Plant Physiol. (1992) 100, 630-6360032-0889/92/1 00/0630/07/$01 .00/0

Received for publication February 24, 1992Accepted May 27, 1992

On the Mechanism of Resistance to Paraquat inHordeum glaucum and H. Ieporinum'

Delayed Inhibition of Photosynthetic 02 Evolution after Paraquat Application

Christopher Preston, Joseph A. M. Holtum, and Stephen B. Powles*Department of Crop Protection, Waite Agricultural Research Institute, The University of Adelaide,

PMB 1, Glen Osmond S.A. 5064, Australia

ABSTRACT

The mechanism of resistance to paraquat was investigated inbiotypes of Hordeum glaucum Steud. and H. leporinum Link. withhigh levels of resistance. Inhibition of photosynthetic 02 evolutionafter herbicide application was used to monitor the presence ofparaquat at the active site. Inhibition of photosynthetic 02 evolu-tion after paraquat application was delayed in both resistant bio-types compared with the susceptible biotypes; however, this dif-ferential was more pronounced in the case of H. glaucum than inH. leporinum. Similar results could be obtained with the relatedherbicide diquat. Examination of the concentration dependence ofparaquat-induced inhibition of 02 evolution showed that the re-sistant H. glaucum biotype was less affected by herbicide comparedwith the susceptible biotype 3 h after treatment at most rates. Theresistant H. leporinum biotype, in contrast, was as inhibited as thesusceptible biotype except at the higher rates. In all cases photo-synthetic 02 evolution was dramatically inhibited 24 h after treat-ment. Measurement of the amount of paraquat transported to theyoung tissue of these plants 24 h after treatment showed 57% and53% reductions in the amount of herbicide transported in the caseof the resistant H. glaucum and H. leporinum biotypes, respec-tively, compared with the susceptible biotypes. This was associatedwith 62% and 66% decreases in photosynthetic 02 evolution ofyoung leaves in the susceptible H. glaucum and H. Ieporinumbiotypes, respectively, a 39% decrease in activity for the resistantH. Ieporinum biotype, but no change in the resistant H. glaucumbiotype. Photosynthetic 02 evolution of leaf slices from resistantH. glaucum was not as inhibited by paraquat compared with thesusceptible biotype; however, those of resistant and susceptiblebiotypes of H. leporinum were equally inhibited by paraquat.Paraquat resistance in these two biotypes appears to be a conse-quence of reduced movement of the herbicide in the resistantplants; however, the mechanism involved is not the same in H.glaucum as in H. leporinum.

The bipyridylium herbicides paraquat and diquat are non-selective contact herbicides that act by intercepting electronsfrom the photosynthetic electron transport chain at PSI. Thisreaction results in the production of bipyridyl radicals thatreadily react with 02 to produce superoxide and then,

l Partially supported by an Australian Research Council Postdoc-toral Fellowship (C.P.).

through a series of reactions, produce H202 and the hydroxylradical. These toxic oxygen species cause extensive lipidperoxidation leading to loss of cell membrane integrity andrapid desiccation (2, 12).

Resistance to these herbicides has appeared in at least 10weedy species after repeated exposure to the herbicides (see8 for review). The mechanism of paraquat resistance has beeninvestigated in six of these species (6, 7, 9, 13, 16, 19);however, the mechanism of resistance has not been deter-mined unambiguously for any biotype. In two resistant bio-types, Conyza bonariensis (16) and Lolium perenne (9), it hasbeen proposed that the mechanism of resistance is a result ofincreased levels of enzymes that detoxify active 02 species.In other cases where the mechanism of paraquat resistancehas been investigated, evidence has been presented purport-ing to show reduced movement of the herbicide in the leafof the resistant plants (3, 7, 19). These studies mainly havebeen performed by feeding ['4C]paraquat through the petioleor leaf bases of detached leaves and observing its spread byautoradiography. In a paraquat-resistant biotype of Hordeumglaucum Steud., it has been shown that there is no differencein the susceptibility of protoplasts isolated from resistant andsusceptible individuals to inhibition by paraquat, suggestingthat, in this biotype, resistance might be conferred by theexclusion of herbicide from the cell (15).Here we have reinvestigated the mechanism of paraquat

resistance in a biotype of H. glaucum (3, 15) previouslyexamined and compared it with a paraquat-resistant biotypeof H. leporinum Link. We have used photosynthetic 02 evo-lution as an indicator of the appearance of paraquat at theactive site and have confirmed reduced movement of para-quat within the leaves of the resistant biotypes of H. glaucumand H. leporinum. In both cases there is reduced translocationof paraquat to the young tissue in the resistant biotypes thatremains photosynthetically active and continues to grow,whereas that of the susceptible biotypes is killed.

MATERIALS AND METHODS

Plant Material and Herbicide Application

Seeds of paraquat-resistant Hordeum glaucum Steud. andH. leporinum Link. were obtained from plants originally col-lected from two separate alfalfa fields in Victoria, each with

630

www.plant.org on August 5, 2015 - Published by www.plantphysiol.orgDownloaded from Copyright © 1992 American Society of Plant Biologists. All rights reserved.

MECHANISM OF PARAQUAT RESISTANCE IN Hordeum SP.

a long history of paraquat use. The susceptible biotypes werecollected from pastures that had no history of herbicide use.

The response to paraquat of the paraquat-resistant and pa-

raquat-susceptible biotypes has been described elsewhere (14,20). Plants were grown in soil in 18-cm pots and kept in a

growth room with a 14 h/10 h, 20°C/150C light/dark cyclewith a light intensity of 300 ,uE m-2 s-'. The plants were usedbetween 4 and 6 weeks after sowing, at which stage theywere actively tillering.

Plants were sprayed with herbicide using a custom-builtlaboratory spray cabinet (20). This was a moving-belt (1 ms-1), twin-nozzle unit that applied a total volume of 118 Lha-' at 250 kPa pressure. The herbicide products appliedwere Gramoxone (a.i.,2 200 g L` paraquat) and Reglone (a.i.,200 g L` diquat). To all spray treatments 0.2% nonionicsurfactant (Agral 600) was added. Plants were sprayed in themoming and placed in the dark immediately after treatment.

02 Evolution Measurements

02 evolution of leaf segments was measured with a Han-satech leaf disc 02 electrode at 250C. Leaf segments (2.5 cmlong) were cut from young, expanded leaves under waterand placed in an irrigated brass clip (to counter desiccation)in the chamber. The leaves were illuminated at 700 ,uE m-2s-1 in an atmosphere of 21% (v/v) 02, 5% (v/v) CO2, 74%(v/v) N2 for 10 min before measurements. The leaf segmentsused for measurements were taken from the central third ofthe leaf. The leaf area of the segments was determined afterthe O2 evolution measurements by placing the leaf segmentson photographic paper and exposing them to light. The leafarea was then calculated from the weight of a known area ofphotographic paper. For paraquat concentration dependenceexperiments, one plant in a 10-cm pot with at least threetillers was used for each treatment.

Paraquat Determinations

Paraquat in the leaf tissue was determined spectrophoto-metrically by the method described by Calderbank and Yuen(5). Plants that had been grown in the growth room were

sprayed with 200 g of paraquat per hectare then placed inthe dark. Twenty-four hours after spraying, the plants were

harvested, and the parts that had not been directly exposedto herbicide, young leaves not yet emerged, and the bases ofolder leaves were selected. These were pooled for each pot(five plants) and were boiled in 1 N H2SO4 for 4 h. Aftercooling, the solution was filtered and the paraquat separatedusing a Duolite 225 column (BDH Pharmaceuticals, London).The recovery of paraquat in the extraction process was esti-mated from control experiments, where known amounts ofparaquat were added immediately before boiling, at greaterthan 85% for all samples, and there were no differencesbetween resistant and susceptible biotypes.

2 Abbreviation: a.i., active ingredient; I50, concentration requiredfor 50% inhibition.

Photosynthesis by Leaf Slices

Leaves were sliced into approximately 0.5- to 1.0-mm-widestrips under buffer consisting of 50 mM Hepes-KOH, pH 7.6,500 mM sorbitol, and 2 mm CaCl2. The leaf slices were thentransferred to a Clark-type 02 electrode in 3 mL of the samebuffer with 10 mm NaHCO3 added. The leaf slices wereincubated in the presence of paraquat (supplied as the dich-loride salt) for 10 min in the dark and then illuminated for 5min at 1000 AE m-2 s-' when the rate of 02 evolution wasdetermined. Chl was extracted from the leaf slices by grindingin 80% acetone and the Chl concentration was determinedas described by Amon (1).

RESULTS

Paraquat Inhibition of 02 Evolution

The presence of paraquat in the chloroplast in the light hasthree important consequences. First, paraquat will acceptelectrons from PSI and inhibit CO2 fixation. Second, theparaquat radical thus formed will react with 02 to producesuperoxide and will, therefore, reduce net 02 evolution.Third, the toxic oxygen species produced as a result of para-quat action, in particular the hydroxyl radical, will rapidlydestroy chloroplast membranes, rendering the chloroplastinactive. We have used measurements of net 02 evolution toestimate the time taken for paraquat to appear in the chlo-roplasts of leaves. Paraquat-susceptible and paraquat-resist-ant plants of H. glaucum and H. leporinum were sprayed inthe laboratory sprayer and placed in the dark; photosynthetic02 evolution was then measured up to 4 h after spraying.Paraquat, when applied at 100 g a.i. ha-', rapidly enters thechloroplast of the susceptible biotypes, and appreciable in-hibition of photosynthetic 02 evolution of leaf segments canbe observed 40 min after spraying the plants (Figs. 1C and2C). Reducing the herbicide concentration delayed the onsetof inhibition of 02 evolution such that at 20 g of a.i. ha-',inhibition of 02 evolution was not observed until 2 h afterspraying of the susceptible biotypes (Figs. 1A and 2A). Ateach paraquat concentration, the inhibition profiles of thetwo paraquat-susceptible biotypes were similar.The paraquat-resistant H. glaucum biotype showed a delay

in the onset of inhibition of 02 evolution after paraquatapplication, compared with the susceptible biotype at all ratesof herbicide application (Fig. 1). The biggest difference be-tween the two biotypes was observed at 50 g of a.i. ha-',where inhibition of 02 evolution of the resistant biotype wasdelayed 1.5 h compared with the susceptible biotype (Fig.1B). In contrast to the situation observed for paraquat-resist-ant H. glaucum, the differences in onset of inhibition of 02evolution between paraquat-susceptible and paraquat-resist-ant H. leporinum were small (Fig. 2). The largest difference intime of onset of inhibition of 02 evolution was observed at50 g of a.i. ha-', where inhibition of 02 evolution of theresistant H. leporinum biotype was delayed by 0.5 h comparedwith the susceptible biotype (Fig. 2B).The inhibition of 02 evolution in response to paraquat dose

was examined in a series of experiments in which plants weresprayed with herbicide and then placed in the dark. Photo-synthetic activity of leaf segments from each plant was meas-

631

www.plant.org on August 5, 2015 - Published by www.plantphysiol.orgDownloaded from Copyright © 1992 American Society of Plant Biologists. All rights reserved.

Plant Physiol. Vol. 100, 1992

20R A

15

10

'.5

0 . 20 g a.l. ha"0 __B

20E 15

C

.2102 3%ft.ie fersraig h

0 5

2(AS0(,10)g f.i.ha-'B 20cCDu

15R

10S

5

100 g a.l. ha0 -

0 1 2 3 4Time after spraying (h)

Figure 1. Time course of inhibition of photosynthetic 02 evolutionin paraquat-susceptible (0) and paraquat-resistant (0) biotypes ofH. glaucum after application of paraquat. Paraquat was applied at20 (A), 50 (B), or 100 (C) g of a.i. ha'. Each point is the mean (±SE)of four experiments.

ured before spraying and 3 h after spraying. Three hoursafter spraying, the plants were retured to the growth roomand a third measurement was taken 24 h after spraying(Fig. 3A). Photosynthetic02 evolution of the susceptible H.glaucum biotype was inhibited by paraquat 3 h after sprayingat all herbicide concentrations applied and was completelyinhibited by application of 200 g of a.i. ha-'. In contrast tothe susceptible biotype, the lower (0-30 g of a.i. ha-') con-centrationsof paraquat had little impact on photosynthetic02 evolution of the resistant H. glaucum biotype, however, athigher paraquat concentrations 02 evolution was inhibited,almost completely so, by 200 g of a.i. ha-'. At most herbicideconcentrations, 02 evolution activity of the paraquat-resistantH. glaucum biotype was less inhibited by the herbicide appli-cation than was that of the paraquat-susceptible biotype.Photosynthetic 02 evolution of both biotypes of H. glaucumwas dramatically inhibited 24 h after herbicide application atall paraquat concentrations (Fig. 3B). There was no differencebetween the paraquat-susceptible and paraquat-resistant bio-types except at the lowest two (10 and 20 g of a.i. ha-')

concentrations, where the resistant biotype had a net positive02 evolution rate, whereas the susceptible biotype had a netnegative rate.The same experiment performed on paraquat-susceptible

and paraquat-resistant H. leporinum biotypes produced aslightly different result. Photosynthetic 02 evolution wasequally inhibited in the two biotypes 3 h after paraquattreatment except at the highest two rates (100 and 200 g ofa.i. ha-') (Fig. 4A). By 24 h after treatment, photosynthetic02 evolution had been greatly inhibited for both biotypes atall herbicide concentrations (Fig. 4B). No recovery of photo-synthetic activity occurred in the 24 h after paraquat treat-ment for any of the biotypes examined here.

Diquat Inhibition of 02 Evolution

The paraquat-resistant biotypes of H. glaucum and H. le-porinum show considerably greater tolerance to the relatedbipyridyl herbicide diquat than do the susceptible biotypes(14, 20). Diquat inhibition of photosynthetic 02 evolutionwas examined in the same way as for paraquat and, like

20

15

10

,0 5'(ACo

0h 200E 15

co 10-a

%.-0c 20cCu

15

10

5100 ga.1. ha'l s\,

a

\NO -.-0 1 2 3

Time after spraying (h)

Figure 2. Time course of inhibition of photosynthetic 02 evolutionin paraquat-susceptible (0) and paraquat-resistant (0) biotypes ofH. leporinum after application of paraquat. Paraquat was applied at20 (A), 50 (B), or 100 (C) g of a.i. ha-'. Each point is the mean (±SE)of four experiments.

632 PRESTON ET AL.

www.plant.org on August 5, 2015 - Published by www.plantphysiol.orgDownloaded from Copyright © 1992 American Society of Plant Biologists. All rights reserved.

MECHANISM OF PARAQUAT RESISTANCE IN Hordeum SP.

I-

Cl

E

000E

0._

0

0S-00-Ww:

25

20

15

10

5

0

-525

20

15

10

5

0

-50 50 100 150 200

Paraquat applied (g a.i. ha-1)

Figure 3. Concentration dependence of paraquat inhibition of 02evolution in paraquat-susceptible (0) and paraquat-resistant (0)biotypes of H. glaucum 3 h (A) and 24 h (B) after herbicideapplication. Each measurement is the mean (±SE) of four experi-ments. Values below 0 denote net uptake of 02.

20

v-# 15co

10E

coC O

O -5._

> 15

0 10> 150

-5

-10

0 50 100 150 200

Paraquat applied (g a.i. ha-1)

Figure 4. Concentration dependence of paraquat inhibition of 02

evolution in paraquat-susceptible (0) and paraquat-resistant (0)biotypes of H. Ieporinum 3 h (A) and 24 h (B) after herbicideapplication. Each measurement is the mean (±SE) of four experi-ments. Values below 0 denote net uptake of 02.

20

w-

'0 15

0 10o0

E 5

0: 20

0 15N0S.-o 100

cc 5

00 1 2 3

Time after spraying (h)4

Figure 5. Time course of inhibition of photosynthetic 02 evolutionin paraquat-susceptible (0) and paraquat-resistant (0) biotypes ofH. glaucum (A) and H. leporinum (B) after application of diquat.Diquat was applied at 200 g of a.i. ha-'. Each point is the mean(±SE) of four experiments.

paraquat, diquat rapidly inhibited 02 evolution in both sus-ceptible biotypes (Fig. 5). The concentration of diquat re-quired to elicit the same response was greater than for para-quat, consistent with the reduced efficacy of diquat on grassspecies (4). For both paraquat-resistant biotypes there was adelay in the inhibition of photosynthetic 02 evolution of theresistant compared with the susceptible biotypes; however,this delay was greater for H. glaucum than for H. leporinum(Fig. 5). The responses of the two paraquat-resistant biotypesto paraquat and diquat are similar, which suggests that themechanisms of resistance to the two herbicides are likely tobe the same within each biotype.

Paraquat Transport

The 02 evolution data presented above suggest that thereis reduced movement of paraquat in the leaves of the para-quat-resistant individuals. Field observations are that theparaquat-resistant individuals of H. glaucum and H. leporinumsuffer injury from the herbicide, but then grow back fromundamaged tissue, whereas the susceptible biotype does notregrow. We tested the hypothesis that paraquat resistance isa consequence of reduced translocation of herbicide to thegrowing points by directly measuring the amount of paraquatin the young tissue 24 h after the application of 200 g of a.i.ha-' paraquat to intact potted plants. Using a spectrophoto-metric method for detecting paraquat residues in plant tissue(5), a technique that does not detect the photochemical deg-radation products of paraquat (18), we observed that the

633

www.plant.org on August 5, 2015 - Published by www.plantphysiol.orgDownloaded from Copyright © 1992 American Society of Plant Biologists. All rights reserved.

Plant Physiol. Vol. 100, 1992

amount of paraquat translocated to the young tissue wasreduced in the paraquat-resistant biotypes of H. glaucum andH. leporinum by 57% and 53%, respectively (Table I). Moreparaquat was detected in the young tissue of H. glaucum thanin H. leporinum, but the reason for this is not known.

In a separate experiment, resistant and susceptible plantswere treated with paraquat and placed in the dark for 24 h.The young, unsprayed leaves were dissected out of the leafsheath, and photosynthetic 02 evolution was measured usingthe leaf disc 02 electrode. The results presented in Table Ishow that photosynthetic 02 evolution of these leaves fromthe resistant H. glaucum biotype was largely unaffected bythe application of paraquat, and that of the resistant H.leporinum biotype was inhibited by 39%. Young leaves fromthe susceptible biotypes showed 62% and 66% decreases inphotosynthetic 02 evolution for H. glaucum and H. leporinum,respectively.

Leaf Slice Photosynthesis

No difference in susceptibility of photosynthetic 02 evo-lution to inhibition by paraquat between the resistant andsusceptible biotypes of H. glaucum was observed at the iso-lated protoplast level (15), yet there is a difference when leafsegments are used (Fig. 1). Powles and Comic (15) suggestedthat the cell walls of H. glaucum may be involved in resistanceto paraquat. After unsuccessful attempts to isolate intact cellsfrom these species, this hypothesis was tested using thin(0.5-1 mm) slices of leaf tissue exposed to different concen-trations of paraquat. Paraquat was readily able to inhibit 02evolution activity of the susceptible H. glaucum biotype withan I50 of about 100 ,M (Fig. 6A). Leaf slices from the resistantH. glaucum biotype proved to be less susceptible to inhibitionby paraquat compared with the susceptible biotype and hadan Iso of >500 LM. Photosynthetic 02 evolution of leaf slicesfrom the susceptible H. leporinum biotype was as sensitive toparaquat as was that from the susceptible H. glaucum biotype(Fig. 6B). In contrast to the situation in H. glaucum, thephotosynthetic 02 evolution of leaf slices from the resistantH. leporinum biotype was equally sensitive to paraquat com-pared with the susceptible biotype. These data suggest that

Table I. Paraquat Content and Photosynthetic 02 Evolution of theYoung Tissue of Paraquat-Resistant and Paraquat-SusceptibleBiotypes of H. glaucum and H. leporinum 24 h after Treatment with200 g of a.i. ha-' ParaquatThe measurements of paraquat content and 02 evolution were

obtained in separate experiments. Values are the means (±SE) offour samples.

Species Biotype Paraquat Content Photosynthetion2Jg (g fresh weight)' 1urmol (mg Chl)` h-

H. glaucum Susceptible 3.45 ± 0.20 125 ± 22 (38)Resistant 1.54 ± 0.19 329 ± 14 (93)

H. leporinum Susceptible 1.78 ± 0.05 132 ± 28 (34)Resistant 0.84 ± 0.08 214 ± 20 (61)

Values in parentheses are percentage of those obtained fromcontrol plants treated with wetting agent only.

80 H. gwucum

;; 60

0O0

20

N 100 B

80 H. leporinumco

60 s

40

20R

00 100 200 300 400 500

Paraquat concentration (pM)

Figure 6. Paraquat inhibition of 02 evolution of leaf slices fromparaquat-susceptible (0) and paraquat-resistant (0) biotypes of H.glaucum (A) and H. leporinum (B). Each point is the mean (±SE) offour experiments. One hundred percent activity was 75 + 4, 69 +2, 45 + 6, and 47 ± 3 ,Amol of 02 evolved (mg Chl)-' h-1 for theparaquat-susceptible and paraquat-resistant H. glaucum biotypes,and paraquat-susceptible and paraquat-resistant H. leporinum bio-types, respectively.

there is a barrier to movement of the herbicide from outsidethe cell to the chloroplast in the paraquat-resistant biotypeof H. glaucum, a barrier that is not present in any of the otherbiotypes.

DISCUSSION

Previous investigations on the mechanism of resistance toparaquat in a biotype of H. glaucum have shown that resist-ance is not due any change at the active site or to changes inpermeability of the chloroplastic or plasma membranes to theherbicide (15). In addition, levels of the enzymes that detoxifyactive 02 species were not increased in the resistant biotypecompared with the susceptible biotype (15). Herbicide pene-tration of the cuticle was also not different between theresistant and susceptible biotypes (3). It was suggested thatparaquat resistance in this biotype was due to exclusion ofthe herbicide from the cytoplasm through sequestration inthe apoplast (3, 15).

Paraquat is a photosynthetic herbicide that subverts elec-tron transport. This property of paraquat was used to trackmovement of the herbicide in the leaves of the resistant andsusceptible biotypes of H. glaucum and H. leporinum. Theresults of these experiments demonstrate that paraquat takeslonger to reach an inhibitory concentration in the chloroplastsof the resistant biotypes compared with the susceptible bio-types. The time differential for paraquat to inhibit photosyn-thetic 02 evolution between the resistant and susceptible

PRESTON ET AL.634

www.plant.org on August 5, 2015 - Published by www.plantphysiol.orgDownloaded from Copyright © 1992 American Society of Plant Biologists. All rights reserved.

MECHANISM OF PARAQUAT RESISTANCE IN Hordeum SP.

biotypes was greater in the case of H. glaucum than H.leporinum. Photosynthetic 02 evolution, once inhibited,stayed inhibited for at least 24 h after treatment. The simi-larity of the results obtained with paraquat and diquat sug-gests that a single mechanism might provide resistance toboth herbicides in each species. The response of photosyn-thetic 02 evolution of the resistant biotypes of H. glaucumand H. leporinum to paraquat application (Figs. 1-4) is incontrast to that reported for other paraquat-resistant weedbiotypes. Paraquat-resistant biotypes of C. canadiensis (13)and Lolium perenne (10) showed little or no inhibition ofphotosynthetic activity, measured as CO2 fixation, up to 4 hafter treatment with paraquat, whereas photosynthetic activ-ity of the paraquat-susceptible biotypes was rapidly andpermanently inhibited. In contrast, photosynthetic activity,measured as 14C02 fixation, was reported to be transientlyinhibited and then to recover in a paraquat-resistant biotypeof C bonariensis (11, 17). Increased levels of the enzymesthat detoxify active 02 species have been implicated in pa-raquat resistance in C bonariensis (16) and L. perenne (9), butnot in C canadiensis (13) or in other studies on C. bonariensis(21). On the basis of differences in response of photosyntheticactivity to herbicide application, we suggest that the mecha-nism of resistance to paraquat in H. glaucum and H. leporinummust differ from that in C bonariensis, C. canadiensis, and L.perenne.The delay in inhibition of photosynthetic 02 evolution of

the resistant biotypes of H. glaucum and H. leporinum afterparaquat or diquat application suggests that reduced herbi-cide movement may be a mechanism of resistance. Anyreduced movement observed within leaf segments should,on a larger scale, result in reduced movement of paraquat tothe meristematic tissue. This hypothesis was confirmed bymeasuring the amount of herbicide in the young tissue 24 hafter treatment. Paraquat contents of this tissue were foundto be reduced by 53% to 57% in the paraquat-resistantbiotypes compared with the paraquat-susceptible biotypes(Table I). Reduced herbicide movement already has beenreported in paraquat-resistant biotypes of C bonariensis(7), Erigeron philadelphicus (19), and H. glaucum (3) where['4C]paraquat has been fed through the petiole or leaf basesof detached leaves. Here, in contrast, we have demonstrateda significantly reduced movement of paraquat in a basipetaldirection in paraquat-resistant plants treated and grown un-der conditions and herbicide dosages relevant to the fieldsituation. This reduced translocation of paraquat is correlatedwith a reduced inhibition of photosynthetic activity of theyoung leaves in the resistant biotypes. Photosynthetic activityof the young leaves 24 h after spraying of both susceptiblebiotypes was inhibited by more than 60%, whereas that ofthe resistant H. glaucum biotype was unaffected by paraquatapplication and that of the resistant H. leporinum biotype wasinhibited by 39%.The paraquat that was observed in the young leaves of the

resistant H. glaucum biotype had not reached the active sitebecause no inhibition of 02 evolution was observed. Incontrast, a proportion of the translocated herbicide in thesusceptible biotypes and the resistant H. leporinum biotypehad reached the active site, as indicated by the reduced levelsof 02 evolution. The differences in photosynthetic 02 evo-

lution of the young leaves of the two resistant biotypes afterparaquat application is indicative of a possible difference incompartmentation of the herbicide between the two resistantbiotypes.The biochemical mechanism that reduces movement of

paraquat in the resistant biotypes of H. glaucum and H.leporinum has not yet been elucidated; however, the mecha-nism may be different in the two resistant biotypes. Studieswith leaf slices (Fig. 6) show that for H. glaucum there isreduced efficacy of herbicide movement across the cell walland into the chloroplast. Whether this is due to increasedbinding of herbicide in the cell wall as proposed by Bishopet al. (3) or whether some other mechanism is involvedremains to be seen. In contrast, the resistant H. leporinumshows no restriction of paraquat movement into the cell, sothe cell wall does not contribute to resistance in this species.Instead, the mechanism for resistance in this biotype mayinvolve reduced movement of paraquat into the vasculartissue, resulting in reduced export of herbicide from the leaf;however, this remains to be determined. In conclusion, re-sistance to paraquat in the biotypes of both H. glaucum andH. leporinum is a result of reduced paraquat movement fromthe treated leaves; however, the way this is achieved appearsto be different between the two biotypes.

LITERATURE CITED

1. Arnon DI (1949) Copper enzymes in isolated chloroplasts. Po-lyphenoloxidase in Beta vulgaris. Plant Physiol 24: 1-15

2. Babbs CF, Pham JA, Coolbaugh RC (1989) Lethal hydroxylradical production in paraquat-treated plants. Plant Physiol90: 1267-1270

3. Bishop T, Powles SB, Cornic G (1987) Mechanism of paraquatresistance in Hordeum glaucum. II. Paraquat uptake and trans-location. Aust J Plant Physiol 14: 539-547

4. Calderbank A, Slade P (1976) Diquat and paraquat. In PCKeamey and DD Kaufman, eds, Herbicides. Chemistry, Deg-radation and Mode of Action, Vol. 2. Marcel Dekker Inc., NewYork, pp 501-540

5. Calderbank A, Yuen SH (1965) An ion-exchange method fordetermining paraquat residues in food crops. Analyst 90:99-106

6. Carroll EW, Schwarz OJ, Hickock LG (1988) Biochemical stud-ies of paraquat-tolerant mutants of the fern Ceratopteris ri-chardii. Plant Physiol 87: 651-654

7. Fuerst EP, Nakatani HY, Dodge AD, Penner D, Arntzen CJ(1985) Paraquat resistance in Conyza. Plant Physiol 77:984-989

8. Fuerst EP, Vaughn KC (1990) Mechanisms of paraquat resist-ance. Weed Technol 4: 150-156

9. Harper DB, Harvey BMR (1978) Mechanism of paraquat toler-ance in perennial ryegrass. II. Role of superoxide dismutase,catalase and peroxidase. Plant Cell Environ 1: 211-215

10. Harvey BMR, Fraser TW (1980) Paraquat tolerant and suscep-tible perennial ryegrasses: effects of paraquat treatment oncarbon dioxide uptake and ultrastructure of photosyntheticcells. Plant Cell Environ 3: 107-117

11. Jansen MAK, Malan C, Shaaltiel Y, Gressel J (1990) Mode ofevolved photooxidant resistance to herbicides and xenobiotics.Z Naturforsch 45c: 463-469

12. Kunert KJ, Dodge AD (1989) Herbicide-induced radical damageand antioxidative systems. In P. Boger and G. Sandmann, eds,Target Sites of Herbicide Action. CRC Press Inc., Boca Raton,FL, pp 45-63

13. Polos E, Mikulas J, Szigeti Z, Matkovics B, Hai DQ, ParduczA, Lehoczki E (1988) Paraquat and atrazine co-resistance inConyza canadiensis (L.) Cronq. Pestic Biochem Physiol 30:142-154

635

www.plant.org on August 5, 2015 - Published by www.plantphysiol.orgDownloaded from Copyright © 1992 American Society of Plant Biologists. All rights reserved.

PRESTON ET AL.

14. Powles SB (1986) Appearance of a biotype of the weed, Hordeumglaucum Steud., resistant to the herbicide paraquat. Weed Res26: 167-172

15. Powles SB, Cornic G (1987) Mechanism of paraquat resistancein Hordeum glaucum. I. Studies with isolated organelles andenzymes. Aust J Plant Physiol 14: 81-89

16. Shaaltiel Y, Gressel J (1986) Multienzyme oxygen radical de-toxifying system correlated with paraquat resistance in Conyzabonariensis. Pestic Biochem Physiol 26: 22-28

17. Shaaltiel Y, Gressel J (1987) Kinetic analysis of resistance toparaquat in Conyza. Evidence that paraquat transiently inhibitsleaf chloroplast reactions in resistant plants. Plant Physiol 85:

Plant Physiol. Vol. 100, 1992

869-87118. Slade P (1966) The fate of paraquat applied to plants. Weed Res

6: 158-16719. Tanaka Y, Chisaka H, Saka H (1986) Movement of paraquat in

resistant and susceptible biotypes of Erigeron philadelphicusand E. canadiensis. Physiol Plant 66: 605-608

20. Tucker ES, Powles SB (1991) A biotype of hare barley (Hordeumleporinum) resistant to paraquat and diquat. Weed Sci 39:159-162

21. Vaughn KC, Fuerst EP (1985) Structural and physiologicalstudies of paraquat-resistant Conyza. Pestic Biochem Physiol24: 86-94

636

www.plant.org on August 5, 2015 - Published by www.plantphysiol.orgDownloaded from Copyright © 1992 American Society of Plant Biologists. All rights reserved.