Plant Growth Regulation 24: 91–99, 1998. 91c 1998 Kluwer Academic Publishers. Printed in the Netherlands.

Detection and identification of gibberellins in needles of silver fir (Abies albaMill.) by combined gas chromatography-mass spectrometry

Alexander Christmann1� & Patrick Doumas2

1Institut fur Botanik, Universitat Hohenheim (210), Garbenstraße 30, D-70593 Stuttgart, Germany;2INRA, Biochimie et Physiologie Moleculaire des Plantes, F-34060 Montpellier Cedex 1, France

Received 6 May 1997; accepted in revised form 6 October 1997

Key words: Abies alba, conjugates, GC-MS, gibberellins, identification, needle senescence

Abstract

Extracts of mature silver fir (Abies alba Mill.) needles, current-year, and one-year old (A) and seven to nine-yearold (B), were purified by reversed and normal phase HPLC. Gibberellin (GA)-like compounds were detected bythe Tan-ginbozu dwarf rice micro-drop bioassay and corresponding fractions were analyzed by GC-MS. GA9

was present in small amounts, while a major component was a cellulase-hydrolysable GA9 conjugate which wasassumed to be GA9 glucosyl ester. It is proposed that GA9 glucosyl ester plays a key role in the regulation ofendogenous GA levels in silver fir needles.

Abbreviations: DW = dry weight; FW = fresh weight; GC-MS = combined gas chromatography-mass spectrometry;LC-MS = combined liquid chromatography-mass spectrometry; HPLC = high performance liquid chromatography;RT = retention time; SIM = selected ion monitoring

1. Introduction

Senescence of leaves is a well-regulated processinvolving probably all classes of phytohormones [23].Christmann and Frenzel (1994) suggested that needlesof silver fir (Abies alba Mill.) belonging to differentage-classes might be particularly well suited for thestudy of how senescence is regulated by changes inendogenous levels of different phytohormones. Whileabscisic acid is probably less involved in the regula-tion of fir needle senescence [7], IAA and ethyleneseem to play an important role. With increasing needleage, rising levels of the senescence-promoting hor-mone ethylene [5] are balanced by a concomitant risein levels of IAA [6]. In the very oldest needles, how-ever, this balance disappears prior to abscission withIAA levels declining in the presence of still elevatedlevels of ethylene [5, 6].

Actually nothing is yet known about simultane-ous changes in levels of cytokinins or gibberellins(GAs) with increasing needle age. GAs seem to beinvolved in the regulation of many aspects of growth

and development of coniferous trees [9], yet the roleof GAs in the regulation of senescence of conifer nee-dles is completely obscure. In leaves of dicotyledo-nous species, the application of GAs often retardedchlorophyll loss and has been shown to inhibit RNAand protein breakdown (see [23] for a list references)all of which are processes associated with senescence.Correspondingly, endogenous GA levels were found todecline steadily in senescing dicotyledonous leaves [1,2, 10], indicating that GAs are presumably involved inthe regulation of senescence in such leaves. Accord-ing to these results, it seems possible that GAs play arole in the control of conifer needle senescence. How-ever, an influence of GAs on associated physiologicalprocesses in leaves may not be exerted by mere changesin concentration but also by changes in GA pattern (cf.[33]) or by changes in tissue sensitivity (cf. [31]). We,therefore, chose to start our work on GAs and silverfir needle senescence with a study of the GA pattern inmature silver fir needles of differing ages.

Mature silver fir needles more than three years oldenter an early phase of senescence, which is character-

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ized by a gradual loss of photosynthetic capacity and agradual decline of chlorophyll and protein content dur-ing the course of about six years [3, and Christmann,unpublished results]. This phase of slow senescence isfollowed by a phase of rapid needle senescence.

In order to compare the endogenous GA pattern weused non-senescing mature needles and mature needleswhich had already entered the early phase of senes-cence.

2. Material and methods

2.1 Plant material

From a 30-year-old healthy silver fir tree (Abies albaMill.) growing in the northern Black Forest, a firstorder twig from the sun crown with needles up to tenyears old was harvested in summer 1994,several weeksafter the formation of current-year needles was com-pleted. Mature current-year needles and one-year oldneedles (sample A) and seven, eight and nine-yearold needles (sample B) were pooled and immediatelyfrozen in liquid nitrogen. Samples were left in liquidnitrogen until freeze-dried. Homogenisation of freeze-dried needles was carried out in a hammer mill (Retsch,Haan, Germany) which was cooled with liquid nitro-gen. Final particle size was 0.2 mm. The ground nee-dles were again freeze-dried overnight to remove anytraces of water condensed during homogenisation andwere finally stored at - 20 �C over silica gel.

2.2 Extraction and purification of plant material

Ground needles of 25 g dry weight were extracted in500 ml methanol:0.02 M phosphate buffer pH 7.0 (4:1;v:v) containing 0.01% (w/v) 2,6-di-t-butyl-4-methyl-phenol as an antioxidant under nitrogen for 8 hoursin the dark. Samples were stirred during extractionand kept at 4 �C. After suction filtration through ateflon filter (5 �m), the residue was re-extracted with500 ml of the extraction mixture for 16 hours underthe same conditions as above and filtered. From thecombined filtrates of the needle extracts, lipophilicpigments were removed by freezing, thawing and re-filtration. Methanol was then removed in vacuo at35 �C. The aqueous residue was acidified to pH 2.7with 6 M HCI and extracted 5 times with 100 ml water-saturated ethyl acetate and 5 times with 100 ml water-saturated n-butanol according to Doumas et al. (1992).The organic phases were combined and the organic

solvents were reduced in vacuo at 35 �C to 200 ml vol-ume. This residue was shaken 4 times against 10 mlwater, adjusted to pH 2.7 with 0.1 M HCI, to removephosphoric acid formed after acidification of phosphatebuffer [11]. The organic phase was then evaporated todryness under reduced pressure. The residue was tak-en up in a small volume of methanol and transferredto a beaker containing 1 g of Celite [14]. Methanolwas then removed under reduced pressure with gentlestirring. Further purification of the extracts soaked upby the Celite was achieved on a 12� 200 mm columnfilled with silica (32–63 �m, 60 A, ICN, Eschwege,Germany) preconditioned with hexane:ethyl acetate(5:95; v:v) saturated with 0.5 M formic acid. Gib-berellins were eluted with 200 ml of the conditioningsolvent. The eluate was evaporated to dryness underreduced pressure and stored at –20 �C.

2.3 HPLC separations

Purification of extracts was achieved by reversed-phase HPLC according to Moritz et al. (1989)with minor modifications on a 250 � 4.6 mm i.d.LiChrospher 100 (5 �m) RP-18 column (Merck,Darmstadt, Germany) with a Gynkotek HPLC sys-tem (Gynkotek, Germering, Germany). Flow ratewas 1.5 ml min�1. Solvent A was methanol:aceticacid:water (10:1:89; v:v:v) and solvent B wasmethanol. After 10 minutes of 100% A, a linear gradi-ent to 70% B was formed in 30 minutes. 70% B wasmaintained for further 10 minutes, then the solventcomposition was switched to 100% B and kept at 100%B for a further 30 minutes. Eighty 1.5 ml fractions werecollected and evaporated to dryness in a vacuum con-centrator (Bachofer, Reutlingen, Germany). Fractionswere dissolved in methanol and 1:50 aliquots of eachfraction were tested for GA-like activity with the Tan-ginbozu dwarf rice micro-drop bioassay [22] using riceachenes which were a gift from Prof. Pharis, Calgary.The residues of each fraction which exhibited GA-likeactivity were combined and purified by normal phaseHPLC with the same equipment as described aboveaccording to Moritz et al. (1989) on a 150 � 4.6 mmi.d. Nucleosil-NO2 (5 �m) column (Interchrom, Paris,France), which was kept at 38 �C. Flow rate was2 ml min�1. Solvent A was n-heptane saturated with1 M formic acid:n-heptane (1:1; v:v) and solvent Bwas ethyl acetate:water:formic acid (98.5:1:0.5;v:v:v).After 2 minutes of 100% A a linear gradient to 100% Bwas formed in 60 minutes. 100% B was maintained fora further 18 minutes. Eighty 2 ml-fractions were col-

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lected and evaporated to dryness as described above.Fractions were dissolved in methanol and 1:50 aliquotsof each fraction were tested for GA-like activity in thedwarf rice bioassay. The residues of those fractionswhich exhibited GA-like activity in the bioassay afternormal phase HPLC were methylated with a solutionof diazomethane in diethylether according to Schlenkand Gellerman (1960) and stored at –20 �C prior toGC-MS analysis.

2.4 Cellulase treatment and HPLC of GA9 methylester

After normal phase HPLC, fractions which putativelycontained GA9 in a conjugated form were combinedand evaporated to dryness. They were then hydrolyzedwith cellulase according to Doumas et al. (1992). Thehydrolyzed fraction was adjusted to pH 2.7 with 6 MHCI and extracted 5 times with 2 ml ethyl acetate.The organic phases were combined, evaporated to dry-ness and methylated with a solution of diazomethanein diethylether. Reversed phase HPLC separation wasperformed on a 250 � 4.6 mm i.d. LiChrospher 100(5 �m) RP-18 column (Merck, Darmstadt, Germany)with a Waters HPLC system (Waters Associates, Mil-ford, MA, USA). Flow rate was 1 ml min�1. SolventA was methanol:water (10:90; v:v) and solvent B wasmethanol. After 10 minutes of 70% A a linear gradi-ent to 100% B was formed in 30 minutes. 100% Bwas maintained for further 25 minutes. Eighty 1 ml-fractions were collected. Fractions corresponding tothe retention time of [3H]-GA9 methyl ester were com-bined, evaporated to dryness under reduced pressureand subjected to GC-MS analysis.

2.5 Gas chromatography-mass spectrometry

Bioactive fractions were trimethylsilylated using 50 �lof pyridine and 50 �l of N,O-bis(trimethylsilyl)tri-fluoroacetamide with 1% (v:v) trimethylchlorosilane(Pierce Europe, The Netherlands) for 30 min at 70 �C.Samples were reduced to dryness under a gentle streamof nitrogen and were dissolved in 5 �l of chloroformbefore being injected splitless onto a fused silica cap-illary column (AT-1 chemical bonded phase 0,25 �m,0,35 �m id � 30 m, Heliflex) installed in a MFC500gas chromatograph (Fisons Instruments, UK) linked toa QMD 1000 mass spectrometer (Fisons Instruments,UK). Oven temperature was 60 �C during the injection,with an injection port temperature of 270 �C. After adelay time of 1 min the oven temperature was increased

by 15 �C min�1 to 200 �C and then by 5� min�1 to275 �C. The carrier gas flow rate (He) was 2 ml min�1.Interface temperature was kept at 280 �C and elec-tron energy was 70 eV. Full scan mass spectra wererecorded of each fraction exhibiting GA-like activity.Those fractions where amounts of GA-like compoundswere too low to give full scan mass spectra were re-runusing SIM. Kovats’ retention indices were determinedusing n-alkanes C23-C28 [15].

2.6 Estimation of concentrations of GA9 and acellulase-hydrolysable GA9 conjugate

Estimation of concentrations of GA9 and a cellulase-hydrolysable GA9 conjugate was based on a compari-son of the response in the dwarf rice bioassay of appro-priate fractions obtained after normal phase HPLC withthe response to different concentrations of GA9 stan-dard.

3. Results

After reversed phase HPLC of needle extracts severalfractions exhibited GA-like activity in the dwarf ricebioassay. Some fractions corresponded to RTs of [3H]-standards of GA1=3 and GA9 (Figure 1). The pattern ofGA-like activity was almost identical in correspondingfractions from needles of a different age (Figure 1).

After normal phase HPLC of reversed phase HPLCfractions with GA-like activity, three peaks of GA-likeactivity were found with two of them corresponding toRTs of [3H] standards of GA9 and GA4. A third peakwas observed with a RT of about 50 min, indicatingthe presence of a GA or a GA conjugate with chro-matographic properties slightly different from those of[3H]-GA20 standard (Figure 2). Again, the pattern ofGA-like activity was similar in corresponding fractionsfrom needles of a different age.

All fractions with GA-like activity obtained afternormal phase HPLC were subjected to GC-MS analy-sis, and, in full-scan mode, GA9 was detected in thefraction with RT 49 min, sample B. Fractions withGA-like activity in the bioassay were re-analyzed byGC-MS in the SIM mode. In fractions RT 23–25 min,which correspond to the RT of [3H]-GA9 standard (Fig-ure 2), GA9 was also detected in low amounts in bothsample A and sample B (Table 1). From these contrast-ing results, it was supposed that a conjugate of GA9

was present in fraction RT 49 min which had to someextent been hydrolysed during sample derivatization.

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Figure 1. Detection of GA-like activity by Tan-ginbozu dwarf rice assay in methanol extracts of mature current-year, and one-year old (A) andseven to nine-year old (B) silver fir needles after LiChrospher 100 C18 reversed phase HPLC. Solid horizontal line: methanol control.��� =RT of tritiated GA standards.

Hydrolysis with cellulase of the fractions RT 48–52min (sample A) and RT 50–51 min (sample B) indeedliberated GA9, which after methylation and reversed-phase HPLC purification was identified by comparisonof the full-scan mass spectrum obtained with the spec-trum of GA9 methyl ester standard (Figure 3).

Concentration of GA9 and of the cellulase-hydrolysable GA9 conjugate in the needles was esti-mated by comparing GA-like activity of the fractionsobtained after normal phase HPLC (cf. Figure 2) withthe activity of different concentrations of GA9 standardin the bioassay. Concentration of GA9 was estimated

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Figure 2. Detection of GA-like activity by Tan-ginbozu dwarf rice assay after Nucleosil NO2 normal phase HPLC of active fractionsfrom methanol extracts of mature current-year, and one-year old (A) and seven to nine-year old (B) silver fir needles chromatographed byreversed-phase HPLC. Solid horizontal line: methanol control.��� = RT of tritiated GA standards.

at 12 (A, RT 24–26 min) and 6,8 pmol g�1 DW (B,RT 23–25 min). Concentration of the conjugate, whichwas assumed to be GA9 glucosyl ester, was estimatedat 150 (A, RT 48–52) and 250 pmol g�1 DW (B, RT49–51 min).

4. Discussion

The spectrum of GAs usually found in conifers(Table 2) is consistent with an early non-hydroxylationpathway of GA biosynthesis in such species [19, 25]

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Figure 3. GC-MS scan of authentic GA9 methyl ester standard (A) and of fractions from silver fir needles (B; mature current-year, one-yearold, and seven to nine-year old needles combined) obtained by reversed-phase HPLC after normal phase HPLC, hydrolysis by cellulase andmethylation.

with GA9, which was found in all conifer species stud-ied so far, probably being the last non-hydroxylatedGA on the pathway. Our findings that GA9 and a GA9

conjugate are present in needles of Abies alba suggestthat in this species too, biosynthesis of GAs via theearly non-hydroxylation pathway is probably the mainroute.

In studies with other species of members of thefamily Pinaceae, hydroxylated GAs that are likelyto derive from GA9 were also identified in differentorgans, including the needles (cf. Table 2). While GA9

was the primary GA present in young (2- to 3-month-old) Pinus radiata needles [25], Wang et al. [34] failedto detect GA9 in very young (3 to 4 mm long) needles

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Table 1. GC-MS (SIM-mode) of authentic GA9-methyl esterstandard and fractions after normal phase HPLC of silver firneedle extracts

Standard compound orfraction identification

Five characteristicions m/z (relativeintensities)

Kovatsretentionindex

Authentic GA9 methylester

330(8), 298(100),270(91), 243(91),227(70)

2349

Methyl ester of 25min zone after normalphase HPLC

330(7), 298(100),270(94), 243(95),227(70)

2345

Table 2. Gibberellins in conifers, positively identified by GC-MSor LC-MS

Gibberellin Species Plant organ Reference

A3, A4, A7 Pinus attenuata pollen [13]

A1, A3, A4, A9,A12, A15, A20,A29, A34, A51

Picea abies shoots [19]

A1, A3, A9 Picea abies shoots [24]

A1, A3, A4, A7,A9, conjugatesof A9 and A15

Picea sitchensis shoots [20, 21]

A1, A3, A4, A7,A8, A9, A15, A20

Pinus contorta shoots [35]

A1, A3, A4, A7,A8, A9, A15

Pinus radiata shoots [35]

A1, A3, A4, A7,A9, conjugate ofA9

Pseudotsugamenziesii

shoots [8]

A1, A4, A7, A9,A20

Pinus sylvestris cambialregion

[30]

A4, A7, A9 Pinus sylvestris cambialregion

[32]

iso-A9,conjugate of A9

Picea sitchensis needles [16, 17]

A9 glucosyl ester Picea sitchensis needles [18]

A1, A3, A4, A9,conjugate of A9

Picea sitchensis needles [21]

A1, A3, A4, A7,A8, A9, A20

Pinus contorta needles [35]

A9 Pinus radiata needles [35]

of Pinus sylvestris, but were able to identify GAs 1,3 and 4. These contrasting results point to changes inthe pattern of endogenous GAs during needle devel-opment. This pattern further appears to be influencedby flowering since in a comparison of Picea sitchensisclones, GA9 dominated in young needles (harvestedwhen shoot extension was approximately 95% com-plete) of good-flowering clones while needles of poor-flowering clones contained high levels of GA1 in theneedles [21]. However, in needles, high levels of GAsother than GA9 have only been reported for young,growing needles. Thus, major changes in conifer nee-dle GA pattern might be confined to growing nee-dles, whereas a GA pattern seems to be maintainedin mature needles with GA9 dominating. In our study,only mature needles of differing ages were comparedand these needles did not differ inasmuch as GA9 wasthe primary GA present both in the current-year toone-year old and in the seven to nine-year old needles.With the dwarf rice bioassay, which responds to bothfree and conjugated GAs [12, 29], we were also able todetect a conjugated form of GA9. The chemical identityof the sugar moiety of this GA9 conjugate still has to beconfirmed, but according to its hydrolysis by cellulaseand due to the fact that no glucosides can be formedfrom GA9 which lacks hydroxyl groups, the conju-gate presumably is GA9 glucosyl ester (GA9-GE). InPicea sitchensis shoots and needles, the identity ofa GA9 conjugate that had previously been tentative-ly identified as GA9-GE after enzymatic hydrolysisand GC-MS identification of the aglycone [16, 20] hasmeanwhile been confirmed by LC-MS [18].

Since only one other conjugated form of a gib-berellin, a conjugated form of GA15, has been detectedup until now in one conifer (Picea sitchensis; [20]),whereas a conjugated form of GA9 has also been iden-tified in Picea sitchensis and Pseudotsuga menziesii(cf. Table 2), conjugation of GA9 might play a dom-inant role in GA conjugation of conifers. ConjugatedGA9 might serve as a storage product that is occasion-ally exported from the needles, or might also play arole in the regulation of endogenous levels of free GA9

in needles (cf. [27]). In maize seedlings [28], a rapidexchange among pools of GA glucosyl ester, GA glu-coside and free GAs was found, supporting the viewthat in regulation of free GA levels in plants, reversibleconjugation/hydrolysis of GAs is important.

Besides conjugation of GAs and hydrolysis of GAconjugates, several other sites for control of GA levelsexist in plants and have also been demonstrated asbeing important in conifers, e.g., the 3ß-hydroxylation

98

step leading to the formation of GA4 from GA9 and ofGA1 from GA20 [19]. However, the particular impor-tance of the different regulation steps in control of freeGA levels in conifers remains to be elucidated.

We have estimated the levels of free and conjugatedGA9 in fractions obtained after normal phase HPLCusing the dwarf rice bioassay in which GA9 glucosylester is almost as active as GA9 [16]. However, sincewe could not account for losses of GAs during sampleclean up and since no correction of probably variablebackground inhibition of dwarf rice growth was made,this estimation is only preliminary and needs to beverified by quantitative analysis in the future. Levelsof GA9 seemed far lower than those reported for stillgrowing needles of Picea sitchensis ([21]: 7–28 ng g�1

FW), while levels of the putative GA9 glucosyl esterin fir needles seemed higher ([21]: 4–12 ng g�1 FW).In mature fir needles, levels of GA9 seemed to furtherdecline with increasing needle age, whereas levels ofconjugated GA9 rose.

Our estimation of GA9 and the tentatively identi-fied GA9 glucosyl ester in silver fir needles suggeststhat levels of free GA9 were very low while levels ofthe GA9 conjugate were comparatively high. This isin contrast to the results of Moritz et al. [21], whoestimated that in needles of Picea sitchensis concen-trations of free GA9 approximately doubled concen-trations of a cellulase-hydrolysable GA9 conjugate.However, Moritz et al. [21] used young, still growingneedles while we analysed mature needles. Thus, theobserved differences might be attributed to differencesin needle age.

A decline of concentration of GA9 with increasingneedle age would be consistent with the view that GAsare involved in hormonal regulation of needle sense-cence. In conifers, GA9 is thought to be a precursorof GA1, GA3 and GA4 which may be active per sein shoot growth regulation [33]. Since mature needlesno longer grow, other functions must be attributed toGAs in such needles and one of these might be thatGAs play a role in maintaining basic physiologicalprocesses and thereby in retarding senescence. It is notyet clear whether GA9 may be active per se in thiscontext or if very low levels of more polar GAs, thatcould not be detected by our analytical procedures, areneeded to maintain basic needle metabolism.

The level of GA9, nevertheless, might be a measureof the physiological activity of mature silver fir nee-dles. Reliable quantification of GA9 and of the GA9

conjugate in needles of a different age are thus desir-able and might help to gain more insight into the phys-

iological function of the GA9 conjugate in young andold needles and help to elucidate the possible role ofGA metabolism during needle maturation and duringdifferent phases of needle senescence. We shall, there-fore, try to quantify the GAs in silver fir needles ofdiffering age in the future.

Acknowledgements

We are indebted to Marc Bonnet-Masimbert for invit-ing A. Christmann to his laboratories, to Per-ChristerOden for a gift of GA9 standard and of tritiated GA9

and to Richard P. Pharis for a gift of dwarf rice ach-enes. We thank the EUROSILVA research cooperationfor financial support and for organizing the stay of A.Christmann at INRA, Orleans.

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