HORMONES IN PLANTS BEARING NITROGEN- FIXING ROOT NODULES: THE DISTRIBUTION OF CYTOKININS IN VICIA FAB A L.

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1 . (1976) 76, HORMONES IN PLANTS BEARING NITROGEN- FIXING ROOT NODULES: THE DISTRIBUTION OF CYTOKININS IN VICIA FAB A L. BY I. E. HENSON AND C. T. WHEELER ; -'"'' Botany Department, University of Glasgow >,,"'''''" {Received 22 September igy 5) ''- ' ',.. ', S U M M A R Y.:'..', -,..-'-.: ; : :[: ' Cytokinin activity in extracts of nodules, roots, stems and leaves of Viciafaba L. was estimated using the soybean callus bioassay. High levels of cytokinins were present in nodules and leaves while the amounts detected in roots and stems were much lower. Cytokinin levels in the root nodules were as much as twelve to thirteen times those detected in the roots. At least three kinds of cytokinins were present in the plant, two of which had similar chromatographic properties to the cytokinin zeatin and its riboside. The third cytokinin had properties which distinguished it from any of the known cytokinins. This peak was predominant in the leaves while the zeatin riboside-like peak was the main cytokinin detected in the root nodules and roots. INTRODUCTION The root nodules of nitrogen-fixing plants are important sinks for photosynthetic assimilates (e.g. Small and Leonard, 1969; Wheeler, 1971; Lawrie and Wheeler, 1973). The reduction in nitrogenase activity and in the supply of photosynthetic assimilates to the nodules of Pisum sativum during fruit development prompted the suggestion (Lawrie and Wheeler, 1974) that movement of assimilates to the nodules might be subject to hormonal control in a manner analogous to the 'hormone-directed' transport of nutrients in stems to such sinks as fruits and apical buds (e.g. Seth and Wareing, 1967; Patrick and Wareing, 1973). Root nodules contain substantial levels of auxins (e.g. Link and Eggers, 1940; Pate, 1958; Dullaart, 1967; i97oa,b) and gibberellin-like substances (Radley, 1961; Dullaart and Duba, 1970) while cytokinin activity has been detected in root nodules oi AInus glutinosa (Rodriguez-Barrueco and de Castro, 1974) and in nodules oi Phaseolus vulgaris (Puppo, Rigaud and Barthe, 1974). The latter workers found also that equivalent amounts of root tissue of Phaseolus contained little cytokinin activity. Phillips and Torrey (1970, 1972) showed that in its free-living form Rhizobium is capable of cytokinin production and can secrete cytokinins into the surrounding medium. This suggests that nodule cytokinins could originate, at least in part, from the endophyte and their possible importance in the initiation and development of the nodule has been considered (Torrey, 1961; Libbenga et al., 1973). The present paper examines the qualitative and quantitative distribution of cytokinins in nodulated plants of the broad bean, Vicia faba, as part of a wider investigation into the nature and role of plant hormones in the nitrogen fixing root nodule. :.,,:.-(.- i; r ^ * v DHT -h- rn;!? '^ H ' 433

2 434 I- E. HENSON AND C. T. WHEELER MATERIALS AND METHODS Plant material Plants oi Vicia faba L. 'Exhibition Long Pod', were grown in plastic pots (i6 cm diam.) in Peralite (British Gypsum Co. Ltd). The seed was inoculated prior to sowing with Wageningen Strain PN-27 of Rhizobium leguminosarum. The plants were grown in a controlled environment room with a photoperiod of 16 h ( hours) at I9 C and an 8-h dark period at i5 C, conditions which favour nodulation. The plants were supplied with nutrients (Crone's nitrogen-free formula) and continually deflowered during growth to minimize effects of fruit formation on nodule development and function. The plants were harvested 13 weeks after sowing. Extraction and partial purification of cytokinins The procedure used is summarized in Fig. i. The fresh plant material was homogenized and subsequently extracted in methanol: water (4:1 v/v) using 10 ml/g fresh weight. The extracts were stirred continuously at about i C for h, filtered and the -.i. ",.-. Tissue extracted.m.1 ='. ' - in methanol:water (4:1 v/v) ; -..'-: ' ' ' : -,,.. i \ :;:,,,-.,_,,. : - - " extract reduced to aqueous, '. -., ' i. ' i '^''f' ' " '. ' centrifuged '' ' '..,, -.. : ^.^ :. -, :.., ', ^ i -. - :. '., i.,, :,j,,:,!'. ' - -: cation-exchange,,. '. r -.. ; v^.' column.....,..' I I ' water wash (effluent) ammonia wash (eluate) i _ : i partitioned 4 x ethyl partitioned s x n-butanol acetate at ph 2.5 at ph 8.0 I - I i \ i 1, 1 ethyl aqueous- aqueous n-butanol acetate partitioned, (discard) reduced to dryness, (discard) 3 x n-butanol. redisolved in 100 ml at ph 8.0 distilled water, r;., I,;. partitioned 3 x ethyl ^ n-butanol aqueous- acetate at ph 2.5 (discard) treated with alkaline phosphatase, aqueous ethyl acetate partitioned 3 x (Fraction I) (Fraction IT) n-butanol at ph 8.0 ac(ueous n - b u t a n o l - ^,..,,'.....,.*. -..'.,!,..-:! :V..4 i.-. ~ (discard) (Fraction III) Fig. I. Outline of procedure used for the partial purification of cytokinins from extracts of Vicia faba..., ' ' residues re-extracted a further two times. The filtrates were combined and reduced to the aqueous phase (c. 100 ml) with a rotary film evaporator at 30 C. The extracts were adjusted to ph 3.5 with o.i N HCl and centrifuged (30 min, 20,000 x g, 2o C). The supernatants, at ph 3.5, were passed through a column of the cation-exchange resin Zerolit 225 (SRC 14, mesh, NHJ form) using 2.0 ml resin per gram initial fresh weight. The column was washed sequentially with ten volumes distilled water, six volumes 3.0 N NH4OH and two volumes distilled water. The ammonia and final water washes were

3 Cytokinins in Yicia (aha 435 combined and reduced to dryness on a rotary evaporator at 30 C. The residue, in 100 ml water at ph 8.0 was partitioned five times against equal volumes of water-saturated n-butanol. The aqueous phase was discarded. The n-butanol phase was reduced to dryness, the residue redissolved in 100 ml water and partitioned three times against equal volumes of ethyl acetate at ph 2.5. The aqueous phase was readjusted to ph 7.0 and reduced to dryness (Fraction I). The ethyl acetate phase was also reduced to dryness to give Fraction II. The initial water wash from the cation-exchange column was reduced to 100 ml, partitioned four times against equal volumes of ethyl acetate at ph 2.5 and then three times against water-saturated n-butanol at ph 8.0. The remaining aqueous phase was reduced to dryness then redissolved in 50 ml water containing 100 mg MgClj 6H2O and 10 mg alkaline phosphatase (calf intestinal mucosa, Sigma Chemical Co Ltd). The solution was incubated at ph 9.0 for 5.5 h at 37 C in a shaking water bath; this treatment converts cytokinin nucleotides, which are insoluble in butanol, to butanol-soluble nucleosides. The ph was then readjusted to ph 8.0 and the extract partitioned three times against equal volumes of water-saturated n-butanol. The combined n-butanol phases were evaporated to dryness and constituted Fraction III. ) ' ^ " Chromatography Paper chromatography was performed using 23-cm wide strips of Whatman 3 MM paper which were developed in a descending manner with isopropanol:o.88 sg NH4: water (10: i: i v/v) to a distance of about 30 cm. The papers were divided into 10 x0.1 i?p zones for bioassay after drying in an air stream for several hours at 30 C. Partition chromatography on Sephadex LH20 (Armstrong et al., 1969) was conducted with a 2.5 X90 cm column which was eluted with ethanol: water (7:13 v/v) at a flow rate of 30 ml per h. 30-ml fractions were collected and reduced to dryness in the bioassay flasks in an air stream at 40 C. Chemicals All solvents were redistilled before use. Zeatin and zeatin riboside were obtained from Calbiochem, N*'-(A^-isopentenyl) adenine and its riboside from Sigma Chemical Co Ltd, kinetin from British Drug Houses Ltd. Zeatin-9-^-D-glucoside was a gift from Dr R. Horgan. Bioassay The soybean cotyledon callus bioassay (Miller, 1963, 1968) was used to test fractions for cytokinin activity. Flasks, 100 ml conicals with 25 ml basal medium were inoculated with three callus explants (about 20 mg each). The cultures were maintained at 26 C under weak fluorescent light for days before fresh weights were recorded. Cytokinin levels were calculated by reference to a standard series of flasks containing kinetin which were cultured concurrently with each assay. RESULTS Bioassay of paper chromatograms of the three fractions obtained from nodule, root, stem and leaf extracts showed that cytokinin activity was confined mainly to Fraction I.

4 436 I. E. HENSON AND C. T. WHEELER No activity was detected in Fraction II in any plant part. Also no activity was detected in Fraction III, with the exception of stem extracts which contained about ng kinetin equivalents per kg fresh weight. This cytokinin activity was presumed to originate from ribonucleotides present in this fraction. Paper chromatography of Fraction I resolved two peaks of cytokinin activity, at about Rp 0.3 and 0.6. These are referred to as peaks I and II respectively. The relative amounts of activity in these peaks, as well as the total cytokinin activity, differed in the various plant parts (Table i). The root nodules contained the greatest cytokinin activity, closely followed by the leaves, while roots and stems had much lower levels. Cytokinin activity in the nodules was about times that detected in the roots and stems and about 30-40% higher than in the leaves. Peak II activity predominated in nodule extracts and was the only peak detected when 5 g fresh weight equivalent was bioassayed. Stem and root extracts also contained more activity in peak II than peak I, but the leaves contained, by contrast, considerably more activity in peak I than peak II. The Rp of peak II was similar to that of zeatin, zeatin riboside, N -(A^-isopentenyl) adenine (i^ade) and N*- (A^-isopentenyl) adenosine (i^ado) which are not well separated by this system. Peak I did not chromatograph with any of the cytokinin standards but had a similar Rp to that of adenine and adenosine. Table i. Levels of cytokinms in various plant parts as determined hy the soybean callus bioassay following paper chromatography of Fraction I in isopropanol:ammonia:water (10:1:1 w/w). Cytokinin levels are given as ixg kinetin equivalents per kg fresh weight. Results based on the analysis of 25 g fresh weight equivalents of tissue, except for first bioassay of nodules for which only 5 g was used. Plant part Nodules Roots Stems Leaves..- Peak I N.D. N.D. 3-8 First bioassay Peak II II.O Total II.O Peak I 55-4 ft' Second bioassay Peak II Total \','", N.D. = Activity not detected. ^., _ The nature of the cytokinins present in Fraction I was investigated further by elution of the biologically active zones from the paper chromatograms with methanol: water (4:1 v/v) and chromatography of the combined eluates on a Sephadex LH-20 column. Three main peaks of cytokinin activity were resolved (Fig. 2A-D). These are termed a, b and c in order of increasing elution volume and were found to elute off the column in the same volumes of eluate as authentic samples of zeatin-9-glucoside, zeatin riboside and zeatin, respectively. Although peaks b and c behaved in all respects like zeatin and its riboside, the more polar peak a is unlikely to be zeatin-9-glucoside since the latter compound migrated to Rp on paper chromatograms (isopropanol: ammonia: water, 10:1:1 v/v), a region which did not coincide with cytokinin activity in the extracts. Bioassay of the Sephadex LH20 column eluates showed that in the nodules and roots activity present at peak b exceeded that of peaks a and c. In the leaves, most activity was associated with peaks a and c and the zeatin riboside-like peak b was not detected (Table 2).

5 Cytokinins in Vicia faba 437 ZG7R (A) 25 n i S ZGZR Z i 5 r t/i (B) I'O Q. O» callus WeigtIt of 2-5 r (D) '25 id Elufion volunne (ml) 900 Kinetin (/^g/l) n Elution volume (ml) Kinetin (;UgA) 900 Fig. 2. Soybean callus bioassays of Fraction I of extracts of nodules (A), roots (B), stems (C) and leaves (D) of Vicia faba passed through a Sephadex LH20 column eluted with ethanol:water (7:13) at 30 ml per h. The positions of elution of zeatin-9-glucoside (ZG), zeatin riboside (ZR), zeatin (Z), N*-(A^-isopentenyl)adenosine (i''ado) and N''-(A^-isopentenyl)-adenine (i"'ade) are indicated by horizontal bars. Table 2, Levels of cytokinins in various plant parts as determined by the soybean callus bioassay following partition chromatography of Fraction I on a column of Sephadex LHzo eluted with ethanol: water (7:13 vjv). Cytokinin levels are given as jxg kinetin equivalents per kg fresh weight. Results based on the analysis of ^o g fresh weight of tissue, except for the nodules for which 20 g was used. :ak a b c Elution volume (ml) Total Nodules Roots N.D ' ' '' N. D. = Activity not detected. Stems N.D, 2-5 2,2 4-7 Leaves 36.8 N.D, '<] -rr-.^hl :,-i II \ DISCUSSION The root nodules of Viciafaba evidently contain high levels of cytokinins. Although the nodules comprised only a small part of the total root system (^some 0,55 g per plant compared to 16,1 g per plant for the roots proper) they contained, nevertheless, a substantial proportion of the cytokinin activity associated with the root system. Thus out of a total of 269 ng per plant kinetin equivalents present in the root system (nodules

6 438 I. E. HENSON AND C. T. WHEELER plus roots) 83 ng per plant was present in the nodules, as estimated following paper chromatography of the extracts. These results agree with the findings of Puppo, Rigaud and Barthe (1974) for Phaseolus vulgaris and are also supported by investigations on cytokinins in Alnus glutinosa, currently in progress (Henson and Wheeler, unpublished results). It appears, therefore, that all three groups of the major growth-promoting hormones are concentrated in the nodules, since it is known already (see Introduction) that root nodules contain higher levels of auxins and gibberellins than normal root tissue. The preliminary chromatographic analysis of cytokinin activity suggests that at least three cytokinins are present in Vicia. Two of the cytokinins (Fig. 2, peaks b and c) behaved in all respects like zeatin and its riboside. The possible nature of peak a is unknown. The cytokinin complement of Vicia nodules is different, therefore, from that of Phaseolus nodules in which Puppo et al. (1974) reported the presence of cytokinin activity which chromatographed close to the position of i^ade and i^ado. As these two cytokinins may be recovered readily from an aqueous solution, even at low ph, by partitioning witb ethyl acetate (Hemberg, 1974; Letham, 1974) they would have appeared, if present, in Fraction II. None of the extracts of Vicia showed activity in this fraction. Further analysis is required to establish fully whether the cytokinins with identical chromatographic properties from different plant organs are indeed the same compound(s) and also whether each peak contains more than one compound. The high levels of cytokinin activity in the leaves, relative to that found in root and stem extracts (Tables i and 2), is of interest since it is thought that leaf cytokinins may originate in the roots and be transported to the leaves in the transpiration stream (Kende, 1971). The reduction in the numbers of meristematic sites on the Vicia stems as a result of deflowering (see Methods) may have contributed to the high levels of leaf cytokinin activity as Engelbrecht (1972) found that cytokinins, in the form of a polar peak of activity, accumulated in the leaf blades of Phaseolus leaf cuttings lacking a shoot meristem. Similarly, Beever and Woolhouse (1974) found that removal of buds from the stems of Perilla lead to a large increase in cytokinin-like activity in the xylem sap. It is also conceivable that the root nodules may contribute compounds with cytokinin activity to the shoot. Translocation of nodule cytokinins could have special effects on the physiology of the plant and might be responsible for some of the differences which have been noted between nodulated and non-nodulated plants of the same species. Several workers have demonstrated effects of cytokinins on chloroplast development and chlorophyll synthesis (e.g. Adepipe, Hunt and Fletcher, 1971; Farineau and Roussaux, 1975) and it is of interest, therefore, that Sironval, Bonnier and Verlinden (1957) found that the chlorophyll content of young leaves of nodulated soybeans was higher than that of uninoculated plants fed combined nitrogen. Further studies to elucidate some of these problems are currently in progress at Glasgow.,. i,-,ij i tr.*;?; ''I '7-. ^ i M, ACKNOWLEDGMENTS We thank Dr R. Horgan for a gift of zeatin-9-glucoside. Professor M. B. Wilkins for the loan of UV monitoring equipment and Mrs M. E. McLaughlin for help with the culture of the plants. Strain PN27 of Rhizobium leguminosarum was kindly supplied by Dr A. Houwers of Landbouwhogeschool, Wageningen. The work was carried out with the aid of grant B/RG/71340 from the Science Research Council., ' Mnmiyni.::-. ads '!v

7 Cytokinins in Yicia iaha. 439 REFERENCES ADEPIPE, N. O., HUNT, L. A. & FLETCHER, R. A. (1971), Effects of benzyladenine on photosynthesis, growth and seneseenee of the bean plant. Physiologia PL, 25, 151. ARMSTRONG, D. J., BURROWS, W. J., EVANS, P. K. & SKOOG, F. (1969). Isolation of cytokinins from RNA. Biochem. Biophys. Res. Commun., 37, 451. BEEVER, J. E. & WooLHOUSE, H. W. (1974). Increased eytokinin export from the roots oi Perilla frutescens following disbudding or floral induction. Roy. Soc. New Zealand Bull., 12, 681. DuLLAART, J. (1967). Quantitative estimation of indoleacetic aeid and indolecarboxylic aeid in root nodules and roots of Lupinus luteus L. Acta. bot. neerl., id, 222. DuLLAART, J. (1970a). The bioproduction of indole-3-acetic acid and related compounds in root nodules and roots of Lupinus luteus L. and by its rhizobial symbiont. Acta. bot. neerl., 19, 573. DuLLAART, J. (1970b). The auxin eontent of root nodules and roots oi Alnus glutinosa (L.) Vill.jf. exp. Bot., DuLLAART, J. & DuBA, L. I. (1970). Presence of gibberellin-like substances and their possible role in auxin bioproduction in root-nodules and roots of Lupinus luteus L. Acta bot. neerl., 19, 877. ENGELBRECHT, L. (1972). Cytokinins in leaf cuttings of Phaseolus vulgaris L. during their development. Biochem. Physiol. Pflanz., 163, 335. FARINEAU, N. & RoussAUX, J. (1975). Influence de la 6-benzyl-aminopurine sur la differenciation plastidiale dans les cotyledons de concombre. Physiologia PL, 33, 194. HEMBERG, T. (1974). Partitioning of cytokinins between ethyl acetate and acid water phases. Physiologia PL, 32, 191. KENDE, H. (1971). The cytokinins. Int. Rev. CytoL, 31, 301. LAWRIE, A. C. & WHEELEB, C. T. (1973)- The supply of photosynthetic assimilates to nodules of Pisum sativum L. in relation to the fixation of nitrogen. New Phytol., 72, LAWRIE, A. C. & WHEELER, C. T. (1974). The effects of flowering and fruit formation on the supply of photosynthetic assimilates to the nodules of Pisum sativum L. in relation to the fixation of nitrogen. New Phytol., 73, iijg. LAWRIE, A. C. & WHEELER, C. T. (1975). Nitrogen fixation in the root nodules of Vicia faba L. in relation to the assimilation of earbon. i. Plant growth and metabolism of photosynthetic assimilates. Neio Phytol., 74, 429. LETHAM, D. S. (1974). Regulators of cell division in plant tissues. XXI. Distribution coeflicients for cytokinins. Planta, 118, 361. LiBBENGA, K. R., IREN, F. VAN, BoYERS, R. ]. & SHRAAG-LAMERS, M.F. (1973). The role of hormones and gradients in the initiation of cortex proliferation and nodule formation in Pisum sativum L. Planta, 114, 29. LINK, G. K. K. & EGGERS, V. (1940). Avena eoleoptile assay of ether extracts of nodules and roots of bean, soybean and pea. Bot. Gax., ioi, 650. MILLER, C. O. (1963). Kinetin and kinetin-like compounds. In: Modern Methods of Plant Analysis. (Ed. by H. F. Linskens and M. V. Tracey), pp Springer-Verlag, Berlin. MILLER, C. O. (1968). Naturally occurring cytokinins. In: The Biochemistry and Physiology of Plant Growth Substances. (Ed. by F. Wightman and G. Setterfield), pp Runge Press, Ottawa. PATE, J. S. (1958). Studies of the growth substances of legume nodules using paper chromatography. Aust. y. biol. Sci., 11, 516. PATRICK, J. W. & WAREING, P. F. (1973). Auxin-promoted transport of metabolites in stems oi Phaseolus vulgaris L. J. exp. Bot., 24, PHILLIPS, D. A. & TORREY, J. G. (1970). Cytokinin production by Rhizobium japonicum. Physiologia PL, 23, PHILLIPS, D. A. & TORREY, J. G. (1972). Studies on cytokinin production by Rhizobium. PL Physiol., Lancaster, 49, 11. Puppo, A., RiGAUD, J. & BARTHE, P. (1974). Sur la presence de cytokinines dans les nodules de Phaseolus vulgaris L. Cr. hebd. Seanc. Aead. Sci., Ser. D., 279, RADLEY, M. (1961). Gibberellin-like substances in plants. Nature, Lond., 191, 684. RODRIGUEZ-BARRUECO, C. & DE CASTRO, F. (1974). Cytokinin-induced pseudonodules on Alnus glutinosa. Physiologia PL, 29, 277., SETH, A. K. & WAREING, P. F. (1967) Hormone-directed transport of metabolites and its possible role in plant senescence. J. exp. Bot., 18, 65. SiRONVAL, C, BONNIER, CH. & VERLINDEN, J-P. (1957). Action of day length on nodule formation and chlorophyll content in soybean. Physiologia PL, 10, 697. SMALL, J. G. C. & LEONARD, D. A. (1969). Translocation of '"'C-labelled photosynthate in nodulated legumes as influenced by nitrate nitrogen. Am.J. Bot., 56, 187. TORREY, J. G. (1961). Kinetin as a trigger for mitosis in mature endomitotic plant cells. Expl. Cell Res., 23, WHEELER, C. T. (1971). The causation of the diurnal changes in nitrogen fixation in the nodules of Alnus glutinosa. New Phytol., 70, 4.8y. _....-,

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