TheRole of Phytic Acid in the Wheat Grain1

Plant Physiol. (1970) 45, 376-381

The Role of Phytic Acid in the Wheat Grain1Received for publication December 23, 1968

S. G. WILLIAMS2Department of Agricultural Biochemistry, Waite Agricultural Research Institute, University of Adelaide,Adelaide, South Australia

ABSTRACT

The concentrations of adenosine triphosphate and phyticacid in testa, embryo plus scutellum, aleurone, and endo-sperm fractions from grain of Triticum vulgare cv. Insigniahave been determined during development under both nor-mal conditions and those of water stress. Phytic acid wasnot detected in the endosperm. In the embryo plus scutel-lum and aleurone fractions there was a rapid build-up ofphytic acid, but the adenosine triphosphate level did notchange markedly at this time. These results are not con-sistent with physiological roles previously suggested forphytic acid other than the role of phytin as a phosphorusand cation store for the germinating seed.

Phytic acid is a major phosphorus component of many seeds.In mature grain of Triticum vulgare cv. Gabo this compound,which accounts for about 88% of the acid-soluble phosphorusand 53% of the total phosphorus, is largely concentrated in thetesta-pericarp fraction (11). Despite the abundance of this ma-terial in the seed, very little is known of its physiological role. Asuggestion by Atkinson and Morton (2) that phytic acid may beable to phosphorylate nucleotide diphosphates to triphosphateshas some support from the work of Morton and Raison (13) andBiswas and Biswas (4), but this evidence is not unequivocable.Recently Sobolev and Rodionova (20) reported that phytic acidwas synthesised by a mixture of aleurone grains and mitochondriaisolated from ripening sunflower seeds when myoinositol andsuccinate were present. On addition of hexokinase and glucose tothis mixture the amount of adenosine triphosphate in the mediumfell, and no phytic acid was produced. Both sets of findings implythat the rate of synthesis of phytic acid is closely linked to theATP level in the cell. For this reason the amounts of ATP andphytic acid in several morphologically distinct components of thewheat grain were determined from shortly after anthesis throughto ripening.

MATERIALS AND METHODS

Plant Material. Triticum vulgare cv. Insignia was grown in18-cm pots containing sterilized potting soil. Four plants weregrown in each pot, and only one tiller from each was allowed todevelop. The date of anthesis of each ear was recorded. Oneplanting was maintained in a glasshouse and ripened in October

I Financial assistance from the Australian Commonwealth WheatIndustry Research Council is gratefully acknowledged.

2 Present address: Wellcome Research Laboratories, Beckenham,England.

(maximum temperature 27 C). Water was given regularly every2 days. A second planting was maintained in a phytotron (daytemperature 22 C, night temperature 10 C, day length 10 hrincreased to 12 hr shortly before anthesis) and watered as beforeexcept that no water was given between the 10th day after anthesisand the 17th day when the flag leaf showed distinct signs ofwilting.Wheat bran was commercial material available locally.Chemicals. Adenosine triphosphate and desiccated firefly tails

were purchased from the Sigma Chemical Company, St. Louis,Missouri. A solution of ATP (10-4 M) in 0.01 M phosphate buffer(pH 7.4) was prepared and stored at -15 C. Electrophoresis ofthis solution, by the method described under the determinationof phytic acid phosphorus, indicated that it contained 2.7% ofadenosine diphosphate and was stable for several months.

"Carrier-free" 32p inorganic phosphorus (32pi) was obtainedfrom the Australian Atomic Energy Commission, Lucas Heights,N.S.W., Australia. The other chemicals were analytical gradereagents, and all water used was double distilled from glass.

Dissection. Two heads which had flowered on the same daywere cut and immediately placed upright in water. The top thirdof each head was removed and discarded, and 16 to 20 grainsfrom the center of the head were removed one at a time and everyalternate grain (8 to 10 in number) was quickly dissected at roomtemperature with a needle and forceps. The pericarp above thecross cells and tube cells was first removed from the grain (testafraction, T) and then the embryo plus scutellum (E + S) wasdissected as a unit. Next a longitudinal cut was made in the grainopposite to the crease and the aleurone layer, together with thechlorophyll layer when present, was peeled off (aleurone fraction,A). The endosperm fraction (En) which remained included about20% of the aleurone layer which had not been removed from thecrease. Those grains not dissected formed the whole grain fraction(WG).Determination of Fresh Weight and Dry Weight. Fractions from

one of the heads were placed in weighed sample tubes which werestoppered and reweighed. The tubes were then opened, heated at110 C for 16 to 20 hr, and cooled in a desiccator before beingweighed again.

Extraction. Fractions from the other head were immediatelyplaced in liquid nitrogen. Each fraction was then poured into astone mortar, and when the nitrogen had evaporated the plantmaterial was homogenized by hand with a chilled pestle. Either2 or 4 ml of 0.4 N perchloric acid were added so that the whole ofthe homogenate was wetted before the acid froze. When themortar had warmed to room temperature, the suspension waspoured into a centrifuge tube.

Total Phosphorus Determination. Portions of the suspension(0.1-0.5 ml) were placed in glass tubes and dried at 110 C. Theresidues were digested in 2 or 4 ml of 70%0 (w/v) perchloric acidaccording to the method of Galanos and Kapoulas (6), and phos-phorus was determined as described by Bartlett (3).

Inorganic Phosphorus Determination. The crude extracts werecentrifuged at 2000g, and aliquots of the supernatant fractions

376

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PHYTIC ACID IN WHEAT GRAIN

Fig 1(a) Fig I (b)

s0

60-

~40-E

20

I I ____ ____ ___ ____ __*_ _ WG| __ l __l

Fig 2(a) Fig 2(b)

80

60 En '

40 0

240-701~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4

Fig 3(a) Fig 3(b)

200-

200101~~~ ~ ~ ~~~~~~~E

100 /A

-7/ WG~~~W

Days DaysFIG. 1. Fresh weight (@), dry weight (a), and moisture content (A) of the whole grain (WG) from the normal (a) and water-stressed (b)

plants plotted against the- time after anthesis. The period ("a" to "b") during which water was withheld from the latter group of plants is indi-cated in Figure lb.

FIG. 2. Fresh weights of the fractions testa (T), embryo plus scutellum (E + S), aleurone (A), and endosperm (En) for the normal (a) andstressed (b) grain are plotted against time after anthesis. That portion of the fresh weight equivalent to the dry weight is represented by the cross-hatched areas. The remainder corresponds to the moisture content. Data for the whole grain (WG) taken both before and after dissection waspossible are also presented.

FIG. 3. Total phosphorus (PT) content of the fractions T, (E + S), A, and En for the normal (a) and stressed (b) grain plotted against timeafter anthesis. That portion of PT which was phytic acid phosphorus is represented by the cross-hatched areas. Data for WG taken both beforeand after dissection was possible are also presented.

Plant Physiol. Vol. 45, 1970 377



Plant Physiol. Vol. 45, 1970

were adjusted to 4 ml with water. Then 6 ml of isobutanol and1 ml of acid molybdate reagent were added(24), and the methodof Jennings and Morton was followed (11).

Phytic Acid Phosphorus. Portions (0.20 ml) of the centrifugedextracts were applied in a narrow transverse band 4.0 cm longacross a Whatman 3MM chromatography paper which had beenpreviously washed first in 0.1 M oxalic acid and then with diluteammonia solution. Two such bands together with phytic acidstandards were spaced across a 15-cm strip of paper which wassubjected to electrophoresis at 2 kv for 45 min in the apparatusdescribed by Tate (23). When dry the electrophoretograms werepassed through the 95%O acetone dip reagent of Harrap (8) andheated at 65 C for 15 min before irradiating with an ultravioletlamp. The blue spots which indicated phytic acid were cut fromthe paper (areas less than 10 cm2) and digested with 10 ml of70% (w/v) perchloric acid in Kjeldahl flasks fitted with coldfingers, and the phosphorus was determined as described (3).The recovery of standard phytic acid solutions in these procedureswas at least 90%.

Luciferin-Luciferase Assay for ATP. To 0.1 ml of each of thecentrifuged extracts was added 0.9 ml of water, and 0.1-mlaliquots of this solution were analyzed for ATP by the methodof Strehler and Totter (22) as modified by Stanley and Williams(21). Internal standards were incorporated in all assays whichwere performed within a few hours of the extraction.Exchange of Inorganic Phosphate with Phytic Acid. Phytase was

prepared by ammonium sulphate precipitation of a wheat branextract according to the method of Nagai (14). The dialyzed solu-tion contained 12.5 mg of protein per ml. In 1 %- (w, v) sodium

0

.2

0Io

phytate solution buffered to pH 5.5 with acetic acid this enzymepreparation liberated 3.8 ,umoles of inorganic phosphate per hrper mg of protein at 20 C. The exchange of 32p inorganic phos-phate (32Pi) with phytic acid was examined under these condi-tions.A solution (pH 5.5) containing 1 l sodium phytate (w/v),

phytase (1.2 mg/ml), and 2.2 ,uc of 32Pi per ml was incubated at20 C. Samples were removed at intervals, and the reaction wasstopped by adding perchloric acid (0.05 ml of 70% [w/v] HCl04per ml). The precipitated protein was removed by centrifuging,and the phytic acid in aliquots (0.05 ml) of the supernatant was

Table I. Effect ofDissectioni oii Various Parameters of Wheat GraillIn each case values for the four dissected fractions were summed

and expressed as a percentage of the value of the parameter ob-tained from a similar sample of whole grain. This figure indicatesthe change in a given parameter brought about by dissection.

ITotalPhytate In-

Parameter Fresh Dry ToaPhytat Phos-AT oraiPWeight Weight phohsoPhossA Phorgaphorus phorus phorus

Numbers of dissections 17 17 15 11 17 17Range of recovery (%) 83-108 89-110 87-11260-129 46-98 80-128Mean recovery (%) 90 96 100 92 73 109Standard deviation of ±9 +5 ±9 ±20 ±16 ±14mean recoveries (%)

Days DaysFIG. 4. ATP content of the fractions T, (E + S), A, and En for the normal (a) and stressed (b) grain is plotted against time after anthesis.

Values for WG taken both before and after dissection was possible are also presented.FIG. 5. Inorganic phosphorus content (Pi) of the fractions is plotted in the same manner.

378 WILLIAMS




separated by the method described for determining phytic acid.The area of the paper containing phytic acid was cut from thechromatogram and counted in a Beckman low $11 gas flowproportional counter, and then the phosphorus was determinedas before.A similar experiment was performed except that the incubation

mixture contained 20 mg/ml of wheat bran which had beenhomogenized in liquid nitrogen in place of the sodium phytateand purified phytase enzyme.

RESULTS

Accuracy of the Data. The changes in fresh weight and dryweight during grain development are shown in Figure la. Similarresults for the whole grain fraction (WG) were obtained on twoother occasions from wheat grown under field conditions, and thesame pattern is observed when wheat is grown under optimumconditions in the phytotron (P. E. Stanley, personal communica-tion). All are very similar both quantitatively and qualitativelyto the results recorded by Jennings and Morton (10). Althoughour data were obtained from samples of only 10 grains from asingle head, it appears, therefore, that for components of thegrain which exhibit little variation from day to day this smallsample size introduces a negligible sampling error. The differencesbetween the normal growth pattern (Fig. la) and the one forgrain grown with an interrupted water supply (Fig. lb) aretherefore real differences.

Before the 7th day after anthesis the grain was too small todissect, and after the 35th day it is too dehydrated. Examinationof the individual morphological fractions was therefore restrictedto the central period of the grain's development.For each variable which was recorded for the dissected fractions

the same one was recorded for a similar sample of whole graintaken from the same head. The sum of the values for the testafraction (T), the embryo and scutellum fraction (E + S), thealeurone fraction (A), and the endosperm fraction (En) wasexpressed as a percentage of the value for WG. Percentage re-coveries for each component, after each dissection, were deter-mined in this way, and a summary of the results is given in Table I.Recovery of dry material and total phosphorus was about 100%.However, there was a small loss of fresh weight probably causedby evaporation of water during dissection. Small changes in theinorganic and phytic acid phosphorus are consistent with theaction of phosphatases. The recovery of ATP is definitely low,however, and must reflect the high level of ATPase in thesetissues. A loss of between 12 and 33% of the ATP present in theintact wheat grain was reported by Jenner (9) although hisdissection procedure was simpler and more rapid than the onedescribed here.

In order to compensate for this loss of ATP the values for thefractions T, (E + S), A, and En obtained after each dissectionwere corrected according to the percentage recovery of ATP notedfor that dissection. Values of the other components were correctedin a similar manner.The variation in the data shown in Table I is small relative to

the changes most of the variables exhibit during development(Figs. 1-5). Errors of this size do not, therefore, obscure anyover-all trend or marked changes which occur.

Presentation of the Data. The changes in the testa, embryo plusscutellum, aleurone, and endosperm of fresh weight, dry weight,total phosphorus (PT), phytate phosphorus (Ps), ATP, and inor-ganic phosphorus are shown in Figures 2 to 5. For the dissectedfractions the values obtained for T were first plotted and a curvedrawn through these points. The results for (E + S), A, and Enwere superimposed similarly. When the grain was either too smallor too mature for dissection the data for the whole grain fractionhas been plotted.

Variation in Fresh Weight and Dry Weight. Figure 2 shows thatthe effect of the water stress is to induce a low fresh weight andmoisture content between days 14 and 31 and also to depress therate of accumulation of dry matter during this period. Theseeffects are most pronounced in the endosperm and testa whichappear to buffer the aleurone and embryo from this change in theenvironment.

Distribution of Phytic Acid Phosphorus (PO) and Total Phos-phorus (PT). The rate of accumulation of phytic acid in thealeurone layer is maximal at day 23 in the stressed grain and atday 28 in the normal grain (Fig. 6, a and b) although both grainsfinally dehydrate at approximately the same time (Fig. 2). Similareffects are noted in the embryo plus scutellum fraction althoughthey are not so marked (Figs. 3 and 6).

It would appear from Figure 3 that the endosperm containssignificant quantities of phytic acid, but at several stages duringdevelopment both before and after day 30 no trace of this com-pound was found in endosperm which had been carefully dis-sected from the cheeks of the grain so that contamination withthe aleurone layer was avoided. This result is in accord with thework of Rijven (17). It is concluded, therefore, that significantamounts of phytic acid do not occur in the endosperm and thatthe levels recorded in Figure 3 merely represent contamination ofthe endosperm fraction by the aleurone layer.

Distribution of ATP and Inorganic Phosphorus. Figure 4 showsthe amounts of ATP found in the fractions. The water stressproduced a marked decrease in the total ATP level which reached

DaysFIG. 6. a: Values for the molar concentration of ATP (@), in the

aleurone fraction (A) and the embryo plus scutellum fraction (E + S)from the normal grain were calculated by dividing the ATP contentof these fractions (Fig. 4) by the respective moisture content (Fig. 2a)and are plotted against time after anthesis. The relative rate of changein the phytic acid phosphorus (PO) with time (dP,/dt) for these frac-tions is similarly plotted (U). This function was determined graphicallyfrom a plot of Ps against time for these tissues (Fig. 3a). b: Data for thestressed gain analogous to those presented in part a for normal grainare displayed.

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Plant Physiol. Vol. 45, 1970

a minimum near day 24 but increased to greater than normalvalues by day 31. This effect was least in the embryo and scutellumfraction.

Little difference in the distribution pattern of Pi between thetwo treatments was seen, however (Fig. 5).

Relationship between Concentration of ATP and Phytic Acid.The water content of each fraction at any stage during develop-ment may be read from the graphs (Fig. 2), as can the quantityof ATP present (Fig. 4). If at a given point on the time scale thequantity of ATP is divided by the corresponding water content,then a parameter is obtained which has the dimensions of con-centration and represents the mean concentration of ATP in thegiven fraction at that time. Results obtained in this way havebeen plotted for the embryo plus scutellum and aleurone fractions(Fig. 6, a and b). The graphs also show the rate at which phyticacid accumulates (d P4 /dt). This was determined for each fractionby drawing a curve through a plot of phytic acid phosphorusagainst time and then determining graphically the gradient ofthis curve at various points.The ATP concentration is similar in the various components,

and for the normal grain there is no marked change in this levelduring the accumulation of phytic acid although in both the Aand (E + S) fractions the ATP concentration falls markedlyshortly afterwards (Fig. 6a). For the water-stressed grain, theincrease in phytic acid coincides with a low ATP concentrationin fraction A and an increasing ATP level in fraction (E + S).Exchange of Inorganic Phosphate with Phytic Acid. The ex-

change of 32Pi with phytic acid in the presence of an activephytase was examined. The results obtained with a purifiedphytase preparation are shown in Table II, and those obtainedwith a crude bran homogenate are shown in Table III. In neithercase was a significant exchange reaction observed.

Table II. Exchanzge of 32Pi with Phytic AcidExchange of 32Pi (2.2 ,c/ml) with phytic acid (1% w/v) in the

presence of purified wheat bran phytase (1.2 mg/ml) of specificactivity 3.8 ,umoles of Pi liberated per hr per mg in acetate buffer,pH 5.5, at 20 C. At the times indicated the phytic acid in an aliquotof the incubation mixture was separated by electrophoresis andcounted, and then the phosphorus in this fraction was determined(see "Materials and Methods").

Time Phytic Acid Phosphorus Net Radioactivityper 0.05 ml in Phytic Acid

hrj pg cpin

0 240 02 112 04 20 06 12 0

Table III. Exchanige of 32Pi with Entdogenious Phytic AcidExchange of 32Pi (2.2jAc/ml) with the endogenous phytic acid in

homogenized wheat bran (20 mg/ml) suspended in sodium acetatebuffer (0.1 M), pH 5.5, at 20 C. At the times indicated the mixturewas analyzed as described in the legend of Table II.

Phytic Acid Phosphorus Net RadioactivitvTime per 0.05 ml in Phytic Acid

hr pg Cpt?l

0 18.0 30.33 14.0 20.67 12.5 31.00 13.0 41.50 8.0 62.00 4.0 6

DISCUSSION

Three physiological roles suggested for phytic acid in seeds are(a) a phosphorus store (7), (b) an energy store (4, 13), (c) itsrapid synthesis near maturity represents such a drain on ATPthat the metabolism of the seed is inhibited and dormancy ensues(20).The first role has been discussed elsewhere (7). Since phytic

acid accounts for such a large proportion of the phosphorus pres-ent in mature wheat grain (11) and that this compound is un-doubtedly hydrolysed to inorganic phosphate during the firstdays of germination (12) the role of this compound in providingthe growing plant with a significant pool of inorganic phosphorusat this time cannot be denied. The real question is whether phyticacid fulfills any role additional to this one and we shall thereforeturn our attention to the other possibilities.The postulated high phosphoryl transfer potential of phytic

acid (2) has led to the suggestion by Morton and Raison (13)that phytic acid is involved in protein synthesis in the wheatendosperm and also led Biswas and Biswas (4) to suggest thatphytic acid may provide an energy store on which a seed candraw for the very first processes of germination. Our results show,however, that there is no significant level of phytic acid in thewheat endosperm, in agreement with the results of Rijven (17).This conclusion does not support the work of Morton andRaison. Phytic acid may well have been present in their prepara-tion, but this would probably have arisen by contamination ofthe endosperm plastids with aleurone grains from the aleuronelayer. This tissue could easily have been damaged sufficiently inthe rolling process by which the endosperm was obtained torelease considerable quantities of aleurone grains.The suggestion of Biswas and Biswas implies that the enzymes

responsible for the utilization of the energy contained in phyticacid during germination must be present in the mature seed sinceenergy would be required for their synthesis de novo.The enzyme phytase, which can be purified from the mature

seeds and is responsible for the degradation of the phytin duringthe first few days of germination, has, however, all the charac-teristics of a nonspecific phosphomonoesterase (14). If this en-zyme mediates ATP-linked reactions of the type

Phytic acid v/

ADP ATP

inositol pentaphosphate 14-- -. inositol tetraphosphateADP ATP

then it might be expected that inorganic phosphate would ex-change with the phytic acid via exchange reactions with the ATP.No such evidence for the postulated phosphotransferase proper-ties of phytase was observed with either the purified enzyme orthe crude bran homogenate.

There is, therefore, no substantiated evidence to support thehypothesis that the phytin in seeds supplies energy for the proc-esses of germination. On the contrary, recent work has shownthat respiration in wheat grain begins very early during germina-tion and certainly before substantial amounts of phytic acid havebeen hydrolyzed (15). It is unlikely, therefore, that stored energyis required during this period.

If Sobolev's postulate (c) is true, then the level of ATP shouldfall substantially with the rise in phytic acid. In fact, the resultspresented in Figure 6 show that this is not so since the concen-tration of ATP does not fall to low levels until approximately7 days after the maximum rate of phytic acid formation (Fig. 6).Nevertheless, in rice grain (1) and in the potato tuber (18) asharp rise in phytic acid occurs shortly before maturity, whichsuggests that the synthesis of phytic acid is associated with theonset of dormancy.

380 WILLIAMS




Comparison of Figure 6a with 6b shows that maximum rate ofaccumulation of phytic acid in the aleurone layer of the normalgrain occurred later (day 28) than in the stressed grain (day 23).This suggests that a premature synthesis of phytin is part of thereaction to the environmental stress and may reflect a generalmechanism by which the synthesis of phytin plays a part in theslowing of metabolism prior to dormancy. The results describedhere indicate that removal of the ATP pool is not the mechanismfor such a reaction. An alternative mechanism may be that syn-thesis of the strongly chelating phytic acid (16) exerts an effecton the cellular metabolism by combining with multivalent cations(19) which are known to play a significant part in the control ofmany cellular processes, particularly those involving phospho-transferases on which energy metabolism depends (5).

Acknowledgments-The author would like to thank Professor D. J. D. Nicholas forhis continuous encouragement throughout this work and the Australian Common-wealth Wheat Industry Research Council, who gave generous financial assistance.Thanks are also due to Miss P. Lim of this department for a gift of purified wheat branphytase and to Mr. Geoffrey Clarke for skillful technical assistance.

LITERATURE CITED

1. ASADA, K. AND Z. KASAI. 1962. Formation of myo-inositol and phytin inripening rice grains. Plant Cell Physiol. 3: 397-406.

2. ATKINSON, M. R. AND R. K. MORTON. 1959. In: M. Florkin and H. S. Mason,eds., Comparative Biochemistry, Vol. 2, Part 1. Academic Press, New York.

3. BARTLETT, G. R. 1959. Phosphorus assay in column chromatography. J. Biol.Chem. 234: 466-468.

4. BiswAs, S. AND B. B. BIswAs. 1965. Enzymatic synthesis of guanosine tri-phosphate. Biochim. Biophys. Acta 108: 710-713.

5. BYGRAVE, F. L. 1967. The ionic environment and metabolic control. Nature214: 667-671.

6. GALANos, D. S. AND V. M. KAPOULAS. 1966. A rapid method for the determina-tion of organic nitrogen and phosphorus based on a single perchloric acid deter-mination. Anal. Chim. Acta 34: 360-366.

7. HALL, J. R. AND T. K. HoDGEs. 1966. Phosphorus metabolism of germinatingoat seeds. Plant. Physiol. 41: 1459-1464.

8. HARRAP, F. E. G. 1960. The detection of phosphate esters on paper chromato-grams. Analyst 85: 452.

9. JENNER, C. F. 1968. The composition of soluble nucleotides in the developingwheat grain. Plant Physiol. 43: 41-49.

10. JENNINGS, A. C. AND R. K. MORTON. 1963. Changes in carbohydrates, proteinand non-protein nitrogenous compounds of developing wheat grain. Aust. J.Biol. Sci. 16: 318-331.

11. JENNINGS, A. C. AND R. K. MORTON. 1963. Changes in nucleic acids and otherphosphorus-containing compounds of developing wheat grain. Aust. J. Biol.Sci. 16: 332-341.

12. MIHMLOVIC, M. L., M. ANTIC, AND D. HADzIjEv. 1965. Chemical investigationof wheat. VIII. Dynamics of various forms of phosphorus in wheat during itsoogenesis. The extent and mechanism of phytic acid decomposition in germinat-ing wheat grain. Plant Soil 23: 117-128.

13. MORTON, R. K. AND J. K. RmsON. 1963. A complete intracellular unit for in-corporation of amino acid into storage protein utilizing adenosine triphosphategenerated from phytate. Nature 200: 429-433.

14. NAGAI, Y. AND S. FUNAHASHI. 1963. Phytase from wheat bran. Agr. Biol. Chem.27: 619-624.

15. NEGVOGOROVA, L. A. AND N. M. BoRisovA. 1967. Trigger mechanism of ger-minating seeds. Soviet Plant Physiol. 14: 65-71.

16. POSTERNAK, T. 1965. The Cyclitols. Holden-Day Inc., San Francisco. p. 223.17. RIJvEN, A. 1964. Distribution of phosphorus during wheat grain development.

Aust. J. Biol. Sci. 17: 572-574.18. SAMOTUS, B. AND S. SCHWIMMR. 1962. Phytic acid as a phosphorus reservoir in

the developing potato tuber. Nature 194: 578-579.19. SEN, T. L. N. AND A. M. ALTsCHUL. 1967. Isolation of globoids from cotton-

seeds aleurone grain. Arch. Biochem. Biophys. 121: 678-684.20. SOBOLEv, A. M. AND M. A. RoDIoNovA. 1966. Phytin synthesis by aleurone

grain in ripening sunflower seeds. Soviet Plant Physiol. (Eng. Transln.) 13:958-961.

21. STANLEY, P. E. AND S. G. WILLIAMS. 1968. Use of the liquid scintillation spec-trometer for determining adenosine triphosphate by the luciferase enzyme. Anal.Biochem. 29: 381-392.

22. STREHLER, B. L. AND J. R. TOTTER. 1952. Firefly luminescence in the study ofenergy transfer mechanisms. Arch. Biochem. Biophys. 40: 28.

23. TATE, M. E. 1968. Separation of myo-inositol pentaphosphates by movingpaper electrophoresis. Anal. Biochem. 23: 141-149.

24. WEIL-MALHERBE, H. AND R. H. GREEN. 1951. The catalytic effect of molybdateon the hydrolysis of organic phosphorus bonds. Biochem. J. 49: 286-292.

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TheRole of Phytic Acid in the Wheat Grain1

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