Some Factors Influencing Activity of 12 PhenoxyAcids on Mesquite Root Inhibition 1 2Richard Behrens3 & Howard L. Morton
Department of Plant Sciences, Texas Agricultural Experiment Station, College Station & Crops Research Division,Agricultural Research Service, U.S. Department of Agriculture
The biological activity of phenoxy acids is con-sidered greatest with 4-chloro, 2,4-dichloro, 2,4,5-trichloro, and 2-methyl-4-chloro ring substitutions(14. 26). Phenoxy acids with an even number ofcarbon atoms in the side chain attached directly tothe oxygen are generally more active than those withan odd number due to degradation of the side chainby beta oxidation (11, 20,26). Lowering pH of thesolution in which they are applied increases the bio-logical activity of given concentrations of phenoxyacids. This effect has been attributed to an increas-ed concentration of undissociated molecules in the ex-ternal medium (2, 5, 7, 10), a change in intracellularpH (2, 5), an effect on membrane permeability (6,22), or a competition between phenoxv ancl buffermolecules (28).
Objectives of this study were A, to compare therelative activity of 12 phenoxy acids as indicated bygrowth inhibition of the roots of mesquite seedlingsfollowing applications to the root-bathing medium orto the cotyledons, and B, to examine some relationsof structure, solubility, dissociation and pH to theinhibitory activity of these compounds.
Materials & MethodsThe nmethods used were the same as those de-
scribed in a previous paper (4). Followving germi-nation in petri dishes, mesquite [Prosopis juliflora(Swartz) var. glandulosa (Torr.) Cockr.] seedlingswere suspended in 250-ml jars containing a tap waterroot-bathing medium and exposed to incan(lescentlight at 1,000 to 1,500 ft-c for 16 to 24 hours beforetreatment.
When solution treatments were used, the lengthof individual roots was determined just prior to place-ment in tap water containing 0.00, 0.12, 0.50, or 2.00ppm of the test compounds. Buffering with 0.0065 -mphosphate aided in maintaining the solutions at pH6, 7 or 8. The bathing solutions were replaced byequivalent solutions after 24 hours to minimize pHdrift. Root lengths were remeasured after 48 hours.Growth in length, expressed as percentage of growth
periment Station.3 Present address: Department of Agronomy & Plant
Genetics, University of Minnesota, St. Paul 1.
of check, was used as an indication of inhibitory ac-tivitv. Three replicates of 12 seedlings each wereused in a completely randomized design.
WN hen cotyledonary applications were used, theprocedure was much the same. After the initial root-length measurement, the test compound was appliedto one cotyledon of each seedling at rates of 0.00,0.10, 0.41, or 1.65 Ag in a 2.05-IA drop of 75 %ethanol. In a single test, 0.00, 0.08, or 0.33 /Ag wereapplied in a 60 % acetone-40 % nontoxic oil (Sova-spray 100, a saturated, straight-chain, paraffin oil,supplied by the Socony-Vacuum Oil Co.) carrier.The second root-length measurement was made after24 hours. The root growth, expressed as percentageof growth of check, was used as a measure of in-hibitory activity. Six replicates of 12 seedlings eachwere included when cotyledonary applications wereused.
Ring substitutions, 4-chloro, 2,4-dichloro, 2,4,5-trichloro, and 2-methyl-4-chloro, were studied be-cause many phenoxy compounds with these substitu-tions are known to be highly active growth regula-tors. Phenoxy acids with acetic, 2-propionic and 4-butyric acid side chains were studied for the samereason. All 12 combinations of ring substitutionsand side chains were included in the experiments4.Compounds except 2,4,5-T were obtained from themanufacturer in chemically pure form; 2,4,5-T was
4 Abbreviations and sources of compounds used: 2-methyl-4-chlorophenoxyacetic acid. MCPA (Source:Mathieson Chemical Corp., Baltimore, Md.). 2-(2-methyl-4-chlorophenoxy) propionic acid. 2- (MCPP) (Source:Dow Chemical Co., Midland, Mich.). 4- (2-methyl-4-chlorophenoxy) butyric acid. 4- (MCPB) (Source: May& Baker Ltd., Daggenham, England). 4-chlorophenoxy-acetic acid. 4-CPA (Source: Eastman Organic Chemi-cals, Rochester 3, N.Y.). 2- (4-chlorophenoxy) propionicacid. 2- (4-CPP) (Source: Dow Chemical Co., Mid-land, Mich.). 4- (4-chlorophenoxy) butyric acid. 4- (4-CPB) (Source: May & Baker Ltd., Daggenham, Eng-land). 2,4-dichlorophenoxyacetic acid. 2,4-D (Source:Eastman Organic Chemicals, Rochester 3, N.Y.). 2-(2,4-dichlorophenoxy) p r o p i o n i c a c i d. 2- (2,4-DP)(Source: Dow Chemical Co., Midland, Mich.). 4- (2,4-dichlorophenoxy) butyric acid. 4- (2,4-DB ) (Source:May & Baker Ltd., Daggenham, England). 2,4,5-trichlorophenoxyacetic acid. 2,4,5-T (Source: DowChemical Co., Midland, Mich.). 2- (2,4,5-trichloro-phenoxy) propionic acid. 2- (2,4,5-TP) (Source: DowChemical Co., Midland, Mich.). 4-(2,4,5-trichlorophen-oxy) butyric acid. 4- (2,4,5-TB) (Source: May &Baker Ltd., Daggenham, England).
165 www.plantphysiol.orgon July 10, 2020 - Published by Downloaded from
purified by recrystallization of the technical gradeproduct from 30 % ethanol.
Dissociation constants (pK's) of the 12 phenoxyacids were determined at concentrations of 500 ppmin water at 60 C. This temperature was necessaryto achieve a 500 ppm concentration of the less solublecompounds. A Beckman, model H-2, pH meter wasused to make the necessary pH measurements. Itwas standardized with 0.05-molar potassium phthalatebuffer taken as pH 4.10 at 60 C. The pH value ofthe buffer at this temperature was calculated accord-ing to the procedures outlined by Dole (9).
Water solubilities of the 12 phenoxy acidls weredeterminedl by adding an excess to distilled Nater andholding at 50 C for 24 hours. Then, after standingfor 48 hours at 25 C the undissolved residue was re-moved by filtration. The amount of a compound re-maining in solution was determined by titration to aphenolphthalein end point with sodium hydroxide.
Results & DiscussionCompounds with the 2-methyl-4-chloro ring sub-
stitution were most effective in reducing root elonga-tion when added to the root-bathing medium follow-ed in order of decreasing activity by the 4-chloro-,2,4-dichloro-and 2,4,5-trichlorophenoxy aci(ds (tableI). Deviations froml this pattern were noted in the
approximately equal activity of the 4-chloro-and 2,4-dichloro substitutions in the acetic acid series and thelow activity of 2-(2,4-DP).
If the side chain effect on inhibitory activity isconsidlered, compounds with the 2-propionic sidechain wvere the least active when added to the root-bathing medium. This might be expected since the2-phenoxypropionic acids are stereoisomers and avail-able infornmation (1. 19, 21, 27) indicates that onlyone isomer has high biological activity. The activeisomers of 2-(2,4,5-TP) and 2-(2,4-DP) have ap-proximiiately twice the biological activity of the racem-ic comlpouni(ls (3. 21). By analogy it is assumed thata simlilar relationship exists for the remaining 2-propionic acids. 2- (fACPP) and(l 2- (4-CPP'). If thisis true, the 2-phenoxypropionic aci(ls consi(lerilng theactive isomler concentration olly, would have ap-proximiately twice the activity listedl in table I an(lthe phenoxyacetic acidls must be consi(lered less activethan the active isomiiers of the 2-phenoxypropionicaci(ls.
Comiipounids with the 4-butyric aci(l side chalin ex-cept 4-(2.4.5-TB) iniduce(d the higlhest levels of rootgrowth inlhibition (table I). The concenitration ofthe active 4-phenoxybutyric aci(ls in the root-bathingmedium necessary to incduce 70 cl growth inhibitioinaverage(l less than 0.23 ppm versus 0.70 plpil for the
Table IEffect of pH on Concentrations of 12 Phenoxy Acids Necessary to Ilnduce 70 % Inhibition
of Mesquite Root Growth When Added to the Root-Bathing Medium
Acid Side ChainRing substitution
2-Methyl-4-chloro
pH
678
Mean
4-Chloro 678
Mean
2,4-Dichloro 678
Mean
2,4,5-Trichloro
Mean
678
Aceticppm
0.230.350.40
0.33
0.430.530.550.50
0.490.450.43
0.46
0.471.722.30
1.50
2-Propionicppm
0.300.500.55
0.45
0.450.751.250.82
>2.00>2.00> 2.00
>2.00
0.501.641.93
1.36
4-Butyricppm
<0.120.120.30
<0.18
<0.120.200.35
<0.22
0.170.300.40
0.29
>2.00>2.00>2.00
>2.00
Ring substitution meanpplml
< 0.32
<0.51
>0.91
> 1.62
Side chain mean 0.70 > 1.15>0.58*
0.67 (approx.)< 0.23**
* Approximate mean based on an estimated active isomer concenitrationi of 50 (-** Approximate mean with the inactive 2,4,5-trichloro substitution excluded.
phenoxyacetic acid group and 0.58 ppm of the activeisomer- for the 2-phenoxypropionic acids. Greaterinherent activity of the 4-phenoxybutyric acid com-pounds on an equimolar basis is not a tenable hypoth-esis since the reduction of their side chain to aceticacid has been demonstrated as a necessary prerequisiteof activity (11, 20, 26). If equimolar concentrationsof the 4-phenoxybutyric acids cannot be more activethan the homologous phenoxyacetic acids, their ap-parent higher activity is likely clue to a greater up-take which results in enhanced accumulation of theactive homolog (following beta oxidation) at thesite of action. This possibility will be discussedfurther below.
The relatively low inhibitory activity of 4- (2,4,5-TB), 50 % reduction in mesquite root growth at3.0 ppm versus 50 % inhibition with 4-(MCPB) and4- (2,4-DB) at .06 ppm, was to be expected sinceSynerholm and Zimmerman (,0) found 4- (2,4,5-TB)to be inactive in tomato epinasty tests. Also, Wainand Wightman (26) showed that the phenoxy serieswith the 2,4,5-trichloro ring substitution except 2,4,5-T was inactive in tomato-epinasty and pea-curva-ture tests but active in a wheat cylinder elongationtest. They attributed the low activity of 4- (2,4,5-TB) and the higher homologs with an even numberof carbon atoms in the side chain to the failure ofpea and tomato stem beta-oxidation enzymes to func-tion in the presence of the 2,4,5-trichlorophenyl ring.The low activity of 4- (2,4,5-TB) in the mesquiteroot inhibition tests is due, most likely, to the samecausal factor.
The relationship of the dissociation constants andinhibitory activity was examined by using calculatedpK' values for the 12 phenoxy acids listed in tableII. It should be emphasized that the pH values usedin the calculation of pK' were determined at 60 Cbecause of the limited water solubility of some of thetest compounds at room temperature. However, thisslhouldl not invalidate comparisons of calculated pK'values on a relative basis. For reference, the rangeof pK' values obtained by other workers are includedin table II.
Table IIDissociation Constants, pK', of 12 Phenoxy Acids
Dissociation ConstantsCompounds Found
(pK' at 60 C) LiteratureMCPA 3.22 3.28(13)3.49(6)2-(MCPP) 3.38 3.2(15)4- (MCPB) 4.864-CPA 3.47 2.36(16)2- (4-CPP) 3.434- (4-CPB) 4.872,4-D 3.22 2.64-3.31(10,12,
There is no apparent relationship between the de-grees of dissociation of the 12 phenoxy acids andtheir ring substitutions. However, a relationshipbetween the degree of dissociation and the side chainis evident. The pK' values of phenoxy acids withthe 4-butyric acid side chain, pK' 4.58 to 4.87. differ-ed markedly from those of compounds with aceticacid and 2-propionic acid side chains, pK' 3.22 to3.47 (table II). Compounds with the 4-butyric acidside chain, as previously mentioned, were the mostactive in the root-growtlh inhibition tests. This sug-gests an inverse relationship between inhibitory ac-tivitv and dissociation among these compounds.
On the basis of the pK' values (table II), the con-centrations of un-dissociated molecules in root-bath-ing solutions containing the 4-phenoxybutyric acidsat pH 6 to 8 are 10 to 50-fold greater than in equi-molar solutions of the phenoxyacetic and 2-phenoxy-propionic acids. If it is assumed that un-dissociat-ed molecules are absorbed with greater ease than dis-sociated ones (2. 7), the higher concentration of theundissociated molecules in the root-bathing solutionscontaining the 4-phenoxybutyric acids would resultin greater accumulation within the roots of com-pounds with this side chain than of compounds withthe 2-propionic or acetic side chains. As mentionedpreviously, this might account for their greater ac-tivity in the root-inhibition tests.
For all compounds tested except 2,4-D a givenconcentration in the root-bathing solution was mosteffective in inducing root-growth inhibition at pH 6anid least effective at pH 8 (table I). The greatest1H response was exhibited by 2,4,5-T. A 0.47-ppmccncentration of 2,4 5-T was required to cause a70 % reduction in root growth at pH 6 whereas a2.30-ppm concentration was necessary at pH 8, ap-proxinmately a fivefold difference. There was noapparent association of a particular ring substitutionor side chain with the magnitude of the pH effect.
Audus (2), Wedding et al. (29), Blackman andRobertson-Cuninghame (5) and Erickson, et al.(10) are among those reporting that the biologicalactivity of 2,4-D is affected by external pH. In thepresent experiments, 2,4-D was the only compoundthat did not induce a different degree of root in-hibition when the pH of the root-bathing medium waschanged. When the test was repeated, the lack ofresponse of 2.4-D to changes in pH was demonstrat-ed again. No obvious reason for the failure of pHto influence 2,4-D activity was evident. A possibleexplanation might be based on the suggestion ofSimon and Beevers (18) that the pH effect on bio-logical activity may differ with the specific test andthe organism.
An increase in the hydrogen-ion concentration ofthe phenoxy solutions from pH 8 to 6 increased theconcentration of undissociated growth regulatormolecules in the root solution almost 100-fold andresulted in greater inhibitory activity (table I).Yet, equivalent increases in root growth inhibitioncould be achieved with, at most, a fivefold increase
in the total concentration of growtltakes 20 timles as great a clhange iiundissociatedl molecules to equal theabout by a variation in the total ccomlpundl, some effect of pH othegree of (lissociation of the pheinoxNpear to be the major determiinianitroot absorption.
The beta oxidlation of 4-phenoxthe correspon(ling phenoxyacetic afor biological activity. Keeping thiparison of the undissociated miiolecuin e(lui-effective solutions of aaci(d and its active homiiolog, phmliglht provi(le further evidenlce onthe conicentration of undissociatedducing root-growth inhibition. Ecentrationis of undissociated 4- (2,4require(d for 70 % root-growth inlfor the 3 pH levels, were 4.41 X10- ', respectively. The un(lissocikof 4-(2,4-DB) wvas 20 timles as greaindicatiing a considerably lower effbasis of undissociated 4-(2,4-DB).againi suggests the limnited imiiportartration of undissociate(d imioleculesof root-growtlh inhibitioin. On theis a possibility that this (lifferencelowv efficiency of mles(luite roots, (betaoxi(lation of 4-(2,4-DB3) to 2,4-
There was no evi(lent relationsubstitutioins of the 12 phenoxy cc(legree of root inhibition that deviledonary applications (table III)tivity of 2-(4-CPP ) was striking.plicatioln in(luced root-growth inhilgreater than that induce(d by the ot4 to 16 tinmes this application rate.
A correlation of si(le-clhain strttory activity was apparent when ccatioins were used. The 2-phenoxyl
This difference duced considerably more root-growth inhibition thaInce of the concen- the phenoxyacetic acids if only the active stereo iso-as a determinant miier of the 2-phenoxypropionic acids is considere(l.other hand, there Comiipounds with the 4-butyric acid sidle chaini except
2 couldl be (lue to 4-(M\ICPB) Nere least active.about 5 CC, in the It is apparenit fromii the activity rankings in table-D. INT that the site of application hlad ain imiiportanit in-between the ring fluence on the relative inhibitory activity of the 12)mpounds and the comiipounds. WVhile 4-(M\ICPBR), 4-(4-CPB) and 4-eloped after coty- (2.4-DB) rankedl 1. 2 and 3, respectively, in r1oot
The high ac- solutioin applications, they ranke(d 5, 10 and 11, re-A 0.103-,ug ap- spectivelv, when cotyledonary applications were use(l.
bition equal to or Numerous other clhanges in activity rankilng werehlier compounds at evident.
The addition of test comiipoundls to the root solii-icture and inhibi- tion places them acdjacelnt to the elongation region ofotyledlonary appli- the root which is in all probability the area of re-propionic acids in- sponse. Reaching the area of response requires onlly
aibsorption andl diffusion througlh a few layers ofcells, a (listance of a few miillinmeters at the mlost.On the other hand, cotyledonary applications place
ving Cotyledonarythe conmpounds 60 to 80 mm from the responsive
:y Acids area of the root. In addition, the cuticle is a barrierinol to cotyledonary uptake. After penetration of the
cuticle, the compound must move througlh the coty-cotyledon (,ug) ledon to the plhloem. Translocation througlh the1.648 Mean phloemn to the root is followed by diffusion through-
out the root. In the previously dlescribed pathways,55 71 (lifferences in cuticular absorption an(l phloem trans-98 102 location or phloem translocation alone, could account74 84 for the observedl variations in activity arising from52 80 the miiethod of application.83 94 The influence of the cuticle on absorption of the39 50 phenoxy compounds was indicated in the following93 96 experiment. The 2,4,5-trichloro-substituted series of57 75 phenoxy acids was applied to the cotyledons of nmes-75 82 (luite seedllings in an acetone-nontoxic oil carrier66 79 known to induce greater absorption thlan 70 S4 ethanol
(4). Eightyfold increases in in}w-ere obtained when this carrier wasNevertheless this group of compousame relative activity ranking as vcarrier was used. Apparently, thrise to the differences in inhibitorycompounds were not influenced by tIof absorption induced by the acetcarrier.
Solubility of a compound may Ithe degree of cuticular absorption (Overbeek et al. (24) associated tlgroup of maleimides directly withThey explained this relationshipgreater water solubility being assolipoid solubility and maintained thawith high water solubility did notthe lipoid phases in the plant andinactive. The possibility of a simthe 12 phenoxy acids was examined.used are listed in order of decreasingin table IV. They are ranked alsotheir relative inhibitory activity inroot solution applications. A corrsolubility and the inhibitory activity c
acids is not evident for either applic
Table VIRoot Growth as % of Check Follom
Applications of 2,4,5-T & 2-(4-CPFLevels for the Root-Bathing
Amount pH of root 1Compound applied
(Ag) 6
2,4,5-T 0.103 850.412 731.648 53Mean* 70
2-(4-CPP) 0.103 860.412 580.648 35Mean** 60
* LSD for pH means at P .01 = 13** LSD for pH means at P .01 = 14
hibitory activityused (table V).nds retained therhen the ethanole factors givingactivity of thesehe greater degreetone-nontoxic oil
greatly influence(8, 22, 25). Vanhe activity of awater solubility.on the basis ofciated with lowt the maleimidesreadily penetrate
wvf-ri- thbpr-fnrsr
The influence of the hydrogen-ion concentrationof the root-bathing medium on the relative inhibitionof root growth induced by phenoxy compounds ap-plied to the cotyledons of mesquite seedlings was ex-amined. By this technique the possible effect of ex-ternal pH on the inhibitory activity of phenoxy com-pounds within the root could be examined withoutroot absorption being a factor. Twvo test compounds,2,4,5-T and 2-(4-CPP), were selected because theyinduced significant increases in inhibitory activityof root-bathing solutions as pH was lowered. Thedlata in table VI show that the pH of the root-bath-ing nmedium in most instances was not correlated withroot-growtth inhibition when cotyledonary applica-tions were used. The one exception, 2,4-5-T combin-ed with pH 7 of the root-bathing solution, indlucedsignificantly greater root-growth inhibition than pH6 or pH 8. WVhile this effect is of interest, it is un-like the response to pH obtained when 2,4,5-T wasadded to the root-bathing medium (table I). In thatcase, root-growth inhibition was inversely correlatedw-ith pH, a trend that is not evident in this test.
The fact that the influence of pH on inhibitory ac-tivitv cannot be wholly attributed to dissociation ofthe compound in the external mediunm or to an effectwithin the cells of the root draws attention to thecell menmbrane as a likely site of the response to pH.The mlost probable effect of pH on the cell membranewould be on its permeability as suggested by Brianand Rideal (6) and van Overbeek (22).
Summary
icla trlatVIofo The activity of 12 chlorinated phenoxy acids was
The compounds determined in root-growth inhibition tests by apply-> water solubilitv ing the compounds to the roots or to the cotyledonson the basis of of mesquite [Prosopis jutliflora (Swartz) var. glan-
cotyledonary andl dulosa (Torr.) Cockr.] seedllings. In the root ap-elation of water plications the 2-methyl-4-chloro ring substitution in-'f the 12 phenoxy duced the greatest inhibition followed by the 4-chloro,a,4-dichloro and 2,4,5-trichloro substitutions in ordernof decreasing activity. Relative inhibitory activity
did not appear to be correlated with the ring sub-stitution when cotyledonary applicaticns were used.
ving Cotyledonary The 4-butyric aci(l sidle chain except 4-(2,4.5-TB)Medium imparted the greatest inhibitory activity in root solu-
tion applications followed by the active stereoisomersbathing medium of the 2-propionic aci(ds and(l the acetic acids in order
of decreasing activity. The greater inhibitory ac-7 8 tivity of the 4-phenoxybutyric acids was associated70 89 with less dissociation. However, inhibitory activity59 72 was not proportional to the concentration of undis-51 58 sociated molecules in the root-bathing solution. In57 73 the cotyledonary applications, the active stereoiso-
niers of the 2-propionic acids induced the greatest dIe-75 77 gree of root-growth inhibition followed by compounds50 w4with acetic acid and 4-butyric acidI side chains in32 39 order of decreasing activity. The use of another52 57 carrier, acetone-nontoxic oil, greatly increasecl the
inhibition induced by cotyledonary applications of 4-butvric acids indicating that absorption rather than
translocation may limit the action of these com-pounds.
In root solution applications, root inhibition wasusually increased by decreasing pH. However, theincrease in inhibition was not proportional to the in-crease in concentration of undissociated phenoxy acidmolecules. This strongl) suggests that pH has amore important role than its effect on phenoxy aci(ddissociation. The pH of the root-bathliing solutiondi(d not influence the (legree of root inhibition in-duced by cotyledonary applications which nullifiesthe possibility of intracellular influences of externalpH.
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