.0

Ado

ZOG cZ tZR DHZR i6Ade ,6Ado

Total

1.2 0.65

I.l

0.75 1.3

1.9

1.9

0.7 0.4 4.6

0.7 0.45 4.9

0.5

1.2

2.9

11.0 2.0 17.5

1.2

5.R

1.2

with acid and re-chromatographed on HPLC. Both co-eluted with Ade. The metabolite co-eluting with DHZR at 6 h was treated with acid, after which the radioactiv-

ity coincided with the elution position of dihydrozeatin (DHZ) (Fig. 4). The radioac-

tive peaks co-eluting with i6Adeli 6Ado were treated as described for the roOt extract and ~4!paration of the base and riboside was achieved (Fig. 4). Differential rates of uptake and metabolism by the organs

The amounts of identifiable radioactive cytokinins recovered were quantified and have been expressed as the percentage of the total radioactivity supplied at the com-

mencement of the experiment (Table 1). Uptake of ['H]i'Ade was greatest in the stem

tissue, with much lower levels of [JH]i6Ade being recovered from root, bud and leaf explants. While levels of PH]i6 Ade were found to increase in the stem and bud explants with time, this compound remained constantly low in the roots and was found to decline in the leaves. Recovery of radioactive i6 Ado appeared to follow the same trends as for PH]ifiAdc, but the percentage recovery was far lower. Of the radioactive zeatin metabolites recovered, hoth tZR in the roots and DHZR in the leaves remained constant, while cZ in the stem was found to increase with time. The amounts

of Ade recovered follow closely the pattern of recovery of ['H]i'Ade. while the amounts of Ado recovered can be correlated to the levels of i6Ado in both the root and stem tissues. Discussion

Comparison of the uptake and metabolism of ['H]i'Ade by different organs of Pisum indicates that different types of cytokinin metabolites are associated with each organ. In the roots, tZR appeared to be the major metabolite. Contrary to a previous report (Scott and Horgan, 1984), ZOG, considered to be a major endogenous cytokinin in bean roots, wa... not recovered. Since glucosylation is considered to represent a storage or inactivation form (Van Staden and Davey, 1979), it is likely that supraoptimallevels of tZR had not yet been achieved in the root tissue. Minor quantities

J.

Plant P/rysiol. Vol. 131. pp. 181-190 (1987)

188

R. A. KING and J.

VAN

STADEN

of ribosylzeatin have been recovered from root tissue (Scott and Horgan, 1984), and it is likely that it is in the ribose form that cytokinins are exported from the root to

the shoot (Palmer et al., 1981 a). The hydroxylation of exogenous i'Ade to zeatin me· tabolites has been reported for Actinidia roots (Einset, 1984). In addition, j6Ade and

i'Ado have been extracted from pea roots (Wightman et al., 1980), although in many higher plants i'Ade and i'Ado are not detectable (Scott et aI., 1980; Palmer et aI., 1981 a). It has been suggested (Stuchbury et aI., 1979) that if i'Ade compounds are

intermediates in the production of zeatin, then the enzymes that catalyze their hydroxylation must be very active. An enzyme which cleaves the isopentenyl sidechain

from j6Ade and j6Ado to yield Ade and Ado, respectively, has been characterized (Whitty and Hall, 1974). The relative values of i'Ade to Ade and i'Ado to Ado recovered suggest that i6 Ado is a likely intermediate in the conversion of j6Ade to tZR in this system, since hydroxylation of i'Ade and i'Ado is stereospecific (Palni and Horgan, 1983). Metabolites a and b, identical for both time intervals failed to shift position following acid treatment, indicating that they could possibly be glucosylated

derivatives of i6Ade or i6 Ado. Recovery of relatively large amounts of tZR in the roots would imply that this me-

tabolite represents the form in which cytokinins are transported from the roots to the leaves. Indeed, the major cytokinins extracted from the stems of decapitated bean

plants have been found to be the ribosides and nucleotides of zeatin and dihydrozeatin (DHZ) (Palmer et aI., 1981 a). However, the major radioactive cytokinins recovered from the stem tissues supplied with [3H]i6Ade were found to be cZ and

ZOG. This demonstration of the ability of isolated stem tissue to hydroxylate i' Ade to a zeatin metabolite supports recent evidence that cytokinins, including i6 Ade are

synthesized in pea leaves and stems in addition to the reported root site (Chen et aI. , 1985). In addition, it is clear that glucosylation of zeatin is not restricted to the leaves or roots of plants. Minor quantities of ZOG have been detected in bean stem tissue

(palmer et aI., 1981 a), and the ability of stem tissues to glucosylate cytokinins could explain the cytokinin-like effects that some stems appear to have on in vitro bud growth and development (Woolley and Wareing, 1972; Peterson and Fletcher, 1975). Apart from the identifiable cytokinin metabolites discussed, the stem tissues were found to accumulate six unidentified radioactive metabolites. Significance of these metabolites is not known, but it is likely that portions of these metabolites involve glucosylation of i6 Ade. Glucosylation of i6 Ade at the 7 position has been shown in to-

bacco cells, but significant glucosylation of i'Ado has not been detected (Laloue et aI., 1977). These results suggest that the stem may play an active role in the biosynthesis of cytokinins transported in the shoot.

Bud explants comprising a node bearing an inhibited lateral bud, the stipule and small stem segment attached, yielded few radioactive metabolites. The possibility ex-

ists that inhibited lateral buds were unable to hydroxylate i'Ade to zeatin. It has been demonstrated (Woolley and Wareing, 1972) that inhibited lateral buds lack the capacity for cytokinin biosynthesis, perhaps through inhibition of this terminal hydroxylation step. Unidentified metabolites i and j responded to acid and ,B-glucosidase treatment respectively by shifting to the elution positions of i6 Ade and i6 Ado. How-

J.

Plant Plrysiol. Vol. 131. pp. 181 - 190 (1987)

Metabolism of N6-{~2-isopentenyl} of Pisum sativum

189

ever, it is unlikely that these metabolites represent 7 or 9-glucosylated forms of j6Ade and i6Ado, since these forms are resistant to enzymic degradation (Letham and Palni, 1983). Metabolite k, not recovered in the extract of other organs could not be shifted on acid treatment and remained unidentified. The major radioactive cytokinin metabolite recovered from leaf extracts was DHZR. It has been previously demonstrated (Wang et al., 1977; Wang and Horgan, 1978) that the major endogenous cytokinins in bean leaves are the riboside and O-glucoside DHZ. Since isolated pea leaves have been shown to synthesize i6Ade from supplied ["C]Ade (Chen et aI., 1985) it would appear that cytokinins associated with leaf tissue need not represent translocated forms from the root. Studies on the metabolism of DHZ and zeatin by bean leaves (Wareing et aI., 1977; Palmer et aI., 1981 b) have indicated that while the leaf lamina is the site of cytokinin glucosylation, ribosylation of DHZ occurs in the petiole. However, recovery of DHZR from the leaf tissue of Pisum indicates that this ribosylation can occur in the absence of petiole tlssue. These studies on Pisum reveal that the roots, stems and leaves each have the ability to metabolise j6 Ade to different cytokinin metabolites. The capacity of the different tissues to promote or inhibit this hydroxylation step may prove to be a controlling factor in the regulation of cytokinin activity in the whole plant. Acknowledgements The financial assistance of the C.S.I.R. , Pretoria, is acknowledged.

References BURROWS, W. J.: Incorporation of 3H-adenine into free cytokinins by cytokinin-autonomous tobacco callus tissue. Biochem. Biophys. Res. Commun. 84, 743-748 (1978). CHEN, C. M., J. R. ERn, S. M. LEISNER, and C. C. CHANG: Localization of cytokinin biosynthetic sites in pea plams and carrot roots. Plant Physio!. 78, 510 - 513 (1985). CHEN, C. M. and B. PETSCHOW: Cytokinin biosynthesis in cultured rootless tobacco plants.

Plant Phpiol. 62, 861-865 (1978).

DYSON, W. H.,]. E. Fox, and J. D. MCCHESNEY: Shon term metabolism of urea and purine cytokinins. Plant Physio!. 49, 506-513 (1972). EINSET, J. W.: Conversion of N6-isopentenyladenine to zeatin by Actinidia tissues. Biochem.

Biophys. Res. Commun. 124, 470-474 (1984).

LALOUE, M., C. TERRINE, and

J.

GUERN: Cytokinins: metabolism and biological activity of N 6_

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