of apical growth in seedlings of S. pentandra growing in LD (8,. 10). The action .... to a VG Micromass 7070H mass spectrometer with a DEC PDP. 8A computer ...
Plant Physiol. (1985) 78, 473-476 0032-0889/85/78/0473/04/$O 1.00/0
Identification of Endogenous Gibberellins from Salix pentandra1 Received for publication June 25, 1984 and in revised form January 15, 1985
JOHN K. DAVIES2, EINAR JENSEN, OLAVI JUNTTILA*, LAURENT RIVIER, AND ALAN CROZIER Botany Department, The University, Glasgow GJ28QQ, Scotland (J.K.D., A.C.); Institute of Biology and Geology, University of Tromso, P.O. Box 3085 Gukeng, N-9001 Tromso, Norway (E.J., O.J.); and Institute ofPlant Biology and Physiology of the University, 1015 Lausanne, Switzerland (L.R.) ABSTRACr
Gibberellins A,, Al,, A2n, and A2. have been identified by sequential high-performance liquid chromatography retention time (Rt) and combined ps chromatography-mass spectrometry (Rt and characteristic mass spectra) from elongating shoots of Salix pentaadra L. Gibbereilins A, and Al, were also detected in purified extracts from male and female flowers (catkins) of S. pentandra.
Cessation of apical growth of young seedlings of deciduous, temperate-zone woody plants, such as Salix pentandra, is usually induced by SD (7-9, 22). It has been shown that this SD effect can be overcome by exogenous GA3 but not auxin or cytokinin treatments (8, 11, 14, 18). Whereas ABA has only a limited effect, growth retardants such as CCC and B-995 induce cessation of apical growth in seedlings of S. pentandra growing in LD (8, 10). The action of both CCC and B-995 is antagonized by a simultaneous application of GA3 (8). Preliminary analysis of shoot extracts have shown a significant decrease in GA-like activity prior to cessation of apical growth in S. pentandra (12). Before the possible involvement of GAs in this process can be investigated at the biochemical level, it is necessary to identify the major endogenous GAs in S. pentandra. In this paper, we report on the analysis of several GAs in elongating shoots and male and female flowers of S. pentandra. MATERIALS AND METHODS Plant Material. The main study used about 3 kg fresh weight (approximately 70% moisture content) of elongating shoots of Salixpentandra L. harvested in early June 1980 from adult trees growing near the University of Troms0 (69° 39'N lat.). Two other samples of about 0.9 kg fresh weight were collected from elongating seedlings growing in a greenhouse. Samples of male (0.9 kg) and female (0.6 kg) flowers (catkins) were collected outof-doors at the time of anthesis in 1982. There were small variations in the extraction and purification prodedures for the 'Supported in part by a grant from the Norwegian Research Council for Science and the Humanities, a Nansen Foundation grant, and the British Council. 2 Present address: Lehrstuhl fur Pflanzenphysiologie, Ruhr-Universitiit Bochum, Postfach 102148, D-4630 Bochum, Federal Republic of Germany. 3Abbreviations: GA, gibberellin; B-995, 2,2-dimethylhydrazide; CCC, 2-chloroethyltrimethylammonium chloride; GACE, gibberellin methoxycoumaryl ester, GAMeTMS, gibberellin methyl ester trimethylsilylether; GC-SICM, gas chromatography-selective ion current monitoring. 473
different samples. Extraction and Purification of the 3 kg Sample of Elongating Shoots. The tissue was homogenized and extracted twice with 5 L of 80% aqueous methanol. The methanolic filtrates were combined and 1 ng (1,2-3H)GA, (0.78 TBq/mmol), 10 ng [1,23H]GA4 (0.05 TBq/mmol), 5 ng [I-3H]GA5 (0.16 TBq/mmol), 1 ng [2,3-3H]GA9 (1.32 TBq/mmol), and 5 ng [2,3-3H]GA20 (0.05 TBq/mmol) were added before reduction to dryness at 30°C in vacuo. The dried extract was dissolved in 500 ml pH 8.0, 0.5 M phosphate buffer, and partitioned five times against 250ml volumes of toluene to remove impurities. The aqueous phase was then slurried with a 1:1 100 mg ml1' mixture of insoluble PVP and polyamide (1,6), filtered through Whatman No. 1 filter paper, adjusted to pH 2.5, and filtered through cellulose powder before being partitioned five times against 200-ml volumes of ethyl acetate. The acidic, ethyl acetate extracts were combined, and water was removed by freezing and treatment with anhydrous sodium sulfate prior to being reduced to dryness in vacuo at 35°C in the presence of toluene. The acidic, ethyl acetate-soluble extract was purified by DEAESephadex A25 anion exchange chromatography according to procedures described by Crozier (1). Consecutive 50-ml fractions were collected and aliquots were assayed for radioactivity by liquid scintillation counting. The [3H]GA internal standards eluted as discrete peak. The appropriate fractions were combined, reduced to the aqueous phase in vacuo at 35C, adjusted to pH 2.5, and partitioned five times against two-fifths volumes of ethyl acetate. The ethyl acetate extracts were combined, water removed, and reduced to dryness in vacuo. The sample was dissolved in 1.5 ml of tetrahydrofuran and injected onto two 1000- x 25-mm Biobead 5SX-4 columns connected in series and eluted with tetrahydrofuran at a flow rate of 2 ml min-' (19). A single peak of radioactivity (Rt 450475 ml) was detected and taken to dryness in vacuo. This fraction was subjected to preparative HPLC as noted in Reeve et al. (20). Seventy 10-ml fractions were collected and aliquots were assayed for radioactivity by liquid scintillation counting, and GA-like activity in the barley aleurone (50-fold dilution), Tan-ginbozu dwarf rice microdrop (50-fold dilution), and lettuce hypocotyl (30-fold dilution) bioassays (5, 16, 17). Fractions containing GAlike activity were analyzed further by analytical HPLC and/or GC-MS. Extraction and Purification of the Other Samples. Tissues were extracted and partitioned as described above except that only [1,2-3HJGA, was added to the extracts as an internal marker. The acidic, ethyl acetate-soluble fraction was chromatographed on a 450- x 10-mm i.d. PVP-Sephadex LH-20 column (21). The GA fraction (35-100 ml) was extracted (Sx) into ethyl acetate at pH 2.5 and further purified by DEAE-Sephadex A25 (1). Preparative HPLC was then carried out on a 250- x 9-mm i.d. Partisil 10 column with a 1.0 N acetic acid stationary phase according to procedures described by Reeve et al. (20). The
474
DAVIES ET AL.
Table I. Capillary GC-SICM ofAuthentic GA,MeTMSi, GA 19MeTMSi, GA29MeTMSi, and MeTMSi Derivatives of Purified Samples from Elongating Shoots and Male and Female Flowers of S.
pentandra Splitless injection. Open oven for 50 s, then 70 to 240C at the rate of 15C min-'. (GAI, GAIg, and GA29 samples were developed with intervals of several weeks. No effort was made to exactly reproduce the helium carrier gas flow. The retention times of GA,, GA19, and GA29 may therefore not reflect the elution order of these GAs when analyzed under identical chromatographic conditions.) Sample Retention Time m/z (% Relative Intensity) 11.39 506 (100) 491 (22) 448 (44) 377 (45) GA,MeTMSi Shoots Not analyzed Male flowers 11.37 506 (100) 491 (13) 448 (26) 377 (33) Female flowers 11.36 506 (100) 491 (14) 448 (27) 377 (36) GA19MeTMSi 12.19 434 (100) 402 (66) 375 (63) Shoots 12.21 434 (100) 402 (52) 375 (63) Male flowers 12.20 434 (100) 402 (51) 375 (62) Female flowers 12.19 434 (100) 402 (49) 375 (63) GA29MeTMSi 11.43 506 (100) 491 (17) 375 (42) Shoots 11.43 506 (100) 491 (17) 375 (43) Male flowers 11.43 Not detected Female flowers 11.43 Not detected
`5000 0 4000 0.
c 3000
02000
EE
50
,
40
30 co
20 co
column was eluted with a 76-min gradient of 5 to 100% ethyl acetate in hexane at a flow rate of 3 ml min-'. The equipment used comprised a Waters 6000A and M45 solvent delivery unit, a Waters 660 solvent programmer, and a Waters model U6K injector. Fifty 6-ml fractions were collected and assayed for radioactivity and for GA-like activity in the Tan-ginbozu dwarf rice bioassay. Biologically active fractions were purified further on a 100- x 8-mm i.d. 5 ,um Waters Rad-Pak C-18 column which was eluted with a 20-min gradient of 10 to 70% methanol in 20 mm (pH 3.5) acetic acid buffer. Twenty five l-ml fractions were collected, assayed for radioactivity, and fractions with the Rt of GA1 and expected Rts of GAI9 and GA29 were taken to
dryness and analyzed by GC-SICM. Analytical HPLC of Methoxycoumaryl Esters. Aliquots of bioactive fractions were derivatized by Crown ether catalysis to form methoxycoumaryl esters (2). Following purification by steric exclusion chromatography (3), the derivatized samples were analyzed by reversed- and normal-phase HPLC (4). The equipment used comprised an Altex model 332 liquid chromatograph linked to a Perkin-Elmer 650- 1Os spectrofluorimeter (excitation, 320 nm; emission, 400 nm) fitted with a 1 6-pu flow cell. Samples were introduced off-column via a Perkin-Elmer 7105 sample valve. Reversed-phase separations were carried out on a 250- x 5-mm i.d. column packed with 5 ,um ODS-Hypersil and eluted at 0.75 ml min-' with 30 min gradient of 60 to 100% methanol in 20 mm (pH 3.5) ammonium acetate. A 250- x 5mm i.d.-5 ,um CPS Hypersil column eluted isocratically at 1 ml min-' with hexane/dichloromethane (8:2) in 3% ethanol was used for normal phase analyses. Combined GC-MS. The instrument used comprised a HewlettPackard 5710A gas chromatograph linked via a jet separator (packed colum GC) or by a direct inlet (capillary column GC) to a VG Micromass 7070H mass spectrometer with a DEC PDP 8A computer system. Samples were methylated and silylated before being introduced via an injector at 250°C onto a 2-m x 2-mm i.d. glass column packed with 1 % SP 2100 on Supelcoport (100-120 mesh) support. The column temperature was held at 150°C for 2 min and then programmed to 230°C at 8°C min-'. The He carrier gas flow was 30 ml min-'. The temperature of the jet separator was 240°C and that of the ion source 220C. Positive ion electron impact spectra were recorded at 70 ev with a 2.0 s decade-' scan rate and a 1.0 interscan delay. Alternatively,
Plant Physiol. Vol. 78, 1985
c
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7
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5 4
a
3
0 0
I1
I 40 30 Fraction
FIG. 1. Bioassay of preparative HPLC fractions of the semi-purified, acidic ethyl acetate-soluble extract from elongating shoots of S. pentandra. Fractions tested in the barley aleurone bioassay, the Tan-ginbozu dwarf rice bioassay, and the lettuce hypocotyl bioassay. Retention of [3H]GA standards indicated by horizontal bars.
the derivatized extracts were introduced onto a 15-m x 0.25mm i.d. fused silica capillary column with DBI stationary phase (0.25 gm film thickness) via a split/splitless injector operating at 250°C. The helium carrier gas flow rate was about 1 ml min-'. Slightly different temperature programs were used for the various fractions (for details, see figure and table legends). Positive ion 70 ev electron impact spectra were recorded with a 1.0 s decade-' scan rate and 1.0 s interscan delay. RESULTS AND DISCUSSION Bioassays of aliquots of the semipurified acidic ethyl acetate fraction from 3 kg of elongating shoots of S. pentandra, following preparative HPLC, revealed the presence of three major zones
of GA-like activity (Fig. 1). Fractions 21 to 22. With the aliquots used, these fractions (which co-chromatographed with [3H]GA9) were active only in the lettuce hypocotyl bioassay. Gradient elution of the methoxycoumaryl ester derivatives on reversed-phase HPLC revealed the presence of a number of fluorescent peaks, none of which had the retention characteristics of GA9 methoxycoumaryl ester. Similarly, GC-MS analysis of the MeTMSi derivatives did not detect the presence of GA9 or any other component recognizable as a known GA.
Authentic GA20 MeTMSi
m/z 418
.,
ENDOGENOUS GIBBERELLINS IN SALIX
475
GA2oMeTMSi (Fig. 2),and a full scan mass spectrum was also obtained (data not shown). Fractions 44 to 48. These fractions contained the largest
Putative GA20 MeTMSi from Salix
amount of GA-like activity, especially fraction 45. Reversedphase HPLC of the methoxycoumaryl esters of fraction 45 revealed the presence of a number of fluorescent peaks, one of which cochromatographed with GAICE. No GA3CE-like peak was detected in this fraction. The GA,CE-like component when
m/z 418
analyzed by normal-phase HPLC, again cochromatographed
with GA,CE (see Ref. 2 for HPLC traces). A GC-MS analysis of the MeTMSi derivatives of another aliquot revealed a multicomponent total ion current trace, and mass spectra obtained from m/z 403 m/z 403 0) minor peaks gave spectra which were in good agreement with the standard spectra of GA, (Fig. 3) and GA,9 (data not shown). c A spectrum which was in good agreement with the standard spectrum of GA29 (except for the m/e 303 ion, which we believe was missing due to a temporary computer failure during that part of the mass spectrum) was also obtained. This indicate the mlz 375 ml/z 37spresence of GA29 in the sample. Extracts from 0.9 kg of elongating shoots and 0.9 kg of male and 0.6 kg offemale flowers were also analyzed. The semipurified acidic, ethyl acetate extracts were run on preparative HPLC and m/z 359 m/z 359 the GA,-like zone was collected and purified further by reversed, a, I, I, IX,,l, l, l, Il, l, l, l, l, l, phase HPLC to separate GA,, GA,9, and GA29 (13). Fractions 12 with the Rts of Gal, Gaig, and Ga29 were collected and analyzed 8 4 8 12 4 0 0 by capillary GC-SICM as the MeTMSi derivatives (Table I). This Retention time (min) confirmed the presence of GA,9 and GA29 in elongating shoots FIG. 2. Capillary GC-SICM of authentic GA2oMeTMSi and putative of S. pentandra, while GA, and GA,9 but not GA29 were detected GrA2zMeTMSi from S. pentandra. Split injection (1:10). Column tem- in both male and female flowers. Salix pentandra thus contains a C20 GA, GA,9, and the C,9 perature was held at 200'C for 2 min, increased then to 260°C at the rate 0*If 5C min-'. GAs GA20, and GA,. In view of the available information on GA biosynthesis pathways in other tissues (15), it is likely that Fractions 33 to 36. This zone was the most active in the lettuce GA,9 is the immediate precursor of GA20 which undergoes 3-alhypocotyl bioassay and also induced a response in the Tan- hydroxylation to yield GA,, or 2-,B-hydroxylation to form GA29.
O-
g1nbozu. Reversed phase HPLC (50J-l100Y or- methanol in ZU
mM [pH 3.5]acetic acid buffer) yielded a single bioactive fraction (lettuce hypocotyl bioassay) which was analyzed by capillary GCSICM and GC-MS. Capillary GC-SICM at m/z 359, 375, 403, and 418 revealed the presence of a compound with chromatographic and fragmentation properties identical to those of 10
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.
73 -'3
