Phytic Acid Metabolismin Lily (Liliumlongiflorum Thunb.) Pollen'

Plant Physiol. (1987) 83, 408-4130032-0889/87/83/0408/06/$01.00/0

Phytic Acid Metabolism in Lily (Lilium longiflorum Thunb.)Pollen'

Received for publication May 8, 1986 and in revised form October 3, 1986

JIH-JING LIN2, DAVID B. DICKINSON*, AND TUAN-HUA DAVID HoDepartment ofHorticulture, University ofIllinois, 1301 W. Gregory Dr., Urbana, Illinois 61801 (J.-J.L.,D.B.D.); and Department ofBiology, Washington University, St. Louis, Missouri 63130 (T.-H.D.H.)

ABSTRACT

The accumulation of phytic acid during development of lily (Liliumlongiflorum Thunb.) pollen and its degradation during germination havebeen studied. A substantial amount of phytic acid accumulates in lilypollen by 5 days before anthesis, and little change occurs during subse-quent maturation. Mature lily pollen contains 7 to 8 micrograms phyticacid per milligram pollen. Considerable degradation of phytic acid occursby 15 minutes of incubation in glucose culture medium, and very little isleft by 3 hours. No partially phosphorylated myo-inositol accumulatesduring germination. The breakdown of phytic acid proceeds at a constantrate during this time period. The rate is calculated to be 0.037 microgramphytic acid/milligram pollen/minute. Two phytases are detected in ger-minated lily pollen extract using high performance liquid chromatographywith an anion exchange column (diethylaminoethyl-5PW). The resultssuggest that one of the phytases is already present in mature ungermi-nated lily pollen and the other one is newly synthesized during germina-tion from a long-lived, pre-existing mRNA.

Phytic acid, myo-inositol 1,2,3,4,5,6 hexakisphosphate, is ofwidespread occurrence in seeds (5). It appears to function as astorage form of phosphorus in seeds (4, 5), typically accountingfor from 50 to 80% ofthe mature seed's total phosphorus. Phyticacid has also been reported in roots and tubers (24), and inorganic soils (2). Recently, phytic acid has been identified inpollen of many plant species (16). Significant quantities (0.05-2.1% of dry weight) of phytic acid were observed in pollen fromplants with style lengths greater than 5 mm, while little or nophytic acid was found in pollen from composites and grasseswith very short styles. Helsper et al. ( 14) reported that the myo-inositol moiety released during phytic acid degradation in ger-minating petunia pollen is utilized for synthesis of phosphatidylinositol and pectic polysaccharide, both of which are needed inlarge amounts for pollen tube assembly as pollen tube elongationgets under way. Therefore, the degradation of phytic acid duringpollen germination is important because it not only supplies Pibut also replenishes the pool of myo-inositol. The rate of phyticacid mobilization in germinating petunia pollen was also re-ported to be controlled by incompatibility (17).

Phytic acid metabolism in seeds has been studied extensively

I Supported by National Science Foundation Grants 79-22686 to D.B. D. and DCB 83161319 to T.-H. D. H. and Project 65-341 of theAgricultural Experiment Station, College of Agriculture, University ofIllinois at Urbana-Champaign.

2 Present address: Department of Biology, Washington University, St.Louis, MO 63130.

(6); however, literature about phytic acid metabolism in pollenis very limited. The only information available so far is frompetunia pollen (14, 17, 18). Because of its importance as a reservematerial, we studied the accumulation of phytic acid duringdevelopment of lily pollen, and its degradation during germina-tion and tube growth. The information is needed for understand-ing why prematurely harvested lily pollen is unable to germinate(20). We also isolated and studied the enzyme, phytase, which isresponsible for phytic acid degradation in vivo.

MATERIALS AND METHODS

Total P and Phytic Acid Determination in Developing andGerminating Lily Pollen. Easter lilies (Lilium longiflorum Thunb.cv Ace) were grown in the greenhouse. Pollen at various devel-opmental stages was removed from anthers (20), dried for 24 hat room temperature, and then stored in the -80°C freezer beforeuse. Mature pollen was germinated in a 0.29 M glucose culturemedium (8) for various times and was harvested by filteringthrough nylon cloth (20-40,um). A modification of the methodsof Early and Deturk (10) and Deboland et al. (7) was used forthe routine determination of phytic acid. Pollen samples (40-60mg) were placed in 50 ml polypropylene centrifuge tubes towhich 15 ml of 0.4 M HCI in 0.7 M Na2SO4 was added. Magneticstirring bars were placed in the tubes, and samples were stirredfor 18 to 24 h at room temperature. Following centrifugation(10,OOOgat 0°C for 15 min), 10 ml ofeach extract was transferredto a 30-ml Corex centrifuge tube, diluted with 10 ml of glassdistilled H20, treated with 5 ml of 15 mm FeCl3 in 0.2 M HCIcontaining 0.35 M Na2SO4, and heated for 30 min in a boilingwater bath. The ferric phytate precipitate obtained after centri-fugation (10,OOOg for 15 min) was washed once with 10 ml of0.2 M HCI, completely digested on a hot plate (Lindberg Co.,model 53014, set at high) with 1 ml concentrated H2SO4 andH202 (as needed until digest was clear), and then diluted to afinal volume of 8 ml with glass distilled H20. P in the digestswas determined colorimetrically (3, 4). The level of phytic acidin pollen samples was calculated based on a P:phytic acid molarratio of 6: 1.For total P determination, pollen samples (10-20 mg) were

placed in 30-ml Corex centrifuge tubes, digested on a hot platewith 1 ml concentrated H2SO4 and H202 as needed to givecomplete digestion, and diluted to 8 ml with glass distilled H20.P in the digests was determined colorimetrically (3, 4).

Ion-Exchange Chromatography. 1. Separation ofmyo-InositolPolyphosphate and Pi using an Ammonium Formate LinearGradient. Pollen samples were extracted in 15 ml 0.4 M HCI withconstant stirring for 18 to 24 h at room temperature. The extractswere centrifuged (l0,OOOg for 15 min). Ten ml of the clearsupernatant fluid was diluted to 50 ml with glass distilled H20and loaded onto a 0.7 cm by 1.3 cm column containing 5 ml ofDowex I-X8 (formate) resin (pH 5.2). The effluent collected

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PHYTIC ACID METABOLISM IN LILY POLLEN

during column loading was tested for P to ensure completebinding. A duplicate column was prepared for the standards andloaded with 0.1 ml of 10 mM KH2PO4 and 0.5 ml 1 mg/ml Naphytate solution. The columns were eluted at 1 ml/min with alinear gradient (0.24-1.2 M) ofammonium formate (pH 7.0) (7),to a total of 400 ml, and 5 ml fractions were collected. Theconductivities of the fractions were measured with a conductivitybridge equipped with a platinum-iridium electrode (cell constant= 1.0). The concentrations of ammonium formate in the frac-tions were calculated from a standard curve. After collection, thefractions were transferred to separate digestion tubes with 1 mlconcentrated H2SO4 and H202 (as needed) on the hot plate. Thedigests were neutralized with 5 ml of 5 N NaOH and diluted to12.5 ml with glass distilled H20. Total P was then determinedcolorimetrically (3, 4). Elution profiles of pollen extracts werecompared with the elution of the standards.

2. Separation ofmyo-Inositol 2-Phosphate, myo-Inositol Poly-phosphates and Pi using an HCl Gradient. Pollen extract pre-pared as described above was passed through a 0.7 cm by 13 cmcolumn containing 5 ml of AG 1-X8 (Cl-form, 200-400 mesh)(pH 4.0). Again, effluent collected during column loading wastested for P to ensure complete binding. A duplicate column wasprepared for the standards which contained equal amounts ofmyo-inositol 2-phosphate and Pi (1 mmol of each) and 500 ,ugof myo-inositol pentakisphosphate and Na phytate. The columnwas eluted at 1 ml/min with a linear gradient of HCI (0-1.0 N)to a total of 600 ml, and 5 ml fractions were collected. Totalphosphorus was then determined as above.

Phytase Isolation and Assay. Pollen samples (including maturedry and germinated) were ground for approximately 5 min in amortar with 0.1 M Na acetate buffer (pH 5.2) at 0°C. The clearsupernatant after two centrifugations at l0,000g for 10 min wasdialyzed against 1 L of 0.1 M Na acetate buffer (pH 5.2) for 18to 24 h with one change of the same buffer. After dialysis, theextract was centrifuged (10,000g for 10 min at 4°C) and thenplaced in a 59°C water bath for 2 min and then immediatelychilled in ice water and centrifuged at l0,OOOg for 15 min at 4°C.Solid (NH4)2SO4 (277 mg/ml) was added to give 45% of satura-tion level. The extract was centrifuged after 15 min (l0,OOOg for15 min at 4°C). The clear supernatant then received 134 mg

(NH4)2SO4/ml to reach 65% of saturation and centrifuged againafter 15 min. The precipitate collected was dissolved in anappropriate amount of 0.1 M Na acetate buffer (pH 5.2) anddialyzed overnight against 1 L of 0.02 M Tris acetate buffer (pH7.5) at 0°C. Two hundred /A of the dialyzed fraction was injectedonto a DEAE-5PW column in a Waters HPLC. The column(purchased from Waters Co.) was washed with 0.02 M Tris-acetate (pH 7.5) at 1 ml/min during the first 10 min to removeunbound materials, and the bound proteins were eluted at 1 ml/min with a linear gradient of Tris-acetate (pH 7.5)(0.02-0.9 M)in a total volume of 30 ml. One ml fractions were collected.Phytase and phosphatase activities of each fraction were deter-mined using Na phytate and p-nitrophenyl phosphate (p-NPP)as substrate, respectively. The reaction mixture for phosphataseassay contained 2.5 mM MgCl2, 2.5 mm p-NPP, 10 yA of eachfraction, and 0.1 M Na acetate buffer (pH 5.0) in a total volumeof 0.5 ml and was incubated at 30°C for 30 min. The reactionwas terminated by adding 2 ml of 10% NaOH. The A400 valueof the assay mixture was then measured, and jAmol of p-nitro-phenol (p-NP) produced was calculated from a standard curve.

For assaying phytase activity, the reaction mixture contained 2.5mM MgCl2, 0.5 mm Na phytate (pH 7.0) in 0.1 M Na Pipes buffer(pH 6.5) or 2.5 mm Na phytate (pH 5.0) in 0.1 M Na acetatebuffer (pH 5.0) and 100 of enzyme fraction in a total volumeof 0.5 ml. The reaction proceeded at 30°C for 8 h, and was

terminated by the addition of 50 uA of 50% TCA. The clarifiedreaction mixture (0.3 ml) was used for P determination as

described earlier. The phytase activity was expressed as nmol Pireleased/assay tube.

RESULTS

The content of phytic acid in lily pollen increased between 4and 5 d before anthesis and then stayed constant at approxi-mately 7 gg per mg pollen till anthesis (Table I). Phytic acid Paccounted for 21 to 30% of total P.Mature lily pollen contained 8.14 ± 0.74 ,ug phytic acid per

mg pollen (mean ± SD, n = 4) according to the iron precipitationprocedure described under the "Materials and Methods" section.Considerable degradation of phytic acid already occurred in thefirst 15 min of incubation in the glucose culture medium, andonly a trace was left by 31/3 h (Fig. 1). The breakdown of phyticacid proceeded at an approximately constant rate during thisperiod (0-3Y/3 h), and a linear regression gave a good fit to thedata with a calculated rate of 0.037 Mg phytic acid/mg pollen.min (Fig. 1). Total P stayed constant as expected. Total P andphytic acid were determined in the culture medium from whichthe germinated pollen had been removed by filtration, and thefiltrate contained no detectable phosphorus.Anion exchange chromatography ofa 1.2% HCl pollen extract

(mature ungerminated) with a linear ammonium formate gra-dient yielded only one major peak containing phosphate (Fig.2), and it corresponded to authentic phytic acid. This peakcontained the equivalent of 8.65 Mg phytic acid per mg pollen,

Table I. Phytic Acid and Total P Content ofDeveloping Lily PollenTime before Tota P PhyticAci& PhyticAcid PAnthesis

d jug/mg pollen % oftotal P5 5.71 ± 0.135 4.22 ± 0.593 21.54 5.34±2.214 7.10± 1.429 25.93 6.83 ± 1.636 7.13 ± 1.919 30.42 7.43 ± 1.007 7.19 ± 1.552 28.11 7.35 ± 1.158 6.66 ± 1.560 26.40 8.89 ± 0.953 7.01 ± 1.473 22.1

a Phytic acid and total P are expressed on a dry weight basis. Eachvalue represents mean SD of 3 to 5 samples.

10 1 I11 1Tota P

10

8 CZ

f 6-' Rate -0.037E1g PhyticAcid/mg pollen/min

I~~~~~

% 4

2 - Phytic acid'42

0 60 120 180

Time (min)FIG. 1. Total P and phytic acid content of germinating lily pollen.

Pollen was germinated in 0.29 M glucose medium for the times indicated.Duplicate flasks were used for each time point, and there were duplicatedeterminations on each flask. The vertical lines represent the extent ofvariation between pairs of flasks.

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Plant Physiol. Vol. 83, 1987

60

50

40

30

20

in .2

0.00 - '$o CCCCXCCtCC fC O 0

4 . Sxst4 53 mMho0.40 60

0.30

0.20 A ~~~~~~~~~40

0K20.10 Phytic 20

I I~~%

10 20 30 40 50Fricion no.

FIG. 2. Anion-exchange chromatography on Dowex 1 -X8 of an acidextract ofungerminated pollen and phosphorus standards. Samples wereeluted with a linear gradient of ammonium formate (0.24-1.2 M), and 5ml fractions were collected. Above, the column was loaded with 10 mlof extract equivalent to 13.34 mg ungerminated lily pollen; below, thecolumn was loaded with I mmol Pi and 0.77 gmol phytic acid.

in good agreement with the results of the iron precipitationprocedure. The anion-exchange chromatography used here isable to separate all of the partially phosphorylated myo-inositolsfrom phytic acid except for myo-inositol pentakisphosphate.

Experiments were conducted to determine whether any par-tially phosphorylated myo-inositol was present in germinated lilypollen. Anion-exchange chromatography with a linear HCI gra-dient, which is able to resolve myo-inositol pentakisphosphateand phytic acid, revealed that no partially phosphorylated myo-inositol accumulated during the first 15 min of incubation. Bothextracts of 15 and 90 min germinated pollen contained majorphosphate peaks which corresponded to authentic phytic acid(Fig. 3). Based on total P recovered in the phytic acid peak, the15 min pollen sample was calculated to contain 6.93 ug phyticacid per mg pollen which was comparable with the results of theiron precipitation procedure (6.75 ,ug phytic acid/mg pollen).Similarly, good agreement ofthe two methods was obtained withthe 90 min sample.The protein synthesis inhibitor cycloheximide (100 ,g/ml) and

the RNA synthesis inhibitor cordycepin (150 ,ug/ml) were addedto pollen culture medium separately to see if either of them hadan inhibitory effect on phytic acid mobilization. Cordycepin hadno effect on phytic acid degradation during germination (Fig. 4)despite its well known ability to inhibit RNA synthesis in plants(15). In a separate experiment (data not shown), we demonstratedthat cordycepin, at the concentration used in this study, causeda 30 to 40% inhibition of the incorporation of [3H]uridine intothe nucleic acid fraction in the first 90 min of lily pollen germi-nation; after 90 min, the inhibition was complete. A differentresult was observed when cycloheximide was added to the culture

CZ

0i

-iO 5

0~~~~~~~~~~~~~~~20 40 60 80

Fraction Number

FIG. 3. Anion-exchange chromatography on AG l-X8 ofacid extractsof germinated pollen samples and phosphorus standards. Samples wereeluted with a linear gradient of HCI (0-1.0 N), and 5 ml fractions werecollected. Above, the column was loaded with 10 ml of extract frompollen germinated for 90 min. The extract was equivalent to 160 mgpollen. Middle, the column was loaded with 10 ml extract from pollengerminated for 15 min. The extract was equivalent to 26.67 mg pollen.Bottom, the column was loaded with I mmol myo-inositol 2-phosphate,1 mmol Pi, and 500 gg each of phytic acid and myo-inositol pentakis-phosphate.

10I

-C-ordycep'n -

0 60 120 180Time (min)

FIG. 4. Effect of cordycepin on phytic acid degradation during lilypollen germination. Pollen was germinated in 0.29 M glucose mediumwith or without 150 Ag/ml cordycepin. The vertical lines represent therange between duplicate flasks. Duplicate determinations were done onthe extract from each flask.

medium (Fig. 5). Phytic acid degradation proceeded at a normalrate for the first 60 min, but further degradation was suppressedby cycloheximide (Fig. 5). Pollen tube growth and germinationpercentage were checked at 60 and 210 min under a dissectingmicroscope. The percentage of pollen germination was approxi-mately 52% in the presence of cycloheximide or cordycepin,which was not different than the control (55%) after 60 min of

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PHYTIC ACID METABOLISM IN LILY POLLEN

0 60 120Time (min)

180

FIG. 5. Effect of cycloheximide on phytic acid breakdown during lilypollen germination. Cycloheximide (100 ug/ml) was added to the glucosemedium. Each point represents a mean of duplicate determinations onextracts from duplicate flasks. Vertical lines are the range between themeans of the duplicate flasks.

germination. Extensive pollen tube growth (1.52 mm), whichresembled the control (1.44 mm), was observed in the presenceof 150 ,g cordycepin/ml after 210 min. However, pollen tubegrowth was drastically inhibited by cycloheximide, and averagelength was approximately 26 ,m.

It has been reported that there may be two different phytasespresent in lily pollen (9). One of them (pH 5.0 phytase) ispreexisting in mature ungerminated pollen and has a pH opti-mum at pH 5.0. The other one (pH 6.5 phytase) is newlysynthesized during lily pollen germination and has highest activ-ity against phytate at pH 6.5. We therefore analyzed phytaseactivity in enzyme extracts from pollen germinated for varioustimes on an HPLC with a DEAE-5PW column using a Tris-acetate gradient (0.02-0.9 M, pH 7.5) to study the appearance ofpH 6.5 phytase during germination. Results are presented inFigure 6 and Table II. The results clearly showed that only asingle peak of phytase activity (the pH 5.0 phytase) was presentin ungerminated lily pollen (Fig. 6, top). The elution profilesrevealed that a second phytase (the pH 6.5 phytase) had appearedby 30 min of incubation (Fig. 6, middle). A noticeable rise inactivity of pH 6.5 phytase was observed after 60 min of germi-nation (Fig. 6, bottom), and the activity of this phytase increasedas germination proceeded (Table II). After 5 h of germination,considerable phytase activities were still present in lily pollen andthe pH 6.5 phytase remained predominant.

Phytase activity was dramatically repressed by cycloheximide(100 ,g/ml) (Table III), while cordycepin (150 ,g/ml) had noeffect (Fig. 7), in agreement with the effects on phytic aciddegradation observed earlier. Residual pH 5.0 phytase activity(2.44 and 3.55 versus 10.31 and 14.61 nmol Pi released/mgpollen -h) was still present in the pollen germinated 60 and 180min in the presence of cycloheximide; a little pH 6.5 phytaseactivity (0.5 and 1.14 versus 10.17 and 28.12 nmol Pi released/mg pollen.h) was also detected in both cases. The results ofDEAE chromatography also indicated that pollen germinated 90min in the presence o-f 100 Mg/ml cycloheximide contained thepH 5.0 phytase only (Fig. 7). The appearance ofpH 6.5 phytasewas inhibited by cycloheximide. However, both phytases werepresent in 90 min germinated pollen with 150 Mg/ml of cordy-cepin (Fig. 7). In the presence of cordycepin, phytase activitieswere comparable to control values.

Pi is the degradation product of phytic acid. Preliminarystudies had shown that 25 mM KH2PO4 was not harmful to lilypollen tube growth (data not shown). The present work revealed

.8

a

Po

:e

I

l

£

w0

0

0

16 1 8 20 22 24 26 28 30

Fracton no.FIG. 6. DEAE chromatography of ungerminated pollen and pollen

germinated in glucose medium for 0, 30, and 60 min. Phytase activity atpH 5.0 and 6.5 were determined for each fraction according to theprocedures described under "Materials and Methods."

Table II. Activity ofpH 5.0 andpH 6.5 Phytases at VariousGermination Times

Phytase Activity8Germination Time

pH 5.0 pH6.5

min nmol Pi released/mgpollen. h0 2.60 0.0430 1.86 0.1660 1.81 0.7190 1.95 2.85120 2.50 6.48180 3.27 13.12300 2.68 7.25

a Values are taken from the experiment shown in Figure 6. Fractions18 to 23 contained the pH 5.0 phytase; fractions 25 to 27 contained the6.5 phytase with most or all of the activity in fractions 25 and 26.

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Plant Physiol. Vol. 83, 1987

Table III. Effect ofCycloheximide on Phytase ActivityAssay condition Phytase Activity

Germination 100o g/mlTime pH Phytic acid cycloheximide

Added Not added

h mMnmol Pi released/mg

pollen * h1 5.0 0.5 2.24 8.19

5.0 2.5 2.44 10.316.5 0.5 0.51 10.176.5 2.5 0.36 6.52

3 5.0 0.5 3.26 21.535.0 2.5 3.55 14.686.5 0.5 1.14 28.126.5 2.5 0.63 12.45

0.60.04

0.40.02

0.2

*

0.06

0.04 o0.6

0.40.02 z .

.0.2

0.00 - 0.0 ~

0.06

0.04

0.6

0.02 0.4

00 0.2

o.oo o.o

15 20 25 30

Frection no.

FIG. 7. Comparisons of DEAE chromatography of enzyme extractsprepared from 90 min germinated pollen with or without cycloheximideand cordycepin. Above, enzyme extract equivalent to 400 mg pollengerminated in the presence of cycloheximide (100 ,ug/ml) was loadedonto the column; middle, enzyme extract equivalent to 200 mg pollengerminated in the presence of cordycepin (150 jug/ml) was loaded ontothe column; bottom, enzyme extract equivalent to 400 mg pollen ger-minated without inhibitors.

Table IV. Effect ofPi on Phytic Acid Breakdown in Vivo and PhytaseActivity

Phytase Activity8Treatment Phytic Acid Content!

pH 5.0 pH 6.5

Pi released/mgag/mgpollenpolnhpollen - h2 h germination+25 mM KH2PO4 1.51 12.87 23.43-25 mM KH2PO4 3.09 10.05 22.46

aDuplicate determinations were done on each pollen sample.

that 25 mM KH2PO4 had no effect on phytase activity (TableIV). Furthermore, the degradation ofphytic acid was significantlystimulated by 25 mM KH2PO4 during the 2 h germination period(Table IV).

DISCUSSION

The present investigation shows that a substantial amount ofphytic acid has accumulated in lily pollen by 5 d before anthesis,and little change occurs during subsequent stages of maturation.This pattern is different from that of petunia pollen in which thehighest phytic acid level is reached just before anthesis ( 14).There is no lag between phytic acid degradation and the onset

of lily pollen germination, and the presence of considerablephytase activity in the mature ungerminated lily pollen couldaccount for this observation (9). The phytase activity (10 nmolPi released/mg pollen-h) at optimal phytic acid concentration(9) obtained from ungerminated lily pollen is not very differentfrom the observed in vivo rate of phytic acid degradation (0.037,ug/mg pollen-h or 16.82 nmol Pi released/mg pollen h). Thediscrepancy may be due to the presence of inhibitory materialsin the crude extract, because (NH4)2SO4 fractionation gave a67% increase in activity compared to the crude (data not shown).However, enough phytase activity (16.2 nmol Pi released/mgpollen - h) appears in crude extracts ofpollen germinated for 1 .5h(9) to account for the observed rate of phytic acid breakdown,and activity further increases to 29.2 nmol Pi released/mg pol-len *h after 3 h of germination. The increased phytase activity isprobably due to the formation of new enzyme molecules trans-lated from a long-lived, preexisting mRNA, since its appearanceis prevented by cycloheximide but not by cordycepin. Similarresults were also reported in petunia pollen (18). It has long beenknown from studies with inhibitors ofRNA and protein synthesisthat the ungerminated pollen grain at anthesis contains storedstable mRNA (22). The poly(A)+ RNA from ungerminated Tra-descantia pollen has been extracted, translated in a cell freesystem, and shown to code for similar proteins as are synthesizedduring pollen germination (12). It seems likely then that the pH6.5 phytase is among these newly synthesized enzymes.Two phytases are identified in germinated lily pollen (9). They

differ in optimal pH. The low pH phytase (pH 5.0 phytase) ispresent in mature ungerminated pollen, but the pH 6.5 phytaseappears to be newly synthesized during germination. The resultsofDEAE anion exchange chromatography show that the low pHphytase remains relatively constant during germination, whilethe activity of the pH 6.5 phytase increases markedly. After 5 hof germination, considerable phytase activity is still noticeableeven though there is no phytic acid left. A similar phenomenonis observed in Phaseolus vulgaris L. (21, 26), and pea seeds (13).It is not understood how the phytase activity is regulated duringgermination. Perhaps these results are due to a slow turnoverrate ofthe enzyme, or possibly phytase has other as yet unknownfunctions. Unlike lily pollen, two phytases are already present inmature ungerminated petunia pollen (1). The existence of twophytases has also been noted in lettuce seed (23), cotton (1 1),

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PHYTIC ACID METABOLISM IN LILY POLLEN

and wheat bran (19).The results of the cycloheximide experiment suggest that the

low pH phytase of lily pollen may be effective on phytic aciddegradation only during early germination, and pH 6.5 phytaseis the enzyme required for the further breakdown of phytic acid.Furthermore, the apparent higher substrate affinity of the pH6.5 phytase (9) is consistent with its acting later in germinationwhen the phytic acid is low.Ten mm Pi completely represses the low pH phytase activity

in vitro, but does not have an effect on the pH 6.5 phytase (datanot shown). However, a high concentration of Pi (25 mM) in theculture medium exerts no effects on either pollen tube growth orappearance of the pH 6.5 phytase in germinating lily pollen, anddegradation of phytic acid is accelerated by the added Pi. Sensi-tivity of the low pH phytase to Pi may reduce the in vivoeffectiveness of this enzyme if Pi accumulates during germina-tion. In that case, the pH 6.5 phytase would become increasinglyimportant as germination proceeds. Or the failure of Pi to haveany effect on the in vivo breakdown may be due to exclusion ofthis ion from the site of breakdown. Different results werereported for petunia pollen (18) where phytic acid degradationas well as tube growth are significantly inhibited by 3 and 10 mMKH2PO4, respectively. The in vitro phytase activity of petunia isalso dramatically reduced by Pi, but the in vivo effect may notbe direct since growth is also inhibited. The inhibitory effect ofPi on phytase isolated from bean seeds, mung beans, and wheatbran was also reported (6).Mature lily pollen has single-membrane organelles with darkly

stained contents (25). Their contents as well as the organellesthemselves disappeared during germination, indicating a mobi-lization of reserve materials. These organelles might be proteinbodies that contain phytic acid but evidence supporting thisnotion is still lacking. Further research on the localization andmetabolism of phytic acid in pollen is necessary to gain insightsinto the regulation of phytic acid degradation during germina-tion.

Acknowledgments-Dr. Victor Raboy is thanked for the advice on phytic acidisolation and column chromatography, and Steve Hudson is thanked for the adviceon column chromatography.

LITERATURE CITED

1. BREMEIJER GMM 1971 Latent glutamate dehydrogenase in pollen of Petuniahybrida. Acta Bot N&erl 20: 119-131

2. CALDWELL AG, CA BLACK 1958 Inositol hexaphosphate III. Content in soils.Soil Sci Soc Am Proc 22: 296-302

3. CHEN LH, TY TORIBARA, H WARNER 1956 Microdetermination of phospho-rus. Anal Chem 28: 1756-1758

4. CHERYAN M 1980 Phytic acid interactions in food systems. Crit Rev Food SciNutr 13: 297-335

5. COSGROVE DJ 1966 Chemistry and biochemistry of inositol polyphosphates.Rev Pure Appl Chem 16: 209-224

6. COSGROVE DJ 1980 Inositol Phosphates: Their Chemistry, Biochemistry andPhysiology. Elsevier, Amsterdam, p 191

7. DEBOLAND AR, GB GARNER, BL O'DELL 1975 Identification and propertiesof "phytate" in cereal grains and oilseed products. J Agric Food Chem 23:1186-1189

8. DICKINsoN DB 1967 Permeability and respiratory properties of germinatingpollen. Physiol Plant 20: 118-127

9. DICKINSON DB, J LIN 1985 Phytases ofgerminating lily pollen. In DL Mulcahy,GB Mulcahy, E Ottaviano, eds, Pollen Biology and Technology. Springer-Verlag, New York, pp 357-362

10. EARLY EB, EE DETURK 1944 Time and rate of synthesis of phytin in corngrain during the reproductive period. J Am Soc Agron 36: 803-814

11. FONTAINE EDW, A PoNs, GW IRVING 1946 Protein phytic acid relationshipin peanuts and cottonseed. J Biol Chem 164: 487-507

12. FRANKIS R, JP MASCARENHAS 1980 Messenger RNA in the ungerminatedpollen grains: a direct determination of its presence. Ann Bot 45: 595-599

13. GUARDIOLA JL, JF SUTCLIFFE 1971 Mobilization of phosphorus in the cotyle-dons of young seedlings of the garden pea (Pisum sativa L.). Ann Bot 35:809-823

14. HELSPER JPFG, HF LINSKENS, JF JACKSON 1984 Phytate metabolism inpetunia pollen. Phytochemistry 23: 1841-1845

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