The Role of Phytic Acid in the Wheat Grain

Author(s): S. G. Williams
Source: Plant Physiology, Vol. 45, No. 4 (Apr., 1970), pp. 376-381
Published by: American Society of Plant Biologists
Stable URL: http://www.jstor.org/stable/4261994
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Plant Physiol. (1970) 45, 376-381

The Role of Phytic Acid in the Wheat Grain1

Receivedfor publicationDecember23, 1968

S. G. WILLIAMS2
Department of Agricultural Biochemistry, Waite Agricultural Research Institute, University of Adelaide,
Adelaide, South Australia

ABSTRACT (maximumtemperature27 C). Waterwas given regularlyevery

2 days. A second plantingwas maintainedin a phytotron(day
The concentrations of adenosine triphosphate and phytic
temperature22 C, night temperature10 C, day length 10 hr
acid in testa, embryo plus scutellum, aleurone, and endo- increasedto 12 hr shortlybeforeanthesis)and wateredas before
sperm fractions from grain of Triticum vulgare cv. Insignia exceptthat no waterwas givenbetweenthe 10thday afteranthesis
have been determined during development under both nor- and the 17th day when the flag leaf showed distinct signs of
mal conditions and those of water stress. Phytic acid was
wilting.
not detected in the endosperm. In the embryo plus scutel- Wheat bran was commercialmaterialavailablelocally.
lum and aleurone fractions there was a rapid build-up of Chemicals.Adenosinetriphosphateand desiccatedfireflytails
phytic acid, but the adenosine triphosphate level did not were purchasedfrom the Sigma ChemicalCompany,St. Louis,
change markedly at this time. These results are not con- Missouri.A solutionof ATP (10-4 M) in 0.01 Mphosphatebuffer
sistent with physiological roles previously suggested for (pH 7.4) was preparedand stored at -15 C. Electrophoresisof
phytic acid other than the role of phytin as a phosphorus this solution, by the method describedunder the determination
and cation store for the germinating seed. of phytic acid phosphorus,indicatedthat it contained2.7% of
adenosinediphosphateand was stablefor severalmonths.
"Carrier-free"32p inorganicphosphorus (32Pi)was obtained
I from the AustralianAtomic EnergyCommission,LucasHeights,
N.S.W., Australia. The other chemicalswere analytical grade
reagents,and all waterused was double distilledfrom glass.
Dissection.Two heads which had floweredon the same day
Phytic acid is a majorphosphoruscomponentof many seeds. were cut and immediatelyplaceduprightin water.The top third
In mature grain of Triticum vulgare cv. Gabo this compound, of each head was removedand discarded,and 16 to 20 grains
which accountsfor about 88% of the acid-solublephosphorus from the centerof the head wereremovedone at a time and every
and 53% of the total phosphorus,is largelyconcentratedin the alternategrain(8 to 10 in number)was quicklydissectedat room
testa-pericarpfraction (11). Despite the abundanceof this ma- temperaturewith a needle and forceps. The pericarpabove the
terialin the seed, very little is known of its physiologicalrole. A cross cells and tube cells was firstremovedfrom the grain (testa
suggestionby Atkinsonand Morton (2) that phyticacid may be fraction,T) and then the embryoplus scutellum(E + S) was
able to phosphorylatenucleotidediphosphatesto triphosphates dissectedas a unit. Next a longitudinalcut was madein the grain
has some supportfrom the work of Mortonand Raison (13) and opposite to the creaseand the aleuronelayer, togetherwith the
Biswas and Biswas (4), but this evidenceis not unequivocable. chlorophylllayerwhenpresent,was peeledoff (aleuronefraction,
RecentlySobolev and Rodionova (20) reportedthat phytic acid A). The endospermfraction(En) whichremainedincludedabout
was synthesisedby a mixtureof aleuronegrainsand mitochondria 20% of the aleuronelayerwhichhad not been removedfrom the
isolated from ripening sunflowerseeds when myoinositol and crease.Thosegrainsnot dissectedformedthe wholegrainfraction
succinatewerepresent.On additionof hexokinaseand glucoseto (WG).
this mixturethe amountof adenosinetriphosphatein the medium Determinationof Fresh Weight and Dry Weight. Fractions from
fell, and no phyticacid was produced.Both sets of findingsimplyone of the headswereplacedin weighedsampletubes whichwere
that the rate of synthesisof phytic acid is closely linked to the
stopperedand reweighed.The tubes werethen opened,heatedat
ATP level in the cell. For this reason the amountsof ATP and 110 C for 16 to 20 hr, and cooled in a desiccatorbefore being
phyticacid in severalmorphologicallydistinctcomponentsof the weighedagain.
wheatgrainweredeterminedfrom shortlyafteranthesisthrough Extraction.Fractionsfrom the other head were immediately
to ripening. placed in liquid nitrogen.Each fractionwas then pouredinto a
stone mortar,and when the nitrogenhad evaporatedthe plant
MATERIALS AND METHODS materialwas homogenizedby hand with a chilled pestle. Either
2 or 4 ml of 0.4 N perchloricacid wereaddedso that the wholeof
Plant Material. Triticum vulgare cv. Insignia was grown in the homogenatewas wetted before the acid froze. When the
18-cm pots containingsterilizedpotting soil. Four plants were mortar had warmedto room temperature,the suspensionwas
grownin each pot, and only one tillerfrom each was allowedto pouredinto a centrifugetube.
develop. The date of anthesis of each ear was recorded.One Total Phosphorus Determination. Portions of the suspension
plantingwas maintainedin a glasshouseand ripenedin October (0.1-0.5 ml) were placed in glass tubes and dried at 110 C. The
residuesweredigestedin 2 or 4 ml of 70% (w/v) perchloricacid
' Financialassistancefromthe AustralianCommonwealth Wheat accordingto the methodof Galanosand Kapoulas(6), and phos-
IndustryResearchCouncilis gratefully
acknowledged. phoruswas determinedas describedby Bartlett(3).
2Presentaddress:WellcomeResearchLaboratories, Beckenham, InorganicPhosphorusDetermination.The crude extractswere
England. centrifugedat 2000g, and aliquots of the supernatantfractions
376
Plant Physiol. Vol. 45, 1970 PHYTIC ACID IN WHEAT GRAIN 377

Fig 1(a) Fig

60

._
c40
E

Fig 2 (a) Fig 2(b)

Fig 3 (a) Fig 3(b)

0
Days Days
FIG. 1. Fresh weight (a), dry weight (a), and moisturecontent (A) of the whole grain (WG) from the normal (a) and water-stressed(b)
plants plotted againstthe time after anthesis.The period ("a" to "b") duringwhich water was withheldfrom the lattergroup of plants is indi-
cated in Figure lb.
FIG. 2. Fresh weights of the fractionstesta (T), embryo plus scutellum(E + S), aleurone (A), and endosperm(En) for the normal (a) and
stressed(b) grainare plottedagainsttime after anthesis.That portionof the freshweightequivalentto the dry weight is representedby the cross-
hatchedareas. The remaindercorrespondsto the moisturecontent. Data for the whole grain (WG) taken both before and after dissectionwas
possibleare also presented.
FIG. 3. Total phosphorus(PT) content of the fractionsT, (E + S), A, and En for the normal (a) and stressed (b) grain plotted againsttime
after anthesis.That portion of PT which was phytic acid phosphorusis representedby the cross-hatchedareas.Data for WG taken both before
and afterdissectionwas possibleare also presented.
378 WILLIAMS Plant Physiol. Vol. 45, 1970

were adjusted to 4 ml with water. Then 6 ml of isobutanol and phytate solution buffered to pH 5.5 with acetic acid this enzyme
1 ml of acid molybdate reagent were added(24), and the method preparation liberated 3.8 ,moles of inorganic phosphate per hr
of Jennings and Morton was followed (11). per mg of protein at 20 C. The exchange of 32p inorganic phos-
Phytic Acid Phosphorus. Portions (0.20 ml) of the centrifuged phate (32Pi) with phytic acid was examined under these condi-
extracts were applied in a narrow transverse band 4.0 cm long tions.
across a Whatman 3MM chromatography paper which had been A solution (pH 5.5) containing 1% sodium phytate (w/v),
previously washed first in 0.1 M oxalic acid and then with dilute phytase (1.2 mg/ml), and 2.2 ,c of 32Pi per ml was incubated at
ammonia solution. Two such bands together with phytic acid 20 C. Samples were removed at intervals, and the reaction was
standards were spaced across a 15-cm strip of paper which was stopped by adding perchloric acid (0.05 ml of 70% [w/v] HC104
subjected to electrophoresis at 2 kv for 45 min in the apparatus per ml). The precipitated protein was removed by centrifuging,
described by Tate (23). When dry the electrophoretograms were and the phytic acid in aliquots (0.05 ml) of the supernatant was
passed through the 95% acetone dip reagent of Harrap (8) and
heated at 65 C for 15 min before irradiating with an ultraviolet
lamp. The blue spots which indicated phytic acid were cut from Table I. Effect of Dissection on Various Parameters of Wheat Grain
the paper (areas less than 10 cm2) and digested with 10 ml of In each case values for the four dissected fractions were summed
70% (w/v) perchloric acid in Kjeldahl flasks fitted with cold and expressed as a percentage of the value of the parameter ob-
fingers, and the phosphorus was determined as described (3). tained from a similar sample of whole grain. This figure indicates
The recovery of standard phytic acid solutions in these procedures the change in a given parameter brought about by dissection.
was at least 90%.
Luciferin-LuciferaseAssay for ATP. To 0.1 ml of each of the In-
Total Phytate
centrifuged extracts was added 0.9 ml of water, and 0.1-ml Parameter FreshDry organic
Weiht Phos- Phos- ATPo Phos-
Weight Weight
weight
aliquots of this solution were analyzed for ATP by the method phorus phorus phorus
of Strehler and Totter (22) as modified by Stanley and Williams
(21). Internal standards were incorporated in all assays which Numbers of dissections 17 17 15 11 17 17
were performed within a few hours of the extraction. Range of recovery (%) 83-108 89-110 87-112 60-12946-98 80-128
Exchange of Inorganic Phosphate with Phytic Acid. Phytase was Mean recovery (%) 90 96 100 92 73 109
prepared by ammonium sulphate precipitation of a wheat bran Standard deviation of ±9 ±5 ±9 ±20 =-16 =14
extract according to the method of Nagai (14). The dialyzed solu- mean recoveries (%)
tion contained 12.5 mg of protein per ml. In 1% (w/v) sodium

I-

!<
o

-i

DaysDays
FIG.4. ATP content of the fractionsT, (E + S), A, and En for the normal (a) and stressed (b) grain is plotted againsttime after anthesis.
Values for WG taken both before and after dissectionwas possibleare also presented.
FIG.5. Inorganicphosphoruscontent (Pi) of the fractionsis plotted in the same manner.
Plant Physiol. Vol. 45, 1970 PHYTIC ACID IN WHEAT GRAIN 379

separatedby the method describedfor determiningphytic acid. Variationin FreshWeightandDry Weight.Figure2 showsthat
The area of the paper containingphytic acid was cut from the the effect of the waterstressis to inducea low fresh weightand
chromatogramand counted in a Beckman low 3II gas flow moisturecontentbetweendays 14 and 31 and also to depressthe
proportionalcounter,and then the phosphoruswas determined rate of accumulationof dry matter during this period. These
as before. effects are most pronouncedin the endospermand testa which
A similarexperimentwas performedexceptthat the incubation appearto bufferthe aleuroneand embryofromthis changein the
mixture contained 20 mg/ml of wheat bran which had been environment.
homogenizedin liquid nitrogenin place of the sodium phytate Distributionof Phytic Acid Phosphorus (P4) and Total Phos-
and purifiedphytaseenzyme. phorus(PT). The rate of accumulationof phytic acid in the
aleuronelayeris maximalat day 23 in the stressedgrain and at
RESULTS day 28 in the normalgrain(Fig. 6, a and b) althoughboth grains
finallydehydrateat approximately the sametime (Fig. 2). Similar
effectsare noted in the embryoplus scutellumfractionalthough
Accuracyof the Data. The changes in fresh weight and dry
weightduringgraindevelopmentare shownin Figurela. Similar they are not so marked(Figs. 3 and 6).
resultsfor the whole grainfraction(WG) were obtainedon two It would appearfrom Figure 3 that the endospermcontains
otheroccasionsfromwheatgrownunderfieldconditions,and the significantquantitiesof phytic acid, but at severalstages during
same patternis observedwhen wheat is grown under optimum developmentboth beforeand after day 30 no trace of this com-
conditionsin the phytotron(P. E. Stanley,personalcommunica- pound was found in endospermwhich had been carefullydis-
sected from the cheeks of the grain so that contaminationwith
tion). All are very similarboth quantitativelyand qualitatively the aleuronelayerwas avoided.This resultis in accordwith the
to the resultsrecordedby Jenningsand Morton (10). Although
our data were obtainedfrom samplesof only 10 grains from a work of Rijven (17). It is concluded,therefore,that significant
amountsof phytic acid do not occurin the endospermand that
single head, it appears,therefore,that for componentsof the the levelsrecordedin Figure3 merelyrepresentcontaminationof
grain which exhibit little variationfrom day to day this small the endospermfractionby the aleuronelayer.
samplesize introducesa negligiblesamplingerror.The differences Distributionof ATP and InorganicPhosphorus.Figure4 shows
between the normal growth pattern (Fig. la) and the one for
the amounts of ATP found in the fractions.The water stress
grain grown with an interruptedwater supply (Fig. lb) are
thereforereal differences. produceda markeddecreasein the total ATP levelwhichreached
Before the 7th day after anthesisthe grain was too small to
dissect,and afterthe 35th day it is too dehydrated.Examination
of the individualmorphologicalfractionswas thereforerestricted
to the centralperiodof the grain'sdevelopment.
For eachvariablewhichwas recordedfor the dissectedfractions
the same one was recordedfor a similarsample of whole grain
taken from the same head. The sum of the values for the testa
fraction (T), the embryo and scutellumfraction (E + S), the
aleurone fraction (A), and the endospermfraction (En) was
expressedas a percentageof the value for WG. Percentagere-
coveriesfor each component,after each dissection,were deter-
minedin thisway,anda summaryof theresultsis givenin TableI.
Recoveryof dry materialand total phosphoruswas about 100%.
However,therewas a small loss of freshweightprobablycaused
by evaporationof waterduringdissection.Small changesin the
inorganic and phytic acid phosphorusare consistent with the
action of phosphatases.The recoveryof ATP is definitelylow,
however, and must reflect the high level of ATPase in these
tissues.A loss of between12 and 33% of the ATP presentin the
intact wheat grain was reported by Jenner (9) although his
dissectionprocedurewas simplerand more rapid than the one
describedhere.
In orderto compensatefor this loss of ATP the valuesfor the
fractionsT, (E + S), A, and En obtainedafter each dissection
werecorrectedaccordingto the percentagerecoveryof ATP noted
for that dissection.Valuesof the othercomponentswerecorrected
in a similarmanner.
The variationin the data shown in Table I is small relativeto
the changes most of the variablesexhibit during development
(Figs. 1-5). Errorsof this size do not, therefore,obscureany 10
over-alltrendor markedchangeswhich occur. Days
Presentationof the Data. The changesin the testa, embryoplus FIG. 6. a: Values for the molar concentrationof ATP (0), in the
scutellum,aleurone,and endospermof fresh weight,dry weight, aleuronefraction(A) andthe embryoplusscutellum fraction(E + S)
totalphosphorus(PT), phytatephosphorus(P<), ATP, and inor- fromthe normalgrainwerecalculatedby dividingthe ATPcontent
ganic phosphorusare shown in Figures2 to 5. For the dissected of thesefractions(Fig.4) by the respective
moisturecontent(Fig.2a)
fractionsthe values obtainedfor T werefirstplottedand a curve andareplottedagainsttimeafteranthesis.Therelativerateof change
in the phyticacidphosphorus (Ps)withtime (dP,/dt)for thesefrac-
drawnthroughthese points. The resultsfor (E + S), A, and En tionsis similarlyplotted(I). Thisfunctionwasdetermined
were superimposedsimilarly.Whenthe grainwas eithertoo small graphically
froma plotof Ps againsttimeforthesetissues(Fig.3a).b: Dataforthe
or too maturefor dissectionthe datafor the whole grainfraction stressedgainanalogousto thosepresentedin parta for normalgrain
has been plotted. aredisplayed.
380 WILLIAMS Plant Physiol. Vol. 45, 1970

a minimum near day 24 but increased to greater than normal DISCUSSION

values by day 31. This effect was least in the embryo and scutellum
fraction.
Little difference in the distribution pattern of Pi between the
two 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 quantity
of ATP present (Fig. 4). If at a given point on the time scale the
quantity 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 the
given fraction at that time. Results obtained in this way have
been plotted for the embryo plus scutellum and aleurone fractions
(Fig. 6, a and b). The graphs also show the rate at which phytic
acid accumulates (d Po/dt). This was determined for each fraction
by drawing a curve through a plot of phytic acid phosphorus
against time and then determining graphically the gradient of
this 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 level
during the accumulation of phytic acid although in both the A
and (E + S) fractions the ATP concentration falls markedly
shortly afterwards (Fig. 6a). For the water-stressed grain, the
increase in phytic acid coincides with a low ATP concentration
in 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 active
phytase was examined. The results obtained with a purified
phytase preparation are shown in Table II, and those obtained
with a crude bran homogenate are shown in Table III. In neither
case was a significant exchange reaction observed.
Table II. Exchange of 32Pi with Phytic Acid
Exchange of "2Pi(2.2 jc/ml) with phytic acid (1% w/v) in the
presence of purified wheat bran phytase (1.2 mg/ml) of specific
activity 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 aliquot
of the incubation mixture was separated by electrophoresis and
counted, and then the phosphorus in this fraction was determined
(see "Materials and Methods").
Three physiological roles suggested for phytic acid in seeds are
(a) a phosphorus store (7), (b) an energy store (4, 13), (c) its
rapid synthesis near maturity represents such a drain on ATP
that 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 first
days of germination (12) the role of this compound in providing
the growing plant with a significant pool of inorganic phosphorus
at this time cannot be denied. The real question is whether phytic
acid fulfills any role additional to this one and we shall therefore
turn 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 wheat
endosperm and also led Biswas and Biswas (4) to suggest that
phytic acid may provide an energy store on which a seed can
draw for the very first processes of germination. Our results show,
however, that there is no significant level of phytic acid in the
wheat endosperm, in agreement with the results of Rijven (17).
This conclusion does not support the work of Morton and
Raison. Phytic acid may well have been present in their prepara-
tion, but this would probably have arisen by contamination of
the endosperm plastids with aleurone grains from the aleurone
layer. This tissue could easily have been damaged sufficiently in
the rolling process by which the endosperm was obtained to
release considerable quantities of aleurone grains.
The suggestion of Biswas and Biswas implies that the enzymes
responsible for the utilization of the energy contained in phytic
acid during germination must be present in the mature seed since
energy 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 during
the 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 < , >

ADP ATP
Time Phytic Acid Phosphorus Net Radioactivity
per 0.05 ml in Phytic Acid
inositol pentaphosphate < , , > inositol tetraphosphate
hr Pug cpm ADP ATP
0 240 0
2 112 0 then it might be expected that inorganic phosphate would ex-
4 20 0 change with the phytic acid via exchange reactions with the ATP.
6 12 0 No such evidence for the postulated phosphotransferase proper-
ties of phytase was observed with either the purified enzyme or
the crude bran homogenate.
Table III. Exchange of 32P with EndogenousPhytic Acid There is, therefore, no substantiated evidence to support the
Exchange of 32Pi(2.2 ,c/ml) with the endogenous phytic acid in hypothesis that the phytin in seeds supplies energy for the proc-
homogenized wheat bran (20 mg/ml) suspended in sodium acetate esses of germination. On the contrary, recent work has shown
buffer (0.1 M), pH 5.5, at 20 C. At the times indicated the mixture that respiration in wheat grain begins very early during germina-
was analyzed as described in the legend of Table II. tion and certainly before substantial amounts of phytic acid have
been hydrolyzed (15). It is unlikely, therefore, that stored energy
Time Phytic Acid Phosphorus Net'Radioactivity
in Phytic Acid
is required during this period.
per 0.05 ml
If Sobolev's postulate (c) is true, then the level of ATP should
hr Ag cpm fall substantially with the rise in phytic acid. In fact, the results
0 18.0 3 presented in Figure 6 show that this is not so since the concen-
0.33 14.0 2 tration of ATP does not fall to low levels until approximately
0.67 12.5 3 7 days after the maximum rate of phytic acid formation (Fig. 6).
1.00 13.0 4 Nevertheless, in rice grain (1) and in the potato tuber (18) a
1.50 8.0 6 sharp rise in phytic acid occurs shortly before maturity, which
2.00 4.0 6 suggests that the synthesis of phytic acid is associated with the
onset of dormancy.
Plant Physiol.Vol. 45, 1970 PHYTIC ACID IN WHEAT GRAIN 381

Comparisonof Figure6a with 6b showsthat maximumrate of 8. HARRAP, F. E. G. 1960. The detection of phosphate esters on paper chromato-
accumulationof phytic acid in the aleuronelayer of the normal grams. Analyst 85: 452.
9. JENNER, C. F. 1968. The composition of soluble nucleotides in the developing
grainoccurredlater (day 28) than in the stressedgrain (day 23). wheat grain. Plant Physiol. 43: 41-49.
This suggeststhat a prematuresynthesisof phytinis part of the 10. JENNINGS,A. C. AND R. K. MORTON. 1963. Changes in carbohydrates, protein
reaction to the environmentalstress and may reflect a general and non-protein nitrogenous compounds of developing wheat grain. Aust. J.
mechanismby which the synthesisof phytin plays a part in the Biol. Sci. 16: 318-331.
11. JENNINGS,A. C. AND R. K. MORTON. 1963. Changes in nucleic acids and other
slowingof metabolismprior to dormancy.The resultsdescribed phosphorus-containing compounds of developing wheat grain. Aust. J. Biol.
hereindicatethat removalof the ATP pool is not the mechanism Sci. 16: 332-341.
for such a reaction.An alternativemechanismmay be that syn- 12. MIHAILOVIC, M. L., M. ANTIC,ANDD. HADZIJEV.1965. Chemical investigation
of wheat. VIII. Dynamics of various forms of phosphorus in wheat during its
thesis of the stronglychelatingphytic acid (16) exerts an effect
oogenesis. The extent and mechanism of phytic acid decomposition in germinat-
on the cellularmetabolismby combiningwith multivalentcations ing wheat grain. Plant Soil 23: 117-128.
(19) which are known to play a significantpart in the control of 13. MORTON, R. K. ANDJ. K. RAISON. 1963. A complete intracellular unit for in-
many cellular processes,particularlythose involving phospho- corporation of amino acid into storage protein utilizing adenosine triphosphate
transferaseson which energymetabolismdepends(5). generatedfrom phytate. Nature 200: 429-433.
14. NAGAI, Y. AND S. FUNAHASHI. 1963. Phytase from wheat bran. Agr. Biol. Chem.
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Thanks are also due to Miss P. Lim of this departmentfor a gift of purifiedwheat bran 16. POSTERNAK, T. 1965. The Cyclitols. Holden-Day Inc., San Francisco. p. 223.
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Aust. J. Biol. Sci. 17: 572-574.
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