Gibberellins in developing fruits of Prunus avium

Pergamon

GIBBERELLINS

0031-9422(93)EOO99-Z

IN DEVELOPING

Pbytochemimy, Vol. 35, No. 6, pp. 1383 1386, 1994 Copynght Q 1994 Elscvier Science Ltd Pmted in Great Britm. All nghts reserved KU-9422,94 S6.00+0 00

FRUITS OF PRUNUS A’VIUM

PATRICK S. BLAKEand GORDON BROWNING Horticulture Research International, East Malling, West Mailing, Kent ME19 6BJ, U.K. (Receioedin revised form 14 October 1993)

Key Word Index-Prunus

avium; Rosaceae; gibberellins; identification;

developing fruits.

Abstract-Gibberellins present in sweet cherry fruitlets (Prunus auium cv Stella) were analysed by combined capillary gas chromatography-mass spectrometry after purification using a procedure designed to retain polyhydroxylated acids. Comparison of full-scan mass spectra of the TMSi ethers of the methyl esters and Kovats retention indices (KRIs) with those of standards revealed the presence in extracts of GA,, GA,, GA,, GA,,, GA,, and GA,,, and comparison with published mass spectra and KRIs GA,,, GA,, and GA,,. With the presence also of the previously identified GA,,, cherry fruitlets thus contained the C-12, and C-12, C-15 hydroxylated derivatives of both GA, and GA3.

INTBODUCIION Except for the recent identification of GA,, (12c+OHGA,) [l] in developing fruits of Prunus avium, gibberellins (GAS) present in this species have not previously been characterized. The only GAS unequivocally identified by full-scan GC-MS in other Prunes species are GA,, GA,, and GAs6, which were detected in peach seed extracts [2,3]. GC-SIM evidence for the presence of GA,, GA,, GAP9 and GA,, in immature seeds of apricot (P. armenica) [4], and for GA,, in sour cherry fruitlets (P. cerasus) [SJ has also been reported, as has GC-SIM evidence that cell suspension cultures and immature seeds of apricot can metabolize 3HGA, and *HGAs to free labelled GAI, GA,, GA,, GA, and GA,, [6], and GA,, GA, and GA, [7], respectively, in addition to various conjugates. The presence in Prunus species of GAs, and GA,, is of considerable interest because of reports that in Lolium temulentum polyhydroxylated active GAs, e.g. GA,,, could be specifically involved in the control of flowering [S, 91. In P. avium, pattern and degree of GA hydroxylation influences the ability of exogenously applied GA to inhibit floral initiation. Thus, GA, and GA,, were inactive, while GA, and GA4 inhibited by ca 15%. GA, (With a C-2,3 double bond) inhibited floral initiation by IO%, GA, and GA, (each with C-1,2 double bond) inhibited by 70% and 40%, respectively. Similarly, growth of P. aoium seedlings was greater when they were treated with GA, or GA, than when treated with GA, or GA,. Mature shoots (including those of rooted mature cuttings of similar size to the seedlings) were less responsive to GA treatment, but the response was similar for GA,, GA,, GA, and GA,, while spur shoots on mature trees only responded to GA, or high doses of GA, [lo]. Preparatory to a study of GAS in germinating seeds, seedlings and mature vegetative tissue of P. avium in

relation to juvenility, we have analysed by full-scan capillary GC-MS other possible active GAs in sweet cherry developing fruitlets, choosing to characterize these first because immature seeds can contain up to x 1000 larger concentration of GAS than vegetative tissues [ll].

RF$ULTsAND DI!KU!%SION The method described for GA purification was baaed on that reported for the isolation of GA,, [l]. Immediate filtration of the 80% methanol followed by overnight extraction in pure methanol resulted in less highly coloured extracts, after evaporation of the methanol and centrifugation to remove insoluble material, than longer periods of extraction in 80% methanol. For further purification, columns of insoluble PVPP, charcoal:celite, and QAE Sephadex were used. Use of this method yielded in preliminary experiments, cleaner extracts, richer in GAS at the GC-MS stage than conventional solvent partitions, which also had the disadvantage of requiring n-butanol partitions for polyhydroxylated GA recovery. Lettuce hypowtyl bioassay of fractions collected after Cis reverse-phase HPLC revealed strong GA-like activity in fractions 13 and 14, and 16-18, with some activity in fractions 23-25 and 30-35 (data not shown). Groups of two-four of the biologically active or other HPLC fractions were combined as appropriate for SIM and full-scan GC-MS, or for further purification of the Me esters by TLC. A total of nine GAS was identified either by comparison of full-scan mass spectra and KRIs of derivatized extracts with those we obtained for authentic protio or deuteriated standards, or by comparison with published mass spectra and KRIs [12] (Table 1). Thus, GA, (fractions 24 and 25), GA, (fractions 23 and 24), GA, (fractions 31 and 32), GA,, (fractions 34-36) and GAao

1383

1384

P. S. BLAKE and G. BROWNING

(fractions 30 and 31) were identified by comparison with protio-GA standards, GA,, (fractions 18 and 19) by comparison with [17-‘H,]GAz, ([17-2H2]GAs differ from those of the corresponding protio-GAs by a KRI of ca 2 under our conditions), and GA,, (fractions 13 and 14), GA,, (fractions 17 and 18) and GAs6 (fractions 13 and 14) by comparison with published information [12]. The KRI values we obtain for authentic GA,, GA,, GA2c. GA, and GA,, are greater by 21,24,35,36 and 36, respectively, than the values published in ref. [12]. GA, was not unequivocally detected, but its presence could have been obscured by the presence of large amounts of GAs5, which shares the same molecular ion (M+ at m/z 594) and differs in KRI value by only 5. Comparison of TIC traces indicated that GA,,, GA,, and GA,, were the most abundant of the GAS detected. Specific SIM searches, using standards as references, for the early 13hydroxylation pathway precursors GAhL and GA,, [13] were unsuccessful. Similar searches for 12-OH-GA,, and 12-OH-GA,, possible pre(GA&, 12-OH-GA,, cursors for the 12-hydroxylated GAS detected, were also unsuccessful, although a reference standard was available only for 12~OH-GA,. Consequently, no evidence was obtained for the pathway of synthesis of GA,, or for the 12-hydroxylated GAS detected, and in particular, whether there was an early 12-hydroxylation pathway in cherry [14]. Of the GAS detected in cherry fruitlets, both GA1 and GA, strongly promote shoot growth, depending on juvenility status, and GA, strongly inhibits floral initiation, whereas GA, and GA2, are much less active [lo]. The presence of the C-12, and C-12, C-15 hydroxylated derivatives of both GA, and GA, in cherry fruitlets thus raises interesting questions about the possible biological role of these polyhydroxylated GAS, especially given the prominent abundance of GA,, and GA,,. EXPERIMENTAL

Plant material. Rapidly expanding, immature fruitlets were collected 40-50 days after anthesis (May 1991/1992) from trees of the self-fertile sweet cherry cv Stella on Colt rootstocks planted (4.5 x 3 m spacing) in 1982, and maintained in an orchard kept weed-free with overall applications of the herbicides simazine and paraquat. The trees were fertilized and sprayed for pest and disease control according to the East Malling standard programme for sweet cherries. The fruitlets were plunged into liquid N, in the field, and stored at -20” until required. Extraction and purijcation. Frozen samples (200 g fr. wt) were homogenized with 800 ml of cold (4”) 80% MeOH containing 20 mg 1-i BHT and 833 Bq of [l, 2 -3H]GA, (1406GBq mmol-‘; Du Pont de Nemours GmbH, NEN Division, Dreiech, Germany). The extract was immediately filtered, the aq. MeOH retained, and the residue re-extracted with MeOH overnight at 4” and filtered. MeOH was removed at 35” from the pooled filtrates under red. pres. on a rotary film evaporator (RFE). An equal vol. of 0.5 M KPi buffer pH 8.2 was added to the aq. residue, which was then frozen, thawed and centrifuged for 20 min (10 000 g; 4”). The supernatant

was passed through a column of insoluble PVPP (10 g), this was washed with 50 ml of KPi buffer, the pooled eluates adjusted to pH 3, and passed through a column (3.5 x 12 cm) of charcoal:celite (1: 2 w/w). The column was washed with 400 ml H,O (pH 3), and GAS eluted in 400 ml 80% Me,CO, which was reduced to the aq. phase (RFE), and the pH adjusted to 8.0. The extract was then passed through a column (2 x 13 cm) of QAE-Sephadex A25, the column washed with 150 ml H,O pH 8, and GAS recovered in 100 ml 0.2 M HCOOH, which was passed directly through two primed Crs Sep-Pak cartridges in series. After washing the cartridges with 5 ml H,O pH 3, GAS were eluted in 20 ml 80% MeOH, and this taken to dryness (RFE), the extract dissolved in 200 /.d 10% MeOH in 5% HOAc and injected (500 /11injection loop) into a C,, reverse phase HPLC (Hewlett Packard series 1050) column (4.6 mm i.d. x 250 mm) containing Hypersil 5 ODS. The column was eluted at a flow rate of with 5% MeOH for 5 min, followed by a 1 mlmin-’ linear gradient to 100% MeOH obtained over 45 min (solvents contained 50 pll-’ HOAc). An aliquot from each of the 50 1 ml frs collected was taken for bioassay of GA-like activity with the lettuce (Lactuca sativa L. cv Arctic King) hypocotyl test. The remainder was taken to dryness on a centrifugal vacuum concentrator (CVC), redissolved in 100 $ MeOH and methylated with excess ethereal CH,N,. The Me esters were taken to dryness under a stream of O,-free N, and for GC-MS redissolved in 25 d Tri-Sil/BSA, heated to 100” for 5 min, evapd to dryness and dissolved in 10 ~1 BSTFA, or for purification by TLC, dissolved in lSO$ anhydrous CH,Cl,. For TLC, extracts were applied in a narrow band to 20 x 20 cm aluminium-backed, silica gel coated (0.2 mm; Merck 5553), pre-washed plates and Me-GA standards applied within a scored strip next to each vertical edge of the plate. The plates were developed using CHCl-MeOH (9 : 1) as the solvent, the scored strips cut from each side of the plate, which were sprayed with 10% H,SO, in EtOH, heated at 110” for 10 min, and the position of GAS marked under UV light (375 nm). Silica gel was removed in broad bands from the remainder of the plates as appropriate, placed in plugged pasteur pipettes and eluted with EtOH, which was then taken to dryness (CVC), and the extract trimethylsilylated for GCMS as above. Capillary column GC-MS. TMSi ethers of the Me esters were analysed using a VG TRIO 1 MS coupled to an HP 5890 CC equipped with a split/splitless injector. The CPSIL 5 CB capillary column (25 m long x 0.25 mm i.d.) was coupled directly to the ion source with an interface temp. of 275” and He carrier gas inlet pressure at 40 kPa. The MS source was at 200” and electron energy 70 eV. The injector was used in the splitless mode at a temp. of 275”. After injection of samples (1 pl), the CC oven was maintained at 50” for 0.7 min with the splitter closed, after which the splitter (50 : 1) was opened, and 0.3 min later the oven temp. increased at 15” min- ’ to 220”, and then at 2” min-’ to 300”. Mass spectra were acquired 20 min after injection, using the VG Lab-Base data system, by scanning every 0.9 set from 50 to 650 amu. Samples were co-

Gibberellins in Prunes a&m

1385

Table 1. Comparison of Kovats retention indices (KRI) and relative intensities of characteristic ions for MeTMSi derivatives of ~b&rellins in nature fruits of Pnutus &urn with those of standard compounds (or literature values for standard compounds) Iden- Kovats tified retention GA index

Diagnostic ions (m/z) with abundance in reference and sample

32

IOIl

2969 2979 86 2975 2984 87 2865 2865 85 2814 2831 29* 2684 2698 3 2713 2692 2712 1 2693 2669 2692 20 2517 2482 2517 5 2513 2471 2512 19 2627 2596 2629

Lit. reference Sample Ion Lit. reference Sample Ion Standard Sample Ion Lit. reference Sample Ion Lit. reference Sample Ion Standard Lit. reference Sample Ion Standard Lit. reference Sample Ion Standard Lit. reference Sample Ion Standard Lit. reference SampIe Ion Standard Lit. reference sample

680 [M]* 44 5 682 [Ml+ 12 592 [M] + 12 12 594 [M] + 48 : CM]+ 100 z&i,+ 100 100 100 506 [M] + 100 100 100 418 [M] + 100 100 100 416 [M] * 100 100 100 462 [M-j+ 4 9 4

665 12 17 667 9 11 548

579 9 3 491 13 5 489 11 8 11 491 11 11 11 403 18 18 17 401 20 22 24 434 96 loo 91

590 100 100 592 loo 100 502 7 8 550 9 6 447 8 5 475 15 14 17 448 20 21 25 389 7 4 8 385 2 4 4 402 52 38 39

546

31 14 548 11 8 489 21 21 504 59 29 315 16 100 445 19 14 16 377 24 13 22 375 71 47 a4 312 S 9 6 375 84 46 12

HPLC fraction 500 41 19 533 12 14 355 12 8 491 100 100 303 22 9 387 14 12 16 376 27 17 22 359 21 14 25 257 19 26 23 374 96 64 100

456 23 12 302 29 28 238 14 10 445 13 6 281 5 9 370 26 2s 22 313 33 11 18 301 27 14 26 343 16 24 17 345 45 26 44

397 25 21 463 7 11 193 38 32 375 42 27 235 13 16 355 19 12 23 235 21 7 19 235 18 9 10 299 43 60 53 285 38 23 33

339 48 76 339 13 7 147 100 100 348 17 15 207 38 13 208 100 39 95 207 45 25 72 207 96 32 44 207 40 5.5 45 208 100 34 45

13,14

13,14

16,17

17,18

l&l9

23,24

24,25

30,31

31,32

34-36

*Spectrum was unsnap by extmneo~ ions. The identifi~tion was made on the basis of most characteristic ions rna~~~ng intensity at correct KRI. KRI of authentic &GA,, was 2697.

injected with 0.1 ~1 of a parafilm extract for calculation of KRIs [IS]. Specific GC-SIM searches undertaken were for 12-OH-GA,, [GAJ (m/z 506 EM]‘, 491,462,418, 403), 12a-OH-GA, (504 [Ml’, 489,460,416,401) and 12OH-GA,, (550 [Ml’, 522,447,419), GA,, (432 [M-J+, 417, 373,238,207) and GA,, (448 EM]“, 389,251,241, 235). Samples of GA,, GA,, GA,,, GAao, GA&., 12a-OHGAS, GA*,, [17-2H,]GA, and [17-2H,]GA29 were obtained from Professor L. N. Mander (Australian National University, Canberra, Australia), Cl*-GAS3 from Dr P. Hedden (AFRC Institute of Arable Crops Research, Long Ashton, Bristol), and GA, from Zeneca Crop Protection. Acknowledgements--We

thank Mandy Marchese for ex-

cellent technical assistance, and Professor L. N. Mander

and Dr P. Hedden for generous gifts of 12a-OH-GA, and GA,,, and C!‘4-GA,3, respectively. This work was funded by the U.K. Ministry of Agriculture, Fisheries and Food. REFzXEffcEs 1. Blake, P. S., Browning, G., Chu, A. W. L. and Mander, L. N. (1993) Pkytockemistry 32, 781. 2. Yamaguchi, I., Yokota, T., Murofushi, N., Takahas~, N. and Ogawa, Y. (1975) Agric. Biot. Chem. 39,239X 3. Bhaskar, K. V., Chu, W. L. A,, Gaskin, P. A., Madder, L. N., Murofushi, N., Pearce D. W., Pharis, R. P., Takahashi, N. and Yamaguchi, I. (1991) Tetrahedron Letters 32,6203. 4. Bottini, R., Bottini, G. A., Koshioka, M., Pharis, R. P.

and Coombe, B. G. (1985) ~~~~? P~ys~o~.78,417.

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P. S. BLAKEand G. BROWNING

5. Bukovac, M. J. and Yuda, E. (1979) Plant Physiol. 63, 129. 6. Koshioka, 7. 8. 9.

10.

M., Pharis, R. P., Matsuta, N. and Mander, L. N. (1988) Phytochemistry 27,3799. Bottini, G. A., Bottini, R., Koshioka, M., Pharis, R. P. and Coombe, B. G. (1987) Plant Physiol. 83, 137. Pharis, R. P., Evans, L. T., King, R. W. and Mander, L. N. (1987) Plant Physiol. 84, 1132. Evans, L. T., King, R. W., Chu, A., Mander, L. N. and Pharis, R. P. (1990) Planta 182, 976. Oliveira, C. M. and Browning, G. (1993) Plant Growth Regulation 13, 55.

11. Hedden, P. (1987) in Principles and Practice ofPlant Hormone Analysis (Rivier, L. and Crozier, A., eds), Vol. 1, p. 20. Academic Press, London. 12. Gaskin, P. and MacMiIlan, J. (1991) GC-MS of the Gibberellins and Related Compounds: Methodology and a Library of Spectra. Cantocks, University of

Bristol, Bristol. 13. Graebe, J. E. (1987) Ann. Ret;. PIant Physiol. 38,419. 14. Lange, T., Hedden, P. and Graebe, J. E. (1993) P~anta 189,340.

15. Gaskin, P., MacMillan, J., Firn, R. D. and Pryce, R. J. (1971) Phytochemistry 10,1155.