PHYSIOL. PLANT. 54: 34-40. Copenhagen 1982

The absorption and translocation of didofop-methyl andamitrole in wheat and oat roots

Andrew Jacobson and Richard H. Shimabukuro

Jacobson, A. and Shimabukuro, R. H. 1981. The absorption and translocation ofdiclofop-methyl and amitrole in wheat and oat roots. -Physiol. Plant. 54: 34^0.

The absorption and translocation of diclofop-methyl (methyl 2-[4(2',4'-dichloro-phenoxy)phenoxy]propanoate) was examined by using a specially designed treatmentapparatus that separated excised roots or roots of seedlings into four zones. ["C]-Diclofop-methyl was absorbed along the entire root length of both wheat {Triticumaestivum L.) and oat {Avena sativa L.). In both species, absorption was greatest in theapical region of the root. Absorption by the apical region of wheat roots was morethan three times greater than the basal portions, and more than twice as great as theapical region of oat roots. Less than 5% ofthe absorbed diclofop-methyl was translo-cated in both wheat and oat roots. Diclofop-methyl and diclofop(2-[4(2',4'-dichlorophenoxy)phenoxy]propanoic acid) were the predominant translocatedforms. The absorption and translocation of amitrole (3-amino-l,2,4-triazole) werealso examined. Amitrole was absorbed along the entire length of wheat roots andtranslocated primatily in the basipetal direction. The usefulness of the specially de-signed apparatus for biochemical and physiological studies is discussed.

Additional key-words: Avena sativa, Triticum aestivum. .

A. Jaeobson and R. H. Shimabukuro, Agronomy Department, North Dakota StateUniversity, and Metabolism and Radiation Research Laboratory, Agricultural Re-search, Science and Education Administration, U.S.D.A., Fargo, North Dakota58105, USA; A. J., (present address) Rohm and Haas Co., Research Laboratories,Springhouse, PA 19002, USA.

Introduction

The herbicide, diclofop-methyl, shows great promise for observed between tolerant and susceptible species-selective weed control in cereal crops. Wheat {Triticum However, a difference in the metabolic pathway wasaestivum L.) is resistant and barley {Hordeum vulgare observed between wheat and oat (Shimabukuro et al-L.) is moderately tolerant to the herbicide at concentra- 1979). This suggested that metabolism was the basis foftions that will control wild oat {Avena fatua L.) and herbicide selectivity.several species of foxtails {Setaria sp.; Andersen 1976, Various methods have been utilized for studying ab-Miller and Nalewaja 1974). Diclofop-methyl inhibits sorption and translocation of herbicides (Hay 1976)'root elongation in cultivated oat {Avena sativa L.; However, these methods cannot be used to analyze ab-Hoerauf and Shimabukuro 1979, Shimabukuro et al. sorption by different morphological regions in root of1978) and shoot growth in wild oat (Donald and shoot tissue. Recently, Singh and Jacobson (1977) pub-Shimabukuro 1980, Hoerauf and Shimabukuro 1979). lished a novel method for examining the absorption andNo differences in foliar absorption (Donald and translocation of inorganic ions applied to various zonesShimabukuro 1980, Shimabukuro e/«/. 1979, Todd and of barley root segments. We have modified this system,Stobbe 1977) or translocation (Boldt and Putman 1980, and in this communication present data examining theBrezeanu et al. 1976, Shimabukuro et al. 1979, absorption and translocation of diclofop-methyl by dif'Shimabukuro and Walsh 1979) of diclofop-methyl were ferent morphological regions of wheat and oat roots.

Received 15 June, 1981.

34 0031-9317/82/010034-07 $03.00/0 ©1982 Physiologia Plantarum Physiot. Plant. 54,

•Materials and methods

Six-day-old wheat {Triticum aestivum L. cv. Waldron)and seven-day-old oat seedlings {Avena sativa L. cv.Garry) were used in this study. Cultivated oat instead ofwild oat were used for reasons stated previously(Fleteher and Drexler 1980). Seeds were allowed toimbibe water for 24 h and then were placed between'ayers of absorbent paper toweling (Blankendaal et al.1972) saturated with a nutrient solution consisting of 1" ^ each of MgSO4, KH2PO4 and Ca(NO3)2. At theend of the growth period, 6-day-old wheat and 7-day-old oat seedlings were washed and excised at the seed-root interface. At this stage of growth, roots from bothspecies were 16 to 18 cm long.

A plexiglas treatment apparatus, referred to as achamber (Fig. 1) and similar to that described previ-ously (Singh and Jacobson 1977), was used to treatroots. The chamber consists of four compartments, each2-5 cm long by 3.5 cm wide and with a volume of 15 ml.Solutions in adjacent compartments were separated by|wo barrier spaces with an intervening air space. Thebarrier spaces were filled with a mixture of anhydrous'anolin U.S.P. and white petroleum jelly (1:1 w/w). Five1-mm slits were cut perpendicularly in each barrier. Theslits in the barriers were aligned throughout the lengthot the chamber. Each slit accommodated one root.Apices of excised wheat and oat roots were placed incotnpartment 1, and basal ends were placed in com-partment 4. To prevent desication during the treatmentperiod, roots were maintained in a moist environment,j-hambers, containing roots, were placed in a rectangu-ar glass dish lined with moist paper towelling and fittedith a plexiglas cover. The covered glass dish was

placed in a seed germinator to maintain an environment°f high RH and a temperature of 25°C.

h

an

Senair '̂

g s treatment apparatus used in this study. The's divided into 4 compartments (labeled 1, 2, 3, and 4)

'°"8. ̂ '''^ ^ volume of 15 ml. The apical portion ofroot is placed in compartment 1 and the cut basal

'" """"^P^rtment 4. Solutions bathing root segments were'̂̂ ''y 3 double lipophilic barrier (A) and an intervening

(B). For a further description see Materials and

Roots were treated with either 10 \iM amitrole(3-amino-l,2,4-triazole-5-^''C; New England Nuclear,sp. act. 3.8 mCi/mmol) or 10 [iM diclofop-methyl(methyl 2-[4-(2',4'-dichlorophenoxy-U- ^''Qphenoxy]propanoate; New England Nuclear, sp. act., 2mCi/mmol). The labeled compound was placed in oneof the four compartments and the remaining three con-tained water. Amitrole was soluble in water, but it wasnecessary for diclofop-methyl to be dissolved initially inacetone and brought up to volume with the slow addi-tion of water (final acetone concentration, 1% v/v). Thetreatment period for all experiments was 24 h.

Qualitative determination of absorption and translo-cation was accomplished by autoradiography. Treatedroots were washed in water, gently dried on absorbenttoweling, glued to matting paper, frozen, andlyophilized for 24 h. Dry, mounted roots were au-toradiographed on Kodak No-Screen X-ray film.

The absorbed and translocated radioactivity wasquantified by liquid scintillation spectrometry. After a24 h treatment period, roots were cut into four piecesby excising along the intervening air spaces between thelipophilic barriers, washed in water, and frozen in liquidN2. Root segments were lyophilized and then com-busted in a Model 306 Packard Tri-Carb sampleoxidizer. The resulting ^"COj was counted by liquid sci-ntillation. Solutions from each compartment were alsocounted for radioactivity.

Thin layer chromatography (TLC) was performed onglass plates coated with 250 nm silica gel HF activatedat 110°C for 2 h. Chromatograms were developed intoluene/absolute ethanol/acetic acid (150:7:7). Theradioactive zones on TLC plates were detected with aradiochromatogram scanner, and the zones werescraped and quantified by liquid scintillation counting in10 ml of Instagel (Packard Instrument Co.) and 3 ml ofwater.

Results and discussion ;

Amitrole absorption and translocation. Amitrole ab-sorption and translocation has been extensively studied(Hay 1976, Ashton and Crafts 1973), and for thisreason we initially employed it to examine the useful-ness of these chambers. As a control, chambers wereprepared without roots, and 15 ml of "C-amitrole wasplaced in one compartment and water added to threeother compartments. After 24 h the amount of radioac-tivity was determined in all four compartments as wellas the lanolin-petroleum jelly mixture within the bar-riers. The compartment where the label was initiallyplaced contained more than 98% of the applied label.No radioactivity was detected in the other compart-ments, and only a small amount of radioactivity, if any,was detected in the lipophilic mixture within the bar-riers adjacent to the compartment containing thelabeled amitrole.

• Ptant. 54, 1982 35

Fig. 2. Absorption and translocation of '''C-amitrole by excisedwheat roots. Treated roots are shown on the left and au-toradiographs are shown on the right. "C-Amitrole was placedin compartment 1 (apex of root; A) and in compartment 3 (B).

Examination of Figure 2 and Table 1 shows thatamitrole was absorbed along the entire length of wheatroots. Throughout this study, absorption is defined asthe sum of radioactivity found in the five root segments

Tab. 1. Absorption and translocation of "C-amitrole by different morphological regions of wheat roots. Excised rootspositioned with apices in compartment 1 and basal ends in compartment 4. Roots were treated in either compartment 1, 2, 3, or''with 10 (iM "C-amitrole for 24 h. The three untreated compartments contained water. The radioactivity reported for th'treatment compartments represents only radioactivity absorbed by the five root segments within that compartment whereas value*in nontreated compartment represent radioactivity translocated into the five root segments within that compartment an''radioactivity in the water bathing these root segments. The values in parenthesis represent the percentage + SD of the tota'radioactivity recovered. Data are averages from duplicates of three experiments.

within the four compartments and the solutions of thethree nonlabeled compartments. Figure 2A and B showthat absorption and subsequent translocation occurredwhen labeled amitrole was placed in the first (apical)and third compartments. Similar results were obtainedwhen labeled amitrole was placed in compartments 2and 4 (autoradiographs not presented). One must becareful when analyzing this data since absorption ofapplied amitrole by plant material is very small (Hay1976, Ashton and Crafts 1973). In our study only 0.6 to0.7% ofthe initially applied radioactivity was absorbed.Translocation occurred readily throughout wheat rootsas has been previously reported in other species (Hay1976). Our results indicate that translocation was pre-dominantly in the basipetal direction. This suggests thatin wheat, root applied amitrole moves principally in thexylem.

Diclofop-methyl absorption and translocation. Hav-ing demonstrated from the experiments with amitrolethat the root-treating system works as expected, de-tailed studies on the absorption and translocation ofdiclofop-methyl were performed using the same system-A control without roots was run for diclofop-methyl asdescribed previously for amitrole. Initially the amountof ^''C-diclofop-methyl recovered from the treatmentcompartment ranged from 25 to 60%, with the remain-der of the radioactivity detected in the lipophilic mix-ture contained within the barriers adjacent to the treat-ment compartment. If the prepared chamber was pre-conditioned by adding water 12 h prior to the additionof ^''C-diclofop-methyl, the recovery of radioactivity iOthe treatment compartment ranged from 48 to 61%'The more consistent recoveries obtained by precondi-tioning the chamber were probably the result o'anhydrous lanolin absorbing water and reducing thspartitioning of diclofop-methyl into the lipophilic mat-rix. In all cases no radioactivity was detected in th^water of the nontreated compartments. Similarly, i'

Treatment dpm/compartment

1

2

3

4

1

4967(57.5±5.2%)

526(5.3±3.1%)

95(1.7±0.2%)

76(2.2+0.1%)

2

1204(24.5 + 3.5%)

2323(24.5±2.1%)

420(7.4±3.3%)

90(2.6±0.6%)

3

308(3.6±1.8%)

3396(35.6±0.3%)

1576(28.9±4.1%)

400(11.8±2.9%)

4

1250(14.4+1.0%)

3262(34.6±3.0%)

3397(62.0±5.1%)

2888(83.4±3.7%)

36 Physiol. Plant. 54,

BASE

APEXJ

BASE

1

wmAPEX

\

I 1

f'8- 3. Absorption and translocation of "C-diclofop-methyl by Fig. 4. Absorption and translocation of '''C-diclofop-methyl byxcised wheat roots. Treated roots are shown on the left and excised oat roots. Treated roots are shown on the left and"toradiographs are shown on the right. "C-Diclofop-methyl autoradiographs are shown on the right. "''C-Diclofop-methyl3s placed in compartment 1 (A) and in compartment 3 (B). was placed in compartment 1 (A) and in compartment 2 (B).

ater was placed in the intervening air space, noradioactivity was detected in adjacent compartments.

Results in Figures 3 and 4, and Table 2 show thatJ sorption of diclofop-methyl occurred along the entire^ngth of wheat and oat roots. Autoradiographs ofheat roots showed absorption of the herbicide by eachorphologieal root segment isolated within a particular

cotnpartment (Fig. 3A, compartment 1; Fig. 3B, com-^j^^ 3). Similar results were obtained for oat rootsj ^ 4 A , compartment 1; Fig. 4B, compartment 2).

oot segments were combusted and the radioactivityquantified by liquid scintillation spectrometry (Tab. 2).

osorption of diclofop-methyl by the apical wheat seg-ents was more then three times the absorption in each'he basal segments. Absorption in the three basipetalgnients was nearly equal. Oat apices absorbed nearlylee the amount of herbicide as the root tissue in each

°̂ the two adjacent zones (3.5 to 11.0 cm). These resultsgree with the concept that the apical portion of theot, 0.5 to 5.0 cm from the root tip, is the primary site

Or herbicide absorption (Bukovac 1976, Shimabukuro"d Walsh 1979). Results in Table 2 also show that theheat root apices absorb twice as much "C-diclofop-

l as oat root apices. The difference between wheat

and oat roots may be due to specific differences in ab-sorption, translocation (explained below), ormetabolism. The most likely explanation involves adifference between the two species in the metabolism ofdiclofop-methyl. Diclofop-methyl and its free acidmetabolite, diclofop, are toxic to oat roots (Shimabuk-uro et al. 1979). A water-soluble ester conjugate ofdiclofop formed by oat roots may be reversibly hyd-rolyzed to the phytotoxic acid form (diclofop). Tolerantwheat roots are capable of detoxifying diclofop-methylto a water-soluble phenolic conjugate. The productionof phytotoxic metabolites probably injures oat roots andreduces the absorption of diclofop-methyl since oatroots are not able to detoxify the metabolite completely.In wheat roots, absorption of diclofop-methyl is main-tained at a higher rate than oat roots because of therapid detoxication of the herbicide. The production inboth wheat and oat roots of water-soluble metabolitesimplies the absorption of the compound into the planttissue. Additionally, more radioactivity was found in themethanol-insoluble residue of wheat (7.0%, 28,423dpm/g fresh weight) than oat (2.2%, 9,640 dpm/g freshweight) after 24 h. This incorporation of the herbicideinto a bound, insoluble form may facilitate its continued

•«°I-Ptam. 54, 1982 37

Tab. 2. Absorption and translocation of "C-diclofop-methyl in wheat and oat roots. Excised roots were positioned with apices incompartment 1 and basal ends in compartment 4. Roots were treated in either compartments 1, 2, 3, or 4 with 10 [M ^"C-diclofop-methyl for 24 h. The radioactivity reported for the treatment compartments represents only radioactivity absorbed bythe five root segments within that compartment whereas values in nontreated compartments represent radioactivity translocatedinto the five roots segments within that compartment and radioactivity in the water bathing the root segments. The values inparenthesis represent the percentage ± SD deviation of the total radioactivity recovered. Data are averages from duplicates ofthree experiments.

Species

Wheat

Oat

Treatmentcompartment

1

2

3

4 •

1

2

3

4

1

1959(99.1±1.4%)

12(1.6±2.1%)

3(0.6+0.8%)

10(1.9+2.1%)

904(99.3±0.7%)

16(2.3±3.2%)

6(1.6±2.2%)

5(1.5±2.5%)

dpm/compartment

2

11(0.8+1.1%)

622(94.3±8.9%)

10(1.3+2.1%)

1(0.1±0.1%)

5(0.4±0.4%)

515(95.9±4.6%)

4(0.4+0.4%)

2(0.6±1.3%)

3

0(0.0+0.0%)

26(3.3 ±5.4%)

457(97-5±3.2%)

1(0.2±0.5%)

2(0.1+0.2%)

8(1.7±1.9%)

647(97.3±2.2%)

3(1.0±2.1%)

4

1(0.1+0.3%)

7(0.8±1.7%)

4(0.6±1.0%)

519(97.8±1.9%)

3(0.2 + 0.3%)

2(0.2±0.3%)

(0.7±1.0%) "

362(96.9+5.9%)

uptake from the treatment solution. Differences in ab-sorption between the apical segments of wheat rootsand its basal neighboring segments may be due to sec-ondary thickening of older root tissue or to metabolicdifferences. It is possible that the apical portion containshigher activities of the enzymes necessary to metabolizediclofop-methyl.

Examination of Figures 3 and 4, and Table 2 indicatesthat unlike amitrole, diclofop-methyl was not translo-cated to any appreciable degree. Between 93% to 99%

Fig. 5. Absorption and translocation of "C-diclofop-methyl by5 day wheat seedlings. The shoot-seed-root interface regionwas treated with '''C-diclofop-methyl herbicide. The plantmaterial is on the left and the autoradiograph is on the right.

of the absorbed radioactivity in wheat and oat rootsremained in the treated zones. Of the total appliedradioactivity (approximately 600,000 dpm/15 ml), lessthan 0.4% was absorbed and translocated, and less than0.002% was recovered in the solution containing thebasal cut end. The lack of translocation in excised rootswas not because of the absence of transpiration result-ing from excision of the shoots. Seedlings treated with^''C-diclofop-methyl in the region of the root-seed-shoot interface showed no translocation of the herbicide(Fig. 5). These results are similar to those observed pre-viously in wheat, oat, and soybeans (Boldt and PutmaH1980, Brezeanu et al. 1976, Shimabukuro and Walsh1979).

To determine the chemical form of the herbicide tha'was translocated, the radioactivity in the solution con-taining the excised basal end (compartment 4) wa*examined. Previously, Shimabukuro et al. (1979)showed that for both wheat and oat the radioactivitytranslocated from the roots was present in the shoot*mainly as a water-soluble conjugate(s). However, thes^observations did not indicate the translocated form "'the herbicide. In the present experiments, '̂̂ C-diclofop'methyl was placed into compartment 1,2, or 3. After 2''h the radioactivity translocated into the solution ""compartment 4 (from eight chambers) were combined'evaporated, and subjected to thin layer chromato'graphy. Analysis showed (Table 3) that in wheat mof̂than 90% ofthe radioactivity recovered from compafl'

38 Physiol. Ptant. 54, 1

Tsb- 3. Metabolism and translocation of '"C-diclofop-methyl'n wheat and oat roots. Excised roots were positioned withspices in compartment 1 and basal ends in compartment 4. 10^ "C-diclofop-methyl was placed in either compartment 1,2, or 3 and water placed in the three remaining compartments.After 24 h, solutions from eight chambers were combined, andthe radioactivity qualitatively determined by TLC and quanti-tated by liquid scintillation spectrometry. Data are averagesirom duplicates of three experiments.

Compound % of '"C in each TLC fraction

Treatmentcompartment

Basal endcompartment

Wheat

hyl 7.390.9

lofop 1.00.8-

Oat

34.364.4

0.10.70.5

Wheat

7.982.7

0.28.90.3

Oat

31.763.5

2.40.81.6

Diclofop-mcDiclofopRing-OH diConjugatesUnknown

nient 4 was the parent and free acid components. Theremaining major component (8.9%) was a water-soluble'̂ onjugate(s). In wheat, the radioactivity in the treat-•iient compartment, as well as in the compartment con-aining the excised basal end, was predominantly ther̂ee acid, diclofop, after 24 h. In oat, over 95% of the

radioactivity recovered from the solution containing thee x c d basal end was diclofop-methyl and diclofop,

diclofop accounting for about 64%. In the treat-compartments, diclofop-methyl and diclofopsed over 98% of the radioactivity recovered in

*he solution with diclofop accounting for 65%. Theprobable reason for the quantitative differences be-ween data reported here and that presented earlierl^himabukuro et al. 1979) is due to differences in ex-V rimental procedures. The root tissues used for treat-"^^ , the method of treatment, and the treatment times

vs. 46 h) were significantly different. In this pre-^ investigation we examined the radioactivity trans-

lated from the treatment compartment into the sol-'On in the compartment containing the cut basal end.

^ the earlier study (Shimabukuro et al. 1979), wholeot tissue and the treatment solution bathing the roots*̂ re examined. The movement of "C-diclofop-methyl

vveen compartments by diffusion did not occur as'cated by the previously described control experi-nts. The outward diffusion of water-insoluble and

^ u b l e metabolites from excised roots or roots ofings bathed in 10 yM ^^C-diclofop-methyl hasobserved (A. Jacobson, unpublished results). The

^ of water-soluble metabolites seemed to occur^ y when a saturated and thus constant level of water-j . "oie metabolite was reached within the treated root

sue. These results suggest that diclofop-methyl and its^. ^oolites are translocated in very limited amountsg ^'clofop-methyl and/or diclofop as the predomin-

translocated form(s). However, these results do not

indicate whether translocation occurs in the symplast orapoplast.

Our results support earlier studies (Boldt and Putman1980, Brezeanu et al. 1976, Donald and Shimabukuro1980, Shimabukuro ef a/. 1979, Todd and Stobbe 1977)demonstrating that neither absorption nor translocationof diclofop-methyl appears to be the basis for its selec-tivity. A previous report (Shimabukuro et al. 1979)showed that the metabolism and degradation ofdiclofop-methyl differed between tolerant wheat andsusceptible oat. Wheat formed an acidic aryl-O-glycoside while oat formed a neutral glycosyl ester. Aninteresting question arises as to why certain organiceompounds can be translocated and others cannot. Anumber of suggestions have been presented (Crafts andCrisps 1971). One possibility is that compounds thatcan be translocated are able to enter the steele in rootswhile those that cannot be translocated remain in thecortex.

In conclusion, we have described a novel system forinvestigating a number of physiological processes. Wemeasured conveniently the quantitative and qualitativeaspects of diclofop-methyl absorption and translocationin wheat and oat roots. The uniqueness of this system isthat a particular morphological region of a root or shootmay be isolated and treated separately from the rest ofthe tissue or plant. Thus, we were able to examine ab-sorption of the herbicide, diclofop-methyl, along theentire length of wheat and oat roots. At the same time,we were able to examine the translocation of the her-bicide and identify the compound(s) being translocatedand released by the cut basal end. This system can bereadily applied to other physiological investigationsconcerning growth and metabolism.

Acknowledgements - The authors thanks Ms. J. Pender for hertechnical assistance and Mr. T. Hlavaty for his photographicassistance.

Mention of a trademark or proprietary product does notconstitute a guarantee or warranty of the product by the U.S.Department of Agriculture and does not imply its approval tothe exclusion of other products that may also be suitable.

Cooperative investigation by the Agric. Exp. Stn., NDSU,Fargo, ND and the USDA-SE-ARS. Published as Agric. Exp.Stn. J. Article No. 1132.

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Edited by L.E.

40 Physiol. Plant. 54,