RESPIRATION AND PHOSPHORYLATION OF MITOCHONDRIA FROM NORMALAND CROWN-GALL TISSUE CULTURES OF TOMATO 1' 2

TAIKI TAMAOKI, A. C. HILDEBRANDT, R. H. BURRIS, A. J. RIKER, AND B. HAGIHARA3DEPARTMENTS OF PLANT PATHOLOGY AND BIOCHEMISTRY AND INSTITUTE FOR ENZYME RESEARCH,

UNIVERSITY OF WISCONSIN, MADISON

Knowledge of the initiation mechanism of thecrown-gall disease incited by Agrobacterium tuime-faciens (Smith & Town.) and the subsequent growthof the abnormal tissue is fundamental to understandingdiseased growth (2, 18, 24). The nutritional require-ments for normal and gall tissues have been intensivelystudied (25), and the necessity for proper balance ofamino acids and growth substances in the gall forma-tion has been emphasized (2, 23). Braun (3) hassuggested that conversion of a normal cell into acrown-gall cell is accompanied by the permanent ac-tivation of a series of growth-substance-synthesizingsystems, which are precisely regulated in all normalcells. Similarly, Klein (17) has proposed stepwiseactivation of metabolic systems, such as respirationas well as protein and nucleic acid syntheses, duringthe period of transformation of a normal cell into atumor cell.

Biochemical analyses are needed to clarify furtherthe metabolic characteristics of the diseased growth.Experiments are especially interesting with isolatedenzymes or enzyme systems from normal and diseasedtissues. Recent results with plant tissue cultures instudies of cell cytology and physiology (16), andpathology (12, 36) have made attractive the use ofsuch cultures for biochemical analyses.

Earlier respiratory studies of normal and crown-gall tissues were concerned largely with tissue slicesor suspensions of intact cells from tissue cultures atvarious stages of growth. In general, reduced res-piratory levels were observed with crown-gall tissue(24). The results, however, were not always con-sistent; they were dependent upon the basis for ex-pressing the respiratory activity (2).

The purpose of the present paper is to report the

1 Received May 12, 1960.2 This work was supported in part by the Rockefeller

Foundation, by the American Cancer Society, by NationalInstitutes of Health, and by the Research Committee ofthe Graduate School from funds supplied by the WisconsinAlumni Research Foundation. Published with the ap-proval of the Director of the Wisconsin Agricultural Ex-periment Station.

3 Present address: Department of Biology, Osaka Uni-versity, Osaka, Japan.

The authors are indebted to Mr. Eugene Herrling forassistance in preparing illustrations.

oxidative and phosphorylative properties of mitochon-dria isolated, respectively, from normal and crown-gall tissue cultures of tomato. Portions of the workalready have been summarized (31, 33).

MATERIALS AND METHODSTISSUE CULTURES: The tissue cultures were orig-

inally isolated from normal stem or crown gall onstems of tomato plants (Lycopersicuin esculentum Millvar. Bonny Best). They had been cultivated for 5years on a White's agar medium (35) modified byHildebrandt et al (13), and supplemented with 2,4-dichlorophenoxyacetic acid and coconut milk (29).Both normal and crown-gall tissue cultures grew atsimilar rates on the above medium. Normal tissuecultures were unable to grow in the absence of coco-nut milk, whereas crown-gall tissue cultures main-tained growth at a reduced rate (30).

PREPARATION OF MITOCHONDRIA: Tissue cultures15 to 20 days old were used as a source of mitochon-dria. During this period, tissues were actively grow-ing and had a relatively constant respiratory level[avg Qo, (N), 80] and respiratory quotient (avgR.Q., 0.9) (30). About 50 g of tissue were washedin 1 liter of AI/200 phosphate buffer, pH 7.4, fora few minutes to remove agar, salts and other com-ponents of the medium which might be on the surfaceof the cells. The suspension of cells was thenfiltered on four layers of cheesecloth. Any necrotictissue was removed. The washed cells were groundin a glass homogenizer with a Teflon pestle in a solu-tion containing 0.5 M mannitol, 0.005 M cysteine,0.02 M KH,PO4 and sufficient tris-hydroxymethyl-aminomethane (tris) to bring the pH to 7.4. Thehomogenate was centrifuged at 500 G for 10 minutesto remove cell debris. The supernatant was centri-fuged further at 5,000 G for 10 minutes, and thepellet was suspended in 0.4 M mannitol of pH 7.8adjusted with tris. The suspension again was centri-fuged first at low and then at high speeds. The finalpellet from the high speed centrifugation was re-suspended in a small amount of the 0.4 M mannitol,and this suspension was used as the mitochondrialpreparation in the following experiments.

A high percentage of particles in the preparationwas similar in appearance to mitochondrial particlesseen with phase optics in living tomato cells in cul-

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TAMAOKI ET AL-NORMAL AND CROWN-GALL TISSUE CULTURES

ture. These particles were dark-brown, sphericalbodies that ranged in size from 0.5 to 2 A. No mor-

phological differences were observed between mito-chondrial particles from the normal and crown-galltissue cultures. With a fraction spun down at 12.000G for 10 minutes in 0.5 M sucrose solution, roughlyhalf of the particles stained with Janus green B.

DETERMINATION OF OXYGEN CONSUMPTION:Oxygen consumption was measured with an oxygen

electrode apparatus (9) which consisted of a rotating,polarizing, platinum electrode and a recording milli-voltmeter. The change in concentration of oxygen inthe reaction solution was detected as the change inthe cathode reaction current which was convertedinto voltage and recorded automatically. The reac-

tion cells used (9) were of 2.0 to 2.5 ml capacity withtwo narrow necks, one for inserting the electrode andthe other for adding reaction components and subse-quent stirring with a glass rod. Normally the reac-

tion took place with the following amounts of ma-

terials per ml of the reaction mixture: 5.0 ,Lmoles ofinorganic orthophosphate, 0.5 /Amoles of ethylenedia-mine tetraacetic acid, 5.0 ,.'moles of MgCl2, 10.0 Amolesof KCl, 0.25 jAmoles of mannitol, 2.5 Asmoles of sub-strate and a mitochondrial suspension that contained130 to 200 ug protein nitrogen. The concentrationof oxygen in the air-saturated, isotonic medium was

regarded as 240 mA M at 26° C (6).

DETERMINATION OF PHOSPHORYLATION: The re-

action mixture contained, in addition to the basal com-

ponents mentioned above, radioactive inorganic or-

thophosphate (60,000 to 100,000 cpm/ml), adenosinetriphosphate (ATP, 1 Mmole/ml) and sufficient hexo-kinase and glucose. An aliquot of 0.1 ml of the re-

action mixture was withdrawn soon after adding themixture of ATP, glucose and hexokinase. The sec-

ond and third 0.1 ml aliquots were taken at the endof the reaction after all oxygen in the reaction mix-ture had been consumed. Esterified p32 in each ali-quot was analyzed according to the chromatographicmethod of Hagihara and Lardy (10). The oxygenconsumption between the first and second (or third)samplings was usually 300 to 400 mAatoms, and thetime elapsed was 3 to 6 minutes. From a few reactionmixtures with a low rate of respiration, the secondand third aliquots were taken before oxygen con-

sumption was completed to avoid introducing an error

from air diffusion.

PREPARATION OF ASCORBIC ACID OXIDASE:Ascorbic acid oxidase was extracted from the celldebris fraction of crown-gall tissue cultures by treat-ment with 0.8 M (NH4)9SO4 (pH 7.0 with NH4OH)for 12 hours at 5° C. After extraction the cell debriswas removed by centrifugation. Ammonium sulfatewas added to the supernatant until the concentrationwas 1.6 M. The precipitate was discarded. Thesoluble fraction was dialyzed against M/15 phosphatebuffer, pH 7.0, and then used as the ascorbic acid oxi-dase preparation.

RESULTS

Typical respiratory responses of mitochondriafrom normal and crown-gall tissue cultures to varioussubstrates, to cofactors and to inhibitors as traced bythe oxygen electrode apparatus are illustrated infigure 1 with the same right to left direction as therecord sheet. Starting at the upper right edge, theoxygen consumption by the endogenous respirationof the preparations was 0.8 mAatoms of oxygen/minute/ml of the reaction mixture. Upon additionof succinate, the rate was increased to 13.6 and 11.7mlatoms, respectively, for normal and crown-gallparticles. These rates were accelerated 2.4- and 1.4-fold, respectively, by adding adenosine diphosphate(ADP). Malonate inhibited the succinate oxidationcompletely. Oxidation of citrate occurred in thepresence of malonate and was enhanced by addingdiphosphopyridine nucleotide (DPN). Reduced di-phosphopyridine nucleotide (DPNH) was oxidizedrapidly, as indicated by a sharp change in slope ofthe curves after its addition. Antimycin A stronglyinhibited DPNH oxidation, whereas ascorbate was

oxidized rapidly in the presence of Antimycin A.Figure 1 shows that the mitochondrial preparations

from normal tissue cultures oxidized succinate, citrateand DPNH more rapidly, but ascorbate more slowly,than the mitochondrial preparations from crown-galltissue cultures, as discussed later.

o o:~~~~~~~~~~~~~~~~o0.0 32..i- 0?CO7D1400

2. 2 0.. ADF I MTClO0I~O

75.6A

MAOASUCCINATE

CITRATC 300NAE

20. ON 8

200~LANTIMYCIN A

32 ASCORSATE 100

I '$nTI~~MME ONT 0 0

FIG. 1. Respiration curves at 260 C of mitochondriafrom normal and crown-gall tissue cultures of tomato.Recordings with Varian recorder. Figures in m,uatoms/min/mi. Total reaction mixture 2.2 ml. Recording ofthe reactions was from right to left as traced from theoriginal records.

A: Respiration curve of normal tissue mitochondria.Mitochondria, 134 utg N in 0.28 ml; succinate, 2.8 ,umoles/ml; ADP: 0.2,umole/ml; malonate, 14 ,umoles/ml; citrate,2.8,usmoles/ml; DPN; 0.5 ,umole/ml; DPNH: 0.7,utmole/ml; Antimycin A, 0.4, zg/ml; ascorbate, 2.8 sLmoles/ml.B: Respiration curve of crown-gall mitochondria. Mitochondria, 146 cgN in 0.28 ml. All others same as above(A).-

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PLANT PHYSIOLOGY

OXIDATION OF KREBS CYCLE ACIDS AND DPNH:Table I shows rates of oxidation of DPNH, ascorbicacid, and the principal acids of the Krebs cycle bymitochondria from normal and crown-gall tissue cul-tures. These figures are based on several measure-ments for which a single substrate was tested to avoidthe effects of other substrates, inhibitors, or changein oxygen tension which might be encountered in anexperiment employing multiple compounds as in figure1. Mitochondria from normal cultures oxidized theKrebs cycle acids tested more rapidly than mitochon-dria from crown-gall tissue cultures. With bothnormal and crown-gall particles the rates of oxidationof citrate, a-ketoglutarate, malate, and glutamatevaried from preparation to preparation, but usuallyfell in the range of 0.4 to 0.8, when the rate of suc-cinate oxidation was set to 1.0. DPNH was oxidizedrapidly (about 7 times faster than succinate) both bynormal and crown-gall mitochondria.

Mitochondria from both normal and crown-galltissue cultures responded similarly to the cofactorsand inhibitors tested (figs 1 & 2). Adding DPNenhanced oxidation of citrate, malate, a-ketoglutarateand glutamate 1.4- to 2-fold depending upon the prepa-ration. The oxidation of DPNH was not affectedby adding DPN or cytochrome c (fig 2, B), althoughthe latter enhanced the oxidation rate about twofoldwhen mannitol was omitted from the reaction mixture.Cytochrome c added to sucrose preparations of normal

C H E V~~~~~~O=.

500 s

- 4 ° ° 40

c*/+Tll ! | 300 °

200

5008

=;~~~~~~, MI -400°

FIG. 2. Effect of ADP, cytochrome c, Antimycin A,

and oxygen concentration on oxidation of succinate,

DPNH, and ascorbate. Recordings with Brown recorder.

Time scale for B, C, and D same as for A. Figures in

m,uatom m/min/.. Others same as in figure 1.

A: Oxidation of succinate by normal tissue mito-

chondria. B: Oxidation of DPNH by normal tissue

mitochondria. C: Oxidation of ascorbate by crown-gall

mitochondria. D: Oxidation of ascorbate by ascorbic

acid oxidase extracted from cell debris fraction of crown-

gall tissue cultures.

TABLE IOXIDATION OF VARIOUS SUBSTRATES BY MITOCHONDRIA

FROM NORMAL AND CROWN-GALL TISSUECULTURES OF TOMATO

NORMAL TISSUE CROWN-GALL

SUBSTRATEAT(MI]

SuccinateCitrate**a-Ketoglutarate**Malate**

Glutamate**DPNH 1Ascorbate 1

02PTAKEM/LOMS/N/MGN

18014211085

RELA-TIVE

ACTIVI-TY*

1.00.790.610.47

02UPTAKE

M,UATOMS/MIN/MGN

1541259475

RELA-TIVE

ACTIVI-TY*

1.00.810.610.49

125 0.69 117 0.76.,250 6.9 1,090 7.1,560 8.7 6,940 45.0

* Relative activity = Oxidation rate of substrate/Oxidation rate of succinate.

** In the presence of DPN.

and crown-gall mitochondria increased the rate ofoxidation of all the Krebs cycle acids tested by about10 %. The oxidations of DPNH and of the Krebscycle acids tested were inhibited about 90 % by Anti-mycin A (0.4 jg/ml) and cyanide (0.1 Amole/ml).

OXIDATION OF ASCORBIc ACID: Mitochondriafrom normal and crown-gall tomato tissue culturesoxidized ascorbate very rapidly; nine times fasterthan succinate by normal tissue mitochondria and 45times faster by crown-gall mitochondria (table I).In contrast to the oxidation of Krebs cycle acids,ascorbic acid was oxidized by mitochondria fronmcrown gall 4.5 times faster than by mitochondria fromnormal tissue. The mitochondrial oxidation of as-corbate was neither enhanced by DPN, ADP, orcytochrome c, nor inhibited by Antimycin A (fig2,C). The addition of cyanide inhibited ascorbateoxidation. The minimum concentration of cyanideto cause maximum inhibition of ascorbate oxidationwas 1.1 jumoles/ml, a concentration about ten timesthat required for maximum inhibition of DPNH oxi-dation (fig 3,A & B). Since the cell debris fractionof the tomato tissue cultures contained high ascorbicacid oxidase activity, ascorbic acid oxidase was ex-tracted from this fraction. The sensitivity of mito-chondrial oxidase to cyanide was similar to that ofextracted ascorbic acid oxidase (fig 3,B & C).

The rate of ascorbic oxidation by the mitochon-drial preparations decreased as the partial pressureof oxygen in the reaction mixture decreased (fig2,C). Extracted ascorbic acid oxidase reacted in thesame fashion (fig 2,D). This indicates a low affinityof the catalyst for oxygen. This contrasted with theoxidation of succinate or DPNH, in which the rateof oxidation was indepencdent of the oxygen tension(fig 2.A & B).

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TAMAOKI ET AL-NORMAL AND CROWN-GALL TISSUE CULTURES

Illllllllllll/lt ITlv lli jl30000

- ! 0

220

1000 MlT INIA OXIDASE

400°KCN,.-

KCNF 300 z

corder Figure inmutm/i/m.K :KN

K0CN,u KC CN 0 , lKCN

IMIN. IO eooz

0

FIG. 3. Effect of cyanide on oxidation of DPNH andascorbate by tomato tissue culture mitochondria and puri-

fied ascorbic acid oxidase. Recordings with Brown re-

corder. Figures in mAatoms/min/ml. KCNa: KCN,

0.06 /Amole/ml; KCNb: KCN, 0.48 u.mole/ml. Others

same as in figure 1.

A: Oxidation of DPNH and ascorbate by crown-gall mitochondria. B: Oxidation of ascorbate by crown-

gall mitochondria. C: Oxidation of ascorbate by ascor-

bic acid oxidase extracted from cell debris fraction of

crown-gall tissue cultures.

EFFECT OF PHOSPHATE ACCEPTOR: Adding ADPor a mixture of ATP, glucose, and hexokinase in-

creased the rate of oxidation of the substrates listed

in table I except for DPNH (fig 2,B) and ascorbate

(fig 2,C). With normal tissue particles, the increases

were 1.6- to 2.5-fold (respiratory control ratios) forsuccinate (fig 1, fig 2,A), and 1.3- to 2-fold for theother substrates. In two experiments, 3.3- and 3.6-fold increases were observed for succinate oxidation.In comparison with the above, crown-gall particlesgave lower respiratory control ratios; less than twofor succinate oxidation.

The acceleration of respiration by ADP continueduntil all the oxygen in the reaction mixture was con-sumed, and there was no cessation of the enhancedrespiration after the added ADP was supposedly ex-hausted by phosphorylation.

OXIDATIVE PHOSPHORYLATION: Table II showsphosphorylating efficiencies of mitochondrial prepara-tions, respectively, from normal and crown-gall tissuecultures supplied succinate and citrate as substrates.With each substrate, particles from crown-gall gaveconsiderably lower P/O ratios than did particles fromnormal tissue.

The P/0 ratio in DPNH oxidation was less thanhalf that obtained in succinate oxidation. No ap-preciable phosphorylation was observed to be coupledwith oxidation of ascorbate.

DISCUSSIONIn biochemical work on plant mitochondria, fast

growing tissues from seedlings generally have beenemployed as the source of the particles (7). In thepresent study mitochondria from tomato cells culti-vated in vitro were sufficiently active to examinerespiratory and phosphorylative properties with theaid of an oxygen electrode apparatus.

A comparison of oxidation rates of Krebs cycleacids indicated that mitochondria from crown-galltissue cultures had lower activity than mitochondriafrom normal tissue cultures. Crown-gall mitochon-dria also were lower in phosphorylating efficiency;the P/0 ratio of crown-gall mitochondria was about0.6 that of normal tissue particles. The lower oxi-dative and phosphorylative activities of crown-gallmitochondria contrast with the active growth of thetissue. This raises possibilities that A, crown-galltissue cultures more efficiently utilize the energy pro-duced than do normal tissue cultures, or B, crown-galltissue cultures obtain energy other than through theenergy-yielding system coupled with the oxidation ofthe Krebs cycle substrates.

The increase in respiration rate after ADP orphosphate acceptor (respiratory control) indicatedtight coupling of the respiratory system to the phos-phorylating system. This enhancing effect of a phos-phate acceptor decreased with aging of mitochondria,and its decrease preceded a decrease in P/0 ratio(11). Thus, the ability to exhibit respiratory con-trol was a good indication of the qualitv of a mito-chondrial preparation. The respiratory control hasbeen shown with liver and kidney mitochondria (4-to 10-fold increase) (6, 11, 19), and to a lesser ex-tent with particles isolated from heart muscle (4. 26,27), ascites tumor cells (5), and certain plant species(7, 20). In succinate oxidation by mitochondriafrom normal tomato tissue cultures, the control ratioeasily exceeded two; a value of 3.6 was obtained inone case. With crown-gall particles, on the otherhand, the ratio rarely exceeded two.

TABLE IICOMPARISON OF PHOSPHORYLATING EFFICIENCIES OFMITOCHONDRIA FROM NORMAL AND CROWN-GALL

TISSUE CULTURES OF TOMATO

P/0 RATIOExp. No. SUBSTRATE NORMAL

TISSUE CROWN-GALL

I Succinate 1.0 0.66Citrate 1.1 0.67

II Succinate 0.93 0.45Citrate 1.0 0.65

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PLANT PHYSIOLOGY

With rat liver mitochondria (6, 11) the ADP-enhanced respiration returned to the original ratewhen the added ADP was phosphorylated. Bonnerand Ito (1) reported deceleration of respiratory ratewith cauliflower mitochondria. This respiratorydeceleration was not observed in the present experi-ments.

The rapid oxidation of ascorbate by mitochondriafrom normal and crown-gall tissue cultures deservesattention. In contrast to the Krebs cycle acids tested,ascorbate vas oxidized more rapidly by mitochondriafrom crown-gall tissue cultures than by those fromnormal tissue cultures. The oxidation of ascorbatediffered from the oxidation of the Krebs cycle acidsor DPNH in A, insensitivity to Antimycin A, B,higher resistance to cyanide, and C, low affinity foroxygen (34). Thus, ascorbic acid oxidase seemedinvolved in this oxidation. In fact, purified ascorbicacid oxidase behaved similarly in a comparative ex-

periment. Ascorbic acid oxidase has long been con-

sidered a soluble enzyme and has been highly purifiedfrom solution; however, recently at least some of itsactivity has been found to be associated with the cellsurface (14, 22), and with mitochondria (21, 37).Since the cell debris fraction oxidized ascorbic acidrapidly, the mitochondrial oxidation of ascorbate fromcontaminating cell debris deserved consideration.However, the observations throughout the experi-ments supported the view that the mitochondria per se

carried ascorbic acid oxidase.Mitochondria from both normal and crown-gall

tissue cultures oxidized DNPH rapidly (7 times as

fast as succinate). Rapidl oxidation of DPNH alsohas been reported with mitochondria from skunk cab-bage (8), from lupine (15), and from many otherplants (28). Spectrophotometric studies on DPNHoxidase, DPNH-cytochrome c reductase, cytochromec oxidase, and diaphorase activities in mitochondriafrom normal and crown-gall tissue cultures are dis-cussedl in a forthcoming paper (32).

It may be concluded that the respiratory mechan-isms of mitochondria from normal and crown-galltissue cultures are quite similar. The only differencesnoted in their oxidative and phosphorylative activitieswere of a quantitative rather than a qualitative nature.

SUMMARYOxidative and phosphorylative activities of mito-

chondria from normal and crown-gall tissue culturesof tomato were studied with an oxygen electrode.Mitochondria from crown-gall tissue cultures oxidizedreduced diphosphopyridine nucleotide (DPNH), glu-tamate, succinate, citrate, malate, and a-ketoglutaratemore slowly (about 0.9 times), but ascorbic acid more

rapidly (4.5 times) than mitochondria from normaltissue cultures.

The phosphorylating efficiency of crown-gallmitochondria (P/O ratio, about 0.6 with succinate)was lower than that of normal tissue mitochondria(P/O ratio, about 1.0 with succinate). Adding a

phosphate acceptor (adenosine diphosphate or glu-

cose-hexokinase-adenosine triphosphate mixture) in-creased the rates of oxidation of the Krebs cycle acidstested, but not of DPNH and ascorbic acid. In suc-cinate oxidation by normal tissue mitochondria, theenhancing effect by the phosphate acceptor was gen-erally more than twofold (respiratory control ratio).With crown-gall mitochondria, on the other hand, theratio rarely exceeded two.

The oxidation of ascorbic acid by the mitochondriafrom normal and crown-gall tissue cultures was com-pared to the oxidation by an ascorbic acid oxidasepreparation; all the evidence obtained indicated thatthe mitochondria from botlh tissue cultures containedascorbic acid oxidase.

Mitochondria from both normal and crown-galltissue cultures responded similarly to cofactors (di-phosphopyridine nucleotide, cytochrome c) and in-hibitors (cyanide, Antimycin A).

In conclusion, the mitochondria from normal andcrown-gall tissue cultures differed in their oxidativeand phosphorylative activities in rather small quanti-tative rather than qualitative characteristics. Fur-thermore, mitochondria isolated from tomato tissuecultures, except for ascorbic acid oxidase activity,were similar in their respiratory properties to thosefrom intact higher plants and animals.

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'TAMAOKI ET AL-NORMAL AND CROWN-GALL TISSUE CULTURES

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