mechanisms of hydroquinone-induced growth reduction in leafy spurge

Journal of Chemical Ecology, Vol. 25, No. 7, 1999

MECHANISMS OF HYDROQUINONE-INDUCEDGROWTH REDUCTION IN LEAFY SPURGE

RICHARD R. BARKOSKY,1,* JACK L. BUTLER,2

and FRANK A. EINHELLIG3

1 Department of Biology, Minot State UniversityMinot, North Dakota 58707

2Department of Biology, Central Missouri State UniversityWarrensburg, Missouri 64093

3 Southwest Missouri State UniversitySpringfield, Missouri 65804

(Received March 16, 1998; accepted March 14, 1999)

Abstract—Field observations indicate leafy spurge (Euphorbia esula) isinhibited by the presence of Antennaria microphylla. Hydroquinone (HQ), oneof several compounds isolated from A. microphylla has been shown to inhibitleafy spurge seed germination, root elongation, and callus culture growth. Thepresent study was designed to analyze the effects of HQ on water relations andphotosynthesis of leafy spurge. Plants grown in 0.25 mM HQ had consistentlyhigher leaf diffusive resistance and lower transpiration rates than control plants(P < 0.05). Chlorophyll fluorescence was significantly lower than controls (P <0.05) towards the end of the treatment period. At the end of the treatment,tissue from 0.25 mM HQ plants had higher levels of 13C, indicating therehad been a sustained interference with stomatal function. These data suggestthat a disruption of the plant water balance is one mechanism of leafy spurgeinhibition by A. microphylla.

Key Words—Hydroquinone, allelopathy, plant water balance, photosynthesis,chlorophyll fluorescence, 13C isotopes, leafy spurge, Euphorbia esula, smalleverlasting, Antennaria microphylla Rydb.

INTRODUCTION

Leafy spurge (Euphorbia esula L.) is a Eurasian plant species that was intro-duced onto the North America continent in the late 1800s. Since that time,

*To whom correspondence should be addressed.

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0098-0331/99/0700-1611$16.00/0 © 1999 Plenum Publishing Corporation

leafy spurge has become well established and currently infests extensive areasof rangeland throughout much of the northern Great Plains. In North Dakotaalone, leafy spurge infestations reduced the carrying capacity of grazeableland, impacting regional economies at an annual cost of about US$75 million(Leistritz et al., 1992). Much of the weed control effort focuses on conventionalcontrol/eradication programs utilizing herbicides, an approach that is expensiveand therefore limited to areas of high productivity. Alternative approaches tocontrol leafy spurge include biological insect control (Gagne, 1990; Gassmanand Shorthouse, 1990; Pecora et al., 1989, 1991) and encouraging lambs andgoats to graze preferentially on leafy spurge (Walker et al., 1992).

Leafy spurge dominates and displaces many native plant species in a vari-ety of habitats. Belcher and Wilson (1989) found that leafy spurge significantlydecreased the frequency of five common native mixed-grass species in Mani-toba, Canada. Within portions of the North Dakota Badlands, native grasslandsdeteriorate further when heavy infestations of leafy spurge cause shifts in habi-tat utilization by native ungulates (Trammel and Butler, 1995). That researchdemonstrates that bison, elk, and mule deer shift their foraging away from areasof leafy spurge infestation and towards remaining noninfested areas. Increasedgrazing of these noninfested areas could facilitate further encroachment of leafyspurge.

While many native plants are displaced by leafy spurge, small everlasting(Antennaria microphylla Rydb.), a common native forb, is phytotoxic to andresists encroachment by leafy spurge (Selleck, 1972). It is unlikely that compe-tition alone explains the inhibition of leafy spurge due to the fact that the plantis a deep rooted vigorous clonal species, whereas small everlasting is shallowrooted and grows relatively slowly. In the field, small everlasting inhibited thegrowth of leafy spurge while soil removed from around small everlasting had asimilar effect (Selleck, 1972). In this same study, plant and soil extracts of smalleverlasting inhibited leafy spurge seed germination and seedling development.Extraction and isolation of compounds from small everlasting yielded hydro-quinone (HQ), arbutin (a monoglucoside of HQ), caffeic acid, and benzoquinone(Manners and Galitz, 1985). This experiment also demonstrated that HQ was themost phytotoxic of the extracted compounds to seed germination and root elon-gation of leafy spurge. In another study, it was shown that media extracts fromcallus cell cultures of small everlasting and exogenously supplied HQ inhibitedboth leafy spurge callus tissue and suspension culture growth (Hogan and Man-ners, 1990).

Allelopathy refers to the process by which certain compounds (allelochemi-cals) produced by plants are released into the environment where they can inter-fere with the growth of other plants. In direct contrast to competition, allelo-pathic interactions involve the addition of chemical substances into the environ-ment. It has been well documented that interference by certain donor species,

1612 BARKOSKY, BUTLER, AND EINHELLIG

particularly by the release of phenolic compounds, is related to disruption of cer-tain physiological processes of target plants (Einhellig, 1986; 1995). The allelo-pathic influence of HQ on the growth of soybean has been linked to changesin plant water relations (Barkosky, 1988). One primary mechanism of interfer-ence may be a perturbation of cell membranes, which would likely influenceplant water relations and lead to a reduction in overall plant growth (Einhellig,1986, 1995). Determination of intermediate or secondary physiological effects,including interactions with plant hormones, ion uptake, mitochondrial respira-tion, and photosynthesis, could lead to further insight into the mode of action ofallelochemicals.

The focus of this investigation was to study the allelopathic mechanism ofaction of HQ on leafy spurge by relating any changes in growth to effects onwater relations and photosynthesis.

METHODS AND MATERIALS

Lateral root sections of leafy spurge, collected from a local infestation nearVermillion, South Dakota, were transplanted into 4-quart pots rilled with com-mercial potting soil and allowed to regrow. Cuttings were made by followingthe procedure outlined below that was developed by Lyn (1992) and modifiedby Barkosky (1997). Stem tips 75-80 mm long were cut from individual plantsand all but two or three of the uppermost leaves removed. The cut end wasthen dipped in commercial root hormone and placed into vermiculite moistenedwith reverse-osmosis water. After two days, cuttings were transferred to indi-vidual 400 ml opaque vials filled with 0.5 strength Hoagland's nutrient solutionfor 28 days. HQ treatments began on day 31 by transferring cuttings into nutri-ent medium amended with HQ, and solutions were replaced every three daysthroughout the treatment period to ensure consistent nutrient levels and expo-sure to HQ. In a preliminary 14-day experiment, plants were treated with 0.25,0.5, and 0.75 mM HQ in order to determine the amount of HQ needed for growthinhibition. From this experiment it was determined that 0.25 mM HQ was thelowest concentration that significantly reduced the growth of leafy spurge withinthis system. Plants were then treated for a longer duration (30 days) using 0.25and 0.1 mM HQ.

Plants were exposed to greenhouse conditions throughout the propagationand treatment period. Baseline diffusive leaf resistance and transpiration readingswere taken the day before treatment began (day 30 of the propagation period)and then every three days between 12:00 and 15:00 hr throughout the treat-ment period using a Li-Cor 1600 steady-state porometer. Measurements weremade on all six plants per treatment group on the abaxial surface of three ofthe newest leaves per plant that were sufficiently large to completely fill the

HYDROQUINONE EFFECT ON LEAFY SPURGE 1613

porometer cuvette. Chlorophyll fluorescence was chosen to evaluate the effectsof HQ on photosynthesis and was measured on the same three leaves chosenfor measurement with the porometer. Krause and Weis (1991) summarized theevidence and method showing chlorophyll fluorescence to be a valuable toolfor measuring PSII efficiency. Baseline fluorometer readings were obtained theday before treatment began and then every six days during the experiment usingan Opti-Sciences-modulated fluorometer. Leaves were dark-adapted for 15 minprior to illumination with the fluorometer. Data are expressed as the Fv/Fm ratio,a value that is an important measure of the physiological state of the photosyn-thesis activity.

At the end of the experiment, plants were harvested by removing leavesfrom the plant to obtain leaf area. All tissue was oven-dried at 105°C for 72 hr.Leaf area was determined by first photocopying all leaves from each plant andthen using a Panasonic Video Digitizer with Digital Image Analysis System com-puter software (Decagon Device, Inc., Pullman, Washington). Leaf tissue wasanalyzed for carbon isotope ratio (13C: 12C) with an isotope-ratio mass spec-trometer at the Duke University Phytotron, Durham, North Carolina. Sampleswere prepared for analysis by grinding oven-dried leaves in a Cyclone SampleMill fitted with a 0.4-mm screen. Results of this analysis are expressed as a D13Cvalue obtained from equation 1 (O'Leary, 1988).

RESULTS

Leafy spurge cuttings exposed to 0.5 mM and 0.75 mM HQ in the 14-day preliminary experiment exhibited stunted growth and had visible signs ofdamage including chlorotic looking or dead older leaves, dark purple youngerleaves, and black slimy roots. Although growth reduction occurred in plants inthe 0.25 mM HQ group, they did not exhibit the same visible signs of damage asdid plants subjected to higher concentrations. Plants in all treatment groups had


The effect of HQ on growth and carbon isotope discrimination was analyzedby using one-way analysis of variance (ANOVA) with means separated by Dun-can's multiple range test. Porometer and fluorometer data were analyzed usinga repeated measures design with treatment (HQ) and day of treatment (0-30) asindependent variables. Sidak's t test was used to compare experimental groups.All analyses were conducted using the Statistical Analysis System (SAS Institute1992).


significantly lower leaf area, leaf weight, root weight, and shoot weight whencompared to control plants (Table 1).

Data from the preliminary experiment established 0.25 mM HQ as the con-centration that would inhibit growth of leafy spurge, but also allow leaves todevelop to sufficient size for measurement with the porometer and fluorometerover a 30-day treatment period. At the end of the 30-day experiment, both the0.1 mM and 0.25 mM HQ treatments reulted in significant reductions in growthwhen compared with controls (Table 2). At both treatment levels, all growth vari-ables measured were significantly lower than control values, and all but shootweight were significantly lower between the 0.1 and 0.25 mM HQ treatmentgroups.

Beginning early in the second week of the experiment (day 9), leafy spurge

TABLE 1. EFFECT OF HYDROQUINONE (HQ) ON GROWTH OF LEAFY SPURGE AFTER14-DAY TREATMENTa

Plant variable

Leaf area (cm2)Leaf wt. (mg)Root wt. (mg)Shoot wt. (mg)Plant wt. (mg)

HQ treatment

Control

113 (5) a354 (26) a58 (5) a

223 (18) a635 (4) a

0.25 mM

76 (4) b227 (16) b

27 (4) b139 (9) b393 (3) b

0.5 mM

70 (2) bc157 (8) c

17 (2) b132 (9) b306 (1) b

0.75 mM

64 (3) c144 (10) c26 (6) b

115 (6) b285 (2) c

aMean (SE) values (N = 6) within rows not followed by the same letter are significantly different(P < 0.05), ANOVA with Duncan's multiple range test.

TABLE 2. EFFECTS OF HYDROQUINONE (HQ) ON GROWTH OF LEAFY SPURGEAFTER 30-DAY TREATMENTa

Plant variable

Leaf area (cm2)Leaf wt. (mg)Root wt. (mg)Root length (cm)Shoot wt. (mg)Shoot length (cm)Plant wt. (mg)

HQ treatment level

Control

289 (10) a985 (48) a225 (11) a23 (1) a

903 (102) a43 (2) a

2113 (80) a

0.10 mM

204 (4) b710 (46) b164 (15) b

12 (1) b384 (62) b

29 (1) b1258 (119) b

0.26 mM

90 (7) c371 (24) c

88 (6) c7 (1) c

259 (8) b20 (1) c

718 (33) c

aMean (SE) values (N = 6) in a row not followed by the same letter are significantly different(P < 0.05), ANOVA with Duncan's multiple range test.


FIG. 1. Effect of hydroquinone (HQ) on leaf diffusive resistance in leafy spurge over 30days of treatment. Each value is the mean of six plants; different letters indicate signifi-cance between groups, ac = 0.03, with Sidak's t test.

plants treated with 0.25 mM HQ had significantly higher diffusive resistance thancontrols (Figure 1). These high resistances continued throughout the duration ofthe treatment period. By day 12, plants in both treatment groups had significantlyhigher diffusive resistance than controls (Figure 1). Following a similar pattern,transpiration rates of treated plants were significantly lower than control plantsat both treatment levels by the third day of treatment (Figure 2).

Photosynthesis activity in the 0.25 mM treated plants began to declinebelow that of controls starting in the third week of the experiment, as indicatedby the Fv/Fm ratio (Figure 3). In the last week of treatment these plants also hadsignificantly lower Fv/Fm values than plants treated with 0.1 mM HQ. Plantstreated at the lower concentration of HQ also had significantly lower Fv/Fm

ratios than controls by the end of the treatment period (Figure 3).Treatment for 30 days with 0.25 mM HQ resulted in plants that had dis-

criminated less against 13C over the duration of the experiment, as indicated bya less negative D13C (Table 3). Plants treated with 0.1 mM HQ had D13C valuessimilar to controls (Table 3).


FIG. 2. Effect of hydroquinone (HQ) on transpiration in leafy spurge over 30 days oftreatment. Each value is the mean of six plants; different letters indicate significancebetween groups, ac = 0.03, with Sidak's test.

FIG. 3. Effect of hydroquinone (HQ) on chlorophyll fluorescence in leafy spurge over 30days of treatment. Each value is the mean of six plants; different letters indicate signifi-cance between groups, ac = 0.03, with Sidak's t test.


TABLE 3. EFFECT OF HYDROQUINONE (HQ) ON STABLE CARBON ISOTOPE RATIOSIN LEAFY SPURGEa

Plant parameter

D 13C (%)

HQ treatment

Control

-28.14 (0.10) a

0.10 mM

-28.16 (0.16) a

0.25 mM

-25.81 (0.09) b

aMean (SE) values (N = 6) in a row not followed by the same letter are significantly different(P < 0.05), ANOVA with Duncan's multiple range test.

DISCUSSION

These experiments clearly demonstrate that exposure to as little as 0.1 mMHQ inhibits the growth of leafy spurge at the whole plant level. Furthermore,given the close parallel between effects of growth on water status, a disrup-tion in water relations appears to be the primary mode of action that leads toreductions in overall growth. In past research, changes in water relations havebeen associated with the allelopathic action of several plants. Extracts of Kochiascoparia have been shown to increase leaf diffusive resistance and reduce leafwater potential in soybean and sorghum (Einhellig et al., 1982). In a relatedstudy, extracts of velvetleaf (Abutilon theophrasti Medic.) increased diffusiveresistance and lowered leaf water potential and relative water content in soy-bean (Colton and Einhellig, 1980). In earlier work, we determined that treat-ment with 0.25 mM HQ increased stomatal resistance, lowered transpiration,and reduced the growth of soybean seedlings (Barkosky, 1988). Data from thecurrent experiment show that leafy spurge exposed to either treatment levelsexperienced changes in stomatal function early in the experiment as evidencedby high resistances and low transpiration rates. As the experiment progressed, itis likely that this water stress led to the decline in the chlorophyll fluorescenceof treated plants.

The Fv/Fm ratio, typically 0.75-0.85 for nonstressed plants, is highly cor-related with the quantum yield of net photosynthesis (Araus and Hogan, 1994).A reduction in Fv/Fm can either indicate damage to thylakoid membranes, par-ticularly those associated with PSII reaction centers, or inhibition of excitationenergy transfer from antenna to reaction centers (Krause and Weis, 1984). Ithas been shown that disruption of photosynthetic activity can occur when avapor pressure deficit-induced stomatal closure reduces transpiration and uptakeof CO2 (Ben et al., 1987). It is also possible that, in this particular experiment,a chronic reduction in the amount of available CO2 during the 30-day exposureto HQ resulted in lower Fv/Fm values for treated plants.

Diffusional limitation of CO2 through the stomata decreases discrimina-tion against the heavy isotope, while factors influencing enzyme activity tend to

increase the discrimination (Berry, 1989; Farquhar et al., 1989; O'Leary, 1988).The carbon isotope ratios of plants treated with 0.25 mM HQ are consistentwith stomatal diffusion limitations. If HQ had negatively impacted photosyn-thetic activity directly (e.g., reducing the activity of photosynthetic enzymesor damaging thylakoid membranes), then the carbon isotope ratio would havelikely shown more discrimination against 13C and chlorophyll fluorescence mayhave declined earlier in the treatment period. Plants treated with 0.1 mM HQdid exhibit some changes in stomatal function and overall growth compared tountreated plants, but the effects were much less than the results in leafy spurgetreated with 0.25 mM HQ. Likewise, the 0.1 mM HQ plants did not discriminateagainst 13C differently than controls.

Although there is no direct evidence from this experiment, changes in waterbalance of treated plants could have been initiated at the site of action, theroot/treatment solution interface, by inhibition of ion uptake across root plasmamembranes. It has been suggested that perturbations of cell membranes by phe-nolic compounds may cause physiological changes that combine to reduce plantgrowth (Einhellig, 1986, 1995). In this model, membrane perturbations repre-sent the "the common denominator" in an array of physiological changes thatreduce plant growth. Glass and Bohm (1971) demonstrated that HQ interfereswith 86Rb+ ion uptake in barley roots, suggesting a disruption of active transport.It is possible that disruption of leafy spurge root membranes led to the physio-logical changes observed in this experiment by inhibiting ion and water uptake.According to Einhellig (1986, 1995), allelochemicals can alter membrane per-meability by disrupting structural associations, modifying membrane channels,and reducing the activity of membrane-bound carrier proteins. Baziramakengaet al. (1995) reported that the benzoic and cinnamic acids caused changes inthe content of sulfhydryl groups within membrane proteins. Any of these mem-brane effects would affect ion transport and water relations and could ultimatelyinfluence stomatal function and photosynthesis.

It is also possible that direct disruption of stomatal function, at the level ofguard cell membranes, could account for the observed changes in plant water bal-ance and that this ultimately affected ion and water uptake. Perhaps HQ directlyinfluenced guard cell membranes by a similar mechanism, as noted above forperturbation of root membranes, which then led to stomatal closure. It has alsobeen shown that allelochemicals can affect ABA levels. ABA is a phytohormonethat has a regulatory role in stomatal function and so influences water relations(Holappa and Blum, 1991).

This experiment documents some of the phytotoxic effects of HQ on thephysiology and growth of leafy spurge. This and other experiments tend to sup-port the field observation that small everlasting resists encroachment by leafyspurge. In the absence of a recognized ecological interaction, this experimentwould simply represent a bioassay-type experiment using leafy spurge as the


test species. A bioassay of this type could be useful in a program to producenaturally occurring herbicides, but would perhaps lack ecological validity. Fur-thermore, the foundation of this and other experiments lies with the premisethat HQ is released in sufficient quantities by small everlasting to inhibit thegrowth of leafy spurge. The most likely avenue of release is by way of seasonaldecomposition of small everlasting, although rot exudation may play a role. Theargument also could be made that other factors, such as interspecific competi-tion, are responsible for the observed interference, but in light of the pronouncedmorphological dichotomy between the two species, the relative contributions ofboth competition and allelopathy have to be determined to fully understand theinteraction.

In conclusion, these experiments demonstrate that HQ inhibits the growthof leafy spurge and that a disruption of plant water relations may be an importantmode of action that leads to the reduction in growth.

Acknowledgments—This material is based upon work supported by the Cooperative StateResearch Service, U.S. Department of Agriculture, under agreement No. 94-38300-0282.11.

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mechanisms of hydroquinone-induced growth reduction in leafy spurge

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