Planta (1993)189:312 320

Pl~__t)~ 9 Springer-Verlag 1993

Immunoaffinity co-purification of cytokinins and analysis by high-performance liquid chromatography with ultraviolet-spectrum detection Bj6rn Nicander, Ulf Stfihl, Per-Olof BjBrkman, and Elisabeth Tillberg Department of Plant Physiology, Swedish University of Agricultural Science, P.O. Box 7047, S-75007 Uppsala, Sweden; Fax: 46(18) 67 29 30 Received 23 September; accepted 10 October 1992

Abstract. A rapid methodology for the simultaneous analysis of a large number of cytokinins is presented. The cross-reactivity of a mixture of polyclonal antibodies against zeatin riboside and isopentenyladenosine was exploited in a protocol that can be used for immunoaffinity purification of 23 additional cytokinins. Ligands include the cytokinin bases zeatin, dihydrozeatin, isopentenyladenine, benzyladenine and kinetin, and their corresponding nucleoside, nucleoside-5'-monophosphate, and 9-glucoside derivatives, as well as cis-zeatin, cis-zeatin riboside, the 2-methylthiol derivatives of isopentenyladenosine and zeatin riboside, and benzyladenine-3-glucoside. Mixtures of cytokinins could be retained with high recoveries of all the components. Immunoaffinity purification of extracts of Arabidopsis thaliana (L.) Heynh, and Solanum tuberosum L. gave fractions clean enough, as verified by gas chromatographymass spectrometry (GC-MS), to allow analysis of endogenous cytokinins using a single high-performance liquid chromatography (HPLC) step with on-line UV-spectrum detection. The detection limit was 4-6 pmol. The procedure described forms a routine assaying technique that is faster and simpler, yet yields better qualitative and quantitative information than the commonly used procedure of immunoassaying of HPLC fractions. Key words: Arabidopsis- Cytokinin - Immunoaffinity chromatography- Solanum Zeatin

Introduction Cytokinins are a class of plant growth substances, so named because they induce cell division in plant tissue IAC=immunoaffinity chromatography; ipA= isopentenyladenosine, 9-fl-D-ribofuranosyl-N6-(AZ-isopentenyl)adenine; G C - M S = g a s chromatography-mass spectrometry; HPLC = high-performance liquid chromatography; PBS = Phosphate-buffered saline; ZR=zeatin riboside; 9-fl-D-ribofuranosylzeatin Abbrevations:

(Skoog et al. 1965). Generally they promote growth, induce bud formation and bud development, break apical dominance, and counteract senescence. The beststudied group of compounds with cytokinin action found in plants are adenines substituted at N 6 with either isoprene, a modified isoprene, a benzyl group, or an o-OHbenzyl group. Among the metabolites are nucleosides, nucleotides, and a number of conjugates at positions 7, 9 and at the hydroxylated isoprene-derived sidechain, most often with glucose (Letham and Palni 1983). A family of modified bases found in the transfer RNA of plants, animals and microorganisms are also cytokinins. Some of these compounds have been reported as free molecules in plants at levels where they can have physiological effects (Louis and Durand 1978; Takagi et al. 1989). Investigations have indicated roles for various cytokinins as being active forms, transport forms, storage forms, or inactivated forms (Letham and Palni 1983; McGaw et al. 1984), but much remains to be learned about the roles of the components of this complex metabolic system, both on the physiological and the biochemical levels. Fast and simple assays for most of the cytokinins have not been available. The popular enzyme-linked immunosorbent assay (ELISA) and radio immunoassay (RIA) techniques are usually used to assay for only a few, namely zeatin, zeatin riboside (ZR), isopentenyladenine and isopentenyladenosine (ipA). In a few cases, immunoassays have been devised for cytokinins other than these (Turnbull and Hanke 1985; Badenoch-Jones et al. 1987; Smart et al. 1991). The latter make use of the ability of antibodies raised against ZR or ipA to crossreact to a larger or smaller degree with other, structurally similar, cytokinins (Weiler 1980; Barthe and Stewart 1985). When antibodies against ZR or ipA were used in immunoaffinity chromatography (IAC), several cytokinins other than the immunogens were also retained (McDonald and Morris 1985; Davies et al. 1986; McDonald et al. 1986; Wang et al. 1987; Morris et al. 1991).

B. Nicander et al. : Co-purification of cytokinins

Here we report that at least 25 cytokinins have enough affinity for a mixture of anti-ZR and anti-ipA antibodies to make immunoaffinity purification of them possible. Immunoaffinity chromatography purification followed by HPLC with on-line UV-spectrum analysis is a rapid procedure suitable for the routine analysis of large numbers of samples. Materials and methods Chemicals. Deuterium-labeled and unlabeled cytokinins were from Apex Organics (Leicester, U K ) ; eis-zeatin, cis-zeatin riboside, kinetin, kinetin riboside, benzyladenine and benzyladenosine from Sigma (St. Louis, Mo., USA); ADP, ATP, and 2',3"-dideoxyadenosine from Pharmacia (Uppsala, Sweden). Other purines, 1,3-diphenylurea, N-(2-chloro-4-pyridyl)-N'-phenylurea, polylysine-Lagarose, and Crotalus 5'-phosphatase (N--4005) were from Sigma. Tritiated ipA ( ~ 1.8 T B q - m m o 1 - 1 ) was synthesized from [2,5", 8-3H-]adenosine (2.15 T B q . m m o l - 1 ; Amersham, UK) as described by Laloue and Fox (1987). 6-N-(3-Methyl-3-hydroxybutylamino)purine was prepared as described by Robins (1967).

Preparation of the immunoaffinity gel. Zeatin riboside or ipA were coupled to bovine serum albumin (Serva, Heidelberg, F R G ; Weiler 1980). Antisera against the conjugates were induced in New Zealand White or New Zealand Black rabbits supplied by the National Veterinary Institute (Uppsala, Sweden). Anti-cytokinin antibodies were affinity-purified with polylysine-agarose columns to which the respective cytokinin had beer~ bound (McDonald and Morris 1985). Anti-zeatin riboside and anti-ipA antibodies were bound to Affi-gel 10 (Bio-Rad, Richmond, Calif., USA). Each IAC column consisted of 1 ml of gel with 0.5 mg of anti-ZR and 0.5 mg of anti-ipA antibodies packed in a 3-ml polypropylene tube (J.T. Baker, Philipsburg, N.J., USA). The IAC columns were stored in phosphatebuffered saline (PBS) with 0.2% sodium azide at 4 ~ C.

Immunoaf['inity chromatography. A precolumn containing 1 ml Sepharose 6B (Pharmacia) in a 3-ml polypropylene tube was fitted on top of each IAC column. Sample, eluents and columns were taken to 25 ~ C. The flow-rate was kept between 1-2 ml per rain during sample loading and washing. A maximum of 10 ml of sample was applied to each column assembly. The coupled column sets were each washed with 2 x 2.5 ml 25~ PBS. The precolumns were removed. The IAC columns were each washed with 5 x 2.5 m125 ~ C PBS. Each column was eluted with (i) 3 ml water, (ii) 2 ml 30% methanol and (iii) 3 ml 100% methanol, and the eluates pooled. The IAC columns were immediately washed with a further 2 x 2.5 ml methanol, 2 x 2.5 ml water and 2 x 2.5 ml PBS, and were then ready for another cycle of purification. Water and methanol were HPLC grade. The precolumns did not bind any measurable amount of cytokinins. The IAC eluate was concentrated to approx. 300 gl by vacuumevaporation (Evapotek; Haake-Biichler Instruments, Saddle Brook, N.J., USA). An aliquot of 100 pmol of dideoxyadenosine was added to serve as an internal standard in the subsequent HPLC step. In the tests to determine which cytokinins bind and column recoveries, standards were diluted in 2 ml PBS and applied to IAC columns without precolumns. The washing and eluting were as described above. In the HPLC step, monitoring was at 265 nm only, giving a detection limit of < 0.5 pmol. High-performance

liquid chromatography. Cytokinins were separated on a Supersphere RP-select B HPLC column (250 mm long, 4 mm i.d.; Merck, Darmstadt, FRG). The eluents contained 2% HPLC-grade acetic acid (J.T. Baker) in water, and the column was eluted with a gradient from 1 to 40% acetonitrile (HPLC grade; Rathburn, Walkerburn, Scotland, UK). A stepped linear gradient

313 from 1 to 40 % acetonitrile with the following profile was used: 0 rain 1% acetonitrile, 2 min 3.8%, 8 min 7%, 16 min 7%, 20 rain 13%, 28 min 15%, and 40 min 40%. The pH changed from approx. 2.7 to approx. 2.8 during the gradient. The flow rate was 1 ml per rain, and the column temperature 30~ C. The HPLC pump was a MerckHitachi 655A-12 with a 6-ml mixing volume between gradient mixer and column top. Radioactivity of the ipA fraction was determined by scintillation counting in Ultima Gold (Packard Instruments, Meriden, Conn., USA).

Analysis of UVspectra. Spectra were obtained with a Spectra-Focus scanning spectrophotometric HPLC detector (Spectra-Physics, San Jose, Calif., USA). The interval between 240 (eluent cut-off limit) and 320 nm was scanned approximately twelve times a second, yielding three averaged "time slices" per second, each slice with an absorbance value for every 5 nm. Spectra were produced by interpolation of these values using instrument software or, for small peaks a spreadsheet program (Synergy software, Reading, Pa., USA) where up to 100 time-slices were averaged to produce spectra of higher accuracy. Plant materials and treatment. Potato (Solarium tuberosum L., cv. Bintje) tubers were taken from cold storage and allowed to sprout in the dark at room temperature. Sprouts 1-2 cm long and underlying tuber tissue (2-5 mm) were removed with an 1 I-ram-diameter cork borer. Arabidopsis thaliana L. (Heynh.) was cultivated in pots with soil in a growth chamber at 22~ and a 16-h photoperiod for four weeks. Flowering plants were harvested 4 h after the beginning of the photoperiod. The materials were immediately frozen in liquid nitrogen and either immediately extracted or stored at - 80 ~ C.

Extraction. Frozen plant material was ground to a fine powder under liquid nitrogen. The powder was divided into aliquots corresponding to maximally 10 g fresh weight plant tissue. Each aliquot was extracted by sonication (Laloue and Pethe 1982) in ice-cold methanol:chloroform:formic acid:water 60:15:5:20 (by vol.) (Bieleski 1964). The solvent volume was approximately six times that of the plant material. As an internal standard, 1 pmol [3H]ipA (670 M B q - retool-1) was added. I After standing on ice for 20 min, insoluble material was removed by centrifugation, and was extracted once more. The two supernatants were pooled. To remove chloroform, water (25 % of supernatant volume) was added, and the sample briefly centrifuged to separate the chloroform phase. The upper phase was evaporated under vacuum (Evapotec; HaakeBfichler Instruments) to remove remaining chloroform and the methanol. The remainder, formic acid in water, was diluted with water to the original volume of extraction solution used, giving approx. 1 M formic acid, passed through columns of insoluble polyvinylpolypyrrolidone (Sigma) at 4 ~ to reduce contents of phenols and quinones (Nieman and Clark 1984), and lyophilized.

Treatment with 5'-phosphatase. High-performance liquid chromatography fractions that yielded UV peaks were collected, evaporated to dryness, and dissolved in 100 m M Tris-HC1 (pH 8.5). Samples were incubated with 0.01 U of 5'-phosphatase in 150 l-tl 100 mM Tris-HC1 (pH 8.5) for 60 min at 37 ~ C. Gas chromatography-mass spectrometry (GC-MS). The HPLC eluent containing peaks of interest was collected, evaporated to dryness and dissolved in methanol. Amounts of 250 pmol of [2Hs]zeatin, [2Hs]ZR, [2Hs]zeatin-9-glucoside, and [2H6]ipA were added to samples believed to contain the corresponding compounds. The samples were permethylated as described previously (Horgan and Kramers 1979; Horgan and Scott 1987). Gas chromatographymass spectrometry was performed using a Hewlett Packard 5890 system with a mass-selective detector (Hewlett Packard 5970) working in electron-impact-ionization mode at 70 eV, and a

314

B. Nicander et al.: Co-purification of cytokinins

HN/R R Table 1. Relations between structure and affinity to IAC gels containing anti-ZR and anti-ipA antibodies. An a m o u n t of 100 or 200 pmol of a compound was applied to an IAC column, and binding determined. All ligands ( + ) showed binding > 9 0 % at 100 or 200 pmol, all non-binding ( - ) < 0 . 1 % binding

I R

I R

Compound

Substituent at N 6

Adenine substituted at

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Peak No."

Isopentenyladenine Isopentenyladenosine (ipA) Isopentenyladenosine-5'-monophosphate Isopentenyladenine-9-glucoside 2-Methylthioisopentenyladenosine

~ ~

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16 17 8 14 21

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5 9 1 3

Zeatin Zeatin riboside (ZR) Zeatin riboside-5'-monophosphate Zeatin-9-glucoside Zeatin-7-glucoside 2-Methylthiozeatin riboside Zeatin-O-glucoside Zeatin-O-glucoside riboside Dihydrozeatin Dihydrozeatin riboside Dihydrozeatin riboside-5'-monophosphate Dihydrozeatin-9-glucoside

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18 19 12 15

cis-Zeatin cis-Zeatin riboside

~

Benzyladenine Benzyladenosine Benzyladenosine 5'-monophosphate Benzyladenine-9-glucoside Benzyladenine-7-glucoside Benzyladenine-3-glucoside

f ~

Kinetin Kinetin riboside Kinetin riboside 5"-monophosphate K inetin-9-glucoside

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20

13

-

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12 m x 0.2 mm x 0.33 ~tm HP-1 methyl silicon column, also from Hewlett Packard. Helium flow was 0.74 ml 9min 1. The temperature was 60 ~ for the first 2 min, then increased at 30 ~ per min until 280 ~ was reached, and kept constant for another 10 min. The retention time of zeatin was 9.52 min, Z R 13.19 rain, zeatin-9-glucoside 13.44 rain, ipA 11.82 rain, and isopentenyladenine-9-glucoside 12.7 rain. Zeatin was monitored at 230 and 235, Z R at 216, 221,390 and 395,zeatin-9-glucoside at 216, 221,434 and 439, and ipA at 217, 223, 391 and 397. For the putative 9-glucoside of isopentenyladenine, the mass range between 100 and 450 was recorded.

Results

Ligands of the IAC columns. I n p r e l i m i n a r y e x p e r i m e n t s , IAC columns with antibodies against ipA or ZR were u s e d in s e q u e n c e t o p u r i f y c y t o k i n i n s f r o m p l a n t e x t r a c t s . It was found that the two types of column were able to bind the same compounds, but with different capacities (data not shown). To find out what other cytokinins could be purified with the columns, we screened commercially available cytokinins. The two types of antibody used, affinity-purified rabbit anti-ipA and anti-ZR, were

B. Nicander et al.: Co-purification o f cytokinins

mixed to increase the range of binding. The capacity per ml of the mixed antibody-gel used throughout this paper to retain the two immunogens was 4 nmol for Z R and 20 nmol for ipA. A cytokinin standard of 100 or 200 pmol was applied to an IAC column, and the binding determined. Of 30 cytokinins tested, a total of 25 bound (Table 1). These included the natural cytokinin bases zeatin, dihydrozeatin, isopentenyladenine, and benzyladenine (6-benzylaminopurine), and the nucleoside, 5'-mononucleotide, and 9-glucoside derivatives of these. All the commonly assayed-for cytokinins are found in this group. Other ligands were the transfer-RNA cytokinins cis-zeatin, cis-zeatin riboside and the 2-methylthiolated derivatives of ipA and ZR, as well as the 3-glucoside of benzyladenine and the synthetic cytokinin kinetin and four of its metabolites. The isopentenyladenine derivative, 6-N-(3-methyl-3hydroxybutylamino)purine (not shown in Table 1), that could arise as an artefact during extraction, also showed high affinity. The structurally similar cyclohexyladenosine (and several related compounds, data not shown), used to classify types of adenosine receptors, also bound. Recoveries for all ligands were above 90% Cytokinins with no affinity (detection limit < 0.5 pmol) for the IAC gels included O-glucosides of zeatin and 7-glucosides. ATP and other common adenine derivatives did not bind. The non-purine cytokinin diphenylurea was not recovered, but a few of the 100 pmol of N-(2-chloro-4-pyridyl)-N'-phenylurea applied were (data not shown). Summing up, the gels were able to recognize modifications at positions 2, 3 and 9, but not at 7 or at the hydroxyl group of the N 6 sidechain.

Bindin9 capacity of the IAC columns. For IAC of cytokinins from plant extracts to be useful, the recoveries of the ligands must be high even when several cytokinins are present. To test the capacities of the columns for the binding of different cytokinins, and effects on those capacities from competition between cytokinins with different relative affinities, mixtures of cytokinins were applied to the IAC columns and recoveries determined. Figure 1 shows an example of an experiment in which increasing loads of equimolar amounts of 12 different cytokinins were applied. Six ligands, including the immunogens ZR and ipA, were retained with high recoveries over the concentration range tested. The other six showed concentration-dependent losses. The four dihydrozeatincontaining compounds of the mixture showed the largest losses, and thus the lowest relative affinity (Fig. 1b). The nucleotides of isopentenyladenine and zeatin started to be lost at higher loads, exhibiting the lowest affinity of the isopentenyladenine and zeatin derivatives (Figs. 1a, c). The dihydrozeatin nucleotide experienced a shortage of binding sites already at the 12 x 40 pmol load (Fig. lb). In similar experiments where fewer or more cytokinins were used, the points at which recoveries of ligand began to go below 80% were higher or lower, respectively (data not shown). However, the antibody/gel ratio was considered to have an abundant capacity for the use of co-purification of cytokinins, since the sensitivity of de-

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Fig. la-e. Affinity of IAC columns for individual cytokinins in mixtures of cytokinins. Increasing amounts of a mixture containing equal quantities of 12 cytokinins were applied to IAC columns, and recovery of each of the ligands determined. The amount of each cytokinin in the mixture is plotted against percentage recovery, a Zeatin compounds: 9 5'-monophosphate of ZR; s ZR; +, zeatin; D, zeatin-9-glucoside, b Dihydrozeatin compounds; o, 5'-monophosphate of dihydrozeatin riboside; • dihydrozeatin riboside; +, dihydrozeatin; D, dihydrozeatin-9-glucoside,e Isopentenyladenine compounds; o, 5'-monophosphate of ipA; A ipA; +, isopentenyladenine; cz, isopentenyladenine-9-glucoside

tection of cytokinins (as shown below) makes it possible to analyze plant materials that will give cytokinin loads well below 12 x 40 pmol. More extensive washing than in the procedure given in the Materials andmethods section, i.e. the use of higher volumes of PBS, washing with buffers of high salt concentration, or with water (MacDonald and Morris 1985) lowered recoveries of some cytokinins, especially of the nucleotides. These experiments indicate that the columns had different recoveries and maximum capacities for different

316

B. Nicander et al. : Co-purification of cytokinins

0.004 0.003 134

0.002

1,9

12

Fig 2, Separation by of HPLC cytokinins that bind to IAC columns. Column: 250 mm long, 4 mm i.d; Supersphere RP-select B. Flow rate: 1 m l - m i n -1. Eluent: 2% acetic acid in water, with a gradient from 1 to 40% acetonitrile. The injected mixture contained 20 pmol each of 21 cytokinins, and 100 pmol dideoxyadenosine (designated by *), which served as an internal standard. Peak numbers 1 to 21 are explained in Table 1

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Retention time (min)

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240 250 260 270 280 290 300 310 320 Wavelength (nm) Fig. 3. Comparison of UV spectra of 21 cytokinins. Each spectrum

is from a 20-pmol peak in a chromatogramlike the one in Fig. 2. Modifications of the adenine ring were at the followingpositions: atN6(~),atN6and9( . ),atN6and3( ...... .),atN 6, 9 and 2 (- . . . . ) cytokinins, depending on what other ligands were present in the sample. As a further possible complication, plant materials may contain substances that affect antibody-ligand interactions. To circumvent these problems, a routine of repurifying the flow-through from IAC purifications to check for additional binding material was adopted.

Analyses using H P L C and UV spectra. To be able to analyze an immunopurified fraction from a plant extract, an HPLC system to separate IAC-binding cytokinins was developed (Fig. 2). Since kinetin-containing compounds have never been isolated from nature, they were not included. Two kinetin metabolites co-eluted with other cytokinins (data not shown). The HPLC eluate was continually scanned between 240 (eluent cut-off limit) and 320 nm with an on-line UV-spectrum detector. The UV spectra of the 21 cytokinins collected from the chromatogram in Fig. 2 are plotted ing Fig. 3. The spectra fall into four groups, the compounds of each group all being modified at the same

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positions in the purine ring (Leonard et al. 1965). The cytokinin bases, adenines modified at position N 6, had very similar spectra, with km,x approx. 269 nm. There was little effect of the composition of the substituents, including the aromatic benzyl chain. A further modification at position 9 (cytokinin nucleosides, nucleotides, and 9-glucosides) gave spectra with maximal approx. 263 nm. Again, the sidechains contributed little to the spectra. The addition of a methylthiol group at position 2 on a n N 6 + 9 compound gave a maximum at 280 nm, and the single tested cytokinin modified at position 3, benzyladenine-3-glucoside, had a peak at 285 nm. The small differences seen within the groups in Fig. 3 were caused by structural differences and changes in solvent composition as the gradient progressed. The N 6 sidechain of dihydrozeatin compounds, which lack a double bond compared with the other cytokinins (see Table 1), had their spectra shifted 1-2 nm downwards relative to the other members of the groups. The feature was shared by 6-N-(3-methyl-3-hydroxybutyl-amino)purine, which also lacks the double bond (data not shown). Other cytokinins modified at N6+9 besides those used for Fig. 3 are known, and yet others may await isolation. If present in an extract, it is quite possible that such a compound will go undetected because of identical retention times and UV spectra. The smallest N 6 and N 6 + 9 peaks that gave useful spectra from one scan corresponded to approx. 30-40 pmol. By averaging many scans, the quality of spectra from smaller peaks was improved, pushing the limit for obtaining a good spectrum (+ 2 % of one scan of 200 pmol) down to 4-6 pmol (data not shown).

Plant extracts. The procedure was tested on plant extracts. Figure 4 shows the HPLC chromatogram of the IAC-purified fraction from an Arabidopsis extract. Three larger peaks eluted at the retention times of the 5'-monophosphate of ZR, zeatin-9-glucoside (9-[3-D-glucopyranosylzeatin), and the 5'-monophosphate of ipA (designated 1, 3 and 8, respectively in Fig. 4). The UV spectra of all three peaks are typical of N 6 q'- 9 modified adenines (Fig. 5). Substances causing the peaks 1 and 8 were collected and treated with 5'-phosphatase. A new HPLC analysis of each peak yielded a single UV-absorbing peak

B. Nicander et al.: Co-purification of cytokinins

317

0.004 A6 0.003 1

3

0.002

A4 A5

Fig. 4. Separation by HPLC of IACpurified extracts of Arabidopsis. An amount corresponding to 7 g fresh weight was injected. Numbers above the trace indicate peaks with retention times and UV spectra corresponding to peaks with the same number in Fig, 2 and Table 1 ; HPLC conditions as in Fig. 2

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Immunoaffinity co-purification of cytokinins and analysis by high-performance liquid chromatography with ultraviolet-spectrum detection.

A rapid methodology for the simultaneous analysis of a large number of cytokinins is presented. The cross-reactivity of a mixture of polyclonal antibo...
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