16,17-dihydro 16,17-dihydroxy gibberellin A9: A metabolite of [3H]-gibberellin Ag in chloroplast sonicates from Pisum sativum var. «Alaska»

Bot an y Dep artment , Rhodes University, Grahamsto wn, South Afri ca

16,17-Dihydro 16,17-Dihydroxy Gibberellin A9 : A Metabolite of [3H]-Gibberellin Ag in Chloroplast Sonicates from Pi sum sativum var, «Alaska» I. D. RAILTON With 5 figures Received August 26, 1976 . Accepted September 7, 1976

Summary Tritium labell ed gibberellin A9 ([ 3H ]- GA 9) wa s metabolized by chloroplast sonicat es from shoots of «Alaska» pea seedlings into 16, 17 dihydro 16,17-dihydro xy GA 9 • Id ent ificat ion was made by combined gas chromatography - mass spectrometry (GC-M3). Key w ords: gibberelli n A 9 , 16,17-didydro 16,17-dihy d rox y GA 9 , chloropl ast, Pisum satiuum,

Introduction Recent studies ha ve shown that chloroplast preparations fro m sever al higher plants, including barley (REID, 1967; STODDART, 1968 ), kale (STODDART, 1968), potato (RAILTON, 1971 ; RAILTON and WAREING, 1973) pea (RAILTON and REID, 1974 a) and tom ato (RAILTON, unpublished), possess high levels of gibberellin-like (G A-like) activi ty. Circumstan tial evidence (STODDART, 1969) suggests th at such p repar at ions are capa ble of G A synthesis. Th e autonomy of th e chloroplast with regard to GA biosynthesis is unknown but recent evidence (RAILTON and REID, 1974 a, b) suggests that this or ganell e could exert a compartrnentation effect on GA met abol ism in the pea shoot to selectively locali ze a GA 2o-like GA wh ilst allo wing th e passage of an other pea GA , probably GA 29 , into th e cytopl asm. It is possible th erefore th at pea chloroplasts coul d serve to «package» parts of th e GA biosynthetic pathway and so exert some form of metabolic control over GA biosynthesis in pea leaves. Studies on the effects of da yl ength on GAs in potato shoots (RAILTON and WAREING, 1973 a, b, c) have shown th at levels of po tato chloroplast GA-like substances are under photoperiodic control and are th erefore regulated by photochrome. It is apparent that chloroplasts could serve as a useful tool for th e study of GA met abol ism and its environment al control in higher plants. It is necessary therefore to envestigate further th e ability of these or ganelles to biosynthesize and met abolize GAs . In a pre vious pap er (RAILTON and REID, 1974 b) Z. Pflanzenp hysiol. Bd . 81. S. 323-329.1977.

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it was shown that an in vitro system derived from chloroplasts of Pisum sativum could metabolize 17[3H]-GA o and 2,3[3H]-GA 20 into more polar, acidic products. I now report further on the characterization of one of the products of [3H]-GA g metabolism in this system. Materials and Methods Seeds of Pisum sativum L. (cv. Alaska) were soaked in running tap water for 12 hours and were planted in moist vermiculite and maintained under continuous illumination at 26°C for 8-10 days. Chloroplasts were isolated from pea shoots by grinding excised, pre-cooled shoot tissue in a chilled mortar with an ice-cold grinding medium (sucrose, 0.33 M; HEPES (N-2-hydroxyethyl-piperazine-N l-2-ethanesulphonic acid) 10 mM; dithiothreirol, 1.0 mM; MgCI 2 ' 6 HP, 15 mM; KCl, 100 mM; at pH 8.0). The slurry was filtered through 8 layers of cheese-cloth and centrifuged at 1,200 rev/min for 2 min. The supernatant was decanted and recentrifuged in an identical manner. After discarding the pellet, the supernatant was centrifuged at 3,200 rev/min for 4 min at 0-2 °C, the pellet resuspended in fresh buffer and recentrifuged under the same conditions. The final chloroplasts pellet was suspended in an incubation medium consisting of extraction medium plus ATP, 0.4 ,uM; phospho-enolpyruvate, 0.5,uM; pyruvate kinase, 20/1g; GTP, 0.02,uM; NADPH, 0.1 ,uM; at pH 8.0, and sonicated whilst the tubes were embedded in crushed ice. Preparation of [3H]-GA g

Synthesis was carried out in a manner similar to that described by CROSS et al. (1968). GAg methyl ester was treated with osmium tetroxide and periodic acid to yield 16--oxo17-nor GAg. GAg nor-ketone so formed was converted to 17 [3H]-GAg in the Wittig reaction using triphenylphosphine and [3H]-methyl iodide. The product was crystallized to constant specific activity from acetone-light petroleum (b.p, 60-80°) and stored at - 20°C in absolute ethanol. [3H]-GAg chromarographed as a single peak by gas chromatographyradio counting. Non-radioactive GAg, to be used as a «carrier», was purified by partition column chromatography on Sephadex LH-20 (MACMILLAN and WELS, 1973) in order to remove small amounts of GA 4 and GA 7 contaminants. By gas chromatography this sample was 99.98 % pure. Incubations

[3H]-GAg, together with 2 mg purified «carrier» GAg dissolved in 15,uL of ethanol, were added directly to sonicates of pea chloroplasts. Incubations were carried out in a Gallenkamp shaking water bath at 27°C under high intensity illumination. Reactions were terminated after 40 mins. by the addition of ice-cold methanol and methanolic solutions were clarified by centrifugation. The protein pellets were washed thoroughly with absolute methanol until colourless. Extraction and Chromatography

All methanol was removed in vacuo at 35°C and an equal volume of 0.5 M phosphate buffer (pH 8) was added to the residual aqueous phase. After adjusting to pH 9 with 1 N KOH the extract was partitioned (5 X) against equal volumes of diethyl ether, adjusted to pH 3 with 1 N HCl, and then partitioned against ethyl acetate (5 X). The acidic, ethyl acetate fraction, which contained almost all the radioactivity was examined further. The total ethyl acetate fraction was strip loaded onto a thin-layer plate of silica gel Hand chromatographed Z. Pflanzenphysiol. Ed. 81. S. 323-329.1977.

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in a solvent system consisting of ethyl acetate, chloroform, formic acid (50: 50 : 1 v/v). Following thin layer chromatography (TLC), the plate was divided into Rf strips and the levels of radiocativity associated with each strip determined by liquid scintillation counting. The gel was scraped directly in scintillation vials and was eluted with 0.5 ml absolute methanol. Following addition of 10 mls of cocktail (5 grm 2,5 diphenyl oxazole per litre toluene) samples were counted in a Packard Tricarb scintillation spectrometer. Gas chromatography-radiocounting (GC-RC)

For GC-RC, Rf strips 6 and 7 (see figure 1) were combined and eluted with watersaturated ethyl acetate. The sample was reduced to dryness and the methyl ester, trimethyl silyl ether derivatives (TMSiMe derivatives) prepared as described previously (CAVELL et al., 1967). GC-RC was carried out as described 4) by MACMILLAN and WELS (1974) using 2 0/ 0 SE-33 as liquid stationary phase on Gaschrom Q (80-100 mesh) wh ch was temperature programmed (180-230 DC at 3°/min). Combined gas chromatography-mass spectrometry (GC-MS)

GC-MS was performed on an AEI MS 30 mass spectrometer coupled to a Pye 104 gas chromatograph through a silicone-membrane separator. Mass spectra were obtained at 24eV with a source temperature of 210 0 and a separator temperature of 190 0 • The mass spectra were processed on-line by a Line 8 computer.

Results and Discussion The distribution of radioactivity on TLC following chromatography of the acidic, ethyl acetate fraction from incubates of [3H]-GA g with chloroplast sonicates from pea is shown in Figure 1. Two metabolite zones produced from [3H]-GA g were apparent at Rfs 0.0-0.1 (zone 1) and Rfs 0.5-0.7 (zone 2) and in previous work (RAILTON and REID, 1974 b) both these zones were found to exhibit biological activity in the dwarf rice var. GA9

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Tan-ginbozu bioassay. Sonicates which were held at 100 DC for 2 minutes prior to the addition of [3H]-GA o showed a much reduced incorporation of label into metabolite zo ne 2 suggesting an enzyme-controlled conversion (Figure 2). Metabolite zone 2 was analyzed further by GC-RC and by GC-MS. Three radioactive mass peaks were obtained from this zone by GC-RC and these are indicated on the total ion current trace (TIC) shown in Figure 3.

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Fig. 3: Total ion current trace of the TMSiMe derivatives of acidic compounds from TLC zone 2. Peaks A( e), B(e ) and q e ) correspond to radioactive peaks detected by GC-RC. Z. Pjlanzenphysiol. Ed. 81. S. 323-329.1977.

16,17-Dihydro 16,1 7-Dihydroxy GAg

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Th e large mass peak, A was shown by GC-MS to have an identical mass spectrum to th at publ ished for GAg (Fig. 4, I) (BINKS et aI., 1969). Th e second peak, B at lon ger retenti on time contained mainly phthalate plasticizer and attempts to characterize the [3H] _ compound were unsuccessful. Th e pre sence of a fragment ion at ml e 156 in th is peak wa s characteristic of 15-hydroxylated GAs (d . GA 32) and suggested th e presence of 15-0H GAg (Figure 4, II ), recentl y chara cterized from Pyru s communis and now given the «A» number, GA 45 (BEARDER et al., 1975). However, th e absence of a molecular ion at M + 418 characteristic of TMSiMe derivati ves of monohy droxy C-19 GA s, pr ecluded th is possibility. Th e third radioact ive mass peak , C, cont ained a compound with an ident ical mass spectrum (Figure 5) to that of th e known GAg derivati ve 16,17-dihydro 16,17-dihydroxy GAg (Figure 4, lIT) (CROSS et aI., 1968). Interestingly thi s compound had a greater mobility on

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TLC than would be expected for a molecule with this polarity, and it is possible that it existed as an epoxide which was opened on derivatization. Recent studies (BEARDER et al., 1976) on the microbial hydroxylation of GA 9 and its methyl ester by mutant BI-41 a of Gibberella fujikuroi and by Rhizopus nigricans have shown that these fungi readily hydroxylate GA 9 to several GAs including GA 45 and 16,17-dihydro 16,17-dihydroxy GA 9 • These results suggest, therefore, that both fungi and pea chloroplasts have the ability to hydroxylate at C-16 in the ent-gibberellane skeleton. Although C-16 hydroxylation has been demonstrated before in pea seedlings i.e., GA i 4 -+ GA 42 (DURLEY et al., 1974) and GA 9 -+ GA lO (RAIL TON et al., 1974), this is the first time that hydroxylation at C-17 has been shown in higher plants. The ability of plant tissues to hydroxylate the exocyclic methylene group suggests some possible difficulties using GAs labelled in this readily accessible group. Should oxidation at this site prove to be a prerequisite for the eventual removal from the ent-gibberellane skeleton of the exocyclic methylene function, then metabolites produced from GAs by this process may not necessarily still carry tritium label and may not be detected. This would certainly lead to errors in estimating the amounts of applied GAs metabolised by plant tissues and the nature of the metabolites themselves. The present results show that chloroplast sonicates can hydroxylate applied GA 9 but the relevance of this to GA biosynthesis in the intact plant remains to be investigated further. Acknowledgements I am extremely grateful to Dr. J. MACMILLAN, School of Chemistry, University of Bristol (U.K.) for use of GC-MS equipment and to Mr. Paul Gaskin for his skill in recording the mass spectra. This work was supported in part by grants from Rhodes University C.S.1.R., Pretoria and the Atomic Energy Board of South Africa.

References BEARDER, J. R., V. M. FRYDMAN, P. GASKIN, 1. K. HATTON, W. E. HARVEY, J. MACMILLAN, and B. O. PHINNEY: Fungal products. Part XVIII. Microbiological hydroxylation of gibberellin A 9 and its methyl ester. J. Chern. Soc. Perkin 1,178-183. (1976). BEARDER, J. R., F. G. DENNIS, J. MACMILLAN, G. MARTIN, and B. O. PHINNEY: A new gibberellin (A 45 ) from seed of Pyrus communis L. 9, 669-670. (1975). BINKS, R., J. MACMILLAN, and R. J. PRYCE: Plant hormones. VIII. Combined gas chromatography-mass spectrometry of gibberellins Ai to A 24 and their trimethyl silyl ethers. Phytochemistry 8, 271-284. (1969). CAVELL, B. D., R. J. PRYCE, J. MACMILLAN, and A. C. SHEPPARD: Thin layer and gasliquid chromatography of the gibberellins: direct identification of the gibberellins in a crude plant extract by gas-liquid chromatography. Phytochemistry 6, 867-874. (1967). CROSS, B. E., K. NORTON, and J. C. STEWART: The biosynthesis of the gibberellins. III. J. Chern. Soc. c., 1054-1063. (1968). DURLEY, R. c., I. D. RAILTON, and R. P. PHARIS: The metabolism of gibberellin Ai and gibberellin A 14 in seedlings of dwarf Pisum sativum. In: S. Tamura. Proc. 8th Int. Conf. Plant Growth Substances. (Ed.) Hirokawa Press Tokyo. 1974.

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MACMILLAN, J., and C. M. WELS: Detailed analyses of metabolites from mevalonic lactone in Gibberella fu jikuoi. Ph ytochemistr y 13, 1413-1417. (1974). - - Part ition chromatography of gibberellins and related diterpenes on columns of Sephadex LH-20. ]. Chromatog. 87, 271-276. (1973). RAILTON, I. D.: Studies on t he effects of day len grh on endo genous gibberellins in potato . Ph . D. The sis. Un iversity of Wale s. (1971). R AILTON, I. D. , and D . M. REID: Stu dies on gibberellins in shoots of light grown peas. 1. A re- evaluation of t he data. Plant Sci. Lett. 2, 157-163. (1974 a). - - Studies on gibberellins in shoots of light grow n pea s II I. Interconver sion of [3H]_ G Ag and [3H ]-GA 2o to othe r gibberelli ns by an in v itro system der ived from chloroplasts of Pisum satiuum, P lant Sci. Lett . 3, 303-308. (1974 b). RAILTON, I. D., and P . F. WAREING: Effects of da ylength on endogenou s gibberellins in leaves of Solanum andigena. 1. Changes in level s of free acidic gibberellin-l ike substances. Physio!' Plantarum. 28, 88-94. (1973 a). - - Effects of da ylength on endo genou s gibberellins in Solanum andi gena. II. Metabolism of gibberellin Al by potato shoots. Ph ysio!. Plantarum. 28, 127-1 31. (1973 b). - - Effects of day length on endo genous gibberellins in Solanum andigena. III. Gib berellin production by the leaves. Physio!. Plantaru m 29, 430-433 (1973 c). RAILTON, I. D. , R. C. D URLEY, and R . P. PHARIS: Metabolism of t ritiat ed gibberellin A o by shoots of dark-grown dwarf pea cv. Meteor. Plant Physio!' 54, 6-12. (1974). REID, D. M.: Stu dies on th e sites of synt hesis and mode of act ion of gibbere llins in plants. Ph . D . Thesis. Q ueen's Univ. Belfast. Northern Ir elan d. (1967). STODDART, ]. L. : The association of gibberellin-like activity with th e chloroplast fraction of leaf homogenates. P lanra (Bed.) 81, 106-111. (1968). - Incorporat ion of kaurenoic acid into gibberellins by chloroplast preparations of Brassica oleracea. Ph ytochemistry 8, 831-837. (1969). 1. D . RAILTON, Department of Botan y, Rhodes University, P.O. Box 94, Grahamstown 6140, South Af rica.

Z. Pfl anzenphysiol. Bd . 81. S. 323-329. 1977.