plant physiology · studies on the absorption, transport and metab-olism of synthetic growth...

PLANT PHYSIOLOGYVOLUME 32 JULY, 1957 NUMBER 4

STUDIES OF CARBOXYL-C14-LABELED 3-INDOLEACETIC ACIDIN PLANTS1" 2, 3

S. C. FANTG AND JOSEPH S. BUTTSDEPARTMIENT OF AGRICULTURAL CHEMISTRY, OREGON AGRICULTURAL EXPERIMENT STATION,

OREGON STATE COLLEGE, CORVALLIS, OREGON

Studies on the absorption, transport and metab-olism of synthetic growth regulators using radioactivetracers have been carriecd out by many workers. Incontrast, little work has been reported regarding thenatural auxin, indoleacetic acid, wlhich is of great in-terest to plant physiologists. This paper reports theresults of experiments on the absorption and trans-location of IAA-1-C'4 in bean plants, and also the ef-fects induced by 2,4-D treatment and light on the de-struction of IAA in vivo.

MATERIALS AN D METHODSThe synthesis of IAA-1-C14 was carried out ac-

cording to the follow-ing scheme:

1c3IjrH2 2N(CH3)2

H

NaCl4NH20

Graminae

CH,2 C1OONa

H

Indoleacetate

This method is essentially that of Stutz et al (10).The following procedure gave the highest yield basedon NaC14N In a 25-ml Pyrex test tube, 350 mg(2 mMI) of gramine (purchased from Nutritional Bio-chemicals Corporation), 49 mg of NaCM4N (1 mM\I)and 40 mg of NaOH (1 mM113) were dissolved in 10 mlof 50 % methanol solution. After cooling the solutionin a dry iee bath, the tube was momentarily evacu-ated, flushed with nitrogen gas, and sealed. The mix-ture was then autoclaveed at 20 lb pressure for 19hours. At the end of the reaction, the seal was brokenand the contents were transferred to a flask. The so-lution was first evacuated to remove the methanoland was then extracted four times with five-ml por-tions of ether to remove the excess gramine. Afterchilling to 0° C and adjusting the pH to 3.0 the IAAprecipitated directly from the aqueous solution. Theprecipitate was filtered immediately and washed witha small amount of ice water. A yield of 105 mg with

1 Received November 20, 1956.2 Technical Paper -No. 1007. Oregon Agricultural Ex-

periment Station.3 This work was supported by contract No. AT (45-1)-

304 United States Atomic Energy Commission.

an activity of 6.3 x 107 cpm/mMl was obtained, M.P.166°-16&° C after recrystallization. The sample wasalso chromatographed using isopropanol-water-aim-monium hydroxide (10: 1: 1) as the developing sol-vent. The position of radioactive spot coincided withthat of pure IAA.

Bean plants (Phaseolus vulgaris L., var. BlackV'alentine), corn plants (Zea Slays L., var. GoldenCross) and pea plants (Pisum sativum L., var. Alaska)were grown under greenhouse conditions in flats con-taining Chehalis sandv loam soil. A 95 % ethanolsolution containing 0.1 % IAA-I-C14 and 0.2 %Tween-20 was used for treatments. The bean plantswere treated when the primary leaves were almostfully expanded and the terminal buds were still verysmall. The solution was applied quantitatively toeach plant along the midrib of the primary leaf bymeans of a microsyringe. After harvesting, all plantswere sectioned into leaf, stem and roots; the sectionswere pooled and homogenized with 80 % ethanol.The corn plants were treated when they had grownto the fourth leaf stage. The pea plants were used 10to 14 days after germination.

Forty uniform bean plants were selected andtreated with 43 ,ugm IAA-1-C14 per plant in the mid-rib of one primary leaf. Ten plants were harvested1, 3, 7, and 14 days after treatment. Eight plantsfrom each group were weighed, sectioned, compositedand homogenized with 80 % ethanol while the remain-ing two were used for making radioautograms. Oneml aliquot of the ethanol solution from each com-posite was dried in cupped planchets and the C14 ac-tivitv was measured with a thin mica window G-Mcounter (1.9 mg/cm) .

Since the increase in rate of oxidation of IAA invitro by light has been demonstrated (5), an experi-ment was designed to study the effect of light on thedestruction of IAA in vivo. Eight uniform beanplants were treated with IAA-1-C14 (50 jugm perplant) on both primary leaves. The plants wereplaced in bell jars, four plants to each jar. One jarwas covered with light-proof black paper, while theother was exposed to fluorescent light (1200 ft-c in-tensity) continuously except for the period from 10P.i\I. and 6 A.IL The respiratory CO2 was sweptout continuously from the system, either with CO.-free air or with 02 and collected in 'NaOH solutions.

253

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PLANT PHYSIOLOGY

The trapping solutions were replaced periodically withfresh NaOH. The carbonate in the solutions was pre-

cipitated as BaCO3 and the radioactivity was meas-

ured in the usual manner. This type of experimentwas carried out twice with bean plants, once withcorn plants, and once with pea seedlings. In each case

eight plants were used.The enhancement of IAA oxidation in vitro by

2,4-D has been reported previously (4, 6). Recently,it was shown that the apparent enhancement of oxi-dation of IAA by 2,4-D was caused by an impurity(2,4-dichlorophenol). With pure 2,4-D no effect of2,4-D on the oxidation of IAA in vitro has been re-

ported (7). This experiment was carried out as an

in vivo study to determine whether or not the 2,4-Dexerts its action on the enzyme systems controllingthe destruction of IAA in plants. Several experi-ments were performed. In the first run, 80 uniformbean plants were divided into four groups of 20 plantseach. Each plant was treated with 21.8 ,ugm of IAA-J_C14 on one of the primary leaves. After comple-tion of IAA treatment, three groups of plants were

again treated on the same leaf with either 5, 10, or

20 ,ugm of non-radioactive 2,4-D (recrystallized twicefrom benzene and showing no effect on the oxidationof IAA in vitro), respectively, while the 4th group re-

ceived no 2,4-D treatment. Four plants from eachgroup were harvested at various intervals, and were

divided into leaf, stem and roots samples on whichradioactivities were determined. A second experi-ment was carried out with 16 plants in each group.

The IAA-1-C14 was applied at the same level. Eachplant in the four groups received 0, 1, 5, and 50 jugmof 2,4-D, respectively. In order to demonstrate fur-ther the effect of 2,4-D an experiment with a directmeasurement of radioactive respiratory CO2 fromIAA-1-C14 treated bean plants was carried out. Eachof eight uniform bean plants received 50 /%gm of IAA-l_C14. Four of these plants received an additional50 ugm of non-radioactive 2,4-D per plant. Imme-diately after the application of chemicals, these two

FIG. 1. The accumulation pattern of C1 in the bean

plant. The primary leaf, on the right, received 43 ,ugmof indoleacetic acid-i-C" the previous day.

groups of plants were put separately into two belljars and the amounts of radioactive CO2 given offwere collected and measured periodically as described

TABLE IABSORPTION AND TRANSLOCATION OF C" IN BEAN PLANTS WHICH RECEIVED 43 /LGM OF

3-INDOLEACETIC ACID-1-C'4 TREATMENT PER PLANT

1 DAY 3 DAYS 7 DAYS 14 DAYS

ToT 'L RCVR TOTAL RECOVERY TOTAL RECOV'ERY TOTAL RECOVERYACTIVITY RECOVERY ACTIVITY ACTIVITY ACTIVITY

cpm % cpm / cpm 6X0 cpm C

Treated leaves 52250 87.5 48750 81.3 39300 65.5 33850 56.4Petioles, treated leaves 6700 11.2 4450 7.4 4000 6.7 5050 8.4Untreated leaves 50 0.1 50 0.1 0 0 0 0Petioles. untreated leaves 0 0 0 0 0 0 0 0Terminal buds 200 0.3 700 1.2 1050 1.8 1100 1.8First internodes 700 1.2 900 1.5 600 1.0 700 1.2Hypocotyls 450 0.8 650 1.1 400 0.7 600 1.0Roots 250 0.4 400 0.7 250 0.4 500 0.8

Totals 101.5 93.3 76.1 69.6

Plants were harvested 1, 3, 7, and 14 days after treatment. Total activity applied to each group of 8 plants=60,000 cpm. All results were counted by a thin mnica window G-M counter (1.9 mg/cm2).

254



FANG AND BUTTS-IND

previously. The same type of experiment was re-peated once, using corn as test plants.

RESULTS AND DISCUSSIONThe results of absorption and translocation of

IAA-1-CL4 in bean plants are shown in table I. Ex-cluding the activity from the blade of the treatedleaves, between 10 to 14 % of the C14 activity was

OLEACETIC ACID-1-C14 255

absorbed and transported in all four groups. Theamount of total recovery of C14 decreased with time,indicating that part of the applied IAA had beenmetabolized, the radioactivity appearing in the res-piratory C02. The concentration of C14 in the petioleof the treated leaf, first internode and hypocotyl,reached the maximum in the first day. The accumu-lation of C14 in the terminal bud continued and it

o accumulated activityx specific activity

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5 10 15 20 25 30

Corndark O accumulated activity

X specific activity

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Time in Hours

FIG. 2. Effect of light on the accumulative and specific activity of respiratory CO2 in plants which receivedindoleacetic acid-i-C" treatment. At arrow, black paper was removed and the plants were exposed to light.

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256 PLANT PHYSIOLOGY

TABLE IIEFFECT OF 2,4-D ON THE ABSORPTION AND TRANSLOCATION OF C14 IN BEAN PLANTS WHICH

RECEIVED 21.8 ,GM OF 3-INDOLEACETIC ACID-1-C'4 TREATMENT PER PLANT. (IAA AND2,4-D EtERE APPLIED SIMULTANEOUSLY ON ONE OF THE PRIMARY LEAVES)

3 DAYS 4 DAYS 7 DAYS 11 DAYS 17 DAYS

PLANT PART TOTAL RE- TOTAL RE- TorAL RE- TOTAL RE- TOTAL RE-ACTIVITY, COVERY, ACTIVITY, COVERY, ACTIVITY, COVERY, ACTIVITY, COVERY, ACTIVITY, COVERY,CPM C CPM %O CPM % CPM % CPM %

ControlLeaf 3600 24.6 3250 22.2 2300 15.8 1000 6.9 550 - 3.8Stem 550 3.8 750 5.1 700 4.8 650 4.5 750 5.1Root 0 0 0 0 0 0 0 0 0 0

5 Agm 2,4-DLeaf 5100 34.9 5300 36.3 4200 28.8 3200 21.9 2600 17.8Stem 300 2.1 100 0.7 300 2.1 300 2.1 150 1.0Root 0 0 0 0 0 0 0 0 0 0

10 /Agm 2,4-DLeaf 6550 44.9 6200 42.5 4550 31.2 2950 20.2 3100 21.2Stem 500 3.4 300 2.1 100 0.7 400 2.7 250 1.7Root 0 0 0 0 0 0 0 0 0 0

20 ,ugm 2,4-DLeaf 5450 37.3 4050 27.7 4400 30.1 3500 24.0 2800 19.2Stem 150 1.0 600 4.1 350 2.4 300 2.1 200 1.4Root 0 0 0 0 0 0 0 0 0 0

Total activity applied to each group of 4 plants = 14,600 Cpm. A thin mica window G-M counter (1.9 mg/Cm2)was used.

did not reach a maximum until the 7th day after been treated for 1 day with 1AA-1-C14 is shown intreatment. No activity or only! a trace of C14 was figure 1. When these results are compared with anoted in the untreated primary leaves. The accumu- previous study of the rate of translocation of C14 inlation pattern of C14 in bean plants after having bean plant receiving labeled 2,4-D (2) it is clear that

TABLE IIIEFFECT OF 2,4-D ON THE ABSORPTION ANTD TRANSLOCATION OF C4 IN BEAN PLANTS WHICH

RECEIVED 21.8 ,uGM OF 3-INDOLEACETIC ACID-1-C' TREATMENT PER PLANT. (IAA AND2,4-D WEERE APPLIED SIMULTANEOUSLY ON THE SAME PRIMARY LEAF)

7 DAYS 11 DAYS 14 DAYS 18 DAYS

PLANT PART TOTAL RECOVERY, TOTAL RECOVERY, ToTAL RECOVERY TOTAL RECOVERYACTIVITY, ACTIVITY, ACTIVITY, ACTIVITY,CPM / CPM % CPM CPM %

ControlLeaf 3700 25.0 2250 15.2 2850 19.3 2050 13.8Stem 1200 8.1 1400 9.5 500 3.4 1050 7.1Root 0 0 0 0 0 0 0 0

1 Agm 2,4-DLeaf 4100 27.7 2600 17.6 2850 19.3 2300 15.5Stem 950 6.4 1400 9.5 1350 9.1 500 3.4Root 0 0 0 0 0 0 0 0

5 ,ugm 2,4-DLeaf 4400 29.7 3900 26.4 3350 22.6 2300 15.5Stem 1400 9.5 950 6.4 1050 7.1 1450 9.8Root 0 0 0 0 0 0 0 0

50 Agm 2,4-DLeaf 7200 48.6 5700 38.5 5200 35.1 5000 33.8Stem 1450 9.8 2000 13.5 1700 8.7 950 6.4Root 0 0 0 0 0 0 0 0

Total activity applied to each group of 4 plants = 14,800 cpm. A thin mica window C-M counter (1.9 mg/cm2)was used.



FANiG AN'D BUTTS-IN DOLEACETIC ACID-1-C14

o accumulated activity

n specific activity

.-I0*..0'

D 10 15 20 25 30

Corn2,4-D treated

O accumulated activityx specific activity

404

so(

)t if " X10~~ ~ ~ ~ ~ 4

5o 155 10 15 20 25 30

20(

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Beancontrol

x

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v .0

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ted activityactivity

O__---

o0

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Bean2,4-D treated

O accumulated activitya specific activity

Time in Hours

FIG. 3. Effect of 2,4-D on the accumulative and specific activity of respiratory CO2 in plants which received

indoleacetic acid-i-C14 treatment.

the C14 of the IAA does not translocate as readily.A larger portion of it remained in the treated leafand may be graduallv metabolized. Chromatographicstudy of 80 % alcoholic extract of IAA-1-C14 treatedbean leaves revealed the presence of six compoundscontaining carbon-14. The RF of these compoundsagree quite well with the values reported by Andreaeand Good (1) and Good et al (8). Detailed study ofthese IAA metabolites will be reported in a followingpaper.

The effect of light on the destruction of IAA in

plants is represented in figure 2. It is clearly shownthat the plants which were kept in darkness did notproduce radioactive CO2 from IAA-1-C14 at the samerate as the plants expose to light. As soon as theplants in the dark were exposed to light, the rate ofradioactive CO2 evolved was greatly accelerated. Thedestruction of IAA in corn plants depends completelyon light, as negligible amounts of C14'O were givenoff by the plants during the dark period. On the

contrary, the pea and bean plants which were kept inthe dark continuously destroyed the IAA at a slowerrate during the first 12-hour period. Since the same

amount of IAA-1-C14 was used in the treatment ofplants in all three experiments, the difference in theaccumulated activitv as CO2 during a 30-hour periodshould provide a good indication that a species differ-ence on the rate of destruction of IAA might exist.The specific activity of the respiratory CO2 duringillumination reached a maximum in approximately 4hours in pea and bean plants, and in 8 hours in cornplants. The considerably higher specific activity ofrespiratory CO, in corn plants, as compared to thosefrom pea and bean plants, suggests that a differentpathway of IAA metabolism may exist in different

plant species or possibly a different rate of destruc-tion. This also indicates that the destruction of IAAby plants in vivo was dependent on light. If the bio-synthesis of IAA in plants is not light dependent, thenthe concentration of this chemical will generally be

Corncontrol

257

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PLANT PHYSIOLOGY

higher at nighttime; therefore, more growth of plantsin nighttime can be expected.

The results on the effect of 2,4-D on the rate ofdestruction of IAA in bean plants are shown in tablesII and III. The total recoverv of radioactive carbonwas low in these two experiments (carried out duringApril and i'vIay) as compared to the previous one (car-ried out dutring January), indicating that factors suchas temperature, light intensity and photoperiod mayaffect the rate of IAA destruction. As indicated intable II, in the first groups which received no 2,4-Dtreatment, the recoverv of radioactivity declined from28.4 % in the third-day- sample to 8.9 % in the sev-enteenth-day sample. These data indicate that al-most three-fourths of the applied IAA was metabo-lized in the first three davs. In the case of 2,4-Dtreated plants, the total recovery of C14 was consist-ently higher than that of the corresponding untreatedplants. However, the (lifference in effect of 2,4-D be-tween three levels usedI (5, 10, and 20 jAgm) is notsignificant. Table III shows the results from the sec-ond experiment in wlhich the dosages of 2,4-D used inthe treatment, were chainged to 1, 5, and 50 ligm, re-spectively. No inhibitive effect was foundl in plantsreceiving 1 ugm of 2,4-D, as the total recoveries ofradioactivity were practically identical as compared tothat of the corresponding control groups. In the caseof 5 tgm 2,4-D, the total recoveries of radioactivitywere significantly higher than in the control group.The (lestruetion of IAA was areatlv reduced in thegroup which received 50 agm of 2,4-D during the en-tire experimental perio(l.

The effect of 2,4-D on the destruction of IAA-1-C'4in bean ancd corn plants as measured from the accu-mulated activity of respiratory CO2 is shown in figure3. The inhibitive effect of 2,4-D treatment again wasdemonstratedl on the destruction of IAA in both beanand corn plants because the radioactivity in the re-spiratory CO,2 was always found to be less when 2,4-Dwas appliedl. In the case of bean plants, the signifi-cant effects induced by 2,4-D were observed after 4hours of treatment, while in corn plants the inhibi-tive effect was not observed until the 12th hour. Theproduction of CO, was greatly increased and theradioactivity was decreased in both 2,4-D treatedplants, as indicated by the specific activity curves(fig 3). This result agrees with the data from tissueslice stu(lies (3, 9) that at low concentration 2,4-Dstimulates- rate of respiration.

In the study of the relationship) of 2,4-D to theendogenous auxin of the plant, Weintraub (11) foundthat the auxin content of 2,4-D treated bean budswas very mutch smaller than that of the normal buds.This difference is increased by increasing the amountof 2,4-D applied. They suggested that the lowerauxin content is either (lue to a diminished rate ofprodtuction or an augmented rate of destruction. Theinhibitive effect of 2,4-D on the oxidation of exogen-ous IAA in both bean and corn plants, as found inthese experiments, would suggest that the mechanismof action of 2,4-D which brings abotit a diminished

content of extractable auxin in Weintraub's experi-ment is probably due to a diminished rate of produc-tion. It seems likely that the action of 2,4-D onexogenous IAA is probably due to an indirect effectpossibly inhibiting the synthesis of an enzyme or otherco-factors concerned with destruction of IAA. It isalso possible that 2,4-D may stimulate the re-utiliza-tion of radioactive carbon from IAA-1-C14 and leadto the same end result. However, chromatographicstudy of alcohol extracts from leaf tissues receivingIAA-1-C14 alone or IAA-1-C14 plus non-radioactive2,4-D revealed that both extracts contain six identicalradioactive compounds. Therefore, it is doubtful ifthe lower production of respiratory C1402 irn the 2,4-Dtreated plants is due to re-utilization of radioactivecarbon. Nevertheless, as a major hypothesis the selec-tive toxicity of 2,4-D against broadleafed plants canno longer be explained simply on the basis of the dis-turbance of IAA metabolism in plants.

SUMMARYIndoleacetic acid-1-C14 with an activity of 6.50 x

107 cpm/mM has been synthesized.The rate of the absorption and translocation of

C14 after receiving IAA-1-C14 treatment on the pri-mary leaf was studied in bean plants. Only 10 to14 % of the C14 activity was absorbed and trans-ported to the other parts during 14 days experimentalperiod. A larger portion remained in the leaf whichreceived IAA treatment.

The effect of light on the destruction of IAA-1-C14was studied in kidney bean, pea and corn plants. Thedestruction of IAA-1-C14 was greatly inhibited in thedark as measured by the production of respiratoryC1402. The bean and pea plants destroyed IAA butat a slower rate during the first 12-hour period in theclark, while the destruction of IAA in the corn plantswas completely light dependent.

The effect of 2,4-D on the destruction of exogenousIAA has also been demonstrated in the bean and cornplants. 2,4-D treatment decreased the rate of de-struction of IAA in both plant species.

LITERATURE CITED1. ANDREAE, W. A. and GOOD, N. E. The formation of

indoleacetylaspartic acid in pea seedlings. PlantPlmysiol. 30: 380-382. 1955.

2. FANG, S. C., JAWORSKI, E. G., LOGAN, A. V., FREED,V. H. and BUTTS, J. S. The absorption of radio-active 2,4-dichlorophenoxyacetic acid and translo-cation of C"4 by bean plants. Arch. Biochem. Bio-phys. 32: 249-255. 1951.

3. FRENCH, R. C. and BEEVERS, H. Respiratory andgrowth riesponses induced by growth regulatorsand allied compounds. Amer. Jour. Bot. 40: 660-666. 1953.

4. GALSTON, A. W., BONNER, J. and BAKER, R. S.Flavoprotein and peroxidase as constituents of theindoleacetic acid oxidase of peas. Amer. Jour.Bot. 37: 677-678. 1950.

5. GALSTON, A. W., BONNER, J. and BAKER, R. S.Flavoprotein and peroxidase as components of the

25I")8



FANG AND BUTTS-INDOLEACETIC ACID-_-Cl4

indoleacetic acid oxidase system of peas. Arch.Biochem. Biopihys. 42: 456-470. 1953.

6. GOLDACRE, P. L. On the mechanism of action of2,4-dichlorophenoxyacetic acid. Australian Jour.Sci. Research 2: 154-156. 1949.

7. GOLDACRE, P. L., GALSTON, A. W. and WEINTRAuB,R. L. The effect of substituted phenols on theactivity of the indoleacetic acid oxidase of peas.Arch. Biochem. Biophys. 43: 358-373. 1953.

8. GOOD, N. E., ANDREAE, W. A. and VAN YSSELSTEIN,M. WX. H. Studies on 3-indoleacetic acid metabo-lism. II. Some products of the metabolism of

exogenous indoleacetic acid in plant tissues. PlantPhysiol. 31: 231-235. 1956.

9. KELLY, S. M. and AVERY, G. S., JR. The effect of2,4-dichlorophenoxyacetic acid and other physio-logically active substances on respiration. Amer.Jour. Bot. 36: 421-426. 1949.

10. STUTZ, R. E., ATKINSON, D. E. and GORDON. S. A.Synthesis of 3-indoleacetic acid-2-C"4. ANL-4710:1-12. 1951.

11. WEINTRAUB, R. L. 2,4-D, mechanisms of action.Jotur. Agr. Food Chem. 1: 250-254. 1953.

ESTERIFICATION OF PHOSPHATE IN RIPENING FRUIT1

JOY D. MARKS,2 ROBERT BERNLOHR2 AND J. E. VARNERDEPART-MEN-T OF BIOCHEMISTRY, COLLEGE OF AGRICULTURE, THE OHIO STATE UNNIVERSITY.

COLUMBUS 10, OHIO

The biochemical evidences of changes which markthe physiological development of fruit are associatedwith distinct periods in the life-history of the fruit.These periods are: 1) cell division; 2) cell enlarge-ment; 3) maturation; 4) "autogenous climacteric";and 5) senescence. The climacteric (4) refers to arespiratory pattern observed in many fruit followingmaturation, which is characterized by a sudden, sharpincrease in respiratory rate to the "climacteric maxi-mum." This maximum is reached by some kinds offruit, while attached to the plant, or by other fruit asa post harvest phenomenon. The period of senes-cence, which terminates the development of all fruits,precedes final deterioration' and decay of the naturalproduct.

Robertson and Turner (7) proposed that the occur-rence of limited respiration during the pre-climac-teric period of fruit development may be due to theavailability of phosphate acceptors. Thus, theseworkers implicated a controlling role for the processof oxidative phosphorylation during the later periodsof fruit ripening. Pearson and Robertson (6) andMillerd, Bonner, and Biale (5) demonstrated that 2,4-dinitrophenol (DNP), an uncoupler of oxidative phos-phorylation, increased respiration in pre-climactericfruit (apple slices and avocado slices), but had noeffect on fruit at climacteric and post climactericstages. Analogous to the effect of DNP on avocadoslices is the effect of naturally occurringf uncouplersisolated from climacteric avocados by Millerd et al(5). These substances depressed P/0 ratios of iso-lated mitochondria, and caused a corresponding in-crease in respiration. In v-iew of these effects on res-piration in both pre-climacteric fruit and isolatedmitochondria, the hypothesis was proposed (5) thatfruit ripening results from an uncoupling of oxidativephosphor'ylation.

In view of the evidence cited above, it was antici-pated that a measurement of the status of phosphate

1 Received November 20, 1956.2 Charles F. Kettering Foundation predoctoral fellows.

esterification in intact fruit during the sequence ofstages in its life-history would show whether a loss ofphosphorylative capacity did, in fact, precede ripen-ing. The ripening process is considered, in this paper,to embrace the periods of climacteric and senescence.

Measurements of the incorporation of radioactivephosphate into organic phosphate esters has shown sig-nificant esterification by tomato fruit during matura-tion, post climacteric, and senescent periods. It waspossible to prevent phosphorylation by the uncouplingaction of dinitrophenol, but complete disappearance ofphosphorylative capacity did not occur naturally un-til the termination of senescence (apparent in tomatofruit from deep red skin coloration and the soft, struc-tural condition of a completely ripenedI fruit). Thesefindings, therefore, have established the occurrence ofphosphorylation in whole fruit beyond the climactericmaximum of respiration and well into the post climac-teric period of fruit ripening. Quantitative informa-tion concerning the phosphorylative capacity of to-mato fruit are presented in this paper as a basis forthe evaluation of the role of phosphorylation in theripening processes of fruit.

AIATERIALS AND 'METHODSTomato fruit were used for the experiments re-

ported here. These were obtained from local gardensand from greenhouse plants (Ohio Wilt Resistant)grown under the supervision of the Ohio State Uni-versity Department of Horticulture. Solutions usedfor the various treatments were injected into thelocules of the tomatoes by means of a hypodermicsyringe fitted with a two-inch needle. All incubationswere at room temperature. Bruising injury was pro-duced by rolling the fruit (under some mechanicalpressure) prior to injection of labeled phosphate.Care was taken not to puncture the skin of the fruitduring this treatment. After incubation, the fruit wassectioned and extracted by grinding in a mortar withsufficient trichloroacetic acid to bring the final con-centration to 3 %. The clear supernatant fraction ob-

259



plant physiology · studies on the absorption, transport and metab-olism of synthetic growth...

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