Plant Physiol. (1970) 45, 658-662

Metabolically Active Glucosides in Oleaceae Seeds

I. THE EFFECTS OF GERMINATION, GROWTH, AND HORMONE TREATMENTS

Received for publication December 15, 1969

E. SONDHEIMER, G. E. BLANK, EVA C. GALSON, AND F. M. SHEETSChemistry Department, State University College ofForestry at Syracuse University, Syracuse, New York 13210

ABSTRACT

The seeds of six woody species of Oleaceae representingthree genera, contain high concentrations of water-solubleglucosides, with major absorption maxima below 240 nano-meters. In Fraxinus americana seeds three of these com-pounds, designated GL-3, GL-5, and GL-6, account foralmost 10% of the dry weight. They are found in the endo-sperm and embryo but not in the pericarp. While the levelof GL-5 is not particularly influenced by the physiologicalstate of the embryo, that of GL-3 and GL-6 decreases as aresult of germination and growth during a 10-day period.As the concentrations ofGL-3 and GL-6 decrease, new ultra-violet-absorbing compounds are formed. The changes inthe concentration of the ultraviolet-absorbing glucosidesduring cold temperature after-ripening, prior to germina-tion, are small. When germination of dormant embryos isinduced with gibberellic acid, the concentrations of GL-3and GL-6 decrease in a manner similar to that observed withnondormant embryos. In the presence of abscisic acid nolosses of GL-3 and GL-6 were observed. It is suggested thatGL-3 and GL-6 fulfill some definite functions in the germi-nation and growth of F. americana embryos, and thatgibberellic acid and abscisic acid can exert a regulatoryeffect on the metabolism of these glucosides.

The germination of white ash seeds, Fraxinus americana, nor-mally requires the breaking of dormancy through cold tempera-ture after-ripening. Embryos excised from dormant seeds will notgerminate at 22 C under the same conditions that permit germina-tion of embryos from nondormant seeds. Gibberellins and cyto-kinins allow germination and growth of dormant embryos whileABA' prevents these processes (9). The endogenous levels of thelatter hormone reflect the physiological states of the embryos,lower levels being found in nondormant seeds (10).To gain insight into the mode of actions of these hormones,

the changes in certain storage nutrients during germination ofF. americana seeds were followed, and the effects of exogenouslyadded GA3 and ABA were determined. Attention was focused oncarbohydrates since evidence is accumulating that at least onerole of GA3 in germination and development of many seedsinvolves the breakdown of starch (1, 5).Examination of dry F. americana seeds showed that while they

are free of starch they do contain high concentrations of water-

1 Abbreviations: ABA: abscisic acid; TLC: thin layer chromatog-raphy.

soluble, ultraviolet-absorbing, dialyzable glucosides. Three ofthese glucosides, designated GL-3, GL-5, and GL-6-to indicatethat they are glucosides and can be distinguished by their R,values- have been purified sufficiently to permit structural work.The quantitative determination of the glucosides is based on theirintense absorption near 240 nm and purification by TLC. Thismethod has been used to determine the distribution of GL-3,GL-5, and GL-6 in six species, representing three genera of theOleacea family. Changes in concentration of the glucosides inF. americana seeds as a function of cold temperature after-ripen-ing, germination, embryo growth, and hormone treatments werestudied. The levels of sucrose, glucose, and fructose under theseconditions were also followed.

METHODS

Isolation of the Glucosides, GL-3, GL-5, and GL-6. F. ameri-cana seeds, purchased from F. W. Shumacher, Sandwich, Massa-chusetts, were depericarped and ground in a Wiley mill with a20-mesh screen. This meal was extracted at 4 C with 70% metha-nol, and the extract was concentrated below 40 C to one-tenththe original volume. The concentrate was extracted with ether,and the organic phase was discarded. Extraction of aqueousphase with methyl ethyl ketone concentrates GL-5 and GL-6 inthe upper phase. Extraction of the lower phase with n-butanolyields crude GL-3 on concentrating of the butanol extract on arotary evaporator. Further purification of GL-3 is achieved withsilicic acid column chromatography with water-saturated n-butanol as the mobile phase. GL-5 and GL-6 are purified byfractionation of the methyl ethyl ketone extract with a 30-platecountercurrent distribution apparatus with a two-phase water,2-pentanol system. Best results are obtained with mixtures thathave a low GL-3 content. GL-6 is found in fractions 4 to 11 andGL-5 in fractions 18 to 25.GL-3 crystallizes from n-butanol; melting point 136 to 138 C;

[a]25 = _149° (c 0.22, water); E ,2 = 212. The compoundis soluble in water, ethanol, and water-saturated n-butanol. Thecompound is dialyzable against water. Solubility in ether, ben-zene, ethyl acetate, and anhydrous n-butanol is low. The crystal-linity of GL-3 was confirmed by x-ray powder diagram. GL-5crystallizes from methyl ethyl ketone; melting point 115 to 118 C,E11n ,235nm = 217. GL-6 was crystallized from 2-pentanol, melt-ing point 92 to 94 C; [a] = _106° (c 0.34, water);E1S ,277nm= 28 and El ,226nm 244. GL-6 also has a shouldernear 240 nm. All ultraviolet spectra were obtained in 95%ethanol.

Determination of Concentration of the Glucosides, GL-3, GL-5,and GL-6. A 40 to 50-mg sample of vacuum-dried seeds wasground with sand and 70% ethanol. One-tenth of the total ex-tract was concentrated and streaked on a plate coated 0.5-mmthick with Silica Gel GF2b4. The plates were developed with

658

METABOLICALLY ACTIVE GLUCOSIDES

water-saturated n-butanol. The glucosides appeared as violetbands under a low wave length (254 nm) ultraviolet light. Thedesired regions were scraped off and eluted with methanol. Theconcentration of each substance was determined spectrophoto-metrically by the use of the E 17, values listed above. In orderto make a more positive identification, the absorption spectrumbetween 210 and 300 nm was obtained for each band eluted fromthe TLC plates. In every case where the concentration of gluco-side was above 0.4%7, of the dry weight, the spectrum of the elutedband had the same characteristics as that of the correspondingpurified glucoside. For the data in Table I, where we comparedcompounds in different species, the TLC eluates were hydrolyzedwith acid, and the products were chromatographed. Comparisonof these products with those obtained from the hydrolyzed, puri-fied compounds provided additional identifying criteria.

Determination of Carbohydrates and Amylase. The simplecarbohydrates, sucrose, glucose, and fructose, present in 70%ethanol extracts were determined by quantitative paper chroma-tography with ethyl acetate-pyridine-water (8:2: 1) as developingsolvent and reaction with o-aminodiphenylhydrochloride forquantitative spectrophotometric determination (8). Mannitol,which is very difficult to separate from glucose, was detectedqualitatively by removal of glucose as the osazone, paper chroma-tography with the above solvent system, and visualization withsilver nitrate-sodium hydroxide reagent (12). Starch was detectedwith iodine (13). The procedure of Chrispeels and Varner (2)was used for the detection of amylase activity. Standard extractionprocedures were used for the isolation of the structural polysac-charides (3).

Treatments of biological materials. For cold temperature after-ripening, seeds were held at 5 C in moist vermiculite. Fraxinusand Syringa samaras were depericarped by hand. Olea seeds werecracked, and the endocarps were discarded. Fraxinus seedswere soaked 24 hr in tap water at room temperature prior toexcision of the embryos. For germination studies, 10 embryoswere placed on filter paper in 5-cm Petri dishes containingsufficient liquid to keep the embryos moist. Embryos wereincubated with 0.01 M phosphate buffer, pH 6. The Petridishes were placed into growth chambers under a 12-hr lightcycle at 300 ft-c, "day" temperature 22 C, "night" temperature16 C. The formation of a curvature at the basal end of theembryo was interpreted as a positive germination response.Germination data are reported as 210 values: the sum ofthe dailypercentage of germination for a 10-day period starting at the dayof excision (12).

RESULTS

Ultraviolet-absorbing Glucosides in Oleaceae Seeds. That theglucosides, here designated GL-3, GL-5, and GL-6, are majorconstituents of F. americana seeds can be shown by a comparisonof the ultraviolet absorption spectrum of an 85% ethanol extractof defatted seeds with the spectra of the purified substances(Fig. 1). The similarities in the shape of curves 1 and 2 to curve 3clearly indicate that the three glucosides must be major contribu-tors to the total absorption of the crude extract. The high con-centration of these substances prompted us to look for them inseeds of five additional species belonging to Oleaceae (Fig. 2).GL-3 and GL-6 are present in all the seeds we examined, butsignificant amounts of GL-5 have been detected in only threespecies. Other compounds with related ultraviolet absorptioncharacteristics were also found, and one compound with an RFnear 0.1 is present in each extract (Fig. 2). Quantitative data forthe amounts of GL-3, GL-5, and GL-6 found in the dry seedsare tabulated in Table I. The total varied from approximately2c,% in F. excelsior to 12%o in Olea europaea. The trend in therelative concentrations found in dry seeds is GL-3 > GL-6 >

0

g

Wavelength ( nm)

FIG. 1. Ultraviolet absorption spectra in 95% ethanol. 1: 32 mg/liter GL-3 or GL-5; 2: 37 mg/liter GL-6; 3: 96 mg/liter residue fromthe 85% ethanol extract of defatted F. americana seed meal.

GL-5, but F. excelsior seeds do not conform to this pattern. Howmuch variation is due to such factors as the age of the seed, thestorage conditions, and the environment in which the seed isproduced remains to be determined.

In mature, dormant F. americana samaras, the species withwhich we have worked most extensively, these glucosides are notfound in the pericarp. Embryos and endosperm have about thesame percentage composition, but the larger mass of the endo-sperm results in 75% of the total being found in the latter.

Since GL-3 is the predominant glucoside in the dry seeds offive of six species listed in Table I, it was selected for furtherstudy. However, the similarities in spectral and solubility proper-ties, as well as glucose release, indicate a close relationship be-tween GL-3, GL-5, and GL-6. GL-3 contains 53.6% carbon,6.6% hydrogen, and 40.9% oxygen. Standard chemical proce-dures show the presence of esters or lactones or both, vinylgroups, 8.1% methoxyl, and 3.8% C-methyl. Acetate, aldehyde,and carboxylic acid groups are absent. Hydrolysis at 100 C for48 hr with 2 N sulfuric acid yields glucose as the only monosac-charide and in maximum yield of 43%. But refluxing with 0.5%oxalic acid for 45 min gives 26% glucose. Since under the latterconditions little or no starting material remains, it seems likelythat at least two glucose moieties with different chemical linkagesare present. The fact that treatment of GL-3 with emulsin yieldsglucose indicates that at least one of these bonds is a f-glucosidelink.Major F. americana Seed Constituents. The high concentration

of the three ultraviolet absorbing glucosides raises the possibilitythat these compounds have a nutritional function. This made itdesirable to learn more about the typical reserve nutrients presentin these seeds. Careful extractions and iodine detection failed toshow the presence of starches, and amylase activity could not bedemonstrated in seeds or in endosperm that had been incubated48 hr with GA3. The seeds contain 35% lipids, and the proteincontent calculated from Dumas nitrogen is 19%. The lattervalue is apt to be high since it would include free amino acids,peptides, nucleic acids, and other nitrogen-containing substances.

659Plant Physiol. Vol. 45, 1970

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SONDHEIMER ET AL. Plant Physiol. Vol. 45, 1970

-1.0

-.8

-.6

-4

Rf

-0

FIG. 2. Ultraviolet-absorbing glucosides from Oleaceae seeds. Chromatogram of 70OcJ ethanol extracts. TLC plates were coated with Silica Gel

GF254 and developed with water-saturated ni-butanol. Compounds were detected with a short wave length, 254 nm filter, ultraviolet source. 1:

Syringa vulgaris. 2: F. excelsior; 3: F. penniisylvanica; 4: F. americania; 5: F. ornius; 6: Olea europaea.

Table I. Distributionl of GL-3, GL-5, alitc GL-6 in DrySeed of Six Oleaceae Species

ConcnSpecies'

GL-3 GL-5 GL-6

% dry wt

F. americania 6.3 = 0.8 1.6 -+- 0.2 1.7 - 0.1F. ornus 4.2 ± 2.5 <0.2 3.2 i 0.4F. pennsylvanica 5.8 -- 0.8 2.4 -- 0.4 2.8 -- 0.1F. excelsior 0.5 it 0.1 0.4 -- 0.1 1.0 it 0.2Syringa vulgaris 2.7 it 0.5 0.7 ±4 0.1 2.5 -- 0.4Olea europaea 7.6 +t 3.2 <0.2 4.7 -- 1.7

1 Pericarp has been removed from the samara prior to analysis.

Attempts to determine the polysaccharide content of these seedsby the use of successive extraction procedures (3) were compli-cated by the large amounts of extractable nitrogen-containingsubstances. After correcting for contaminating substances bymultiplying the nitrogen content by 6.25, the following values areobtained: hemicelluloses 2%0/; xylans 3%; glucomannans 2%O;and pectins, gum, and mucilages about 1 %.

Information concerning the low molecular weight carbohy-drates was obtained by examination of the ethanol extracts.Quantitative paper chromatography showed that the seeds con-tained 1% sucrose and 0.2%o glucose. If fructose and galactoseare present, the concentration of each is below 0.02%. By far thelargest fraction of the 85% ethanol extractables is the group ofsubstances to which GL-3, GL-5, and GL-6 belong. The largenumber of bands shown for F. americana in Figure 2 suggeststhe presence of a series of related compounds, but since GL-3,GL-5, and GL-6 are the only representatives for which extinctioncoefficients are available we cannot give an estimate of the total.The data in Table I indicate that the three isolated compoundsaccount for almost 10%o of the dry weight. When this value iscompared to that obtained for sucrose, for the water-solublepolysaccharides, and for the various structural carbohydrates, it is

obvious that the ultraviolet-absorbing glucosides are the majorcarbohydrate-containing constituents.

Effects of Physiological State on Concentration of GL-3, GL-5,GL-6, and the Simple Sugars. The occurrence of such largeamounts of the ultraviolet-absorbing glucosides in the dry seedsmade it desirable to determine whether the concentration of thesecompounds is affected by changes in the physiological state of theembryo. These studies were conducted with F. americana seeds.Table II shows the effects of low temperature after-ripeningtreatments on the glucosides and simple sugars. After 3 monthsof storage at 5 C little if any radicle protrusion is observed, butexcised embryos are fully germinable. Comparison of endospermand embryos from dormant and after-ripened seeds shows littlechange in the concentration of the glucosides during after-ripen-ing. Possibly the level of GL-3 decreases during cold treatment,but this effect is not statistically significant at the 90% confidencelevel. If, however, the seed is kept at 5 C with moisture for 12months, the embryos grow quite extensively and the endospermis essentially absorbed. In these seedlings the concentration ofGL-5 does not change much, but GL-3 and GL-6 decrease to very

low levels. On the other hand, the concentrations of the sugars

increased greatly, with the largest changes occurring in the glu-cose and fructose levels (Table II). These seedlings are also theonly specimens in which we have been able to detect starch andmannitol.

In Table III are listed data on the changes in the concentrationof the three glucosides and simple sugars during 10-day develop-ment of excised embryos. A number of trends become apparent,particularly if the content of glucosides is compared before andafter germination. When dormant embryos are incubated for 10days, there is little germination, the fresh weight increase is low,and the concentration of the three glucosides does not changesignificantly from that of freshly excised dormant embryos.However, the same treatment of embryos, excised from non-

dormant seeds, leads to high germination rates, large fresh weightincrease, and a very significant decrease in the levels of GL-3 andGL-6. GA3 is effective in breaking dormancy of embryos excisedfrom dormant seeds (9), and the data in Table III show that theseembryos also lose GL-3 and GL-6 during a 10-day growth period.

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METABOLICALLY ACTIVE GLUCOSIDES

Table II. Effect of Cold Temperature After-ripeninzg on Concentrationt of GL-3, GL-5, GL-6, and the Simple Sugarsin F. americana Seeds

Storage Conditions of Seeds Physiological State Tissue Extracted GL-3 GL-5 GL-6 Sucrose Glucose Fructose

% dry wt % dry w

Dry, no cold temperature Dormant seed Endosperm 5.9 ± 0.7 1.6 ± 0.2 1.5 ± 0.1 0.9 0.15 <.02after-ripening Embryo 7.3 + 1.0 1.7 + 0.3 2.0 ± 0.2 1.1 0.20 <.02

3 months at 5 C with mois- Nondormant Endosperm 4.5 4 0.9 1.1 ± 0.2 1.2 ± 0.4 0.6 0.07 0.02ture seed Embryo 5.5 -+ 1.2 2.0 ± 0.5 2.4 i 1.0 0.7 0.02 <.02

12monthsat5Cwithmois- Etiolatedseed- Entire <.02 1.4 <0.2 2.4 10.2 4.9ture ling seedling

Table III. Chaanges in GL-3, GL-5, GL-6, and the Simple Sugars in F. americana Embryos durinzg hzicubationi

Glucoside Levels' Sugar LevelsTreatment' Fresh Wt E10Increase

GL-3 GL-5 GL-6 Sucrose Glucose Fructose

°%o °%0 dry wt % dry wt

Dormant embryosNone 60 40 6.5 + 0.5 2.0 ± 0.2 2.0 i 0.1 1.2 0.3 0.110- M GA3 210 420 3.0 ± 0.4 2.4 ± 0.3 <0.2 0.6 0.3 0.22 X 105M ABA 10 0 88.0 ± .3 2.2 0.2 2.3 + 0.5 ... ...

Nondormant embryosNone 230 500 3.1 4 1.0 3.1 i 0.5 <0.2 0.8 0.7 0.510-5 M GA3 290 720 1.9 ± 0.6 2.4 i 0.7 <0.2 0.4 0.4 0.22 X 10-5M ABA 13 0 10.3 2.4 2.8 0.4 0.2 <0.02

1 Embryos were incubated for 10 days in Petri dishes on Whatman No. 1 filter paper in test solutions. Ten embryos were used perdish. The incubator was on a 12-hr light cycle, 300 ft-c, 22C day temperature, 16C night temperature.

2 The glucoside level of the dormant embryos before incubation was: GL-3, 7.3 ± 1.0; GL-5, 1.7 ±- 0.3; GL-6, 2.0 ± 0.2. The levelin the nondormant embryos was: GL-3, 5.5 ± 1.2; GL-5, 2.0 ± 0.5; GL-6, 2.4 + 1.0.

The converse holds, too. The addition of (R,S)-ABA at a con-centration at which it interferes with germination of nondormantembryos will also lead to retention ofGL-3 and GL-6 (Table III).GL-5 behaves quite differently from GL-3 and GL-6 in that itsconcentration is much less effected by changes in the physiologi-cal state of the seed (Tables II and III).

DISCUSSION

The glucosides, GL-3, GL-5, and GL-6, described here aremajor constituents of seeds belonging to the Oleaceae family.They are nonfluorescing, ultraviolet-absorbing, low molecularweight compounds. All three have pronounced absorption max-ima below 240 nm, and GL-6 has an additional small peak near280 nm. These characteristics indicate that aromatic chromo-phores either are absent or have such low extinction coefficientsthat they make only minor contributions to the total absorption.From our present knowledge, it appears likely that these are un-reported compounds and differ from the types of substancespreviously isolated from Fraxinus, Syringa, and Olea.A series of papers by Lisitsyn on the carbohydrate metabolism

of ash and lilac have a bearing on our results. Lisitsyn, who didnot use chromatographic procedures, found what he called "acid-labile" glycosides in F. excelsior seeds (7), in leaves of this species(6), and in Syringa vulgaris leaves (6). He defined acid-labileglycosides as compounds that release glucose or fructose duringhydrolysis for 5 min with 2%7 hydrochloric acid at 70 C. Thisauthor's data indicate that roughly half of the total glucose, about6%, is in the acid-labile fraction. There is a high probability thatthe acid-labile glycoside fraction contains some of the compounds

described here since glucose is also released from GL-3 underextremely mild acidic conditions.

Lisitsyn claimed that starch is absent not only from Fraxinusseeds but also from leaves in this species and Syringa. We alsofound no starch in resting seeds or in embryos held for 10 daysat a 22 C/16 C temperature cycle. However, we did detect starchin seeds that had germinated at 5 C, and Gates and Simpson (4)reported starch in Syringa leaves. From these results we concludethat, while starch can be found in this family, it does not play aprominent role in germination and early development of F. ameri-cana embryos.The high percentage of lipids and low levels of nutritionally

important polysaccharides indicate that the primary carbonsource required for germination and growth of F. americanaembryos is the lipid fraction. What the role of the ultraviolet-absorbing glucosides is in the over-all economy of the seeds isstill uncertain. These compounds are present in high concentra-tions in the dry seeds and show a complex pattern of changes as aresult of germination and growth. On a percentage basis, the GL-6levels are affected the most and GL-5 the least. The origin of theultraviolet-absorbing compounds formed during growth is notknown, but it is possible that these substances are conversionproducts of GL-3 and GL-6. The chromatographic patterns ob-tained when germination is induced either by GA3 or by coldtemperature after-ripening are very similar, and the addition ofABA to nondormant embryos causes them to behave similarlyto dormant specimens. We, therefore, propose that GL-3 andGL-6 fulfill definite functions in the germination or growth orboth of F. americana embryos, and that GA3 and ABA, either

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662 SONDHEIMER ET AL.

directly or indirectly, can exert a regulatory effect on theirmetabolism.

Acknowledgments-This study was supported by United States Department ofAgriculture Forest Service Grant 3-4040 and National Science Foundation GrantGB4262

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5. JULIANO, B. 0. AND J. E. VARNER. 1969. Enzymic degradation of starch granulesin the cotyledons of germinating peas. Plant Physiol. 44: 886-892.

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6. LIsITsYN, D. 1. 1952. The role of labile glucosides in plants. Biokhimiya 17: 320-328; Chem. Abstr. 46: 10310 (1952).

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