STUDIES IN PLANT METABOLISM. IV. COMPARATIVE EFFECTS OF2,4-DICHLOROPHENOXYACETIC ACID AND OTHER PLANT

GROWTH REGULATORS ON PHOSPHORUS METABO-LISM IN BEAN PLANTS 1,2,3

S. C. FANG AND JOSEPH S. BUTTSDEPARTMENT OF AGRICULTURAL CHEMISTRY, OREGON AGRICULTURAL EXPERIMENT STATION,

OREGON STATE COLLEGE, CORVALLIS, OREGON

Accumulated evidence has indicated that thephysiological effect of 2,4-dichlorophenoxyacetic acid(2,4-D) in plants may function through its effect onprocesses of respiration (1, 2, 3, 4, 5) as well as onphotosynthesis (6). Since the precise mechanism bywhich plant cells respond to 2,4-D has yet to beelucidated, and the participation of phosphorus inbiological oxidation-reduction reactions had beenfound in both higher animals and microorganisms,an investigation was initiated to study the compara-tive effects of 2,4-D and other plant growth regula-tors on phosphorus metabolism in order to clarify, inpart, the herbicidal effect of 2,4-D.

The immediate objectives of the work reportedhere are as follows: the comparative effects of 2,4-Dand other plant growth regulators on (1) the phos-phorus uptake, (2) the movement and the distribu-tion of phosphorus, and (3) the incorporation ofradioactive phosphorus into some intermediate com-pounds in bean plants.

MATERIALS AND METHODSGROWTH REGULATOR SOLUTIONS: Immediately

prior to use, 95 % ethanol solutions, each containing0.1 % plant growth regulators (2,4-D acid, indole-acetic acid, indolebutyric acid or a-napthaleneaceticacid) and 0.5 % Tween-20, were prepared and wereused throughout this experiment.

TREATMENT OF PLANTS: Bean plants (Phaseolusvulgaris, var. Black Valentine) were grown in sandculture under greenhouse conditions unless otherwisenoted. The nutrient solution of Biddulph (7) wasemployed. In several trials, the plants were random-ized and divided into four groups. Three groupswere treated with 10 ,g, 50 ug or 100 ,ug of 2,4-Dper plant on the midrib of one of the primary leaveswhich was almost fully expanded but whose terminalbud was still small. The fourth served as controls.The same nutrient solution containing 10 to 50 ,ucP32 per liter was then applied daily to the mediumin which the plants were growing. All plants wereharvested after 7 days, and the roots were carefullywashed under running water to remove the unab-sorbed radioactive P. All samples were composited,weighed and homogenized in a Waring Blendor with20 times their weight of water. An aliquot of each

1 Received February 8, 1954.2This work was supported by a grant from the

Atomic Energy Commission.3Approved for publication as Technical Paper No.

834 by the Director of the Oregon Agricultural Experi-ment Station. Contribution of the Department of Agri-cultural Chemistry.

homogenate was taken out with a pipette and (Iriedin a 1 inch diameter stainless steel cup under aninfra-red lamp. The radioactivities of all sampleswere measured directly with a thin mica windowG-MA counter (1.7 mg/cm2). They represent a rela-tive fraction of the total P absorbed and accumu-lated during the experimental period. Other aliquotswere centrifuged with a Servall angle centrifuge toremove the residue and equivalent amounts of thesupernatants were dried in the same manner and theradioactivities of these samples which represent thewater soluble P were again measured. A typical setof data is shown in table I.

In the study of the effect of 2,4-D and otherplant growth regulators on P metabolism, 60 bean

TABLE IEFFECT OF VARIOUS AMOUNTS OF 2,4-D TREATMENT

ON PHOSPHORUS UPTAKE IN BEAN PLANTS(EACH GROUP CONSISTS OF 5 PLANTS)

2,4-D TREATMENT, ,UG

0 10 50 100

Total P3' activity per 25 mg freshsample, cpm

Leaf ......... 490 370 190 150Stem.330 425 350 370Root .9800 9850 9550 9000

Water soluble P3' activity per 25 mg freshsample, cpm

Leaf ......... 348 310 148 115Stem ........ 295 345 255 282Root ........ 8050 7570 9300 8800

plants were grown in the same manner in 2 flats anddivided into 6 randomized groups consisting of 10plants each. The first and fourth groups received notreatment. The second group received 50 ug 2,4-Dtreatment to each plant (the chemical was appliedon one of the primary leaves) and the 3rd, 5th and6th groups received 250 ,ug of indoleacetic acid, 250,ug indolebutyric acid and 250 ,ug a-naphthaleneaceticacid per each plant respectively. A nutrient solutioncontaining 10 Mtc per 1 of p32 was then given daily tothe plants after 24 hours of treatment. Any differ-ence in absorption and distribution of p32 in thebean plants and in the incorporation of p32 into theP intermediate compounds if found between growthregulator treated and control bean plants would bethe result obtained from the physiological effectswhich occurred after the treatment. All plants were

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PLANT PHYSIOLOGY

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FIG. 1. Radioautograph of control bean plant showing the distribution of PTM after a 7-day absorption period.

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FANG AND BUTTS-2,4-D ON PHOSPHORUS6

harvested after 7 days. Two plants from each groupwere used for making radioautographs to show thedistribution of P32 The remaining 8 plants were(livided into leaf and stem portions, the root beingdiscarded. The samples were composited, weighed,and homogenized with 20 times their weight of water.The homogenates were decanted into flasks andsteamed immediately for 10 minutes to inactivateany enzymes present. An aliquot was centrifuged toremove the residue, and the supernatant was pre-served under a layer of toluene and kept in a refrig-erator. These samples were subjected later to chem-ical analyses for inorganic P, organic P, and measure-ment of radioactivity. Chromatographic separation,identification and determination of the P intermedi-ate compounds of these samples were also performed.

MEEASUREMENT OF RADIOACrIvITY: A Tracerlab 64scaler equippedl with a thin mica window G-M\counter, or a windowless gas flow counter was useddepending upon the degree of activity of the sample.Equal amounts of supernatant solutions from growthregulator treated or from control bean plants wereused in every case in order to minimize the errorfrom the self-absorption. No correction for self-ab-sorption was made in any of the measurements. Inmany instances, an Autoscaler equipped with anautomatic sample changer was used for measuringthe radioactivity- of paper chromatograms. All sam-ples were measured to within 5 % probable error.

RADIOAUTOGRAPHY: The radioautographs wereprepared by exposing x-ray film to the plants con-taining P32, for 22 to 48 hours, then developed witha Kodak D-772 developer.

CHROMATOGRAPHIC SEPARATION: One-dimensionalchromatograms of the supernatant fluids were pre-pared in the usual way. The supernatant liquid wasadded dropwise with a glass dropper in a small spotto paper strips, about 6 cm from one end of thestrip, until the desired amount had been applied.Whatman #1 paper strips (1" x 22") were used.The strips were developed with a mixture of iso-propyl ether and 90 % formic acid in a volume pro-portion of 9: 6, respectively (8). After developmentthe chromatographic strips were thoroughly dried inair and cut into 1-cm sections, starting from the spotand consecutively numbered. Each section wasplaced in a stainless steel, cupped planchet and theradioactivity was measured. The relative radioac-tivity of the spots was then computed as describedpreviously (9).

In other strips, after enough radioactivity fromthe supernatant liquids had been applied on thestrips, known amounts of phosphoglyceric acid, glu-cose-l-phosphate and hexosediphosphate (about 50/lg each) were also applied to the spot. After de-

velopment, the chromatograms were sprayed with aperchlorate-molybdate reagent. The technique ofHanes and Isherwood (10) was followed in producingthe blue color of the P chromatograms. After re-cording the positions of P spots, the strips were againcut into 1-cm sections and the radioactivity of eachsection was measured in the same manner. The lo-cations of blue P spots and the radioactive spotswere compared. Several radioactive spots were iden-tified by this process as inorganic phosphate, phos-phoglyceric acid, glucose-i-phosphate and hexose-diphosphate.

DETERMINATION OF TOTAL AND INORGANIC PHOS-PHORUS: The method of Fiske and Subbarow (11)was followed. The color of the solution was meas-ured with a Coleman electric photometer and wascompared with standards producedl from knownamounts of P.

RESULTS AND DISCuSSIONTHE EFFECT OF ½ ARIOUS AMOUNTS OF 2,4-D ON

THE UPTAKE AND DISTRIBUTION OF P32 IN- BEANPLANTS: Data regarding the P32 activities per 25 mgfresh samples of leaf, stem and root from the 2,4-Dtreated and the control bean plants are representedin table I. In all cases, the P32 activities of leavesfrom 2,4-D treated plants were much less when com-pared to the leaves from the control plants, while theactivities in either the stem or root from these twogroups of plants showed no significant differences.When 2,4-D treatments were increased from 10 ug to100 ug per plant, the accumulation of p32 activity inthe leaf from different treatments decreased accord-ingly. This observation indicated that 2,4-D treat-ment affected the upward movement and the supplyof P to the leaves. Consequently, the insufficientsupply of P in the leaf may directly or indirectly af-fect both the important photosynthetic and respira-tory processes of the plant.

THE EFFECT OF 2,4-D AND OTHER PLAN-T GROWTHREGULATORS ON THE DISTRIBUTION OF P32 IN BEANPLANTS: As shown in figures 1 to 4, the distributionof p32 in growth regulator treated plants differedfrom that found in the control plants. The radio-autograph of the IBA treated plants was quiite simi-lar to that of the IAA treated one; therefore it wasnot included. In general, the p32 was concentratedin the petiole of the treated leaf, the midrib of thetreated leaf, and in the first internode. The concen-tration of p32 was extended to part of the hypocotylin a-naphthaleneacetic acid treated plants. The ac-cumulation and the distribution of p32 in the beanplants are probably determined by the plant growthregulator. The distribution pattern of p32 in 2,4-Dtreated plants is very similar to the acctmulation

FIG. 2. Radioautograph of 2,4-D treated bean plant showing the distribution of P' after a 7-day absorptionperiod. (50 Ag of 2,4-D were applied on the left primary leaf.)

FIG. 3. Radioautograph of indoleacetic acid treated bean plant showing the distribution of PS2 after a 7-dayabsorption period. (250 ,ug of IAA were applied on the left primary leaf.)

FIG. 4. Radioautograph of anaphthaleneacetic acid treated bean plant showing the distribution of P' aftera 7-day absorption period. (250 ,ug of NAA were applied on the right primary leaf.)

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pattern of 2,4-D (12). This observation may sug-gest that the distribution of P32 is associated withthe distribution of the growth regulators. In 1948,Brunstetter et al (13) demonstrated that when beanplants were treated with 3-indoleacetic acid, the Pcontent in the treated stem increased about 5 timesover that of the control after 148 hours. Our resultshowed that the accumulation of P32 in IAA treatedstems was the same as compared to the control stems.However, the total P content in the treated stemswas increased about 60 % over that of the controlsand presumably this increase was due to the trans-port of P from the leaf. Whether or not the physio-logical effects of 2,4-D and of other growth regulatorson bean plants, in regard to the distribution of radio-active P, are the same has yet to be studied. How-ever, the growth regulator treatment will certainlymodify the distribution and accumulation pattern ofphosphorus in bean plants.

THE EFFECT OF 2,4-D AND OTHER GROWTH REG-ULATORS ON THE INCORPORATION OF P32 INTO SOMEPHOSPHORUS INTERMEDIATES: It is obvious that boththe total water soluble P32 activity and the specificactivity decreased in the leaves of all bean plantstreated with growth regulators. The effect was morepronounced in 2,4-D treated plants. The total P32activity was found to be greatly increased in 2,4-Dtreated stems over that of the control, slightly de-creased in the IBA treated stem, and without signifi-cant changes in IAA- and NAA-treated stems. Thepercentage of inorganic P was found to be higher inthe leaf and lower in the stem of 2,4-D treated plantsas compared to the control. Also, the incorporationof P32 into glucose-i-phosphate and hexosediphos-phate was less in the leaf and greater in the stem of2,4-D treated plants. A great increase in hexosedi-phosphate was observed only in the 2,4-D treatedstem while no significant, or slight, change was foundwvith the other plant growth regulator treatments.The results demonstrated clearly that all of thegrowth regulators affected the incorporation of P32into one or more P intermediates. 2,4-D has thegreatest effect over the other growth regulators onthe incorporation of P32 into P intermediates.

SUMMARYThe effects of 2,4-D and of other growth regula-

tors on the P metabolism in bean plants was in-vestigated. It was found that 2,4-D treatmentsgreatly reduced the upward movement of radioac-tive P to the leaves; the degree of reduction wasproportioned to the amount of 2,4-D supplied.

Treatment with 2,4-D, IAA, IBA, or NAA modi-fied the distribution and accumulation pattern ofradioactive P in bean plants.

The incorporation of p32 into some P intermedi-ate compounds in the water soluble fraction was af-fected by plant growth regulator treatment. Thegreatest effect was observed in 2,4-D treated plants.

LITERATURE CITED1. BROWN, J. W. Effect of 2,4-dichlorophenoxyacetic

acid on the water relations, the accumulation anddistribution of solid matter, and the respiration ofbean plants. Bot. Gaz. 107: 332-343. 1946.

2. HSUEH, Y. L. and Lou, C. H. Effects of 2,4-D onseed germination and respiration. Science 105:283-285. 1947.

3. SMITH, F. G. The effect of 2,4-dichlorophenoxy-acetic acid on the respiratory metabolism of beanstem tissue. Plant Physiol. 23: 70-83. 1948.

4. KELLY, SALLY 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.

5. MITCHELL, J. E., BuRRs, R. H., and RIKER, A. J.Inhibition of respiration in plant tissues by callusstimulating substances and related chemicals.Amer. Jour. Bot. 36: 368-378. 1949.

6. FREELAND, R. 0. Effects of growth substances onphotosynthesis. Plant Physiol. 24: 621-628. 1949.

7. BIDDULPH, 0. Absorption and movement of radio-phosphorus in bean seedlings. Plant Physiol. 15:131-135. 1940.

8. BEAUDREAU, G. S. The use of P' in the study ofphosphate compounds in gladiolus leaves. A thesis.Oregon State College. 1952.

9. FANG, S. C. and Burrs, J. S. Studies in plant me-tabolism. III. Absorption, translocation and me-tabolism of radioactive 2,4-D in corn and wheatplants. Plant Physiol. 29: 56-60. 1954.

10. HANES, C. S. and ISHERWOOD, P. A. Separation ofthe phosphate esters on filter paper chromato-grams. Nature 164: 1107-1112. 1949.

11. FISKE, C. H. and SUBBAROW, Y. The colorimetricdetermination of phosphorus. Jour. Biol. Chem.66: 375400. 1925.

12. FANG, S. C., JAWORSKI, E. G., LOGAN, A. V., FREED,V. H., and Buirs, J. S. The absorption of radio-active 2,4-dichlorophenoxyacetic acid and thetranslocation of C14 by bean plants. Arch. Bio-chem. Biophys. 32: 249-255. 1951.

13. BRUNSTETTER, B. C., MYERS, A. T., MITCHELL, J. W.,STEWART, W. S., and KAUFMAN, M. W. Mineralcomposition of bean stems treated with 3-indole-acetic acid. Bot. Gaz. 109: 268-276. 1948.

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