metabolism - pdfs.semanticscholar.org · absorption, distribution, & metabolism...

12
ABSORPTION, DISTRIBUTION, & METABOLISM OF 2,2-DICHLOROPROPIONIC ACID IN RELATION TO PHYTOTOXICITY. II. DISTRIBUTION & METABOLIC FATE OF DALAPON IN PLANTS.' CHESTER L. FOY DEPARTMENT OF BOTANY, UNIVERSITY OF CALIFORNIA, DAVIS P'atterns of absorption and initial translocation of ClI"'-and C'4-laheled 2,2-dichloropropionic acid (dala- ponl) in both tolerant and susceptible species wrere dlescribe(d earlier (8). Of equal or perhaps more importance in understanding the physiological action of growth regulators are the long-term translocation, re-distribution, accumulation, and metabolic fate of the compound in association with plant tissues. The recent emphasis on pesticide residues in food and feed products has given the latter considerations an addi- tional order of importance. Despite the foregoing, perhaps our greatest ignorance of herbicides todav concerns their ultimate fate in plants. The tendency of a herbicide to persist in an active form or to be detoxified rapidly is likely to be of primary im- portance, whatever the precise mechanism(s) of herb- icidal action. Alll of a given herbicide that enters a plant, e.g. via the leaves, is not necessarily available for transloca- tion and/or herbicidal action. Possible mechanisms accounting for the immobilizationi, inactivation, or dis- appearance of the compound are adsorption on colloid- al surfaces, true accumulation by living cells, metabolic degradation followed by loss or incorporation of breakdown products, aln(d reduced movement due to phase distribution effects as on a chromatogram (6. 29, 30, 31). The direct loss of various substances fronm roots or other plant parts may also occur (4, 5, 8, 14, 16, 26). Considerable researchi has been conducted on the loss or persistence of 2,4-dichlorophenoxyacetic acid (2,4-D) in both active and (lormant tissues (17. 18. 19, 22, 29, 31). Several papers are summarized in a recent review (33). Radioactive 2,4-D is known to be metabolized to varying degrees in plant tissues, with the resultant incorporation of C'4-labeled com- pounds and the evolution of C140.. Strong evidence exists also for relatively long persistence of 2,4-D in plants, which leads to syniptonmatic expression in suc- ceeding seasons (perennials) or succeeding genera- tions (annuals) by transmission through dormant buds or seeds. This latter phenomenon has been ob- served following both foliar and root applications. 2.4-D is known to persist in plants for longer periods tlhaln exogenous indoleacetic acid. 1 Received revised manuscript May 10, 1961. Prior to the present series of experiments (6, 8), none of the foregoing topics had been investigated Nwith respect to dalapon. Herein are reported the results of several studies on the distributional and metabolic fate of dalapon in association with plant tissues. Finally, these results, as well as experimental findings from other sources, will be discussed in rela- tion to possible mechanisms of herbicidal selectivity. MATERIALS & METHODS The general techniques and materials employed in these studies have been described (8, 9). Radioau- tography, counting, extraction and fractionation. and paper partition chromatography were combined to yield both qualitative and quantitative results. As in the studies reported earlier (8). the follow- ing radiolabeled chemicals were used in treating plants or plant material: A: purified 2,2-dichloropropion- ic acid-Cl36 in acetone (12.78 uc/nmmole converted to the sodium salt in aqueous solution before application to plants; B: 96 %; Na-2,2-diclhloropropionate-2-CI4 (0.98 mc/mM); 78 % Na-2,2-dichloropropionate-2- C14 (0.98 mc/mM). In addition, Na-2.2-dichloro- propionate-2-C14 (containing on a radioactivity basis over half LiCl36), monochloropropionate-2-C4., and 2,2,3-trichloropropionate-2-C14 were used as standards in chromatography. Extracts were spottedl approximately one centi- meter diameter on Whatman No. 1 filter paper by use of a micropipette. After equilibration, the chro- matograms were developed one-dimensionally over- night using n-butanol and 1.5 N ammonium hydroxide (6,9,25). In one phase of the study, dried, ground fruits of cotton were extracted and fractionated according to the scheme shown in figure 1. Further procedural details are given in the Results section. RESULTS & DIscussIoN DEGRADATION BY MICRO-ORGANISMS: Herbicidal activity may be diminished in soils by volatilization, leaching, fixation by adsorption to soil colloids, chem- ical or photochemical decomposition, and micro- biological breakdown. The last of these is perhaps of greatest importance in the disappearance of most organic herbicides tested thus far. Other effects such 698 www.plantphysiol.org on October 15, 2017 - Published by Downloaded from Copyright © 1961 American Society of Plant Biologists. All rights reserved.

Upload: others

Post on 12-Sep-2019

3 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: METABOLISM - pdfs.semanticscholar.org · absorption, distribution, & metabolism of2,2-dichloropropionic acid in relationtophytotoxicity. ii. distribution & metabolic fate of dalapon

ABSORPTION, DISTRIBUTION, & METABOLISM OF 2,2-DICHLOROPROPIONIC ACIDIN RELATION TO PHYTOTOXICITY. II. DISTRIBUTION &

METABOLIC FATE OF DALAPON IN PLANTS.'CHESTER L. FOY

DEPARTMENT OF BOTANY, UNIVERSITY OF CALIFORNIA, DAVIS

P'atterns of absorption and initial translocation ofClI"'-and C'4-laheled 2,2-dichloropropionic acid (dala-ponl) in both tolerant and susceptible species wreredlescribe(d earlier (8). Of equal or perhaps moreimportance in understanding the physiological actionof growth regulators are the long-term translocation,re-distribution, accumulation, and metabolic fate ofthe compound in association with plant tissues. Therecent emphasis on pesticide residues in food and feedproducts has given the latter considerations an addi-tional order of importance. Despite the foregoing,perhaps our greatest ignorance of herbicides todavconcerns their ultimate fate in plants. The tendencyof a herbicide to persist in an active form or to bedetoxified rapidly is likely to be of primary im-portance, whatever the precise mechanism(s) of herb-icidal action.

Alll of a given herbicide that enters a plant, e.g. viathe leaves, is not necessarily available for transloca-tion and/or herbicidal action. Possible mechanismsaccounting for the immobilizationi, inactivation, or dis-appearance of the compound are adsorption on colloid-al surfaces, true accumulation by living cells, metabolicdegradation followed by loss or incorporation ofbreakdown products, aln(d reduced movement due tophase distribution effects as on a chromatogram (6.29, 30, 31). The direct loss of various substancesfronm roots or other plant parts may also occur (4,5, 8, 14, 16, 26).

Considerable researchi has been conducted on theloss or persistence of 2,4-dichlorophenoxyacetic acid(2,4-D) in both active and (lormant tissues (17. 18.19, 22, 29, 31). Several papers are summarized ina recent review (33). Radioactive 2,4-D is knownto be metabolized to varying degrees in plant tissues,with the resultant incorporation of C'4-labeled com-pounds and the evolution of C140.. Strong evidenceexists also for relatively long persistence of 2,4-D inplants, which leads to syniptonmatic expression in suc-ceeding seasons (perennials) or succeeding genera-tions (annuals) by transmission through dormantbuds or seeds. This latter phenomenon has been ob-served following both foliar and root applications.2.4-D is known to persist in plants for longer periodstlhaln exogenous indoleacetic acid.

1 Received revised manuscript May 10, 1961.

Prior to the present series of experiments (6, 8),none of the foregoing topics had been investigatedNwith respect to dalapon. Herein are reported theresults of several studies on the distributional andmetabolic fate of dalapon in association with planttissues. Finally, these results, as well as experimentalfindings from other sources, will be discussed in rela-tion to possible mechanisms of herbicidal selectivity.

MATERIALS & METHODS

The general techniques and materials employed inthese studies have been described (8, 9). Radioau-tography, counting, extraction and fractionation. andpaper partition chromatography were combined toyield both qualitative and quantitative results.

As in the studies reported earlier (8). the follow-ing radiolabeled chemicals were used in treating plantsor plant material: A: purified 2,2-dichloropropion-ic acid-Cl36 in acetone (12.78 uc/nmmole converted tothe sodium salt in aqueous solution before applicationto plants; B: 96 %; Na-2,2-diclhloropropionate-2-CI4(0.98 mc/mM); 78 % Na-2,2-dichloropropionate-2-C14 (0.98 mc/mM). In addition, Na-2.2-dichloro-propionate-2-C14 (containing on a radioactivity basisover half LiCl36), monochloropropionate-2-C4., and2,2,3-trichloropropionate-2-C14 were used as standardsin chromatography.

Extracts were spottedl approximately one centi-meter diameter on Whatman No. 1 filter paper byuse of a micropipette. After equilibration, the chro-matograms were developed one-dimensionally over-night using n-butanol and 1.5 N ammonium hydroxide(6,9,25).

In one phase of the study, dried, ground fruitsof cotton were extracted and fractionated accordingto the scheme shown in figure 1.

Further procedural details are given in the Resultssection.

RESULTS & DIscussIoN

DEGRADATION BY MICRO-ORGANISMS: Herbicidalactivity may be diminished in soils by volatilization,leaching, fixation by adsorption to soil colloids, chem-ical or photochemical decomposition, and micro-biological breakdown. The last of these is perhapsof greatest importance in the disappearance of mostorganic herbicides tested thus far. Other effects such

698

www.plantphysiol.orgon October 15, 2017 - Published by Downloaded from Copyright © 1961 American Society of Plant Biologists. All rights reserved.

Page 2: METABOLISM - pdfs.semanticscholar.org · absorption, distribution, & metabolism of2,2-dichloropropionic acid in relationtophytotoxicity. ii. distribution & metabolic fate of dalapon

FOY-2,2-DICHLOROPROPIONIC ACID & PHYTOTOXICITY

DRY PLANT MATERIALTON)ETHEREXTR ACT3X

LIPIDS,PIGMENTS,ORGANIC ACIDS

WASH, 10%N.2C0C

AQUEO S ETHER

LIPIDS, PIGMENTS

ORGANIC (CHLOROPHYLLS, CAROTENES.ACIDS XANTHOPHYLLS, ETC.)

EVAP. ETHERTO SMALL VOL.ADD PET. ETHERWASH 80%EtOH

H..O " PET.

EXTRACT80% EtOH

2X,60 C, 3 HRS.

SUGARS,ORGANIC ACIDS,AMINO ACIDS, ETC

CONC.IN VACUO55- 65-C6 HRS.

FILTRATE IN'

RESIDUE

H2O

EXTRACT

S OLUBLE SOLUBBLEEETHANOL ETHER PPT. PROTEII

SEPARATELIPIDS, CAROTENES, SUCCESSIVELYXANTHOPHYLLS ETC. ON ION

EXCHANGERESINS

PRECIPITATE SUIIo 2.*

SEPARATION SEPARATION REMAINDER

ANION RESIN CATION RESIN NEUTRALDUOLITE A-2 DUOLITE C-3 (PASSED BOTH RESINS)

ELUTE ELUTENH4OH (I N) H2S04 (I N)pH 10-11 pH

RESIN ANIONIC ACIDS RESIN CATIONIC NEUTRAL SUGARS,(METABOLIC ACIDS, AMINO ACIDS, STEROIDS, PLASTIDPHOSPHORYLATED ALKALOIDS, PIGMENTS, ETC.CPDS, ETC. - ALSO RELATED CPDS.DALAPON)

HCIpH 1.5CONTI NUOUSETHER EXTRACT8 HRS.

AQUEOUS ETHER10%Ne2CO3ANDDE-IONIZEDH20

ETHER AQUEOUS(DALA P ON)

6--IV. RESIDUENS, ETC.

IPER NATANT

ENZYMATICDIGESTION WITHAMYLASES ANDPROTEINASES TOTEST FOR ACTIVITYIN STARGH, INSbLUBLEPROTEINS ANDRESIDUALCARBOHYDRATES

FIG. 1. Analytical scheme followed in categorizing radioactive chemical constituents from the fruits and leavesof cotton 10 weeks after treatment with 96 % Na 2,2-dichloropropionate-2-C'4.

699

www.plantphysiol.orgon October 15, 2017 - Published by Downloaded from Copyright © 1961 American Society of Plant Biologists. All rights reserved.

Page 3: METABOLISM - pdfs.semanticscholar.org · absorption, distribution, & metabolism of2,2-dichloropropionic acid in relationtophytotoxicity. ii. distribution & metabolic fate of dalapon

PLANT PHYSIOLOGY

as temperature, moisture, organic matter content,nutrient levels, pH, etc. appear to be principally in-direct manifestations of the level of activity of soil-borne micro-organisms.

Dalapon has little residual effect under conditionsfavorable to leaching and/or high micro-biologic ac-tivity (13, 27), but it is known to persist in the soilfor long periods under less favorable conditions.

During the present studies, one of the first aliquotsof pure dalapon-CI36 hecame contaminated by micro-organislms (lespite storage in a refrigerator. Thechaniges wlhich occurred in pure stock solution offeredpositive proof that some species of fungi, possiblyAlternaria sp., are capable of using dalapon as a solesubstrate to synthesize new substances. The numberof C136-labeled metabolites formed was somewhat sur-prising. Average Rf X 100 values and tentativeidentification of some of the spots were as follows:8 (inorganic Cl36), 19, 34 (monochloropropionate),42, 51 (dalapon), and 62. No special attempt wasmade to identify the other three substances, but theseresults were confirmed many times. Dissolving thecompound in alcohol rather than water preventedbreakdown, but since alcohol is not a normal com-ponent of herbicidal sprays, and might exert someunknown effects upon permeability, its routine usewas discounted. It was virtually impossible to useaseptic techniques in making numerous applicationsin the greenhouse from the same stock solution vialsand it was not feasible to re-purify repeatedly. Thisserious technique difficulty was overcome by keepingall stock solutions frozen except (luring transfers.In order to avoid errors due to volume changes, thetubes were warmed each time to approximatelv 25 C,the temperature at which they were prepared.

METABOLISM BY HIGHER PLANTS: 1. Prelimiiinaryobservations. The primary interests in these studieswere A: to determine whether dalapon is translo-cated and accumulated as the intact molecule or isdegraded rapidly in tolerant and/or resistant species;B: to determine what metabolic changes occurred(if any), and C: to relate such information to itsherbicidal properties. In all short term experiments(3 days or less), in which expressed plant sap waschromatographed directly, only dalapon (Rf 0.4-0.6)or dalapon plus impurities present in the treatmentsolution were found. Also, by the liquid extractionof corn leaf disks with water or ethanol 3 hours aftertreatment (6, 9) no new compounds were detected onchromatograms of the extracts and the ratios of dala-pon to radioactive impurities already present werenot altered by any (or all) of the following treat-ments: A: freezing overnight and thawing; B:extracting in an oven (24 hr at 50 C); C: adlding0.1 % Vatsol OT to the extracting nmedlium. The Rfvalues of dalaponi and contaminants (acetate., pyruv-ate, monochloropropionate, & 2,2,3-trichloropropion-ate) were lower, in general, than those obtained bySmith (25), using ascending chromatography.

The foregoing, plus results reported earlier (8),indicated that dalapon was metabolized very slowly,

if at all, in cotton, sorghum, and corn; however,. othertests using more refined techniques were considerednecessary.

2. Reaction with tissue homogenates. Eitherdry or fresh whole cotton or sorghum plant materialwas homogenized in a ground glass tissue homo-genizer at room temperature. Weights were com-puted to be equivalent to 0.2 g dry wt/10 ml H2O.Then 1 ml aliquots, comparable on a density of drymatter per milliliter basis, were placed in unstopperedbacteriological culture tubes with 10 Al 96 % dalapon-2-C14 for 3 hours at 25 C. Some aliquots were boiledand re-cooled before adding dalapon; others werefrozen and thawed; still others were added directlyto fresh or dry tissue. After 3 hours, the contentsof all tubes were frozen rapidly, then thawed one byone, centrifuged, 200 !l of the supernatant chromato-graphed and another 200 ,u aliquot plancheted andcounted. The test was designed to determine whetheror not rapid and obvious enzymatically-controlledchanges occur in the dalapon molecule while in as-sociation with tissue homogenates from higher plants.Also, the counts obtained from an aliquot (multipliedby a factor and subtracted from the total activityadded) yielded the amount of dalapon held in or onplant material. Furthermore, any that remained withthe plant residue after exhaustive washing with sev-eral hundred volumes of water was presumed to bemetabolically incorporated in a non-diffusible con-stituent.

No new compounds appeared in any treatment.If dalapon was transformed into other compounds,the concentrations were too low for detection by themethod used. The lowest possible limits of detect-ability by the radioautographic method were notestablished, however, levels of activity correspondingto 13.3 Ag dalapon or 2.8 lAg C136 were discernibleas images on the X-ray film and by counting (scan-ning chromatograms). Therefore the sensitivity ofthe method was as great or greater than these levels.Approximately 30 to 40 % of the dalapon was looselyadsorbed on plant material, but no appreciable differ-ences were noticeable among treatments or betweenspecies, and all of the activity was easily removed bywashing with water, thus indicating that no metabolicincorporation into insoluble macromolecules occurred.The experiment was repeated using dalapon-CI36,and the same general results were obtained.

3. Chromatographry of extracts of various planttparts. Cotton and sorghum plants were treated asin previous tests with 100 Al of either dalapon-2-C"4(96 %) or dalapon-C136. The plants were grown ina light cabinet (500-600 ft-c) for 7 days, washedfree of surface radioactivity and sectioned as follows:Cotton, 1 ) cotyledons (treated); 2) epicotyl (lessstem below 1st leaf) ; 3) stem (epicotyl & hypocotyl),and 4) roots; Sorghum, 1) second and third leaves(treated), 2), first, fourth, fifth, and sixth leaves(including sheaths), 3) younger leaves andI stems,and 4) roots. The plant sections were homogenizedlas in the preceding test and aliquots of these aqueous

700

www.plantphysiol.orgon October 15, 2017 - Published by Downloaded from Copyright © 1961 American Society of Plant Biologists. All rights reserved.

Page 4: METABOLISM - pdfs.semanticscholar.org · absorption, distribution, & metabolism of2,2-dichloropropionic acid in relationtophytotoxicity. ii. distribution & metabolic fate of dalapon

FOY-2,2-DICHLOROPROPIONIC ACID & PHYTOTOXICITY

extracts and of the nutrient solutions were chromato-graphed. See (9) for detailed explanation of tech-niques. Rf values of the radioactive compounds ex-

tracted from all plant parts of both species were iden-tical with those of authentic dalapon. Concentrationsof activity were greatest in the treated sections andin the epicotyls (of cotton). This is in agreementwith other tests. Although the spots on the chromato-grams were sometimes displaced by high solute con-

centrations in the drop, it was clear that dalapon andno other prominent radioactive species was recoveredfrom all plant parts of cotton and sorghum, and fromboth of the nutrient solutions. Representative co-

chromatograms are shown in figure 2. There was

some indication of a slight increase in the inorganicchloride spot (a trace was already present as an

impurity in this aliquot). Although not quantitative-ly convincing, this may have some importance in in-terpreting the initial breakdown steps of dalapon inplants. In comparison with the preceding test it isof interest to notice that a small amount of radio-activity, highest in the treated sections and the epi-cotyl (of cotton), but present in all plant sections,was not removable. Thus it appears that the equiv-alent of 3 to 300 cpm/sections was metabolically in-corporated into insoluble constituents after 1 week incotton or sorghum. In the case of C14-labeled dala-pon, this would correspond to approximately 0.002 Ac

jor.#_. i- lllsS::'3 ':i} :': !: _! _ _i'b.3 . ;:.- 3 w:, wr: :: .:::. _r _X .} . r .. . -:::; . . : ....... ._..::.. :: .Y ::::* < i.i: :.Y; ' . _..:.: '::. ::;.:'.. _ej '::. !:: .. .. ,¢E_::..,eH'.. }:: :. .::.: .:: ;: : .....: ::: :: i}.

.. .: ..::: .. .::

:: :... ^.. ,:_

.. ..

:: ..

::, .. .:

.:. .: :::.: . .... :.:' :. . .::} i .:*:: : .....: : : : . . . .: . :. .: : : - i .s.: :::. . . : : ::iX Li: .. _ .':::..::: .:. ::_.... ' jJw, m.:: ::.,.: .. :::. :: . : ........ _* _ .. : . . : S_ ........ i_' Ww

:::_i..: .... i-:9_.@_

... ...:: ._r...: s_

.: :- :: EEr-_-

*.:'..',::.....

i. :.:;t

:tz

-..b

A 8 C D E F G H

FIG. 2. Radioautograms of representative co-chro-matograms. A: 5 ,ul 96 % dalapon-2-C14 stock solution.B: 5 Al impure 2,2,3-trichloropropionate-2-Cl4 (TCP).C: Dalapon plus TCP, 5 ul each. D: Aqueous extractfrom roots of dalapon-treated cotton plants. E: Extractin D plus dalapon. F: Aqueous drop (from ether ex-

tract) of nutrient solution in which dalapon-treated cottonwas grown. G: Extract in F plus dalapon. H: Aque-ous extract from dalapon-treated leaves of sorghum. I:Extract in H plus dalapon. See text for more completeexplanation.

(1.1 Ag dalapon) per sorghum plant, and 0.01 uc (5.3ILg dalapon) per cotton plant when the applied dosewas 1.0 Ac (530 ,Ag dalapon) in each case.

To complement these experiments, dried plants orparts of plants known to contain high activity wereselected from experiments reported earlier (8), andwere extracted and chromatographed in a similarmanner. Essentially the same results were obtainedas follows: A: dalapon was recovered repeatedly;B: there was a suggestion of an increase in theCl36 spot after 2 weeks, but again this was incon-clusive; C: after 6 to 8 days, a small amount ofradioactivity was not removable from the plant residueby homogenizing and washing exhaustively. Theseresults all show that essentially the only radioactiveproduct extractable with water after treatment periodsof 8 days or less was dalapon; however, a very smallamount of activity seems to be chemically bound thusindicating a very slow incorporation of the labeledatom (s) into insoluble compounds.

4. Distributional and metabolic fate of dalaponin fruiting cotton plants. With the foregoing resultsin mind, it was considered desirable to conduct longer-term studies along similar lines. This experimentwas outlined with two objectives as follows: A: todetermine distribution and accumulation patterns ofdalapon in flowering and fruiting cotton plants, whichcould be correlated with field investigations (somealready reported, and others to be mentioned in thissection); B: to determine whether or not dalaponundergoes measurable change after long periods (9-10 weeks) in cotton, and if any is carried non-metabo-lized into the fruits. Cotton was grown in the green-house to the early stages of reproductive growth andtreated by the cut petiole method already described(8). This insured the rapid introduction of a rela-tively large amount of pure radioactive compound intothe xylem stream, simulating uptake through the soil,but without the complications of soil dilution, adsorp-tion, etc. Also, this technique eliminated the possi-bility of micro-biological breakdown outside the plantand subsequent absorption of the degradation prod-ucts, which is known to occur from nutrient solutionsafter several weeks. The gross distribution of dala-pon in the vegetative plant body followed the patternsalready established. In this case, there were addi-tional sinks or regions of utilization of food materialsinto which dalapon was also transported and held.These organs, the reproductive structures, are ofmost interest in this study (See fig 3 A through D).

It was clear from the autographs that radioactivitywas present in some reproductive structures, boththose already formed at the time of treatment andthose developed after treatment. This observation,plus the fact that the amounts of activity present ap-peared to be correlated with the movement of as-similates into organs of different ages and relativerates of growth, indicate that accumulation in youngflowers, fruits, and seeds was principally by re-dis-tribution in the phloem with the inward movement offood materials. This position is further strengthened

701

www.plantphysiol.orgon October 15, 2017 - Published by Downloaded from Copyright © 1961 American Society of Plant Biologists. All rights reserved.

Page 5: METABOLISM - pdfs.semanticscholar.org · absorption, distribution, & metabolism of2,2-dichloropropionic acid in relationtophytotoxicity. ii. distribution & metabolic fate of dalapon

PLANT PHYSIOLOGY

by the fact that although the entire vegetative topsof the plants are known to be rather uniformly floodedwith dalapon via the transpiration stream within 6hours (8), none of the so-called squares (i.e., bothpre-bloom ovaries & immature fruit stages) whichabscised within 7 days after treatment containedradioactivity. (The special term "square" refers tothe angular appearance create(d by- the leaf-like bracts

* ..-A.4 q

.1

:A :.1

.:1

AU..I '*9'0~~~~~~~~~~~~~~~~~~~~:,Ij~~~~~~~~~~~~~~~~~~~~~~~

S

I

subtending the flower or fruit.) Little or no move-ment into a mature leaf below the treated node oc-curred. Radioactivity was present in all flower parts,including bracts, calyx, corolla, and especially thestigma, staminal column, and individual anthers.Young fertilized ovaries and immature fruits werealso high in activity. In the case of older fruits (fig3 C) it is also clear that activity is widely distributed,

Al

-l K - .' U. -.' W ff

9* . 0

.V.

FIG. 3 A through C. Distributionof C4 in reproductive organs of cottonresulting from introduction of dalapon-2-C14 through cut petioles. (A & A')treated petiole and flower parts whichabscised two weeks after treatment.Note high activity in bracts, calyx,fertilized ovary, stigma, staminalcolumn, and anthers. (B & B')Young and almost mature fruits 9 to10 weeks after treatment. Note ac-tivity in all parts with highest concen-tration in the seeds. (C & C')Treated petiole, nodal region, leaf,fruit stalk, and portions of maturefruit 9 to 10 weeks after treatment(upper half). Note high concentra-tions in fruit wall and in the embryos,with only traces of activity in theovule coats. (Lower half of photo-graph from an untreated check plant).

4@

*1 ft '

S.DVA4

- ................ -.5

.. ..

, S; (Y@ 5:-Yg.

A... FEq F: :: :...t w. ^

} v>?, |C

702

.... I ..'. V "-?o mi",k -,

www.plantphysiol.orgon October 15, 2017 - Published by Downloaded from Copyright © 1961 American Society of Plant Biologists. All rights reserved.

Page 6: METABOLISM - pdfs.semanticscholar.org · absorption, distribution, & metabolism of2,2-dichloropropionic acid in relationtophytotoxicity. ii. distribution & metabolic fate of dalapon

FOY-2,2-DICHLOROPROPIONIC ACID & PHYTOTOIICITY

e.g., in the outer fruit walls, carpellary partitions, andseeds, and there was a slight indication of activityeven in the fiber. It is especially noteworthy thatwithin the mature or nearly mature seeds, radioactivi-ty is highly concentrated in the embryo, with onlytraces in the ovule coat. This agrees well with thedeep-seated nature of an observed delay in germina-tion (6) and the recovery and identification of actualdalapon from cotton seeds following field application(7).

In the second phase of this study, whole treatedfruits were pooled, dried, ground, extracted, and frac-tionated according to the scheme shown in figure 1.Similar samples of older leaf tissue and from non-treated plants were included for comparison. Sinceactivity was too low in the mature leaf samples forvalid appraisal (i.e., considerably less than in thefruits on a dry weight basis), and since no activitywas detected in non-treated plants, only the resultswith the fruit samples are presented. Since thesesamples were not accurately corrected for self-absorp-tion loss, the amounts of radioactivity recovered inthe various fractions as shown in table I are onlyrelative (not absolute). Nevertheless, some trendswere obvious. Most of the activity followed theroute which dalapon would take in the extractionand fractionation scheme, i.e., into the ethanol extract,then into the anionic fraction. This fraction con-tained only one prominent radioactive species whichcorresponded in Rf to dalapon. Also activity in thedistillate and in the neutral fraction is considered tobe dalapon, at least in part. Chromatograms of theneutral fraction gave somewhat anomalous results dueto the high concentration of solutes in relation to theactivity required to produce an image. Althoughnot positively identified, there were at least two spots,one of which appeared to be dalapon. (This could

TABLE I

CHEMICAL DISTRIBUTION OF C14 RADIOAcTIVITY IN COTTONFRUITS 10 WEEKS AFTER INTRODUCING DALAPON-2-C'14

THROUGH SEVERED PETIOLES*

FRACTION cpm

Ether extractNaC03 Wash of ether extract(After separation of the ether extractbetween 95 % EtOH and petroleum etheractivity was about equal in the two fractions.)

Ethanol extractDistillate (from concentrating)Insoluble oily residue (upon concentration)Anionic fraction 2Cationic fractionNeutral fraction

Plant Residue (after ether & ethanol extraction)Water wash of residue

380

12793

252421

448290

* Dried ground cotton fruits were extracted and frac-tionated according to figure 1. All fractions which con-tained radioactivity are shown. Data are relative only(not absolute values).

be attributed to less than quantitative removal ofdalapon on the anion exchange resin.) Althoughperhaps 85 to 90 % of the radioactivity recovered wasstill represented as dalapon, the following results pointto the existence of other C14-oontaining compounds;therefore a slight amount of metabolic decompositionmust have occurred within the cotton plant. A:Radioactivity was present in the ether soluble fraction.This activity resisted removal upon re-solubilizing inether and washing with Na,CO3. This step shouldhave removed any organic acids or dalapon, yet no

DFIG. 3D. Flower at lower right in (A & A') enlarged and rotated 900. Radioactivity is light against dark

background in this photograph only.

703

www.plantphysiol.orgon October 15, 2017 - Published by Downloaded from Copyright © 1961 American Society of Plant Biologists. All rights reserved.

Page 7: METABOLISM - pdfs.semanticscholar.org · absorption, distribution, & metabolism of2,2-dichloropropionic acid in relationtophytotoxicity. ii. distribution & metabolic fate of dalapon

PLANT PHYSIOLOGY

activity was obtained. Upon separation of the ether-soluble portion between 95 %, ethanol and petroleumether, activity was about equally distributed in thetwo fractions. This fact points to the association ofsmall amounts of C14 with both lipids and pigments.B: Spot(s) other than dalapon, albeit faint, wereproduced by the neutral fraction. C: The cationicfraction showed slight activity. D: A small amountof C14 (approximately 1 % of the total recovered)was retained by the plant residue, (lespite exhaustiveextraction. This agrees with earlier observations fol-lowing 6- to 8-day exposures of cotton and sorghumto dalapon-2-C14 and -C131.

Due to the very low radioactivity in the plantresidues, enzymatic digestion w-ith amylases and pro-teinases proved unfruitful.

Although not presented in detail, the followingobservations on monocotvledons should be includedin this discussion. Preceding experiments haveshown conclusively that dalapon, non-metabolized forthe most part, is accumulated in the seeds as wellas other fruit parts of cotton, and the capacity to re-tard germination may persist througlh years of storage(6, 7). No growth regulator symptoms were pro-duced that were definitely ascribable to dalapon. Itis also evident (from other studies) that the char-acteristic dalapon symptoms, i.e., the active stimulus,may persist over long periods in dormant or quiescentorgans of grasses. Examples are A: rhizome budsof Johnson grass (10), B: seeds of wild oat (1),and C: wheat and barley (to be described). Inthese instances, it was not established whether thedelayed or protracted responses are due to dalaponitself or to a biologically active transformation prod-uct. In the absence of such information. the factthat typical symptoms develop in grasses and not incotton, could lead to interesting speculation (seediscussion). The actual time interval over whichdalapon (or the stimulus) persists is probably not toosignificant per se, since it may represent a periodin which the tissues being sampled are in a relatively

inactive or dormant state. Furthermore, measuringeffects of persistence in vegetative tissues may becomplicated by the reproductive phase (as shown incotton) because fruiting results in reduced vegetativegrowth. With a curtailment of activity in vegetativemeristems and a concurrent shift in loci of high me-tabolism to the reproductive structures, dalapon wouldmove into these organs. If, after having accumulateda high concentration of dalapon, the seeds (or buds)mature or become dormant and all metabolic activityis drastically reduced it is logical that the chemicalwould not be dissipated rapidly, but would be presentwhen growth and more active metabolism were re-sumed. The following carry-over of the dalaponstimulus through the seeds is believed to illustratethis phenomenon. At rates as low as 4 lb/acre, ap-plie(d as a pre-plant treatment in the winter (underconditions unfavorable either for leaching or break-down by micro-organisms), wheat and barley wereseverely inhibited as a result of absorption of dalaponthrough the roots (see Acknowledlgments). Figure-4 shows carry-over of the stimulus through the seedsinto the second and third generations of wheat.Notice that the spikes produced on the first culniswere most severely affected in the second generation,thus indicating that re-translocation (from 1st gen-eration seed) and accumulation in the second genera-tion was on a first come, first served basis. Theyoung developing grains that had first access to there-translocated dalapon (or stimulus) accumulatedthe greatest portion, thereby leaving only smallamounts to be translocated into succeeding spikes.Abnormalities also developed in the third generationseedlings grown from spikes of plants which showedmost severe symptoms in the second generation.Some seedlings were simply retarded in growth tovarying degrees but otherwise appeared normal;others grew normal coleoptiles but produced no roots;still others produced long roots but no top growth.Although it has been experimentally proven only incotton, the carry-over effect through the seeds of both

FIG. 4. Carry-over effect of characteristic dalapon symptoms in A (left) second generation, and B (right) thirdgeneration wheat following inhibition of first generation plants by pre-planting application of dalapon at 4 lb/acre.Groups of heads in A are arranged left to right in order of occurrence on the same plant. In B the seedling atleft is normal and others show inhibition or other anomalies.

704

www.plantphysiol.orgon October 15, 2017 - Published by Downloaded from Copyright © 1961 American Society of Plant Biologists. All rights reserved.

Page 8: METABOLISM - pdfs.semanticscholar.org · absorption, distribution, & metabolism of2,2-dichloropropionic acid in relationtophytotoxicity. ii. distribution & metabolic fate of dalapon

FOY-2,2-DICHLOROPROPIONIC ACID & PHYTOTOXICITY

dicotyledons and grasses is presumed to be due, prin-cipally, to non-metabolized dalapon. This statementshould be experimentally confirmed or refuted withrespect to seeds and dormant or quiescent buds ofgrasses as well.

MECHANISMS OF DALAPON ToXICITY: This sec-tion is aimed primarily toward integrating the ex-perimental findings (including those of otherworkers) into a discussion of selective dalapon phy-totoxicity.

I. Non-selective versus selectize toxicity. Dala-pon is capable of producing both an acute (contact)burn and delayed systemic growth regulatory re-sponses following translocation. The relative degreeof expression of each type of injury is apparently de-pendent upon the concentration of dalapon in thecells penetrated and the inherent biochemical suscepti-bility to the growth regulating action of dalapon atlower concentrations. Wilkinson (32) observed thatas the rate, concentration, or time of exposure (eachindicative of levels of dalapon in the tissues) is in-creased the sequence of dalapon responses in plantsis successively formative-toxic-lethal. Lower con-centrations are thus required to produce formativeeffects than to cause the death of a single cell, tissue,or plant. The two effects intergrade but are separate-ly recognizable at the extreme levels of each. Severalphysico-chemical properties of dalapon have been re-viewed in relation to their probable contribution to-ward phytotoxicity (6). The acute toxic effect isbelieved to be due to the action of the herbicide asa strong acid and as a protein precipitant (21. 23).Membrane destruction undoubtedly accounts for thewater-soaked appearance of tissues following leak-age of the cellular contents, in a manner suggestiveof oil toxicity. High concentrations of H ions ortoxic surfactants may also contribute to acute toxicity(6). In cotton, acute toxicity has occurred in re-gions remote to the site of foliar application as wellas following root uptake. Presumably the mechanismis the same, i.e., transport of amounts non-toxic tothe tissues traversed, followed by concentration totoxic levels in certain other tissues. On cursory ex-amination, the end result of this non-selective effect(e.g. cupping of leaves, death of terminal buds, andmalformation of auxillary buds) may be misconstruedto be the same growth regulating action seen in sus-ceptible species. Irregular growth patterns resulted,however, from necrosis in certain areas (e.g., sec-tions of the main vascular tissues) and stresses result-ing from continued growth in adjacent areas (smallerveins and intercoastal regions). The veins werenormal near the junction of the petiole and lamina;therefore substances were still able to traverse theaffected region. Although the effects described arenoticeably different from the proliferation of tissues,etc., seen in grasses, it should be mentioned againthat selectivity of action is most frequently not ab-solute, but relative.

II. Selective herbicidal action of dalapon. Thesecond response, apparently resulting from lower con-

centrations (in meristematic tissues only), appears toexemplify true biochemical selectivity among species.Acute toxicity can reduce or prevent the expressionof the systemic growth regulator type of herbicidaleffect by obstruction of the translocation process; thisresponse is dependent upon translocation into regionsof presently or potentially high metabolic activity.

Initially (6, 8), four factors were considered cap-able of determining or quantitatively regulating selec-tive herbicidal action among species: A: permeability,including stomatal and cuticular absorption and pene-tration of membranes; B: translocation, includingrestriction enroute, and distributional and accumula-tional patterns; C: metabolic decomposition or inac-tivation of the herbicide; D: selective biochemicalinterference of the toxicant with normal metabolicprocesses.

A. Permeability. Initial absorption into leaveshas been discussed in some detail (6, 8). In thesestudies no unexplained differences were noticed in thepenetrability of susceptible and tolerant species byfoliar or root applications of dalapon. [Pre-emer-gence selectivity, however, may be partially explainedby differences in permeability of seed coats and thepenetrating abilities of various formulations ofherbicides (20).]

B. Translocation. The principal difference de-tected between tolerant and susceptible species was inthe degree of retention of dalapon along the trans-port route. Several workers have attached signifi-cance to the apparent restriction of 2,4-D in grassleaves in determining species selectivity (see litera-ture cited in 6, 8). Critical work by Weintraub et al.(30) has shown consistently higher amounts of C14(labeled 2,4-D) exported out of treated leaves ofsusceptible plants than tolerant plants. It is reasonedthat since the exported 2,4-D brings about the mor-phological responses on the basis of which plants arejudged resistant or susceptible, insufficient amountsmay be exported out of the treated leaves of resistantplants to produce a systemic toxic action. Wilkinson(32), on the other hand, attempted to use the samereasoning, in reverse, to explain the increased toxicityof dalapon to grasses following root absorption, bytrapping dalapon or hindering its movement out ofthe leaves. In the present investigation (6, 8) it wasquantitatively demonstrated that the translocation ofdalapon (a grass-selective herbicide) is restricted inthe basal sections of (particularly immature) grassleaves in much the same manner as 2,4-D. In at-tempting to reconcile these views it must be recog-nized that the secondary actions of both 2,4-D anddalapon are expressed in the same metabolically ac-tive tissues, and it is still the herbicide exported outof the mature leaves, principally, which is respon-sible for systemic herbicidal action. Retention ofdalapon in treated leaves would seem to be particular-ly disadvantageous (from the standpoint of obtainingrapid systemic action) in the case of perennialgrasses, where transport not only to the shoot apicalmeristem of the treated plant, but into the rhizome

705

www.plantphysiol.orgon October 15, 2017 - Published by Downloaded from Copyright © 1961 American Society of Plant Biologists. All rights reserved.

Page 9: METABOLISM - pdfs.semanticscholar.org · absorption, distribution, & metabolism of2,2-dichloropropionic acid in relationtophytotoxicity. ii. distribution & metabolic fate of dalapon

PLANT PHYSIOLOGY

buds, is desired. That herbicidal effectiveness is re-duced by disking, mowing, and burning [reviewed in(6)], all of which remove top growth, suggests theslow buildup of dalapon in the rhizomes. Separationfrom the mother plant too soon by either of thesemeans removes not only the apical dominance effect,but also a large portion of the dalapon which hadpenetrated but not yet reached the rhizome buds intoxic concentrations. Accumulation occurs in as-sociation with the transport of food materials intoregions of high metabolic activity. Penetration andtranslocation can also be greatly protracted, providingno acute toxicity occurs (8). Accumulation does notoccur appreciably in (lormant or quiescent organs(mature seeds, dormant buds, etc)., but dalapon isfound there after it has moved in during periods ofactive assimilation. Therefore the treated tops, ifallowed to remain undisturbed, still are able to exertan apical dominance effect and continue to serve asa reservoir for the release of additional dalapon,should high metabolic activity begin or be resumed.This is in agreement with the observation (2) that acontinued exposure to TCA was essential for con-tinued dormancy and probably for subsequent death.The process described probably accounts for thesuperiority of deep plowing in the field control ofperennial grasses. Because of its spectacular nature,other factors may be involved in the case of recoveryfrom dalapon-induced dormancy after burning. Re-striction of movement in grass leaves is not likelya blockage of herbicide transport, peculiarly, but anormal consequence of the compound's movement intothese regions of high metabolic activity (assimilateutilization) with the flow of food materials and reten-tion therein. Thus any of a number of solutes wouldbe expected to follow a similar pattern, to a greateror lesser degree, depending upon their adsorptiveproperties, solubilities, tendency toward metabolic de-composition, etc.

C. Metabolic decomposition or inactivation ofdalapon. Dalapon was not rapidly metabolized ineither tolerant or susceptible plants; therefore thispossibility would not seem of much importance indetermining selectivity. In relation to the smallamount of breakdown which does apparently occur,however, several points may be considered. 1: Someradioactive species, freely re-translocatable in thexylem, were discovered in the guttate from hyda-thodes of sorghum following leaf drop treatment withdalapon-C136, but not with dalapon-2-C14. 2: Cl36 isreadily removed metabolically, at least by micro-or-ganisnis. 3: There was a slight indication of thepresence of inorganic Cl36 on chromotograms of ex-tracts from higher plants 6 to 8 days after treatment.4: All of these studies employed low tracer levels ofdalapon, i.e., perhaps subtle metabolic conversionswould occur more noticeably at higher levels. 5:"Propionate inhibits growth by competing with ,8-alanine for attachment within the yeast cell, therebypreventing the coupling of the pantothenic acidmoieties" (15). 6: Pantothenic acid synthesis is one

of the most sensitive metabolic processes known to beaffected by dalapon and related compounds (12).The ability of the molecule (propionic acid) to com-pete with /8-alanine is decreased by successive in-creases in chlorination. Conversely, the toxicity ofdalapon increased (i.e., approached that of propionicacid) through aging on the shelf for 3 years (presum-ably undergoing decomposition). (The increase ininhibitory action upon aging is suggestive of the be-havior of oils which form organic acids that arehighly toxic in the non-dissociated state.)

From the foregoing observations, it is postulatedthat the delayed growth-regulatory effect manifestedafter translocation into meristematic tissues of highmetabolic activity, may be correlated with the initialsteps in decomposition (i.e., de-halogenation) of thedalapon molecule. Thus, instead of constituting adetoxification mechanism, the first ( ?) steps ofdalapon breakdown would tend to increase its poten-tialities as a competitive inhibitor in pantothenic acidsynthesis. After disturbance of the normal function-ing of pantothenic acid and coenzyme A, it mightthen be further metabolized by the common or per-haps by a modified pathway of propionate oxidation.If dalapon-2-C14 were thus degraded, radioactivitymight logically appear in almost any fraction of com-pounds where carbon would normally go. Thisprobably occurred for a small percentage of the ap-plied dalapon after long exposures (9-10 weeks) incotton. Further investigation is required to establish,with certainty, whether this hypothesis has real sig-nificance in relation to either mechanisms of actionor selectivity. That such differences are not obviousfrom herbicidal responses after foliar applications ofpropionate, mono-, di-, and trichloropropionate, doesnot detract from the credibility of the hypothesis.Wide differences in penetration and translocation tothe cellular site of action may exist within the seriesof compounds. Species resistant to TCA commonlycontain higher amounts of the herbicide in their cellsap than susceptible species (2, 3, 28) indicating thatthe latter use TCA in their metabolism. That growthreduction and morphological responses are correlatedwith metabolism of TCA in the tissues seems perti-nent to the present discussion.

D. Biochemical interference with normal meta-bolic processes. Disturbance of energy metabolismcould logically lead to the observed herbicidal effectsof growth regulators. All anatomical and morpho-logical changes in plants are preceded by biochemicalchanges. As inferred earlier, no clear cut relation-ship exists between formative effects and lethality.Attempts to explain mechanisms of action of a sub-stance which produces formative effects must takecognizance of the influence of such compounds uponthe normal processes of growth and differentiation,which are themselves incompletely understood. Thespecific point of attack of a toxicant might be on theproduction of a substrate, on the enzymes which at-tack a substrate with the release of energy, and/oron the enzymes involved in the utilization of the

706

www.plantphysiol.orgon October 15, 2017 - Published by Downloaded from Copyright © 1961 American Society of Plant Biologists. All rights reserved.

Page 10: METABOLISM - pdfs.semanticscholar.org · absorption, distribution, & metabolism of2,2-dichloropropionic acid in relationtophytotoxicity. ii. distribution & metabolic fate of dalapon

FOY-2,2-DICHLOROPROPIONIC ACID & PHYTOTOXICITY

energy produced. The final toxic result, however,may actually be produced by a complex series of se-quential and consequential reactions. This may ac-count for the fact that the mechanisms(s) of actionis (are) known for so few compounds, if indeed anyare known with certainty.

The most plausible explanation of the mode ofaction of dalapon as a growth regulator seems to re-volve around pyruvate metabolism, which occupies akey position in relation to other metabolic crossroads.Both competitive and non-competitive inhibition ofenzymatic reactions have been attributed to dalapon,and Redemann and Meikle (24) concluded that theymay occur simultaneously. The first was thought toresult from competition between pyruvate and dalaponfor attachment to pyruvate-attacking enxymes, where-as the second (which became noticeable at concentra-tions of inhibitor of 3.5 X 10-4 molar) is probablydue to its action as a protein precipitant, perhaps pre-cipitating the pyruvate-enzyme complex. 'Based oncalculations using expected pyruvate concentrationsin plant tissues, these workers concluded that if suchan inhibition is not responsible for the herbicidal ac-tion of dalapon. it certainly could contribute to thestunting effect following translocation.

Perhaps an equally (or more) important primarysite of action, still involving pyruvate indirectly, isthe competitive inhibition of pantothenic acid svn-thesis (12). First indications were that dalaponcompeted with ,l-alanine, but later results with pureenzyme preparations have suggested that competitionis with pantoic acid rather than ,&-alanine. In eithercase, the synthesis of pantothenic acid, and thereforethe production of functional coenzyme A would beimpaired. Most all the pantothenic acid in animalsand micro-organisms is reportedly present as CoA, al-though it is also found in nature in other combinedforms such as pantetheine and pantethine. Less isknown of its activity and occurrence in higher plants,however several edible reproductive structures areknown sources of the vitamin. It is probably syn-thesized in the leaves and transported into these stor-age organs during periods of rapid development. Thespecific effects of pantothenic acid or CoA upon celldifferentiation are little known.

The improper functioning of CoA in plants couldlead to drastic changes in plant growth and develop-ment. For example, some of the important processesbelieved to be mediated by CoA are A: pyruvateoxidation, B: citrate synthesis, C: a-keto-glutarateoxidation in the citric acid cycle, D: fatty acid andsteroid synthesis and breakdown, and perhaps E: auxinaction-auxins may act through an ester with CoA(auxinyl CoA). Hence CoA is a key compound inplant metabolism and growth through its control ofthe energy in the linking ester bond. Interference ofdalapon with the citric acid cycle in any way, e.g., bycompeting with pyruvate for enzyme attachment orwith f3-alanine for attachment to the other moiety ofpantothenic acid could indirectly cause disturbancesin ancillary processes, such as nitrogen metabolism.

(The dark green appearance of dalapon-treatedplants, delayed maturation, and prolongation of vege-tative growth are characteristic of plants having highlevels of available nitrogen.)

It is entirely probable that dalapon exhibits morethan one primary site of action. Note that ,8-alanine yields pyruvic acid on deamination. It ispossible that dalapon and related compounds (per-haps increasingly with decreased chlorination) areable to compete with several metabolites that arestructurally similar, e.g., 8-alanine, pantoic acid,pyruvic acid-perhaps variably among plant species.

The low response in roots cannot always be at-tributed to a lack of accumulation of dalapon. as wasshown in this study. It seems unlikely that the prin-cipal action is in competition with pyruvate, becausethis substance certainly occurs, albeit fleetingly, in allregions of high respiratory activity which would in-clude root tips as well as shoot apical meristems. Ifthe principal mechanism is the interference withpantothetic acid synthesis, one possible explanationis that synthesis occurs in the shoots, requiring theproducts of photosynthesis, and if the process shouldbe altered the meristematic areas of the shoots, (byvirtue of their closer proximity) would show thedeficiency most readily. Abnormalities can occur inroots under certain conditions, as shown by the workof Grigsby et al. (11). This suggests the indirect in-volvement of light, which is consistent with other ob-servations. Also, it seems safe to assume that light(and elevated temperatures) may indirectly exert aneffect by causing an increase in accumulation ofdalapon in the tops from soil or nutrient solutionthrough an increase in transpiration.

Differences in susceptibility to dalapon amongspecies and among tissues of a given plant are seem-ingly dependent upon the presence or absence of keyenzymes or enzyme precursors. Further experi-mentation into the mechanisms of herbicidal selectivity should emphasize biochemical distinctions betweensusceptible and resistant species.

SUMMARY & CONCLUSIONSI. Dalapon-2-C'4 and _C136 were further em-

ployed in tracer and metabolic studies on cotton,sorghum, and wheat to determine the distributionaland metabolic fate of the herbicide in relation to phy-totoxicity. Radioautography and counting yieldedboth qualitative and quantitative data. Extraction,fractionation, and paper chromatographic procedureswere used to detect dalapon and metabolic degrada-tion products.

II. Pure dalapon-CI3fi in aqueous stock solutionwas metabolized over a period of several months inthe refrigerator by some species of micro-organ-ism(s), possibly Alternaria sp., which produced fivenew C136-labeled substances. Two of the substanceswere tentatively identified as inorganic Cl36 andmonochloropropionate-C136. Similar conversions areprobably operative in soils, accounting for the disap-

707

www.plantphysiol.orgon October 15, 2017 - Published by Downloaded from Copyright © 1961 American Society of Plant Biologists. All rights reserved.

Page 11: METABOLISM - pdfs.semanticscholar.org · absorption, distribution, & metabolism of2,2-dichloropropionic acid in relationtophytotoxicity. ii. distribution & metabolic fate of dalapon

PLANT PHYSIOLOGY

pearance of dalapon toxicity under favorable condi-tions.

III. Ready absorption and translocation ofdalapon, including redistribution and accumulation(e.g., in vegetative meristems, flowers, fruits, &seeds) in response to shifts in loci of high metabolicactivity were confirmed.

IV. Dalapon was absorbed, translocated, re-dis-tributed, and accumulated in higher plants, principal-ly as the intact molecule or dissociable salt thereof,and remained essentially non-metabolized for longperio(ls especially in dormant or quiescent tissues.

V. Seven days after foliar treatment of cottonand sorghum, for example., dalapon, and no otherprominent radioactive substance, was recoverablewith water or ethanol from all plant parts and fromthe nutrient solutions in which the plants were grown.

VI. Ten weeks after treatment (through severedpetioles) approximately 85 to 90 % was recoverableas dalapon from cotton fruits of various ages. Theremainder of the radioactivity was associated with theether-soluble portion, the neutral and cationic frac-tions of an ethanol extract, and the insoluble plantresidue.

VII. Some slow metabolic decomposition resultedin the release of C136 (from dalapon-C136) or the in-corporation of C'4 (from dalapon-2-C'4) into othercompounds. Eventual breakdown possibly involvesan initial dehalogenation followed by normal ormodified propionate oxidation.

VIII. Dalapon stimulus was traced to the thirdgeneration in wheat; it was transmitted in the seedsafter exposure of first generation seedlings to pre-plant applications of dalapon (4 lb/acre) in the field.

IX. Two physiologically distinct types of action(acute toxicity and delayed growth regulatory re-sponses) were confirmed. The former is believedto be due to the action of the herbicide as a fairlystrong acid and protein precipitant, which non-selec-tively causes disruption of the plasma membranes anddestruction of cellular constituents. The second re-sponse resulting from lower concentrations in meri-stematic tissues exemplifies true biochemical selec-tivity among species.

X. Experiments indicated that neither pene-trability, translocatability, nor metabolic inactivationof dalapon is preeminently responsible for herbicidalspecificity. The key to the moderately selectivegrowth regulating activity of dalapon still seems un-questionably to reside in the protoplasm. Results arenot at variance with hypotheses that dalapon inhibitspantothenic acid synthesis and disturbs coenzyme Aand pyruvate metabolism.

ACKNOWLEDGMENTS

Encouragement and guidance were provided byvarious members of the botany dlepartment staff, Uni-versity of California at Davis. Recognition is al-odue the Dow Chemical Co. for gratuitously supplyingthe radioactive chemicals, and for offering continuedinterest and cooperation. Finally, the assistance ofMessrs. Luther Jones and C. A. Suneson in supplyingexperimental samples of seed from dalapon-affectedwheat is gratefully acknowledge(l.

LITERATURE CITED1. ANDERSEN. R. N. & E. A. HELCESON. 1954. Some

effects of post-emergence applications of dalaponto wild oats. Weeds 3(4) : 351-358.

2. BARRONS, K. C. & R. W. HIJMMER. 1951. Basicherbicidal studies with derivatives of TCA. Agric.Chem. 6(6): 48-50.

3. BLANCHARD, F. A. 1954. Uptake, distribution, &metabolism of carbon 14-labeled trichloroacetate incorn & pea plants. Weeds 3(3): 274-278.

4. CLOR, M. A. & A. S. CRAFTS. 1957. Comparativetranslocation of C'4-labeled 2,4-D, amino triazole,& urea in cotton plants & subsequent leakage fromroots. Plant Physiol. suppl 32: xliii.

5. CURTIS, 0. F. & L. M. CLARK. 1950. An Intro-duction to Plant Physiology. McGraw-Hill Co.,New York City. 752 pp.

6. Foy, C. L. 1958. Studies on the absorption, dis-tribution, & metabolism of 2,2-dichloropropionicacid in relation to phytotoxi-city. Ph.D. thesis,University of California, Davis.

7. Foy, C. L. 1959. The accumulation & persistenceof dalapon in the seeds of cotton & small grains.Res. Prog. Report. West. Weed Control Conf.Pp. 83-84.

8. Foy, C. L. 1960. Absorption, distribution, & metab-olism of 2,2-diclhloropropionic acid in relation tophytotoxicity. I. Penetration & translocation ofC36- and C14-labeled dalapon. Plant Physiol. 36:688-697.

9. Foy, C. L. 1960. The adaptation of qualitative &quantitative techniques for determination of radio-active dalapon in plant tissues. Hilgardia 30(5)153-173.

10. Foy, C. L. & J. H. MILLER. 1955. Chemical con-trol of Johnson grass. Proc. South Weed Conf.9: 197-203.

11. GRIGSBY, B. M., T. M. Tsou, & G. B. WILSON. 1955.Some effects of herbicides on growth & cytologicalprocesses. Proc. South. Weed Conf. 8: 279- 283.

12. HILTON, J. L., L. L. JANSEN, & W. A. GENT-NER.1958. Beta alanine protection of yeast growthagainst the inhibitory action of several chlorinatedaliphatic acid herbicides. Plant Physiol. 33(1):43-45.

13. HOLSTUN, J. T., JR. & W. E. LooMIs. 1956. L-each-ing & decomposition of 2,2-dichloropropionic acidin several Iowa soils. Weeds 5 (3) : 205-217.

14. JACKSON, W. A. 1957. Carbon dioxide fixation byplant roots & its influence on cation absorption.N. C. State Coll. Pub. No. 21895, Diss. Abstracts17(8): 1650.

708

www.plantphysiol.orgon October 15, 2017 - Published by Downloaded from Copyright © 1961 American Society of Plant Biologists. All rights reserved.

Page 12: METABOLISM - pdfs.semanticscholar.org · absorption, distribution, & metabolism of2,2-dichloropropionic acid in relationtophytotoxicity. ii. distribution & metabolic fate of dalapon

FOY-E2,2-DICHLOROPROPIONIC ACID & PHYTOTOXICITY

15. KING, T. E. & V. H. CHELDELIN. 1948. Panto-thenic acid studies. IV. Propionic acid & $-alanine utilization. J. Biol. Chem. 174: 273-279.

16. LINDER, P. J., J. C. CRAIG, JR., & T. R. WALTON.1957. Movement of C' 4-tagged alphamethoxy-phenylacetic acid out of roots. Plant Physiol.32(6) : 572-575.

17. MCILRATH, N. J. & D. R. ERGLE. 1951. Persistenceof 2,4-D stimulus in cotton plants with referenceto its transmission to the seed. Botan. Gaz. 112:511-518.

18. McILRATH, N. J. & D. R. ERGLE. 1953. Furtherevidence of persistence of the 2,4-D stimulus incotton. Plant Physiol. 28: 693-702.

19. MILLER, J. H., J. A. WILKERSON, & V. T. WALHOOD.1958. Carryover response of 2,4-dichlorophenoxy-acetic acid through seed of cotton. Proc. South.Weed Conf. 11: 72-73.

20. MUNAKATA, K., K. YOKAYAMA, T. SHIBATA. A.HARADA, & F. HARA. 1959. Herbicidal activitiesof halogenoalkylcarboxylic acid esters. I. Germi-nation inhibition activities. Weeds 7(4): 470-473.

21. OLSSON, E. A., JR. 1957. Selective herbicidal ac-tivity of 2,2-dichloropropionic acid. M.S. Thesis.Colorado State University, Fort Collins.

22. PRIDHAM, A. M. S. 1947. Effect of 2,4-D on beanprogeny seedlings. Science 105: 412.

23. REDEMANN, C. T. & J. HAMAKER. 1954. Dalapon(2,2-dichloropropionic acid) as a protein precipi-tant. Weeds 3 (4): 387-388.

24. REDEMANN, C. T. & R. W. MEIKLE. 1955. Theinhibition of several enzyme systems by 2,2-dichloro-propionate. Arch. Biochem Biophys. 59(1): 106-112.

25. SMITH, G. N. 1958. Counting of radioactive dala-pon in biological systems. Down to Earth 14(2):3-6, 12.

26. SWEET, D. V. 1956. Histological & nutrient lossesfrom plant foliage under mist propagation. Mich.State Univ. Pub. No. 20,087. Diss. Abstr. 17(6):1182.

27. THIEGS, B. J. 1955. The stability of dalapon insoils. Down to Earth 11 (2): 2-4.

28. TIBBETS, T. W. & L. G. HOLM. 1954. Accumulation& distribution of TCA in plant tissues. Weeds3(2): 146151.

29. WEINTRAUB, R. L., J. W. BROWN, M. FIELDS, &J. ROHAN. 1952. Metabolism of 2,4-dichloro-phenoxyacetic acid. I. C1402 production by beanplants treated with labeled 2,4-dichlorophenoxy-acetic acids. Plant Physiol. 27: 293-301.

30. WEINTRAUB, R. L., J. H. REINHART, & R. A.SCHERFF. 1956. Role of entry, translocation, &metabolism in specificity of 2,4-D & related com-pounds. U.S. Atomic Energy Comm. TID 7512,Prof. Conf. Radioisotopes Agric. 203-208.

31. WEINTRAUB, R. L., J. H. REINHART, R. A. SCHERFF,& L. C. SCHISLER. 1954. Metabolism of 2,4-dichlorophenoxyacetic acid. III. Metabolism &persistence in dormant plant tissues. PlantPhysiol. 29: 303-304.

32. WILKINSON, R. E. 1955. The physiological ac-tivity of 2,2-dichloropropionic acid. Ph.D. thesis,University of California, Davis.

33. WOODFORD, E. K., K. HOLLY, & C. C. MCCREADY.1958. Herbicides Ann. Rev. Plant Physiol. 9:311-358.

709

www.plantphysiol.orgon October 15, 2017 - Published by Downloaded from Copyright © 1961 American Society of Plant Biologists. All rights reserved.