the, changes in these variouis substances relative to each other during germination has not been made. Phytate (Ca, MAfg, K salt of inositol phosphoric.
Plant Physiol. ('1966) -/1, 1459-1464
Phosphorus Metabolism of Germinating Oat Seeds' J. R. Hall and T. K. Hodges Department of Horticulture, University of Illinois, Urbana Received July 18, 1966.
Summary. An investigation has been made of the changes in the major phosphorus containing stubstances in Avena sativa during the first 8 days of dark germination. The endosperm, roots, and shoots were analyzed separately for acid soluble-P, phytic acid-P, inorganic-P, lipid-P, nucleic acid-P, and protein-P. Phytic acid-P comprised 53 % of the total seed phosphate, while the sum of lipid-P, nucleic acid-P and protein-P comprised 27 % of the seed phosphate. All these reserve phosphate materials were mobilized and transferred to the developinig axis. The phosphate from phytic acid appeared almost entirely as inorganic-P in the roots and shoots. A close stoichiometry existed between the rate of loss of nucleic acid-P from the endosperm and its rate -Of appearance in the roots and shoots. Thus no net synthesis of nucleic acid occurred during the 8-day period examined. The rate of synthesis of lipid-P in the roots and shoots exceeded its rate of disappearance from the endosperm during the first 4 days of germination. Protein-P increased in the roots and shoots during germination, btit at a rate less than its rate of disappearance from the endosperm. The results provide a relatively complete description of the over-all aspects of phosphorus metabolism associated with germination of oats. The major metabolic processes associated with seed germination are the mobilization of storage materials in the reserve tissule and their subsequLent transfer to and utilization by the developing embryonic axis. This mobilization, transport, and ultilization has been studied for a variety of substances (cf. 15, 23), buit primarily has been concerned with the various N-containing suibstances suich as proteins and nucleic acids. In light of the importance of various phosphorylated substances in metabolism it seemed desirable to characterize the germinatioln process with respect to the time sequence of changes in the major phosphorylated substances. Althoulgh specific aspects of phosphoruis metabolism dturing germination have been examined such as changes in phytic acid, nucleic acid, phospholipids, etc. (1, 3, 11, 20) an over-all description or balance sheet of the, changes in these variouis substances relative to each other during germination has not been made. Phytate (Ca, MAfg, K salt of inositol phosphoric acid) is generally believed to be the primary reserve phosphate in the seed (3, 16). It has been shown that phytase, the enzyme which catalvzes the hydrolysis of phosphate from phytic acid, increases markedly dturing the first few days of germination (19). The liberated phosphate then
of
fun1ds granlted to the University Illilnois anid by National Science Founl(dation Grant
1 Supported by federal
GB-2281.
1459
presuimably enters into various synthetic reactioniv occuirring in the developing axis. However, the extent of this inorganic-P uitilization during (lark germination for synthetic reactions is uinclear. This stems primarily from the uncertainty of the natture of the mobilization, transfer and tutilization of other reserve phosphate containing compouinds. For example, some investigators believe that ntucleic acids are transported from reserve tissule to axis intact or as the nticleotides (13, 17, 18) while others feel that some de novo synthesis of nucleic acids does occur in the axis (4, 7, 11). Similarly, it is not c!ear whether phospholipids move from the reserve tissue to the axis or whether they are synthesized de novo in the axis, althotugh the latter appeared to be the case for cotton germination (5 ). It has also been shown that phosphoproteins occuir itn seeds (5, 12, 24), however, their mobilization and utilization dulring germination has not beeni examined. Thuis, the relative contribution of variou s phosphorylated materials suich as nucleic acids, phospholipids, phosphoproteins, etc., in relation to phvtic acid for contribuiting phocphate for the various synthetic processes in the axis needs examinlationi. In the present work an attempt was made to obtain an over-all description of phosphortus metabolism associated with germination of oats. We have followed the time sequence of changes in acid soltible-P, phytic acid-P, inorganic-P, lipid-P, nuc'eic acid-P and protein-P ill the endosperm, roots, an(d shoots duiring the first 8 days of germinatior.
1460
PLANT PHYSIOLOGY
Materials and Methods
and
placed in
boilin1g water bath for 10 minuites. of the cleared extract was measuire of phosphorylated protein a
Inorganic phosphate
Approximately 100 seedls of Avena sativa Var. Goodfielcl were planted on paper towels saturated with 100 ml of 0.1 mm CaCl.. in 25.4 X 35.6 cm pyrex baking dishes which w,vere covered with perforated Saran wrap. The dishes were placed in a (lark germinator at 280 for the (desired times. Plants were harvested at 2, 4, 6 and 8 days by carefuilly removing the lemma and palea anid then separating the roots and shoots, with the attached remnants of the embryo, from the romnlainder of the seed. The remainiing part of the seed incltuded both the endospermii anid sctitelltum as well as the other associate(l tisstues. This entire grouip of tissues will be referred to as 'endosperm'. The roots and shoots wvere separated at the tranisitioin zone vith the embryo remnants remaining with the shoot. The tissuie xx-as kept on ice tuntil extractioin. For data at 0( days, the entire dry seed, exclulding lemma anld palea, were analyzed. No attempt was made to separate the embryo. Dry weights were (letei-niiied by drying the tissule after (lissection at 700 for 24 hours. For determination of total-P disappearance from the en(losperm an(d its appearance in the roots andl shoots, the variouis organs were thorouighly homogenized in (leionized water vith a power-driven, conical ,lass homogenizer Ali(quiots of the homogenate were wet washed in 10 (N stulfturic acid. Final clearing was accomplishedl with hydlrogen peroxide. The digested samples were (lillitedi and boiled for 10 mintUtes to break pyrophosphate bonds and then assayedl for total-P according to the methodI of Fiske and SuibbaRow (6). L1xtraction Procedutre. The various plant parts were extracted according to the proceduire of Schneider (21) with slight modifications. The 4 fractions examined were the following: I. Acid Soluble-P. The tisstie was homogenized in ice cold 0.2 N perchloric acid with a power (Iriven conical glass homogenizer. The extract was held on ice 15 minuites prior to clearing by centrifulgation. The residtue was re-extracted twice for 15 minu1tes in ice cold 0.2 perchloric acid. The cleared, acid soluble extracts were combined and analyzed for total-P, inorganlic-P, an(I phytic acid-P. II. Lipid-P. The acid insoluble residtue was extractedl 3 times at room temperatture with ethanol: ether: chloroform (2: 2: 1 vX x). The cleare(d extracts were combined and ain aliquot removedl for N
total-P (leterminationi.
III. Niucleic Acid-P. The (lefatted residuie was washed with ice cold 5 % trichloroacetic acid for miniuttes and then extractedl with an additional portion of % trichloroacetic acid at 900 for 15 minlLtes. The cleared extracts were combined and sample taken for total-P dleterminatioln. I Protcinl-P. The hot trichloroacetic acid insollhlde fraction was suispend(led in Nx NaOH a
1
taken as a (8,21). The final residule was analyzed for total-P and never exceeded 0.5 % of the total-P of the tisstue. Total-P in each of the fractions was determine(d as described above for the total-P contelnt of the variouis organs. Direct estimation of inorganic-P in the acid soltuble extract was determined ou a sample prior to digestion. Phytic acid-P of the acid soluble extract was determined by the method of Asada aind Kasai (2) which first inxvolved bringing the acid soluble fraction to 20 mm with NaEDTA prior to neutralization to preveent coprecipitation of phytic acid (22). The cleared,
neuitralized extracts were then applied to coluiinns (1.0 X 10 cm) of Dowex 1-X-8 chloridle resin (200-400 mesh). The coluimn was washed with
wvater and then eluted with a linear gradienit of HCl. Six ml fractions were collectedl andl total-P
of each fraction
was
determined. Two major
phosphate peaks occuirred from the variouis seed extracts. The first peak (tubes 8-12) was in1organic-P and the second peak (tubes 55-65) eltuted at the same place as an auithentic sample of Na-phytate (Sigma Chemical Company). The inorganic-P peak was not quantitative since the effluent and wash also containe(d inorganic-P. The difficuilties encountered in the qulantitative extraction an(1 estimation of the variouis phosphorylated substances in plants are grelat (7, 9, 10) and as Ingle ( 10) has pointed ouit none of the commonly used extraction proceduires appears to be completely satisfactory for the estimatioii of nulcleic acids. Thuis, the variouis fractions described above are considered to represent only a semi-quiantitative estimate of phospholipids, nutcleic aci(ds, phosphoproteins, etc.
Results and Discussion Figuires 1 alnd 2 show the chainges in fresh anid dry weight of the various plant parts at the sampling times employed. It is evident from figuire 1 that approximately 24 hoturs were re(quiired for the seeds to become fully imbibed. After 24 houirs the endosperm (incluiding the scuitellulm and other associated tissuies) decreased in fresh wveight until (lay 4 and then remained fairly constant. The total plant dry weight (fig 2) showed a marke(d decrease between days 2 and 4 and this corresponds to the most rapid rate of loss of (dry weight from the endosperm. By comparing figuires 1 ad( 2 it can be seen that the fresh weight of the shoot material increases muich more rapidly thain the dIry
weight. Disappearanice of total-P from the en(dosperm ancI its appearance in the roots andl shoots are shown in figuire 3. These changes correspoind (Iquite
closelv to the changes in (lrv wteight except for the
HALL AND HODGES-PHOSPHORUS
I FRESH WEIGHT
3 TOTAL PHOSPHATE
2 DRY WEIGHT
.12
.c
I-
a.
a.:3-
\\Entire Plont
f~~~~~~~Soots
a- .09~ _
41
Endosperm
aa-
\4%
1461
METABOLISMN OF GERMINATING OATS
.06.
Ca-
Endosperm
01 -
Roots
2 -hos
C
.'
Cl)
Shoot
4I
E -
Li
Endo2 erm
.03
Roo
1 S5~~~~~
0
n
4 ENDOSPERM
1
Ir4a.
k\
rotol Ph0sphate. Determined sepo Addvd froctions
%%%\'rs
3
v
6 SHOOTS
5 ROOTS
F.
3_
.
roto/ Phosphote
Deftrmined seporoteiy2 Added froctions d/.
z
-J a-
2
0)
rotal hospate.:
t_
.tz-,~~~~~~~~~~~~~
a.
N\K
Acid soluble-P
\
;
E
l
ME_
Nucleic ocid-P
Ii
Lipid-P
-o-L-
/ /O,
2
Deftermined separately Adoded fretions
~~~~~~~~fif~t
bP
Eli IL
-1 L
(-I v
~
ie" N
I
-
tL~ipid =
Acdso/luble- P
Protein-P
$/,A c ocid- p I
(Iv
'
8 ACID SOLUBLE-P
3r 7a ENDOSPERM
/I ~ ~ //
Protein-P
//fcec idF-P
s
9 INORGANIC-P
ACid soluble-P
2 Plytic odd-P
3_
I--
a: a-
Pi~
7b SHOOTS
z
-Q a-
~
I~
a.
;
(aIV
Aecidsoluble orgonc-P
4-7c ROOTS 2
.6
e~~~~~~~
.A~~~~~~A
o
0) E
//~~~~~
Entire pieIt/
77)ow
2
4
4*t 10 NUCLEIC ACID-P
Ro
Endosperm
1
0.
11 LIPID-P
K ".
---
,
-
Entire p/ont
-i.4 /
,A---__
Endosperm
.4-
"I
0~
.3 *
Endosperm
.2 \ / ~~Shoot I 2~~~~~~~~~~~~. .2-)
E
Roots
s
MeShoots
:!.e1 R
I1
0
0
2
4
6
8
0
2
4
6
8
0
2
4
6
TIME, DAYS TIME, DAYS TIME, DAYS FIGS. 1-12. The changes of various components of the 'endosperm', roots and shoots of oats over mination period. The data are averages of from 2 to 6 separate experiments.
8 ani
8-day
ger-
140)2
I'LANT PHYSIOLOGY
rapid iincrease in phosphate in the shoots which is simil'ar to the rapi(d inicrease in fresh weight of the shoots (fig 1). The distribution of total-P between acid soluble-P, lipid-P, nuicleic acid-P and protein-P anld the time sequlence of chaniges in these fractions in the endosperm, roots, aIId shoots are showni in figuires 4. 5, antd 6, respectively. The (lata are presenite(l in this fashion ini order that olne can readily comppare the relatixe amounllts of these substances. Note, however, that the root (lata are presente(d on an expainde(d scale as compared to the (n(losperm and shoot (lata. Also shown in these figuires are the total-P (leterminei( by homogeniization of the tissuie in water (fig 3) anid the total-P as (letermine(l by addinig the separate fractioIns. In general, close agreemenit was fouindl between these totals except in the case of the root tissuie w hich varied con msiderably, especially at (lav 2. In the case of the endospermi all fractionis (lecreased (1,iring the germination period. Most of the phosphate at all samplinlg times was aci(d soluible ad(l comprised 75 to 85 % of the total-P. The nuicleic acid-P made tip about 12 % of the total-P at (la\ (J an(d decreased graduially, to about 6 % b) the eighth (lay. The lipid-P ma(le uip abotut 6.5 to 8.5 %, of the total seed-P andl the percentage in the endosperm remaine(l fairly constant (diring the germination period. The protein-P conitent decreased from ani initial level of 6 % to only 2 % Onl (lay 8. The large anmoLullt of acid soluble-P in the enid)osperm was comprised anlmost entirely of phytic aci(l-P' and( inorgainic-P (fig 7a). Phytic acid-P dlecrease(d at a rate niearly identical to the decrease in aci(i soluble-P while the iniorganiic-P coniteint of the endosperm actually increased slightly dulring the germinationi period. In the (dry seed, phytic acid-P represemits abouit 74 % of the acid soluble-P or 53 % of the total-P. The percenlt of phytic acid-l-P in the eindosperm also decreases rapilly (duiring germinationl ind(licating its preferenitial breakdown. Thuis, for the 5 samplinlg times the percenit of total-P in phytic aci(d in the enidosperm is 53, 50, 31, 22 and 3 % respectively. Conversely, sinice the inorganiic-P contenit of the en(losperm inicreases slightly (Iiiring germiFlationi, as a percentage of total-P in the ell(losperm it increases markedlly. Thuis by the eighth day nearly all the end(osperm phosphate is inorganic-P (cf. figs 4, 7a). I n both the roots antd shoots all the phosphate fraction,rs inicrease(l wvith germiniationi time (figs 5, 60). Aci(d soluble-P accotunited for most of the phosphate. On a perceintage basis the acid soluible-P increase(d gra(llially over the 8 day germincation perio(d from 58 % to 74 % in the roots, and 32 % to 72 %-c in the shoots. The aci(l soluible-P consisted primarily of inorganic-P in the roots (fig 7c). This Av as truie to a somewhat lesser extent in the shoots (fig i . The niatuire of the acid soluble orgainic-P (cacu1. se by dlifferenice in acidl soluible-P and
inorganic-P) was not determinied .it it prestimablv consists primarily of sugar phosphates and(l nulcleotides. Neither the roots or shoots were foulnd( to
contain any phytic acidl. Figutres 8 to 12 presenit these same resuilts in terms of the decrease in specific phosphate fractions of the encdosperm tissule and the concomitaint increase in these phosphate fractions in )0oth the root anid shoot tissuie. The increase in aci(d soluble-I' in
the roots
andl
shoots appears to occtur at the
expense of acid soluble-P in the endosperm (fig 8). However, the (lata in figture 9 as well as figuire 7, showx that the increase in inorganic-Il of the roots andl shoots does not occuir at the expense of inorga nic-P in the en(losperm. As already pointed ouit by others ( 15, 20, 23) phytic acid breakdown in the endosperm accotunts primarily for the inlcrease in inorganic-P of the roots and shoots. This is more clearly (lepicte(l in figuire 13 which shows a near stoichimetric loss in phytic acid-P from the en(losperm anid gain in iniorganiic-I' of the roots an(l shoots. This genieral patterni has been oYbserved repeate(llv by) other invxestigators uisinig (lifferenit extraction techniqules anid species (1, 5). The rate of nticleic acid-P loss from the enldosperm occuirs at nearly the same rate as nutcleic acid-P increases in the roots and shoots (fig 10). The fact that nio net gain in ntucleic acid-P occturs in the entire plant is differenit from the restults obtaiined with corn bl) Ingle and Hlageman (11) and for wheat b))y AIatsuishita (14). However, it
2.0
13 PHYTIC ACID OF ENDOSPERM a Pi OF ROOTS AND SHOOTS
1I.5 Fz
1I.O 0~ 0I
E)
-L~~~~~
0
2
4 6 TIME, DAYS
8
Fi(;. 13. The chlaniges in phlftic acid-P of the 'eiidlosperm' ani(l inor-aiic-P of tbe roots plus shoots ox-er an
8-dax )eriodc otf erlimnationl.
IIALL AND HODSES-lHOSPHIORUS METABOLISMI OF GERMINATING OATS
1463
appears to participate in the metabolism associated with germination. A suimmary of the data are shownv in figure 14. This figure shows the percentage of total-P of the entire plant for the various fractions examined at each of the sampling times. It is quiite apparent that inorganic-P is derived primarily from phytic acid and that a net syinthesis of the other phosphorylated substances does not occulr except for phospholipids.
80 -14 % P IN VARIOUS SUBSTANCES OF THE ENTIRE PLANT 60 LLJ
Discussion z LLI 40
0:
20
Nucleic acid - P
_r__oid- P Protein-
0 0
2
4 6 8 TIME, DAYS
FIG. 14. The changes in percentage of total-P of the
enltire plant for various phosphorus containing substances over an 8-day period of germination. is similar to restults obtained for barley by Ledoulx and Huart (13) and for bean by Oota and Takota (18). These restults wouild then stuggest that either nucleic acids moved intact from the reserve tissule into the developing axis or that nucleic acid synthesis in the axis was solely at the expense of nitcleotides derived from the reserve nuicleic acids. It is possible that some de novo synthesis of nucleotides or-nucleic acids may have occturred in the roots and shoots, btut the present data do not permit an evaluation of this possibility. In
contrast
to the
nuicleic
acid-P,
figuire
l1
shows that a slight net synthesis of phospholipidl occurs dturing the first 4 days of germination. This increase is followed by a graduial decrease in total lipid-P over the next 4 day period buit even at the eighth day more total lipid-P exists than was present in the dry seed. Similar resuilts were obtained 1y Ergle and Gtuinn (5) for cotton seed germination. Figure 12 shows the changes in protein-P dturing the germiniation period sttudied. Although the amouint of protein-P increases in the roots and shoots the entire plant exhibits an over-all decrease. Althouigh the protein-P of the plant represents a small fraction of the total plant-P it certainly
This study provides a fairly complete balance sheet of the changes which occutr in the major phosphorylated stubstainces dturing the germination of oats. The results confirm that phytic acid represents the primary storage form of phosphate in oat seeds (about 53 % of the total-P). However, the actulal participation of the phosphate derived from phytic acid, which appears in the roots an¶1 shoots as inorganic-P (fig 14), in various synthetic reactions in the roots and shoots dturing the dark germination is somewhat qtuestionable. That is, the very fact that the phosphate remains as pools of inorganic-P in the roots and shoots raises the qutestion of its tuse in synthetic events in the developing axis. Althoutgh phytic acid represents approximately 53 % of the seed-P, the combination of nucleic acid-P, lipid-P and protein-P also make tip a sizeable portion of the seed-P (about 27 %). Fturthermore these stubstances are also rapidly mobilized in the endosperm and it woutld appear that there is nearly a direct coniversion of these materials into nucleic acid-P, lipid-P, and protein-P, respectively in the developing roots and shoots. This is especially stuggeste(d in the case of ntucleic acid (figs 10, 14). WVhether the ntucleic acids move as macromolecules (13, 17, 18) or are first degraded and resynthesized in the embryonic axis (4, 7,1 1, 14) is impossible to ascertain from this typo of data. Information on 3P or nucleotide incorporation into nucleotides and nucleic acids are needed to clarify this point. From the variable reports in the literature (4, 7, 11, 13, 14, 17, 18), however, it wouild appear that some species simply possess the capacity for a net synthesis of nutcleic acids duiring dark germination while others do not. In the case of lipid-P it also appears that a transfer from endosperm to axis may occuir bLut in addition some de novo synthesis in the axis mutst also occuir since the rate of increase in phospholipid content of the roots and shoots exceeds the rate of loss from the endosperm (fig 11). This synthetic process is most rapid between days 2 and 4 and is most prevalent in the shoots. The souirce of phosphate for this additioinal synthesis couild have come from inorganic-P originating from phvtic acid or perhaps to some extent from the breakdowtn of phosphoprotein. This latter possibility arises since
PLANT PHYSIOLOGY
1464
the rate of synthesis of protein-P in the roots and shoots does not keep pace with its rate of loss from the endosperm (fig 12). Thtis in the case of phosphoproteins a de novo synthesis during dark germination must also be cquiite limited. The actual fuinction of phosphoprotein in plant metabolism is uinknowin. Its formation (luring wheat seed ripening has been shown by Jennings and Morton (12) and Ergle and Guinn (5) have showni its disappearance from cotton seeds (lutring germination. Cuirrent stuidies are directed toward an evaluation of its possible participation in energized ion transport. As mentioned earlier the fact that the majority of phosphate derived from phytic acid remains as pools of inorganic-P makes one question its metabolic significance in the case of dark germinating oats. This is fturther suggested by the nearly stoichiometric losses from the endosperm and gains in the roots + shoots of nuicleic acids, phospholipids and phosphoproteins. It is possible that plant species such as corin, which possess the capacity for net synthesis of nuicleic acids during germination (11) can immediately uitilize this soturce of inorganic-P. In the present case, it is possible that only in the presence of light wouild the inorganic-P b)e uised for synthesis of organic phosphate substances in the developing axis. A comparative study tising species which exhibit a net gain in nticleic acids in the axis (for example corn) ancl those which do not exhibit a net gain (for example oats) when germinating under light and dark con(litions shouldlc help to clarify this problem.
Literature Cited 1. ALBAUm, H. G. AND WV. UMBRIET. 1943. Phiosphorus transformations during the development of the oat embryo. AAm. J. Botany 30: 553-58. 2. ASADA, K. AND Z. KASAI. 1962. Formation of mwo-inositol and phytin in ripening rice grain. Plant Cell Physiol. 3: 397-406. 3. ASHTON, W. M. AND P. C. WILLIAMS. 1958. The phosphorus compounds of oats. I. The content of phytate phosphoruis. J. Sci. Food Agr. 9: 50511.
4. BARKER, G. R. AND T. DOUGLAS. 1960. FuInctioIn of ribonclclease in germinating peas. Nature 188: 943-44. 5. ERGLE, D. R. AND G. GUINN. 1959. Phosphorus compounds of cottonl embryos and their changes during germination. Plant Physiol. 34: 476-81. 6. FISKE, C. H. AND Y. SUBBAROW. 1925. The colorimetric determination of phosphorus. J. B iol. Chem. 66: 375-400. 7. HOLDGATE, D. P. AND T. W. GOODWIN. 1965. Metabolism of nticleic acids duiring early stages of the germination process in rxye (Secale cercale). Phytochemistry 4: 845-50.
8. HUGGINS, C. G. AND D. V. COHNX. 1959. Studies concerning the composition, distribution, and turnover of phosphorus in a plhosphotide-peptide fraction from mammaliani tissuie. J. Biol. Chem. 234:
257-61. 9. HUrTCHINSON, Vt. C. AND H. N\. NIONRO. 1961. The determinationi of nucleic acids in biological
material. Analyst 86: 768-813. 10. INGLE, J. 1963. The extraction and estimation of nucleotides andl nucleic acidls from plant material Phytochemistry 2: 353-70. 11. INGLE, J. AND R. H. HAGEM1AN. 1965. 'Metabolic chaniges associated Mwith the germination of corn. II. Nucleic acid metabolism. Plant Phvsiol. 40: 48-53. 12. JENNINGS, A. C. AND R. K. MORTON. 1963. Changes in nucleic acids and other phosphorus coIntaining compounds of developing wheat grain. Australian J. Biol. Sci. 16: 332-41. 13. LEDOUX, L. AND R. HUART. 1962. Nucleic acid and(t protein metabolism in barley seedlings. IV. Translocation of ribonucleic acids. Biochinm. Biophy s. Acta 61: 185-96. 14. MATSUSHITA, S. 1958. Studies oni the nucleic acids in plants. III. Clhaniges of the nucleic aci(i contenits during germinationi stage of the rice plant. Mem. Res. Inst. Food Sci., Kyoto Univr. 17: 23-28. 15. MAYER, A. M. AND POLJAKOFF-MAYBER. 1963. The germinationi of see-ls. The MacMillani Company, New York. 16. M\IHAILOVIC, M. LJ., 'M. ANTRO, AND D. HADGIJEN. 1965. Chenical investigations of wheat. 8. Dy namics of various forms of phosphorus in wheat duiring its ontogeniesis. The extent and iieclianislni
of phytic acid decomposition in germinatinig wheat grain. Plant and Soil 23: 117-28. 17. OOTA, Y. AND S. OSA\\-A. 1954. Migrationi of "Storage PNA" from cotyledons inlto growine; organs of bean seed embryo. Experimentia 10:
254-56. 18. OOTA, Y. AND K. TAKATA. 1959. Changes in microsomal ribonucleoprotein in the time course of the germination stage as revealed by electrophoresis. Physiol. Planitaruim 12: 518-52. 19. PEERS, F. G. 1953. The phytase of wlheat. Biochemii. J. 53: 102-10. in i 20. ROVAN., K. S. 1966. Phosphorus m11etabolism l)lants. Intern. Rev. of Cvtol. 19: 301-90. 21. SCH NEIDER, W. C. 1945. Phosphorus comiipounds inl animal tissues. I. Extraction and estimation of deoxvpentose nucleic acids and pentose nuticleic acids. J. Biol. Chem. 161: 292-303. 22. TURNIER, D. H. AND J. F. TURNER. 1961. The uise of perchloric acid in the extraction of phosplhoric compounds from planit tissules. Biochim. Biophys. Acta 51: 591-93. 23. VARNER, J. 1965. Seed (levelopment and germina;ltion. In: Plant Biochelmistry. J. Bonnler and J. Varner, eds. Academii Press, Ne+-7 York, p 76390. 24. Woo, NI. L. 1919. Clicmklcal conistituenits of Aiiiaranthust rctroflferts. Bota-tn. Gaz. 68: 313-44.
