IN OF TREES - plantphysiol.org · strained off through a nvlon net on a funnel into test tubes immersed in ice. Theglass and debris were re-turned to the mortar with ... each tube

ZIMMERMANN-SUGARS IN EXUDATE OF TREES

10. PARTRIDGE, S. M. Partition chromatography and itsapplication to carbohydrate studies. Biochem. Soc.Symposia (Cambridge, Engl.) No. 3: 52-61. 1949.

11. WXANNER, H. Die Zusammensetzung des Siebr6hen-saftes: Kohlenhydrate. Ber. schweiz. bot. Ges.63: 162-168. 1953.

12. WILD, G. M. and FRENCH, D. The galactan seriesof oligosaccharides. Proc. Iowa Acad. Sci. 59:226-230. 1952.

13. ZIEGLER, H. Untersuchungen uiber die Leitung undSekretion der Assimilate. Planta 47: 447-500.1956.

RESPIRATION OF THE MIYCELIA AND MITOCHONDRIA OF THEFILA'MENTOUS WATERMOLD, ALLOMYCES MACROGYNUS1"2

BRUCE A. BONNER3 AND LEONARD MACHLISDEPART-MENT OF BOTANY, UNIVERSITY OF CALIFORNIA., BERKELEY, CALIFORNIA

The immediate purpose of this investigation ofrespiration in the watermold Allomyces macrogynuswas to provide a basis for the ultimate explanation ofthe effect of various nutritional conditions on the utili-zation of mannose and fructose by this mold (28, 29,37). Its primary significance, however, is the isola-tion from one filamentous fungus of mitochondriawhich oxidize the various intermediates of the Krebscycle. Only mitochondria from y-east, among thefungi, have been shown to contain an organized sys-tem associated with mitochondria for carrying outthese reactions (24, 32). The reader is referred toBonner (4) for a comprehensive review of pathwaysof carbohydrate metabolism in the fungi and the re-lated actinomycetes.

MATERIALS AN-D METHODSGROWTH AND PREPARATION OF THE FUNGUS: The

organism used was the Burma 1 Da strain of Allo-myces macrogynus (10). It was grown in liquid, agi-tated culture in 50 ml of medium in 125-ml Erlen-meyer flasks as described by AMachlis (27). Thecomplete medium, as used in this study, is the sameas the minimal medium of Machlis (27) except forthe addition of 10i4 M L-glutamic acid. Low-sugarmedium differs only in that the concentration of glu-cose is 0.05 % instead of 0.5 %. Each flask of me-dium was inoculated with 0.5 or 1.0 ml of mitosporesand then incubated in the dark (except when roomlights were turned on for observations) on a rotaryshaker at 250 C for 48 to 96 hours. The mitosporeinoculum was prepared as previously described (26).

For the manometric determinations, the flasks ofspherical plants were emptied over a wire screen or

1 Received December 18, 1956.2 This work was supported, in part, by Research

Grant G-1291 from the 'National Science Foundation tothe second author.

3 During the progress of this work the first authorheld University Fellowships and a terminal NationalScience Foundation Fellowship. The research was sub-mitted by him in partial fulfillment of the requirementsfor the Ph.D. in Botany at the University of Californiaat Berkeley. Present address: Laboratoire de Genetique,Physiologioue du CNRS, Gif-sur-Yvette, S. e. O., France.

nylon cloth, the occasional large plaints removed, andthe remaining retained plants rinsed with three orfour washes of 0.04 M potassium phosphate buffer atpH 7.0. The rinsed plants from the requisite num-ber of flasks were then composited, susl)ended in thesolution to be used in the vessels and transferred tothe Warburg vessels with a calibrated pipette havingan opening approximately 5 mm in diameter.

PREPARATION OF 'MITOCHONIDRIA: Initial trials toobtain mitochondria were unsuccessful using homogen-izers of the Ten-Broeck and Potter-Elvehjem type,carborundum of several mesh sizes, and washed sand.At the suggestion of Dr. V. WV. Cochrane, groundPyrex glass was tried and mesh sizes 20 through 50were found to work well. The screened glass waswashed with acid followed by distilled water untilall acid was removed. It was then oven-dried.

For a typical experiment 40 flasks of plants grownin complete medium for 80 to 96 hours were used.They were removed from the growth medium andwashed on a nylon cloth over a Biuchner funnel withapproximately 500 ml of cold 0.04 MI potassium phos-phate buffer (pH 7.0 or 7.4). Excess buffer waspressed from the plants which were then ground ina cold room with 50 to 60 gm of ground glass and afinal total of 150 to 180 ml of a solution containing0.5 M sucrose, 0.1 M potassium phosphate, and0.001 M potassium ethylenediamine tetraacetate at apH o 7.0 or 7.4. Both pH 7.0 and 7.4 gave identicalresults.

The plants, glass, and about a third of the buffersolution were ground in a cold mortar on ice for 6to 8 minutes until a smooth paste was formed. Morebuffer was added, mixed with the paste and the liquidstrained off through a nvlon net on a funnel into testtubes immersed in ice. The glass and debris were re-turned to the mortar with the remainder of the buf-fer, reground for 2 or 3 minutes, then filtered throughthe net again, and the easily removable liquid squeezedout. The crude, filtered homogenate was then cen-trifuged for 10 minutes at 500 x g in a refrigeratedangle centrifuge held at - 20 to + 2° C. This removedthe cellular debris and all but the finest of the glassparticles. The supernatant was decanted into more

291

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PLANT PHYSIOLOGY

50-iiil tubes and centrifuged for 15 minutes at 10,000x g. The next supernatant was discarded and ap-proximately 10 ml of sucrose-buffer solution added toeach tube. This solution differed from that used ingrinding only in that the phosphate concentrationwas lowered to 0.01 AI. The particles were loosenedwith a plastic pestle and resuspended in a small glasstissue homogenizer with the plastic pestle rotated byhandl. This suspension was consolidlated into twotubes and again centrifuged at 10,000 x g for 15 min-utes. The supernatant was decanted and the par-ticles resuspended as above in 5 to 10 ml of a solutionof 0.5 M sucrose, 0.001 MI potassium EDTA, and0.001 'M potassium phosphate buffer (pH 7.0 or 7.4).One-lhalf-ml aliquots of the cold suspension were thenad(led to the Warburg vessels containing the buffer,cofactors, and substrates in the main compartment.The vessels were put on the Warburg manometers asquickly as possible and placed in the water bath.After an 8- to 10-minute equilibration period thetaps were closed and readings taken for a periodusually of thirty minutes. An aliquot of the particu-late suspension was taken for nitrogen determinationby the micro-Kjeldahl method.

MANOMETRIC AND CHEMICAL DETERMINATIONS:Manometric determinations were by the direct methodof Warburg with corrections for the retention of CO2in the buffer (38). The temperature was 25 or 300 Cin experiments with intact plants and 300 C withmitochondria.

Manometric data involving absolute rates of gasexchange of intact plants are expressed on the basisof the dry weight of plant material in each Warburgv-essel at the end of the experiment. This was donebecause no satisfactory method was found for intro-ducing uniform samples into the vessels. Fragment-ing the mycelium destroys most of the respiratoryactivity (17), presumably because A. macrogynius is acoenocyte and therefore will not break up into theshort chains of cells Darby and Goddard (8) wereable to use with Myrothecium verrucaria. Thismethod of expressing results has the disadvantagethat within an experiment different treatments maydifferentially affect the final dry weight.

In certain experiments uniformly labeled glucosewas fed to intact plants for varying periods of time.After the exposure, the plants were rinsed quicklywith distilled water and dropped into 80 % ethanol(4 to 6 mg dry weight of plants to 20 ml ethanol).The plants were centrifuged down, re-extracted withapproximately 20 ml of 20 % ethanol, recentrifugedand the two extracts combined. The combined ex-tract was concentrated first in a vacuum still andthen by blowing nitrogen gas over it. The extractswere examined by the chromatographic and radio-autographic methods (1) used by the Bio-OrganicGroup of the Radiation Laboratory of the Universityof California at Berkeley where this phase of thework was done through the courtesy of Dr. M. Calvin.The uniformly labeled glucose-C14 was prepared pho-tosynthetically. It and the Polidase-S (Schwarz Lab-

oratories, Inc.), an aci(I phoslphatase preparation,were kindly supplied by Dr. J. A. lBassham of the Bio-Organic Group named above.

EXPERIMEXNTAL RESULTSGENERAL CHARACTERISTICS OF RESPIRATION: Pre-

liminary experiments on the oxygen uptake of intactmvcelia indicated that the QO, wA-as depressed whenthe dry weight of mycelium per Warburg vessel ex-ceeded the low amount of approximatelv S mg. Itappeared that excess plant material prevented thefree movement of liquid (luring the shaking and hencethe access of oxygen to the plants. To clarify therelation, mycelium of varying anmounts of dry wciglhtwas added to vessels with liquid volumes of 1.5, 2.7and 4 ml yielding a range of ratios of dry weight inmg to liquid volume in ml as indicated in figure 1.As the results show, the measured Q02 decreases atratios greater than 2.5. When the mean Qo2 forthose flasks with ratios greater than 2.5 was com-pared with the mean of those with ratios of 2.0 orless, the means were significantly different by the ttest at a level of 99 %. Similar results were obtainedat a temperature of 300 C. When the ratio was wellabove 2.0, increasing the shaking rate increased theQ02,; with ratios less than 2.0, there was no effect ofan increased rate of shaking. In all subsequent ex-periments a ratio of less than 2.0 was used.

The composition and pH of the suspension me-dium are known to profoundly affect both the endog-enous respiration and the respiratory responses toadded substrates and inhibitors. To test the effect ofthe composition of the suspension medium, the growthmedium excluding organic components was used asthe control.

If only the phosphate buffer was used, there was arapid decline of the endogenous Qo2 (table I) Uponadding back the omittedl inorganic constituents, themaintenance of the Q02 equal to the control wasfound to depend on the presence of the calcium ion.Magnesium did not substitute for the calcium andcalcium could not reverse the decline in Qo2 if addedafter 40 minutes in the phosphate buffer alone.

40

30

l0220

10

n

* 40 ml buffer per vessel

0 2.7ml buffer per vessel& 1.5ml buffer per vesse

0.6 1.0 L4 la 2.2 2.6 3.0 3.4 3.8 4.2Mg Dry Weight per Milliliter of Fluid in the Worburg Vessel

FIG. 1. The relationship of dry wt and fluid vol tothe Qo2. The plants were grown for 78 hrs in completemedium and suspended in 0.5 % glucose, 0.015 M potas-sium phosphate at pH 7.0, 0.5 mM CaCl2 and 0.5 mMMgCl2.

292



BONNER AND MACHLIS-RESPIRATION IN ALLOMYCES

TABLE I

THE EFFECT OF THE COMPOSITION OF THE SUSPENDINGMEDIUM ON THE RESPIRATION OF ALLOMYCES MACROGYNUS

GROWN 83 HRS IN COMPLETE MEDIUM

SUSPENDING MEDIUM Q02

Control, inorganic constituents of thegrowth medium 23

0.015 M Potassium phosphate, pH 7.0 90.001 M CaCl2 and 0.015 M potassium

phosphate, pH 7.0 250.001 M MgC12 and 0.015 M potassium

phosphate, pH 7.0 120.0005 M CaC12, 0.0005 M MgC12 and

0.015 M potassium phosphate, pH 7.0 24Distilled water 19

The effect of the pH of the suspending mediumwas assessed using phosphate and tartrate buffers.Although the two buffers were tested in separate ex-periments, the plants were comparable; hence, theresults are presented in a single graph (fig 2). A pHof 4.6 or less markedly inhibits respiration; however,about 20 % of the growth of the plants is made at apH below 4.6 because the pH of the medium dropscontinuously during growth. In the experiment justdescribed the pH of the growth medium at the timethe plants were used was 6.0. If, however, the plantswere permitted to grow until the culture mediumreached pH 4.6, then transfer to a suspension mediumat 4.6 was not inhibitory. Thus, if the plants accom-modate to the low pH by growth, the pH is not in-hibitory.

Allomyces, like many other fungi, has a high en-dogenous respiration which in the past has interferedwith respiratory studies (171, 41). Figure 3 shows,for plants 61, 71 and 86 hours old, the progress of theendogenous respiration and the effect of adding po-

30 [ 0 Phosphote

201-

Q02

101-

* Tortrete 0 ------_>

/ \0~~~~~

t '.~~~~~~t

I 8~~~~~~~I

tassium acetate. The endogenous activity declines,more so in the younger plants, and acetate does littlemore than prevent the decline.

In an attempt to reduce the endogenous respira-tion and elicit larger responses to added substrate,plants were starved by holding them in buffer solu-tion on the shaker. There was no increase in re-

30F

20p

Q02

0 0~

j o

1O-

C

30F

20k

lo1

C

30F20F

1O0

0L0

-o

____.--0----.o o

it°OOAcetote* * Endogenous

60 120 180Minutes.

FIG. 3. The endogenous respiration and the effect ofadded substrates. Plants grown for 61 (upper), 71 (mid-dle) and 86 (lower) hrs in complete medium and sus-

pended in the inorganic components of the completemedium. Acetate conc, after tipping, was 0.01 M.

v34 4.0 4.6 52 58 64 70 7.6 sponse upon adding substrate. The enzymatic capaci-

pH ties apparently declined as rapidly as the endogenousFIG. 2. The effect of pH on respiration. The plants Qo2. A somewhat different approach was more suc-

were grown for 84 to 86 hrs in complete medium and cessful. Plants were grown in low-sugar medium tosuspended in 0.015 M potassium phosphate or potassium prevent the accumulation of reserve foods. Figure 4tartrate adjusted to the indicated pH values with KOH shows that the endogenous respiration was consider-or HCl, 0.5 mM MgCl2 and 0.5 mM CaC12. ably reduced in the older plants and that the addi-

293

--I'



PLANT PHYSIOLOGY

50 [

40 - // / _30 [

20

10

Qrrn

t

48 Hours

* .* Glucose50 A-.-A Acetote

o----o Endogenous

40

30

20

10

0

54 Hours

1150

*1:./''/

0....~~~~~~~~~~~~~/.-.-

A.....~~~

5 o

51 Hours

o~~~~~~~0----~~~~~~~ ---

I 57 Hours

115Minutes

FIG. 4. The endogenous respiration and the effect of added substrates on mycelium grown in low-sugar medium.Plants were grown in low-sugar medium for the times indicated and suspended in the growth medium less glucose.After tipping, the conc of glucose was 2.75 mM and that of acetate was 8.25 mM.

tion of glucose increased the Qo2 138 to 158 % of theinitial rate. Still older plants could not be used be-cause they become covered with mitosporangia whichdischarge large numbers of mitospores upon transferto the Warburg vessels.

The R.Q. of the endogenous respiration was con-sistently 0.8 to 0.9. The addition of glucose raisedthe R.Q. to slightly over 1.0. Actual values with my-celium grown in complete medium for 61 to 63 hoursand then suspended in fresh complete medium were1.22 with sugar and 0.88 without during the first 70minutes and 1.09 and 0.82 during the first 130 min-utes. In view of copious oil globules in the mycelium,there may possibly be fat oxidation in the absence ofglucose and fat synthesis in the presence of glucose.

THE EFFECTS OF INHIBITORS: The effects of thefive inhibitors, iodoacetate, fluoride, arsenite, fluoro-acetate, and malonate, were studied. The most likelyenzymes inhibited by these poisons are triosephos-

phate dehydrogenase by iodoacetate (19), enolase byfluoride (39), pyruvate and a-ketoglutarate oxidationby arsenite (20, 21, 33), aconitase by fluoroacetate(31) and succinic dehydrogenase by malonate (34, 35).

The results with iodoacetate are compiled in tableII. The endogenous rate of low-sugar plants wasmeasured over a 30-minute period and served as thebasis for comparison between the various treatments.At the end of this period either substrate in buffer orbuffer alone was added from a sidearm. After 15minutes a second rate measurement was made overthe 50- to 80-minute time period. At the end ofthis period inhibitor in buffer or buffer alone wastipped in from the other sidearm and another ratemeasurement made over the period 110 to 140 min-utes. Changes are calculated as a percentage of theinitial endogenous Qo2 to eliminate differences betweenQo2 values based on the final dry weights of theplants which probably reflect increases in dry weight

294

`2



BONNER AND MACHLIS-RESPIRATION IN ALLONIYCES

TABLE IIIODOACETATE INHIBITION OF RESPIRATION IN THE PRESENCE A<ND ABSENCE OF ADDED SUBSTRATES *

ENDOGE-NOUS GLUCOSE, 2.75 mM ACETATE, 8.25 MM

MIN, CONTROL IODOACETATE, CONTROL IODOACETATE. COJNTROLIODOACETATE,Q02 % Qo2 %c Qo2 % Qo2 (/ Qo2 % Qo2 %

0-30 31.7 100 32.3 100 27.3 100 34.9 100 25.9 100 29.8 10050-80 29.5 93 30.7 95 39.4 144 50.8 145 37.2 144 43.1 145110-140 24.3 77 5.2 16 39.2 144 5.4 15 44.0 170 35.9 120

Inhibition, %c 79 90 29

* The main compartment contained 3.2 ml of growth medium less sugar*with 3.6 to 6.8 mg dry wt of plantsgrown 52 hrs. Potassium acetate and iodoacetate were in 0.4 ml of buffer in separate sidearms. All values basedon the average of 2 or 3 vessels.

during the experiment when substrate was presentand decreases wlhen substrate was absent. The per-

cent inhibition is calculated using the comparablevalues from the control vessels lacking iodoacetate.

Iodoacetate at 1Qa3 M inhibits both the endogenousrespiration and the increased respiration made pos-

sible by glucose. Acetate effectively bypasses theiodoacetate inhibition. When lower levels of iodoace-tate are used (104 and 5 x 104 M, causing 59 and89 % inhibition, respectively) the bypassing of theinhibition by acetate is virtually complete.

These levels of iodoacetate are in the concentra-

60

4-

-

._

a

-aac0

Z

c

50

40

30

20

10

0

tion range which cauises inhibition of triosephosphatedehydrogenase (19). AMtuch higher levels are requiredto inhibit succinic (lehvdrogenase (36) while alcoholdehydrogenase inhibition (9) would appear not to beinvolved since the organism neither uses nor producesethanol (18). The respiration of glucose thus ap-pears to be blocke(d at triosephosphate dehydrogenasebv iodoacetate. The insensitivitv of the acetate me-tabolism to iocloacetate inhibition supports this inter-pretation.

Sodium fluioride wi-as added to low-sugar plantssuspended in complete medium with and without theglucose. The Qo'. after 35 minutes exposure to thefluoride was inhibited 27 and 18 % by 0.01 and0.005 M sodium fluoride, respectively, in the absenceof glucose and 76 and 25 %, respectively, in the pres-ence of glucose. Thuis, the endogenous respiration isrelativelv insensitive to fluoride, while glucose metab-olism is strongly inhibited. These data are con-sistent with the interpretation that enolase is in-volved in glucose metabolism.

The effect of the arsenite on low-sugar plants sus-pended in medium containing 0.1 % glucose is shownin figure 5. Fifty percent inhibition (calculated asthe percent reduction of the initial rate) is effectedby approximately 103 AI arsenite, indicating the res-piration of glucose involves the pyruvate and a-keto-glutarate oxidase systems.

Fluoroacetate inhibits both the endogenous andthe glucose-stimulated respiration as would be ex-pected if respiration is mediated by a typical Krebs

TABLE IIIMALONATE INHIBITION *

-4 -2Log of Molor Concentration

of ArseniteFIG. 5. Arsenite inhibition of oxygen consumption in

the presence of glucose. Plants grown in low-sugar me-

dium for 58 hrs and suspended in complete medium con-

taining 0.1 t% glucose.

TREATMENT QO2 AS %c OF CONTROL %G INHIBITION

Control 100 00.005 M Malonate 59 410.01 M Malonate 31 690.05 M Malonate 33 67

* The plants were grown in complete medium for 84hrs and suspended in 0.01 M potassium tartrate, 0.5 mMMgCl2, 0.5 mM CaCI2 at pH 4.8 in the Warburg vessels.

I

295

-

-



PLANT PHYSIOLOGY

cycle. The endogenous rate is re(luced 66 % (from aQo2 of 26.5 to .9.4) by 0.001 MI sodium fluoroacetateand 74 % by 0.005 MI while the rate with 0.5 % glu-cose present is reduced 40 % (Qo, of 41.1 to 24.7)by 0.001 -M and 47 % by 0.005 -M.

M\alonate inhibition and its reversal by succinate,while among the most specific indicators of Krebscycle activity, is very sensitive to pH. In table IIIare the results of an experiment in which plants grownin complete medium were suspended in potassiumtartrate-MNIgCl2-CaCl2 buffer at pH 4.8 with severalconcentrations of the sodium salt of malonate ad-justed to pH 4.8 with HCI. The pH at the end ofthe experiment was 4.9 to 4.95. The inhibition ob-served could not be reversed by succinate since thelatter was inhibitory at concentrations greater than0.01 AI. As will be shown later, a succinate to malo-nate ratio as high as 10 permits only 322 % of thecontrol respiration of isolated mitochondria. Theuse of this or higher ratios with intact mycelia re-sults in nonspecific inhibition by the succinate.

STUDIES WITH LABELED GLUCOSE: In the first ex-periment, plants grown 52 hours in low-sugar me-dium were washed and added to the Warburg vesselsso that each vessel contained 4 to 6 mg dry weightof plant material in 3.6 ml of growth medium lackingglucose. There was no KOH in the center well.After an equilibration period of 10 minutes, the side-arm contents consisting of 0.45 ml of buffer contain-ing 4.5 ,uc of glucose-1-6-C14 (63 ,ugm) was tipped in.After 5 and 20 minutes the plants were collected,rinsed quickly with distilled w.ater andl dropped into20 ml of 80 % ethanol.

The distribution of the radioactivitv after the 5-and 20-minute periods of metabolism is shown in fig-ure 6 andl quantitatively in table IV. In a period of5 minutes the radioactive carbon from glucose wasdistributedl aimong a number of sugar phosphates,nucleotides, and amino acids. After 20 minutes thepattern wa-s much the same with some redistributionof the label away from the sugar phosphates.

TABLE IVDISTRIBUTION OF RADIOACTIVITY ON CHROMATOGRAMS

OF BREAKDOWN PRODUCTS OF GLUCOSE

5-MINEXPOSURETO GLUCOSE

SPOT COMPOUND

A Uridine diphosphateglucose, etc.

B NucleotidesC NucleotidesD HexosemonophosphatesE Phosphoglyceric acidF TriosephosphateG Pentose phosphatesH PhosphoenolpyruvateI Aspartic acidJ IsocitrateK MalateL Glutamic acidM AlanineN ...

Others ...

20-MINEXPOSURETO GLUCOSE

E-- X H X* E- * X

;TSTo oo

2400277018006700370053005501700470

340660

81003001400

6.67.75.0

18.510.214.61.54.71.3

0.91.8

22.40.93.8

137060901600160017002400

8802600270500

27007200....

500

4.720.75.45.45.88.2

3.08.80.91.79.2

24.5

1.7

* Counts per minute are corrected for backgroundbut are not corrected for coincidence which causes lowvalues over 5000 cpm. Because of high activities, count-ing was done with an aluminum foil filter between thespot and the detector tube resulting in a 9-fold reduc-tion of the counts actually obtainable with the geometryof the tube used.

The compounds listed in table IV were identifiedby eluting the spots, treating with polidase, and chro-matographing the hydrolysates with carriers (A, B.C, and G) or by position and spray reactions (spotsE, F, H, I, J, K, L, and AI). Spots A, B, and C ab-sorbed ultraviolet light and were tentatively identi-fiedl as nucleotides. Spot A gave rise to mannose,glucose, ribose, and uiridine as well as some uinidenti-

FIG. 6. Radioautograms of alcohol extracts of Allomyces exposed to uniformly labeled glucose-C14 for 5 min(left) an(d 20 min (righ.t).

296



BON NER AND 'MACHLIS-RESPIRATION 1N ALLOM YCES

fiedl substances; from spot B was identified uridine,ribose, and possibly xanthosine or guanosine; andfrom spot C uridine, ribose and probably xanthosineor guanosine. Spot D, the hexosephosphate areayielded mannose, fructose, glucose and possibly sedo-heptulose or galactose. The pentose phosphate area,G, yielded ribose and arabinose and several unidenti-fied spots.

In table IV it may be seen that between 5 and20 minutes there is a marked decline in the radio-activity in sugar phosphates (D), triosephosphate(F), and phosphoglyceric acid (E), while the radio-activity in glutamic acid (L) and aspartic acid (I)increases. This is suggestive of a flow of radioactivecarbon from glycolytic intermediates into by-productsof the Krebs eycle with the longer incubation period.It is also of interest to note the relatively largeamounts of activity recovered in the nucleotides(spots A, B, and C).A second experiment with radioactive glucose was

designed to test the effects of sodium fluoroacetateand sodium iodoacetate upon the distribution of theradioactivity. Plants were exposed to 5 mM sodiumfluoroacetate, 0.5 mM sodium iodoacetate, or to bufferfor 10 minutes, after which labeled glucose was tipped

TABLE VOXIDATION OF ORGANIC ACIDS BY MITOCHONDRIA

OF ALLOMYCES *

SUBSTRATE ADDITIONAL Q02(N)COFACTORS

Succinate ...... 360Fumarate ...... 98Citrate 0.1 mM TPN 170Cis-Aconitate 0.1 mM TPN 286Isocitrate 0.1 mM TPN 306a-Ketoglutarate ...... 137a-Ketoglutarate 0.1 mM ThPP

0.25 mg CoA 291Acetate 0.1 mM ThPP

0.25 mg CoA - 37Malate 0.1 mM ThPP

0,25 mg CoA -29**Malate (2 mM) 0.1 mM ThPP

0.25 mg CoA 0Pyruvate 0.1 mM ThPP

0.25 mg CoA 20Pyruvate 0.1 mM ThPP+2 mM Malate 0.25 mg CoA 247

None ...... - 43

* The Warburg vessel contained 3.0 ml of reactionmixture, mitochondria equivalent to 0.28 mg nitrogen,and other components in the following concentrations:0.35 M sucrose, 1 mM MgCl2, 0.01 mM cytochrome c,0.1 nriM DPN, 1 mM ATP, 16 mM P04 pH 7.4, 10 mMglucose, and 20 mM substrate (unless otherwise noted).

** Slight positive pressures developed in the manom-eters.

TABLE VIEFFECT OF COFACTORS ON THE SUCCINOXIDASE SYSTEM *

SY STEEM Q02(N)

Complete 378- Cytochrome c 138- DPN 404- ATP 292-MgCl2 361- Suceinate (endogenous) - 9+5 mg Hexokinase 378

* The complete svstem contained these substances inthe concentrations indicated: 0.02 M succinate, 1 mMMgCl2, 0.01 mM cytochrome c, 0.1 mM DPN, 1 mMATP, 0.35 M sucrose, 10 mM glucose, 17 mM potassiumphosphate buffer pH 7.4, and mitochondria equivalent to0.23 mg nitrogen. The center wvell contained 0.2 ml 20 %oKOH.

in. Forty minutes later the plants were waished andextracted. The activity recovered was too small tomake dark enough radioautograms after 4 weeks ex-posure for effective photographic reproduction. Withthe control, 45 % of the recovered activity was inglutamic acid, aspartic acid, and alanine. In con-trast, the chromatograms of plants treated with iodo-acetate showed no activity in these spots althoughthere was sufficient inactive material to give positiveninhydrin reactions. No compounds beyond the tri-osephosphate stage were recovered. The chromato-grams from the plants treated with fluoroacetate dif-fered from the above chromatogram only in having alarge, relatively heavily labeled spot corresponding toJ in figure 6, presumably citrate. Citrate aecumula-tion is characteristic of fluoroacetate poisoning (6).These results served to confirm the usual interpreta-tions of the inhibitions observed manometrically.

RESPIRATION OF 'MITOCHONDRIA: Washed suspen--sions of particles prepared, as described under Meth-ods and Materials, from homogenates of Allomyeesgrown in complete medium will oxidize most of thoseintermediates tested as shown in table V. The con-ditions used in this experiment were set up without aknowledge of optimum conditions for the various re-actions and, thus, may not have resulted in maximumrates. Significantly higher rates have been measuredin other experiments with most of the acids.

The rates of oxidation of fumarate and pyruvateare low, while malate is not appreciably oxidized.However, when malate and pyruvate are both present,the oxygen consumption is enhanced several fold overthat observed with either substrate alone. This spark-ing or priming effect of malate has been found consist-ently; pyruvate alone has never given an appreciableoxygen consumption. Attempts to obtain increasedoxidation of malate and fumarate by the additionof DNP up to 10(3 M or TPN at 1O M were notsuccessful. It was thought that the rate of malateand fumarate oxidation might be enhanced by the re-moval of oxalacetate, the product of the reaction.To this end glutamate was included with these two

297



PLANT PHYSIOLOGY

acids to serve in a transaminating system convertingoxalacetate to aspartate, but the glutamate was oxi-dized appreciably and there was no indication of in-creased oxidation of malate and fumarate. Neitheracetate nor acetyl phosphate were oxidized signifi-cantly in the presence or absence of malate in severaltrials. The acetate-activating system was apparentlyabsent or inactive under these conditions in the par-ticles.

Several cofactors increase the activity of the suc-cinoxidase system as shown in table VI. -Only theomission of cytochrome c, ATP, and the substrate,succinate, caused appreciable decreases in the ratesof oxidation. No appreciable endogenous respirationwas ever observed with isolated particles. The inhi-bition by DPN indicated above was further studiedand confirmed as shown in table VII. Because of themarked response to cytochrome c, it was included asa matter of course in all experiments at 0.01 mM.Doubling the concentration of cytochrome c did notincrease the rate further. Although the omission of0.001 M magnesium did not greatly affect the rate,the addition of higher concentrations has consistentlyenhanced succinoxidase activity (table VIII). Theparticles used in the experiment reported in tableVIII were isolated in the presence of 0.001 M MgCl2,but particles isolated in the standard manner showedsimilar responses. Both adenylic acid and adenosinetriphosphate exert favorable effects on the rate ofsuccinate oxidation, although it is not known whetherconcentrations higher than 3 mM would result in in-creased rates. The maximum effects of the adenylatesare achieved only in the presence of relatively highconcentrations of magnesium (5 mM).

The oxidation of citrate and cis-aconitate was en-hanced by the presence of 0.1 mM TPN. a-Ketoglu-tarate oxidation was enhanced by the addition of co-enzyme A. Thus, the Qo2 (N) in its absence was185 which was increased to 268 with 0.125 mg CoAper 3 ml of reaction mixture and to 313 with 0.25mg CoA.

Coenzyme A and tlbiamin pyrophosphate werenormally added to the reaction mixture for pyruvateoxidation. In one experiment these additives were

omitted resulting in a five-fold decrease in activityfrom a Qo2 (N) of 150 to 31. Whether both or oneof these cofactors was necessary was not determined.

TABLE VIIINHIBITION OF SUCCINOXIDASE BY DPN IN PRESENCE AND

ABSENCE OF ADELYLATES *

ADENYLATE ADDED WITH 0.1 MM DPNT WITHOUT DPN

None 76 1861 mM AMP 138 2241 mM ATP 203 317

*Each vessel contained mitochondria equivalent to0.58 mg of nitrogen, 17 mM PO, buffer pH 7.4, 0.05 mMcytochrome c, 20 mM succinate, 1 mM MgCl2, 10 mMglucose, 0.35 M sucrose, in a total volume of 3.0 ml. Thecenter well contained 0.2 ml 20 % KOH.

TABLE VIIITHE EFFECT OF MAGNESIUM, FLUORIDE, AND ADENYLIC

ACID ON SUCCINOXIDASE *

ADENYLIC ACID MAGNESIUM FLUORIDECONC CONC CONC QO2(N)0.001 0.001 ..... 4230.001 0.002 . 4770.001 0.003 . 4500.001 0.005 ..... 5320.001 0.001 0.0067 3680.001 0.005 0.0067 4230.003 0.001 ...... 7500.003 0.005 ..... 900

*Each vessel contained mitochondria equivalent to0.23 mg of nitrogen, 16 mM P04 buffer (pH 7.4), 0.01mM cytochrome c, 20 mM succinate, 10 mM glucose,and 0.35M sucrose in a total volume of 3.0 ml. Theisolation medium contained 0.001 M MgCl2 in additionto 0.5M sucrose, 0.1 M phosphate, and 0.001 M EDTA.

A number of variations in the incubation mixtureand isolation medium were made in attempts to pro-long the linear rates of oxidation beyond the 25 to35 minutes usually observed. None of these weresuccessful, including the use of 10 mM EDTA, 1 mMMgCl2, 1 mM cysteine, 1 mM BAL, 2 to 20 mM so-dium fluoride, 0.1 to 0.5 mM manganese chloride or0.05 to 0.5 mg of hexokinase in the reaction mixture.

The oxidation of succinate by these particles isinhibited by sodium malonate. In a reaction mixturecontaining 20 mM succinate, the Qo2 (N) was 493.This was reduced to 227, 156, 70, and 38 by malonateconcentrations of 0.001, 0.002, 0.005, and 0.010 M,respectively. The inhibition with the highest malo-nate concentration was 92 %.A number of attempts were made to obtain phos-

phorylation coupled to the oxidation of succinate anda-ketoglutarate by the particles. The disappearanceof inorganic phosphate -from the reaction medium wasmeasured. Two experiments indicated that inclusionof 1 mM BAL in the isolation and reaction mediapreserved enough activity to measure a small amountof phosphate disappearance in the following mixture:0.01 mM cytochrome C, 0.1 mM DPN, 0.1 mM ThPP,0.1 mM TPN, 1.0 mM ATP, 0.2 mM MnCl2, 1.0 mMBAL, 0.12 mg CoA, 10 mM phosphate buffer, pH 6.8,10 mM glucose, 20 mM substrate when added, 0.5 Msucrose, 0.5 mg hexokinase, and 0.53 mg mitochon-drial nitrogen in 3.0 ml. The phosphorylation wasequivalent to P/0 ratios (micromoles inorganic phos-phate disappearing per microatoms oxygen consumed)of up to 0.47. Although phosphorylation was ob-served much more must be known of the effect ofpossible variables in the isolation procedure and re-action medium and their effect on the stability of thephosphorylating system before higher P/0 ratios areobtained.

DIscussIoNThe results of this study permit the discussion

of certain aspects of the physiology and metabolism

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BONNER AND MACHLIS-RESPIRATION IN ALLOIMYCES

of Allonmyces. with respect to previous work on Allo-myces, other members of the aquatic Phycomycetes,other fungi, and to classical metabolic systems.

No means was found to manipulate Allomyces intothe condition commonly referred to as a "resting cell"with a low endogenous metabolism and a large in-crease in respiration upon the addition of respirablesubstrate. When the mveelium is removed from thegrowth medlium containing the usual 0.5 % glucose, itinitially respires at almost the same rate in the ab-sence as in the presence of substrate. The endoge-nous respiration slowly diminished in the younger my-celia, while the older ones apparently had enoughendogenous reserves to maintain near maximal ratesfor several hours, and then the rate diminished as inthe younger mycelia. This slow decline accompaniedby decrease in the ability to respond to added sub-strates has been noted in various fungi: Ashbya gos-sypii (30) and several species of dermatophytes (2).Other fungi may be starved by aeration in buffer, aprocess resulting in low endogenous rates and quitemarkedI responses to substrates (8).

The use of a limited supply of carbohydrate,0.05 % glucose, was a moderately successful methodfor growing cells which had high respiratory rates,and yet, were capable of measurable responses to ex-ternal substrates. This technique has been used withNeurospora (13). Mycelium grown in this mannerundergoes a more rapid decline in endogenous respira-tion than in capacity to respire, probably because ofa more limited reserve of readily respirable material.However, with even the most favorable material, theendogenous rate is more than half the maximal rateattainable with substrate, and any attempt to assessthe quantitative relationship between externally sup-plied substrates and gas exchange may involve a largecorrection for the endogenous respiration. It ispossible (by growing cells labeled with C14) to deter-mine whether or not the external substrate sup-presses the use of internal substances (7, 40), al-though with some fungi this requires an extensiveseries of experiments to establish the degree of sup-pression for each substrate used and the variation ofsuppression with time (3).

There are some clues to the nature of the endoge-nous substrate. Lynch and Calvin (25) found that aconsiderable amount of radioactivity was incorpor-ated into a water-soluble polysaceharide when Allo-myces arbuscula was suspended in a labeled bicar-bonate solution-indicating that this polysaccharidewas metabolically active. It may serve as an endoge-nous substrate. In the present experiments the re-spiratory quotient of 0.8 to 0.9 indicates that somesubstrate other than carbohydrate is quantitativelyimportant or that there is some incomplete oxidation.

The initial steps involved in the breakdown ofglueose have not been ascertained by the experimentsreported here. However, the conversion of radioac-tive glucose into significant amounts of radioactivegollucose and fructose monophosphates suggest that thepathway is phosphorylative. The recovery of labeled

pentosephosphates and hexosediphosplhate (loes notallow a differentiation between some form of a directoxidative pathway and the Embden-Meyerhof-Parnasscheme (15). Since triosephosphate, phosphoglyce-rate, and phosphenolpyruvate become strongly la-beled, and the inhibitors of triosephosphate dehydro-genase (iodoacetate) and of enolase (fluoride) areeffective in suppressing glucose oxidation, it is prob-able that the Embden-.Meyerhof-Parnas pathway isfollowed in the conversion of triosephosphate to py-ruvate.

Of the many possible conversions wlhich pyruvatemay undergo, there is now evidence for several inAllomyces. Ingraham and Emerson (18) have shownthat A. arbuscula may convert large quantities ofglucose to lactic acid, but Mlachlis (27) has showinthat, under the highly aerobic conditions of agitatedculture, only a small amount of lactic aeid is pro-duced by the A. macrogynus used in his studies. Theprobable direct formation of alanine from pyruvate(14) accounts for the large quantities of alanineformed from radioactive glucose and recovered on thepaper chromatograms. The third, and probably mostimportant fate of pyruvate under aerobic conditionsis its oxidation via the Krebs cycle. The evidencefrom studies with inhibitors, tracers, and cell-freepreparations, points to the importance of this se-quence of reactions. Its quantitatively important na-ture is demonstrated by the effect on intact cells offluoroacetate, an inhibitor of aconitase action (31).Although the suceinic dehydrogenase inhibitor, malo-nate, was not used in the presence of glucose with in-tact cells, it did depress the endogenous oxygen con-sumption of intact mycelia and the oxygen uptake ofmitochondria to a considerable extent. The labelingof glutamic and aspartic acids also suggests thatpyruvate is metabolized via the Krebs cycle.

The third type of evidence for the participationof the Krebs cycle in glucose oxidation is the abilityof isolated mitochondria to oxidize the intermediatesof the cycle. Mitochondria from Allomvees have ac-tivity, expressed on a nitrogen basis, of the same or-der as that of particulate preparations from a widevariety of higher plant tissues (12, 16) and fromyeast (24). Although the oxygen consumption ob-served with malate and fumarate was low in compari-son to that with other acids tested, malate readilyserved as a sparker for pyruvate oxidation, suggestingthat malate was being oxidized to oxalacetate. Theresults of Brown and Cantino (5) are of interest inrelation to these results with malate and fumarate.They found that homogenates of the closelv relatedBlastocladiella will dismutate malate to lactate with-out appreciable oxygen consumption. The lactic de-hvdrogenase system was apparently a better electronacceptor than was oxygen under the conditions usedfor the assay. It is very possible that the mitochon-drial preparations from Allomyces behave in the samemanner.

No attempt was made to define the cofactor re-quirements for the oxidation of each acid, but it was

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PLANT PHYSIOLOGY

shown that TPN stimulates the oxidation of citrateand cis-aconitate and that added CoA increases therate of a-ketoglutarate oxidation in the presence ofThPP. Furthermore, the addition of CoA and ThPPmarkedly enhanced pyruvate oxidation in the pres-ence of malate. These results are consistent with thecofactor requirements of the classical system (22).The beneficial effect of adenylates upon oxidationrates of mitochondria has been observed in many sys-tems and has been discussed by Laties (23) in termsof the function of adenylates as phosphate acceptorsand as coenzymes in the transfer of esterified phos-phate. The stimulation of succinoxidase by adenylicacid and ATP observed in this work might fall intoeither or both of these categories. A consistent effectof Mig" ion in increasing oxidation rates may be in-volved in maintaining the integrity of the phosphory-lating systems (11).

From the work reported here, the metabolism ofglucose by Allomyces appears to involve the reac-tions of the Embden-Meyerhof-Parnas scheme fromtriosephosphate to pyruvate and those of the Krebscycle from pyruvate to CO2 and H20. While themitochondria from several fungi have been shown tocontain a succinoxidase system only those from yeasthlave previouslyr been shown to oxidize the variousinterme(liates of the Krebs cycle (24). It is now evi-dent that in at least one filamentous fungus, Allo-myces, there is an organized system associated withthe mitochondria for carrying out these reactions.

SUMMARYCertain aspects of the respiratory metabolism of

the aquatic Phycomycete, Allomyces macrogynus,were studied.

Allomyces, grown with 0.5 % glucose, was found tohave an initial high rate of endogenous respirationwhich declined slowly with time. The effect of addedsubstrates was to restore the oxygen consumption tothe initial rate or slightly higher and to prevent the(lecline which took place in endogenous controls. Bylowering the glucose concentration in the growth me-dium to 0.05 %, plants were obtained which exhibitedan appreciable response to added substrates. The re-spiratory quotient of the endogenous respiration wasfound to be from 0.82 to 0.88, while that of respira-tion in the presence of glucose was from 1.07 to 1.22.

Glucose oxidation was inhibited by iodoacetate,sodium fluoride, sodium fluoroacetate and sodium ar-senite. Acetate oxidation was not inhibited by iodo-acetate.

When intact plants metabolized labeled glucose for5 and 20 minutes, the alcoholic extracts containedmostly sugar phosphates, triosephosphate, phospho-glycerate, phosphoenol pyruvate, glutamic acid, aspar-tic acid, alanine, and nucleotides.

Mitochondria were isolated and found to oxidizecitrate, cis-aconitate, isocitrate, a-ketoglutarate, suc-cinate, fumarate, malate and pyruvate. The oxida-tion of succinate was inhibited by malonate. A slightamount of oxidative phosphorylation was observed.

LITERATURE CITED1. BENSON, A. A., BASSHAM, J. A., CALVIN, M., GOOD-

ALE, T. C., HAAS, V. A. and STEPKA, W. The pathof carbon in photosynthesis. V. Paper chromatog-raphy and radioautography of the products. Jour.Amer. Chem. Soc. 72: 1710-1718. 1950.

2. BENTLEY, MARIAN L. Enzymes of pathogenic fungi.Jour. Gen. Microbiol. 8: 365-377. 1953.

3. BLUMENTHAL, H. J., LEWIS, KATHARINE F., andWEINHOUSE, S. An estimation of pathways of glu-cose catabolism in yeast. Jour. Amer. Chem. Soc.76: 6093-6097. 1954.

4. BONNER, B. A. Respiration of the mycelia and mito-chondria of the filamentous watermold, Allomycesmacrogynus. Doctoral dissertation, University ofCalifornia, Berkeley 1956.

5. BROWN, D. H. and CANTINO, E. C. The oxidation ofmalate by Blastocladiella emnersonii. Amer. Jour.Bot. 42: 337-341. 1955.

6. BUFFA, P., PETERS, R. A. and WAKELIN, R. W. Bio-chemistry of fluoroacetate poisoning. Isolation ofan active tricarboxylic acid fraction from poisonedkidney homogenates. Biochem. Jour. 48: 467-477.1951.

7. COCHRANE, V. W. and GIBBS, M. The metabolismof species of Streptomyces. IV. The effect of sub-strate on the endogenous respiration of Strepto-myces coelicolor. Jour. Bacteriol. 61: 305. 1951.

8. DARBY, R. T. and GODDARD, D. R. Studies of therespiration of the mycelium of the fungus Myro-thecium verrucaria. Amer. Jour. Bot. 37: 379-387.1950.

9. DIXON, M. Action of iodoacetate on dehydrogen-ases and alcoholic fermentation. Nature 140: 806.1937.

10. EMERSON, R. and WILsO-N, C. W. Interspecific hy-brids and the cytogeneties and cytotaxonomy ofEuallomyces. Mycologia 46: 393-434. 1954.

11. ERNSTER, L. and LOW, H. Reconstruction of oxida-tive phosphorylation in aged mitochondrial sys-tems. Exptl. Cell Research, Suppl.3: 133-153. 1955.

12. FREEBAIRN, H. T. and REMMERT, L. F. Oxidativeactivity of subeell particles from a number ofplant species. Plant Physiol. 31: 259-266. 1956.

13. GIESE, A. C. and TATUM, E. L. The effects ofp-aminobenzoie acid, pantothenic acid and pyri-doxin upon respiration of Neurospora. Arch. Bio-chem. 9: 1-13. 1946.

14. GREEN, D. E., LELOIR, L. F. and NOCITO, V. Trans-aminases. Jour. Biol. Chem. 161: 559-582. 1945.

15. GUNSALUS, I. C., HORECKER, B. L. and WOOD, W. A.Pathways of carbohydrate metabolism in micro-organisms. Bacteriol. Rev. 19: 79-128. 1955.

16. HACKETT, D. P. Recent studies on plant mitochon-dria. Intern. Rev. Cytol. 4: 143-196. 1955.

17. INGRAHAM, J. L. Nutritional and metabolic studieswith the aquatic phycomycete, Allomyces. Thesis,Univ. of California, Berkeley, California 1951.

18. INGRAHAM, J. L. and EMERSON, R. Studies of thenutrition and metabolism of the aquatic phyco-mycete, Allomyces. Amer. Jour. Bot. 41: 146-152.1954.

19. JAMES, W. 0. The use of respiratory inhibitors.Ann. Rev. Plant Physiol. 4: 59-90. 1953.

20. KREBS, H. A. Untersuchungen iiber den Stoffwechselder Aminosauren im Tierkorper. Zeits. Physiol.Chem. 217: 191-227. 1933.

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BONNER AND 'MACIILIS-RESPIRATION IN ALLOIMYCES

21. KREBS, H. A. WVeitere LTntersuchungen uiber denAbbau der Aminosauren im Tierkorper. Zeits.Physiol. Chem. 218: 157-159. 1933.

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

23. LATIES, G. G. The dual role of adenylate in themitochondrial oxidations of a higher plant. Physiol.Plantarum 6: 199-214. 1953.

24. LINNANE, A. W. and STILL, J. L. The isolation ofrespiring mitochondria from Baker's yeast. Arch.Biochem. Biophys. 59: 383-392. 1955.

25. LYNCH, V. H. and CALVIN, M. Carbon dioxide fix-ation by microorganisms. Jour. Bacteriol. 63: 525-531. 1952.

26. MACHLIS, L. Growth and nutrition of water moldsin the subgenus Euallomyces. I. Growth factorrequirements. Amer. Jour. Bot. 40: 189-195. 1953.

27. MACHLIS, L. Growth and nutrition of water moldsin the subgenus Euallomyces. II. Optimal com-position of the minimal medium. Amer. Jour. Bot.40: 450-460. 1953.

28. MACHLIS, L. Growth and nutrition of water moldsin the subgenus Euallomyces. III. Carbon sources.Amer. Jour. Bot. 40: 460-464. 1953.

29. MACHLIS, L. Effect of certain organic acids on theutilization of mannose and fructose by the fila-mentous watermold, Allomyces macrogynus. Jour.Bacteriol. 73: 627-631. 1957.

30. MICKELSON, M. N. The metabolism of glucose byAshbya gossypii. Jour. Bacteriol. 59: 659-666. 1950.

31. MORRISON, J. F. and PETERS, R. A. The inhibitionof aconitase by fluorocitrate. Biochem. Jour. 56:36-37. 1954.

32. NOSSAL, P. M., KEECH, B. and UTTER, M. F. Oxida-tive phosphorylation of isolated yeast granules.Federation Proc. 15: 321. 1956.

33. PETERS, R. A., SINCLAIR, H. M. and THOMPSON,R. H. S. An analysis of the inhibition of pyruvateoxidation by arsenicals in relation to the enzymetheory of vesication. Biochem. Jouir. 40: 516-524.1946.

34. QUASTEL, J. H. and WXOOLRIDGE, W. R. Some proper-ties of the dehydrogenating enzymes of bacteria.Biochem. Jour. 22: 689-702. 1928.

35. QUASTEL, J. H. and WHEATLEY, A. H. M. Biologicaloxidations in the succinic acid series. Biochem.Jour. 25: 117-128. 1931.

36. SHEPHERD, C. J. The enzymes of carbohydrate me-tabolism in Neurospora. I. Succinic dehydrogen-ase. Biochem. Jour. 48: 483-486. 1951.

37. SISTROM, DOROTHY E. and MACHLIS, L. The effectof D-glucose on the utilization of D-mannose andD-fructose by a filamentous fungus. Jour. Bact.70: 50-55. 1955.

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39. WARBURG, 0. and CHRISTIAN, W. Isolierung undKristallisation de Garungsferments Enolase. Bio-chim. Zeits. 310: 384-421. 1942.

40. WIAME, J. M. and DOUDOROFF, M. Oxidative assimi-lation by Pseudomonas saccharophila with C'4-labeled substrates. Jour. Bact. 62: 187-193. 1951.

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KINETICS OF GROWTH INHIBITION BY HERBICIDES1,2

R. E. FRANS,3 E. F. LIND AND W. E. LOOMISDEPARTMENT OF BOTANY, IOWA STATE COLLEGE, AMES, IOWA

The use of herbicides has expanded phenomenallyin the decade since the effectiveness of certain growthsubstances was first demonstrated. Research in thefield, however, has been more concerned with prac-tical problems of chemicals and rates than with con-

siderations of physiological action. The similarity ofthe most used herbicide, 2,4-dichlorophenoxyacetic acid(2,4-D), to indoleacetic acid in structure and in ac-

tion at low concentrations has led to the develop-ment of theories of the interference of 2,4-D with nor-mal auxin functions (19, 21). The possibility ofauxin-2,4-D relationships is strengthened by Hoff-mann's work (7) showing that certain chlorinated in-doleacetates were highly toxic, and that 2,4-6-trichloro-phenoxyacetic acid inhibited normal auxin actions intomato, although it was itself devoid of either auxinor herbicidal action (8).

1 Received December 27, 1956.2Journal paper No. J-3102 of the Iowa Agricultural

Experiment Station, Ames, Iowa. Project No. 944.3 Present address: Department of Agronomy, Uiliver-

sity of Arkansas, Fayetteville, Arkansas.

If the herbicides act upon the normal auxin siteswithin the cell, the more extensive literature on auxinaction becomes pertinent, particularly the numerousand apparently successful attempts (5, 12, 13, 14) tocharacterize auxin action by the method first devel-oped by Michaelis and Menten (15) to describe en-zyme reactions. The application of these principlesto growth processes has been criticized on the basisof certain techniques and assumptions (3, 9) and gen-erally by Audus (2) who questions the advisability ofattempting to relate complex growth systems to simpleenzymic processes. Leopold (10), however, feels thatthis analysis offers the best explanation of auxin ac-tion thus far available. It is the purpose of this in-vestigation to attempt to show that growth inhibitioninduced by certain herbicides and growth substancescan be characterized quantitatively by a kinetic anal-ysis of the experimental results.

MATERIALS AND METHODSEXPERIMENTS WITH SOYBEANS: Hawkeye soybean

seedlings were grown in the greenhouse in 4-inch pots

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