Plant Physiol. (1969) 44, 789-795

Vol. 44 No. 6

Respiratory Changes During Seed Germination'Histological Distribution of Respiratory Enzymes and Mobilizationof Fat Reserves in Castor Bean Endosperm and Peanut Cotyledons

Paul Castelfranco, John Lott, and Naama SabarDepartment of Botany, University of California, Davis, California 95616

Received July 21, 1967.

Abstract. Germinating peanut cotyledons and germinating castor bean endosperm havebeen compared with respect to their rates of fat dissimilation and with respect to the anatomicaldistribution of respiratory activity. The lipid mobilization is much slower in peanut cotyledonsthan in castor bean endosperm. Light hais essentially no effect on either system. As germi-nation progresses, the majority of the succinic dehydrogenase and cytochrome oxidase activitiesbecome looalized in the vein regions of peanut cotyledons. In the castor bean endospermthese two activities are uniformly distributed throughout the storage parenchyma and increasewith germination until the organ becomes soft and visibly senescent.

Castor bean endosperm and peanut cotyledonshave been used frequently for biochemical studies onthe fate of storage fats during germination. Recentlysoluble preparations which are able to catalyze the,8-oxidation of higher fatty acvl-CoA derivativeswere obtained from castor bean endosperm (17) andpeanut cotyledons (12, 13, 14). The possibility thatthese soluble preparations might be obtained byrdamaging the mitochondria during isolation has notbeen excluded but this appears unlikely in viewv ofthe mild procedure used, involving low-speed blend-ing in buffers containing hypertonic sucrose. Re-gardless of the actual localization of these enzymes,it seems probable that the utilization of storage lipidsis dependent uipon mitochondrial activity becauseATP is required for the activation of fatty acids andbecause the reduced pyridine and flavin nucleotidesformed during the fl-oxidation pathway must bere-oxidized by the mitochondria in order to produceATP. In resting seeds the mitochondrial activityis extrenmely low. Mitochondrial development hasbeen studied in germinating peanut cotyledons (,6, 8) and castor bean endosperm (1, 2).

In this investigation we have attempted to com-pare the development of mitochondrial activity to thecourse of fat utilization. We have studied the local-ization of mitochondrial respiratory enzymes with

1 Supported by research grants from the United StatesPublic Health Service, GM07532 and ES00054.

respect to the various kinds of tissues which makeup the cotyledons, and we have endeavored to com-pare the course of events in peanut cotyledons andin castor bean endosperm.

Materials and Methods

Peanuts, Arachis hypogaea L., variety VirginiaJumbo, and castor beans, Ricinus comimunis L.,variety Zanzibar, were obtained from the W. AtleeBurpee Company, Riverside, California; castor beans,variety Baker 296. from South Plain Research andExtension Center, Lubbock, Texas; p-NH2 diphenyl-amine (PADA), was obtained from Aldrich andwas crystallized from H.0 and pet-ether; tetranitroblue tetrazolium chloride (TNBT) was obtainedfrom Nutritional Biochemicals.

Peanuts and castor beans were germinated innmoist perlite-vermiculite mixtures as described pre-viously (12). The dry seeds were sorted out foruniform weight, the peanuts weighed 1.0 ± 0.1 g,the castor beans, Zanzibar variety, 0.65 ± 0.07 g andthe castor beans, Baker 296 variety, 0.3 10 ± 0.039 g.The Baker variety was used only for the experimentsummarized in Fig. 7 because at that time the oldcrop of Zanzibar was no longer germinating and thenew crop was not yet obtainable. The peanuts weredusted with Arasan, the castor beans were surfacesterilized by soaking for 3 min in 0.01 % HgCI2,rinsed and soaked for 1 hr in sterile H20 withstrong aeration. Peanuts were grown at two tempera-

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PLANT PHYSIOLOGY

tures, 22 to 230 and 29 to 300, castor beans at thehigher temperature only. Plants wvere grown in thedark or under continuous illuminationi of 550 ft-cfrom a miixture of cool wvhite and incandescent lamps.

Lipid Extractioni. Lipids were extracted with achloroform-methanol-water mixture (4). The coty-ledon or endosperm tissue was chopped coarsely witha razor blade and mixed with enough H,O to bringthe weight up to 8 g. Twenty ml of methanol anid10 ml of chloroform were added and the suspensionwas ground for 2 min in a Virtis homogenizer. Thefine brei was filtered through a Buchner funnlel andthe filter cake was washed with 10 ml 'of chloroform.To the filtrate 10 ml of H.,O and 5 ml of chloroformiiwere added and the heterogenous mixture was clearedby centrifuging in a graduated centrifuge cup at12,000g for 20 min. The volume of the chloroformphase was recorded and an aliquot was evaporatedto dryness, first in a stream of N. and then in avacuum oven at 500. The lipid residue was weighed.

Since the lipid extraction by this procedure gavevalues which were consistently lower than thosefound in the literature (2 to 5 % for castor beans,25 to 30 % for peanutts), the solid residue was driedat room temperature in a vacuum desiccator, trans-ferred to an extraction thimble and extracted in asoxhlet apparatus overnight with anhydrous ether(10). The ether extract was made up to volume, analiquot was evaporated to dryness and the residueweighed. The results of the 2 lipid extractions wereadded together. A preliminary investigation indi-cated that both extracts contained predominantlyneutral lipids and failed to bring out any majordifference in the component fatty acids.

Histochemiiical Tests. Peanut cotyledons and halfendosperms of castor beans were sectioned at rightangles to their long axis vith a sliding microtome.The sections were approximately 120 ju thick. Thehistochemical proceduires were adapted from Averset al. (3).

The stuccinic dehvdrogenase assay utilized aTNBT stock solution prepared from a 0.1 % suspen-sion of the dye in 0.2 mI tris HCI (pH 7.5). Thesuspension was stirred on the steam bath for 15 minand allowed to cool to room temperature. It wasfiltered jtust before use. For the inicubation, theTNBT stock solution was mixed wvith an equalvolume of 0.2 M tris HC1 (pH 7.5) containing Nasuccinate to a final concentration of 0.1 M and, whenneeded, Na malonate to a final concentration of0.2 M.

For the cytochrome oxidase 96 mg o,f PADAwere dissolved in 4 ml of ethyl alcohol and dilutedto 200 ml with 0.2 M tris HCl (pH 7.5). Thissolution was made fresh prior to use; for the incutba-tion this PADA stock solution was diluted with aniequal volume of tris buffer containing, when needed,NaN3 or NaCN to a 1 mM final concentration.

The tissue slices were incubated at room tempera-ture with occasional stirring until adequate stainingwas observed. The PADA treatment usually re-

quired 0.5 to 1 hr, and the TNBT treatment 2 to 3hr. After the incubation the slices were rinsed inbuffer, blotted on filter paper, mounted on a groundglass plate and photographed with illuminatioin fromthe sides.

Spectrophotomvetric Mllethod. The color producedduring the incubation with PADA could be easilyextracted into lipid solvents and measured spectro-photometrically. After the incubation the slices were

rinsed, blotted, transferred to small vials aind frozenwith powdered dry ice. The vials were 'wrapped inaluminunm foil and could 'be stored at -20o forseveral weeks without any loss of color. Five tissueslices were placed in a small glass homogenizer andground with 1 ml of chloroform :methanol (1:1).Half a ml of H,O and 1 ml n-hexane wvere added.The mixtture was shaken thoroughly and centrifugedto separate the phases. The hexane-chlorofornmupper 'phase was transferred to a micro-cuvette, 1 cmin light path,, and the absorbance was read in a Zeissspectrophotometer. The spectrum of the extractablecolor showed a definite maximum at 485 m,. and aminimum at 395 miA. The difference in absorbancebetween these 2 wavelengths was chosen as a relativemeasure of the cvtochrome oxidase activity of agiven tissue, and indicated as AA485-395; this valuewas linear with the number of slices used within thelimits of the experimental error. The extraiction ofthe color was carried out in subdued light to preventphotochemical bleaching of the color. The dryweight of tissue slices was determiiined by overnightdrying under vacuumiat 50°.

Results

Fig. t and 2 show the rate oif lipid breakdown inthe storage organs of peanuts and castor beans. Thefat is broken down in castor bean endosperms muchmore rapidly than in peanut cotyledons (compare

FIG. 1. Lipid content of peanut cotyledons as afunction of germination time at 22 to 230. A-A g oflipid/2 cotyledons; dark grown seedlings. A-A g oflipid/2 cotyledons; light-grown seedling. *-* percentlipid in the cotyledons, fresh wt. basis; dark-grownseedlings. 0-0 percent lipid in the cotyledons, freshwt. basis; light-grown seedlings.

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CASTELFRANCO ET AL.-RESPIRATORY CHANGES

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FIG. 2. Lipid content of castor bean endosperm(Zanzibar variety) as a function of germination timeat 29 to 300. A--A g of lipid/endosperm; dark-grownseedlings. A-A g of lipid/endosperm; light-grownseedlings. percent lipid in the endosperm, freshwt. basis; dark-grown seedlings. 0-0 percent lipidin the endosperm, fresh wt. basis; light-grown seedlings.

Fig. 1 and 2). Light appears to have little effecton the rate of lipid breakdown as far as has beentested. As one might expect, the lipid breakdownis slightly more rapid when peanuts are germinatedat 29 to 300 than at 22 to 230 but the trend is thesame as that presented in Fig. 1.

Fig. 3 and 4 give a visual summary of the histo-chemical observations made during the course of our

study. Each figure is a composite picture showingthe distribution of cvtochrome oxidase (left handside) and succinic dehydrogenase (right hand side)in tissue slices (peanut cotyledon in Fig. 3, castorbean endosperm in Fig. 4), the effect of age on theseactivities, and the effect of substrates and inhibitors.Fig. 3A and 3B, slices 1 through 10 show the resultsof an age study. Peanuts were planted on differentdays and were harvested on the same day, sliced, andincubated simultaneously. The age series show an

increase in the intensity of the histochemical reac-

tions as well as a gradual shift in the distribution ofactivity as germination proceeds from 0 days (slice10) to 21 days (slice 1). There is however, no

difference between the behavior of cytochrome oxi-dase activity (Fig. 3A) and that of succinic dehydro-genase activity i(Fig. 3B) during this proces,s. Bothactivities appear to be relatively uniformly distributedat the earlier ages, although the veins are alwayssomewhat more heavily stained than the surroundingstorage tissue. Later the vein regions become very

dark while the storage parenchyma develops color-

less areas; and finally, at 21 days (slice 1, 3A and3B) both enzyme activities appear to be confined tothe vascular tissue.

Fig. 3C shows the effect of NaN3 and NaCNupon the histochemical test for cytochrome oxidasein 9.5 day old tissue slices. Slice 11 was inculbatedin the standard PADA medium, slice 12 in PADAwith 1 mM NaN3, slice 13 in PADA with 1 mMNaCN and slice 14 in 0.2 M tris buffer. Fig. 3Eshows the effect of the same inhibitors upon 21 dayold peanut slices: slice 15 was incutbated in trisbuffer, slice 16 in PADA with 1 mM NaCN, slice 17in PADA with 1 mm NaN3 and slice 18 in thestandard PADA medium.

Fig. 3D and 3F show the effect of adding malo-nate or omitting succinate upon the histochemicalassay for succinic dehydrogenase. Slices 11 through14 were from 9.5 day old tissue. Slice 11 was incu-bated in the standard TNBT-succinate medium, slice12 in the same medium with 0.2 M malonate, slice 13in TNBT without succinate and slice 14 in tris bufferwith Na succinate. Slices 15 through 18, Fig. 3F,were from 21 day old cotyledon tissue. Slice 15 wasincubated in tris buffer with Na succinate, slice 16in TNBT without succinate, slice 17 in TNBT-suc-cinate with 0.2 'i malonate and slice 18 in standardTNBT-succinate medium.

Fig. 4 shows the behavior of germinating castorbean endosperm tissue slices. The left hand portionof this composite picture summarizes the results ob-tained with the PADA test; the right hand portionsummarizes the results obtained with TNBT. Fig.4A shows a series of slices ranging in age from1 day (slice 5) to 10 days (slice 1). All slices wereincubated simultaneously in the PADA medium.Fig. 4B shows an age series in which the slices wereincubated in the TNBT-succinate medium. Bothseries show that the respiratory activity is very lowinitially and increases with age. This increase isparticularly striking between the second and thefourth day (slice 4 to slice 3 of Fig. 4A and slice 2to slice 3 of Fig. 4B). The castor bean endospermtissue becomes visibly senescent after 7 or 8 days;the obvious decrease in succinic dehydrogenase ac-tivity at 10 days (Fig. 4B, slice 6) is very likelyattributable to incipient necrosis.

The histochemical tests indicate that both cyto-chrome oxidase and succinic dehydrogenase areevenly distributed throughout the thickness of theendosperm. The only cells which appear to havehigher enzymatic activity than the rest are those ofthe epidermal layer.

Fig. 4C shows the effect of the inhibitors NaN3and NaCN on the cytochrome oxidase assay. Slice6 was incubated in tris buffer, slice 7 in PADA con-taining 1 mM NaCN, slice 8 in PADA containing1 mm NaN3 and slice 9 in the standard PADAmedium.

Fig. 4D shows the effect of adding malonate oromitting succinate on the histochemical succinicdehydrogenase assay. Slice 7 was incubated in tris

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buffer with Na succinate, slice 8 in TNBT mlediumii (Fig. 3D.without succinate, slice 9 in TNBT-succinate medium for cvtoclwith 0.2 M malonate, anid slice 10 in the standard extractingTNBT-succinate medium,i. mining th(

Histochenical procedures essentially like ours enzyme acwere employed bv Avers et al. (3) for vital staining NaN3 (5of yeast mitocliondria. The specificity of TNBT for findings csuccinic dehydroge!nase under our experimental con- substrateditions is indicated by the results obtained -with the clhrome oxomission of succinate and the addition of mailonate The cc

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3F, aind 4D). The hiistochemical assay:romle oxidase was easily quantitized bythe tissues with lipid solvents and deter-

e red color spectrophotometricallv. Thisctivity wvas strongly inhibited by 1 mnm;9-85 %) or NaCN (88-96 %). Theseonfirm the validity of using the unnaturalPADA to study the distributionl of cvto-idase.ourse of cytochrome oxidase developlment

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FIG. 3. Summary of the histochemical observations with peanut cotyledon slices. The seeds wvere germinated at22 to 230 PADA tests for cytochrome oxidase are shown on the left hand side of the figure; TNBT tests for succinlicdehydrogenase are shown on the right hand side. The slices were incubated at room temperature, the PADA testsfor 1 lhr and TNBT tests for 2.5 hr. A) PADA test age series. Tissue ages: 1=21 day, 2=19 day, 3=17 day,4=14.5 day, 5=12 day, 6=9.5 day, 7=7 day, 8=4.5 day, 9_1 day, 10=0 day. B) TNBT test age series.1=21 dav, 2=19 dav, 3=17 day, 4=14.5 day, 5=12 day, 6--9.5 day, 7=7 day, 8=4.5 day, 9=2 day, 10=0 day.C) Effect of inhibitors on PADA test: 9.5 day old tissue slices; 11-= standard PADA solution, 12= PADA con-taining 1 mM NaN3, 13= PADA containing 1 mam NaCN, 14= tris buffer. D) Effect of malonate addition and suc-cinate omission upon the TNBT test: 9.5 day old tissue slices; 11 = standard TNBT-succinate solution, 12 =TrNBT-succinate containing 0.2 At Na malonate, 13 = TNBT solution without succinate, 14= succinate-tris buffer. E)Effect of inhibitors on PADA test: 21 day old slices; 15= tris bulffer, 16= PADA solution containing 1 mmNaCN, 17= PADA solution containing 1 mm NaN3, 18= PADA solution. F) Effect of malonate addition andsuccinate omission upon the TNBT test: 21 day old tissue slices; 15-= succinate-tris buffer, 16- TNBT solution with-out succinate, 17= TNBT-succiinate solution containing 0.2 At nmalonate, 18= standard TNBT-succinate solution.

792 PL,ANT PIIYSIOLOGY'

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CASTELFRANCO ET AL.-RESPIRATORY CHANGES

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FIG. 4. Summary of the histochemical observations with Zanzibar castor bean endosperm slices. The seeds weregerminated at 29 to 30°. PADA tests for cytochrome oxidase are shown on the left hand side of the figure; TNBTtests for succinic dehydrogenase are shown on the right hand side. The slices were incubated at room tempera-ture, the PADA tests for 45 min and the TNBT tests for 2 hr. A) PADA test, age series. Tissue ages:1= 10 day, 2= 6 day, 3= 4 day, 4= 2 day, 5= 1 day. B)INB1' test, age series. Tissue ages: 1= 1 day, 2= 2day, 3= 4 day, 4= 6 dav, 5= 8 day, 6= 10 day. C) Effect of inhibitors on PADA test: 6 day old tissue slices;6= tris buffer, 7= PADA solution containing 1 mm NaCN, 8= PADA solution containing 1 mm NaN ,, 9=standard PADA solution. D) Effect of malonate addition and succinate omission upon the TNBT test: 6 day oldtissue slices; 7- succinate-tris buffer, S= TNBT solution without succinate; 9= TNBT-succinate solution con-taining 0.2 At Na malonate, 10= standard TNBT-succinate solutioni.

during germination was followed quantitatively byincu,bating tissue slices with the PADA medium,extracting the slices with organic solvents andmeasuring the color spectrophotometrically. Theseresults are shown in Fig. 5 and 6. Several pointsare worth noting. Peanuts grown at 22 to 230 showa considerable lag in the development of cytochromeoxidase. This observation agrees with the findingsof Breidenbach et al. (6) based on spectral measure-ments on isolated peanut mitochondria. When pea-nuts were grown at 29 to 30°, this lag phase was notapparent (Fig. 5). Another interesting point is thedip in cytochrome oxidase activity between 5 and 7days in peanut cotyledons (Fig. 5). This inflection

was observed repeatedly in separate experiments. Itis probably related to the change in the distributionof respiratory enzymes between the different typesof tissues that make up the cotyledon (Fig. 3).During the first week, the storage parenchymaaccounts for most of the respiratory activity, but ata later stage cvtochrome oxidase and succinic dehy-drogenase become confined to the vein region. Thedip in cytochrome oxidase activity was not observedwith castor bean endosperm which lacks the develop-ment of vascular bundles. Since the experiment withZanzibar castor beans (Fig. 5) was not sufficient todetermine if a dip occurred in cytochrome oxidaseactivity ain additional experiment using Baker 296

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PLANT PHYSIOLOGY

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FIG. 5. Increase in cytochrome oxidase activity incastor bean endosperm and peanut cotyledons as a func-tion of germination time. Zanzibar castor beans were

germinated at 29 to 300. Virginia Jumbo peanuts were

germinated at 22 to 230 and 29 to 300. Tissue slices, 120A thick, were incubated for 1 hr at room temperaturein the PADA medium. The color formed during the in-cubation was extracted and measured as described inMaterials and Methods.

castor beans was undertaken to 'clarify this point(Fig. 6). During the first few days of germinationthe cytochrome oxidase activity increased quiteslowly. This initial phase was followed by a rapidincrease in the activity which continued until theendosperm became soft and obviously senescent.

Discussion

The developmental pictuire which emerges fromour experiments with castor bean endosperm is en-

tirely consistent with the findings of Akazawa andBeevers (1) and Albergoni et al. (2).

The relationship between the development ofmitochondrial respiration and the mobilization ofstorage fat appears to be more complex in peanut

cotyledons than in castor bean endosperm. In thefirst place the process of fat mobilization is consider-ably slower in peanuts (compare Fig. 1 with Fig. 2).This difference may be related to the composition offood reserves in the two seeds since peanuts contain40 to 50 % lipid and 15 to 30 % carbohydrate, whilecastor beans contain 64 % lipid and practically nocarbohydrate (9). One might therefore expect thatthe conversion of storage fats to sucrose is not aprocess of equal urgency in the two seeds. The spe-cific activity of isocitra,te lyase, a key enzyme in thisconversion, is 8 times greater in 5 day old castorbeans than in 7 day old peanuts (7).

Furthermore, there is considerable difference inthe extractability of the lipids from the two sources.Castor bean lipids are easily extracted into chloro-

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castor bean endosperm as a function of germination time.Baker 296 castor beans were germinated at 29 to 300.Tissue slices, 120, thick, were incubated for 45 min atroom temperature. The color formed during the incu-batiQn was extracted and measured as described in Ma-terials and Methods.

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CASTELFRANCO ET AL.-RESPIRATORY CHANGES

form-methanol-water mixtures but diethyl ether isrequired for the complete extraction of peanut lipids.The reasons for this difference are under investiga-tion. We surmise that this difference may be due toeither the chemical structure of the lipid moleculesthemselves, or to their association with proteins,carbohydrates and other cellular constituents.

Breidenbach et al. (5) found that the mitochon-dria increased continuously in peanut cotyledons upto 9.5 days of germination. However, it is importantto realize that in their work the mitochondria fromdifferent anatomical regions of the cotyledons werepooled and that, as we have shown now. the rate ofmitochondrial biogenesis is not uniform in thevarious cotyledonarv tissues. Toward the end oftheir experimental period, which extended from 0 to9.5 days, there must have been a decrease in themitochondrial population in the storage parenchvmacells, which was more than compensated for by themitochondrial increase in the vein regions. Con-sidering the peanut cotyledon as a whole, the lipidbreakdown proceeds at a slow and relatively uniformrate (Fig. 1). Information on the lipid content ofvarious kinds of cells as a function of germinationtime is lackilng. For these reasons it is impossibleat present to correlate the level of mitochondrialactivity with the rate of lipid dissimilation.

Our observations on the change in the distributionof respiratory enzymes between the different tvpesof cotyledonary tissues as germination proceeds, areconsistent with the results of Opik (11) on thecommon bean, Phaseolis vulgaris L. In her cyto-logical study of germinating bean cotyledons sheobserved that the mitochondria of the vascularbundles were more numerous and had a greaterdensity of cristae than those of the storage paren-chvma cells. Furthermore, the vascular bundlesmaintained a high rate of respiratory activitv afterthe storage cells had ceased to respire. Since beansare starch-storing rather than fat-storing seeds, itappears that the respiratory increase in the veinregions is not related to the conversion of lipid tosucrose but rather to the differentiation of vasctularelements and to the transport of sugar (whetherderived from starch or from fat) from the cotyledonsto the growing embryo-axis. Sugar concentration isknowin to exert a profound influence upon the dif-ferentiation of phloem and xylem elements in anumber of higher plant systems (15, 16), althoughthe mechanisms of these effects are still quite ob-scure.

In conclusion, we must stress that peanut cotvle-dons are morphologically and physiologically muchmore complex than castor bean endosperm since theyare in a sense analogous to both castor bean cotvle-dons and castor bean endosperm. While the endo-sperm of germinatinig seeds appears to behave as arelatively simple tissue, cotyledons exhibit a greaternumiber of physiological and morphogenetic processeswhich go on simultaneously and affect one anotherin a variety of ways. At the present time, the fac-tors which control and coordinate the various aspects

of cotyledonary development during seed germinationare still essentially unknown.

Literature Cited

1. AKAZAWVA, T. AND H. BEEVERS. 1957. Mitochon-dria in the endosperm of the germinating castorbean: a developmental study. Biochem. J. 67:115-18.

2. ALBERGONi, F., P. LADo, G. MARZIANI, AND E.MARRE. 1964. Sull' evoluzione dei mitochondrinell' endosperma di semi di ricino in germinazione.Giorn. Bot. Ital. 71: 469-88.

3. AV-ERS, C. J., F. H. LIN, AND C. R. PFEFFER. 1965.Histochemical studies of mitochondrial variationduring aerobic growth of respiration-normalbaker's yeast. J. Histochem. Cytochem. 13: 344-49.

4. BLIGH, E. G. AN-D W. J. DYER. 1959. A rapidmethod of total lipid extraction and purification.Can. J. Biochem. Physiol. 37: 911-17.

5. BREIDENBACH, R. W., P. CASTELFRANCO, AND C.PETERSON. 1966. Biogenesis of mitochondria ingerminating peanut cotyledons. Plant Physiol.41: 803-09.

6. BREIDENBACH, R. W., P. CASTELFRANCO, AND R. S.CRIDDLE. 1967. Biogenesis of mitochondria ingerminating peanut cotyledons II. Changes incytochromes and mitochondrial DNA. Plant Phy-siol. 42: 1035-41.

7. CARPENTER, W. D. AND H. BEEVERS. 1959. Dis-tribution and properties of isocitratase in plants.Plant Physiol. 34: 403-09.

8. CHERRY, J. H. 1963. Nucleic acid, mitochondriaand enzyme clhanges in cotyledons of peanut seedsduring germination. Plant Physiol. 38: 440-46.

9. MAYER, A. M. AND A. POLJAKOFF-MAYBER. 1963.The Germination of Seeds. The Macmillan Com-pany. New York, New York. p 19.

10. Official Methods of Analysis of the Association ofOfficial Agricultural Chemists. 1960. W. Hor-witz, ed. Association of Official AgriculturalChemists, Washington, D. C. p 287-88.

11. OPIK;, H. 1966. Changes in cell fine structure inthe cotyledons of Phaseoluis vuilgaris L. duringgermination. J. Exptl. Botany 17: 427-39.

12. REBEIZ, C. AND P. CASTELFRANCO. 1964. Extra-mitochondrial enzyme system from peanuts cata-lyzing the 3-oxidation of fatty acids. Plant Phy-siol. 39: 932-38.

13. REBEIZ, C., P. CASTELFRANCO, AND A. H. ENGEL-BREcHT. 1965. Fractionation and properties of anextra-mitochondrial enzyme system from peanutscatalyzing the /-oxidation of palmitic acid. PlantPhysiol. 40: 281-86.

14. REBEIZ, C., P. CASTELFRANCO, AND R. W. BREI-DENBACH. 1965. Activation and oxidation ofacetic acid by cell free homogenates of germinatingpeanut cotyledons. Plant Physiol. 40: 286-89.

15. TORREY, J. G. 1963. Cellular.patterns in developingroots in cell differentiation. Symposia of theSociety for Experimental Biology. Academic Press,New York. p 285-314.

16. WETMORE, R. H. AND J. P. RIER. 1963. Experi-mental induction of vascular tissues in callus ofangiosperms. Am. J. Botany 50: 418-30.

17. YA-MADA, M. AND P. K. STUMPF. 1965. Fat me-tabolism in higher plants XXIV. A soluble 3-oxi-dation system from germinating seeds of Ricinutscomnhuinis. Plant Physiol. 40: 653-58.

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