fat metabolism soybeans i. physiology 1'2, 8 · bryos of many plant species, including the soybean,...

FAT METABOLISM IN GERMINATING SOYBEANSI. PHYSIOLOGY OF NATIVE FAT 1'2, 8

VARDA KAHN4, ROBERT W. HOWELL, AND JOHN B. HANSONDEPARTMENT OF BOTANY, UNIVERSITY OF ILLINOIS, CROPS RESEARCH DIVISION, AGRICULTURAL RESEARCH SERVICE,

U. S. DEPARTMENT OF AGRICULTURE, AND DEPARTMENT OF AGRONOMY,UNIVERSITY OF ILLINOIS, URBANA

The fat reserve of seeds constitutes a major sourceof reduced carbon for the growth of germinating em-bryos of many plant species, including the soybean,Glycine max (L.) Merr. Several investigators haveattempted to correlate cytological and morphologicalobservations with physiological and chemical changesduring germination (11, 18, 19, 20, 23). It has beenimplied that esterases play a major role in fat de-gradation (2, 7, 12, 21, 31), but differences in solu-bility of lipids and the components of the aqueouscytoplasm make studies in such systems difficult.Evidence from electron-microscope studies of soy-bean native-fat suggests that in situ the fat may beenclosed in membranous structures 5. These struc-tures collapse as the fat is consumed. Membranescould provide both the hydrophilic and lipophilicproperties required for fat metabolism and might bethe loci of many of the enzymes involved.

Reserve lipids isolated centrifugally are micro-scopically similar to those observed in situ and appearto be homogeneous, uncontaminated by other organ-elles5. The lipid fraction isolated centrifugally wasused in the present study. Physical, chemical, andbiochemical changes occurring in this system duringgermination were investigated.

MATERIALS AND METHODS

Soybean seeds, lightly dusted with chloranil to re-tard fungal growth, were germinated in the dark at210 C in 9 inch X 12 inch pyrex baking dishes, be-tween sheets of absorbent tissue paper moistened with200 ml of distilled water. After various periods ofgrowth, the cotyledons were harvested, separated fromseed coats, rinsed thoroughly with deionized water,and chilled.

Approximately 100 gram lots of cotyledons wereground in 200 ml of chilled grinding medium con-

'Received April 6, 1960.2 The work reported here was taken from a thesis sub-

mitted by the senior author to the graduate college ofthe University of Illinois in partial fulfillment of the re-quirements for the degree of Doctor of Philosophy inBotany.

3 Publication No. 340 of the U. S. Regional SoybeanLaboratory, Urbana, Ill.

4 Present address: Biology Dept., Johns Hopkins Uni-versity, Baltimore, Md.

5 R. F. Bils, personal communication.

sisting of 0.5 M sucrose, 0.07 M tris 6, and 0.004 MEDTA, pH 7.3, for 2 minutes either in a Waringblendor or in a chilled mortar. The homogenate waspassed through four layers of cheesecloth and centri-fuged in the deep freeze at 20,000 X G for 15 minutes.The white fatty layer at the top of the liquid was care-fully removed with a spatula, redispersed in 0.5 Msucrose, 1: 20, and centrifuged at 28,000 X G for 20minutes. The fatty layer was removed and redis-persed in 0.5 M sucrose (1: 5). This fraction is re-ferred to as "native-fat." The washing procedurewas omitted or repeated in certain instances as indi-cated in the data.

PHYSICAL MEASUREMENTS OF WASHED NATIVE-FAT: A: Rewashed native-fat was blotted with a pieceof filter paper for fresh weight determination and thendried in the oven at 50° C to a constant dry weight.Weights were corrected for the occluded sucrose bythe method of Trevelyan and Harrison (30). B:Light-scattering was measured spectrophotometricallyat 520 m/A by the methods of Cleland (6) and Raaflaub(24). C: Viscosity was determined after dialysis inthe cold to equilibrium against various sucrose con-centrations. The corresponding dialyzates were con-sidered as blanks. The determinations were carriedout in an Ostwald viscometer at 28.30 C.

CHEMICAL ANALYSIS OF WASHED NATIVE-FAT:A: Lipid content was determined by extractingweighed samples of washed native-fat three timeswith ethanol: ether: chloroform (2: 2: 1). The sol-vents were evaporated from the pooled extracts, theresidue representing total lipids. B: Lipid phos-phorus was determined on ash solutions. For ashing,50 ml of isopropanol and 0.5 g of Mg(NO3)2 wereadded per gram of lipid. The samples were mixedwith the solvent and a piece of ashless cotton wasadded to the crucible and ignited. The ignition wascompleted with the aid of a strong flame from a bun-sen burner 7. The residue was taken up in diluteHCl and made to volume. Inorganic phosphorus

6 The following abbreviations are used: tris, trishy-droxymethylaminomethane HCI; EDTA, ethylene diaminetetraacetic acid; TCA, trichloroacetic acid; DPN, diphos-phoryridine nucleotide; ATP, adenosine triphosphate;RNA, ribonucleic acid; RNase, Ribonuclease; B.S.A.,bovine serum albumin.

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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

KAHN ET AL-FAT METABOLISM IN GERMINATING SOYBEANS

was determined by the method of Fiske and Subbarow(8). C: The residual pellet of the native-fat (afterlipid extraction) was dissolved in 1 M KOH. Pro-tein content of the pellet was determined by the meth-od of Lowry et al (17) or by digestion with H2SO4and nesslerization (14). D: Nucleic acids were de-termined by the methods of Ogur and Rosen (22).

If DETERMINATIONS OF ENZYMATIC ACTIVITIES As-SOCIATED WITH NATIVE-FAT: A: Lipase: Washednative-fat was dispersed in water (0.5-2.0 ml) andincubated with or without 2 ml of 5 % aqueous tri-acetin for 18 hours in a water bath at 36° to 380 Cwith continuous shaking. The initial pH of the re-action mixture was 7.3. The fatty acids released dur-ing incubation were titrated with 0.004 M standardNaOH. The increase in acid during incubation withtriacetin, less the increase occurring in the absenceof triacetin, was attributed to the activity of endo-genous lipase on the added substrate. The productsobtained in the absence of triacetin were partitionedbetween chloroform or petroleum ether and water andthe fractions were titrated separately. The aminonitrogen liberated during self-digestion was deter-mined by nesslerization of the supernatant after pre-cipitating the incubation mixture with 10 % TCA.

B: Enzymes other than lipase: An acetonepowder of the native-fat was extracted for 30 minuteswith 0.01 M potassium phosphate buffer, pH 7.0,(1: 300 g/ml). The extract was cleared by centri-fuging at 20,000 X G for 10 minutes. Ribonucleaseactivity was determined by measuring changes inRNA by the method of Ogur and Rosen (22). Esterphosphatase, ATPase, and pyrophosphatase activitieswere measured by the amount of inorganic phosphateliberated during incubation of 10 a moles of substratewith the extract in the presence of 10 4 moles ofMgCl2. Lipoxidase activity was assayed by the modi-fied thiocyanate method of Kock et al (15). Peroxi-dase was measured by the method of Lipetz and Gals-ton (16). The presence of proteases was detected byNaOH titration after incubating the extract with 1 %B.S.A., casein, or soybean protein. Dehydrogenaseactivity was measured by incubating 0.5 ml aliquots ofthe extract with 0.33 mg of DPN and the followingsubstrates at 0.05 M final concentration, pH 7.0:potassium a-ketoglutarate, potassium lactate, glycer-aldehyde-3-phosphate, and potassium ,8-hydroxybuty-rate. The change in optical density at 340 myA wasread against blank tubes containing all componentsexcept the substrate.

60

501

S-0

401

301

20A

101

75

70

L-

0-

ap651

60

a a

1 2 3 4 5 6 7Age of Seedlings (Days)

RESULTS

PHYSICAL PROPERTIES OF NATIVE-FAT FRACTION:Water content: The water content of native-fat de-creased rapidly during 7 days of growth (fig 1). The

7This ashing method was suggested by F. I. Collins,U. S. Regional Soybean Lab.

0 0.1 0.3 0.5 0.7 0.9 1.0M glucose

FIG. 1. Water content of washed native-fat as a func-tion of age of soybean seedlings.

FIG. 2. The effect of glucose concentration on thewater content of washed native-fat from cotyledons of3-day-old soybean seedlings.

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8Um m v n --

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a a &

a . A a a a


PLANT PHYSIOLOGY

decrease was generally proportional to the decrease inprotein (fig 4) indicating that the main portion of thewater in the native-fat may be associated with proteinas water of hydration. When the native-fat was dis-persed in solutions of various glucose concentrations,the uptake of water by the native-fat decreased linearlywith glucose concentration (fig 2). This may indi-cate the presence of semi-permeable membranes or ofremoval of water from hydrated surfaces in the native-fat.

Light-scattering: The optical density at 520my decreased linearly with increasing sucrose or glu-cose concentrations, in contrast to what is commonlyfound in mitochondria (24). Changes in opticaldensity are not necessarily an indication of volumechange (28) but may also be due to changes in therefractive indices of either the particles or the dis-persing medium (3). The change in optical densityobserved here was instantaneous following change inmolarity of the medium. This seems to exclude thepossibility of osmotic entry of water (6) so the changein optical density is attributed to changes in the rela-tive refractive indices of the fat droplets atd thesuspending medium. Ball and Barnett (1) have re-ported similar results in experiments with an enzvmepreparation of membranous and vesicular appearancefrom heart muscle.

Relative viscosity: No significant change in therelative viscosity of native-fat dispersions resultedfrom change in osmotic strength of the suspendlingmedium. Since relative viscosity is proportional tovolume in Einstein's equation (13), the viscositymeasurements, like those of light-scattering, did notprovide evidence of a change in volume of particlesfollowing change in osmotic strength of the medlium.

Sonic oscillation (10 kc/sec for 10-30 min), whichis expected to disrupt structural membranes, did notaffect the relative viscosity of native-fat dispersed insucrose solutions.

CHEMICAL COMPOSITION OF NATIVE-FAT: Thenative-fat fraction contained mainly lipids, the rela-tive amount of which increased during the experi-mental period (fig 3). The residual pellet, remain-ing after exhaustive extraction of the lipids, consistedmostly of protein plus traces of nucleic acids.Changes in amounts of nucleic acids, lipid phosphate,and protein with age are shown in figure 4.

Lipoproteins are known to be constituents of bio-logical membranes (4, 26, 27, 29). Phospholipids arereported to be oriented at the surface of fat globules(25). If the fat were enclosed in membranes whichwere not degraded during germination, the ratio oflipid phosphate to protein should be constant through-out. Actually, protein and nucleic acids disappearedsomewhat faster than lipid phosphate, suggesting thatprotein and nucleic acids are preferentially metabo-lized during germination.

ENZYMATIC ACTIVITIES ASSOCIATED WITHNATIVE-FAT FRACTION: Incubatikon of the native-fatdispersions demonstrated the presence of lipase, which

loor

95

0 90

. 88;of

0

-

0residual pellet

I a. . . . a.. .I 2 3 4 5 6 7 8 9 10

Age of Seedlings (Days)

Age of Seedlings (Days)

FIG. 3. Change in total extracted lipids and residualpellet of native-fat as a function of age. The native fatwas extracted three times with ethanol: ether: chloroform(2: 2: 1).

FIG. 4. Chemical composition of native-fat as a func-tion of age.

hydrolyzed the available endogenous substrate to pro-duce free acids. This process is referred to as self-digestion. In addition, the lipase associated with thenative-fat could hydrolyze added triacetin. The ca-pacity for self-digestion increased threefold during 13days of germination and growth. The specific ac-tivity of the native-fat on triacetin increased withrepeated washings (fig 5), indicating that lipase waspreferentially adsorbed on the native-fat while pro-teins other than lipase were effectively removed bythe washing technique. Preferential adsorption of

856


KAHN ET AL-FAT METABOLISM IN GERMINATING SOYBEANS

Z ICP "native fatE

00

z

washinj liquid

number ot successive washings

FIG. 5. Effect of successive washings of native-fatfrom cotyledons of 3-day-old soybean seedlings with 0.5M sucrose (1: 5 v/v) on its capacity for self-digestion andtriacetin hydrolysis.

lipase was verified by dispersing the native-fat in amixture of equal parts of B.S.A. (presumably an inertprotein) and wheat germ lipase 8 (2.4 mg/ml each,pH 7.0). Upon reisolation of the native-fat it wasfound that the specific activity for self-digestion andfor triacetin hydrolysis has been increased appreciably(table I). Activity on triacetin was also increasedby dispersing native-fat in the supernatant fraction,(previously found to be associated with high esteraticactivity) followed by reisolation. On the other hand,the capacity of native-fat for self-digestion decreasedwith repeated washings (fig 5).

The activity of endogenous lipase on triacetin wasalmost completely destroyed by sonic oscillation, while

8 Worthington purified preparation.

the capacity for self-digestion was increased (fig 6).The activity of wheat germ lipase on native-fat wasessentially unaltered by sonic oscillation.

While the acids produced in self-digestion con-sisted of 85 to 90 % free fatty acids, about 10 to 15 %of the acid produced was amino acid.

Enzymes capable of degrading biological mem-branes .were preincubated with native-fat. It waspostulated that after enzymatic degradation of anylimiting membrane, the fat would become more ac-cessible as substrate for added lipase. The rate ofself-digestion of native-fat (incubated for 18 hr at370 C) was increased 1/3 by the action of Crotalusadamanteus venom, consisting mainly of phospho-lipases (1 mg of venom and 100 /Amoles of CaCl2 were

15 0in 0.5

in 0.5 M sucrose

fin water

zl 0

z

0

z

5.5

+ triqcetin self-digestion + wheat germlipose

FIG. 6. Effect of soniic oscillation and tonicity onlipase activity associated with native-fat isolated fromcotyledons of 3-day-old soybean seedlings. The native-fatwas sonicated for 15 minutes at 10 kc/seconds in the cold.Wheat germ lipase 4 mg/reaction mixture at pH 7. In-cubation at 370 C for 18 hours.

BLE I

PREFERENTIAL ADSORPTION OF LIPASE ON NATIVE-FAT OF SOYBEANS

IA eq. NAOH/MG N

NATIVE-FAT PASSED THROUGHE % INCREASEWASHED BSA~4NATIVE-FAT SUPERNATANT B.PS.A. SU- RNAAN B.S.A. ±LIPASESUERNATANT LIPASE

Activity on triacetin 8 3 14 754 11 17 325

Self-digestion 37 13 85 130A dispersion of native-fat wvas thoroughly shaken with either a solution of equal parts of wheat germ lipase and

bovine serum albumin, 2.4 mg/ml each, or clear supernatant obtained after centrifuging a soybean cotyledon homogenateat 20,000 X G for 15 minutes. The native-fat was reisolated from the disprsion by centrifugation at 28,000 X G for20 minutes. After reisolation, the natiVe fat was incubated 18 hours at 370 C. Percent increase was calculated bycomparing activity after passage with activity of washed native-fat in the first coltumn.

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PLANT PHYSIOLOGY

TABLE IIENHANCEMENT OF TRIACETIN AND NATIVE-FAT

HYDROLYSIS BY AMINO ACIDS

SUBSTRATEADDITIVE

TRIACETIN NATIVE-FAT

DL-Lysine 5.7 12.5DL-Histidine 10.0 83.5Glycine 0.2 5.6

DL-Lysine, 6.8 X 10-4moles/ml, pH 8.5; DL-histidine,6.4 X 10-4 moles/ml, pH 6.6; glycine, 1.3 X 10-3 moles/ml, pH 6.6 or 8.5. (Identical results were obtained at thetwo pH values). The reaction mixtures were incubatedfor 18 hours at 370 C. Results reported as net equivalentsNaOH/tmole amino acid added. For comparison, theactivity of purified wheat germ lipase (2.5 mg/ml, pH7.3) on triacetin and on washed native-fat was 2.6 and2.0 net equivalents NaOH/mg protein, respectively.

added/3 ml of reaction mixture), 2/3, by that oftrypsin (1 mg/3 ml of reaction mixture), but wasnot affected by adding RNase (1 mg/3 ml of reactionmixture). The activity of wheat germ lipase onnative-fat (4 mg/3ml of reaction mixture) was notincreased by preincubating native-fat with RNase,trypsin, or phospholipase A.

It has been reported that hydrolysis of triglyceridesis enhanced by low-molecular-weight peptides oramino acids under certain pH conditions (5, 8, 9).Hydrolysis of the native-fat fraction was acceleratedby histidine (pH 6.6), lysine (pH 8.5), or glycine(pH 6.6 or 8.5) in decreasing order of magnitude(table II). Thus, the amino acids produced duringself-digestion may enhance fat hydrolysis.

A phosphate buffer extract of an acetone-powderpreparation of native-fat was used as an enzyme

source for detecting various enzymatic activities(table III). Several enzymes are associated withthe native-fat. The association of these enzymes withthe native-fat may be only superficial and due to con-tamination by other cytoplasmic components, but inviewv of the affinity of these enzymes for the native-fatthey may be associated with it in situ.

DISCUSSIONAn isolated system in which the condition of the

fat is similar to that in situ is particularly advan-tageous for the study of fat metabolism because ofthe contrasting hydrophobic nature of the fat andhydrophilic properties of the surrounding cytoplasm.The isolated native-fat fraction is considered to re-semble the fat in situ with regard to both the lipidmaterial and the enzymes related to its metabolism,since electron-microscope studies indicate that in boththe cotyledons and the native-fat fraction, fat bodiesare surrounded by a limiting layer, possibly a mem-brane. The latter does not disappear in the courseof germination but appears to collapse.

Physical measurements in the present study showthat the fat bodies are limited by a hydrophilic layerfrom which water can be withdrawn osmotically.However, there is no evidence of a volume change, afact suggesting either that there is no internal aqueousphase within the fat bodies or that the membrane doesnot possess semi-permeable properties. The rate ofprotein disappearance from the native-fat fraction isfaster than that of phospholipids. This acceleratedprotein disappearance could be due to chemical altera-tions in the membrane or to superficial occlusion, oradsorption of proteins on the membrane (10). Thelatter would explain the high ratio of protein to phos-pholipid in the native-fat fraction as compared withmembranous structures like mitochondria or nuclei.

TABLE I I IENZYMATIC ACTIVITIES, OTHER THAN LTPASE, ASSOCIATED WITH NATIVE-FAT FRACTION

ENZYMATICACTIVITY

RNAaseEster phosphatase

ATPasePyrophosphatase

LipoxidasePeroxidaseProtease

Dehydrogenase

SUBSTRATE

Yeast RNAGlucose-l-phosphateFructose-1 -6-diphosphateATPPyrophosphate

Linolenic acidPeroxide derivative of linolenic acidBovine serum- albuminCasein

Soybean proteinLactatea-KetoglutarateGlyceraldehyde-3-phosphate,8-Hydroxy butyrate

ACTIVITY/MG PROTEIN/30 MIN

17 ,g RNA Hydrolyzed0.120 Atm Pi ReleasedNone0.704 ,um Pi Released0.710 Am P1 Released

0.045 ,m Peroxide formed1.5 ,tm Peroxide disappeared150 Aeq Peptide bond hydrolyzed140 Aeq Peptide bond hydrolyzed162 ,eq Peptide bond hydrolyzednone

,,

Reaction mixtures were incubated for 30 minutes at 370 C. Enzyme source was a 0.01 M phosphate buffer (pH 7.0)extract of an acetone-powder prepared from native fat.

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KAHN ET AL-FAT METABOLISIY

Such occluded proteins may be functional enzymeswhose close association with the fat bodies makespossible efficient interaction.

Endogenous and added lipase appear to be prefer-entially adsorbed on the native-fat bodies. The activi-ty of the endogenous lipase can account for the majorportion of the acids released during incubation of thenative-fat fraction. The balance of the acids releasedduring self-digestion is made up of amino acids, whichmay function to promote fat hydrolysis. Preincu-batingf the native-fat with enzymes capable of digestingbiological membranes, or physically rupturing thenative-fat, does not render the fat more accessible tohydrolysis by added lipase. Apparently, the mem-brane, if it exists, does not introduce a chemical orphysical hindrance to the hydrolytic action of lipase.

Enzymes such as RNase, protease, lipoxidase, per-oxidase, and ester phosphatase are associated withthe native-fat fraction. The presence of these en-zymes may account for utilizing components such asprotein, nucleic acid, and phospholipids in the native-fat fraction.

SUMMARYThe metabolism of fat in germinating soybeans

during periods up to 13 days was studied. Native-fat was prepared by homogenizing cotyledons in su-crose-TRIS-EDTA followed by centrifugation.

The native-fat initially contained about 50 % waterbut lost water during the growth period. The wateris evidently held either osmotically or by adsorption,since the water content of fat suspended in glucosesolutions was inversely proportional to glucose con-centration.

The dry matter of native-fat initially consisted ofabout 90 % lipids and 10 % residual pellet. The latterwas mostly protein but traces of nucleic acid were alsopresent. After 10 days of germination. practicallyall the residual material had been lost.

The self-digestion activity of native-fat decreasedwith successive washings, but the specific activity ontriacetin increased. Sonic oscillation destroyed ac-tivity on triacetin but enhanced self-digestion. Bothself-digestion and activity on triacetin were increasedby passing native-fat through a wheat germ lipasesolution, indicating adsorption of lipase on the native-fat.

Several enzymes other than lipase were shown tobe associated with the native-fat.

LITERATURE CITED1. BALL, E. C., and R. J. BARNETT. 1957. An inte-

grated morphological and biochemical study of apurified preparation of the succinate and DPNHoxidase system. Jour. Biophys. Biochem. Cytol.3: 1023-1036.

2. BAMANN, E., and E. L. ULLMANN. 1957. In:Encylopedia of Plant Physiology. Vol. VII:109-136.

J IN GERMINATING SOYBEANS 859

3. BARER, R., K. F. A. Ross, and S. TKACZYK. 1953.Refractometry of living cells. Nature 171: 720-724.

4. BURGEN, A. S. V. 1957. The physiological ultra-structure of cell membranes. Can. Jour. Biochem.Physiol. 35: 569L-576.

5. CHESBRO, W. R., and L. R. HEDRICK, 1959. Roleof lysine in catalyzing ester-hydrolysis. Nature183: 994.

6. CLELAND, K. W. 1952. Permeability of isolatedheart sacrosomes. Nature 170: 497-499.

7. CROCKER, W., and L. V. BARTON. 1953. Physiologyof Seeds. Chronica Botanica Corp., Waltham,Mass.

8. FISKE, C. H., and Y. SUBBAROW. 1925. The colori-metric determination of phosphorous. Jour. Biol.Chem. 66: 375-400.

9. GERO, A., and C. L. WITHROW. 1957. Chemicalmodel of a biological reaction. Nature 180: 1354-1355.

10. HANSON, J. B., R. H. HAGEMAN, and M. E. FISHER.1960. The association of carbohydrases with themitochondria of corn scutellum. Agron. Jour. 52:49-52.

11. HOLMAN, R. T. 1948. Lipoxidase activity and fatcomposition of germinating soybeans. Arch. Bio-chem. 17: 459-466.

12. HOYER, E. 1907. Uber fermentative Fettspaltung.Zeits Physiol. Chem. 50: 414-435.

13. JIRGENSONS, B., and M. E. STRAUMANIS. 1954. AShort Textbook of Colloidal Chemistry. P. 148.John Wiley and Sons, Inc., New York.

14. KoCK, F., and T. L. MCMEEKIN. 1929. A newdirect nesslerization microKieldahl method and amodification of the Nessler-Folin reagent for am-monia. Jour. Amer. Chem. Soc. 46: 2066-206(.

15. KOCK, R. B., B. STERN, and C. G. FERRARI. 1958.Linoleic acid and trilinolein as substrates for soy-bean lipoxidase (s). Arch. Biochem. Biophys. 78:165-179.

16. LIPETZ, J., and A. W. GALSTON. 1959. Indoleaceticacid oxidase and peroxidase activities in normaland crown gall tissue cultures of Parthenocissustrienspidata. Amer. Jour. Bot. 46: 193-196.

17. LOWRY, G. H., N. J. ROSEBROUGH, A. L. FARR, andR. J. RANDALL. 1951. Protein measurement withthe Folin-phenol reagent. Jour. Biol. Chem. 193:265-275.

18. MAcLACHLAN, P. L. 1936. Fat metabolism inplants, with special references to sterols. jour.Biol. Chem. 114: 185-191.

19. McALIsTER, D. F., and 0. A. KROBER. 1951. Trans-location of food reserves from soybean cotyledonsand their influence on the development of the plant.Plant Physiol. 26: 525-538.

20. MILLER, E. C. 1938. Plant Physiology. McGraw-Hill Book Co., Inc., New York and London.

21. NICLOUX, M. 1906. Studies on enzyme action-lipase. Proc. Roy. Soc. Series B. 77: 454.

22. OGUR, M., and G. ROSEN. 1950. Nucleic acid ex-tract from lipide free residue. Arch. Biochem.25: 262-276.

23. PACK, D. A. 1925. Dispersion of lipoids. Bot.Gaz. 79: 334-338.

24. RAAFLAUB, J. 1955. Komplexbildner als cofaktorenisolierter Zellgranula. Helv. Chem. Acta. 38: 27-37.


25. Ross, K. F. A., and J. T. Y. CHOW. 1957. Thephysical nature of the lipid globules in the livingneurones of Helix aspersa as indicated by measure-ments of refractive index. Quarterly Jour. Micro-scop. Sci. 98: 341-348.

26. SCHMIDT, W. T. 1939. Der molekular Bau derZelle. Nova Acta Leopoldina 7: 1-20.

27. SEIFRIZ, W. 1955. The physical chemistry of thecytoplasm. In: Encylopedia of Plant Physiology.W. Ruhland, ed. Vol. I: 340-400. Springer-Verlag, Berlin.

28. TEDESCHI, H., and D. L. MARRIS. 1958. Some ob-servations on the photometric estimation of mito-

chondrial volume. Biochem. Biophys. Acta 23:392-402.

29. THOMAS, J. B., M. BUSTRANN, and C. H. PARIS.1952. Participation of Phospho-lipids in mem-branes and structure of chloroplasts. Biochem.Biophys. Acta 8: 90-100.

30. TREVELYAN, W. E., and J. S. HARRISON. 1950.Studies on yeast metabolism. Biochem. Jour. 50:298-303.

31. WALDScHMIDT-LEITZ, E., and A. SCHAFFNER. 1941.Die Methoden der Fermentforschung. In: Bamannand Myrback, Part 2: Pp. 1547-1548. Leipzig, C.thieme. 1941. Academic Press, Inc. New York.1945.

MINERAL DEFICIENCY AND ORGANIC CONSTITUENTS IN TOBACCO PLANTS.I. ALKALOIDS, SUGARS, AND ORGANIC ACIDS1T. C. TSO, J. E. McMURTREY, JR., AND TAMARA SOROKIN

UNITED STATES DEPARTMENT OF AGRICULTURE, AGRICULTURAL RESEARCH SERVICE,CROPS RESEARCH DIVISION, BELTSVILLE, MARYLAND

One of the most fundamental but least understoodphases of plant physiology is the relationship betweenthe quantities of the essential nutrient elements andthe metabolic changes occurring in the plant. A liv-ing plant is a complex biological system composed ofchemical constituents. Most minerals are essentialbuilding materials, and some form parts of indispen-sable catalysts. A deficiency in any mineral affectsthe normal metabolic system and thus disturbs thebalance of the chemical constituents. Abnormal ac-cumulation of certain compounds is associated withthe development of a typical symptom usually recog-nized as due to the abnormal supply of a specificmineral.

In tobacco plants distinctive mineral deficiencysymptoms were demonstrated by McMurtrey (7).The relationship of certain deficiencies to tobaccoalkaloids and some organic fractions has also beenreported (9, 10). It appears desirable for a definitepattern to be established between deficiencies of cer-tain minerals and the organic constituents of thatplant so as to provide basic knowledge.

This paper reports the relative differences in alka-loids, sugars, and organic acids in tobacco plants dueto the deficiency of one of the following minerals:N, P, K, Ca, Mg, S, and B. This is a preliminaryinvestigation of a semi-quantitative nature. Moredetailed and extensive studies are in progress andwill be presented later.

1 Received manuscript April 8, 1960.

MATERIALS AND METHODSNicotiana tabacurn L. var. Connecticut Broadleaf

plants were grown in the greenhouse in 1957 (springand fall), in 1958 (spring), and in 1959 (spring).Nutrient solutions, complete in or lacking N, P, K,Ca, Mg, S, or B, were prepared as described byMcMurtrey (7). Two sources of nitrogen (nitrateand urea) were tested in Ca-deficient experiments.

Each seedling with five leaves and about fourinches tall was grown first in the starter solution withhalf-strength complete nutrient. Three weeks later,after the plant was fully established, it was transfer-red to a partially deficient nutrient solution in a 10-liter jar. Deficiency symptoms usually were evident5 to 8 days later. Calcium or boron-deficiency causedthe drawn appearance of the tobacco plants becausethe buds had ceased to grow, which is referred toas "physiological" topping. As soon as the Ca- or B-deficiency symptom appeared, part of the correspond-ing control plants growing in complete nutrient weretopped (decapitated), and suckers were removedthereafter. Plants were harvested 14 to 18 days aftersymptoms appeared, depending on the conditions ofeach crop season or the severity of the symptom.

There were four replications in each treatment.Composite samples of root, stem, and leaf from fourplants were analyzed separately; the results were ex-pressed on-a per plant basis. All samples were ex-tracted with acetone or ethanol in a Waring Blendor.Samples for alkaloid determinations were extractedwith 50 % acetone and analyzed by both steam dis-tillation and paper chromatography (11). A sepa-

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fat metabolism soybeans i. physiology 1'2, 8 · bryos of many plant species, including the soybean,...

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