Evidence from Studies with Acifluorfen for Participation of a

Flavin-Cytochrome Complex in Blue Light Photoreception forPhototropism of Oat Coleoptiles1 2

Received for publication September 25, 1981 and in revised form March 2, 1982

TA-YAN LEONG AND WINSLOW R. BRIGGSDepartment of Plant Biology, Carnegie Institution of Washington, Stanford, California 94305

ABSTRACT

The diphenyl ether acifluorfen enhances the blue light-induced absorbancechange in Triton X100-solubilized crude membrane preparations frometiolated oat (Avena sativa L. cv. Lodi) coleoptiles. Enhancement of thespectral change is correlated with a change in rate of dark reoxidation ofa b-type cytochrome. Similar, although smaller, enhancement was obtainedwith oxyfluorfen, nitrofen, and bifenox. Light-minus-dark difference spec-tra in the presence and absence of acifluorfen, and the dithionite-reduced-minus oxidized difference spectrum indicate that acifluorfen is actingspecifically at a blue light-sensitive cytochrome-flavin complex. Sodiumazide, a flavin inhibitor, decreases the light-induced absorbance changesignificantly, but does not affect the dark reoxidation of the cytochrome.Hence, it is acting on the light reaction, suggesting that the photoreceptoritself is a flavin. Acifluorfen sensitizes phototropism in dark-grown oatseedlings such that the first positive response occurs with blue lightfluences as little as one-third of those required to elicit the same responsein seedlings grown in the absence of the herbicide. Both this increase insensitivity to light and the enhancement of the light-induced cytochromereduction vary with the applied acifluorfen concentration in a similarmanner. The herbicide is without effect either on elongation or on thegeotropic response of dark-grown oat seedlings, indicating that acifluorfenis acting specifically close to, or at the photoreceptor end of, the stimulus-response chain. It seems likely that the flavin-cytochrome complex servesto transduce the light signal into curvature in phototropism in oats, withthe flavin moiety itself serving as the photoreceptor.

that a flavin is involved in the photoreaction (5, 16, 26). Thepotential biological significance of this photoreduction has beenchallenged by the observation that an artificial system can mimicthe reaction (21), raising questions about specificity of the system.We have shown that the blue light-sensitive, flavin-Cyt complexis associated with the plasma membrane in corn (9). This mem-brane fraction can be purified from corn by a series of differential,sucrose, and Renografm gradient centrifugations. Unlike solubi-lized membrane preparations from Neurospora (14) and Dictyos-telium (17), the flavin-Cyt complex solubilized with Triton XIOOfrom the purified corn membrane fraction retains full blue LIACactivity. This solubilized system has been partially characterized(9, 11).Although the physiological significance of blue LIAC has been

indicated in Neurospora (3, 14, 19), its significance in higher plantsis still obscure. Similarity in the action spectra of the LIAC andphototropism in oats suggests that the blue LIAC in corn and oatsmay be related to the phototropic response, although a goodaction spectrum is available only for Neurospora (14).Diphenyl ether herbicides are known to block electron transport

(4, 13, 18). There is also evidence consistent with possible effectsat the plasma membrane (15, 18). Their herbicide action requireslight absorption (6, 12, 13, 24). We have studied the effects ofthese herbicides on the LIAC in membrane preparations from oatcoleoptiles and on the phototropic and geotropic responses ofintact oat seedlings. We will present evidence here to suggest boththat the LIAC and phototropism are interrelated and that theflavin-Cyt complex is likely to be the photoreceptor for the firstpositive phototropic response of oat coleoptiles. A preliminaryreport of this work has appeared elsewhere (10).

Blue LIAC3 in membrane preparations of Neurospora (1, 14)and corn (1, 7, 9) have been well documented. The absorbancechange is a result of the reduction of a particular b-type Cyt. Themechanism of this reduction is still unknown, although it is clear

' This paper is affectionately dedicated to the memory of Sterling B.Hendricks. In 1963, the junior author spent a sabbatical year with Hen-dricks and the rest of the Beltsville group with the object of applying theingenious spectral techniques devised to detect phytochrome to the prob-lem of the phototropic photoreceptor. The Beltsville group rapidly seducedhim into phytochrome studies instead, thereby opening a major chapter inhis laboratory's research. It is a pleasure to be able to report at long lastsome progress on the original problem, and particularly appropriate to doso in the context of a dedication to Sterling Hendricks.

2Camegie Institution of Washington-Department of Plant BiologyPublication No. 765.

3Abbreviations: LIAC, light-induced absorbance change; 21KP, 21,000g pellet.

MATERIALS AND METHODS

Light-Induced Absorbance Change. Oat seeds (Avena sativa L.cv. Lodi, Lot No. 0170-B, Dakota Seed and Grain Co., Inc.,Watertown, SD) were sown on wet absorbant paper (Kimpack,K-41 Perf'd, Kimberley-Clarke) and allowed to germinate incomplete darkness inside growth cabinets kept at 26°C and 95%humidity. Coleoptiles of 6-d-old seedlings were harvested underdim green light, care being taken to exclude primary leaves. Allsubsequent operations were also carried out under dim green light.Twenty g of the tissue (apical sections approximately 1 cm inlength) were homogenized in 60 ml of extraction buffer whichconsisted of 0.1 M N-morpholinopropane sulfonic acid, 14 mm 2-mercaptoethanol, 3 mm EDTA, 0.25 M sucrose, and 0.1 mM MgC12,adjusted to pH 7.4 with KOH. The homogenate was centrifugedat 5,0O0g for 10 min to remove cell debris, and the supernatantwas then centrifuged at 9,000g for 15 min to sediment most of themitochondria. The supernatant from this centrifugation was thenapplied over 8 ml of 32% (w/w) sucrose in the same buffer and

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LEONG AND BRIGGS

centrifuged at 21,000g for 90 min. The pellet contained most ofthe LIAC activity, whereas membrane particles that remained atthe interface between the sample and the 32% sucrose showedlittle LIAC activity and were probably derived from golgi and ER.The pellet was solubilized in 0.1 M potassium phosphate (pH 6.72),containing 0.25 M sucrose, 3 mm EDTA, 0.1 mM MgCl2, and 0.5%Triton XIOO, clarified by centrifugation at 200,000g for 30 mi,and the supernatant (designated Triton-solubilized 21KP) wasused for LIAC measurements. LIAC measurements and unitswere according to Goldsmith et al. (7). Difference spectra wereobtained as reported elsewhere (9). Replicates of LIAC measure-ments were within 5% within any one preparation. The fluencerate of the actinic light was 1.3 x 10 ,umol m-2s-1, and thesaturating exposure time used was 60 s. Thus, the total fluence iswithin the range of second positive curvature for oat coleoptiles.

Phototropism. Phototropic responses of dark-grown oat seed-lings were determined after the protocol of Zimmerman andBriggs (27). In experiments where acifluorfen was present, theappropriate concentration of herbicide was added to the agar justbefore it was poured into tubes. Thirty-h-old seedings were trans-planted onto the slanted agar surface under dim green light andallowed to grow in dark growth cabinets kept at 26°C and 95%humidity until they were 4 d old. Spraying the seedlings with theacifluorfen solution did not change the results (not shown). Uni-lateral light was from a slide projector fitted with a 500-w, 120-vSylvania projector bulb (DAY) plus a blue filter (Corning 5-60)and suitable neutral density filters (Kodak, Wratten). Fluencerates were measured with a Li-Cor Quantum Photometer (modelLI-185A). A constant fluence rate of l0-3 ,umol m-2s-1was used.Exposure time was regulated with a custom-built sequential gatetiming system equipped with a Uniblitz microsecond 'program-mable' shutter (model No. 225LOAOT5; 25 mm aperture). Rowsof eight seedlings were used for each exposure. Phototropic cur-vatures were allowed to develop in the dark in the growth cabinetfor exactly 100 min after the onset of thelight exposure. Standarderrors never exceeded ±2 degrees.

Geotropism. Geotropic responses were measured in etiolatedoat seedlings grown in the presence or absence of acifluorfen asdescribed for phototropism. Rows of eight seedlings were placedhorizontally and allowed to remain there for a predeterminedtime, after which they were returned to the vertical position.Curvatures were allowed to develop in the dark in the growthcabinet for exactly 120 min following the onset of the gravitationalstimulus. At that time the vials were rotated 900 such that anygeotropic curvature induced would be in the plane of the row ofseedlings. The rows were then shadowgraphed as described else-where (27). Standard errors never exceeded ±2 degrees.

Rate of Dark Reoxidation of the Cytochrome. A typical record-ing of the LIAC induced in the 21KP by a 60-s actinic irradiationis similar to that shown in Figure 1 of Brain et al. (1). In routineexperiments, the LIAC was recorded for 8 min to allow completedark reoxidation of the Cyt. The value of absorbance immediatelyafter the 60 s irradiation was used as 100%/o and that at 8 min as 0%1oreduction of cytochrome. The kinetics of the dark reoxidation ofthe Cyt were then studied over this initial 2-min period. Thepercentage reduction of the Cyt was calculated and the log of thispercentage plotted against time after irradiation. A straight line isobtained since the kinetics are first order during the first 2 min. Alinear regression was done on the points to give the best fittingstraight line through the points (usually r2 0.98). The slope ofthis straightline was then used as the rate of dark reoxidation ofthe Cyt.

Chemicals. Acifluorfen (Blazer), nitrofen (Tok), and oxyfluor-fen (Goal) were kindly provided by Rohm and Haas Co., Phila-delphia, PA, and Bifenox (Modown) was a generous gift from theMobil Chemical Co., Richmond, VA.

RESULTS

Effect of Acifluorfen on the LIAC. The effect acifluorfen on theLIAC when the herbicide is added directly to the Triton-solubi-lized 21KP from etiolated oat coleoptiles is shown in Figure 1.Enhancement of the LIAC is almost linear as the concentration ofacifluorfen is increased from 10-6 to l0-4. However, at concentra-tions higher than l0-4 M, the enhancement effect decreases sharplyuntil there is no enhancement at a concentration of l0-3 M. Thiseffect is quite different from that observed in the membrane-bound 21KP (without Triton X100 solubilization) prepared fromcorn (10) where the enhancement is linear from 10-6 to l0-3 Macifluorfen. This discrepancy may be attributable to nonspecificbinding of the herbicide to lipids of the intact membranes (R.Hertel and E. Schafer, personal communication), but this possi-bility has not been investigated.

Effect of Acifluorfen on the Rate of Dark Reoxidation of theCyt. Since acifluorfen and similar diphyenyl ethers are known toblock electron transport in chloroplasts by inhibiting the oxidationof Cyt f (4), it seemed possible that a similar mechanism mightoccur in the present case: inhibition of reoxidation of the reducedb-type Cyt would cause a greater accumulation of reduced Cyt atsteady-state in the light in the presence of herbicide than in itsabsence.The rate of dark reoxidation of the Cyt in the presence of the

herbicide is expressed as the percentage of the rate of the herbicide-free control (Fig. 2). In the presence of acifluorfen, the rate ofdark reoxidation of the Cyt decreases with increasing concentra-tions up to 4 x l0-' M, where the inhibition is approximately 50%o.At 10-3 M, the inhibition is less pronounced, with a rate approxi-mately 80% of the control. The rate of dark reoxidation (Fig. 2)represents the rate of unloading of electrons from the Cyt, whereasenhancement of the LIAC (Fig. 1) represents the steady-statebalance of electron loading and unloading of the Cyt. If the

40

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Log [acifluorfen, MlFIG. 1. Effects of acifluorfen on LIAC of Triton-solubilized 21KP from

oat coleoptiles. Results expressed as percent buffer control. Each point isaverage of three measurements. Values were within 5% of each other foreach treatment.

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BLUE LIGHT PHOTORECEPTOR IN OATS

herbicide is acting only on dark reoxidation, the curves in Figures1 and 2 should be mirror images of each other. Except for a slightdiscrepancy at a herbicide concentration of4 x 10-4 M, the curvescomplement each other well.Double reciprocal plots of the dose-response curves ofthe LIAC

versus blue light fluence rate for the Triton-solubilized 21KP fromcorn (Fig. 2 in Ref. 9) and oats (results not shown) both yieldstraight lines with similar intercepts. Analysis of the rate of darkreoxidation of the Cyt as a function of fluence rate shows that therate is constant over the range of fluence rates used (9.8-37.1 ,umolm-2s ) (results not shown). This consistency in kinetic behaviorsuggests that a single Cyt is involved in both phenomena.

Specificity of the Action of Acifluorfen. It has been well estab-lished that blue light leads specifically to the reduction of a singleb-type Cyt in the 21KP from corn coleoptiles although there aremany other Cyt present in this fraction (7, 9). To ascertain whetheracifluorfen is blocking oxidation of only that particular b-type Cytor affecting other Cyt as well, light-minus-dark difference spectrawere obtained in the absence or presence of the herbicide (3 x10-5 M) for the Triton-solubilized 21KP from oats and comparedwith the dithionite reduced-minus-oxidized spectrum for the samepreparation (Fig. 3; cf. Ref. 7). The control light-minus-darkdifference spectrum (Fig. 3, top) indicates that blue light leads tothe reduction of a Cyt which has a difference band near 428 nmin contrast to the bulk Cyt present and reducible by dithionite(Fig. 3, bottom), just as was the case with corn preparations (7).Note that as with corn, there is some 20-fold more total Cyt in thepreparation than Cyt reducible by blue light, and the averagedifference band for this total Cyt is 424 nm. The center spectrumin Figure 3 clearly shows that acifluorfen affects only the light-sensitive Cyt system. The light-minus-dark difference peak is stillat 428 nm, and is approximately 30%o larger than the control. Anyeffect of the herbicide on the oxidation rates of other Cyt would

100

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Log [acifluorfen, MIFIG. 2. Effect of acifluorfen on rate of dark reoxidation of Cyt ex-

pressed as percentage of that for herbicide-free control. Each point isaverage of three measurements. Values were within 2% of each other foreach treatment.

a1)C

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Wave length, nm

FIG. 3. Comparison of difference spectra induced in Triton-solubilized2lKP from oat coleoptiles induced by blue actinic light in absence (top)and presence (middle) of acifluorfen (3 x 10-5 M) and by dithionite(bottom) in Soret region. Top two spectra have been multiplied by 20 forcomparison with dithionite spectrum. Difference spectra were obtained as

described by Goldsmith et aL (7). Each spectrum represents the average ofthree scans.

not be detected under these conditions. However, since darkgrowth of the seedlings is unaffected by these herbicide concen-trations (see below), it seems unlikely that respiratory Cyt are

affected.Effect of Different Diphenyl Ethers on the LIAC. The effects of

acifluorfen and three other diphenyl ethers on the LIAC obtain-able from the Triton-solubilized 21KP were compared. The resultsare summarized in Table I. In general, there was an increasingeffect of herbicide in enhancing the LIAC with increasing concen-trations, a maximum, and then a decrease in the effect with thehigher concentrations. The concentration yielding the maximumsignal varied from one compound to the next. Inhibition of theLIAC was commonly observed at the highest concentrations. Ofthe four compounds tested, acifluorfen was by far the mosteffective, and was therefore used in all of the following studiesinvolving diphenyl ethers.

Reversibility of the Effect of Acifluorfen on the LIAC in theUnsolubilized 21KP. The following experiments were designed todetermine whether acifluorfen became tightly associated withmembrane fraction in the course of its action or whether it couldbe readily removed. First, concentrations of the herbicide of 3 xl0-5, 6 x 10-5, 9 X l0-5 and 2.4 x 10-4 M were shown to enhancethe LIAC of the 21KP membranes by 7%, 9%o, 45%, and 35%,respectively. After the measurement with 2.4 x 10-4 M was made,the membranes were diluted 10-fold with buffer and centrifugedfor 45 min at 200,000g to remove the bulk of any free herbicidefrom the preparation. The pellet was then resuspended and itsLIAC measured. A LIAC 87% of the control (the LIAC of thesame preparation before addition of acifluorfen) was recovered.Such a small loss is to be expected when the membranes arerepelleted and then resuspended (7). Clearly, the herbicide doesnot become irreversibly bound, but can readily be removed.

Light minus dark

Light minus dark+ acifluorfen

/ ~~~Reduced minus oxidized

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Table I. Effects of Four Different Diphenyl Ether Herbicides on the LIAC ofthe Triton-Solubilized 21KPfrom OatsResults expressed as percentage of herbicide-free control. Each value is the average of three measurements.

Herbicide ConcentrationCommon Trade NameName 0o-6 4 x 10-6 1.4 x 10-5 4.4 x 10-' l.4xlO4 4.4x104 1.4 x103

M

Acifluorfen Blazer 109 118 130 135 127 100Oxyfluorfen Goal 99.5 107 110 90 98 98 46Nitrofen Tok 105 115 94 115 123 120 88Bifenox Modown 106 105 105 114 118 127 82

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Log [NaN3, M]FIG. 4. Effects of sodium azide on LIAC (0, ) and on rate of dark

reoxidation of the cytochrome (A, - - -) in Triton-solubilized 21KP. ValueofLIAC in absence of azide was 7.41 LIAC units (7). Each point is averageof three measurements.

Effect of Sodium Azide on the LIAC and the Dark Reoxidationof the Cyt. Sodium azide is known to inhibit corn phototropismpresumably by depopulating flavin-excited states (22) and is alsoknown to inhibit the LIAC in corn membrane preparations (5).When increasing concentrations of sodium azide are added to theTriton-solubilized oat 21KP, threshold inhibition of the LIACoccurs at l0-4 M and 50%o inhibition is obtained at approximately6 x l0-3 M (Fig. 4). This inhibitory effect is similar to that foundby Caubergs et al. (5) for corn. When the rates of dark reoxidationof the Cyt are measured at different concentrations of sodiumazide (Fig. 4, upper curve), it becomes clear that the rate ofelectron unloading from the Cyt is not affected significantly byazide. Thus the rate of loading of electrons at the Cyt (which alsodetermines the magnitude of the LIAC at steady-state) must beinhibited. Such inhibition is to be expected if the photoreceptor isa flavin. Similar results (not shown) were obtained with potassiumiodide and phenylacetic acid (concentrations for 50o inhibitionbeing 5 x 10-4 M and 5 X 102 M, respectively). These resultsconfirm previous suggestions that a flavin is involved in the bluelight photoreception for the LIAC (3, 23) and also show that thesite and mode of action of acifluorfen is different from sodiumazide and other inhibitors affecting flavins.

Effect of Acifluorfen on Phototropism. Dark-grown oat seed-lings were irradiated unilaterally with a low fluence rate blue lightsource for varying periods of timd (Fig. 5) and then returned todarkness. Phototropic curvatures were measured 100 min after theonset of the unilateral light exposure. Although the fluence-re-sponse curves show considerable variability, they clearly showfirst positive curvature, second positive curvature, and the dipbetween caused by first negative curvature, as expected fromprevious work (27). However, in the presence of 10- M herbicide(or 10-5, not shown), the peak representing first positive curvatureappears shifted to lower fluences consistently by approximately0.6 log units. This shift was seen in nine of nine experiments.Thus, in the presence of acifluorfen 3-fold less light is required toinduce the same amount of phototropic curvature. Since reciproc-ity is known to hold for first positive curvature in oats (2), theseresults suggest that the herbicide treatment leads to a decrease inthe fluence requirement rather than a decrease in the time neededfor phototropic induction.

Since the fluence-response curves showed significant variability,a single fluence (log ,umol m-2 = -2), at which a maximumherbicide effect might be expected, was used for a series ofreplicate experiments to test the apparent change more rigorously(Table II). At this fluence, the herbicide (at both 10-' and 10-' M)clearly increased the amount ofphototropic curvature as predictedby the fluence-response curves presented above (Fig. 5).A series of phototropism measurements with herbicide-treated

(10-6 to 4.4 x 10-4 M) and control seedlings were conducted asabove, with a fluence at which a strong herbicide effect might beexpected (log umol m-2 = -2). The shape of the resulting concen-tration-response curve (Fig. 6) is remarkably similar to that inFigure 1, indicating that the effect of acifluorfen on the LIAC (anin vitro response to blue light) and on phototropism (an in vivoresponse to blue light) may be closely related.

Effect of Acifluorfen on Geotropism. Although it has been welldocumented that acifluorfen action requires light (6, 12, 13, 24),it is necessary to rule out the possibility that acifluorfen may beaffecting general growth of oat seedlings in the dark. Oat seedlingsgrow normally in acifluorfen (up to a concentration of 10-4 M) inthe dark. When they are transferred to direct sunlight, the treatedplants become wilted within 2 to 3 h. This result is in agreementwith observations made with similar diphenyl ethers (24). Hence,the herbicide effect cannot simply be attributed to some nonspe-cific effect on growth.To test this conclusion further the geotropic responses of etio-

lated oat seedlings grown with or without acifluorfen were com-pared (Fig. 7). Acifluorfen clearly has no significant effect ongeotropism. This result again suggests that the herbicide requireslight for its effect and is acting close to the photoreceptor moietyin the phototropic signal transduction chain.

Finally, the possibility that growth and geotropism might beaffected only under conditions of illumination was tested. Rowsof seedlings grown in different concentrations of the herbicidewere placed in a vertical array with seedlings extending horizon-tally for geotropic induction. Coincident with the onset of geo-

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BLUE LIGHT PHOTORECEPTOR IN OATS

.6 1.0 1.4 1.8 2.2 2.6 3.0 3.4

Log time of exposure (sec)FIG. 5. Fluence-response curves for phototropism in dark-grown oat coleoptiles in absence (0, ) or presence (A, -_- -) of 10-4 M acifluorfen.

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W' Log [acifluorfen, MIFIG. 6. Effects of different concentrations of acifluorfen on phototrop-

ism of dark-grown oat coleoptiles. Log fluence (umol m-2) was -2. Eachpoint represents average of three to five rows of eight seedlings each.Values for individual rows were within 5% ofeach other for each treatment.

tropic induction, each row was given a 10-s exposure of blue lightlaterally at a fluence of l-3'umol m-2 s- as in Table II. Thepurpose of this arrangement was to ensure that phototropic andgeotropic curvatures would occur at right angles to each other,

enabling one to shadowgraph the geotropic response independentof the phototropic one. A mirror was placed behind the row ofseedlings while it was illuminated to minimize any phototropiccurvature that might occur. At 40 min from the onset of geotropicinduction, the seedlings were returned to the vertical position andallowed to develop curvatures for an additional 80 min, at whichtime the geotropic response was shadowgraphed. The results are

shown in Table III. Clearly the geotropic response is unaffectedby acifluorfen even following a blue light treatment that shows a

significant effect of the herbicide on phototropism (cf. Table II).

DISCUSSIONAlthough the action spectrum for first positive phototropic

curvature of oat coleoptiles is remarkably similar to that for theLIAC (14), it is also similar to the absorption spectrum of a

number of other flavoproteins. The action spectroscopy allowsone to state that the flavin-Cyt complex giving rise to the LIACcould be the phototropic photoreceptor, but it is hardly proof thatit is. The present results considerably strengthen the case. Theeffects of azide (Fig. 4), iodide, and phenylacetate clearly implicatea flavin for the LIAC photoreceptor, just as experiments withthese compounds implicated a flavin for the photoreceptor forphototropism of corn coleoptiles (22, 25). Flavins are implicatedin other blue light reactions as well (16, 20). Acifluorfen enhancesboth the LIAC (Fig. 1) and the first positive phototropic response

of oats on the ascending limb of the fluence-response curve (Figs.5 and 6, Table II) to the same extent. Concentration-effect curves

for both processes (Figs. I and 6) have the same shape, showinga clear maximum. However, the optimum effect for phototropismis at a concentration 1 order of magnitude below that for theLIAC. This dicrepancy could be explained if the seedlings actuallyaccumulated the herbicide (they grow on it for 72 h). However,Matsunaka (12) indicates that although these herbicides are indeedtaken up by roots, they are poorly translocated. Whether or notaccumulation can account for the discrepancy mentioned mustawait studies of uptake and distribution of the herbicide in oatseedlings grown under the present conditions. Finally, the lack ofany effect either on geotropism in the dark (Fig. 7) or in the light

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880 LEONG AND BRIUGS riant rnysiol. Vol. /U, 1ybz

Table II. Effect ofAcifluorfen on the Blue Light-Induced Phototropism in Etiolated Oat Seedlings

Phototropism experiments on 4-d-old seedlings were performed as described by Zimmerman and Briggs (26). Acifluorfen was applied in the agarmedium onto which 30-h-old seedlings were transplanted. Data were from lO s x 10-3,mOI m-2s-' exposures. Average values are shown in parentheses.

Curvature Acifluorfen- Curvature Acifluorfen-Treated to Treated to

Control I x 10' M acifluorfen Control Control 1 x 10' M acifluorfen Control

degree ratio degree ratio

11.5 + 1.3 13.5 ± 1.1 1.17 12.7 ± 1.3 18.6 ± 1.6 1.4614.3 ± 1.1 15.9 ± 1.0 1.11 10.9 ± 1.0 15.0 ± 0.8 1.3814.8 ± 1.3 21.5 ± 1.9 1.45 14.0 ± 1.5 14.5 ± 1.7 1.0415.0 ± 1.8 20.8 ± 1.5 1.39 12.1 ± 1.2 13.1 ± 1.1 1.08

12.7 ± 1.5 20.0 ± 1.8 1.57 (12.4) (15.3) (1.24)12.0 ± 1.2 14.1 ± 1.4 1.18

(13.4) (17.6) (1.31)

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Log time of stimulus (sec)FIG. 7. Dose-response curve for geotropism of dark-grown oat coleoptiles in absence (0, ) or presence (A, - - -) of 0'5 M acifluorfen. Each

point represents average of three experiments.

Table III. Test of Effect ofAcifluorfen on the Geotropic Sensitivity ofSeedlings which Received Phototropic Induction (10 s x 10-'umol m-2s-'

fluence)Details in text.

Concentration Acifluorfen Geotropic Curvature

M degree0 12.3 ± 2.24 x 10-6 13.3 ± 2.31.4 x 10- 13.3 1.54.4 x 10- 13.6 1.91.4 x 10-4 12.3 ± 1.9

(Table III) or on elongation of dark-grown seedlings suggests thatthe herbicide must be acting close to the photoreceptor moleculeitself in the reaction chain of events leading to curvature. Its actionin inhibiting dark reoxidation of the b-type Cyt in the presentpreparations (Fig. 3) is consistent with this hypothesis.The exact mode of action of acifluorfen is still unknown. The

effect in green plants is apparently initiated by absorption of light

by xanthophylls (6). As mentioned above, such herbicides inhibitthe unloading of electrons from Cyt f in the chloroplasts. Inaddition, there may be effects at the plasma membrane (15, 18).Some toxic reaction (or reactions) occur resulting in a rapidincrease in membrane permeability and death of the tissue. Haw-ton and Stobbe (8) have proposed that a light-controlled biochem-ical process causes the diphenyl ether (in their case nitrofen) topolymerize, a process that enables it to combine in some way withunidentified cell lipids. Such binding could then affect membraneproperties and lead to the effects described here.The light fluences used for the phototropic experiments were

insufficient to cause any detectable damage to the seedlings, andindeed the only noticeable result was enhancement of phototrop-ism. Whether this effect is related to the damaging effects of farlarger fluences of light remains to be determined. The high lightfluence rates used in the LIAC measurements could conceivablygive rise to acifluorfen effects differing from those induced by lowfluences in the phototropic experiments. However, the correlationbetween effects on the LIAC and on phototropism makes thispossibility unlikely.

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BLUE LIGHT PHOTORECEPTOR IN OATS

Although first positive curvature in oat coleoptiles is known tobe mediated by lateral transport of auxin (see Ref. 2), the mech-anism by which this lateral transport is induced is unknown.However, it seems reasonable that it might be caused by light-induced alteration in the potential across certain plasma mem-branes. Enhancement of phototropism would thus be expectedfrom treatments that would increase this potential. If the electrontransport chain involving flavin-Cyt complex studied here is in-deed oriented across the plasma membrane, inhibition of darkreoxidation could indeed increase the transmembrane potentialcaused by light and lead to phototropic enhancement. There isinsufficient information available to determine whether the effectdescribed here plays any significant role in the herbicidal actionof the diphenyl ether compounds.

Acknowledgments-We thank Mr. S. Graff for his skillful technical assistance. Weare grateful to Ms. D. Mandoli for her careful review of the manuscript. We alsothank Mr. J. Tepperman and Ms. S. Berry for generous help in harvesting oatcoleoptiles. Special thanks are due to Rohm and Haas Co. Ltd., and Mobil ChemicalCo. for generous gifts of diphenyl ethers. The authors are very much indebted to Dr.Wynn John of the Shell Development Co., Modesto, CA for suggesting that diphenylether herbicides might prove useful in our phototropic studies and leading us to thepertinent literature.

LITERATURE CITED

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881

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