Proc. Nati. Acad. Sci. USA Vol. 83, pp. 972-975, February 1986 Botany

Zeatin biosynthesis from N6-(A2-isopentenyl)adenine in and other woody (cytokinin/phytohormones/adenine derivatives/chemotaxonomy) JOHN W. EINSET Arnold Arboretum, Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138 Communicated by Folke Skoog, September 30, 1985

ABSTRACT Tissue cultures of Actinidia kolomikta can be version of i6Ade to io6Ade in Actinidia, which suggest that maintained as callus through repeated passages on a nutrient i6Ade hydroxylation may be, in fact, the natural process of medium devoid ofcytokinin but containing inorganic nutrients, io6Ade biosynthesis in this species. I have measured the sucrose, and other basal organics plus auxin. Under these internal concentrations of i6Ade and io6Ade in autonomous conditions, actively growing callus contained 2 and 0.5 nmol of tissue cultures and compared these values with the apparent the cytokinins zeatin [io Ade; 6-(4-hydroxy-3-methylbut-2- Km for i6Ade hydroxylation. In addition, I demonstrate that enylamino)purine] and N6-(A2-isopentenyl)adenine (i6Ade), re- the hydroxylation is 02-requiring and specific for i6Ade over spectively, per gram (fresh weight). When tissues were trans- the 9-ribonucleoside N6-(A2-isopentenyl)adenosine (i6A). ferred from cytokininless medium to 30 uM i6Ade, endogenous In spite of the evidence for i6Ade as a precursor of io6Ade io6Ade increased linearly to 160 nmol/g (fresh weight) during in plants, including the results in this paper, there are several 8 hr, and i6Ade increased to 5 nmol/g (fresh weight) in 2 hr and reports of experiments involving i6Ade or i6A feeds in which then declined. The apparent Km for i6Ade in A. kolomikta and io6Ade and ribosylzeatin (io6A) levels were unaffected (10) or Actinidia chinensis X Actinidia arguta callus and in tissue slices in which io6Ade and io6A either were not detected as of A. arguta stems was 12 ,uM. In addition, the reaction(s) metabolic products (11) or were detected as only minor converting i6Ade to io6Ade was 02-requiring and specific for metabolites (12). Because of this, I also screened a total of30 i6Ade versus N6-(A2-isopentenyl)adenosine (i6A). When A. different woody species for their abilities to accumulate kolomikta callus was fed 30 MM i6A, io6Ade increased and io6Ade in feeding experiments with i6Ade, finding that io6Ade reached a concentration corresponding to 6 nmol/g (fresh accumulates in only certain defined systematic groupings; weight) in 8 hr. Ribosylzeatin (io A) did not increase. Under N2 this suggests that different mechanisms for io6Ade biosyn- during i6A feeds, i6A accumulated rather than being metabo- thesis may exist among plants. lized to i6Ade, suggesting that i6A normally may be metabolized via i6AMP and i6Ade to io6Ade. A survey of30 species ofwoody MATERIALS AND METHODS plants in 20 families of dicotyledonous angiosperms indicated that the ability to accumulate io6Ade (>10 nmol/g) in 24-hr The methodology for demonstrating the conversion of i6Ade feeds with 30 MM i6Ade was restricted to certain systematic to io6Ade in Actinidia has been reported (9). Basically, it groups -.g., order , families Oleaceae and Rubia- involved incubation of tissue slices on agar medium consist- ceae. This suggests that plants may differ in their pathways for ing of basal inorganic (13) and organic (14) nutrients plus io6Ade biosynthesis and that cytokinin biochemistry has po- i6Ade, followed by fixation of tissues in 2.5 vol of cold 95% tential as a taxonomic character above the species and ethanol per g (fresh weight). After overnight incubation in the levels. cold, the mixture was homogenized, filtered through paper, and the filtrate was concentrated by evaporation in vacuo. Zeatin [io6Ade; 6-(4-hydroxy-3-methylbut-2-enylamino)- Then, after resuspending the residue in 10% ethanol, the purine], which was the first cytokinin to be identified from extract was analyzed for cytokinin content by using HPLC as plants (1, 2), has been found in a broad spectrum of described below. angiosperm species and in gymnosperm species as well. It is In screening woody species for their abilities to accumulate also the most potent naturally occurring cytokinin in several io6Ade during i6Ade feeds, tissue slices of growing stems or bioassays (3). Thus, based on both its distribution and autonomous tissue cultures were incubated with 30 ,uM i6Ade biological activity, either io6Ade or a closely related metab- for 24 hr and then fixed and extracted. Extracts were olite is a prime candidate as the active form of cytokinin in analyzed by HPLC using a Varian 5000 instrument with a plants. 1-ml injector loop and a C18 Micropak MCH-5 column (30 cm Although the biosynthetic pathway(s) producing io6Ade is x 4 mm). The column was developed with a linear methanol unknown, both Letham and Palni (4) and Chen (5) have gradient from 15% to 100% for 85 min, followed by a gradient proposed schemes involving hydroxylation ofNM-(A2-isopent- from 100% to 15% for 30 min. HPLC profiles from i6Ade fed enyl)adenine (i6Ade) and its mononucleotide (i6AMP). More- and nonfed tissues were then evaluated for evidence of over, hydroxylation of i6Ade or related isopentenyl cyto- io6Ade accumulation in feeding experiments. The presence of kinins has been reported in at least five different systems; an absorbance peak in the region corresponding to io6Ade the fungus Rhizopogon (6, 7), immature corn kernels (7), and equivalent to >10 nmol/g (fresh weight) was taken as microsomes from tobacco tissue cultures (5), microsomes tentative evidence for io6Ade accumulation. In each case from cauliflower florets (8), and several different tissues from indicated, the tentative identification of io6Ade was con- Actinidia (9). This paper reports further details of the con- firmed by collecting the presumed peak and showing that its

The publication costs of this article were defrayed in part by page charge Abbreviations: io6Ade, zeatin; i6Ade, N6-(A2-isopentenyl)adenine; payment. This article must therefore be hereby marked "advertisement" io6A, ribosylzeatin; i6A, N6-(A&2-isopentenyl)adenosine; i6AMP, N6- in accordance with 18 U.S.C. §1734 solely to indicate this fact. (A2-isopentenyl)adenine mononucleotide. 972 Downloaded by guest on September 27, 2021 Botany: Einset Proc. Natl. Acad. Sci. USA 83 (1986) 973 RESULTS i6Ade Metabolism in Actinidia. Autonomous tissue cultures of Actinidia kolomikta started from growing stem segments rapidly accumulated io6Ade on a medium supplemented with 30 ,M i6Ade. io6Ade increased linearly from a basal concen- tration of 2 to 160 nmol/g (fresh weight) in 8 hr (Fig. 1). After 100 _ 72 hr, the level of io6Ade reached nearly 500 nmol/g (fresh weight; data not shown). By contrast, internal i6Ade levels increased during only the initial 2 hr of incubation on i6Ade medium and then they declined. Presumably, i6Ade levels ~50 obtained during feeds reflected influx into the tissue minus o 0 metabolism. In fact, when tissues were incubated for 2 hr in 30 ,M i6Ade and then transferred to cytokininless medium (dashed line in Fig. 1), i6Ade levels decreased precipitously, indicating that i6Ade is rapidly metabolized. Under these same transfer conditions, io6Ade content remained relatively level-i.e., once formed in feeding experiments, io6Ade is not 5 Ade~ appreciably metabolized. To determine the apparent Km of i6Ade in the conversion of i6Ade to io6Ade in Actinidia, tissue slices were incubated on different concentrations of i6Ade, and the linear rates of 0 ~~ ~ ~~A------io6Ade accumulation were determined. Fig. 2 shows a 0 2 4 6 8 Lineweaver-Burk plot of io6Ade formation in A. kolomikta Time, hr callus. The apparent Km, calculated from the x intercept, corresponds to 12 AM. Thus, the Km for A. kolomikta is in the FIG. 1. Effect of i6Ade feeds on io6Ade and i6Ade levels in callus range of the endogenous i6Ade level. Callus of Actinidia of A. kolomikta. Tissue slices from 3- to 4-week cultures were transferred to nutrient medium supplemented with 30 ,uM i6Ade. A, chinensis x Actinidia arguta and tissue slices from stems of Cytokinin concentrations in tissues incubated with i6Ade for 2 hr and A. arguta also had a Km for i6Ade of 12 ,M (data not shown). then transferred to cytokininless medium. Data are from a single i6A Metabolism. Fig. 3, which summarizes feeding exper- experiment; the feeding experiment was conducted on three separate iments with 30 ,M i6A, shows that i6A levels increased occasions with similar results. rapidly during the initial 2 hr, declined, and then reached a relatively constant level of 10 nmol/g (fresh weight) by 3 hr. UV spectrum was identical to that of standard io6Ade. By contrast, io6Ade increased slowly and linearly during the Species meeting these criteria are indicated as positive (+). incubation period, reaching approximately 6 nmol/g (fresh Species that did not produce a significant io6Ade peak are weight) by 8 hr. Levels of i6Ade and io6A remained low and indicated as negative (-). undetectable, respectively.

0

y = 0.26x + 0.02 Km = 12 ,tM

1-4

1/S FIG. 2. Lineweaver-Burk plot for io6Ade accumulation in A. kolomikta fed i6Ade. Tissue slices were incubated for 8 hr on nutrient medium supplemented with different i6Ade concentrations and then analyzed for io6Ade accumulation. S, substrate in M-1; v = velocity in nmol'g-1. Downloaded by guest on September 27, 2021 974 Botany: Einset Proc. Natl. Acad. Sci. USA 83 (1986) Table 1. Cytokinin contents of A. kolomikta callus incubated with 30 IAM i6A in air or in a N2 atmosphere Levels in callus, Hours of nmol/g (wet weight) incubation Atmosphere i6Ade i6A io6Ade 0 Air 0.5 ± 0.2 ND 2.1 ± 0.5 - ~~~~00 0 U 1 Air 0.7 ± 0.1 5.2 ± 1.0 2.4 ± 0.4 1 N2 1.3 ± 0.4 12.4 ± 2.3 2.5 ± 0.2 E 2 Air 0.7 ± 0.2 15.2 ± 0.6 3.5 ± 0.4 2 N2 0.6 ± 0.1 38.6 ± 5.1 2.3 ± 0.2 - C Each point represents the mean of four independent determina- tions ± SEM. ND, not detectable-i.e., <0.1 nmol/g (wet weight).

> ~~~~~~0 io6Ade tissues (Table 1). It also prevented the metabolism of i6A to ;50 io6Ade. Presumably, the sequence of reactions converting ~ 0~~~~~ 0~ i6A to io6Ade involves i6A, i6AMP, i6Ade, and io6Ade. Other plausible pathways such as i6A, i6Ade, and io6Ade or i6A, 0O ° i6Ade io6A, and io6Ade are not excluded, but they seem less likely. 0 0 After all, there was no evidence for significant hydroxylation ° , i6A to 0 L8 of i6A to form io6A or for activity that would convert 0 2 4 6 8 i6Ade or for activity converting io6A to io6Ade. If a Time, hr phosphorylase catalyzing the i6A-to-i6Ade reaction were present in significant quantities, for example, it would be FIG. 3. Effect of i6A feeds on io6Ade, i6Ade, and i6A levels in expected to be active, even in a N2 atmosphere (15). callus of A. kolomikta. Tissue slices were incubated on nutrient Comparative i6Ade Metabolism Among Woody Species. medium supplemented with 30 ,uM i6A. Data are from one of three which had similar results. Table 2 shows the results of i6Ade feeding experiments with different experiments, all of tissues of different woody species. It is apparent that io6Ade accumulation occurred in only certain taxonomic groupings. Incubation of callus with i6A in the presence of N2 instead None ofthe species in superorders Magnoliidae and Rosidae, of air resulted in an increased accumulation of i6A within the for example, accumulated io6Ade to significant levels, but all Table 2. Distribution of i6Ade to io6Ade activity among woody plant taxa Superorder Order Family Species Status Magnoliidae Magnoliales Magnoliaceae Magnolia heptapeta x Magnolia acuminata Liriodendron tulipifera Annonaceae Asimina triloba Laurales Calycanthaceae Calycanthus fertilis Dilleniidae Dilleniales Dilleniaceae Hibbertia japonica Theales Theaceae Camellia japonica Franklinia alatamaha Malvales Tiliaceae Tilia platyphyllos Urticales Ulmaceae Ulmus parvifolia Ericales A. arguta + A. chinensis x A. arguta + A. chinensis + A. kolomikta + Ericaceae Rhododendron aurigeranum + Rhododendron jasminiflorum + Clethraceae Clethra alnifolia + Cyrillaceae Cyrilla racemiflora + Rosidae Fabales Leguminosae Robinia pseudoacacia Cornales Cornaceae Cornus kousa Sapindales Aceraceae Acer mono Staphyleaceae Staphylea holocarpa Sapindaceae Koelreuteria paniculata Rutaceae Citrus medica Citrus sinensis Asteridae Gentianales Oleaceae Syringa vulgaris + Forsythia sp. + Scrophulariales Scrophulariaceae Paulownia coreama Rubiales Rubiaceae Cephalanthus occidentalis + Coffea arabica + Damnacanthus indicus + Tissue slices from growing stems were incubated with i6Ade as described. For species of Actinidia, autonomous tissue cultures were also tested. Downloaded by guest on September 27, 2021 Botany: Einset Proc. Natl. Acad. Sci. USA 83 (1986) 975 of the species we tested in order Ericales had high io6Ade plants: those that accumulated significant concentrations of contents after incubation. Mok et al. (11) examined i6A io6Ade [.10 nmol/g (fresh weight)] in tissue slices and those metabolism in Phaseolus vulgaris and Phaseolus lunatus, that didn't. I furtherfound that plants in the two categories were two herbaceous species in Leguminosae, and found no not randomly distributed in relation to systematics. Thus, the evidence for its metabolism to io6A or io6Ade. Again based characteristic may have potential in chemotaxonomy. on Table 2, additional families exhibiting significant io6Ade The example of the genus Actinidia illustrates how accumulation in feeds were Oleaceae and Rubiaceae, both in cytokinin biochemistry might be of value as a taxonomic superorder Asteridae. Paulownia coreama, also in superor- characteristic (18). According to Bessey's landmark descrip- der Asteridae, did not accumulate io6Ade. tion of Angiosperm systematics, Actinidia was placed in family Dilleniaceae, order Ranales. However, our data on DISCUSSION i6Ade metabolism in presumed relatives (i.e., Magnolia, Liriodendron, Asimina, Calycanthus, and Hibbertia) show In this report, a simple but definitive HPLC methodology is that Actinidia can be distinguished from them based on used to study aspects of io6Ade biosynthesis in tissue slices cytokinin biochemistry. Likewise, Actinidia differs with of woody plant species. The evidence indicates again (9) that respect to cytokinin metabolism from Camellia and species of Actinidia rapidly metabolize i6Ade in feeding Franklinia, two genera in order Theales which Hutchinson experiments and that the tissues accumulate io6Ade to considered to be the proper order for his designation offamily concentrations approaching 500 nmol/g (fresh weight) in 72 Actinidiaceae. On the other hand, the data on io6Ade accu- hr on a medium with 30 /iM i6Ade. It is further shown that the mulation are consistent with the placement of Actinidia in i6Ade-to-io6Ade conversion is 02-requiring and that this order Ericales by both Hallier and Cronquist. Hallier con- activity appears to be specific for i6Ade, inasmuch as I did not sidered Actinidia as a genus within family Clethraceae and I detect hydroxylation of i6A in comparable feeding experi- found Clethra alnifolia to accumulate io6Ade. By contrast, ments. By analogy with other hydroxylation systems, both Cronquist placed Actinidia in a separate family-i.e., plant (16) and animal (17), it is possible that i6Ade hydroxyla- Actinidiaceae. tion is mediated by a membrane-bound monooxygenase. Chen (5) and Chen and Leisner (8) have reported on 1. Letham, D. S., Shannon, J. S. & MacDonald, I. R. (1964) microsomal activity from tobacco tissue cultures and cauli- Proc. Chem. Soc. (London), 230-231. flower florets that will hydroxylate i6Ade and i6A. 2. Miller, C. 0. (1965) Proc. Natl. Acad. Sci. USA 54, 1052-1058. Even the mechanism of 3. Skoog, F. & Armstrong, D. J. (1970) Annu. Rev. Plant Phys- though i6Ade hydroxylation is not iol. 21, 359-384. completely understood, the evidence suggests that this ac- 4. Letham, D. S. & Palni, L. M. S. (1983) Annu. Rev. Plant tivity could account for io6Ade biosynthesis in Actinidia Physiol. 34, 163-197. callus cultures based on the presence of i6Ade as a native 5. Chen, C. M. (1982) in Plant Growth Substances 1982, ed. cytokinin in these tissues at a concentration that is in the Wareing, P. F. (Academic, London), pp. 155-163. range of the apparent Km for the hydroxylation reaction. The 6. Miura, G. & Miller, C. 0. (1969) Plant Physiol. 44, 372-376. precursor of i6Ade, however, is not known. I fed i6A in the 7. Miura, G. & Hall, R. H. (1973) Plant Physiol. 51, 563-569. presence of N2 to test whether i6A is the direct precursor of 8. Chen, C. M. & Leisner, S. M. (1984) Plant Physiol. 75, i6Ade via a phosphorylase-type reaction. The rationale ofthe 442-446. experiment was that, if i6A is converted to i6Ade in one step, 9. Einset, J. W. (1984) Biochem. Biophys. Res. Commun. 124, i6Ade should accumulate under N2 because its subsequent 470-474. metabolism to io6Ade would be inhibited. The finding that 10. Einset, J. W., Biol. Plant., in press. 11. Mok, M. C., Mok, D. W. S., Dixon, S. C., Armstrong, D. J. i6Ade did not increase dramatically and that, instead, the N2 & Shaw, G. (1982) Plant Physiol. 70, 173-178. treatment caused i6A to accumulate suggests that metabolism 12. Laloue, M., Terrine, C. & Guern, J. (1977) Plant Physiol. 59, of i6A to i6Ade involves more than a single reaction. A 478-483. plausible sequence would be i6A-* i6AMP-* i6Ade, although 13. Murashige, T. & Skoog, F. (1962) Physiol. Plant. 15, 473-497. I have no direct evidence for it. 14. Linsmaier, E. M. & Skoog, F. (1965) Physiol. Plant. 18, Regardless of the details of cytokinin biochemistry, the 100-127. evidence indicates considerable variability among plants in 15. Ross, C. W. (1981) in The Biochemistry ofPlants, eds. Stumpf, their metabolic responses to i6Ade feeds. Letham and co- P. K. & Conn, E. E. (Academic, New York), Vol. 6, pp. workers have already documented several other instances of 169-204. plant-specific differences in the metabolism of exogenous 16. West, C. A. (1980) in The Biochemistry ofPlants, eds. Stumpf, P. K. & Conn, E. E. (Academic, New York), Vol. 2, pp. cytokinins (cf. ref. 4). In the experiments, slices of actively 317-364. growing tissues were incubated for 24 hr on a nutrient medium 17. Estabrook, R. W. & Werringloer, J. (1978) Methods Enzymol. supplemented with 30 AM i6Ade, and then the tissues were 52, 212-220. extracted and analyzed for their cytokinin contents. Based on 18. Lawrence, G. H. M. (1951) of Vascular Plants these tests, I was able to distinguish two different categories of (Macmillan, New York). Downloaded by guest on September 27, 2021