P l a n t a 9 Springer-Verlag 1983
Cytokinins in transfer RNA of normal and crown-gall tissue of Vinca rosea L.M.S. Palni* and R. Horgan Department of Botany and Microbiology, University College of Wales, Penglais, Aberystwyth SY23 3DA, Dyfed, U K
Abstract. cis-Zeatin riboside was identified in transfer-RNA hydrolysates from both normal and crown-gall tissue of Vinca rosea L. The transisomer was associated exclusively with the crowngall transfer-RNA. The importance of these observations is discussed in relation to biosynthesis of free cytokinins.
Key words: Crown gall - Cytokinin biosynthesis - Transfer R N A - Vinca.
Crown-gall tumour induction results in specific biochemical changes which include the production of elevated levels of phytohormones (auxins and cytokinins). Consequently axenic tumour cells, unlike their normal counterparts, grow on defined media without added auxin and cytokinin. Cytokinin metabolism has been studied extensively in Vinca rosea crown-gall tumour tissue (Horgan et al. 1981). A number of cytokinins have been identified unambiguously in this tissue, including t r a n s - z e a t i n riboside (ZR) which is the major endogenous free cytokinin (402 ng g- 1 fresh weight). In contrast, the normal stem callus tissue of V. rosea contains very low levels of t r a n s - Z R (2.5 ng g-1 fresh weight). Furthermore, we have demonstrated incorporation of [14C]adenine (most probably as Y-AMP) into a number of cytokinins endogenous to V. rosea crown-gall tissue (Stuchbury * Present address and address f o r correspondence: Research
School of Biological Sciences, Australian National University, P.O. Box 475, Canberra City, ACT 2601, Australia Abbreviations: GC-MS = gas chromatography-mass spectrome-
try; HPLC = high-performance liquid chromatography; TLC = thin-layer chromatography; TMSi=trimethylsilyl; t R N A = transfer R N A ; ZR = zeatin riboside
et al. 1979; Palni et al. 1983). There has been considerable controversy regarding the relative importance of a direct biosynthetic route to free cytokinins, and an indirect route via transfer-RNA (tRNA) (for discussion see review by Letham and Palni 1983). Although some evidence exists in support of the direct route, the possibility that free cytokinins are tRNA derived cannot be discounted. Plant tRNAs contain predominantly cisZR as their cytokinin-active riboside, while the considerably more active t r a n s - i s o m e r and its derivatives occur as the dominant free cytokinins in higher plants. This paper reports the identification and measurement of ZR in tRNA hydrolysates from normal and crown-gall tumour tissue of V. rosea and its implications for the biogenesis of free cytokinins. Material and methods All chromatographic solvents used were of reagent grade and were distilled in an all-glass apparatus. Triethylammonium bicarbonate was prepared by saturating a 2.5 M solution of triethylamine (Sigma Chemical Co., St. Louis, Mo., USA) with carbon dioxide. Vinca rosea L. crown-gall tissue was cultured on the medium of Miller (1974) as described by Stuchbury et al. (1979). Normal stem callus tissue of V. rosea L. was maintained under identical conditions on the medium of Wood and Braun (1967) supplemented with 1 mg 1-1 2,4-dichlorophenoxy acetic acid and 2 mg 1-1 N6_benzylaminopurine. Tissue which was six to eight weeks old was harvested, frozen immediately in liquid nitrogen and stored at - 20 ~ C before use. Extraction of t R N A from both normal (1.06 kg) and crown-gall (4.7 kg) tissue, was done essentially as described by Burrows et al. (1971) with some modifications (Vreman et al. 1972). The t R N A samples from both normal (50.2 mg) and crown-gall (35.9 rag) tissue were subjected to polyacrylamide-gel electrophoresis, which indicated the presence of a small amount of DNA. Both t R N A preparations were subjected to enzymic hydrolysis using an established procedure (Hall 1964). The hydrolysates were extracted at pH 8.2 with ethyl acetate (8 x equal volume). The combined ethyl-acetate fractions from each t R N A hydrolysate were reduced to dryness in vacuo
L.M.S. Palni and R. Horgan: Transfer-RNA cytokinins at 30 ~ C, and fractionated on a column of Sephadex LH-20 (80 cm long, 2.5 cm diameter; bead size 25-100 gm, Pharmacia Fine Chemicals, Uppsala, Sweden) eluted with 35% aqueous ethanol (Stuchbury et al. 1979). Eighty fractions of 30 ml were collected at the flow rate of 30 ml h - 1. Suitable aliquots from each fraction were withdrawn for determination of cytokinin activity using both tobacco callus (Murashige and Skoog 1962) and Amaranthus betacyanin (Biddington and Thomas 1973) bioassays. A major peak of cytokinin activity was detected in fractions corresponding to the elution volume of ZR in both samples. These fractions in each sample were combined and one fifth of each was subjected to thin-layer chromatography (TLC) as detailed below, and the remaining sample in both cases was analysed using high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GCMS). The HPLC was performed using equipment and conditions as described by Horgan and Kramers (1979) on a column of Hypersil ODS (150 cm long, 4.5 mm inner diameter; Shandon Southern, Runcorn, UK) eluted isocratically with 12% acetonitrile in water (pH adjusted to 7 with triethylammonium bicarbonate) at 2 ml min - 1. One twentieth of each HPLC fraction was taken for tobacco callus bioassay (Fig. 1). Gas-liquid chromatography of trimethylsilyl (TMSi) derivatives of putative cis- and trans-ZR thus isolated was performed on a Pye 104 gas chromatograph (Pye Unicam, Cambridge, UK) which contained silanised glass column (1.65 m long, 4 mm inner diameter) packed with Gas Chrom Q (100-120 mesh; Applied Science Laboratories Inc., State College, USA) coated with 2% OV-I, held at 285~ with a He flow rate of 30ml rain -~. The gas chromatograph was interfaced through a single-stage silicon-rubber membrane separator to a Kratos MS30 mass spectrometer (Kratos Instruments, Manchester, UK) linked to a DS 50 Data System (Data General Corp., Westboro, Mass., USA). Low-resolution mass spectra were recorded repetitively at 3 s mass decade-1 at 24 eV over all GC peaks. The TMSi derivatives were prepared by heating samples with a 1 : 1 mixture (20 pl) of pyridine and N,O-bis-trimethylsilylacetamide at 90 ~ C for 2 h. In both samples the ZR fractions following Sephadex LH20 chromatography were also subjected to TLC on silica-gel plates (5 x 20 cm; PF2s4; E. Merck, Darmstadt, F R G ) developed three times in chloroform: acetic acid (4:1, v/v). The cisand trans-ZR were resolved completely in this system. Twelve zones were removed from each plate, eluted with 60% ethanol (4 x 1 cm 3) and bioassayed.
Results and discussion
The results of the tobacco callus bioassay following HPLC analyses of ZR fractions obtained after Sephadex LH-20 column chromatography of the normal and crown-gall tRNA hydrolysates, are shown in Fig. 1. The major zone of biological activity in the crown-gall sample (Fig. 1 B) was coincident with a UV-absorbing peak with the same elution volume as trans-ZR. Minor peaks of biological activity co-chromatographed with cis-ZR and transZ. Bioassay, following HPLC analysis of the normal tissue tRNA sample, (Fig. 1 A) showed weak biological activity coincident with a UV-absorbing peak which had the retention time of cis-ZR. No UV absorbance or biological activity was observed at the elution volume of trans-ZR in this sample.
The putative cis- and trans-ZR peaks from the HPLC of the crown-gall tRNA hydrolysate, and the putative cis-ZR peak from the HPLC of the tRNA hydrolysate of normal stem callus were further examined by electron-impact GC-MS as TMSi derivatives. The derivatisation procedure produced a mixture of tetra- and penta-TMSi compounds. Although insufficient material was available to obtain full mass spectra, the major ions for the (TMSi)4- and (TMSi)5-zeatin ribosides were observed at the correct gas-liquid chromatography retention times for cis- or trans-isomers in the three samples. Trimethylsilyl derivatives of cis-ZR elute earlier than those of trans-ZR on GC under the conditions used. The ions characteristic for (TMSi)4-ZR seen in the mass spectra were: 624, 550, 549, 536, 348,320,201, 188, 75, 73. Although molecular ions (639 for tetra- or, 711 for penta-TMSi-ZR) were not observed, an important feature of the mass spectra was the absence of any ions indicating impurities. The bioassay results following TLC also supported our identifications of cis- and-or trans-ZR in the two samples. The UV peaks corresponding to the elution volumes of cis- and-or trans-ZR from the two samples (Fig. 1 A, B) were found to be homogeneous by HPLC in diverse systems (results not shown). By HPLC analyses under identical conditions, after calibration of the instrument with a series of known amounts of trans-ZR standards, it was possible to estimate that crown-gall tRNA contained 42 ng mg-1 cis-ZR and 18 ng mg-1 trans-ZR whilst normal tissue tRNA contained 28 ng mg-1 cis-ZR and no detectable level of trans-ZR. The values quoted here are underestimates caused by extraction losses etc. However, because of the close chemical and physical similarity of cis- and trans-ZR, the relative amount of these two compounds is probably a true reflection of their ratios in the tissue tRNA. The biosynthetic origin of the greatly elevated cytokinin levels in crown-gall tissues is unknown. In view of known low rates of t R N A turnover in plants, the efficiency of [14C]adenine incorporation into trans-ZR and related free cytokinins in V. rosea crown-gall tissue would indicate that modification and turnover of tRNA were not obligatory in the process (Stuchbury et al. 1979; Palni et al. 1983). However, the possibility that the metabolism of tRNA in crown-gall tissues is abnormal cannot be ignored. The levels of cis-ZRpresent in the tRNA of V. rosea crown-gall tissue are slightly higher (1.5 times) than those in the tRNA of normal tissue. However, trans-ZR is associated
L.M.S. Palafi and R. Horgan: Transfer-RNA cytokinins
3 4 "l-~l 1~
4 0 Time
Fig. 1 A, B. Reversed-phase HPLC and tobacco callus bioassay of normal (A) and crown-gall (B) tRNA hydrotysates after initial fracfionation on a column of Sephadex LH-20. The bars indicate elution volumes of cytokinin standards as follows: 1, cis-zeatin riboside; 2, trans-zeatin riboside; 3, eis-zeatin; 4, trans-zeatin. Arrows indicate change in sensitivity setting from 0.1 to 0.05. Dotted line: callus yield in control flasks
exclusively with the crown-gall tRNA. The cis-/ trans ratio (2.3) in the tRNA of this tissue is very low compared with ratios of 50 in Spinacea oleracea (Vreman et al. 1978) and 40 in Pisum sativum tRNAs (Vreman et al. 1974), the only other cases where plant tRNA is known to contain trans-ZR. It is most unlikely that the high levels of trans-ZR (1 mol/800 mol tRNA) in the tRNA of V. rosea crown-gall tissue have resulted from random incorporation of free trans-ZR. This is the major endogenous cytokinin in the tissue and is also released into the culture medium in substantial amounts (Palni and Horgan 1982). In a critical study by Walker et al. (1974) using cytokinin dependent tobacco tissue supplied with an optimal concentration of cytokinin, the maximum incorporation of 1 tool ofcytokinin per 10,000 tRNA molecules was reported. Increasing the cytokinin concentration in the medium tenfold did not significantly alter the level of cytokinin incorporation in tRNA. This is the first report of the isolation ofcytokinins from crown-gall tRNA, and the observed high levels of trans-ZR in V. rosea crown-gall tRNA may be of functional importance. The present study raises the possibility that biogenesis of free trans-ZR in this tissue could result from an extremely rapid turnover of specific tRNA species containing trans-ZR. Abnormally high turnover rates of a subpopulation of tRNA, rich in modified nucleosides, have been reported for animal tumours (Borek et al. 1977). However, an unequivocal answer to the nature and extent of the role
played by tRNA in the biogenesis of free cytokinins will be obtained only by direct measurement of the rate of turnover of trans-ZR in specific tRNA species under conditions where the rates of production of free trans-ZR can be measured. We would like to thank Dr. I. Szir~ki for discussions and Dr. D.S. Letham for reading the manuscript.
References Biddington, N.L., Thomas, T.H. (1973) A modified Amaranthus betacyanin bioassay for the rapid determination of cytokinins in plant extracts. Planta 1il, I83-186 Borek, E., Baliga, B.S., Gehrke, C.W., Kuo, C.W., Belman, S., Troll, W., Waalkes, T.P. (1977) High turnover rate of transfer RNA ir~ tumor tissue. Cancer Res. 37, 3362-3366 Burrows, W.J., Skoog, F., Leonard, N.J. (1971) Isolation and identification of cytokinins located in the transfer ribonucleic acid of tobacco callus grown in the presence of 6-benzylaminopurine. Biochemistry 10, 2J8%2194 Hall, R.H. (1964) Isolation of N6-(Aminoacyl)adenosine from yeast ribonucleic acid. Biochemistry 3, 769-773 Horgan, R., Paini, L.M.S., Scott, I., McGaw, B. (1981) Cytokinin biosynthesis and metabolism in Vinca rosea crown gall tissue. In: Metabolism and molecular activities of cytokinins~ pp. 56-65, Guern, J., P~aud-Leno~l, C., eds. Springer, Berlin Heidelberg New York Horgan, R., Kramers, M.R. (1979) High-performance liquid chromatography of cytokinins. J. Chromatogr. 173, 263-270 Letham, D.S., Palni, L.M.S. (1983) The biosynthesis and metabolism of cytokinins. Annu. Rev. Plant Physiol. 34, 163-197 Miller, C.O. (1974) Ribosyl-trans-zeatin, a major cytokinin produced by crown gall tumour tissue. Proc. Natl. Acad. Sci. USA 71,334--338
L.M.S. Palni and R. Horgan: Transfer-RNA cytokinins Murashige, T., Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497 Palni, L.M.S., Horgan, R. (1982) Cytokinins from the culture medium of Vinca rosea crown gall tumour tissue. Plant Sci. Lett. 24, 327-334 Palni, L.M.S., Horgan, R., Darrall, N.M., Stuchbury, T., Wareing, P.F. (1983) Cytokinin biosynthesis in crown-gall tissue of Vinea rosea. The significance of nucleotides. Planta 159, 50-59 Stuchbury, T., Palni, L.M., Horgan, R., Wareing, P.F. (1979) The biosynthesis of cytokinins in crown-gall tissue of Vinca rosea. Planta 147, 97-102 Vreman, H.J., Schmitz, R.Y., Skoog, F., Playtis, A.J., Frihart, C.R., Leonard, N.J. (1974) Synthesis and biological activity of 2-methylthio-cis- and trans-zeatin and their ribosyl derivatives. Isolation of the cis- and trans-ribonucleosides from Pisum tRNA. Phytochemistry 13, 31-37
181 Vreman, H.J., Skoog, F., Frihart, C.R., Leonard, N.J. (1972) Cytokinins in Pisum transfer ribonucleic acid. Plant Physiol. 49, 848-851 Vreman, H.J., Thomas, R., Corse, J., Swaminathan, S., Murai, N. (1978) Cytokinins in tRNA obtained from Spinacia oleracea L. leaves and isolated chloroplasts. Plant Physiol. 61, 29(~306 Walker, G.C., Leonard, N.J., Armstrong, D.J., Murai, N., Skoog, F. (1974) The mode of incorporation of 6-benzylaminopurine into tobacco callus transfer ribonucleic acid. A double labelling determination. Plant Physiol. 54, 737-743 Wood, H.N., Braun, A.C. (1967) The role of kinetin (6-furfurylaminopurine) in promoting division in cells of Vinca rosea L. Ann. N.Y. Acad. Sci. 144, 244-250 Received 29 March; accepted 21 April 1983