the composition of stigmatic exudate from lilium longiflorumcarbohydrate components of stigmatic...

Plant Physiol. (1970) 46, 150-156

The Composition of Stigmatic Exudate from Lilium longiflorumLABELING STUDIES WITH MYO-INOSITOL, D-GLUCOSE, AND L-PROLINEI

Received for publication February 4, 1970

C. LABARCA, M. KROH,2 AND F. LOEWUSDepartment of Biology, State University ofNew

ABSTRACT

Stigmatic exudate, a secretion product recovered fromthe upper surface of Lilium longiflorum pistils, has beenexamined. Over 99% of the exudate is accounted for as water,carbohydrate, and protein. Exclusive of water, 95% is a highmolecular weight, protein-containing polysaccharide com-posed of galactose, arabinose, rhamnose, glucuronic acid,and galacturonic acid.Detached pistils supplied with myo-inositol-U-14C, myo-

inositol-2-3H, D-glucose-1-14C, or L-proline-U-14C producelabeled stigmatic exudate. When myo-inositol is supplied,the exudate is rich in labeled arabinose and uronic acids,but some label also recycles through the hexose phosphatepool of secreting cells, causing label to appear in galactoseand rhamnose residues. When glucose is provided, galactoseis the major constituent labeled but all of the other carbo-hydrate constituents are also labeled. Proline produces apattern very similar to that obtained with glucose.Stigmatic exudate also contains a small amount of low

molecular weight carbohydrate. If myo-inositol is used tolabel exudate, free labeled myo-inositol cannot be detectedin the low molecular weight fraction until it has been sub-jected to acid hydrolysis. Similarly, if D-glucose is thesource of label, free labeled glucose is found in the lowmolecular weight fraction only after acid hydrolysis.

York at Buffalo, Buffalo, New York 14214

and penetration of pollen tubes into Cruciferae stigmas. Theyfound that pollen tubes of cross-pollinated, self-incompatiblespecies grew inside the cellulose-pectin layer of cell walls of epi-dermal papillae. They suggested that growing pollen tube tipssecrete enzymes which dissolve pectic substances in the transmit-ting tissue of the style, and that the products of this process areutilized by the pollen tube for tube wall formation. Kanno andHinato (6) have also reported on the role of self-incompatibilityin pollen development. They regarded the cellulose-pectin layerrather than cuticle to be the final barrier to tube growth in self-pollinated, incompatible stigmas.Rosen and Gawlik (29) have suggested, from electron micro-

scopic studies, that style secretion product of Lilium longiflorumplays a nutritional role in pollen tube development in compatiblepistils.

Studies by Kroh and Loewus (13) on germination and growthof L. longiflorum pollen in artificial medium containing myo-inositol-2-14C showed that this cycitol is used by developingpollen tubes for biosynthesis of pectic substance. When myo-inositol-U-14C or -2-3H was given to detached pistils of L. Iongi-florwn, a portion of the label accumulated in stigmatic exudate(14). When these pistils were self- or cross-pollinated, the pollentubes that developed and penetrated the labeled exudate werefound to contain radioactive pectin (15). More recently, Krohet al. (12) have succeeded in demonstrating direct utilization ofcarbohydrate components of stigmatic exudate for pollen-tubewall formation. The present report concerns the composition ofstigmatic exudate recovered from plant-attached pistils or fromdetached pistils which have been labeled with myo-inositol-U-'4C,myo-inositol-2-3H, D-glucose-1-14C, or L-proline-U-14C.

During pollen germination and growth, the delicate pollentube becomes embedded in a sheath of exudate or intercellularsubstance while penetrating the style of the pistil. This sheath ispoorly understood regarding both composition and function.Recently, Konar and Linskens (10) reviewed the literature relatedto stigmatic exudate. They found no consistent pattern for thecomposition of stigmatic exudate among various plant species.Moreover, it was evident to them that very little specific informa-tion had been accumulated in earlier studies. In their own workwith Petunia (9, 10), Konar and Linskens showed that stignaticfluid from this plant lacked free water and consisted primarily ofan oil with smaller quantities of amino acids. No protein was de-tected.Kroh (11) and Kroh and Munting (16) have examined growth

1 This investigation was supported in part by a grant (GM-12422)from the National Institutes of Health, United States Public HealthService, and by postdoctoral support to C. L. from the GraduateSchool, State University of New York at Buffalo.

2Present address: Department of Botany, University of Nijmegen,Nijmegen, The Netherlands.

MATERLIL AND METHODS

Unlabeled exudate was collected periodically from stigma sur-faces of flowers ofLilium longiflorum (cv. Ace). Plants were grownunder greenhouse conditions.Labeled exudate was recovered from detached pistils according

to the procedure used in a previous study (14). About 2 hr wererequired for uptake of radioactive solution (0.1 ml) by each pistil(tepals and stamens removed). Over the next 5 to 7 hr, small in-crements of water were added to the vial in which the cut surfaceof the pistil was immersed. Subsequently, a larger volume ofwater was provided to keep the cut surface immersed, and thiswater was replaced daily. Labeled, detached pistils were main-tained in continuous light. With conditions outlined here, sig-nificant amounts of radioactivity appeared in stigmatic exudatewithin 24 hr and continued to be produced for 4 or 5 days. Exu-date was collected with disposable plastic micropipettes three orfour times each day. Exudate was pooled and stored at -20 C.

All ratioactive compounds used in this study were examinedchromatographically to establish radiopurity. myo-Inositol-U-"4C (12.5 c/mole) was purchased from Amersham/Searle. myo-

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STIGMATIC EXUDATE FROM LILIUM

Inositol-2-3H (5 c/mole) was prepared according to Reymond(27). D-Glucose-1-14C (1.4 c/mole) was prepared according toHers et al. (5). L-Proline-U-14C (174 c/mole) was purchased fromNew England Nuclear Corporation.

Gel Filtration. The exudate was diluted 3- to 5-fold with 0.1 Macetic acid and was placed on a column of Sephadex G-100(110 x 1 cm, 1-2 ml per run). Elution was run with 0.1 M aceticacid, and fractions of 1 ml were collected at the rate of 3 ml/hr.The high molecular weight fraction from Sephadex G-100 waslyophilyzed, redissolved in 0.1 M acetic acid (1-2 ml), and furtherfractionated on a column of Sephadex G-200 (210 x 1 cm). Thelow molecular weight fraction from Sephadex G-100 was furtherfractionated on Sephadex G-10 (100 X 1 cm) following the pro-cedure just outlined. Fractions were analyzed for total carbohy-drate (4), protein (23), and radioactivity. Fractions within eachradioactive peak were pooled, lyophilyzed and stored at -20 C.

Acid Hydrolysis. Exudate and its fractions were hydrolyzedwith 2 M trifluoroacetic acid (120 C, 0.5 hr, sealed tube) except asindicated in results. Products were separated by descending paperor thin layer chromatography in (a) ethyl acetate-pyridine-water(8:2:1, v/v), (b) ethyl acetate-pyridine-water (10:6:5, v/v), (c)acetone-water (4:1, v/v), (d) n-butanol-acetic acid-water(63:27:10, v/v), and (e) ethyl acetate-water-acetic acid-formicacid (18:4:3:1, v/v). Acidic components were also separated bypaper electrophoresis in 0.1 M ammonium formate (pH 3.8, 20v/cm, 3 hr). Arabinose was measured quantitatively with orcinolreagent (25), galactose with Galactostat (Worthington Biochem-ical Corp.), and uronic acids with carbazole reagent as modifiedby Knutson and Jeanes (7, 8).Enzymatic Hydrolysis. Exudate fractions were incubated in

0.1 M sodium phosphate (pH 6.5) with Pronase (10 mg, volume1 ml, 37 C, 20 hr). After inactivating the Pronase (10 min, 100 C)and after cooling, Pectinol R-10 (10 mg) and disodium EDTA(1 mg) were added, and the incubation was continued for another6 hr at 37 C. Pectinol was then inactivated (10 min,'100 C), andthe solution was centrifuged. The precipitate was discarded, andresidual protein in the supernatant solution was removed bypassage through a Dowex-50 H+ ion exchange column. The ef-fluent was then loaded on a column of Dowex-1 exchange resin(formate form, 15 x 1 cm, 200 to 400-mesh, 8% cross-linked).Neutral components were recovered from the effluent and sub-sequent water washes of the column. Acidic components wereeluted with two successive formic acid gradients (0-0.1 and 0.1-3 M). Neutral and acidic fractions were further separated by paperchromatography and paper electrophoresis as described above.Measurement of Radioactive Compounds. Radioactive arabi-

nose was diluted with L-arabinose as carrier, oxidized to arabi-nonic acid, and recovered as crystalline potassium arabinonate(3). Radioactive galactose was diluted with D-galactose as carrier,reduced with sodium borohydride to galactitol, and crystallizedfrom aqueous ethanol. Radioactive rhamnose was diluted with L-rhamnose as carrier and recrystallized as the free sugar. Sampleswere dissolved or dispersed in a liquid scintillation-countingmixture of dioxane-naphthalene (18) and were counted in anautomatic liquid scintillation spectrometer at 80% efficiency for14C and 23% for 3H. Paper chromatograms, thin layer strips andpherograms were scanned for radioactivity at an efficiency of20% for 14C and 1% for 3H.

Gas-Liquid Chromatography. Samples to be separated by gas-liquid chromatography were first converted to trimethylsilylethers (32) and were then injected into a column consisting of 3%silicone polymer (cyanoethyl methyl, and dimethyl), XE-60,on Supelcoport (6 ft by 4 mm internal diameter) with N2 ascarrier gas (42 mg/min, 15 p.s.i. backpressure). Components inthe trimethylsilylated mixture were detected by flame ionization.Each injection was measured isothermally at 175 C or a tempera-ture programmed from 130 to 180 C at 3°/min. Radioactive

mixtures were monitored for 'IC by splitting the effluent gases asthey emerged from the column and by trapping portions of ap-propriate peak fractions in chilled glass tubes containing glasswool moistened with n-heptane. The tubes were then transferredto vials containing dioxane-naphthalene counting mixture andassayed for radioactivity.

RESULTS

Fractionation of Unlabeled Exudate. As recovered from thestigma, whole exudate is an aqueous, viscous fluid that contains9%, w/v, solids of which 85 to 90% is carbohydrate and 7% isprotein. When this exudate was fractionated on Sephadex G-100,about 95% of the solids were recovered as carbohydrate andprotein in a single peak of high molecular weight materialidentified as G-100-I in Figure 1A. Two minor Lowry-reactive

G-100-IW ~~~~~~A

s 20 400

M ai0LL0-1

0~~~~~~~~

-J20do ~~~~~~~~~~~G-;00-H0

G-l00-I

BCO

Iz

G100I~~~~G10-1

00

40 60 80

VOLUME OF ELUENT ("L)FIG. 1. Gel filtration of stigmatic exudate through Sephadex G-l00.

A: Unlabeled exudate. Carbohydrate (solid line) and protein (dashedlinle) profiles. B: Labeled exudate. Profiles of radioactivity from pistilslabeled with myo-inositol-U-'4C (solid line) and myo-inositol-2-3H(dashed line). C: Labeled exudate. Profiles of radioactivity from pistilslabeled with D-glucose-1-'4C (solid line) and L-prohine-U-"4C (dashedline).

151Plant Physiol. Vol. 46, 1970

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LABARCA, KROH, AND LOEWUS

~~ ~ ~ ~ ~ ~ ~ ~ ~ 0I-.~~~~~~~~~~~~~~~~~~~~~~~~~~~~L

throug SehdxG202:Unaee0-0-rato.Croyrt

0~~~~~~~~~~~~~~~~~

(solid l) aG-200-I

frcion.2Prolfilesofrationactivity fracpitionLable stighmyo-icnosidtel

U-14C (solid line) and myo-inositol-2-3H (dashed line). C: LabeledG-100-I fraction. Profile of radioactivity from a pistil labeled withL-proline-U-14C.

peaks were retarded by the gel. The slowest, G-100-11, also in-cluded phenol-sulfuric acid-reactive material that amounted toabout 1 to 2% of the total carbohydrate.When further fractionated on Sephadex G-200, G-100-I could

beseparatedintoa peak,G-200-1, thatcontainedabout 15% ofthecarbohydrate and 30% of the protein followed by an unresolvedmnixture of carbohydrate-rich compounds of decreasing molecu-lar weight (Fig. 2A). This latter mixture showed very little varia-tion in monosaccharide composition when fractions in zones a, b,and c were pooled separately, hydrolyzed, and compared on thebasis of monosaccharide products by paper chromatography insolvent A.Paper chromatography of a portion of G-100-I after acid hy-

drolysis indicated that it was a polysaccharide composed ofuronic acids, galactose, arabinose, and rhamnose. Colorimetricassays of G-100-1 gave the composition shown in Table I. Thepresence of both glucuronic and galacturonic acids was confirmedby eluting a portion of the uronic acid region from the pDaper

chromatograph and then reducing the acids with sodiumborohydride. Gulonic and galactonic acids produced by thisreduction were converted to the corresponding lactones,treated with trimethylsilylating reagent and analyzed by gas-liquid chromatography. By this method, the ratio of glucuronicacid to galacturonic acid was 7:1, somewhat higher than theratio of 4:1 obtained by colorimetric analysis of G-100-I by theprocedure of Knutson and Jeanes (7, 8).

Gas-liquid chromatography of acid-hydrolyzed G-100-I, bothbefore and after reduction with sodium borohydride, confirmedthe presence of galactose, arabinose, and rhamnose as the threemajor sugar residues present. These results indicated a muchhigher ratio of galactose to arabinose plus rhamnose (1.9 to 1)than data obtained by colorimetric assay of unhydrolyzedG-100-I. Gas-liquid chromatography also established the absenceof glucose and mannose as major constituents and indicated thatxylose and fucose, if present, must be less than 5% of the totalcarbohydrate.

G-100-11, the low molecular weight mixture recovered by gelfiltration, was separated by paper chromatography in solvent Ainto two alkaline silver nitrate-reactive spots, one at the originand the other as an elongated streak with approximately the sameRF as galactose. On acid hydrolysis, a single spot, coincident withgalactose, was obtained. Positive identification as galactose wasnot obtained because of the limited amount of material available.Unhydrolyzed G-100-I1 had an absorption peak at 250 to 255nm.

Distribution of Radioactivity in Labeled Exudate. Radioactivityrecovered as labeled exudate over a 5-day period after feedinglabeled precursor to a detached pistil is given in Table II. It aver-

Table I. Composition of G-JOO-I Fraction of Stigmatic Exudateafter Acid Hydrolysis

With the exception of rhamnose, all carbohydrates listed herewere determined by colorimetric analysis of whole G-100-I frac-tion. Rhamnose was determined as its trimethylsilyl ether bygas-liquid chromatography after acid hydrolysis of G-100-I frac-tion and separation of hydrolysis products by paper chromatog-raphy in solvent A. Values reported under "Amount" are per-centage of dry weight in whole exudate.

Component Amount

Glucuronic acid 9.5Galacturonic acid 2.5Galactose 30Arabinose 28Rhamnose 12.5Total carbohydrate 82.5Protein 6.6

Table II. Recovery of Label in Stigmatic Exudate

Sourceof Amount of Isotope Isotope RecoveredSource of Label Supplied to Pistil in Exudate

.c %myo-Inositol-U-14C 10 5.5

12.5 5.912.5 8.012.5 3.9

myo-Inositol-2-3H 27 1.0D-Glucose-1-'4C 18 5.0L-Proline-U-14C 10 1.1

Plant Physiol. Vol. 46, 1970152




Table III. Distribution of Radioactivity between High (G-100-1)anid Low (G-JOO-II) Molecular Weight Fractions of Stigmatic

Exudate

Source of Label G-100-I G-100-II RadioactivityRecovered

myo-Inositol-U-14C 86 13 99'71 22 93

myo-Inositol-2-3H 35 25 60D-Glucose-1-l4C 76 12 88L-Proline-U-'4C 84 4 88

1 Losses due to adsorption on Sephadex gel were reduced bycombining labeled exudate with unlabeled exudate (3: 1).

600

z

w

> ~~~~Wu

~ ~ ~ ~ ~ 3

0CL~ ~

tX 200 /U

0 50 00 150VOLUME OF ELUENT

[ML]

FIG. 3. Gradient elution on anionic exchange gel, Sephadex A-25,of G-100-I fraction from a pistil labeled with D-glucose-1-14C. Detailsare given in the text.

aged 5 %o of label supplied in the case of myo-inositol-U-'4C andD-glucose-1-'4C, results which are similar to values obtained pre-viously (14), but only 1 % in the case of myo-inositol-2-3H andL-proline-U-14C. Distribution of radioactivity between peaksG-100-I and G-100-II for each labeled exudate is shown in Figure1,B and C. No radioactivity appeared in the small intermediateprotein peak between G-100-I and G-100-II, even when L-proline-U-14C was supplied to a pistil. Radioactivity recovered from frac-tions under G-100-I and -II is listed in Table III. With oneexception, most of the radioactivity (71-86%o) in the exudate ap-peared in G-100-I. The poor recovery obtained in fractions fromexudate labeled with myo-inositol-2-3H was not examined further.Label not recovered in G-100-I and -II probably was due to lossesby adsorption on Sephadex particles.

Distribution of Radioactivity in G-100-I. Fractionation oflabeled G-100-I on Sephadex G-200 gave results shown in Figure2, B and C. Approximately 15 to 20% of the radioactivity was ex-cluded from the gel. The rest was retarded and was eluted as asecond peak of lower molecular weight compounds. Insufficientmaterial was available for protein assays but comparison withFigure 2A indicated that most protein was associated with labelthat appeared in the excluded volume. Significantly, L-proline-U-14C labeled G-100-I had a Sephadex G-200 profile similar tothat from myo-inositol labeled exudate.The acidic nature of the entire G-100-I fraction was demon-

strated by placing a sample (45,000 cpm) of G-100-I from D-glucose-1-14C labeled exudate on a column of Sephadex A-25anionic exchange gel (14 x 1 cm). No "4C appeared in the effluentor subsequent water wash. When the column was eluted with a

formic acid gradient (250 ml of 4 M formic acid flowing through amixing chamber that initially contained 250 ml of water), 85% ofthe 'IC appeared in a single peak between 44 and 160 ml (Fig. 3).The remaining 15% was not eluted by this gradient.

Acid-hydrolyzed G-100-I fractions from myo-inositol, D-glu-cose, and L-proline labeled exudates gave paper chromatograms(solvent A) with radioactivity at the origin and in regions corre-sponding to galactose, arabinose, and rhamnose. Traces of radio-activity were also found in areas coinciding with mannose andxylose (= fucose) when glucose and proline were used as sourcesof label. G-100-I hydrolysates from exudates labeled with myo-inositol and glucose contained labeled components that moved ator near the solvent front and this radioactivity was recovered ineluted solvent. Acidic components that remained at the origin ofthe chromatogram were separated by paper electrophoresis. Re-sults are gathered in Table IV.

Paper electrophoresis of acidic components in acid-hydrolyzedG-100-I produced three radioactive spots, a slowly migratinggroup of acidic oligosaccharides, an intermediate spot coincidentwith galacturonic acid (but also containing acidic oligosaccha-rides), and a spot corresponding exactly with glucuronic acid.If the same acidic mixture was separated on paper with solvent E,a system that resolves glucuronic acid and 4-0-methyl glucuronicacid, three radioactive peaks were obtained in a radiochromato-gram scan, of which the fastest was a mixture of glucuronic andgalacturonic acids. The latter two are not resolved by this solventsystem (28). No radioactivity was detected in the region of 4-0-methyl glucuronic acid (Rglucuronic acid =2.0). The two slowermoving spots were identified as acidic oligosaccharides. Asjudged by '4C recovered in the fastest moving spot, about 15 to20% of the acidic components in the hydrolysate was free uronicacid.

Free uronic acid from acid-hydrolyzed G-100-I of myo-inositol-U-1'C labeled exudate was separated from other hydrolysis frag-ments with the system described above. This mixture of glucu-ronic acid and galacturonic acid was reduced with sodium boro-hydride, converted to lactones, and treated with trimethylsilylating

Table IV. Distributioni of Radioactivity among Components ofAcid-hydrolyzed G-IOO-I Fraction after Chromatography in

Solventt A

Distribution of Isotope in G-100-I(headings indicate source of label)

Component75yo)- myc- n-Glucose- L-Proline-

Inositol- Inositol- 1-14C U-14CineU-14C 2-3

Origin'Peak 1 4.9 5.1 2.3 4.8Peak 2 8.6 7.7 7.2 9.8Peak 3 3.5 2.9 2.3 2.6Total 19.0 15.7 11.8 17.2

Mannose <1 1.9 1.0 1.5Galactose 16.2 4.9 55.6 53.5Arabinose 53.2 68.7 17.3 21.3Xylose (= fucose) <1 2.6 <1 1.5Rhamnose 3.1 1.0 5.1 4.9Unknowns (eluted)' 8.9 5.2 8.5 1.0

Separated by paper electrophoresis. Peak 1 is a mixture ofacidic oligosaccharides. Peak 2 is a mixture of acidic oligosac-charide and galacturonic acid. Peak 3 is glucuronic acid. Proline-labeled G-100-I had labeled proline in peaks 1 and 2.

2 Radioactivity eluted during chromatography and recoveredin the solvent.

Plant Physiol. Vol. 46, 1970 153




reagent. Analysis by gas-liquid chromatography showed thatthe major components present were tetrakis-O-trimethylsilyl-galactono-1,4-lactone and tetrakis-O-trimethylsilyl-gulono-1,4-lactone in the ratio of 1:8. The radioactivity of these two com-pounds, measured at the same time, was in the ratio of 1: 4. Thus,both galacturonic acid and glucuronic acid were present in theuronic acid spot, both were labeled, and glucuronic acid was themajor acid present, in amount and in content of 14C.

Pistils fed L-proline-U-"4C produced exudate in which radio-active proline was present as such in the G-100-I fraction. Whenthis fraction was hydrolyzed in strong acid (6 N HCl, 18 hr, 105 C,sealed tube) and then separated by thin layer chromatography oncellulose in solvent D (26), most of the radioactivity appeared ina streak just behind the solvent front; however, about 5% ap-peared as a compact spot (RF = 0.52) that coincided with theproline control spot. No 14C was found in hydroxyproline (RF =0.25).

In G-100-I from myo-inositol labeled exudate, more than one-half of the radioactivity appeared in arabinose. Some 14C alsoappeared in galactose and rhamnose, an indication that somemetabolic recycling of label had preceded secretion of exudate.The extent of this recycling was greater than that encountered inprevious studies with other plant tissues (21), and deservesfurther investigation.

In G-200-I from glucose- and proline-labeled exudates, galac-tose was the major radioactive constituent. Glucose residues werenot detected in acid-hydrolyzed G-100-I, labeled or unlabeled.Gas-liquid chromatography of unlabeled acid-hydrolyzed G-100-Ialso failed to reveal the presence of glucose residues.Enzymatic Hydrolysis of G-100-I from myo-Inositol-U-'4C

Labeled Exudate. Previous work with labeled pistils (14) has un-covered the observation that a commercial pectinase (PectinolR-10) commonly used in this laboratory lost its ability to hy-drolyze pectic substance to monosaccharides in ethanol-extractedresidues of pistil when the enzyme mixture was stored at 5 C forprolonged periods of time (in excess of 1 year). Pretreatment ofsuch residues with Pronase gave a product that could now behydrolyzed by pectinase all the way to monosaccharides. Thisprocedure was used to hydrolyze G-100-I from myo-inositol-U-14C labeled exudate. After incubation with Pronase, the reactionmixture was heated to destroy the enzyme and was reincubatedwith pectinase. Products were separated by ion exchange chro-matography. Of the "4C added to the column, 40% appeared inneutral compounds in the effluent. Paper chromatography re-vealed the following radioactive distribution: arabinose, 29%;galactose, 2%; rhamnose, <1%; and unidentified oligosaccha-rides, 9%.Another 40% of the 14C on the column was eluted with formic

acid. Two concentration gradients were used. The dilute gradient(to 0.1 N acid) produced only two small radioactive peaks ac-counting for 3% of the 14C. A more extended gradient (0.1-3 Nacid) released a single radioactive peak that contained 36% of the"C. Hydrolysis of the latter (2 N HCI, 0.5 hr, 105 C, sealed tube)followed by paper electrophoresis of products revealed threeradioactive spots, one close to the point of sample application,another coinciding with galacturonic acid, and a faster spot cor-responding to glucuronic acid. Paper chromatography of a por-tion of the same hydrolysis mixture in solvent A followed byradioactive scanning revealed equivalent amounts of 14C inarabinose and galactose plus a substantial amount of acidic la-beled material that remained at the origin. Results indicated thatenzymatic digestion had stripped off a substantial portion of thearabinosyl groups, leaving an acidic core rich in galactose. Acidhydrolysis removed additional arabinose, galactose, and uronicacid residues.

Distribution of Radioactivfty in Labeled G-100-II. Gel filtra-tion on Sephadex G-10 of G-100-II from myo-inositol-U-"C

labeled exudate produced a single irregular radioactive peak be-tween 42 and 60 ml on a column in which the void volume ap-peared at 32 ml. On the same column, a mixture of authenticgalacturonic acid and arabinose appeared, unresolved, between51 and 56 ml. G-100-II was not resolved with any of the solventsystems used for paper chromatography in this study. There wasno evidence of free myo-inositol, labeled or unlabeled; however,after acid hydrolysis of G-100-II in 2 M trifluoroacetic acid, la-beled myo-inositol was recovered by chromatography. Positiveidentification was made by gas-liquid chromatography (3% OV-1(methyl silicone) on GasChrom Q, 6 ft by 4 mm, 200 C, N2 ascarrier, flame ionization detector, 7:1 split of effluent gases).For this identification, a portion of myo-inositol recovered bythin layer chromatography in solvent C was converted to itstrimethylsilyl ether. Aliquots of the trimethylsilylation mixturewere injected directly on the gas-liquid column. The '4C wasrecovered exclusively in the retention volume corresponding tohexakis-O-trimethylsilyl-myo-inositol.

In a separate experiment, about 18% of the '4C in acid-hy-drolyzed G-100-II was recovered in myo-inositol by carrier dilu-tion. This was equivalent to about 4% of the total '4C in unfrac-tionated exudate.

G-100-II from exudate labeled with D-glucose-1-'4C wasroughly resolved into three radioactive spots (Rglucose = 0.13,0.36, and 0.80) by paper chromatography with solvent A. Themost rapid spot coincided with galactose. After acid hydrolysis,most of the radioactivity appeared in a single peak correspondingto glucose and accompanied by a small, trailing shoulder corre-sponding to galactose.

DISCUSSION

The bulk of organic compounds present in stigmatic exudatefrom L. longiflorum is carbohydrate, and of this, roughly 95% ispolysaccharide. Only about 2% is present as carbohydrate com-pounds of less than 1000 daltons. Of whole exudate, over 90% isaccounted for as water, carbohydrate, and protein. Gel filtrationprovides a convenient and effective procedure for separating thepolysaccharide-protein component from low molecular weightcompounds. Analysis of acid-hydrolyzed polysaccharide fromstigmatic exudate indicates that it is closely related to the generalclass of acidic polysaccharides usually identified as exudate poly-saccharides and complex pectic substances (2). The compositionof stigmatic exudate in Tables I and IV is remarkably similarbdata obtained from other exudate polysaccharides (1, 31). Thehigh ratio of glucuronic acid to galacturonic acid rules out simplepectic substance unless stigmatic exudate consists of a mixture ofacidic polysaccharides, some quite pectic in composition, othersextremely rich in glucuronic acid. Significantly, 4-0-methyl glucu-ronic acid, a component of acidic polysaccharides in cell walls ofmany plant species, is not found in stigmatic exudate.

Results obtained in this study favor the idea of a complex exu-date polysaccharide that contains both galacturonic and glucu-ronic acids. Thus, sequential hydrolysis with Pronase and Pecti-nol R-10 releases most of the arabinose as free sugar but leaves alarge fragment of acidic polysaccharide rich in galacturonic acid,glucuronic acid, arabinose, galactose, and rhamnose. Acid hy-drolysis of this fragment releases most of the arabinose, galactose,and glucuronic acid as monosaccharide residues along with smallacidic fragments, possibly aldobiouronic acids rich in galactui-ronic acid, rhamnose, and galactose. Tentatively, one may con-clude that the high molecular weight component of stigmaticexudate is a heteropolysaccharide with an acidic main chain ofgalacturonic acid residues, possibly interrupted at intervals byrhamnose, with side chains composed of galactose, arabinose, andglucuronic acid. The ease with which arabinose residues are re-

moved by acid may indicate that these residues are in the fura-

154 Plant Physiol. Vol. 46, 1970




nosyl configuration. It may also indicate that these residues arepresent as a highly branched structure external to galactosyl sidechains. It is unlikely that arabinogalactan is present inasmuch asall of the G-100-I is retained on Sephadex A-25 (an anionic ex-change gel). Since G-100-I fraction contains protein tightly boundto polysaccharide and since the pattern of carbohydrate and pro-tein distribution in 45%7O phenol is toward the aqueous phase (20),it is quite possible that much of the stigmatic exudate consists ofglycoprotein. Additional work is necessary to clarify this aspect.

Exudate Polysaccharide from myo-Inositol Labeled Pistils. De-tached pistils placed in a solution of radioactive myo-inositoltransfer the label through vascular bundles to regions of activesecretion of stigmatic exudate, specifically those cells that line thesurface of the stigma and the style canal (14). Recently, Rosen hasexamined the fine structure of those cells (30). Much of the labelfrom myo-inositol that reaches those cells and is eventually con-verted to exudate appears in residues of arabinose, glucuronicacid, and galacturonic acid in the p:olysaccharide. However, somelabel also appears in galactose and rhamnose. Label in pentoseand uronic acid is anticipated on the basis of previous studieswhich showed that these residues arise from glucuronic acid, theprimary product of the oxidative cleavage of myo-inositol inplants (19-21). Its presence in galactose and rhamnose indicatesthat a portion recycles into hexose phosphate either by reentry ofpentose into pentose phosphate metabolism or by photosyntheticfixation of radioactive carbon dioxide which is released duringconversion of uronic acid to pentose. Conceivably, both possi-bilities contribute to the phenomenon. In pistils labeled withmyo-inositol-2-3H, only pentose phosphate can deliver label to thehexose pool (19). Since exudate polysaccharide recovered fromthese pistils contains only one-third as much label in galactoseand rhamnose relative to pentose as that from myo-inositol-U-14Clabeled pistils (Table IV), results favor a dual process.Exudate Polysaccharide from D-Glucose and L-Proline Labeled

Pistils. When glucose is supplied to pistils, the label appears in allcarbohydrate products in exudate, but galactose is most heavilylabeled (56%70). About 30% of the 14C is present in pentose anduronic acid residues as compared with over 70% when myo-inositol-U-14C is source of label. Results indicate that labeledglucose, or for that matter any labeled sugar, entering the hexosephosphate pool in the secreting cells of the pistil will contributelabel to exudate polysaccharides with the distribution pattern ob-tained here. This is borne out by the '4C pattern in G-100-I fromproline labeled pistils.When L-proline-U-14C is used as a source of label, the labeling

pattern of exudate resembles that obtained with D-glucose-1-"4C,although the amount of label incorporated is only one-fifth asgreat. Well over 80% of this label appears in neutral sugar resi-dues after acid hydrolysis. When one compares the relative per-centages of label from D-glucose-1-14C and L-proline-U-14C thatremain at the origin after paper chromatography (Table IV), thenthe latter has 5 or 6% more label, possibly amino acid or peptideresidues. This difference corresponds to the labeled L-proline re-covered after strong acid hydrolysis of high molecular weightG-100-I. Extensive conversion of proline to hydroxyproline, ashas been reported to occur in cell wall bound-protein of higherplants (17), is not observed. Although proline metabolism inplants is still not fully clarified (24), it is obvious from results pre-sented here that a portion of the label derived from this aminoacid does recirculate into carbohydrate products via hexose phos-phate, possibly by processes uniquely associated with cells re-sponsible for secretion of stigmatic exudate.

Functional Relations between Stigmatic Exudate and PollenTube Growth. Smith and Montgomery (31) have commented onthe functional role of exudate polysaccharides in wound-sealingprocesses in plants. It seems quite likely that stigmatic exudatewith its high content of acidic polysaccharide provides a cornm-

parable protection for fragile pollen tubes during germination andgrowth. Further, both the low and high molecular weight frac-tions (G-100-II and -I, respectively) of stigmatic exudate fromL. iongiflorum stimulate pollen germination and tube growth whenadded to suspensions of L. Iongiflorum p )llen grains in artificialmedia (22). The acidic polysaccharide portion of stigmatic exu-date also functions as a source of carbohydrate residues for tubewall biosynthesis during pollen germination and tube develop-ment, in vitro and in vivo (12).

Acknowledgments-The authors gratefully acknowledge the help provided by Mr.Thomas J. Arrigo of Arrigo Greenhouses, Buffalo, New York, in obtaining an ade-quate supply of stigmatic exudate for this study. They also thank Professor W. Rosenfor helpful discussions and Miss Anne Golebiewski for skillful assistance.

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the composition of stigmatic exudate from lilium longiflorumcarbohydrate components of stigmatic...

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