Zeatin-Like Cytokinins in Yeast: Detection by Immunological Methods

J.PlantPhysiol. Vol. 135.pp.385-390(1989)

Zeatin-Like Cytokinins in Yeast: Detection by Immunological Methods PAULA

E. JAMESON 1 and Roy O.

MORRIS

Department of Biochemistry, University of Missouri-Columbia, Columbia, MO 65211 USA 1 Permanent address: Department of Botany, University of Otago, Dunedin, New Zealand Received April 17, 1989 . Accepted July 10, 1989

Summary Immunoaffinity chromatography, HPLC, ELISA and RIA have been employed to detect cytokinins in autoclaved yeast extract and yeast tRNA. Yeast extract contains, in addition to isopentenyladenine and isopentenyladenosine, small but significant amounts of trans-zeatin and trans-zeatin riboside. Purified yeast tRNA contains predominantly isopentenyladenosine but also contains trans-zeatin riboside and other cytokinin-like compounds. Because of the presence of cytokinins in yeast extract, this material should be used with caution in studies of cytokinin production by phytopathogenic bacteria.

Key words: Saccharomyces cerevisiae, tRNA, zeatin, zeatin riboside, isopentenyladenine, isopentenyladenosine, immunoa/finity chromatography, RIA, ELISA. Abbreviations: cZ = cis-zeatin; cZR = cis-zeatin riboside; DZ = dihydrozeatin; DZR = dihydrozeatin riboside; ELISA = enzyme-linked immunosorbent assay; iP = isopentenyladenine; iPA = isopentenyladenosine; PBS = phosphate-buffered saline; RIA = radioimmunoassay; tZ or Z = trans-zeatin; tZR or ZR = trans-zeatin riboside.

Introduction For over 30 years it has been known that yeast is a rich source of factors which promote plant cell division in culture (Miller et aI. 1955). That this activity may be due to cytokinins was indicated by van Staden (1974) who showed that yeast extract (Difco) contained two compounds which were active in the soybean callus bioassay, one of which exhibited chromatographic properties on Sephadex LH-20 similar to trans-zeatin riboside (tZR) while the other was considerably more polar in nature. Riedel et al. (1977) used van Staden's (1974) procedures to confirm the presence of tZR. However, in addition to a polar compound, they detected cytokininactive compounds in yeast extract having chromatographic mobilities on Sephadex LH-20 similar to trans-zeatin (tZ), and on paper similar to isopentenyladenine (iP) and isopentenyladenosine (iP A). It is now well known that certain gall-forming plant pathogenic bacteria (e.g. Agrobacterium tume/aciens and Pseudomonas savastanoi) carry genes which encode cytokininbiosynthetic prenyl transferases. As a result of expression of © 1989 by Gustav Fischer Verlag, Stuttgart

these genes, the bacteria secrete tZ, iP, and other cytokinins (Kaiss-Chapman and Morris 1977, Regier and Morris 1982, MacDonald et al. 1986, Heinemeyer et al. 1987, Powell et al. 1988). Since some of these studies were conducted using media containing yeast extract, we were concerned about the contribution that this component might have made to the total cytokinin activity detected. We now report that yeast extract contains extremely low levels of tZ and tZR but significant levels of iP and iPA. Examination of yeast tRNA showed that it contained, in addition to the expected iPA (Hall et al. 1966, Hecht et aI. 1969), small but measurable amounts of tZR, and three other immunologically active compounds.

Materials and Methods Isolation of cytokinins from 523 medium and yeast extract

Kado and Heskett's (1970) 523 medium was prepared and autoclaved, prior to the removal of 50 ml replicate samples for analysis. An equivalent amount of yeast extract (Difco) was dissolved in

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E. JAMESON and Roy O. MORRIS

water (i.e. 0.2 g/50 ml) and autoclaved prior to analysis. Cytokinins were identified in these samples as outlined by MacDonald and Morris (1985). Briefly, the pH of each sample was adjusted to between 6.5 and 7.0, internal standards ([3H]-tZR and [3H]-iPA trialcohol; 25,000 cpm each) were added, and the extract applied to a DEAE-cellulose column (DE-52, Whatman; 20 ml) which was linked to an octadecyl silica column (Sepralyte, Analytichem International; 2.5 ml). The DEAE-cellulose had been preconditioned according to the manufacturer's instructions, stirred for 3 h with ammonium acetate buffer (2 M, pH 6.5), with periodic adjustments of the pH as necessary, and finally washed with ammonium acetate (40 mM, pH 6.5, Buffer A) and stored in this buffer at 4°C with the addition of 0.1 % sodium azide. Following packing of the DE-52 columns, each was washed with 10 volumes of Buffer A prior to sample application. Octadecyl silica columns were prepared for use by washing with 20 volumes methanol followed by 20 volumes of 40 mM acetic acid adjusted to pH 3.38 with triethylamine and finally with 10 volumes of Buffer A. Following application of the sample, the linked columns were washed with 2 volumes (40 ml) of Buffer A, the DE52 column was removed and discarded, and the octadecyl silica column washed with a further 10 volumes of Buffer A. The octadecyl silica column was purged with nitrogen and the cytokinins subsequently eluted with 2.5 volumes of methanol. The eluate was reduced almost to dryness in vacuo (Savant, Speed-vac), the sample diluted to 1.4 ml in Buffer A, and applied to an immunoaffinity column.

Isolation of cytokinins from yeast tRNA

Baker's yeast tRNA (Sigma, Type X-S, 10,000 A 260 units) was dissolved in water (20 ml). Potassium hydroxide solution (10 M) was added to a final concentration of 0.3 M and the mixture was incubated under nitrogen in a sealed polypropylene container in the dark at 37°C for 24 h. Tris chloride (1 M, pH 7.0, 0.1 vol.) was added and the pH was adjusted to 9.5 by addition of HCI. The resulting nucleotides were dephosphorylated by addition of bacterial alkaline phosphatase (Sigma, Type III, 20 units, centrifuged prior to use to remove ammonium sulfate and resuspended in Tris chloride O.lM, pH 8.5) and MgCh (1M, 200JLI). The mixture was incubated for 6 h, further MgCh (800 JLI) and enzyme (20 units) were added, and the incubation continued for an additional 16h. The precipitated magnesium phosphate was removed by centrifugation (10,000 rpm/l0 min), the supernatant was adjusted to pH 7.0 with HCI (1 M), and the cytokinins were adsorbed onto octadecyl silica essentially as describe above. Cytokinins were eluted with methanol (4 ml), the eluates were dried in vacuo and fractionated by HPLC. HPLC fractions (0.5 ml) were collected and aliquots were assayed by ELISA or RIA as described below.

Immunoaffinity purification of cytokinins

Monoclonal antibodies raised against cytokinins were linked to cyanogen bromide-activated cellulose (MacDonald and Morris 1985) using a modification of Kohn and Wilchek (1982) to activate the cellulose. Two antibody clones were used: clone 16 possessed good affinity for hydroxylated cytokinins such as tZ and tZR. Clone 12 showed less discrimination and was capable of binding a wide range of cytokinins including iP, iPA, tZ, and tZR. Small plastic columns were packed with 3 ml of antibody-cellulose (2 ml immobilized clone 12, layered over 1 ml immobilized clone 16, total capacity 2.5 JLg) and washed with 10 volumes Buffer A prior to sample application. The columns were washed sequentially with 5 volumes Buffer A, 3 volumes Buffer A + 2 % dimethylsulphoxide + 0.5 M NaCI, 10 volumes Buffer A + 0.5 M NaCl, and 5 volumes Buffer A.

The columns were then purged with nitrogen and the cytokinins were eluted with 2.5 volumes of methanol. High performance liquid chromatography

Samples were subjected to HPLC in a triethylamine buffer (40 mM acetic acid, pH 3.38 with triethylamine)/acetonitrile gradient on an octadecyl silica column (Altex 5 JLm, 250 x 4.6 mm). Fractions (0.5 ml) were collected, aliquots were dried in vacuo and assayed. The region of the gradient within which zeatin-like cytokin ins normally elute (8 to 31 min) was assayed by ELISA. The region where less polar cytokinins such as iP and iPA elute (31.5 to 40 min) was assayed by RIA. Enzyme linked immunosorbent assay

The procedure of Maldiney et al. (1986) was followed with modifications. Ninety-six well plates (Nunc # 269620) were coated with tZR-ovalbumin conjugate (7.5 ng/well). Samples and standards (150JLI) were dissolved in PBS-Tween (sodium chloride, O.14M; phosphate buffer, 50 mM, pH 7.0; Tween-20, 0.05 %), added to appropriate wells, followed by 50 JLI of anti-tZR monoclonal antibody (clone 16, ascites fluid, diluted approx. 1: 20,000). The plates were shaken and incubated for 25 min at 37°C. The wells were then washed five times with PBS-Tween and aspirated prior to the addition of 200 JLI of rabbit anti-mouse antibody/alkaline phosphatase conjugate (Sigma) diluted 1: 1,000 in PBS-Tween. The plates were again shaken and incubated for 25 min at 37°C, washed five times with Tris-saline (sodium chloride, 0.14M; Tris chloride, 0.1M, pH 8,0; Tween-20, 0.05 %), the wash liquid was completely removed by aspiration, and the enzyme activity was estimated by addition of 200JLl4-nitrophenyl phosphate solution (1 mg/ml in 10% diethanoamine buffer, pH 9.8) containing MgCh (0.5 mM). The enzyme assay was allowed to proceed at 37°C for 1 h. Absorbance at 405 nm was measured on an automatic ELISA plate reader (Molecular Devices). A response in the ELISA greater than that obtained with 250fmol (88 pg) tZR was considered significantly different from zero. However, standard curves were routinely performed on each plate. Because of the extreme sensitivity of the ELISA it was important to ascertain that the 25,000 cpm of [3H]-ZR added as an internal standard did not cause a response in the ELISA. A control containing 25,000 cpm of [3H]-ZR was run through the entire purification, HPLC and ELISA procedure. There was no response in the ELISA to the CH]-ZR. Radioimmunoassay

The RIA was conducted essentially as described in MacDonald and Morris (1985) but using monoclonal antibody clone 12. The dried samples in 1.5 ml Eppendorf centrifuge tubes were dissolved in 350 JLl RIA buffer [50 mM sodium phosphate buffer with 0.14 M NaCl, pH 7.2, 0.1 % w/v gelatin (Difco), 0.01 % ovalbumin (Sigma grade V)). Addition of 50 JLl of [3H]-iP A trialcohol to the dissolved samples provided between 5,000 to 6,000 cpm per vial. Antibody (diluted in RIA buffer so that the 50 JLl added bound 50 % of PH]iP A trialcohol) was added and the solutions mixed and incubated at room temperature for 20 min. Ammonium sulphate (90 % saturated, pH 6.5 to 7.5 with ammonium hydroxide, 600 JLl) was added, the solutions briefly mixed and allowed to stand at room temperature for 15 min. The tubes were then centrifuged (14,000 rpm, 1 min), the supernatant aspirated and 50 JLl methanol was added to the pellet. The tubes were left to stand for 10 min, mixed briefly, 1 ml scintillation cocktail was added, mixed thoroughly twice, and the level of radioactivity was determined by scintillation counting. The minimum detection level was approximately 0.3 pmol (100 pg) iP A. A standard curve was run with each assay.

Cytokinins in Yeast

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Results and Discussion

Cytokinins in yeast extract J1~i"ing preliminary experiments, we observed that unfractionated 523 medium caused a positive reaction in both the ELISA and the RIA. Subsequently, we fractionated and analyzed both the medium and its individual components for compounds which were retained on anti-cytokinin immunoaffinity chromatography columns and which cross-reacted in either the ELISA (which was specific for hydroxylated cytokinins) or RIA (which was specific for non-hydroxylated cytokinins). The results of such an analysis are illustrated in Fig. 1. Yeast extract contained five compounds which crossreacted with anti-cytokinin antibodies. Two of the compounds were active in the ELISA. They had HPLC retention times coincident with tZ (14.0 min) and with tZR (19.9 min). Two other compounds were detected by RIA. They had retention times coincident with iPA (33.7 min) and iP (35.3 min). The fifth compound, of high polarity, was crossreactive in the ELISA and had a retention time of approximately 8 min. The five compounds were also detected in complete 523 medium (data not shown). Riedel et al. (1977) reported that there were four major cytokinin-active species in yeast extract. In addition there is evidence in his data for the presence of a very polar compound. In contrast, van Staden (1974) reported the presence of a single tZR-like compound and a very polar substance. The methods available to van Staden (1974) and Riedel et al. (1977) did not resolve cis- and trans-zeatin and dihydrozeatin

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or cis- and trans-zeatin riboside and dihydrozeatin riboside. On the basis of the retention of the major species on anticytokinin immunoaffinity columns, of their detection by specific ELISA (hydroxylated cytokinins) or RIA (iP and iPA) and of their HPLC retention times shown in Fig. 1, we conclude that, in fact, tZ, tZR, iP A and iP are present in yeast extract. The nature of the highly polar compound at a retention time of approximately 8 min remains undetermined. Although rigorous quantitation of the cytokinins by gas chromatography-mass spectrometry was not attempted, some quantitative information was provided by the immunological assays (which were performed in conjunction with recoveries of internal radiolabelled standards: ca. 65 % recovery for [3H]-ZR and ca. 35 % recovery for [3H]-iPATA). On this basis, we compared the levels of cytokinins present in 523 medium and yeast extract. The results are shown in Table 1. The levels of tZ and iP are expressed as their tZR Table 1: Cytokinins in 523 medium, yeast extract, and yeast tRNA*. Sample

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Morris 1977) or Pseudomonas savastanoi strain 1006 (MacDonald et al. 1986), which excrete high levels (greater than 20 pmoll ml) of tZ or tZR, they may be a problem for low producers such as Agrobacterium tumefaciens strain NT1 (Regier and Morris 1982) or Corynebacterium fascians (unpublished data). In the case of iP and iPA, the problem of interference is more severe. The amounts of iP and iPA present in 523 are sufficient to interfere significantly in estimates of cytokinin production by pathogenic bacteria. Experiments should be designed to avoid this medium and others which contain yeast extract, especially until it has been determined whether a particular micro-organism can convert the nonhydroxylated cytokinins into hydroxylated forms. To this effect Laloue and Morris (unpublished data) have shown that E. coli cannot achieve this conversion.

and iPA equivalents respectively and are therefore underestimated because of their lesser cross-reactivities in the ELISA (Z approximately SX less than ZR) and RIA (iP approximately 3X less than iPA). Nevertheless, we conclude that tZ and tZR are present, but at extremely low levels (combined approx 35 pmol/g yeast extract). In contrast, iP and iP A are present at substantially greater levels (combined approx 14nmol/g yeast extract). The amounts of tZ, tZR, iP A and iP in 523 medium could be almost totally accounted for by those detected in the yeast extract. From a practical point of view, it should be noted that while the levels of tZ and tZR in 523 are not sufficiently high to cause problems when assaying bacterial strains such as Ag· robacterium tumefaciens strain C58 (Kaiss-Chapman and

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Cytokinins in Yeast

Cytokinins in yeast tRNA An obvious source for the cytokinins present in the yeast extract is yeast tRNA itself. Nuclease action and dephosphorylation could readily release cytokinins from the tRNA during autolysis. However, iPA is the only cytokinin which has been unequivocally identified in yeast tRNA (Hall et al. 1966, Hecht et al. 1969). The origin of the free tZ and tZR in yeast extract is therefore unknown. Because the original identification of iPA in tRNA was done at a time when sensitive techniques for cytokinin detection had not been developed, it seemed reasonable to reinvestigate the nature of the cytokinins present in yeast tRNA. Accordingly, 200 mg purified yeast tRNA was subjected to mild base hydrolysis and enzymatic dephosphorylation. The cytokinins were concentrated on octadecyl silica and fractionated by HPLC. Individual HPLC fractions then were assayed either by ELISA or by RIA. The results are illustrated in Fig. 2. There was a substantial peak of UV-absorbing material at the retention time for iPA (33.7 min, Fig. 2 A). RIA of this fraction indicated that iPA was present (Fig. 2 B). Integration of the peak area and quantitation based on RIA standards indicated that the tRNA contained 97.5 pm 011A 260 unit (based on UV absorbance) and 84.2 pmoll A 260 unit (based on RIA) respectively. The agreement is excellent and indicates that the peak is composed principally of iP A. Two other peaks of immunologically active material were also detected by RIA; one at a retention time of 37.8 min, and a second at 31.9 min. Both compounds cross-reacted with the clone 12 antibody in the RIA to almost the same extent as did iPA. It is possible, therefore, that both possess a non-hydroxylated isoprenoid side chain and that they differ from iP A in modifications on the purine ring. A potential candidate for the compound at a retention time of 37.5 min is 2-methylthio-iPA which is a major component of E. coli tRNA (Burrows et al. 1968). Further, Manachini et al. (1982) reported the detection of a compound by bioassay following hydrolysis of S. cerevisiae tRNA which they ascribed to 2methylthio-iP. The identity of the compound at retention time 31.9 min is not as well established. It is more polar than iPA and may be 2-hydroxy-iP A. The most significant finding was the detection of tZR. Assay of the fractions comprising the earlier portion of the gradient by ELISA showed two zones of activity. The zone at a retention time of 20.8 min corresponded exactly in mobility to authentic tZR. On the basis of its adsorption by an anti-cytokinin immunoaffinity column, its retention time on HPLC, and its cross-reaction in the ELISA with clone 16, a monoclonal antibody raised against tZR, we conclude that this compound is, in fact, tZR. What is surprising is the fact it is the trans-isomer. The predominant cytokinin present in plant tRNA (and some bacterial tRNA) is cZR or its 2methylthio-derivative (Cherayil and Lipsett 1977, Edwards et al. 1981). It is possible that the cis-isomer may be present in yeast tRNA but it has, unfortunately, low cross-reactivity to clone 16 in the ELISA, although the absorbance trace in Fig. 2 A gives little evidence for its presence. The other compound which was detected by ELISA was highly polar. It had a retention time (8 min) similar to that of the polar material detected in yeast extract but its identity at

389

this time is unknown. There have been no reports of cytokinin glucosides in tRNA although polar immunologically reactive compounds have been detected in crown gall tissues (MacDonald et al. 1981). Quantitatively, iPA is the predominant cytokinin in yeast tRNA. There were approximately 84 pmol iP AIA 260 unit tRNA (Tablet). The presence of iPA in yeast tRNA has been reported several times (e.g. Hall et al. 1966). Quantitatively the level reported here is in good agreement with those observed by Laten and Zahareas-Doktor (1985) (78 pmoll A 260 tRNA) and Horvath and Denes (1977) (114pmoll A 260 tRNA). The level of tZR was very much lower (6.5 fmol tZRIA 260 RNA). It would be of interest to determine whether it is present in a unique isoaccepting species or if it is present in the yeast mitochondrial tRNA population. The ratios of hydroxylated to non-hydroxylated cytokinins in yeast extract versus yeast tRNA are clearly different. The basis for this difference is unlikely to be determined easily because of the difficulties in accurate measurement of turnover rates of total tRNA or of individual isoaccepting species. An additional complicating factor is the possible presence of a non-tRNA mediated cytokinin biosynthetic pathway as suggested by Laten and Zahareas-Doktor (1985). Acknowledgements We thank Mr. J. W. Morris for valuable assistance with the HPLC analyses. The work was supported by the Competitive Research Grants Office, Science and Education Administration, U.S. Department of Agriculture (Grant 85-FSTY-9-0146) to R.O.M. and was carried out while P.E.]. was on sabbatical leave from the University of Otago, New Zealand.

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