bioregulatory effects of the fungicidal strobilurin kresoxim-methyl in wheat ( ...

Pestic. Sci. 1997, 50, 11È20

Bioregulatory Effects of the Fungicidal StrobilurinKresoxim-methyl in Wheat (Triticum aestivum)Klaus Grossmann* & Gu� nter Retzlaþ

BASF Agricultural Research Station, D-67114 Limburgerhof, Germany

(Received 22 July 1996 ; revised version received 22 October 1996 ; accepted 21 January 1997)

Abstract : Apart from its fungicidal e†ect, the strobilurin kresoxim-methyl (BAS490 F) was found to induce physiological and developmental alterations in wheat(T riticum aestivum L.) which are seen in connection with improved yield. In aseries of biotests including heterotrophic maize and photoautotrophic algal cellsuspensions, duckweed, isolated mustard shoots and germinating cress seeds,kresoxim-methyl showed a similar response pattern to standard auxins (e.g.indol-3-ylacetic acid, IAA; 2-(1-naphthyl)acetic acid, a-NAA). Auxin-like activityof kresoxim-methyl was also found when stem explants of tobacco were culturedon a hormone-free medium. Kresoxim-methyl stimulated shoot formation, par-ticularly at 10~7 M. The same e†ect was induced by 10~8 M IAA. The determi-nation of phytohormone-like substances in shoots of wheat plants foliar-treatedwith 7] 10~4 M kresoxim-methyl revealed only slightly changed levels ofendogenous IAA, gibberellins and abscisic acid. In contrast, the contents of dihy-drozeatin riboside-type cytokinins increased to 160% of the control, while trans-zeatin riboside- and isopentenyladenosine-type cytokinins remained nearlyunchanged. The most remarkable alterations were the reductions in 1-aminocyclopropane-1-carboxylic acid (ACC) levels and ethylene formation whichwere demonstrated in intact plants, leaf discs and the shoots of wheat subjectedto drought stress. Kresoxim-methyl a†ected the induction of ACC synthaseactivity which converts S-adenosyl-methionine to ACC in ethylene biosynthesis.In shoots from foliar-treated wheat plants, 10~4 M kresoxim-methyl inhibitedstress-induced increases in endogenous ACC synthase activity, ACC levels andethylene formation by approximately 50%. Reductions in ACC synthase activityand ACC levels of 30% were also obtained at low concentrations of a-NAA(10~6 M). In contrast, ACC synthase activity in vitro was not inÑuenced byadding the compounds. In wheat leaf discs, the inhibiting e†ect of kresoxim-methyl, a-NAA and IAA on ethylene formation was accompanied by delayed leafsenescence, characterized by reduced chlorophyll loss. However, in contrast tokresoxim-methyl which showed only inhibitory activity on ethylene synthesisover a wide range of concentrations applied, the auxins stimulated ethylene pro-duction at high concentrations of about 10~4 M. The inhibition of ethylene bio-synthesis by kresoxim-methyl, together with an increase in endogenouscytokinins could explain the retardation of senescence and the intensiÐed greenleaf pigmentation in wheat exposed to this strobilurin.

Key words : 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, auxin activ-ity, cytokinins, ethylene biosynthesis, kresoxim-methyl, leaf senescence

1 INTRODUCTION

Kresoxim-methyl (BAS 490 F) belongs to a new class offungicides which are derived from the fungal secondary

* To whom correspondence should be addressed.

metabolite strobilurin.1,2 The compound is character-ized by its broad-spectrum fungicidal activity againstnumerous foliar pathogens from the ascomycetes,basidiomycetes, deuteromycetes and oomycetes in e.g.pome fruits, grape vine, vegetables and cereals. Theuptake and translocation properties provide protective,

11Pestic. Sci. 0031-613X/97/$17.50 1997 SCI. Printed in Great Britain(

12 Klaus Grossmann, Gu� nter Retzla†

curative, eradicative and long residual disease control.3The mode of action is the e†ective inhibition of mito-chondrial respiration by blocking electron transfer atthe complex. The binding site is thecytochrome-bc1ubihydroquinone cytochrome-c oxidoreductase.1,2,4Studies using mitochondrial preparations from yeast,Botrytis cinerea, house Ñy, rat and maize revealed littlevariation in enzymatic sensitivity to strobilurins at themitochondrial target level.2 The biokinetic properties ofstrobilurins (uptake, transport, metabolism) are decisivefor species selectivity.2

In addition to its direct e†ect on the fungus,kresoxim-methyl has been found to induce physiologi-cal alterations in various crops which are often seen inconnection with a positive inÑuence on yield.5 In wheat,these non-fungicidal, secondary e†ects include a darkergreen appearance of the leaves, retardation of senes-cence with enhanced concentrations of chlorophyll andprotein and increased biomass production withfavoured harvest index.5 Kresoxim-methyl signiÐcantlychanged the carbon dioxide compensation point ofwheat plants in favour of an improved photosyntheticcarbon dioxide uptake as opposed to the respiratorycarbon dioxide release.6 This e†ect could be explainedby a transient inhibition of plant respiration which wasobserved in wheat plants after foliar treatment withkresoxim-methyl (unpublished data). The compounddid not alter the photosynthetic electron transport inphotosystem II, as measured in the Hill reaction withisolated wheat thylakoids (unpublished data). In conse-quence, an increase in shoot growth and thus in thephotosynthetically active green tissues was observed(unpublished results) which led to further increases inthe photosynthetic capacity of treated plants. However,these interactions are not sufficient to account for theall physiological and developmental e†ects elicited bythe compound. Particularly, the delay in senescencecaused by kresoxim-methyl in wheat can kindle specula-tions as to whether changes in the hormone status ofthe plant may be implicated in the underlying mode ofaction. Possible e†ects of kresoxim-methyl on phyto-hormonal metabolism or signalling as well as a bio-regulatory “hormone-likeÏ activity of the compounditself might play a role.

In the present work a set of selected biotests usingmaize cell suspensions, algae, duckweed, isolatedmustard shoots and germinating cress seeds was used toobtain a physiological Ðngerprint of kresoxim-methyl.The response pattern was compared with proÐles ofcompounds with known modes of action to classify thebioregulatory activity of kresoxim-methyl. Since simi-larities to auxins were found, kresoxim-methyl wasfurther characterized in bioassays for auxin-like activity.In addition, phytohormone levels of indol-3-ylaceticacid (IAA), gibberellins, abscisic acid (ABA), cytokininsand ethylene were determined in wheat plants, followingfoliar treatment with the compound. The e†ects, partic-

ularly on 1-aminocyclopropane-1-carboxylic acid (ACC)production in ethylene biosynthesis, were characterizedand compared with the induced physiological alter-ations in biomass production and senescence.

2 MATERIALS AND METHODS

2.1 Chemicals

The fungicide kresoxim-methyl (methyl (E)-methoxy-imino-a-(o-tolyloxy)-o-tolylacetate ; BAS 490 F, Fig. 1)was from BASF AG, Ludwigshafen, Germany. 2-Chloroethylphosphonic acid (ethephon) was obtainedfrom Calbiochem (Frankfurt, Germany), aminoethoxy-vinylglycine (AVG) from Fluka (Neu-Ulm, Germany),indol-3-ylacetic acid (IAA), 2-(1-naphthyl)acetic acid (a-NAA), 2-methyl-4-chlorophenoxyacetic acid (MCPA)and 2,4-dichlorophenoxyacetic acid (2,4-D) from Riedel-de Haen (Seelze, Germany). The other compounds,including 6-benzylaminopurine (BAP) were obtainedfrom Sigma (Mu� nchen, Germany). Stock solutions ofthe chemical compounds in acetone (kresoxim-methyl,IAA, a-NAA, MCPA, 2,4-D) or in water (AVG,ethephon) were diluted 100-fold in the test.

2.2 Bioassays

2.2.1 Maize cell suspension bioassayFreely suspended callus cells from maize (Zea mays L.cv. Black Mexican Sweet) were cultivated in a modiÐedMurashigeÈSkoog medium and the bioassay was per-formed as described previously.7 The cells were sub-cultured at seven-day intervals. Acetone solutions of thecompounds were pipetted into plastic tubes and thesolvent allowed to evaporate before adding 2 ml ofexponentially growing cell suspension. The tubes (threereplicates) were shaken at 400 rev min~1 and 25¡C inthe dark on a rotary shaker. After incubation for eightdays, the conductivity of the medium was measured asthe growth parameter.

2.2.2 Algae bioassayCells of Scenedesmus acutus (culture collection Go� t-tingen, 276-3a) were propagated photoautotrophically.7

Fig. 1. Structural formula of kresoxim-methyl.

Bioregulatory e†ects of kresoxim-methyl in wheat 13

The bioassay was carried out in plastic microtitre dishescontaining 24 wells.7 Before loading the wells with0É5 ml cell suspension each (two replicates), 0É5 mlmedium and compound in acetone solution were added,allowing the organic solvent to volatilize. The 15 addi-tional compartments between the wells were Ðlled withsodium carbonate/bicarbonate bu†er. The dishes weresealed with plastic lids and incubated on a shaker undercontinuous light at 23¡C. After 24 h, the contents ofeach well were removed and cell numbers were deter-mined by a Coulter Counter.

2.2.3 L emna bioassayStock cultures of L emna paucicostata L. were propagat-ed mixotrophically in an inorganic medium containingsucrose.7 The bioassay was conducted under asepticconditions in plastic Petri dishes (5 cm in diameter).7The test compounds were added to the dishes inacetone solution and the organic solvent allowed tovolatilize before loading them with 15 ml mediumwithout sucrose followed by four fronds each. Theculture dishes were then closed with plastic lids andincubated under continuous light (Philips TL whiteneon tubes, c.40 kmol m~2 s~1, 400È750 nm) in agrowth chamber at 25¡C. Eight days after treatment, theincrease of the area covered by the fronds in each dishwas determined as the growth parameter using animage analysing system.7 The area of fronds beforeincubation was subtracted from this value.

2.2.4 Isolated shoot bioassaySeedlings of mustard (Sinapis alba L.) were grown understandardized greenhouse conditions. The shoots wereremoved, weighed and placed upright in plastic vials(25 mm in diameter, 38 mm in height ; Greiner, Nu� rt-ingen, Germany) containing 12 ml double-distilledwater and the test compound added in acetone solution.To avoid evaporation, the vials were closed with plasticcovers with slits into which the shoots were Ðtted (threeshoots per vial). The vials were cultivated in growthchambers with 16/8 h light/dark cycles at 24/22¡C(light : Osram krypton 100-W lamps and Osram univer-sal white neon tubes, c.200 kmol m~2 s~1, 400È750 nm). After three days the changes in fresh weightwere measured by weighing the shoots and subtractingthe value from the weight at the beginning.

2.2.5 Cress germination bioassaySeeds of cress (L epidium sativum L.) were placed inplastic Petri dishes (5 cm in diameter) Ðlled with a ver-miculite substrate. Stock solutions of the test com-pounds in acetone were added, together with 8 mlwater.7 Control seeds were moistened only with waterand acetone. The dishes were incubated in a growthchamber at 25¡C in the dark until the onset of germi-nation (approximately two days). Then, they were kept

for Ðve days at room temperature and normal day/nightconditions in the laboratory. Inhibition of germinationwas evaluated visually (0\ no inÑuence, 100\ totalinhibition).

The results were expressed as percentage inhibition orreduction relative to untreated controls. Mean values ofthree replicates are given as the percentage growth inhi-bition relative to the control. Individual standard errorswere less than 5%.

2.3 Experiments with tobacco stem explants

Tobacco plants (Nicotiana tabacum L. cv. Samsun NN)were raised on a nutrient-supplemented peat-based sub-strate under controlled conditions. Pieces of stem 15 cmin length were excised from the stem approximately20 cm below the shoot apex of plants in Ñower (80 cmin height). The leaves and lateral buds were removedand the stem pieces were cut into 2-cm segments whichwere surface-sterilized with 70% ethanol for 2 min andwith 6% sodium hypochlorite for 10 min. Afterwards,the outer layers of tissue were peeled o† and the seg-ments were cut transversely into discs, each about 2 mmthick. The stem discs were halved and transferred to ste-rilized cylindrical plastic containers (65 mm in diameter,65 mm in height ; Greiner, Nu� rtingen, Germany ; threediscs per container, three replicates) with 65 ml modi-Ðed MurashigeÈSkoog medium with amino acids andvitamins7 and the test compound (in acetone or watersolution) solidiÐed with agar (1É1%). The containerswere sealed with plastic covers and incubated for 10weeks under dim light at 25¡C.

2.4 Experiments with wheat leaf discs

Discs (0É4 cm in diameter) were cut from leaf blades ofwheat (T riticum aestivum L. cv. Kanzler) with a cork-borer and Ñoated for c.1 h in Petri dishes containingdouble-distilled water so that stress ethylene induced bythe excision process could dissipate.8 Twenty random-ized discs were placed adaxially on a Ðlter paper in aPetri dish (5 cm in diameter) moistened with 1 ml MES(2-[N-morpholino]ethanesulfonic acid) bu†er (pH 6É1 ;10 mM) containing the test compounds. In one setof experiments, the leaf discs were incubated in con-tinuous light (c.55 kmol m~2 s~1, 400È750 nm, Osramuniversal white neon tubes) at 25¡C for 20 h. The Ðlterpapers with leaf discs were then rolled cylindrically,placed in plastic tubes (13 mm in diameter, 65 mm inheight) and sealed with rubber caps. After incubation inlight at 25¡C for 5 h, ethylene formation was quantiÐedby gas chromatography.8 In a parallel experiment, leafdiscs in Petri dishes were incubated in darkness at 25¡Cfor three days, harvested and powdered under liquidnitrogen. Total chlorophyll was extracted with cold


acetone (0É2 g plant material in 10 ml of 80% acetone)in triplicate and quantiÐed.9

2.5 Experiments with wheat plants

Young wheat plants (T riticum aestivum L. cv. Kanzler)were raised in vermiculite substrate moistened withhalf-strength LinsmaierÈSkoog10 nutrient solutionunder controlled conditions.11 Uniformly developedplants which had reached the 2nd-leaf stage (approx.14 cm in height) were transferred to 320-ml glass vesselswith half-strength LinsmaierÈSkoog medium and placedin growth chambers with 16/8 h light/dark cycles at25/20¡C and 75% relative humidity (nine plants pervessel, Ðve replications ; light : Osram Powerstar HQI-R250 W/NDL and Osram Krypton 100-W lamps,c.530 kmol m~2 s~1, 400È750 nm.11 The solutions wereaerated throughout the experiments. After one day ofadaptation, plants were sprayed uniformly with 0É5 mlper vessel of an aqueous solution containing kresoxim-methyl (7] 10~4 M, equivalent to 13 kg per plant,300 g ha~1 related to the treated area of the glassvessel) prepared in acetone (16 g litre~1 Ðnalconcentration). In control treatments, aqueous solutionscontaining corresponding acetone concentrations butwithout kresoxim-methyl were applied, with no adversee†ect on the growth of the plants. To avoid possiblekresoxim-methyl e†ects on untreated plants via the gasphase, controls were cultivated in a separate growthchamber set to give identical environmental conditions.At various times after treatment, growth parameterswere measured and shoots and roots from replicatevessels were harvested, immediately frozen in solidcarbon dioxide and stored at [80¡C.

In a further set of experiments, wheat plants wereraised in vermiculite substrate and groups of plantswere directly sprayed with kresoxim-methyl or a-NAA(0É5 ml on 80 plants). After 3 h incubation in growthchambers under the conditions described above, waterstress was initiated by incubating detached shoots at22¡C and 50% relative humidity. After 1 h, shoots wereharvested, immediately frozen in solid carbon dioxideand stored at [80¡C. For determination of ethyleneformation, detached and stressed shoots were subse-quently incubated in glass tubes (17 mm in diameter,200 mm in height ; three shoots per tube) which weresealed with rubber caps. After incubation in light at25¡C for 2 h, ethylene formation was quantiÐed by gaschromatography.8

2.6 Analytical determinations

2.6.1 Determination of ACC and ethylenePlant material was powdered under liquid nitrogen andsamples (100 mg, three replications) were extracted with70% aqueous ethanol. The ACC content was assayed,

following conversion to ethylene,12 by gas chromatog-raphy.8

2.6.2 Determination of ACC synthase activityACC synthase (EC 4.4.1.4) was extracted and assayed asdescribed previously.13 Powdered plant material (1É5 g ;two replicates) was homogenized in 100 mM EPPS (N-[2-hydroxyethyl]piperazine-N@-3-propanesulfonic acid)/KOH bu†er (pH 8É5) containing dithiothreitol (5 mM),pyridoxal phosphate (6 kM), leupeptin (10 kM) and Pefa-bloc SC (4-(2-aminoethyl)phenylsulfonylÑuoride hydro-chloride, Merck, Darmstadt, Germany ; 10 kM). Theextract was centrifuged and the supernatant was passedthrough a Sephadex G 25 column (Pharmacia, Uppsala,Sweden) which had been equilibrated with EPPS bu†er(5 mM ; pH 8É5) containing dithiothreitol (1 mM), pyri-doxal phosphate (6 kM) and Pefabloc SC (10 kM). TheACC synthase assay mixture, with a total volume of0É6 ml, contained 0É3 ml enzyme preparation in 80 mM

EPPS bu†er with 20 kM pyridoxal phosphate and100 kM S-adenosyl methionine. After an incubationperiod of 2 h at 37¡C, the reaction was stopped byadding 20 kmol mercury (II) chloride. Subsequently, theACC produced was determined by chemical conversionto ethylene.12 All assays were performed with four repli-cates. Protein was determined in the enzyme extracts14using the Bio-Rad protein assay reagent (Mu� nchen,Germany) with bovine serum albumin as a standard.For investigation of kresoxim-methyl e†ects on ACCsynthase activity in vitro, ACC synthase was extractedfrom water-stressed shoot material and 60 kl of anaqueous solution containing kresoxim-methyl preparedas a stock solution in dimethyl sulfoxide (50 g litre~1Ðnal concentration in the assay) was added to the assaymixture before incubation.

2.6.3 Extraction and determination of phytohormonesPowdered plant material (1 g) was extracted with 80%methanol (three replicate extractions) and the extractswere passed through a phase prepackedC18-reversedcolumn (SEPPAK; Waters, Ko� nigstein, Germany) asdescribed.15,16 The effluent was concentrated undervacuum and dissolved in double-distilled water (3 ml).T rans-zeatin riboside (ZR)-, dihydrozeatin riboside(DZR)- and isopentenyladenosine (iPA)-type cytokininsand (])-ABA in an aliquot of the extract (1É5 ml) wereseparated by high performance liquid chromatography(HPLC) on a reverse-phase Nucleosil 120-5 km C18column using a linear gradient from methanol] water(5] 95 by volume) containing 0É1 M acetic acid(solution A) to methanol ] water (95] 5 by volume)(solution B). The fractions containing the phytohor-mones were collected and the quantitative determi-nation was performed by enzyme-immunoassay withmonoclonal antibodies 100% reactive against theirrespective antigens.15 For determination of gibberellins


(GAs) and IAA, the residual 1É5 ml of the effluent fromthe SEPPAK column was acidiÐed with 1 M hydro-chloric acid, and extracted with ethyl acetate (3] 3 ml).After concentration to dryness, the extract wasredissolved in methanol (200 kl) and methylated withethereal diazomethane.16 IAA methyl ester content wasdirectly assayed by enzyme-immunoassay, while GAswere quantiÐed by radioimmunoassay with a polyclonalantiserum against methyl ester.15 The antibodiesGA1were kindly provided by Professor E. W. Weiler(University of Bochum, Germany). The detection limitwas c.1 pmol for IAA and 0É1 pmol for all other phyto-hormones, as estimated from standard curves. Allsamples were assayed at least in triplicate and the con-centrations were expressed as the equivalents of phyto-hormone in pmol g~1 fresh weight. Internalperformance controls of assay accuracy and reliabilitywere carried out as described.15,16

All experiments were repeated at least twice andproved to be reproducible. The results of a representa-tive experiment are shown.

3 RESULTS AND DISCUSSION

3.1 Physiological proÐling using bioassays

Recently, we have developed a set of Ðve bioassaysusing heterotrophic maize and photoautotrophic green

algae (Scenedesmus acutus) cell suspensions, duckweed(L emna paucicostata), isolated mustard shoots and ger-minating cress seeds which are useful to characterizeand classify the physiological mode of action of newcompounds in the search for their biochemical targets.7The response pattern, which includes inÑuences on ger-mination and the vegetative growth of cells andorganized tissues, represents a physiological Ðngerprintof a compound. The results can be interpreted directlyor a library of response patterns of compounds withknown mode of action can be screened for similarities.Although this is not an absolutely conclusive way oftesting for any speciÐc mode of action, it does providesome clues which can be used as an aid to direct furtherinvestigations. At the high concentrations appliedkresoxim-methyl inhibited growth of maize cell suspen-sions, L emna and germinating cress seeds (Fig. 2). Nopreference either for the heterotrophic (maize cell sus-pension, germination of cress seeds) or photoautotrop-hic (algae, L emna, mustard shoot) systems was observed.Green algae and isolated mustard shoots showed onlyslight response. In L emna growth inhibition was accom-panied by an intensiÐed chlorophyll pigmentation.Nearly the same type of physiological proÐle in thevarious systems was obtained with natural (IAA) andsynthetic auxins (a-NAA) and auxin herbicides, such asMCPA and 2,4-D (Fig. 2). Maize cell suspensions,L emna and germinating cress seeds were highly a†ected,whereas green algae and mustard shoots showed only

Fig. 2. E†ects of kresoxim-methyl 2-(1-naphthyl)acetic acid (a-NAA), indol-3-yl acetic acid (IAA), 2-methyl-4-chlorophenoxyaceticacid (MCPA) and 2,4-dichlorophenoxyacetic acid (2,4-D) in biotests including maize and algal cell suspensions, duckweed, isolated

mustard shoots and germinating cress seeds. SE of the mean in all cases less than 5%.


low susceptibility. However, in contrast to kresoxim-methyl, the auxin compounds induced epinastic defor-mation of cress seedlings. Auxins are known to regulateplant growth and development by inÑuencing funda-mental processes, such as cell elongation and division.17However, their e†ects on growth are extremely diverseand biphasic. Dependent on the concentration and bio-logical activity, opposite e†ects are observed.18 Withregard to its physiological proÐle, it is suggested thatkresoxim-methyl might possess auxin-like activity. Inaccordance with this conclusion, kresoxim-methyl wasable to alleviate growth inhibition in L emna caused by10~5 M BAP (Fig. 3). This anticytokinin e†ect was alsoobserved after the application of auxins, such as a-NAA(Fig. 3). Furthermore, when stem explants of tobaccowere cultured in vitro on a medium containingkresoxim-methyl, but no phytohormones, morpho-genesis of shoots and subsequently roots was induced,particularly at concentrations of about 10~7 M (Fig. 4).In the control explants only proliferation of callus wasobserved. Explants grown on 10~4 to 10~6 M IAA pro-duced exclusively callus (not shown). At concentrationsof 10~8 M IAA, shoot initiation was induced (notshown). However, it should be mentioned that the stem

Fig. 3. InÑuence of kresoxim-methyl (KM) and 2-(1-naphthyl)acetic acid (a-NAA) on 6-benzylaminopurine (BAP)-inducedinhibition of growth in L emna paucicostata. SE of the mean in

all cases less than 5%.

Fig. 4. InÑuence of kresoxim-methyl (KM) on organ forma-tion in stem explants of tobacco after 10 weeks of incubation

on agar-solidiÐed medium.

explants used in this study included vascular tissuewhich could supply phytohormones such as cytokininsendogenously19 which might have inÑuenced theresponse of the explant tissue to the exogenouslyapplied compounds.

3.2 Phytohormonal changes in wheat plants

In order to obtain a clearer picture of whetherkresoxim-methyl itself has auxin-like activity or thecompound is able to change the auxin status in planta,the e†ects of the compound on the contents of IAA andother groups of phytohormones, including immunoreac-tive gibberellins, cytokinins and ABA in shoots of wheatplants were studied. In addition, the contents of 1-aminocyclopropane-1-carboxylic acid (ACC), the directprecursor of ethylene in its biosynthesis,20 were deter-mined. After foliar treatment with 7] 10~4 M

kresoxim-methyl for 48 h, the levels of IAA, andGA1-ABA-like material were slightly increased in the shoottissue (Fig. 5). Levels of ZR- and particularly DZR-typecytokinins in the shoot increased to 113% and 160% ofthe control, respectively, while iPA-type cytokinins werereduced, though not signiÐcantly. Concomitantly, areduction of nearly 30% in ACC levels, relative to con-trols, was observed (Fig. 5). When plants were incubatedfor a further six days after treatment with kresoxim-methyl, a 12% increase in plant fresh weight comparedto controls was found.

In conclusion, kresoxim-methyl did not markedlystimulate endogenous auxin levels. On the contrary,when determined 24 h after foliar treatment, evendecreased levels of IAA in the shoot tissue wereobserved (data not shown). The compound increasedparticularly the amount of immunoreactive DZR-typecytokinins in the shoot tissue, whereas ACC levelsdeclined. The latter e†ect might be explained by aninhibitory inÑuence of kresoxim-methyl on a step priorto the conversion of ACC to ethylene in its biosyntheticpathway. In accordance with this hypothesis, kresoxim-


Fig. 5. Changes in immunoreactive phytohormone levels and1-aminocyclopropane-1-carboxylic acid (ACC) contents inshoots of wheat plants after foliar treatment with kresoxim-methyl for 48 h. Control values (^SE) representing 100%levels of phytohormone-like material (pmol equivalent g~1fresh weight) were : ACC, 1920(^200) ; ZR, trans-zeatin ribo-side, 8(^1) ; DZR, dihydrozeatin riboside, 7(^1) ; iPA, iso-pentenyladenosine, 102(^4) ; ABA, (])-abscisic acid, 19(^2) ;IAA, indol-3-ylacetic acid (1052^ 21) ; gibberellinGA1, A1,28(^1).

methyl was shown to decrease ACC levels and ethyleneformation by 46% and 26%, respectively, in hetero-trophic cell suspension cultures of sunÑower after treat-ment with 10~5 M of the compound for seven days (F.Siefert and K. Grossmann, unpublished data).

3.3 InÑuence on ACC synthase activity

The key enzyme in ethylene biosynthesis is ACC syn-thase which catalyses the conversion of S-adenosylmethionine to ACC.20 Auxins have beenshown to regulate de-novo synthesis of this enzyme.20Since the levels of ACC synthase activity in youngwheat plants were very low, we stress-induced the enzy-matic activity in order to evaluate more precisely a pos-sible inÑuence of kresoxim-methyl on ACC synthaseactivity in vivo. First, plants were foliar-treated withincreasing concentrations of kresoxim-methyl for 3 h.Subsequently, water stress was initiated by allowingdetached shoots to lose fresh weight under reducedhumidity conditions. During 1 h of stress, the freshweight of detached shoots decreased by approximately12%, while ACC synthase activity increased about80-fold relative to intact, non-stressed shoots. Depen-dent on the concentration, kresoxim-methyl e†ectivelyreduced ACC synthase activity and ACC levels in thetissue by 50% at 10~4 M (Fig. 6). Under these condi-tions, ethylene formation was inhibited by 36% (from1É43 to 0É92 nmol g~1 FW h~1). Foliar treatment ofwheat plants with a-NAA also decreased this enzymaticactivity (Fig. 6). Reductions of up to 30% were obtainedat low concentrations of about 10~6 M. Similar changesin ACC synthase activity per mg protein were induced(not shown). Closely correlated with the reducedenzyme activity, ACC levels in the tissue declined belowthose of controls (Fig. 6). In contrast, additions of

Fig. 6. E†ects of kresoxim-methyl and 2-(1-naphthyl)aceticacid (a-NAA) on 1-aminocyclopropane-1-carboxylic acid(ACC) synthase activity and ACC levels in shoots from wheat.Wheat plants were foliar-treated with the compounds for 3 h.Shoots were isolated and water-stressed. Vertical bars rep-resent SE of the mean. Results of two separate experiments

are shown.

kresoxim-methyl or a-NAA in concentrations of 10~7to 3 ] 10~4 M did not alter the in-vitro activity of ACCsynthase extracted from detached shoots subjected todrought (results not shown). Similarly, the free acid ofkresoxim-methyl, which is the Ðrst metabolite in wheat,was not active in the enzyme assay. Thus, as previouslyshown for auxins,20 kresoxim-methyl could play a regu-latory role in the induction process of de-novo enzymesynthesis. Contrary to auxins, kresoxim-methyl appearsto function only as an inhibiting factor.

3.4 Ethylene formation and leaf senescence

Ethylene is involved in responses to stress and in devel-opmental processes such as germination, ripening andsenescence.21 While cytokinins possess senescence-delaying activity,22,23 ethylene is a well knownenhancer of the senescence syndrome, including chlo-rophyll degradation.21 Experiments with wheat leafdiscs were conducted to show relations between theinÑuence of kresoxim-methyl on ethylene productionand leaf senescence. The e†ects were compared with the


action of AVG, an inhibitor of ACC synthase, the ethyl-ene generator ethephon21 and the auxins IAA anda-NAA (Figs 7, 8). After exposure of wheat leaf discs to10~5 or 10~4 M kresoxim-methyl for 24 h, ethylene for-mation was reduced by up to 40% (Fig. 7). Correspond-ing e†ects on ethylene formation were induced by10~5 M a-NAA and, with less efficiency, IAA. However,at 10~4 M, the auxins stimulated ethylene production,whereas kresoxim-methyl showed a dose-dependent

Fig. 7. E†ects of kresoxim-methyl (KM), 2-(1-naphthyl)aceticacid (a-NAA), indol-3-yl acetic acid (IAA) and amino-ethoxyvinylglycine (AVG) on the formation of ethylene inwheat leaf discs after 25 h of treatment. Vertical bars represent

SE of the mean.

inhibition which was also observed under dark condi-tions (not shown). This result corresponds with thee†ects of the compounds on ACC synthase activity.

When leaf discs were incubated in the dark for threedays, kresoxim-methyl delayed leaf senescence, charac-terized by reduced chlorophyll loss, at concentrations of10~4 M and above (Fig. 8). a-NAA and IAA were activeeven at a concentration of 10~5 M (Fig. 8). AVG, whiche†ectively inhibited ethylene formation (Fig. 7), alsoretarded leaf senescence, whereas ethephon acceleratedthis process (Fig. 8). This indicates that the inhibition ofethylene synthesis caused by kresoxim-methyl appearsto be related to its senescence-delaying activity.

4 CONCLUSIONS

In addition to the fungicidal e†ect of kresoxim-methyl,physiological and developmental alterations have beenobserved which could contribute to an improvedharvest index in wheat.5 High yields and a better cerealquality in wheat have also been reported for thestrobilurin-type fungicide azoxystrobin.24 Kresoxim-methyl is thought to increase the area of photosyntheticgreen tissues and consequently to improve the assimi-lation rate in wheat by favouring the Ðxation of carbondioxide, ultimately through an inhibitory inÑuence onplant respiration (Fig. 9). The latter e†ect is most likelydue to a direct interference of kresoxim-methyl with themitochondrial electron transport in wheat, because stro-bilurins have been shown to block the complex, notbc1only in mitochondrial preparations from yeast, but alsofrom maize.2 In addition, kresoxim-methyl showed

Fig. 8. InÑuence of kresoxim-methyl (KM), indol-3-yl acetic acid (IAA), 2-(1-naphthyl)acetic acid (a-NAA), amino-ethoxyvinylglycine (AVG) and ethephon on total chlorophyll content of wheat leaf discs after treatment for three days. Vertical

bars represent SE of the mean.


Fig. 9. Proposed model of the bioregulatory action of kresoxim-methyl in wheat. The arrows represent a direct or indirect e†ect oncarbon dioxide assimilation and the metabolism of phytohormones and possible relations to growth and yield formation processes.

ACC, 1-aminocyclopropane-1-carboxylic acid ; DZR, dihydrozeatin riboside.

auxin-like properties in a series of biotests and inducedchanges in the hormone status in wheat. The mostremarkable alteration was the inhibition of ethylenebiosynthesis which was demonstrated in leaf discs,intact plants and water-stressed shoots. Kresoxim-methyl a†ected the rate of conversion of S-adenosylmethionine to ACC in the pathway. It isassumed that the compound interfered with the induc-tion process of ACC synthase (Fig. 9), becausekresoxim-methyl inhibited e†ectively the rise in ACCsynthase activity in wheat shoots under drought stress,whereas enzyme activity in vitro was not inÑuenced byadding the compound. Using four gene-speciÐc probesfor ACC synthase25 (a generous gift of Prof. S. F. Yang,University of Hong Kong), it was shown that kresoxim-methyl did not inÑuence the increase in ACC synthasetranscripts in wheat shoots after drought stress (F.Scheltrup, J. Lerchl & K. Grossmann, unpublishedresults). Therefore, regulation of ACC synthase bykresoxim-methyl might be at the post-transcriptionallevel. Increased ethylene production is induced inresponse to a multiplicity of abiotic (e.g. temperatureextremes, drought, excessive water, wounding, bending,chemicals) and biotic (e.g. pathogen infection, insectdamage) environmental stresses.21 Ethylene also playsan important role in the regulation of developmentalprocesses, including plant aging and reproductivegrowth.21 In crops like wheat, stress ethylene impairsyield, particularly through an acceleration of leaf senes-cence and the initiation of premature ripening of thegrain which cut short the production of assimilates andthe duration of grain Ðlling. Consequently, in its func-tion as an inhibitor of ethylene biosynthesis, kresoxim-methyl could alleviate an ethylene-inducedover-reaction of the plant to unfavourable environ-mental stress conditions and thus have a positive inÑu-ence on yield. The compound could maintain or extendthe functioning of the assimilating organs and could

prolong and even intensify the partitioning of photo-synthetic products to the grain which results in a higherharvest index and improved grain weight (Fig. 9). In theexperiments using wheat leaf discs, kresoxim-methylreduced loss of chlorophyll as a parameter for theprogress in senescence. The e†ect was preceded by aninhibition of ethylene formation. A comparison of thee†ects elicited by compounds which interfere with ethyl-ene formation (AVG, ethephon) indicated that, inwheat, a reduced rate of ethylene production is, indeed,causally connected with delayed leaf senescence.

Auxins are well known to regulate ethylene pro-duction at the level of ACC synthesis. Following theliterature, only stimulatory e†ects of auxins on ethylenebiosynthesis through de-novo induction of ACC syn-thase have been reported.20,21 However, at very lowconcentrations of auxins such as a-NAA, we observedan inhibition of ACC synthase activity and ethylene for-mation in wheat. This result correlates with the biphasicbehaviour of auxins in the regulation of plant growth :at low concentrations growth is promoted, whereas, athigher levels, phytotoxic inhibition of growth andstimulation of ethylene synthesis are elicited.18However, in contrast to auxins, kresoxim-methylshowed only inhibitory activity on ethylene synthesisover a wide range of concentrations. In addition, intreated wheat plants, kresoxim-methyl appeared to shiftthe hormonal balance in favour of cytokinins ratherthan of ethylene. Since ethylene has been suggested toaccelerate the breakdown of cytokinins26 throughstimulation of cytokinin oxidase activity,21 reducedlevels of ethylene should therefore result in maintainedor increased cytokinin contents (Fig. 9). Besides theirsenescence-delaying potential in wheat leaves,23 cyto-kinins are also well-known enhancers of light-inducedchlorophyll and thylakoid formation.27,28 On the otherhand, ethylene has been shown to increase chlorophyllcatabolism in various leaves.21 Therefore, the induced


shift in the hormonal balance might also favour chloro-plast development and chlorophyll synthesis whichcould contribute to the intensiÐed green pigmentation(greening e†ect) of wheat plants exposed to kresoxim-methyl (Fig. 9).

ACKNOWLEDGEMENTS

The authors would like to thank Prof. Elmar W. Weiler(Univ. of Bochum) for a generous gift of antibodies fordetermination of phytohormones and Peter Ewert,Sigrid Hennig, Simone Huber, Anneliese Keller, JacekKwiatkowski and Heinrich Siebecker for technicalassistance and Alan Akers for critical reading of theEnglish manuscript.

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