Plant Physiol. (1969) 44, 508-516

Fat Metabolism in Higher Plants XXXVII.Characterization of the P-Oxidation Systems FromMaturing and Germinating Castor Bean Seeds'

D. Hutton and P. K. StumpfDepartment of Biochemistry and Biophysics, University of California, Davis, California 95616

Received October 28, 1968.

A bstract. In the maturing castor bean seed (Ricinus communis), maximum $3-oxidationappears at 28 days after flowering and in the germinating seed, 4 days after germination.Highest specific activities for both 1-oxidation systems and their component enzymes areassociated with cytosomal particles banding at a density of 1.25 g/ml in a sucrose gradient.Substrate specificity studies indicate that of several fatty acidis, ricinoleate is oxidized mostrapidly by the preparation from the maturing seed (28 days after flowering) while palmitateand linoleate are oxidized most rapidly by extracts obtained from tissue germinated for 4 days.The :-oxidation activities observed in both systems reflect the expression of activity of atleast 3 of the component enzymes, crotonase, ,3-hydroxyacyl dehydrogenase and /3-keto-thiolase,which rise and fall coordinately. Acyl thiokinase does not appear to play a limiting role inregulating :-oxidation per se under the conditions employed here.

Earlier work carried out in this lafboratory byYamada and Stumpf (24) with germinating castorbeans reveal,ed the existence of both a soluble and aparticulate /3-oxidation system. The assay svsteminvolved trapping acetyl-CoA produced by /8-oxida-tion of 14C-containing long chain fatty acids withmalate synthetase [L-malate glyoxylate lyase (CoA-acetylating), 4.1.3.2] and added glyoxylate to yieldmalate which via conversion through oxaloacetatewas eventually decarboxylated to yield phosphenol-pyruvate and radioactive carbon dioxide. Radioac-tive CO2 was thus employed as an overall measure of/3-oxidation. The peak of 14C09 evolution observedat 4 days after germination (DAG) could be relatedto several factors including, a rise in /8-oxidationactivity per se, a rise in malate synthetase, and re-lated activities or to both these factors. Beeverset al. (2) have shown that in a variety of oil bearingseed tissues during germination the activities ofmalate synthetase and isocitrate lyase (threo-Ds-Isocitrate glyoxylate lyase, 4.il.3.1), key enzymes ofthe glyoxylate cycle and a prerequisite for the con-version of fat to sucrose, rise and fall simultaneouslywith the peak of lipid utilization.

As part of an investigation in this laboratory intothe developmental and mechanistic aspects of /3-oxi-dation of ricinoleate in the castor bean, /3-oxidationactivity has been assayed directly by measuringacetyl-CoA formation, thereby permitting a directcorrelation of /3-oxidation activity per se as related

1 This investigation was supported by NSF Grant GB5879X and by USDA Grant 12-14-100-7990(74).

to chan-ges in maturing and germinating tissues.The same assay procedure has been further

utilized to investigate unpublished preliminary find-ings (14) that a /8-oxidation system appears to peakaround 28 to 30 days after flowering (DAF) inmaturing castor bean seeds on the plant.

During the investigation, we were in communica-tion with Professor Harry Beevers and his groupwho have also independently observed similar resultsof /3-oxidation in germinating castor bean glyoxy-somes.

Materials and Methods

Castor bean seeds (Ricinus communis L., varietyBaker 296) were obtained from Dr. W. E. Domingoof the Baker Castor Oil Company, La Mesa, Cali-fornia, arranged by Dr. R. D. Brigham of theU.S.D.A., Agricultural Research Service, Luibbock,Texas.

Maturing castor bean tissue was gathered fromripening seed capsules of plants grown either in theopen field during summer, or in the green houseduring winter. Considerable variation in develop-ment was observed when "days after flowering" wasused as the criterion of seed maturation. For thisreason, the morphological features of representativeseeds at various DAF stages were used as a moreaccurate guide to the developmental stage reached bythe tissue. Cross sections of the seed at the releventtimes are depicted in Fig. 1. Seeds gathered subse-quently were thus assigned DAF periods consistentwith the morphological development scheme outlinedabove. Starting material was obtain,ed by removing

508 www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from

Copyright © 1969 American Society of Plant Biologists. All rights reserved.

HUTTON AND STUM\IPF-f-OXIDATION IN CASTOR BEAN SEEDS

pliable, cream opaque lipidwhite seed containing toughening,

coat endosperm purple brown

embryo

translucentucellus

20 DAF 24 DAF

hardening, purple

moistopaque

endosperm

28 DAF

brittle, purpleseed coat

32 DAF 40 DAF

FIG. 1, Cross section views of the maturing castor

bean during development.

the total contents of the seed within the seed coat.For germination studies, castor bean seeds were

grown at 300 in moist vermiculite in the dark. Theendosperm tissue was obtained by removing testae,hypocotyls and roots and where practically feasible,cotyledons as well; the latter could only readily beremoved between the fifth and seventh days aftergermination (DAG).

Cofactors were obtained from the followingsources: NAD+, NADP, ATP from Sigma Chem-ical Company, CoA from P-L. Biochemicals Incor-porated. Crotonic anhydride, crotonic acid and dike-tene were purchased from K and K LaboratoriesIncorporated. Dithiothreitol l(DTT) was obtainedfrom Calbiochem. Condensing enzyme (Citrate ox-

aloacetate lyase [CoA-acetylating], 4.1.3.7) was pur-chased from Calbiochem and malate dehydrogenase(L-Malate :NAD oxidoreductase, 1.1.1.37) fromSigma Chemical Company.

Maturing and germinating tissue was homoge-nized in 0.1 M potassium phosphate buffer pH 7.3,0.4 M sucrose, 0.025 M NaF, 0.010 M DTT, 0.001 M

EDTA, in a Servall Omni-mixer blender at setting100 for 10 sec and 1 min, respectively. For 20 to 28DAF tissue, 1:1 (tissue:buffer) ratio was used; for32 to 40 DAF and for 1 to 7 DAG tissue, 1:2(tissue:buffer) ratio was used. In 1 experimentwhere crotonase (L-3-Hydroxyacyl-CoA hydro-lyase,4.2.1.17) activity was measured in a 12,000g super-natant fraction the DTT concentration was reducedto 0.001 M to minimize any addition across the doublebond of the substrate, crotonyl-CoA.

For those experiments where 12,000g pellets were

prepared for sutbsequent isopycnic centrifugation on

sucrose gradients, tissue was hand chopped with a

razor blade and homogenized in a slightly modifiedversion of the buffer utilized by Breidenbach et al.(3): 0.1 M tris buffer pH 7.5, 0.4 M sucrose, 0.01 M

DTT, 0.1 % BSA, 0.01 M KCl, 0.001 M EDTA,0.0001 M MgCl2,6H2O at the same ratios of tissueto buffer mentioned above. Homogenization was

conducted in a mortar and pestle for 2 min withoutabrasive.

Crude homogenates were passed through 4 laversof cheesecloth and centrifuged at 500g for 10 min.The 5OOg pellet was resuspended in buffer and re-sedimented at 10,000g for 5 min and again taken upin a minimum of buffer. The supernatant from thecrude homogenate was centrifuged at 12,000g for30 min. The 12,000g pellet was washed with bufferby resuspending and recentrifuging for 10 min at20,000g and finally taken up in a minimum of buffer;in those cases where the pellet was to be applied toa sucrose gradient the pellet was resuspended in30 % w/w sucrose after washing in buffer. The12,000g supernatant fraction, when necessary, wassubsequently centrifuged at 105,000g in a SpincoModel L ultracentrifuge for 1 hr. The pellet waswashed by resuspending in buffer and recentrifugedfor 1 hr at 105,000g and finally taken up in a mini-mum of buffer. The supernatant fraction from thisstage was designated the 105,000g supernatant frac-tion.

For sucrose gradient runs, 12,000g pellets sus-pended in 30 % sucrose, were layered (20-30 mg)on linear sucrose gradients consisting of 5 mls 60 %w/w sucrose and 24 mls of sucrose grading froni60% w/w to 30 % w/w linearly in Spinco rotor SW25/1 cellulose nitrate tubes. Gradients were cen-trifuged for 3 hr at 22,500 rpm and sutbsequentlyfractionated into 1 ml fractions with the aid of aGilson fraction collector and drop counter attachment.

All stages during homogenization and preparationof respective fractions were carried out at 0 to 30.

Incubations for a-oxidation assays were carriedout at 300 for 1 or 2 hr in 50 ml Erlenmeyer flasksequipped with serum caps to reduce evaporation.Reactions were terminated through the addition of0.1 ml 4 M perchloric acid.

Fatty acid synthetase assays were incubated in50 ml Erlenmeyer flasks for 2 hr at 30°. The re-action was terminated through addition of 0.2 ml40 % KOH and the flask contents heated at 80° for30 min to saponify the fatty acid thioesters (bothACP and CoA derivatives). Concentrated HCl0.3 ml was then added and the fatty acids extractedas follows. Methanol (7.5 ml) was added to theflasks and the total contents then transferred to large20 cm X 2.5 cm test tulbes. The flasks were rinsedwith 1.5 ml water and the rinsings added to themethanolic reaction mix. After the addition of3.75 ml of chloroform, the tubes' contents were shakento obtain good intimate mixing. A further 3.75 mlof chloroform was added, again the mixture was wellagitated followed by an addition of 3 ml of water.After a final vigorous mixing the 2 phases wereallowed to separate and the upper aqueous layerdiscarded. The lower chloroform layer was washedtwice with 10 ml of 1 M HCl/saturated NaCl solutionto remove traces of contaminating radioactive sub-strates and subsequently dried over anhydrous sodium

509

www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from Copyright © 1969 American Society of Plant Biologists. All rights reserved.

PLANT PHYSIOLOGY

sulphate. Aliquots were counted (after the chloro-form had been evaporated) in a toluene POPOP/PPO based scintillation solution using a NuclearChicago Un.ilux Scintillation Spectrometer.

The estimation of acetyl-CoA and assays of thefl-oxidation enzymes were performed with the aid ofa Cary 14 dual beam recording spectrophotometer.Overall fl-oxidation was ass.ayed by determining theacetyl-CoA produced enzymatically by Decker'smethod (9). The reaction flask contents were de-proteinized for acetyl-CoA analysis according to theprocedure of Decker (10). Crotonase was assavedby the method of Stern (22) while 8-hydroxyacyldehydrogenase (L-3-hydroxyacyl-CoA :NAD oxidore-ductase, 1.1.4.35) and f-keto-thiolase (Acyl-CoA:Acetyl-CoA C-acyl transferase, 2.3.1.16) were as-sayed according to Overath et al. (18). KCN, 3.3mm, was included in the cuvette when assaying,8-hydroxyacyl dehydrogenase to inhibit DPNH oxi-dase activity of mitochondria (3). Crotonyl-CoAand acetoacetyl-CoA were prepared by the methodsof Stern (22) and Decker (8) respectively. Pal-mityl-CoA was prep.ared both by a modified vers.ionof Stern's procedure 1(22) (anhydride dissolved intetrahydrofuran) and by Seubert's method (21) in-volving the acyl chloride.

The marker enzymes which were employed toidentify the various bands obtained in the sucrosegradients wvere fumarase (L-malate hvdro-lyase,4.2.1.2) for mitochondria and isocitrate lyase forglyoxysomes. Fumarase was assayed according toRacker (19) and isocitrate lyase by the method ofDixon and Kornberg (12). Catalase (Hydrogenperoxide:hydrogen peroxide oxidoreductase 1.11.1.6)was assayed according to Beers and Sizer (1).Acetyl-CoA carboxylase [Acetyl-CoA :carbon dioxide

Table I. Locali2ation of /3-oxidation Activity in Sub-cellular Fractions of 28 DAF and 4 DAG

Castor Bean Seeds

Tissue Fraction Activity'

units/mlg unzits/seed %25OOg pellet 0.30 0.29 3.6

2812,000g pellet 10.70 5.92 73.6

DAF105,000g pellet 0.00 0.00 0.0105,000g supernatant

fraction 0.60 1.83 22.8

500g pellet 2.20 9.31 0.14

12,000g pellet 59.30 164.00 4.6DAG

105,000g pellet 5.30 3.61 0.1105,000g supernatant

fraction 121.90 3560.00 95.2

Activity unit = mMmoles acetyl-CoA produced/hr(free palmitate as substrate, using incubation condi-tions given in legend to Fig. 2).

2 % based on units/seed.

ligase (ADP), 6.4.1.3] was determined by the methodof Burton and Stumpf (6) while fatty acid svnthetaseactivity was assayed using Brooks and Stumpf'smethod (5). Glycolic acid oxidase (Glycollate:oxy-gen oxidoreductase, 1.1.3.1) and glvoxvlate reductase(Glvcollate :NAD oxidoreductase, 1.1.1.26) were de-termined by the nmethods of Zelitch (25, 26).

Protein wvas determined by the method of Lowvryetal. (16).

Results

Preliminary experiments indicated that fl-oxida-tion in maturing seeds peaked around 28 days afterflowering and 4 days after germination. The dis-tribution of these activities among the various sub-cellular fractions are given in table I.

In the 28 DAF castor bean tissue, the highestspecific activity is associated with the 12,000g pellet.However a sizable proportion of the activity(22.8 %)', although of lower specific activity (20 foldless), is found in the 105,l000g supernatant fraction.

In contrast, in the 4 DAG castor bean seed, the105,l 0g supernatant fraction contains the greatestcapacity and highest specific activity to oxidize fattyacids; however, although only a minor proportion ofoxidation takes place in the 12,000g pellet, the specificactivity located here is remarkably high (approxi-mately 50 % that of the 105,0)00g supernatant).

Isopycnic sucrose density gradient centrifugationrevealed that fl-oxidation activity, residing in the12,000g pellet, was associated with more than 1 band.As stummarized in table II in both 28 DAF and 4DAG tissue, the gradients 'showed 2 prominent bands,1 centered around a density of 1.19 g/ml and theother, considerably heavier, around 1.25 g/ml.\WThereas the 1.25 band from 4 DAG tissue appearedquite sharp, its 28 DAF counter part usually wasconsiderably more diffuse. In both maturing andgerminating tissues the 1.19 band was iden'tified asmitochondrial since it possessed virtually all thefumarase activitv. The heavier 1.25 band of the4 DAG tissue contained substantial isocitrate lyaseactivity but negligible amounts were found in thecorresponding 28 DAF band. However, catalase,acetyl-CoA carboxvlase and fatty acid synthetasecould be detected in the 1.25 b.and of the 28 DAFtissue; catalase occurred in several fold excess overthe activity detectable in the m.itochondrial band.Neither glycolic acid oxida'se nor glyoxylate reduc-'tase activity was detected in any bands from 4 DAGand 28 DAF tissue, Tentatively these heavier par-ticles probably belong to a population of organelleswhich include cvtosomes, microbodies, glyoxvsomesand peroxisomes. It should be noted that the 1.25band posses'ses significantlv higher specific activityregarding fl-oxidation than any other sub-cellularfraction investigated in both germinating and matur-in!g castor bean tissue.

510

www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from Copyright © 1969 American Society of Plant Biologists. All rights reserved.

HUTTON AND STUMPF-fl-OXIDATION IN CASTOR BEAN SEEDS

The capacities of the 2 systems to oxidize a

num(ber of fatty acids are depicted in Fig. 2 and 3.For the maturing castor bean seed, f-oxidationpeaked for all substrates tested at 28 DAF exceptfor palmityl-CoA where an earlier rise led to a

maximum at 24 DAF which remained as a plateaufor a considerable number of days. However ricino-

leic acid was oxidized at least 3 fold more rapidlythan any other substrate tested. The remaining fattyacids attained peaks of activity which clusteredaround 20 % that of ricinoleate.

During germination of the castor bean, thefl-oxidation system achieves a maximum for all fattyacids tested at 3 DAG (in some experiments the

Table II. Enzzmic Characterization of Bands Obtained by Suicrose Density Gradients

Tissue28 DAF 4 DAG

Enzyme Band density in sucrose gradient (g/ml)activity1 1.19 1.25 1.19 1.25

units/iiig unJtits/1ing0-Oxidation 17.9 42.3 88.5 187.5Fumarase 1040.0 217.0 7660.0 343.0Isocitrate lyase 41.8 27.7 100.0 717.0Catalase 42.9 145.2 67.7 728.0Fatty acid synthetase 23,500 32,900 0 0Acetyl-CoA carboxylase 4.9 6.1 0.0 0.0Glycolic acid oxidase 0.0 0.0 0.0 0.0Glyoxylate reductase 0.0 0.0 0.0 0.0

Units of activity are: for fumarase, isocitrate lyase, glycolic acid oxidase and glyoxylate reductase, m,"moles sub-strate reacted/min: for catalase, Amoles substrate reacted/min; for 0-oxidation, musmoles acetyl-CoA produced/hr(palnmitate as substrate); for acetyl-CoA carboxylase, m,umoles 14CO2 fixed/min; for fatty acid synthetase, cpmof 14C-acetyl-CoA and 14C-malonyl-CoA incorporated into long chain fatty acids/2 hr.

Table III. Speciflic Activities and Percent Total Activity of 83-oxidation and Coiiipofiteitt Enzyiiies in Suicrose Gradientsof 12.000g Pellets From Maturing and Germinating Tissute During Dezelopment

Enzyme specific activities'

Development Band density in 3-Hydroxyacylstage sucrose gradient2 0-Oxidation3 Crotonase dehydrogenase 0-Keto-thiolase

g/ml2 DAG 1.13 15.10 30.70 85.80 14.20

1.19 4.20 (30)4 62.80 (25) 142.00 (25) 11.40 (49)1.25 18.80 (70) 349.00 (75) 773.00 (73) 22.40 (51)

4 DAG 1.13 539.50 967.00 1260.00 64.301.19 88.50 (34) 207.00 (7) 381.00 (11) 10.70 (17)1.25 187.50 (66) 3210.00 (93) 335000 (89) 58.80 (83)

6 DAG 1.13 0.00 157.00 643.00 42.001.19 0.00 (0) 230.00 (32) 629.00 (38) 5.00 (63)1.25 0.00 (0) 526.00 (68) 1125.00 (62) 3.20 (37)1.13 6.23 0.00 76.80 18.00

20 DAF 1.19 3.16 (35) 43.90 (83) 195.50 (91) 1200 (91)1.25 18.00 (65) 27.50 (17) 61.90 (9) 5.04 (9)1.13 3.80 59.40 112.00 35.70

28 DAF 1.19 17.90 (47) 88.00 (36) 188.00 (16) 2.50 (30)1.25 42.30 (53) 323.00 (64) 833.00 (84) 12.30 (70)1.13 2.81 13.20 53.20 10. 0

38 DAF 1.19 2.69 (53) 22.20 (30) 6670 (21) 6.15 (42)1.25 3.80 (47) 83.20 (70) 399 00 (79) 13.20 (58)

1 See legend to table 11 for definitions of activity units.2 Band positions are: 1.13 - top of gradient, 1.19 - mitochondrial, 1.25 - cytosomal.3 Free palmitate employed as substrate.4 Values given in parenthesis in table represent percent total activity i.e. specific activity X mg protein in respective

band found distributed b.tween cytosomal and mitochondrial bands.

511

www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from Copyright © 1969 American Society of Plant Biologists. All rights reserved.

PLANT PHYSIOLOGY

9C

7C

50

"I

z 30

ic

i Ricinole ate

i P. i Pamtt

I ,/

I/ \\ Oae. / * .Pamtlo

/

I /..'I. ---

20 24 28 32 36 40 44DAF

FIG. 2. Substrate specificity for $-oxidation in12,000g pellets from maturing castor beans. Acetyl-CoAproduction expressed as units/mg. (See table II for unitdefinition.) Incubations contained in a final volume of1 ml: -50 ,moles potassium phosphate buffer pH 7.4,0.75 ,tmoles CoA, 1 Amole ATP, 1 ,mole NAD+, 1,umole NADP+, 1 ,umole MnCl, 0.12 /tmole fatty acid(NHW4, or CoA derivative), 0.5 mg 12,000g pellet pro-tein. Flasks were incubated for 2 hr at 300.

peak occurred at 4 DAG) but thereafter there was aprecipitous decline of activity, In contrast to thematuring castor bean, however, both palmitate andlinoleate were oxidized to a greater extent at 3 DAGthan were any of the other fatty acids. In this case,the oxidation activity for palmitate and linoleate onlyamounted to approximately a 2 fold increase overthe other fatty acids and represented a 20 fold in-crease in capacity compared with the oxidation rateat 1 DAG.

Assays of 3 component systems, namely crotonase,/3-hydroxyacyl dehydrogenase and /8-keto-thiolase,during development of both fl-oxidation systems areillustrated in Fig. 4. Clearly all enzymes reach amaximum at approximately the same time as theparent maximae of overall f8-oxidation.

The results of the development studies made ofthe 12,000g pellets on sucrose density gradients ob-tained from 20, 28, and 38 DAF and 2, 4, and 6 DAGcastor beans with regard to fl-oxidation and itscomponent enzymes' specific activities are summar-ized in table III. Specific activities of both f8-oxi-dation and its component enzymes during develop-ment mirror the results obtained with less refinedfractions. All activities in the 1.13 soluible, the 1.19mitochondrial and 1.25 cytosomal bands rise and fallthrough the 28 DAF and 4 DAG peaks. In general,

rises were greater for activities located in the heavy1.25 bands than in the soluble 1.13 and the mito-chondrial 1.19 bands. Although the highest specificactivity with regard to the complete f8-oxidationsy-stem resides in the 1.13 density band of the 4 DAGextract, high specific activities of both fl-oxidationand its component enzymes are associated with theheavy 1.25 band. The anomalous behavior of the,8-keto-thiolase seen in 12,000g pellets from maturingcastor beans is again observed in the heavy 1.25 bandon sucrose gradients where it is seen .to rise between20 and 28 DAF but then maintains this level throughto 38 DAF; this property is not shared by the other2 enzymes which fall off in activity after 28 DAF.

4r

3

2c~J10

z

1

Palmityl- CoA.. .

I,

0 1 2 3 4 5 6 7

DAGFIG. 3. Substrate specificity for :-oxidation in 12,000g

supernatant fractions from germinating castor beans.Acetyl-CoA production expressed as uInits/mg. For in-cubation conditions see legend to Fig. 2, except 12,000gsupernatant fraction protein used. Flasks were incubated1 hr at 300.

,8-keto-thiolase activity in the mitochondrial band ofmaturing castor bean tissue also appears unusual; asharp drop in activity is seen when passing from20 to 28 DAF followed by a partial recovery at38 DAF.

\When particle bound enzyme activities are ex-pressed on a total activity basis i.e., taking intoaccount the total particulate protein found in eachof the 1.25 and 1.L19 bands, the 1.25 band appears tocontain the greatest capaci.ty for f8-oxidation; like-'wise the greatest capacities for the component enzyme

512

i,,

x0

www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from Copyright © 1969 American Society of Plant Biologists. All rights reserved.

HUTTON AND STUMPF-/3-OXIDATION IN CASTOR BEAN SEEDS

activities are found located in the 1.25 band. Onenotable exception exists at 20 DAF when the major

tr-iAainf1n1PInI7yrmP arfiv1tv cnnnpJrJ toLho- lUI..dlPU in

Discussion

1.19 band in contrast to the observation that for Considerable /8-oxidation activity appears to de-rall b3-oxidation per se, the bulk of activity is velop in 2 phases of the life cycle of the castor bean.sent in the 1.25 band. During the maturation phase of the seed, a /3-oxi-

dation system appears which increases to a maximumat 28 DAF. This confirms previous unpublished

*.20t5 studies (14) carried out in this laboratory whichlt showed that 1_14C fatty acids, fed to tissue slices of

InIV@Aa. maturing seeds, were oxidized to 14CO0 which peakedbetween 20 and 40 DAF. Remarkable is the obser-

)4o vation that this system arises and peaks coinciden-/Vtially with the fatty acid synthetase system respon-+/(\sible for synthesizing ricinoleic acid (7, 13, 14).

During the germination phase, a /8-oxidation)I 2 / X \ \ 3 system exhibits a maximum activity between 3 and 4

DAG. The data presented here confirm the findingsof Yamada et al. (24). This /3-oxidation system ispresumably concerned with mobilization of the lipid

D- -8 / \2 stores by conversion of the ricinoleic acid to sucrosefor subsequent embryo development (2).

The particulate site with highest specific activityco both for /8-oxidation and its component enzymes is

4 l1 z associated with an organelle, heavier than mitochon-dria and banding at a density of 1.25 g/ml in thesucrose gradient.

Such particles have received many and diverse20 24 28 32 36 40 44 descriptions ranging from specific terms derived

DAF from enzymes found within them e.g. peroxisomes(11,23) and glyoxysomes (4) to more general termse.g. cytosome (17) and microbodies (20). We pre-fer the general description of cytosome for want ofa better term and will use it to describe any particle

t or population of particles sedimenting in a sucrose'gradient at a density of 1.25 g/ml. The size of

, be these particles coupled with the fact that they all4, may possess only 1 limiting membrane (electron..r-147 micrographs of glyoxysomes (4) and cytosomes (17)oAashow only a single membrane) possibly accounts for

120 12 their fragility. Even when gentle mortar and pestletechniques are employed to maintain as many particles

10 intact as possible, extensive /8-oxidation activity stillappears in the 105,000g supernatant fraction. This

..80 / / \ \8 , is most pronounced in the 4 DAG tissue where 95 %v/\\of the activity is located in the soluble proteins; in/6 n the 28 DAF tissue gentle homogenization gives better/// results with only about 25 % of the activity now

40 4 present in the supernatant fraction. Cytosome fra-gility is also evidenced by the relatively high amounts

2 of /8-oxidation activity and that of its componentenzymes at the top of the sucrose gradients where

_______._____._____._____._____._____.____ solulble proteins presumably remain (see table III)0 1 2 3 4 5 6 7 although the original pellets were thoroughly washed

DAG with buffer to remove presumably all entrapped solu-FIG. 4. Assays of the enzymes of /-oxidation during ble cytoplasmic proteins. It would therefore suggestrelopment of a) maturing and b) germinating castor that /3-oxidation is possibly associated with a discreteLns. Units expressed as m,moles substrate per reacted cytosomal particle which under the conditions pres-min. ently employed is susceptible to rupture. The obser-

theovepre

0z

I1-zI

devbeaper

513

www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from Copyright © 1969 American Society of Plant Biologists. All rights reserved.

PLANT PHYSIOLOGY

vation that differing amounts of fl-oxidation enzymesare associated with the cytosomal and mitochondrialbands' particles would appear to argue against anon-specific entrainment of enzymes from the solublefraction of the cell by "sticky" particles. To validatethis viewF, further attention should be given to iso-lating higher proportion.s of in,tact cytosomes throughutilization of extraction buffers of higher osmolarity.Presumably therefore assuming fl-oxidation is lo-cated in cytosomal elements, preservation of the in-tegrity of the latter should lead to higher recover.iesof f8-oxidation in these particles and less in thesoluble fraction of the cell.

During developmen.t of the maturing and germi-nating tissues of the castor bean the proportion of12,000g pellet protein/seed increases. In matturingcastor beans the increase is prolonged through the20 to 40 DAF per.iod; in the germinating bean theproportion of 12,000g pellet protein/seed reaches apeak at 4 DAG and thereafter declines. Examina-tion of the sucrose gradients at 20 DAF and 2 DAGreveals little or no protein in the 1.25 band or cyto-some region; however a prominent mitochondrialband is present at a density of 1.19 g/ml. At thepeak of fl-oxidation activity in the respective tissues,considerable cvtosomal formation has taken place,particularly in the germinating beani, and a band isnow observed at 1.25 g/ml. Although at latertimes in the respective t.issues, fl-oxidation activityfalls off, considerable protein remains in the cvto-somal band. As development proceeds, fl-oxidationand its component enzymes in the cytosomal band ofboth systenms increase in specific activity and thenfall off once the peak has been reached, a similartrend is seen in the mitochondrial hand but the mag-hitude of the increase, especially for the componentenzymes. is much lo-wer. f8-keto-thiolase appears toexhibit anomalous behavior in the cvtosomal bandof the matturing castor bean system since its activitydoes not fall after 28 DAF; this confirms studies'ising 12,00g pellets from 20 to 40 DAF castorbean.s. The reason for this is unknown. Also ofinterest is the observation that in the mitochondrialbands of both matuiring and germ.inating tissues theflB-oxidation enzymes do not precisely mirror thecorresponding development peaks of overall f8-oxida-tion. Passing from 2 to 6 DAG, crotonase andfi-hydroxyacyl dehydrogenase activities constantlyincrease, while f8-keto-thiolase shows a drop in ac-tivitv; the latter observation possibly is responsiblefor the fall in overall fl-oxidation noted in passingfronm 4 to 6 DAG. A similar trend is not seen inthe mitochondrial bands fromn maturing castor beanti s-sue. Ratlher, only crotonase shows a pronouincedrise and fall in activity, with 8-hydroxyncyl dehy\-drouenase and fl-keto-thiolase each showing decreasesin passinig from 20 DAF to 38 DAF. It is interest-ing to note further that whereas for crotonase andfi-hydroxyacyl dehydrogenase the specific activitylocated at the top of the gradient is practically in all

cases lower than the value in the correspondingcytosomal band, fl-keto-thiolase in most cases has agreater specific activity at the top of the gradientthan in the corresponding cytosomal band. Thisan,omalous distribution of f8-keto-thiolase activity onsucrose gradients cannot be explained at present.

Of further interest, at 20 DAF the major activityof 3 fl-oxidation enzymes lies in the mitochondrialband whereas the activity of overall f8-oxidationappears to reside in the cytosomal band. It is pos-sible th.at at this period of maturation, either themitochon-drial acyl thiokinlases or acvl dehydrogenaseis limiting. Clearly the other 3 enzymes are in highconcentration in the mitochondrial band (see tableIII). As the tissue matures, these limiting factorspartly disappear allowing s.ome increase in overallmitochondrial f8-oxidation activity yet the very greatincreases in cytosomal fl-oxidation enzyme activitiesresult in the cytosomal band ultimately possessinghighest overall fl-oxidation and component enzymeactivities.

It should be emphasized that values of fl-oxida-tion based on acetyl-CoA production will necessarilyyield minimal data when considering oxidation sys-tems either in the mitochondria or cytosomes. Thus,depending on the levels of intermediates and cofac-tors within the intact organelle, acetyl-CoA will bemetabolized further or allowed to accumulate, viz.in mitochondria the TCA cycle would aid in reducingthe level of acetvl-CoA, and likewise in glyoxysomes,the glyoxylate cycle.

Attempts have been made to characterize thecytosomal bands found in sucrose gradients of 12,000gpellets from both maturing and germinating seeds.The band from germinating seeds possesses traces offumara,se (possibly a result of entrainment from themitochondrial band) but substantial amounts of iso-citrate lyase activity. It would appear to be identicalto the glyoxysome band characterized by Breidenbachet al. (4). The possession by these particles of boththe glyoxylate cycle enzymes and also those offl-oxidation would be in the interests of overallcellular efficiency; acetyl-CoA, the product of fl-oxi-dation and the substrate for glyoxylate cycle opera-tion, would be produced in situ. It is temptin(g tosuppos.e the remaining enzymes respons'ible for svn-thesizing sucrose from the products of glyoxylatecycle activity also are localized here. Beevers re-ported recently several gluconeogenesis enzymes tobe associated with a proplastid band in sucrosegradients (15).

The cytosomal band from maturing castor beansshowed negligible fuimirase and isocitrate lvase ac-tivity. However catalase, acetyl-CoA carboxvlaseand fatty acid synthetase were demonstrated present,catalase being present in several fold excess overthat found in the mitochondria. Clearly these cvto-somes are not aggregated mitochondria or gly.oxy-somes, but appear to be concerned with fattv acidmetabolism. The role of a fl-oxidation system, in-

514

www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from Copyright © 1969 American Society of Plant Biologists. All rights reserved.

HUTTON AND STIJMPF-/3-OXIDATION IN CASTOR BEAN SEEDS

creasing and decreasing in the maturing seed at thesame time as is the fatty acid synthetase systemremains an enigma. At this stage in its life cycle,the castor bean is synthesizing and storing vastquantities of ricinoleic acid in lipid micelles; anyfunctional /8-oxidation must logically be relativelysmall. A comparison of the maximally developedcapaci.ties of both /3-oxidation systems shows thegermination system to possess about 165 fold moreactivity on a per seed basis calculated from the12,000g supernatant fraction of 4 DAG and the12,000g pellet from 28 DAF castor beans. Even inintact tissue, Glew (14) observed considerable /3-oxi-dation occurring in maturing tissues. It is conceiv-able that the /3-oxidation system is a scavenigingsystem preventinig the buildup of free fatty acids inthe tissue which could markedly effect the generalmetabolism of the maturing seed. Indeed Galliardet al. (13) concluded a very active thiolesterase wasresponsible for the production of ricinoleate (andnot the CoA derivative) from olevl-CoA with cellfree preparations of 32 DAF castor bean tissue.A comparison of the substrate specificities for

both systems reveals interesting differences. Thegerminating castor bean system oxidizes all fatty acidderivatives tested but shows a slight preference forlinoleate and palmitate (about 2-fold greater rate ofoxidation). The matur,ing castor bean seed on theother hand although oxidizing fatty acids on a scaleat least 2 orders of magnitude lower, shows a markedpreference for ricinoleate (ab.out 4-fold greater ac-'tivity than other fatty acids tested). This observa-tion would support the scaveniging function of the/3-oxidation system in maturing seeds.

Of interest is also the observation that palmityl-CoA oxidation shows a peak in oxidation at 4 DAGand a rise between 20 and 28 DAF. If limitingth'iokinase activity had been solely responsible forthe rises in /8-oxidation observed during developmentwhen ammonium salts of fatty acids were suppliedas substrates, no increases should have been observed'for palmityl-CoA oxidation (this assumes the thio-kinase was of general specificity for a variety offatty acids). The peak of /8-oxidation for palmityl-CoA in the maturing bean however is not nearly aspronounced as that found in germinating castor beanis.A sudden rise is seen 'between 20 and 24 DAF butthereafter the decline, if any, is extremely gradual.Clearly a limiting thiokinase activity cannot be theanswer and the observation that the component en-zymes of /3-oxidation rise and fall as does /-oxida-tion activity itself rather indicates these enzymes tobe the immediate regulators of /3-oxidation.

Although a discrepancy appears to exist for theDAG stage at which the peaks for overall /8-oxida-tion and the component enzymes are reached (3DAG for /8-oxidation, 4 DAG for the enzymes con-cerned), this probaibly reflects merely non-identicalgermination conditions rather than some fundamentalbiochemical asynchrony.

Acknowledgments

The authors thank Mrs. Barbara Weigt for skilledtechnical assistance, Dr. P. Heinstein for performing theacetyl-CoA carboxylase assays, Mr. A. Baur for prepar-ing several of the CoA derivatives, and Dr. W. E.Domingo of the Baker Castor Oil Company, California,for a generous supply of castor bean seeds. The authorsare grateful to Dr. R. W. Breidenbach for technical ad-vice in setting up and fractionating the sucrose gradients.

Literature Cited

1. BEERS, R. F. AND I. W. SIZER. 1952. A spectro-photometric method for measuring the breakdownof hydrogen peroxide by catalase. J. Biol. Chem.195: 133-40.

2. BEEVERs, H. 1961. Metabolic production of sucrosefrom fat. Nature 191: 433-36.

3. BREIDENBACH, R. W. AND H. BEEVERS. 1967. As-sociation of the glyoxylate cycle enzymes in anovel subcellular particle from castor bean endo-sperm. Biochem. Biophys. Res. Commun. 27:462-69.

4. BREIDENBACH, R. W., A. KAHN, AND H. BEEVERS.1968. Characterization of glyoxysomes from castorbean endosperm. Plant Physiol. 43: 705-13.

5. BROOKS, J. L. AND P. K. STUMPF. 1966. Fatmetabolism in higher plants. XXIX. Propertiesof a soluble fatty acid synthesizing system fromlettuce chloroplasts. Arch. Biochem. Biophys.116: 108-16.

6. BURTON, D. AND P. K. STUNIPF. 1966. Fat me-tabolism in higher plants. XXXII. Control ofplant acetyl-CoA carboxylase activity. Arch.Biochem. Biophys. 117: 604-14.

7. CANVIN, D. T. 1965. The biosynthesis of longchain fatty acids in the developing castor bean.Can. J. Botany 43: 49-62.

8. DECKER, K. 1959. In: Die aktivierte Essig Saure,Ferd. Enke, Stuttgart.

9. DECKER, K. 1963. In: Methods of EnzymaticAnalysis. H. U. Bergmeyer, ed. Verlag Chemie,Academic Press. p 423-24.

10. DECKER, K. 1963. In: Methods of EnzymaticAnalysis. H. U. Bergmeyer, ed. Verlag Chemie,Academic Press. p 420.

11. DEDUVE, C. E. AND P. BAUDHUIN. 1966. Peroxi-somes (Microbodies and related particles). Phy-siol. Rev. 46: 323-57.

12. DIXON, G. H. AND H. L. KORNBERG. 1959. Assaymethods for key enzymes of the glyoxylate cycle.Biochem. J. 72: 3 p.

13. GALLIARD, T. AND P. K. STUMPF. 1966. Fat me-tabolism in higher plants. XXX. Enzymatic syn-thesis of ricinoleic acid by a microsomal prepara-tion from developing Ricinus coinmuiZnis seeds. J.Biol. Chem. 241: 5806-12.

14. GLEW, R. H. 1968. Developmental aspects oflipid metabolism in the developing and germinat-ing castor bean seed. Thesis, University of Cali-fornia, Davis.

15. KOBR, M. J. AND H. BEEVERS. 1968. Distributionof gluconeogenic enzymes in the castor beanendosperm. Plant Physiol. 43: s-17.

515

www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from Copyright © 1969 American Society of Plant Biologists. All rights reserved.

PLANT PHYSIOLOGY

16. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND

R. J. RANDALL. 1951. Protein measurement withthe Folin Phenol reagent. J. Biol. Chem. 193:265-75.

17. MOLLENAUER, H. H., D. J. MORRE, AND A. G. KEL-LEY. 1966. The widespread occurrence of plantcytosomes resembling animal microbodies. Proto-plasma 62: 44-52.

18. OVERATH, P., E. M. RAUFUSS, W. STOFFEL, ANDW. ECKER. 1967. The induction of enzymes offatty acid degradation in E. coli. Biochem. Biophys.Res. Commun. 29: 28-33.

19. RACKER, E. 1950. Spectrophotometric measure-ments of the enzymatic formation of fumaric andcis-aconitic acids. Biochim. Biophys. Acta 4:211-14.

20. ROUILLET, C. AND W. BERNHARD. 1956. "Micro-bodies" and the problem of mitochondrial regen-eration in liver cells. J. Biophys. Biochem. Cytol.(Suppl.) 2: 355-0.

21. SEUBERT, W. 1960. In: Biochemical Preparations.H. A. Lardy, ed. J. Wiley and Sons, Incorpo-rated. 7: 80-83.

22. STERN, J. R. 1955. In: Methods in Enzymology.S. P. Colowick and N. 0. Kaplan, eds. AcademicPress. Vol. I, p 559-66.

23. TOLBERT, N. E., A. OESER, T. KISAKI, R. H. HAGE-MAN, AND R. K. YAMAZAKI. 1968. Peroxisomesfrom leaves with enzymes related to glycolatemetabolism. Federation Proc. 27: 344.

24. YAMADA, M. AND P. K. STUMPF. 1965. Fat me-

tabolism in higher plants. XXIV. A soluble,8-oxidation system from germinating seeds ofRicinus communis. Planit Physiol. 40: 653-58.

25. ZELITCH, I. AND S. OCHOA. 1953. Oxidation andreduction of glycolic and glyoxylic acids in plants.I. Glycolic acid oxidase. J. Biol. Chem. 201:707-18.

26. ZELITCH, I. 1953. Oxidation and reduction of gly-colic and glyoxylic acids in plants. II. Glyoxylicacid reductase. J. Biol. Chem. 201: 719-26.

516

www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from Copyright © 1969 American Society of Plant Biologists. All rights reserved.