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

Plant Physiol. (1987) 83, 408-413 0032-0889/87/83/0408/06/$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 Ho Department of Horticulture, University of Illinois, 1301 W. Gregory Dr., Urbana, Illinois 61801 (J.-J.L., D.B.D.); and Department of Biology, Washington University, St. Louis, Missouri 63130 (T.-H.D.H.) (6); however, literature about phytic acid metabolism in pollen is very limited. The only information available so far is from The accumulation of phytic acid during development of lily (Lilium petunia pollen (14, 17, 18). Because of its importance as a reserve longiflorum Thunb.) pollen and its degradation during germination have material, we studied the accumulation of phytic acid during been studied. A substantial amount of phytic acid accumulates in lily development of lily pollen, and its degradation during germinapollen by 5 days before anthesis, and little change occurs during subse- tion and tube growth. The information is needed for understandquent maturation. Mature lily pollen contains 7 to 8 micrograms phytic ing why prematurely harvested lily pollen is unable to germinate acid per milligram pollen. Considerable degradation of phytic acid occurs (20). We also isolated and studied the enzyme, phytase, which is by 15 minutes of incubation in glucose culture medium, and very little is responsible for phytic acid degradation in vivo. ABSTRACT

left by 3 hours. No partially phosphorylated myo-inositol accumulates during germination. The breakdown of phytic acid proceeds at a constant rate during this time period. The rate is calculated to be 0.037 microgram phytic acid/milligram pollen/minute. Two phytases are detected in germinated lily pollen extract using high performance liquid chromatography with an anion exchange column (diethylaminoethyl-5PW). The results suggest that one of the phytases is already present in mature ungerminated lily pollen and the other one is newly synthesized during germination from a long-lived, pre-existing mRNA.

Phytic acid, myo-inositol 1,2,3,4,5,6 hexakisphosphate, is of widespread occurrence in seeds (5). It appears to function as a storage form of phosphorus in seeds (4, 5), typically accounting for from 50 to 80% of the mature seed's total phosphorus. Phytic acid has also been reported in roots and tubers (24), and in organic soils (2). Recently, phytic acid has been identified in pollen of many plant species (16). Significant quantities (0.052.1 % of dry weight) of phytic acid were observed in pollen from plants with style lengths greater than 5 mm, while little or no phytic acid was found in pollen from composites and grasses with very short styles. Helsper et al. ( 14) reported that the myoinositol moiety released during phytic acid degradation in germinating petunia pollen is utilized for synthesis of phosphatidyl inositol and pectic polysaccharide, both of which are needed in large amounts for pollen tube assembly as pollen tube elongation gets under way. Therefore, the degradation of phytic acid during pollen germination is important because it not only supplies Pi but also replenishes the pool of myo-inositol. The rate of phytic acid mobilization in germinating petunia pollen was also reported 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 the

MATERIALS AND METHODS Total P and Phytic Acid Determination in Developing and Germinating Lily Pollen. Easter lilies (Lilium longiflorum Thunb. cv Ace) were grown in the greenhouse. Pollen at various developmental stages was removed from anthers (20), dried for 24 h at room temperature, and then stored in the -80°C freezer before use. Mature pollen was germinated in a 0.29 M glucose culture medium (8) for various times and was harvested by filtering through nylon cloth (20-40,um). A modification of the methods of Early and Deturk (10) and Deboland et al. (7) was used for the routine determination of phytic acid. Pollen samples (40-60 mg) were placed in 50 ml polypropylene centrifuge tubes to which 15 ml of 0.4 M HCI in 0.7 M Na2SO4 was added. Magnetic stirring bars were placed in the tubes, and samples were stirred for 18 to 24 h at room temperature. Following centrifugation (10,OOOgat 0°C for 15 min), 10 ml of each extract was transferred to a 30-ml Corex centrifuge tube, diluted with 10 ml of glass distilled H20, treated with 5 ml of 15 mm FeCl3 in 0.2 M HCI containing 0.35 M Na2SO4, and heated for 30 min in a boiling water bath. The ferric phytate precipitate obtained after centrifugation (10,OOOg for 15 min) was washed once with 10 ml of 0.2 M HCI, completely digested on a hot plate (Lindberg Co., model 53014, set at high) with 1 ml concentrated H2SO4 and H202 (as needed until digest was clear), and then diluted to a final volume of 8 ml with glass distilled H20. P in the digests was determined colorimetrically (3, 4). The level of phytic acid in pollen samples was calculated based on a P:phytic acid molar ratio of 6: 1. For total P determination, pollen samples (10-20 mg) were placed in 30-ml Corex centrifuge tubes, digested on a hot plate with 1 ml concentrated H2SO4 and H202 as needed to give complete digestion, and diluted to 8 ml with glass distilled H20. P in the digests was determined colorimetrically (3, 4). Ion-Exchange Chromatography. 1. Separation of myo-Inositol Polyphosphate and Pi using an Ammonium Formate Linear Gradient. Pollen samples were extracted in 15 ml 0.4 M HCI with constant stirring for 18 to 24 h at room temperature. The extracts were centrifuged (l0,OOOg for 15 min). Ten ml of the clear supernatant fluid was diluted to 50 ml with glass distilled H20 and loaded onto a 0.7 cm by 1.3 cm column containing 5 ml of Dowex I-X8 (formate) resin (pH 5.2). The effluent collected

Agricultural Experiment Station, College of Agriculture, University of Illinois at Urbana-Champaign. 2 Present address: Department of Biology, Washington University, St. Louis, MO 63130. 408

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during column loading was tested for P to ensure complete binding. A duplicate column was prepared for the standards and loaded with 0.1 ml of 10 mM KH2PO4 and 0.5 ml 1 mg/ml Na phytate solution. The columns were eluted at 1 ml/min with a linear gradient (0.24-1.2 M) of ammonium formate (pH 7.0) (7), to a total of 400 ml, and 5 ml fractions were collected. The conductivities of the fractions were measured with a conductivity bridge equipped with a platinum-iridium electrode (cell constant = 1.0). The concentrations of ammonium formate in the fractions were calculated from a standard curve. After collection, the fractions were transferred to separate digestion tubes with 1 ml concentrated H2SO4 and H202 (as needed) on the hot plate. The digests were neutralized with 5 ml of 5 N NaOH and diluted to 12.5 ml with glass distilled H20. Total P was then determined colorimetrically (3, 4). Elution profiles of pollen extracts were compared with the elution of the standards. 2. Separation of myo-Inositol 2-Phosphate, myo-Inositol Polyphosphates and Pi using an HCl Gradient. Pollen extract prepared as described above was passed through a 0.7 cm by 13 cm column containing 5 ml of AG 1-X8 (Cl-form, 200-400 mesh) (pH 4.0). Again, effluent collected during column loading was tested for P to ensure complete binding. A duplicate column was prepared for the standards which contained equal amounts of myo-inositol 2-phosphate and Pi (1 mmol of each) and 500 ,ug of myo-inositol pentakisphosphate and Na phytate. The column was 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. Total phosphorus was then determined as above. Phytase Isolation and Assay. Pollen samples (including mature

dry and germinated) were ground for approximately 5 min in a mortar with 0.1 M Na acetate buffer (pH 5.2) at 0°C. The clear supernatant after two centrifugations at l0,000g for 10 min was dialyzed against 1 L of 0.1 M Na acetate buffer (pH 5.2) for 18 to 24 h with one change of the same buffer. After dialysis, the extract was centrifuged (10,000g for 10 min at 4°C) and then placed in a 59°C water bath for 2 min and then immediately chilled 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 saturation level. The extract was centrifuged after 15 min (l0,OOOg for 15 min at 4°C). The clear supernatant then received 134 mg (NH4)2SO4/ml to reach 65% of saturation and centrifuged again after 15 min. The precipitate collected was dissolved in an appropriate amount of 0.1 M Na acetate buffer (pH 5.2) and dialyzed overnight against 1 L of 0.02 M Tris acetate buffer (pH 7.5) at 0°C. Two hundred /A of the dialyzed fraction was injected onto a DEAE-5PW column in a Waters HPLC. The column (purchased from Waters Co.) was washed with 0.02 M Trisacetate (pH 7.5) at 1 ml/min during the first 10 min to remove unbound 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 determined using Na phytate and p-nitrophenyl phosphate (p-NPP) as substrate, respectively. The reaction mixture for phosphatase assay contained 2.5 mM MgCl2, 2.5 mm p-NPP, 10 yA of each fraction, and 0.1 M Na acetate buffer (pH 5.0) in a total volume of 0.5 ml and was incubated at 30°C for 30 min. The reaction terminated by adding 2 ml of 10% NaOH. The A400 value of the assay mixture was then measured, and jAmol of p-nitrophenol (p-NP) produced was calculated from a standard curve. For assaying phytase activity, the reaction mixture contained 2.5 mM 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 acetate buffer (pH 5.0) and 100 of enzyme fraction in a total volume of 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 clarified reaction mixture (0.3 ml) was used for P determination as

described earlier. The phytase activity was expressed as nmol Pi released/assay tube. RESULTS The content of phytic acid in lily pollen increased between 4 and 5 d before anthesis and then stayed constant at approximately 7 gg per mg pollen till anthesis (Table I). Phytic acid P accounted 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 precipitation procedure described under the "Materials and Methods" section. Considerable degradation of phytic acid already occurred in the first 15 min of incubation in the glucose culture medium, and only a trace was left by 31/3 h (Fig. 1). The breakdown of phytic acid proceeded at an approximately constant rate during this period (0-3Y/3 h), and a linear regression gave a good fit to the data with a calculated rate of 0.037 Mg phytic acid/mg pollen. min (Fig. 1). Total P stayed constant as expected. Total P and phytic acid were determined in the culture medium from which the germinated pollen had been removed by filtration, and the filtrate contained no detectable phosphorus. Anion exchange chromatography of a 1.2% HCl pollen extract (mature ungerminated) with a linear ammonium formate gradient yielded only one major peak containing phosphate (Fig. 2), and it corresponded to authentic phytic acid. This peak contained the equivalent of 8.65 Mg phytic acid per mg pollen, Table I. Phytic Acid and Total P Content ofDeveloping Lily Pollen Time before Tota P PhyticAci& PhyticAcid P Anthesis d jug/mg pollen % of total P 5 5.71 ± 0.135 4.22 ± 0.593 21.5 4 5.34±2.214 7.10± 1.429 25.9 3 6.83 ± 1.636 7.13 ± 1.919 30.4 2 7.43 ± 1.007 7.19 ± 1.552 28.1 1 7.35 ± 1.158 6.66 ± 1.560 26.4 0 8.89 ± 0.953 7.01 ± 1.473 22.1 a Phytic acid and total P are expressed on a dry weight basis. Each value represents mean SD of 3 to 5 samples. 10 1

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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 duplicate determinations on each flask. The vertical lines represent the extent of variation between pairs of flasks.

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Fricion no. FIG. 2. Anion-exchange chromatography on Dowex 1 -X8 of an acid extract of ungerminated pollen and phosphorus standards. Samples were eluted with a linear gradient of ammonium formate (0.24-1.2 M), and 5 ml fractions were collected. Above, the column was loaded with 10 ml of extract equivalent to 13.34 mg ungerminated lily pollen; below, the column was loaded with I mmol Pi and 0.77 gmol phytic acid.

in good agreement with the results of the iron precipitation procedure. The anion-exchange chromatography used here is able to separate all of the partially phosphorylated myo-inositols from phytic acid except for myo-inositol pentakisphosphate. Experiments were conducted to determine whether any partially phosphorylated myo-inositol was present in germinated lily pollen. Anion-exchange chromatography with a linear HCI gradient, which is able to resolve myo-inositol pentakisphosphate and phytic acid, revealed that no partially phosphorylated myoinositol accumulated during the first 15 min of incubation. Both extracts of 15 and 90 min germinated pollen contained major phosphate peaks which corresponded to authentic phytic acid (Fig. 3). Based on total P recovered in the phytic acid peak, the 15 min pollen sample was calculated to contain 6.93 ug phytic acid per mg pollen which was comparable with the results of the iron precipitation procedure (6.75 ,ug phytic acid/mg pollen). Similarly, good agreement of the two methods was obtained with the 90 min sample. The protein synthesis inhibitor cycloheximide (100 ,g/ml) and the RNA synthesis inhibitor cordycepin (150 ,ug/ml) were added to pollen culture medium separately to see if either of them had an inhibitory effect on phytic acid mobilization. Cordycepin had no 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 demonstrated that cordycepin, at the concentration used in this study, caused a 30 to 40% inhibition of the incorporation of [3H]uridine into the nucleic acid fraction in the first 90 min of lily pollen germination; after 90 min, the inhibition was complete. A different result was observed when cycloheximide was added to the culture

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FIG. 3. Anion-exchange chromatography on AG l-X8 ofacid extracts of germinated pollen samples and phosphorus standards. Samples were eluted with a linear gradient of HCI (0-1.0 N), and 5 ml fractions were collected. Above, the column was loaded with 10 ml of extract from pollen germinated for 90 min. The extract was equivalent to 160 mg pollen. Middle, the column was loaded with 10 ml extract from pollen germinated 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 pentakisphosphate. I

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60 120 180 Time (min) FIG. 4. Effect of cordycepin on phytic acid degradation during lily pollen germination. Pollen was germinated in 0.29 M glucose medium with or without 150 Ag/ml cordycepin. The vertical lines represent the range between duplicate flasks. Duplicate determinations were done on the extract from each flask. 0

medium (Fig. 5). Phytic acid degradation proceeded at a normal rate for the first 60 min, but further degradation was suppressed by cycloheximide (Fig. 5). Pollen tube growth and germination percentage were checked at 60 and 210 min under a dissecting microscope. The percentage of pollen germination was approximately 52% in the presence of cycloheximide or cordycepin, which was not different than the control (55%) after 60 min of

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FIG. 5. Effect of cycloheximide on phytic acid breakdown during lily pollen germination. Cycloheximide (100 ug/ml) was added to the glucose medium. Each point represents a mean of duplicate determinations on extracts from duplicate flasks. Vertical lines are the range between the means of the duplicate flasks.

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germination. Extensive pollen tube growth (1.52 mm), which resembled the control (1.44 mm), was observed in the presence of 150 ,g cordycepin/ml after 210 min. However, pollen tube growth was drastically inhibited by cycloheximide, and average length was approximately 26 ,m. It has been reported that there may be two different phytases present in lily pollen (9). One of them (pH 5.0 phytase) is preexisting in mature ungerminated pollen and has a pH optimum at pH 5.0. The other one (pH 6.5 phytase) is newly synthesized during lily pollen germination and has highest activity against phytate at pH 6.5. We therefore analyzed phytase activity in enzyme extracts from pollen germinated for various times on an HPLC with a DEAE-5PW column using a Trisacetate gradient (0.02-0.9 M, pH 7.5) to study the appearance of pH 6.5 phytase during germination. Results are presented in Figure 6 and Table II. The results clearly showed that only a single peak of phytase activity (the pH 5.0 phytase) was present in ungerminated lily pollen (Fig. 6, top). The elution profiles revealed that a second phytase (the pH 6.5 phytase) had appeared by 30 min of incubation (Fig. 6, middle). A noticeable rise in activity of pH 6.5 phytase was observed after 60 min of germination (Fig. 6, bottom), and the activity of this phytase increased as germination proceeded (Table II). After 5 h of germination, considerable phytase activities were still present in lily pollen and the pH 6.5 phytase remained predominant. Phytase activity was dramatically repressed by cycloheximide (100 ,g/ml) (Table III), while cordycepin (150 ,g/ml) had no effect (Fig. 7), in agreement with the effects on phytic acid degradation observed earlier. Residual pH 5.0 phytase activity (2.44 and 3.55 versus 10.31 and 14.61 nmol Pi released/mg pollen -h) was still present in the pollen germinated 60 and 180 min in the presence of cycloheximide; a little pH 6.5 phytase activity (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 of DEAE chromatography also indicated that pollen germinated 90 min in the presence o-f 100 Mg/ml cycloheximide contained the pH 5.0 phytase only (Fig. 7). The appearance of pH 6.5 phytase was inhibited by cycloheximide. However, both phytases were present in 90 min germinated pollen with 150 Mg/ml of cordycepin (Fig. 7). In the presence of cordycepin, phytase activities were comparable to control values. Pi is the degradation product of phytic acid. Preliminary studies had shown that 25 mM KH2PO4 was not harmful to lily pollen tube growth (data not shown). The present work revealed

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FIG. 6. DEAE chromatography of ungerminated pollen and pollen germinated in glucose medium for 0, 30, and 60 min. Phytase activity at pH 5.0 and 6.5 were determined for each fraction according to the procedures described under "Materials and Methods."

Table II. Activity ofpH 5.0 and pH 6.5 Phytases at Various Germination Times Phytase Activity8 Germination Time pH 5.0 pH6.5 nmol Pi released/mg min h pollen. 0 2.60 0.04 30 1.86 0.16 60 1.81 0.71 90 1.95 2.85 120 2.50 6.48 180 3.27 13.12 300 2.68 7.25 a Values are taken from the experiment shown in Figure 6. Fractions 18 to 23 contained the pH 5.0 phytase; fractions 25 to 27 contained the 6.5 phytase with most or all of the activity in fractions 25 and 26.

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Table III. Effect ofCycloheximide on Phytase Activity Assay condition Phytase Activity Germination 100o g/ml Time pH Phytic acid cycloheximide Added Not added Pi nmol released/mg h mM pollen * h 1 5.0 0.5 2.24 8.19 5.0 2.5 2.44 10.31 6.5 0.5 0.51 10.17 6.5 2.5 0.36 6.52 3 5.0 0.5 3.26 21.53 5.0 2.5 3.55 14.68 0.5 6.5 1.14 28.12 6.5 2.5 0.63 12.45

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Table IV. Effect of Pi on Phytic Acid Breakdown in Vivo and Phytase Activity Phytase Activity8 Treatment Phytic Acid Content! pH 5.0 pH 6.5 Pi released/mg ag/mg pollenpolnh pollen - h 2 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 (Table IV). Furthermore, the degradation of phytic acid was significantly stimulated by 25 mM KH2PO4 during the 2 h germination period (Table IV).

DISCUSSION The present investigation shows that a substantial amount of phytic 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 the highest phytic acid level is reached just before anthesis ( 14). There is no lag between phytic acid degradation and the onset 0.6 0.04 of lily pollen germination, and the presence of considerable phytase activity in the mature ungerminated lily pollen could 0.4 account for this observation (9). The phytase activity (10 nmol 0.02 Pi released/mg pollen-h) at optimal phytic acid concentration 0.2 (9) obtained from ungerminated lily pollen is not very different from the observed in vivo rate of phytic acid degradation (0.037 ,ug/mg pollen -h or 16.82 nmol Pi released/mg pollen h). The discrepancy may be due to the presence of inhibitory materials * in the crude extract, because (NH4)2SO4 fractionation gave a 0.06 67% increase in activity compared to the crude (data not shown). q However, enough phytase activity (16.2 nmol Pi released/mg pollen h) appears in crude extracts of pollen germinated for 1 .5h 0.04 o0.6 (9) to account for the observed rate of phytic acid breakdown, and activity further increases to 29.2 nmol Pi released/mg pollen * h after 3 h of germination. The increased phytase activity is 0.4 0.02 z. probably due to the formation of new enzyme molecules trans.0.2 lated from a long-lived, preexisting mRNA, since its appearance is prevented by cycloheximide but not by cordycepin. Similar 0.00 - 0.0 ~ results were also reported in petunia pollen (18). It has long been known from studies with inhibitors of RNA and protein synthesis 0.06 that the ungerminated pollen grain at anthesis contains stored stable mRNA (22). The poly(A)+ RNA from ungerminated Tradescantia pollen has been extracted, translated in a cell free 0.04 system, and shown to code for similar proteins as are synthesized 0.6 during pollen germination (12). It seems likely then that the pH 6.5 phytase is among these newly synthesized enzymes. Two phytases are identified in germinated lily pollen (9). They 0.02 0.4 differ in optimal pH. The low pH phytase (pH 5.0 phytase) is 00 0.2 present in mature ungerminated pollen, but the pH 6.5 phytase appears to be newly synthesized during germination. The results o.oo o.o of DEAE anion exchange chromatography show that the low pH phytase remains relatively constant during germination, while 15 20 25 30 the activity of the pH 6.5 phytase increases markedly. After 5 h Frection no. of germination, considerable phytase activity is still noticeable FIG. 7. Comparisons of DEAE chromatography of enzyme extracts even though there is no phytic acid left. A similar phenomenon prepared from 90 min germinated pollen with or without cycloheximide is observed in Phaseolus vulgaris L. (21, 26), and pea seeds (13). and cordycepin. Above, enzyme extract equivalent to 400 mg pollen It is not understood how the phytase activity is regulated during germinated in the presence of cycloheximide (100 ,ug/ml) was loaded germination. Perhaps these results are due to a slow turnover onto the column; middle, enzyme extract equivalent to 200 mg pollen rate of the enzyme, or possibly phytase has other as yet unknown germinated in the presence of cordycepin (150 jug/ml) was loaded onto functions. Unlike lily pollen, two phytases are already present in the column; bottom, enzyme extract equivalent to 400 mg pollen ger- mature ungerminated petunia pollen (1). The existence of two phytases has also been noted in lettuce seed (23), cotton (1 1), minated without inhibitors. ,

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2. CALDWELL AG, CA BLACK 1958 Inositol hexaphosphate III. Content in soils. and wheat bran (19). Soil Sci Soc Am Proc 22: 296-302 The results of the cycloheximide experiment suggest that the 3. CHEN LH, TY TORIBARA, H WARNER 1956 Microdetermination of phosphoacid low pH phytase of lily pollen may be effective on phytic rus. Anal Chem 28: 1756-1758 degradation only during early germination, and pH 6.5 phytase 4. CHERYAN M 1980 Phytic acid interactions in food systems. Crit Rev Food Sci Nutr 13: 297-335 is the enzyme required for the further breakdown of phytic acid. 5. COSGROVE DJ 1966 Chemistry and biochemistry of inositol polyphosphates. Furthermore, the apparent higher substrate affinity of the pH Rev Pure Appl Chem 16: 209-224 6.5 phytase (9) is consistent with its acting later in germination 6. COSGROVE DJ 1980 Inositol Phosphates: Their Chemistry, Biochemistry and Physiology. Elsevier, Amsterdam, p 191 when the phytic acid is low. AR, GB GARNER, BL O'DELL 1975 Identification and properties Ten mm Pi completely represses the low pH phytase activity 7. DEBOLAND of "phytate" in cereal grains and oilseed products. J Agric Food Chem 23: in vitro, but does not have an effect on the pH 6.5 phytase (data 1186-1189 not shown). However, a high concentration of Pi (25 mM) in the 8. DICKINsoN DB 1967 Permeability and respiratory properties of germinating pollen. Physiol Plant 20: 118-127 culture medium exerts no effects on either pollen tube growth or 9. DICKINSON DB, J LIN 1985 Phytases of germinating lily pollen. In DL Mulcahy, appearance of the pH 6.5 phytase in germinating lily pollen, and GB Mulcahy, E Ottaviano, eds, Pollen Biology and Technology. Springerdegradation of phytic acid is accelerated by the added Pi. SensiVerlag, New York, pp 357-362 tivity of the low pH phytase to Pi may reduce the in vivo 10. EARLY EB, EE DETURK 1944 Time and rate of synthesis of phytin in corn grain during the reproductive period. J Am Soc Agron 36: 803-814 effectiveness of this enzyme if Pi accumulates during germinaEDW, A PoNs, GW IRVING 1946 Protein phytic acid relationship tion. In that case, the pH 6.5 phytase would become increasingly 11. FONTAINE in peanuts and cottonseed. J Biol Chem 164: 487-507 Pi have failure of to Or the proceeds. as germination important 12. FRANKIS R, JP MASCARENHAS 1980 Messenger RNA in the ungerminated any effect on the in vivo breakdown may be due to exclusion of pollen grains: a direct determination of its presence. Ann Bot 45: 595-599 this ion from the site of breakdown. Different results were 13. GUARDIOLA JL, JF SUTCLIFFE 1971 Mobilization of phosphorus in the cotyledons of young seedlings of the garden pea (Pisum sativa L.). Ann Bot 35: reported for petunia pollen (18) where phytic acid degradation 809-823 mM and 10 as well as tube growth are significantly inhibited by 3 14. HELSPER JPFG, HF LINSKENS, JF JACKSON 1984 Phytate metabolism in KH2PO4, respectively. The in vitro phytase activity of petunia is petunia pollen. Phytochemistry 23: 1841-1845 also dramatically reduced by Pi, but the in vivo effect may not 15. Ho THD, JE VARNER 1976 Response of barley aleurone layers to abscisic acid. Plant Physiol 57: 175-178 be direct since growth is also inhibited. The inhibitory effect of 16. JACKSON JF, G JONEs, HF LINSKENS 1982 Phytic acid in pollen. Phytochemistry Pi on phytase isolated from bean seeds, mung beans, and wheat 21: 1255-1258 bran was also reported (6). 17. JACKSON JF, RK KAMBOJ, HF LINSKENS 1983 Localization of phytic acid in the floral structure of Petunia hybrida and relationship to incompatibility Mature lily pollen has single-membrane organelles with darkly genes. Theor Appl Genet 64: 259-262 stained contents (25). Their contents as well as the organelles 18. JACKSON JF, HF LINSKENS 1982 Phytic acid in Petunia hybrida pollen is themselves disappeared during germination, indicating a mobihydrolyzed during germination by a phytase. Acta Bot N&erl 31: 441-447 lization of reserve materials. These organelles might be protein 19. LIM PE, ME TATE 1973 The phytases. II. Properties of phytase fractions F1 and F2 from wheat bran and the myo-inositol phosphates produced by bodies that contain phytic acid but evidence supporting this F2. Biochim Biophys Acta 302: 316-328 notion is still lacking. Further research on the localization and 20. LINfraction J, DB DICKINSON 1984 Ability of pollen to germinate prior to anthesis metabolism of phytic acid in pollen is necessary to gain insights and effect of desiccation on germination. Plant Physiol 74: 746-748 into the regulation of phytic acid degradation during germina- 21. LOLAS GM, P MARKAKIS 1977 The phytase of navy beans (Phaseolus vulgaris L.). J Food Sci 42: 1094-1097 tion.

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

on column

LITERATURE CITED 1.

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

22. MASCARENHAS JP 1975 The biochemistry of angiosperm pollen development. Bot Rev 4: 259-314 23. MAYER AM 1956 The breakdown of phytic acid and phytase activity in germinating lettuce. Enzymologia 19: 1-8 24. MCCHANCE RA, EM WIDDOWSON 1935 Phytin in human nutrition. Biochem J 29B: 2694-2699 25. SOUTHWORTH D, DB DICKINSON 1981 Ultrastructural changes in germinating lily pollen. Grana 20: 29-35 26. WALKER KA 1974 Changes in phytic acid and phytase during early development of Phaseolus vulgaris L. Planta 1 16: 91-98