?ig+, - plant physiology · stanley2,3 and eric e. conn california forest and range experiment...

PLANT PHYSIOLOGY

succinate, while ?Ig+, AMP, and DPN+ were re-quired for the oxidation of citrate.

2. Pyruvate-2-C14 and acetate-2-C14 were metab-olized by the mitochondria.

3. The absorption of water by the endospermtissue was accompanied by the activation of theKrebs cycle enzyme system.

4. The capacity of mitochondrial particles to oxi-dize succinate decreased as the embryos germinated;their ability to oxidize citrate and a-ketoglutarate in-creased.

5. The possible physiological significance ofchanges in particulate enzyme activity, as related towater uptake by the germinating seed, is discussed.

I would like to express my sincere appreciation toDrs. Eric E. Conn and Paul K. Stumpf at whoselaboratories most of this study was carried out.

LITERATURE CITED1. BEAUDREAU, G. S. and REMMERT, L. F. Krebs cycle

activlty of particles from bean seedlings. Arch.Biochem. Biophys. 55: 469-485. 1955.

2. BEEVERS, H. and WALKER, D. A. The oxidative ac-tivity of particulate fractions from germinatingcastor beans. Biochem. Jour. 62: 114-120. 1956.

3. BRINK, R. A. and COOPER, D. C. The endosperm inseed development. Bot. Rev. 13: 423-541. 1947.

4. GOODWIN, B. C. and WAYGOOD, E. R. Succinoxidaseinactivation by a lecithinase in barley seedlings.Nature 174: 517-518. 1954.

5. HACKETT, D. P. Recent studies on plant mitochon-dria. Internat'l. Rev. Cytol. 4: 143-196. 1955.

6. JANSSON, G. An investigation of moisture contentand enzyme activities in barley seedlings in rela-tion to their growth rate. Arkiv Kemi 9: 139-145.1956.

7. KREBS, H. A. The tricarboxylic acid cycle. In:Chemical Pathways of Metabolism, D. M. Green-berg, ed. Vol. I, pp. 109-171. Academic Press,New York 1954.

8. LATIES, G. The physical environment and oxidativephosphorylative capacities of higher plant mito-chondria. Plant Physiol. 28: 557-575. 1953.

9. LIEBERMAN, M. and BIALE, J. Effectiveness of ethyl-ene diamine tetraacetic acid in the activation ofoxidations mediated by mitochondria from broccolibuds. Plant Physiol. 30: 549-552. 1955.

10. MILLERD, A. The complete oxidative degradation ofthe pyruvic acid via the carboxylic cycles. In:Handbuch der Pflanzenphysiologie, Vol. 12, pp. 1-12. Springer-Verlag, Heidelberg, Germany 1955.

11. MILLERD, A., BONNER, J., AXELROD, B. and BAN-DURSKI, R. Oxidative and phosphorylative activ-ity of plant mitochondria. Proc. Natl. Acad. Sci.,U. S. 37: 855-862. 1951.

12. PATON, F. V., NANJI, D. R. and LING, D. R. On thehydrolysis of the endosperm of Phytelephas macro-carpa by its own enzymes. Biochem. Jour. 18:451454. 1924.

13. SPADONI, M. A. and TECCE, G. Action of malonateand fluoroacetate on the respiration of the endo-sperm of seeds of Ricinus communis. (In Italian.)Boll. soc. ital. biol. sper. 30: 724-727. 1954.

14. STANLEY, R. G. Respiratory patterns in germinat-ing seeds of sugar pine, (Pinus lambertiana,Dougl.). Ph.D. Thesis, Univ. of California, Berke-ley, California 1956.

15. STANLEY, R. G. and CONN, E. E. Enzyme activityof mitochondria from germinating seedlings ofsugar pine (Pinus lambertiana, Dougl.). PlantPhysiol. 32: 412418. 1957.

16. WITHROW, A. and WOLFF, J. B. Succinate oxidationby mitochondrial preparations from bean seedlings.Physiol. Plantarum 9: 339-343. 1956.

17. TOOLE, E. H., HENDRICKS, S. B., BORTHWICK, H. A.and TOOLE, V. K. Physiology of seed germiniation.Ann. Rev. Plant Physiol. 7: 299-324. 1956.

ENZYME ACTIVITY OF MITOCHONDRIA FROM GERMINATING SEEDLINGSOF SUGAR PINE (PINUS LAMBERTIANA DOUGL.)1

ROBERT G. STANLEY 2,3 AND ERIC E. CONNCALIFORNIA FOREST AND RANGE EXPERIMENT STATION,4 FOREST SERVICE, U. S. DEPARTMENT

OF AGRICULTURE AND DEPARTMENT OF AGRICULTURAL BIOCHEMISTRY,UNIVERSITY OF CALIFORNIA, BERKELEY, CALIFORNIA.

Recent studies of the respiratory activity of plantmitochondria leave little doubt of the importance ofthese cellular units in plant metabolism. Severalpapers have described the oxidative activities ofmitochondria prepared from Angiosperm species andthe lower groups, bacteria, fungi and algae (1, 15, 29).

1 Received March 26, 1957.2 Lilly Postdoctoral Fellow, National Research Coun-

cil, 1955-1956.3 Study supported in part by the Resources for the

Future, Inc.4 Maintained at Berkeley, California by the Forest

Service, U. S. Department of Agriculture, in cooperationwith the University of California.

Two excellent reviews have recently appeared (8, 10).However, no work on the oxidative nature of particlesfrom a Gymnosperm species has yet been reported.This paper will describe experiments concerned withthe oxidative activities of particulate fractions ob-tained from germinated seedlings and ungerminatedembryos of Pinus lambertiana, a Gymnosperm.

Changes in the levels of enzyme activities andmetabolic routes in maturing plants have been re-ported by several workers. Gibbs and Beevers (7)detected differences in the pathway of glucose dis-similation in tissues of varying ages from castor bean,pea, and other plants. Brummond and Burris (5)noted changes in the Krebs cycle enzymes associated

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STAXLEY AND CONN-ENZYME ACTIVITIES IN PINUS

with mitochondrial particles from green leaves oflupine. Beevers and Walker (4), using mitochondriafrom the endosperm of germinated castor bean,showed significant changes to occur in the levels ofsuccinate oxidation associated with particles removedfrom tissues of different ages. This paper will presentevidence for a decrease in the activities of severalKrebs cycle enzymes during the germination of sugarpine seed. A soluble cytoplasmic factor possibly ef-fecting such changes will be discussed.

MATERIALS AND METHODS

The majority of these studies used sugar pine seedharvested in September, 1954, at Pino-Grande nearPlacerville, California. This seed was supplied by theInstitute of Forest Genetics, U. S. Forest Service,Placerville. The initial experiments, which establishedthe presence of the Krebs cycle, used seeds harvestdin 1953 in the same general vicinity. The cones weredried in a circulating air drying oven at 700 C untilthe cone scales had reflexed and the seeds dropped outor could be shaken out. Seeds were then dewinged,cleaned by hand, and placed in a sealed glass jarwhich contained CaCl2 as a drying agent. The seedswere stored at 50 C until used.

Seeds were surface sterilized by soaking in a 0.75% bromine-water solution for five minutes. Theywere rinsed five times in sterile distilled water and al-lowed to stand in sterile water for ten minutes beforethe hard seed coat, papery membrane and nucellar capwere aseptically removed. The divested seeds, con-taining the embryo surrounded by the female gameto-phvte tissue were then placed in sterile distilled waterat 50 C and air was continuously bubbled through thewater for six hours. At the end of six hours the seedswere planted 1/4 to 1/2 inch below the surface offlats of sterile vermiculite. Enough water was addeddaily to maintain a constant amount of moisture avail-able to the germinating seedlings. After five or sixdays at 300 C + 1° C the radicle protruded about 2cm and the hypocotyl had grown about 0.5 cm. Thisgerminated seedling, separated from residual femalegametophyte tissue, was the material used in most ofthe experiments. When ungerminated embryos wereemployed (tables IV, VI and VII) the embryos wereremoved from the seed after six hours of soaking.The separation of the embryo from the surroundingfemale gametophyte tissue was complete.

Embryos from stratified ungerminated seeds wereused in some experiments (table VI). To obtain theseembryos, intact seeds were stratified without pre-liminary soaking. They were mixed with moistvermiculite and packed in waxed paper quart milkcontainers which were stored at 50 C for varyingintervals of time up to four months. The embryoswere then separated from the surrounding femalegametophyte tissue without preliminary soaking ofthe seed.

ISOLATION OF PARTICLES AND MIEASUREMENT OF

ACTIVITY: Forty to 50 gm samples of germinatedseedlings were rinsed five times in sterile distilled

water. They were then transferred to a chilled mor-tar and homogenized with sand in 150 ml 0.5 M su-crose and 0.1 M phosphate buffer, pH 7.0, forapproximately 30 seconds. The pH was maintainedat 7.0 with 1.0 M NaOH during grinding. The ho-mogenate was filtered through two layers of cheesecloth, and the filtrate was centrifuged for five min-utes at 2,000 x g in an International refrigeratedcentrifuge. The supernatant solution was then cen-trifuged at 20,000 x g for 15 minutes. The precipitateobtained was washed by resuspending in 10 ml of0.5 M sucrose and 0.1 M phosphate buffer, pH 7.0,and centrifuging at 20,000 x g for 15 minutes. Theprecipitate was suspended in sucrose-phosphate bufferof such concentration that, when 0.5 ml aliquots ofthe particulate suspension were added to the reactionvessels, the final concentration of sucrose and phos-phate in the flasks was 0.4 and 0.02 M, respectively.The final suspension contained 1.0 to 4.0 mg of nitro-gen per ml. All preparative steps were carried outat 0 to 50 C.

The particulate fraction containing the microsomeswas prepared by centrifuging the supernatant solu-tion, from which the mitochondria had been removed,at 100,000 x g for one hour. The microsomal pelletobtained was then suspended in 0.5 M sucrose and0.1 M phosphate, pH 7.0.

When ungerminated embryos were used as a sourceof mitochondria, about 15 gm of embryos were ho-mogenized in 50 ml of 0.5 M sucrose and 0.1 M phos-phate, pH 7.0.

Unless otherwise stated, manometric measure-ments were carried out in air in Warburg flasks of ap-proximately 6 ml volume; the center well contained0.04 ml of 5M KOH and filter paper. After mito-chondria were added to complete the reaction mixture,the flasks were attached to manometers, immersedin a water bath at 250 C, and allowed to equilibratefor ten minutes. Oxygen consumption in the pres-ence of various substrates was then compared to thatin the absence of substrates. Values of oxygen up-take, expressed as microliters per hour or microlitersper hour per m, of mitochondrial nitrogen Qo2(N),were calculated on the basis of the reaction occurringduring the first twenty minutes.

The nitrogen content of the mitochondrial prepa-rations was determined by digestion with 12 N H2SO4containing CUSO4 and Na2SeO3. The ammonia wasmeasured by direct Nesslerization.

The amount of C1402 produced metabolically byparticles respiring radioactive substrates was deter-mined by the method of Humphreys, et al (12).

The separation and identification of Krebs cycleacids was done by paper chromatographic procedures.After removal of the KOH, the reaction mixtureswere transferred to centrifuge tubes. Two volumes ofabsolute ethanol were added to precipitate the proteinfraction. The precipitate was then boiled in 10 mlof absolute ethanol to remove any adsorbed organicacid, and this ethanol solution was added to the super-natant solution from the protein precipitation. The

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PLANT PHYSIOLOGY

ethanol solution w-as then evaporated to dryness, theresidue redissolved, and the organic acids extractedby a KHS04 column (24). The ether eluent was

concentrated to 0.5 ml and placed on 6x24-inchstrips of Whatman No. 1 filter paper either as a spotor band. Spots of known acids were placed adjacentto the unknown. Descending chromatograms were

developed with n-butanol-formic acid-water (16). Thechromatograms were dried and placed in contact withKodak no-screen x-ray film for 2 to 5 days, and theradioautographs developed. The chromatograms were

eluted with water, the eluent concentrated and rechro-matographed with the suspected intermediates at ad-jacent spots. Radioautographs of the rechromato-graphed acids were prepared, and the labeled acidsidentified from the adjacent known acids.

REAGENTS: Sodium pyruvate and a-ketoglutaricacid were obtained from Nutritional Biochemical Cor-poration. Fumaric acid (recrystallized) was obtainedfrom Dr. L. Ordin. Citric, L-malic, and succinic acidswere obtained from Eastman Kodak Co.

The following cofactors were purchased from Nu-tritional Biochemical Corporation: diphosphopyridinenucleotide (DPN+), 90 %; triphosphopyridine nucleo-tide (TPN+), 95 %; liver coenzyme concentrate con-

taining 4 % DPN+, 7 % TPN+, and 10 Lipmann unitsCoenzyme A per mg; adenosine-5-phosphoric acid(AMP); adenosine triphosphate (ATP); CoenzymeA (CoA), 70 %; and thiamine pyrophosphate (TPP).Cytochrome c was obtained from Sigma ChemicalCorp.; lipoic acid (Lederle) was obtained from Dr. P.K. Stumpf.

Pyruvamide-2-C14 and acetate-2-C'4 were ob-tained from Tracerlab, Inc. Non-labeled pyruvamidewas the gift of Dr. B. Vennesland. Alanine-1-C14,alanine-3-C14, palmitate-1-C14 and butyrate-1-C'4were obtained from Dr. P. K. Stumpf. Sucrose ofcommercial grade was used. Deionized distilled waterwas used in all metabolic experiments.

RESULTSOXIDATION OF KREBS CYCLE INTERMEDIATES: In

initiating these studies, suitable conditions had to beselected for isolating mitochondria from seedlings ofPinus lambertiana and measuring their enzymatic ac-

tivities. The procedures developed by other workerswith plant mitochondria served as a guide (10). Inpreliminary studies in which the oxidation of a-keto-glutarate and succinate were investigated, homogeni-zation of the seedling with sand in a mortar withpestle proved superior to a chilled Waring blenderrun at maximum speed for 10 seconds. Highest ratesof oxidation were obtained when the seedlings were

homog,enized in 0.5 M sucrose and 0.1 M phosphate,pH 7.0, and when the activity of the particles was

measured in reaction mixtures containing 0.4 M su-

crose and 0.02 M phosphate, pH 7.0. It was neces-

sary to maintain the pH at 7.0 both during homogeni-zation and later during measurement of enzymaticactivity. As was anticipated, magnesium ions andeither AMP or ATP were required to obtain maxi-

TABLE IOXIDATION OF KREBS CYCLE SUBSTRATES BY MITOCHONDRIA

FROM PINUS LAMBERTIANA SEEDLINGS

SUBSTRATE MICROMOLES/VESSEL QO2 (N)

None 8Succinate 20 346Citrate 30 156a-Ketoglutarate 30 111Fumarate 30 100L-Malate 30 100L-Malate 2 27Pyruvate 17 16L-Malate + Pyruvate 2 + 17 139

In addition to the substrates the flasks also contained0.5 ml mitochondria suspension; 5 micromoles MgSO4;7.0 micromoles AMP; 0.05 micromoles cytochrome c in0.4 M sucrose + 0.02 M phosphate buffer, pH 7.0. Theflasks with malate and pyruvate contained, in addition,0.6 micromoles CoA; 0.9 micromoles DPN+; 0.5 micro-moles TPN+; 0.8 micromoles TPP; total volume of fluidwas 1.2 ml. All substrates were adjusted to pH 7.0.

mum rates of oxidation of a-ketoglutarate and suc-cinate by the pine particles. Cytochrome c had noeffect on the rate of oxidation of these substrates.However, cytochrome c and four cofactors (DPN',TPN+, TPP and CoA) were frequently added to reac-tion mixtures to prevent the reaction rates being lim-ited by insufficient concentrations of these substances.

The ability of mitochondria isolated from 5- or6-day-old seedlings of Pinus lambertiana to catalyzethe oxidation of various Krebs cycle substrates isshown in tabfe I. All the substrates tested were oxi-dized. The Qo2(N) for the various substrates wereof the same order of magnitude as reported for otherplant mitochondria (4). The characteristic catalyticaction of malate on pyruvate oxidation described byMillerd et al (17, 18) was also noted. Particles sedi-menting between 20,000 and 100,000 x g did notcatalyze the oxidation of Krebs cycle intermediates.

To determine if oxidation of the acids was pro-

TABLE IIMALONATE INHIBITION OF OXIDATIONS CATALYZED BY

MITOCHONDRIA FROM PINUS LAMBERTIANASEEDLINGS

OXYGEN UPTAKKE

SUBSTRATE CONCEN- + MALO- INHIBI-ADDED TRATION - MALO- NATE TION

NATE (20 MICRO-MOLES)

micro- ull/20 minmoles/vesselNone .. 2 4Suceinate 20 81 21 74L-Malate 2 15 8 47L-Malate and 2 53 29 45Pyruvate 14

a-Ketoglutarate 20 51 17 67

Conditions are as described for table I.

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STANLEY AND CONN-ENZYME ACTIV'ITIES IN PINUS

cee(linfg through a common step, malonate was addedto reaction mixtures which had respired twenty min-utes on various Krebs cycle substrates. The readingswere resumed five minutes after tipping in the in-hibitor from the side arm. Under these conditions,malonate inhibited the oxidation of pyruvate, succi-nate, a-ketoglutarate, and L-malate by the mitochon-dria (table II). Since malonate is known to be aneffective inhibitor of succinic dehydrogenase (14),we may conclude that the oxidation of malate, pyru-vate, and a-ketoglutarate proceeded through the com-mon intermediate, succinate.

METABOLISM OF PYRUVATE-2-C14, ACETATE-2-C14ALANINE-1-C14I AND ALANINE-3-C14: When pyru-vate-2-C14 or acetate-2-C14 was added to mitochon-drial preparations, the particles metabolized thelabeled substrates and the CO2 released was radio-

TABLE IIINIETABOLISM OF PYRUVATE-2-C'4 AND ACETATE-2-C" BY

MITOCHONDRIA FROM PINUS LAMBERTIANASEEDLINGS

EXPT. MITO- INITIAL INITIAL ACTIVITYCHONDRIAL ACTIVITY ACTIVITY RECOVEREDN ADDED IN PYRUVATE IN ACETATE AS CO2

mg Cpm x 10 cpm x 10 %C/o158 1.3 54 ... 13.8154 0.9 90 ... 6.8154 0.9 (heated) 90 ... 0.0158 1.3 .. 144 20.4154 0.9 .. 90 5.5154 0.9 (heated) .. 90 0.0

Each flask contained 14 micromoles pyruvate and 2micromoles of malate, or 0.4 micromoles acetate and2 micromoles of malate added initially. All other con-ditions and cofactors were as listed in table I, exceptthat filter paper was omitted from the center well. Theside arm contained 0.1 ml 10 N H2SO4 which was tippedinto the main compartment at the conclusion of the ex-periment. The flasks containing acetate-2-C'4 also con-tained 0.04 mg lipoic acid. Incubated 2 hrs at 250 C.

active. As shown in table III, the amount of radio-active carbon recovered as CO2 ranged from 5 to20 % of the amount added initially. Radioautogramsshowed that malic, citric, and succinic acids isolatedfrom the reaction mixtures were radioactive.

Recent work has indicated that plant mitochon-dria possess transaminase activity (2, 26, 28). Byadding alanine-1-C14 or alanine-3-C14 and a-ketoglu-tarate to mitochondria from germinated sugar pineseedlings or ungerminated embryos, it was possible todemonstrate the evolution of C1402 (table IV). Inthe case of mitochondria from ungerminated embryos,the production of C1402 was only partially dependentupon the presence of a keto acid. It is possible, there-fore, that alanine oxidation occurred by processes in-volving reactions other than transamination to pyru-vate. However, citric, pyruvic, and succinic acidsisolated from the reaction mixture were found to beradioactive.

TABLE IVMETABOLISM OF ALANINE-1-C" AND ALA.NINE-3-C'4 BY

MITOCHONDRIA FROM PINUS LAMBERTIANA

SOURCE OF SUBSTRATE AND CPM c/o ACTIVITYMITOCHONDRIA INITIAL IN CO RECOVEREDACTIVITY 2 AS CO2

SeedlingSeedling

(heated)Ungerminatedembryo

Ungerminatedembryo

Ungerminatedembryo(heated)

Alanine-3-C'4(54,000 cpm)

Alanine-3-C'4(54,000 cpm)

Alanine-l-C'4(38,200 cpm)

Alanine-l-C'4 *(38,200 cpm)

Alanine-l-C"(38,200 epm)

5,250

28

19,420

14.700

900

9.7

0.05

51.0

38.5

2.4

Conditions as in table III except that substrate wasradioactive alanine (2.6 micromoles) instead of pyruvateor acetate. In addition, each vessel contained 30 micro-moles of a-ketoglutarate instead of malate. Incubated2.5 hours at 25° C.

* Omit a-ketoglutarate.

METABOLISM OF FATTY ACIDS: Studies of the path-ways of fatty acid oxidation have shown the essentialenzymes to be associated either with mitochondria inanimal tissues (9), or with mitochondrial and micro-somal particles in the peanut (13, 25). It was, there-fore, of interest to determine where such activityexists in the pine seedling. Data presented in tableV show that both mitochondrial and microsomalparticles catalyzed the formation of C1402 frompalmitate-1-C14. Butyrate-1-C14 was also metabo-lized by mitochondria from seedlings of P. lamber-tiana. In view of the work of Newcomb and Stumpf(21) on long chain fatty acid oxidation by peanut

TABLE VMETABOLISM OF PALMITATE-1-C" AND BUTYRATE-1-C" BY

PARTICULATE FRACTIONS FROM PINUS LAMBERTIANASEEDLINGS

INITIA.L INITIAL ACTIVITYPARTICLES ACTIVITY ACTIVITY RECOVERED

IN PALMI- IN BUTY-TATE-1-C'4 RATE-i-C'4 AS CO2

cpm cpm?fMitochondria 1,900 .... 7.0Mitochondria (heated) 1,900 .... 0.0Microsomes 1,900 .... 7.8Microsomes (heated) 1,900 .... 0.0Mitochondria .... 7.500 8.5Mitochondria (heated) .... 7.500 0.0

Each flask receiving palmitate (0.1 micromole) alsocontained 0.5 ml mitochondrial suspension; 0.4 M sucrose+ 0.02M phosphate buffer, pH 7.0; 10 micromoles MgSO4;11 micromoles AMP; 0.1 micromole cytochrome c. Theside arm contained 0.3 ml 10N H2SO4; the center wellcontained 0.2 ml 5M KOH; total volume of reactionmixture 3.0 ml; volume of vessels, about 15 ml. Incu-bation time, 1 hr. The flasks receiving butyrate-1-C"(0.1 micromole) in addition contained 0.8 micromoleTPP; 0.6 micromole CoA; 0.9 micromole DPN+; 0.5micromole TPN+; and 15 micromoles malonate.

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PLANT PHYSIOLOGY

microsomes, it appeared that the pine seedling mito-chondria might be contaminated with smaller par-

ticles. Contamination was possible because some

agglutination and sedimentation of the microsomesmight have occurred in the sucrose-phosphate mediumused for extraction (13). However, repeated washingof the mitochondrial preparations of pine seedlingsdid not diminish their ability to metabolize palmi-tate-1-C14. The recent investigation by Stumpf andBarber (25) of the capacity of peanut mitochondriato metabolize fatty acids shows that mitochondria ofpeanut seedling cotyledons also contain enzyme sys-

tems for the /3-oxidation of fatty acids.CHANGES IN KREBS CYCLE ENZYME ACTIVITY

DURING STRATIFICATION AND GERMINATION: As timeof stratification increases up to three or four months,seeds of many plants increase in capacity and rate ofgermination (6). 1\Iirov has shown this to be a char-acteristic of sugar pine seed (19). Therefore it was

desirable to compare the enzymatic activity of mito-chondria isolated from embryos of seeds subjected todifferent periods of stratification. Also, the activityof these particles was compared to that of particlesextracted from the non-stratified, germinated sugar

pine seedlings used in most of the present studies.These results are summarized in table VI.

The mitochondria from embryos of non-stratified,ungerminated seeds possessed a higher level of Krebscycle enzyme activity, expressed as Qo2(N), than themitochondria from germinated seedlings. The valuesin table VI were obtained with seeds harvested in1954. It can be seen that, upon germination, the rateof oxidation of Krebs cycle substrates decreasedabout 60 %. These results are typical of those ob-tained in a dozen or more experiments with 1954seed. In addition, mitochondria from seeds harvestedin 1956 showed a decrease in rate of oxidation of

TABLE VIEFFECT OF GERMINATION AND STRATIFICATION ON OXI-

DATIVE ACTIVITY OF MITOCHONDRIA FROM PINUSLAMBERTIANA EMBRYOS AND SEEDLINGS

SEED TREATMENTCONC, NoT STRATIFIED,

SUBSTRATE MICRO- STRATIFIED UNGERM DMOLES

/VESSEL UN- GERM'D, 60 120

GERM'D 5 DAYS * DAYS DAYS

Oxygen uptake, ul/hr x mg N

Succinate 20 880 330 720 310Citrate 30 410 180 300 220a-Ketogluta-

rate 30 470 200 210 210L-Malate 20 330 180 320 150L-Malate 2 80 55 100 50Pyruvate 17 140 10 120 40Pyruvate+ L-malate 17 + 2 420 220 310 160

Conditions were as described for table I.* This treatment corresponds to the standard germi-

nation procedure described under Methods.

TABLE VIIEFFECT OF SUPERNATANT FRACTIONS ON OXIDATIVE

ACTIVITY OF MITOCHONDRIA FROM GERMI-NATED SEEDLINGS

OXYGEN UPTAKE (/AL/HR)EXPT.

USRTNO. MITOCHONDRIAMITOCHONDRIA+ SUPERNATANT

1 SucCinate 45 167Citrate 53 221None .. 3

2 Citrate 57 226None .. 0

3 Citrate 49 121None .. 42

Each flask contained 0.5 ml mitochondria suspension,10 micromoles MgSO4; 14 micromoles AMP; 0.1 micro-mole cytochrome c and 30 micromoles succinate or citratein 0.4 M sucrose + 0.02 M phosphate buffer, pH 7.0. Theflasks with citrate contained, in addition, 1.2 micromolesCoA; 1.8 micromoles DPN+; 0.9 micromole TPN+ and1.6 micromoles TPP. Total volume, 3.0 ml.

Mitochondria were isolated from 9-day-old seedlings;the seeds had not been stratified. The supernatant solu-tion was that fraction remaining after removal of mito-chondria from homogenates of embryos from unstrati-fied, ungerminated seeds. All seeds were soaked inaerated water at 50 C for six hours. One ml of super-natant solution was added where indicated.

about 80 % after 9 days of germination. Table VIalso shows that the mitochondria from embryos ofnon-stratified, ungerminated seeds possessed a higherlevel of enzyme activity, per mg N, than mitochon-dria from embryos of seeds stratified for 120 days.The results obtained with mitochondria from seedsstratified for only 60 days were intermediate in value.

Some preliminary experiments were performed inan attempt to explain the decreased activities of themitochondria from germinated seedlings. The super-natant solution from germinated seedlings from whichthe mitochondria were removed did not decrease theactivity of mitochondria from ungerminated embryos.This would appear to exclude the presence of an in-hibitor in this supernatant. The supernatant solutionfrom the germinated seedlings had no effect on themitochrondria from the germinated seedling. On theother hand, the supernatant from ungerminated em-bryos had a large stimulatory effect on the mito-chondrial particles from germinated seedlings (tableVII). The stimulatory effect was not lost when thesupernatant was boiled for five minutes at neutrality,but it was lost when the supernatant was boiled forfive minutes in either 1 MI HCl or 1 AI KOH.Neither the microsomes nor the ashed residue of thesupernatant liquid stimulated oxidation. The possi-bility of the stimulatory component being an oxidiz-able substrate or one of the cofactors of the Krebscycle was examined. Doubling the concentration ofsuccinate, citrate, and all of the added cofactors didnot increase the rate of oxidation of suceinate orcitrate in the absence of the supernatant. The addi-tion of flavin coenzymes (flavin mononucleotide and

416


STANLEY AND CONN-ENZYME ACTIVITIES IN PINUS

flavin adenine dinucleotide) had no effect. The addi-tion of a liver coenzyme concentrate was also withoutan effect. It should be pointed out that the seedlingsemployed in the experiment described in table VIIwere 9 to 10 days old. The older age of the seed-lings accounted for the relatively low activity ob-served.

DIscussIoNThe experimental results reported here indicate

that mitochondria of the Gymnosperm species, Pinuslamberitiana, contain the enzyme complex for oxida-tion of intermediates of the Krebs cycle. The prop-erties of these preparations are similar to those re-ported for many other types of plant and animaltissues. The fact that pine embryo and seedlingmitochondria possess enzymatic mechanisms for theoxidation of alanine and fatty acids further extendsthe likeness between pine mitochondria and those ofother plants and animals (8).

Both microsomal and mitochondrial particles fromsugar pine seedlings were found to oxidize palmitate-1-C14. Studies on peanut mitochondria and micro-somes have established the existence of a ,8-oxidationmechanism for fatty acids in the mitochondria and anatypical fatty-acid oxidase in the microsomes (25).The most reasonable interpretation of results ob-tained with pine seedling mitochondria and micro-somes is that corresponding enzyme systems exist inthe pine particles. However, further work includingstudies with palmitic acid labeled in carbon atomsother than C-1 is required to establish this point.

Several hypotheses may be offered to explain whythe activity of the mitochondrial preparations frompine seeds decrease upon germination or stratifica-tion: first, enzymes localized in pine seed mitochon-dria may decrease during stratification and germina-tion. Brummond and Burris (5) showed that an ap-parent change in the localization of malic and suc-cinic dehydrogenases occurred in the mitochondria ofmaturing lupine plants. Second, the fragility of theparticulates may increase with germination. Third, ametabolic inhibitor may accumulate. Fourth, thequantity of proteins or nitrogen containing macro-molecules (nucleic acid) sedimenting with the mito-chondria may increase with germination. This wouldcause an apparent decrease in the Qo2 (N). Finally,the concentration of an essential cofactor may de-crease owing, perhaps, to its being catabolized or re-distributed throughout the cell during germination orstratification. Some evidence for such a factor in non-stratified, ungerminated seed has been presented(table VII).

One purpose of this study was to gain greater in-sight into the germination process of sugar pine seed.It has been observed that although the pine seed issaturated with water, a lag occurs in active germina-tion as evidenced by growth and respiration measure-ments (11, 23). This study has shown that theKrebs cycle enzyme complex is present in ungermi-nated sugar pine seed. V-anecko (27) has also de-

tected Krebs cycle activity in mitochondria extractedfrom water saturated but ungerminated pea seeds.It is possible that the intial lag in germination ofsugar pine seed is due to a lack of enzymatic cofac-tors. The lag could also result from a deficiency ofsubstrates of the Krebs cycle or other enzymes. Bach(3) found all the dehydrogenases present in peaseeds, but concluded that the cofactors were essen-tially absent in the ungerminated seeds. DPN+ hasbeen observed to increase in wheat and bean seedsduring the first four days of germination (22). In-creases in the unsaturated fatty acids and invertsugars have been reported by MIirov during stratifi-cation of pine seeds (20).

SUMMARY1. Particulate cellular fractions were separated

from seedlings and embryos of sugar pine (Pinus lam-bertiana). The particles centrifuging between 2,000and 20,000 x g catalyzed the oxidation of acids of theKrebs cycle. The cofactor requirements, and effectsof malonate on these oxidations indicated that theparticles are similar to mitochondria isolated fromother plants and animals. This is the first such re-port for Gymnosperm species.

2. Pyruvate-2-C14 and acetate-2-Cl4 were metab-olized by mitochondria from sugar pine. 'Malic, cit-ric, and succinic acids isolated from the reaction mix-ture were radioactive.

3. Alanine-1-C14 and alanine-3-C14 were metab-olized by the mitochondria of sugar pine and radio-active CO2 was produced. Pyruvic, citric, and suc-cinic acids isolated from the reaction mixture wereradioactive.

4. The metabolism of palmitate-1-C14 and buty-rate-1-C14 by pine mitochondria and microsomes indi-cated the presence of mechanisms for the oxidation offatty acids.

5. The mitochondria from the embryos of un-germinated pine seeds showed a higher oxidative ac-tivity on various Krebs cycle substrates than particlesfrom 5-day-old germinated seedlings, or particlesfrom embryos of seeds stratified 60 or 120 days.

6. The mitochondria which showed decreasingoxidative activity with seedling development werestimulated by the supernatant from ungerminatedseeds from which the mitochondria were removed.The stimulating factor was not present in the micro-somes or the ash; it was stable to heat, unstable toacid and alkali, and not replaceable by flavin nucleo-tides.

LITERATURE CITED1. AJL, S. J. and WONG, D. T. 0. A reappraisal of the

tricarboxylic acid cycle in the respiration of Esche-richia coli. Arch. Biochem. Biophys. 54: 474-485.1955.

2. AKAZAWA, T., FUNAHASHI, S. and URITANI, I. COU-pling of transamination with the tricarboxylic acidcycle in the soybean seedlings. Jour. Agr. Soc.Japan 27: 849-852. 1953.

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3. BACH, D. Dehydrogenases et coferment chez lePisum sativum. Compt. rend. soc. biol. 127: 175-177. 1938.

4. BEEVERS, H. and WALKER, D. A. The oxidative ac-tivity of particulate fractions from germinatingcastor beans. Biochem. Jour. 62: 114-120. 1956.

5. BRUMMOND, D. 0. and BuRilz, R. H. Reactions ofthe tricarboxylic acid cycle in green leaves. Jour.Biol. Chem. 209: 755-765. 1954.

6. CROCKER, W. and BARTON, L. V. Physiology of Seeds.Pp. 1-267. Chronica Botanica, Waltham, Massa-chusetts 1953.

7. GIBBS, M. and BEEVERS, H. Glucose dissimilation inthe higher plant. Effect of age of tissue. PlantPhysiol. 30: 343-347. 1955.

8. GODDARD, D. R. and STAFFORD, H. A. Localization ofenzymes in the cells of higher plants. Ann. Rev.Plant Physiol. 5: 115-132. 1954.

9. GREEN, D. E. Fatty acid oxidation in soluble sys-tems of animal tissues. Revs. Cambridge Phil.Soc. 29: 330-366. 1954.

10. HACKETT, D. P. Recent studies on plant mitochon-dria. Internat'l. Rev. Cytol. 4: 143-196. 1955.

11. HATANO, K. On the absorption of water by seeds ofLarix Kaempferi and Pinus Thunbergii. Jour.Japan For. Soc. 33: 426-430. 1951.

12. HUMPHREYS, T. E., NEWCOMB, E. H., BOKMAN, A. H.and STUMPF, P. K. Fat metabolism in higherplants. II. Oxidation of palmitate by a peanutparticulate system. Jour. Biol. Chem. 210: 941-948. 1954.

13. KMETEC, E. and NEWCOMB, E. H. Properties of par-ticulate fractions isolated from homogenates ofpeanut cotyledons. Amer. Jour. Bot. 43: 333-341.1956.

14. KREBS, H. A. The tricarboxylic acid cycle. HarveyLectures, 44: 165-199. 1948-49.

15. KREBS, H. A. The tricarboxylic acid cycle. In:Chemical Pathways of Metabolism, D. M. Green-berg, Ed. Vol. I, pp. 109-171. Academic Press,New York 1954.

16. LUGG, V. W. H. and OVERELL, B. T. One- and two-dimensional partition chromatographic separationsof organic acids on an inert sheet support. Aus-tralian Jour. Sci. Res., Ser. A 1: 98-111. 1948.

17. MILLERD, A. Respiratory oxidation of pyruvate by

plant mitoclhondria. Arch. Biochem. Biophys. 42:149-163. 1953.

18. MILLERD, A., BONNER, J., AXELROD, B. and BAN-DURSKI, R. Oxidative and phosphorylative activ-ity of plant mitochondria. Proc. Nat'l. Acad. Sci.U. S. 37: 855-862. 1951.

19. MIRov, N. T. A note on germination methods forconiiferous species. Jour. Forestry 34: 719-723.1936.

20. MIROV, N. T. Possible relation of linoleic acid tothe longevity and germination of pine seeds. Na-ture 154: 218-219. 1944.

21. NEWCOMB, E. H. and STUMPF, P. K. Fatty acidsynthesis and oxidation in peanut cotyledons. In:Phosphorus Metabolism, W. D. McElroy and B.Glass, eds. Vol. 2, pp. 291-300. Johns HopkinsPress, Baltimore, Maryland 1952.

22. RIGGIO-BEVMLARQUE, L. and SCOTTI, R. Functionalactivation of germinating seeds. I. Respirationincrease in synthesis of pyridine adenine nucleo-tide. Atti accad. ligure sci. lettere e arti (Pavia).10: 196-203. 1954. (Chem. Abstr. 49: 11789i.1955.)

23. STANLEY, R. G. Respiratory patterns in germinatingseeds of sugar pine (Pinus lambertiana, Doug.).Ph.D. Thesis, University of California, Berkeley,California 1956.

24. STUMPF, P. K. Fat metabolism in higher plants.III. The enzymatic oxidation of glycerol. PlantPhysiol. 30: 55-58. 1955.

25. STUMPF, P. K. and BARBER, G. Fat metabolism inhigher plants. VII. p-oxidation of fatty acids bypeanut mitochondria. Plant Physiol. 31: 304-308.1956.

26. TAGER, J. M. The oxidation of amino acids by plantcell mitochondria. Annales Acad. Sci. FennicaeSer. A 60: 241-250. 1955.

27. VANECKO, S. On the properties of mitochondria iso-lated from the cotyledons of non-germinating peaseeds. Plant Physiol. 31 (Suppl.): xxitv. 1956.

28. WILSON, D. G., KING, K. W. and BURRIS, R. H.Ti-ansamination reactions in plants. Jour. Biol.Chem. 208: 863-874. 1954.

29. WOLKEN, J. J. and PALADE, G. E. An electron micro-scope study of two flagellates: Chloroplast struc-ture and variation. Annals New York Acad. Sci.56: 873-881. 1953.

RESPIRATION AND THE OXIDATIVE ACTIVITY OF PARTICULATEFRACTIONS FROMI DEVELOPING PEPPER FRUITS

(CAPSICUM ANNUUM L.) 1,2

F. D. HOWARD AND M. YAMAGUCHIDEPARTMENT OF VEGETABLE CROPS, UNIVERSITY OF CALIFORNIA, DAVIS, CALIFORNIA

Organized respiratory activity has been associatedwith mitochondria isolated from various plant tis-sues, and it is now generally believed that mitochon-dria represent a major site of respiratory activity

1 Received April 2, 1957.2 The data reported here are taken from the Ph.D.

thesis of F. D. Howard, presented to the graduate divi-sion of the University of California at Berkieley, Janu-ary, 1957.

(8). Fruits have been used in relatively few mito-chondrial investigations, and only the work of Mil-lerd et al (10) dealt with the relative actvities ofcertain particulate fractions in relation to fruit de-velopment.

The present study was undertaken to investigatethe oxidative activity of particulate fractions from de-veloping pepper fruits and to compare this activitywith that of the intact tissue.

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?ig+, - plant physiology · stanley2,3 and eric e. conn california forest and range experiment...

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