auxin herbicides: current status of mechanism and …passel.unl.edu/image/robles...

11

3

ReviewReceived: 30 June 2009 Revised: 30 July 2009 Accepted: 4 August 2009 Published online in Wiley Interscience: 12 October 2009

(www.interscience.wiley.com) DOI 10.1002/ps.1860

Auxin herbicides: current status of mechanismand mode of actionKlaus Grossmann∗

Abstract

Synthetic compounds that act like phytohormonal ‘superauxins’ have been among the most successful herbicides used inagriculture for more than 60 years. These so-called auxin herbicides are more stable in planta than the main natural auxin,indole-3-acetic acid (IAA), and show systemic mobility and selective action, preferentially against dicot weeds in cereal crops.They belong to different chemical classes, which include phenoxycarboxylic acids, benzoic acids, pyridinecarboxylic acids,aromatic carboxymethyl derivatives and quinolinecarboxylic acids. The recent identification of receptors for auxin perceptionand the discovery of a new hormone interaction in signalling between auxin, ethylene and the upregulation of abscisicacid biosynthesis account for a large part of the repertoire of auxin-herbicide-mediated responses, which include growthinhibition, senescence and tissue decay in sensitive dicots. An additional phenomenon is caused by the quinolinecarboxylic acidquinclorac, which also controls grass weeds. Here, the accumulation of phytotoxic levels of tissue cyanide, derived ultimatelyfrom quinclorac-stimulated ethylene biosynthesis, plays a key role in eliciting the herbicidal symptoms in sensitive grasses.c© 2009 Society of Chemical Industry

Keywords: abscisic acid; auxin herbicides; auxin signalling; cyanide; ethylene

1 INTRODUCTIONIn higher plants, regulation and coordination of metabolism,growth, morphogenesis and responses to biotic and abiotic factorsare mediated by signalling molecules, called phytohormones,which exert their influence by interacting with specific cellularproteins called receptors. Auxins are an important class ofphytohormones, consisting of indole-3-acetic acid (IAA), theprincipal natural auxin in higher plants, and related endogenousmolecules which cause the same responses as IAA.1,2 Because IAAinfluences virtually every aspect of plant growth and development,IAA is thought to act as a ‘master hormone’ in the complex networkof interactions with other phytohormones.3 Auxins generallyregulate cell division and elongation and developmental processesincluding vascular tissue and floral meristem differentiation,leaf initiation, phyllotaxy, senescence, apical dominance androot formation. Auxins are also essential components in tropicresponses.

As early as the 1940s, academic and industrial laboratorieswere able to synthesise an array of derivatives of IAA, including1-naphthalene acetic acid (1-NAA) and the phenoxycarboxylicacids 2-methyl-4-chlorophenoxyacetic acid (MCPA) and 2,4-dichlorophenoxy acetic acid (2,4-D), which were among the mostauxin-active molecules tested at that time.4 – 7 These derivativeselicit the same type of plant responses as IAA, but with a long-lasting and stronger intensity of action, particularly owing to theirhigher stability in the plant. Natural auxins like IAA are subjectedto rapid inactivation through conjugation and degradationby multiple pathways in the plant.1 When present at lowconcentration at the cellular sites of action, they stimulate growthand developmental processes. With increasing concentrationand auxin activity in the tissue, growth is disturbed and theplant is lethally damaged. Consequently, chemical manipulationof the auxin system by these synthetic analogues has gained

considerable importance for probing auxin function in basicresearch8 as well as for applied aspects. Synthetic auxins areused not only as growth regulators for yield improvement inhorticulture and agriculture9 and as media components in tissueculture and plant micropropagation,10 but also as herbicides forweed control.5 – 7 With their worldwide market introduction afterWorld War II, the so-called growth regulator or auxin herbicides2,4-D and MCPA started a new era of weed control in modernagriculture. They exert selective action, preferentially againstdicot weeds in cereal crops, and are translocated systemicallyin the plant. Over the years, various chemical classes of auxinherbicides, with different weed spectra and types of selectivity,have been synthesised and commercially introduced. Currently,these classes include phenoxycarboxylic acids, benzoic acids,pyridinecarboxylic acids, aromatic carboxymethyl derivatives andquinolinecarboxylic acids (Fig. 1). Metabolism to non-phytotoxicmolecules and target-site sensitivity to the compound play themajor role in the selectivity differences of auxin herbicides betweenmonocots and dicots as well as among dicots.4 – 6 As a structuralrequirement for auxin activity, a strong negative charge on thecarboxyl group of the dissociated molecule, which is separatedfrom a weaker positive charge on the planar aromatic ringwith a distinct distance, appears to be essential.11 In additionto concentration effects, the spectrum of biological activities ofsynthetic auxins depends on tissue sensitivity, which is determinedby the type of tissue, physiological stage and plant species, andis probably mediated by differentially elicited signal transductionpathways. When applied as herbicides, synthetic auxins mimic

∗ Correspondence to: Klaus Grossmann, BASF Agricultural Centre Limburgerhof,D-67117 Limburgerhof, Germany. E-mail: [email protected]

BASF Agricultural Centre Limburgerhof, D-67117 Limburgerhof, Germany

Pest Manag Sci 2010; 66: 113–120 www.soci.org c© 2009 Society of Chemical Industry

11

4

www.soci.org K Grossmann

Figure 1. Structures of natural (indole-3-acetic acid) and synthetic herbicides belonging to different chemical classes.

the deformative and growth-inhibiting effects caused by IAA at aconstant very high concentration in the tissue4 – 7 and observedin transgenic, IAA-overproducing plants.12 This phenomenon hasbeen described as an auxin overdose or as an effect of supraoptimalendogenous auxin concentrations which lead to an imbalance inauxin homeostasis and interactions with other hormones in thetissue. However, since Gilbert’s statement from 194613 that auxinherbicides cause susceptible plants ‘to grow themselves to death’,the hypothesis prevailing until recently mainly seized on theobserved growth abnormalities and a subsequent catastrophescenario. Here, a continuous stimulation of plant metabolism wasthought to elicit a deregulation of growth through distorted celldivision and expansion, leading to the collapse of the correlatingplant growth structure.14 However, a more specific mode andmechanism of action underlying growth inhibition and deathwas suggested by the high level of species selectivity of mostauxin herbicides, combined with their rapid and in some casesstereoselective activity (e.g. herbicidally active (+)-D-enantiomersuch as dichlorprop-P) (Fig. 1) at low application rates.6

2 AUXIN OVERDOSE AND THE DEREGULA-TION OF GROWTH2.1 Metabolic and physiological processesWhen dissecting the time course of events with increasing con-centrations in the tissue and distribution (gradient development)in the plant, the deregulation of plant growth by auxin herbi-cides or IAA at high concentrations can be divided into threephases.4,5,7,14 In Fig. 2, these processes are exemplified for thedicot weed cleavers (Galium aparine L.) against the backgroundof reported data in the literature. The first is a stimulation phase,which occurs within the first hours after application. This phase in-cludes the activation of metabolic processes such as stimulation ofethylene biosynthesis through induction of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase in the shoot tissue (1–2 h),followed by symptoms of abnormal (deregulated) growth (3–4 h),including leaf epinasty, tissue swelling and initiation of stem curl-ing. Here, activation within minutes of membrane ion channelsand plasmalemma H+-ATPases is known to be involved in the

cell elongation response. Subsequently, abscisic acid (ABA) accu-mulates, initially detectable in the shoot tissue after 5–8 h. Thesecond phase, which occurs within 24 h, includes growth inhibitionof the root and, to a greater extent, of the shoot, with decreasedinternode elongation and leaf area, and intensified green leafpigmentation. Concomitantly, stomatal closure, paralleled by re-duced transpiration, carbon assimilation and starch formation, andoverproduction of reactive oxygen species (ROS) are observed. Thethird phase is the phase of senescence and tissue decay, whichis characterised by accelerated foliar senescence with chloroplastdamage and progressive chlorosis, and by the destruction of mem-brane and vascular system integrity, leading to wilting, necrosisand finally to plant death. Consequently, the stimulation and sub-sequent inhibition phases both apply to metabolism and growth,ultimately leading to the phytotoxic effect of herbicidal auxins,basically caused by their persistently high intensity of action inthe tissue. From the molecular point of view, extensive percep-tion by auxin receptors, which regulate gene expression as wellas rapidly activating membrane ion channels and plasmalemmaH+-ATPases in the cell elongation response, was assumed tobe the target process in herbicide action as a consequence ofsupraoptimal endogenous auxin concentrations.4 – 7,15

2.2 Auxin perception, signalling and gene expressionOver decades, a goal of research on auxin molecular biologyhas been centred on the identification of receptors that mediatetranscriptional and biochemical responses to auxin. In the 1980s,biochemical approaches revealed auxin-binding protein 1 (ABP1)as a candidate receptor by virtue of its auxin-binding activity.1,2,15

Later, genetic screens for Arabidopsis mutants that are resistantto the action of auxins or auxin transport inhibitors havedefined several genetic loci involved in auxin signalling, includingtransport inhibitor response TIR1.1,2,15 The recent identificationof TIR1 protein as a receptor for auxin perception16 – 19 and thediscovery of a new hormone interaction in signalling betweenauxin, ethylene and the upregulation of 9-cis-epoxycarotenoiddioxygenase (NCED) in ABA biosynthesis20 account for a large partof the repertoire of auxin-herbicide-mediated responses (Fig. 3).

www.interscience.wiley.com/journal/ps c© 2009 Society of Chemical Industry Pest Manag Sci 2010; 66: 113–120

11

5

Auxin herbicides www.soci.org

Figure 2. Three-phase response in auxin herbicide action exemplified for the dicot weed Galium aparine after root uptake and distribution in theplant (shown by coloration in autoradiographs using labelled compound) against the background of reported data in the literature. Modified fromGrossmann.40.

The F-box protein TIR1 is the recognition component ofa Skp1-cullin-F-box protein (SCF) E3 ubiquitin ligase (SCFTIR1)which is part of the ubiquitin–proteasome pathway for proteindegradation.16,18 The substrates for TIR1, Aux/IAA transcriptionalrepressor proteins, are recruited to TIR1 in an auxin-dependentmanner. After binding to TIR1, the repressors are degraded.16,18,21

Crystallographic analysis of this interaction showed that IAA bindsto the base of the same TIR1 pocket that docks the Aux/IAA pro-tein on top of IAA which occupies the rest of the TIR1 pocket.19

In this respect, IAA functions as a ‘molecular glue’ to enhanceTIR1–Aux/IAA protein interaction. In order to validate TIR1 as aprincipal receptor for perception of auxins, the synthetic mim-ics of IAA, 2,4-D and 1-NAA, were additionally tested. TIR1 alsobinds and functionally responds to these synthetic auxins.16,18

Moreover, crystal structures of TIR1 in association with the auxinligands IAA, 2,4-D and 1-NAA revealed that IAA binds to a par-tially promiscuous site at the base of the TIR1 receptor pocket,which can also accommodate the synthetic auxins.19 Interest-ingly, IAA binds to TIR1 with the greatest affinity, and bindinginvolves the side-chain carboxylic group and the ring system.Computer modelling with TIR1 structure revealed that syntheticauxins of the other classes, including the benzoic acid dicambaand the quinoline carboxylic acids quinclorac and quinmerac,also fit into the auxin-binding cavity of the receptor (MietznerT, BASF SE, private communication). Ultimately, binding of IAAor of a synthetic auxin to TIR1 stabilises the interaction be-tween the Aux/IAA repressor and the receptor and causes theSCFTIR1 complex covalently to bind ubiquitin to Aux/IAA pro-tein, which marks it as substrate for degradation by the 26Sproteasome.16,18,19 The loss of Aux/IAA repressor proteins leads toderepression of pre-existing DNA-binding transcriptional activatorproteins, the auxin response factors (ARFs). They continuously ac-tivate transcription of auxin-responsive genes, including those for

1-aminocyclopropane-1-carboxylic acid (ACC) synthase inethylene biosynthesis and Aux/IAA repressors for feedback in-hibition, as long as auxin concentrations in the tissue remainhigh.21,22 The Arabidopsis genome encodes five homologues ofTIR1. Three of them, the auxin-binding F-box proteins AFB1, AFB2and AFB3, also mediate auxin response.17 Consequently, tir1 afb1afb2 afb3 quadruple mutant seedlings of Arabidopsis are auxininsensitive and exhibit severe developmental and morphologicalphenotypes. This suggests that the four homologues collectivelymodulate the plant response to IAA and 2,4-D.17 Moreover, mu-tations in the TIR1 homologue AFB5 have been found to conferresistance, particularly to auxin herbicides from pyridinecarboxylicacid type, with only minimal cross-resistance to 2,4-D or IAA.23 Thisindicates that chemical specificity to the different classes of auxinherbicides can be mediated principally by different auxin recep-tor proteins. Nevertheless, questions remain open as to whetherthe TIR1 receptor family accounts for all of the chemically di-verse signals of auxins and biological activities dependent onthe sensitivity of tissue, physiological stage and plant biotypeand species. This also includes the question as to whether theselectivity of auxin herbicides is based on different receptors inmonocots and dicots. The diversity and tissue specificity of boththe Aux/IAA repressor proteins and ARF transcription factors canexplain the plethora of auxin-specific responses.22,24 In addition,much of the auxin action is mediated through transcription andconsequently can be linked to TIR1 receptor family. However, inthe case of rapid auxin responses, such as auxin-induced ion fluxesin cell expansion, an additional signalling pathway, possibly viathe membrane-bound auxin-binding protein 1 (ABP1) and relatedproteins, has been discussed.2,24 ABP1 may act as a coordinator ofcell division and expansion, with local auxin levels that influenceABP1 effectiveness.25

Pest Manag Sci 2010; 66: 113–120 c© 2009 Society of Chemical Industry www.interscience.wiley.com/journal/ps

11

6


Figure 3. Proposed mechanism and mode of action of auxin herbicides and the phytohormone indole-3-acetic acid (IAA) at supraoptimal endogenousconcentrations in dicot plant species. Auxin herbicides are perceived by TIR1/AFB auxin receptors, a small family of F-box proteins including the transportinhibitor response 1 (TIR1) and homologue auxin-binding F-box (AFB) proteins which are the F-box recognition component for protein substrate of a Skp1-cullin-F-box protein (SCF) E3 ubiquitin ligase. Auxin binding targets Aux/IAA transcriptional repressor proteins to SCFTIR1/AFBs E3 ligase for degradation bythe ubiquitin–proteasome pathway. The loss of Aux/IAA repressors leads to derepression of transcriptional activator proteins, the auxin response factors(ARFs), which activate transcription of auxin-responsive genes. In the shoot tissue, particularly genes of 1-aminocyclopropane-1-carboxylic acid (ACC)synthase in ethylene and 9-cis-epoxycarotenoid dioxygenase (NCED) in abscisic acid (ABA) biosynthesis are overexpressed. Produced ethylene elicits thedownward curvature of leaves (leaf epinasty) and tissue swelling, and regulates auxin levels locally through inhibition of auxin transport. Concomitantly,auxins lead to horizontal stem curvature (stem curling). Here, rapid auxin-induced ion fluxes in cell expansion are assumed to be mediated via themembrane-bound auxin-binding protein 1 (ABP1). Ethylene stimulates NCED activity post-transcriptionally, leading to lasting ABA biosynthesis (dottedlines). NCED catalyses xanthophyll cleavage, leading to increased production of xanthoxin and ABA. ABA is distributed within the plant and mediatesstomatal closure which limits transpiration and carbon assimilation, accompanied with an overproduction of reactive oxygen species (ROS). In addition,ABA directly inhibits cell division and expansion and promotes, together with ethylene, foliar senescence with chloroplast damage and destruction ofmembrane and vascular system integrity. Growth inhibition, tissue desiccation and decay and finally plant death are the consequences. In sensitivegrasses, phytotoxic levels of tissue cyanide accumulated as a coproduct during quinclorac-stimulated ethylene biosynthesis (modified from Dayan et al.8).SAM denotes S-adenosylmethionine; β-CAS denotes β-cyanoalanine synthase.

2.3 Hormone interactions and growth responseThe newly discovered TIR1/AFB receptors link binding of auxinherbicides or IAA at supraoptimal concentrations directly totranscription factor abundance and overexpression of auxin-responsive genes, which in turn leads to the succeeding seriesof biochemical and physiological events associated with herbicideaction (Fig. 3). In this context, excessive stimulation of ACCand ethylene formation through induced ACC synthase inbiosynthesis is a long-known early and ubiquitous responsewhen auxins are applied to sensitive species or overproducedin transgenic plants.5,6,26 Isoforms of ACC synthase are encodedby the multigene ACS family which belongs to the early-auxin-response genes. They are differentially expressed or post-transcriptionally or post-translationally regulated by IAA andauxin herbicides within a few minutes of application. Auxin-induced ACC synthase activity results in elevated concentrationsof ACC, closely paralleled by increased ethylene formation. Inexperiments using transgenic tomato plants, which containedthe ACC synthase antisense gene LE-ACS2,27 and auxin-resistantor ethylene-insensitive lines (Arabidopsis, wild mustard, tomato),herbicide-induced morphological symptoms could be lessened orfully reversed.

Ethylene is a gaseous hormone that is involved in plantresponses to stress and the regulation of senescence and plantgrowth.28 By reorienting microtubules from a transverse to amore longitudinal orientation, ethylene promotes lateral cellexpansion, which leads to swelling of stems and roots. Inaddition, stimulated ethylene mediates auxin effects such asleaf abscission and epinasty, and regulates auxin levels locallythrough ethylene-inhibited auxin transport. Consequently, theauxin-induced ethylene burst contributes to growth abnormalitiesand senescence (Fig. 3).5,6,28

The crucial factor implicated in growth inhibition and the actualphytotoxic response to auxins appears to be the overproductionof ABA.6,29 Following auxin-induced ACC synthase activity andethylene formation, ABA accumulates in root and even morein shoot tissue, up to 70 times the concentration in controls.30

Closely correlated with inhibition of shoot and root growth,induction of ABA was demonstrated for auxin herbicides fromthe different chemical classes and IAA in a variety of dicot species,including members of the Rubiaceae, Solanaceae, Umbelliferae,Fabaceae, Scrophulariacea and Convolvulaceae.31,32 In contrast,crop species with natural tolerance showed no stimulation ofACC synthase activity and ABA levels, which suggests selective


11

7


action, presumably at the receptor site.6,31 Exemplified first forthe highly sensitive dicot G. aparine, IAA and auxin herbicidestrigger de novo ABA biosynthesis. In Galium cell suspensions,ABA concentrations increased as early as 3 h after treatment,which indicates a response directly following hormone signalling.6

Analysis of pathway intermediates showed that ABA biosynthesisis exclusively induced in the shoot tissue by increasing xanthophyllcleavage, leading to enhanced production of the ABA precursorxanthoxin.32 This key regulated step in the pathway is catalysedby the plastid enzyme NCED, which is encoded by a family of NCEDgenes.33,34 Using inhibitors of biosynthesis and tomato mutantsdefective in perception or synthesis of ethylene or ABA, studieshave suggested that auxin-stimulated ethylene appears to be atrigger for ABA accumulation.6,32

Recently, the time course of both auxin-induced GaNCED1gene expression and biosynthesis of ethylene and ABA has beeninvestigated in Galium plants.20 Within 1 hour of root treatment,IAA and auxin herbicides of different chemical classes, includingquinmerac, dicamba and picloram (Fig. 1), led to transientincreases in GaNCED1 mRNA levels exclusively in the shoottissue. Three hours after treatment, transcript levels increasedto maximum values 40-fold greater than in controls; thereafterthe level declined. During this period, the water content andthe osmotic potential in shoot tissue were not changed, whichindicates that GaNCED gene expression was not upregulatedby auxin-mediated turgor changes. ABA began to increase witha lag phase of 2 hours and reached concentrations 24-foldhigher than those in controls after 24 h. Interestingly, increasesin GaNCED1 transcript levels preceded those in ACC synthaseactivity, ACC and ethylene production. Consequently, ethylene orACC are not the primary triggers for gene activation of NCED. Inaccordance with this, applied inhibitors of ethylene biosynthesisonly slightly affected GaNCED1 gene expression by auxin. However,ethylene inhibitors considerably decreased auxin-induced ABAaccumulation, which suggests that auxin-induced ethylene isrequired for ABA accumulation but plays only a minor role inNCED gene expression.20

In conclusion, besides their stimulatory effects on geneexpression in ethylene biosynthesis, IAA and auxin herbicidesare able directly to trigger gene activation of NCED, which in turnis required for upregulation of ABA biosynthesis (Fig. 3). However,the question is still open as to whether auxin signalling in NCEDgene expression also involves SCFTIR1/AFB-mediated degradationof transcription repressors, such as Aux/IAA. Likewise, it has tobe clarified whether induction of NCED gene expression is aubiquitous effect of auxins in susceptible plants. This is mostlikely the case because auxin-induced ABA accumulation has beenobserved in a variety of dicot species.31,32 Whereas auxin is theprimary trigger for NCED gene expression, ethylene appears toenhance ABA biosynthesis, possibly by upregulation of NCEDpost-transcriptionally (Fig. 3).19 Ethylene-mediated upregulationof NCED activity could be based on increasing synthesis, activityand/or stability of the enzyme protein. In accordance withthis, transcriptome analysis in 2,4-D-treated Arabidopsis showedexpression of NCED1 and genes involved in ethylene signallingand biosynthesis.35

Eventually, ABA accumulates in the shoot tissue and issystemically translocated within the plant. Together with ethylene,ABA functions as a hormonal second messenger in the modeof action of auxin herbicides (Fig. 3). ABA is recognised as animportant hormone that promotes leaf senescence and controlsplant growth through effects on stomatal movement and cell

division and expansion. Indeed, the time course of ABA increase inplants closely correlated with stomatal closure, with consequentinhibition of transpiration, carbon assimilation, plant growthand progressive foliar tissue damage.36 The latter effect wasaccompanied with an overproduction of reactive oxygen species(ROS) such as hydrogen peroxide, which appears to be triggeredby the decline of photosynthetic activity owing to ABA-mediatedstomatal closure.36 In addition, ABA-induced activation of plasma-membrane NADPH oxidase could be involved in hydrogenperoxide production.37 Furthermore, the amounts of Cu/Zn-superoxide dismutase increased in the leaf tissue, which furtherpromotes hydrogen peroxide accumulation. Exogenously appliedABA was shown to mimic auxin effects on these processes.36

This is in accordance with the recognised hormonal role ofABA in stomatal closure, growth inhibition and promotion ofleaf senescence. Comparing the effects of auxin herbicides fromdifferent chemical classes, it could be demonstrated that theconcomitant overproduction of ABA and ROS, such as superoxideradicals and hydrogen peroxide, paralleled by tissue damage,are effects common to all auxin herbicides.36,37 An accumulationof hydrogen peroxide is generally considered to contribute tooxidative tissue damage through membrane lipid peroxidationand probably process signalling in senescence.38 Accordingly,treatment of Galium shoots with hydrogen peroxide elicited auxin-herbicide-like phytotoxicity, including progressive leaf chlorosis,followed by wilting, tissue necrosis and plant death.6

Overall, auxin activity alone and auxin-stimulated ethylene andABA appear to be the primary causative factors that elicit thephytotoxic syndrome of auxin herbicides and IAA at supraoptimalendogenous concentrations. In particular, the overproduction ofABA and hydrogen peroxide are the long-searched-for missinglinks between auxin action and induced growth inhibition,senescence and tissue decay. Consequently, using syntheticauxins, new principles have been identified in auxin perceptionand hormone interactions of signalling between auxin and theupregulation of ethylene and ABA biosynthesis in plant growthregulation.8 Moreover, correlative results gave rise to speculationthat this hormone interaction also functions as a module inthe signalling of other auxin-related processes, such as rootgravitropism and inhibition of lateral buds in apical dominance.39

As ethylene and ABA signal transduction chains appear to bepartly overlapping and interfering, interaction between ethyleneand ABA could play a role in phenomena that coincide with a strongstimulation of ethylene biosynthesis. These phenomena includesenescence induced under stress conditions, growth inhibitionelicited by cytokinins, Agrobacterium tumefaciens-induced planttumour development and fruit ripening.6,39

3 THE ROLE OF CYANIDE IN AUXIN HERBICIDEACTION3.1 Quinclorac action in grassesAuxin herbicides preferentially control dicot weeds. An exceptionto this phenomenon is the selective auxin herbicide quinclorac,which additionally controls important grass weeds, such asEchinochloa, Digitaria, Setaria and Brachiaria spp., in rice.40 Growthinhibition with progressive leaf chlorosis, first on the growing areasof the youngest leaf blades, followed by wilting and necrosis of theentire shoot, are the predominant herbicidal symptoms. Studieson the mechanism of action of quinclorac in grasses revealed thatinduced increases in abscisic acid (ABA) and hydrogen peroxidealone are insufficient to elicit these effects.6,40 Findings reported by


11

8


Tittle et al.41 suggested for the first time a role of cyanide, formedas a coproduct during stimulated ethylene biosynthesis, in thephytotoxic mode of action and selectivity of the auxin herbicide2,4-D. Therefore, the hypothesis has been tested that quinclorac,in its function as an auxin herbicide, causes damage to grasses ina similar way.

3.2 Occurrence of hydrogen cyanide in plantsCyanide is ubiquitous in nature and can be formed from a varietyof precursors that are widely distributed.42 – 44 More than 2500plant species have been shown to be cyanogenic, that is, to havethe ability to produce hydrogen cyanide (HCN). Cyanogenesis isfairly common in dicot species (e.g. stone fruits, legumes) andin grasses, including important food plants such as maize, rice,wheat and barley. In most plant species, the mechanism for theproduction of HCN is the degradation of cyanogenic glycoside.More than 60 different cyanogenic glycosides are known andthought to play roles in plant defence against herbivores throughtheir bitter taste and release of toxic HCN on tissue disruption,and as storage compounds of sugar and reduced nitrogen.44 Inaddition, formation of HCN from histidine in the presence of aminoacid oxidase and peroxidase, from hydroxylamine, the possibleintermediate of nitrate assimilation, and from glyoxylate, theproduct of photorespiration, has been reported.45 Since the early1980s, when cyanide was discovered to be formed as a coproductduring phytohormonal ethylene biosynthesis,46 questions havebeen raised on the physiological significance of this metabolite inplants.6,45,47,48

3.3 Cyanide: a coproduct of ethylene biosynthesisCyanide is produced in stoichiometrically equivalent amounts toethylene.47,48 The oxidation of 1-aminocyclopropane-1-carboxylicacid (ACC), catalysed by ACC oxidase, leads to ethylene, CO2

and HCN. The widespread occurrence of ethylene synthesissuggests that this pathway is the principle source of endoge-nous free cyanide in many plants. While ethylene plays animportant role in the regulation of a wide range of develop-mental phenomena in plants, such as growth, senescence andripening,28 cyanide is a phytotoxic agent.43 It is a well-knowninhibitor, particularly of metalloenzymes and of other proteinsthat involve reactions between cyanide and a Schiff-base in-termediate or a substrate to form an inhibitory compound.43

Such enzymes are involved in major plant metabolic processes,such as respiration, carbon and nitrate assimilation, carbohydratemetabolism and defence against toxic-reduced oxygen. Cyanidealso interacts with the Cu-protein plastocyanin, which inhibitsphotosynthetic electron transport. The most cyanide-sensitiveenzymes include nitrate/nitrite reductase, nitrogenase, catalase,Cu/Zn-superoxide dismutase, peroxidase, ribulose-bisphosphatecarboxylase and cytochrome-c oxidase.43 The concentrations ofendogenous cyanide required to cause 50% inhibition of sensitiveenzymes are mostly in the range 5–10 µM.47 In higher plants,the key enzyme for detoxifying HCN is β-cyanoalanine synthase(β-CAS), a pyridoxal phosphate-dependent enzyme that catalysesthe reaction of cysteine and cyanide to form hydrogen sulfide andβ-cyanoalanine (Fig. 3). The latter is subsequently converted toasparagine in a reaction catalysed by β-cyanoalanine hydrolase.High β-CAS activities were found in older plant tissues and ar-eas with meristematic activity and were correlated with cyanideproduction and ethylene concetrations.47 Ethylene and cyanideitself are able to influence cyanide production and metabolism.

Ethylene can affect its own biosynthesis, and consequently alsothat of its coproduct cyanide, either increasing (autostimulation)or decreasing (autoinhibition) the rate of formation.26,48 Ethylenecan regulate ACC synthase and ACC oxidase activity at transcrip-tional and post-transcriptional level26 and is able to induce de novosynthesis of β-CAS.49 Cyanide has been found to upregulate ACCsynthase (ACS6) gene expression,50 catalytically to activate and in-activate ACC oxidase reaction51 and to enhanceβ-CAS.52 However,intracellular ACC oxidase is an exclusively cytosolic enzyme, andconsequently its reaction products, ethylene and HCN, are initiallyreleased in the cytoplasm.53 In contrast, β-CAS is predominantlylocalised in the mitochondria.54 Hence, differential compartmen-talisation of the intracellular site of cyanide release and cyanidedetoxification could result in uneven distribution of cyanide withinthe cell.48 Cyanide removal in cell compartments outside mito-chondria is probably less efficient and could result in transientlyelevated cyanide concentrations localised in cell compartments,such as cytoplasm and chloroplasts.47 Effective detoxification ofcyanide in mitochondria specifically protects cytochrome-c oxi-dase in cyanide-sensitive respiration47 and prevents resulting ROSformation.55 However, other highly sensitive enzymes, such asnitrate reductase and Cu/Zn-superoxide dismutase, are located inthe cytoplasm, while ribulose-bisphosphate carboxylase is com-partmentalised in chloroplasts and catalase in peroxisomes. Thiscould make them more susceptible to cyanide inhibition becausecyanide is highly mobile in the cell. The pKa of hydrocyanic acidin solution is approximately 9.2 at 25 ◦C. As intracellular pH isapproximately neutral, most of the cyanide released during ACCoxidase reaction exists in the undissociated (protonated) form.This form is non-ionic, more lipophilic, and could therefore passthrough membranes into cell compartments more easily.47 Theconsequence of an uneven distribution of HCN at transientlyelevated, phytotoxic concentrations within the cell could be amultiplicity of metabolic effects that could culminate in cell andplant death by cyanide.47,48 However, based on observations thathigh activity of β-CAS was detected in plant tissues that produceethylene at high rates (e.g. 1650 nmol g−1 h−1 in ripe apples) andtissue cyanide concentrations were found below the non-toxicconcentrations for sensitive enzymes of approximately 1 µM, Yipand Yang56 stated that plant tissues possess ample capacity todetoxify HCN formed during ethylene biosynthesis. Consequently,the possibility of a metabolic and physiological role for cyanidein plants was neglected over the years. In the 1990s, studies onthe mode of action of auxin herbicides have re-examined thisquestion and investigated the involvement of stimulated ethyleneand cyanide biosynthesis in the induction of cell and plant death.

3.4 Phytotoxic and regulatory role for cyanideExemplified first for the grass weed barnyard grass [Echinochloacrus-galli (L.) Beauv.], the principal site of quinclorac action islocalised in the root tissue, but endogenous levels of ACC, ethyleneformation and cyanide accumulation increased predominantly inthe shoot, where the phytotoxic symptoms develop.40 Quincloracinduced ACC synthase activity specifically in the root tissue,leading to subsequent increases in ACC as early as 1 h aftertreatment (Fig. 3). Excess ACC is translocated to the shoot, whereit is converted to high levels of ethylene and cyanide by ACCoxidase. The process appears to be self-amplifying because ACCand its product cyanide induce ACC synthase activity in the shoottissue. Treatment of isolated shoots of barnyard grass with ACCvia the vascular system led to an increase in ACC synthase activityand enhanced ethylene and cyanide production in equimolar


11

9


amounts.57,58 ACC synthase activity was stimulated fivefold,relative to the control, 6 h after treatment with 1 mM ACC.58

Tissue cyanide concentrations increased from 5 µM in controls to18 µM during 53 h of incubation.57 In addition, applied cyanidewas able to increase ACC synthase activity in the shoot tissue,58

probably through induction of ACC synthase gene transcription.50

In other susceptible grasses, including large crabgrass [Digitariasanguinalis (L.)], broadleaf signal grass [Brachiaria platyphylla(Griseb.) Nash] and green foxtail [Setaria viridis (L.) Beauv.],cyanide concentrations in the shoot tissue also accumulated.The degree of accumulation was related to the herbicideconcentration and its application time, and closely correlated withherbicidal effects, which included growth inhibition, chlorophyllloss and reduction in carbon assimilation.40,57 Endogenouscyanide concentration increased, e.g. threefold and ninefold inthe shoot tissue of barnyard grass and large crabgrass, andreached a maximum of nearly 30 µM and 50 µM respectivelyafter 96 hours.40,57 Auxin herbicides from other chemical classes,including dicamba and 1-NAA (Fig. 1), caused lower ACC andcyanide concentrations and correlated with lower phytotoxicity.59

Accordingly, studies in smooth crabgrass [Digitaria ischaemum(Schreb.) Muhl.] confirmed that phytotoxic effects of quincloracare mainly caused by an accumulation of cyanide, derivedfrom stimulated ACC synthesis, in the shoot tissue.60 AppliedKCN reproduced quinclorac phytotoxicity with similar amountsof endogenous cyanide. In addition, pretreatment with theACC synthase inhibitor aminoethoxyvinylglycine (AVG) led toreduced quinclorac phytotoxicity and ethylene production.57,60

Concomitantly, in susceptible grasses, the β-CAS was onlyslightly activated. In contrast, quinclorac-resistant grass weedsand biotypes, as well as tolerant rice, showed up to sixfoldhigher β-CAS activity, but no notable changes in ACC synthaseactivity, ethylene and cyanide production in shoot tissues.59,60

Consequently, the selective induction of ACC synthase (possibly viadifferentially sensitive auxin receptors for quinclorac perceptionin grasses or post-transcriptional regulation) and the ability todetoxify cyanide by β-CAS appear to be decisive for the differentsensitivities of grasses towards quinclorac. Contrary to susceptiblegrasses, unchanged cyanide concentrations were found in shootsof sensitive dicots.40 As shown in Galium, only at the onset ofsenescence in treated plants could a threefold increase in HCN,released from the shoots into the gas phase, be detected.30

To sum up, growing evidence suggests that, under distinctcircumstances, particularly when ethylene biosynthesis via theACC pathway is stimulated by the auxin herbicide quinclorac,intracellular cyanide accumulates and mediates phytotoxic effectsthat finally lead to cell and plant death (Fig. 3).40,45,48,60 Thecompartmentalisation of β-CAS in the mitochondria obviouslyprevents it from successfully detoxifying the large amountof cyanide released in the cytoplasm. Here, a multiplicity ofmetabolic effects, which result from inhibition of cyanide-sensitiveextramitochondrial enzymes and proteins, could be implicated.Ultimately, the combined effects of the high ethylene and cyanideconcentrations are responsible for the metabolic and physiologicalresponses observed in sensitive grass weeds such as Brachiaria,Digitaria, Echinochloa and Setaria. A possible ethylene and cyanideindependent effect of quinclorac in causing cell death has beenrecently proposed for maize roots.61 Here, cell death was found tobe accompanied with ROS formation and lipid peroxidation.

A role for cyanide in plants appears not to be restricted toauxin action. More generally, ACC-dependent HCN production issuggested to have a dual, phytotoxic and regulatory, function

in stressed plants.45,47,48 Under conditions of strongly stimulatedethylene biosynthesis, phytotoxic concentrations of cyanide canbe implicated in the induction process of cell death during thehypersensitive response in pathogen infection,62 drought52 andO3-elicited formation of lesions in leaves.55 On the other hand,sublethal concentrations of cyanide, together with ethylene, mayplay a signalling role via specific gene expression, e.g. of ACCsynthase, in acclimation of plants to biotic and abiotic stresses.45

This will be an interesting field for future research to elucidate howcyanide is perceived and transduced into specific downstreamresponses.

ACKNOWLEDGEMENTSThanks are due to Drs Thomas Ehrhardt and John Speakman (BASFSE, Germany) and Franck E Dayan (USDA-ARS, Natural ProductsUtilization Research Unit, University, MS) for critical reading of themanuscript and helpful discussion.

REFERENCES1 Woodward AW and Bartel B, Auxin: regulation, action, and interaction.

Ann Bot 95:707–735 (2005).2 Vanneste S and Friml J, Auxin: a trigger for change in plant

development. Cell 136:1005–1016 (2009).3 Ross JJ, O’Neill DP, Wolbang CM, Symons GM and Reid JB, Auxin–

gibberellin interactions and their role in plant growth. J PlantGrowth Regul 20:346–353 (2002).

4 Cobb AH, Auxin-type herbicides, in Herbicides and Plant Physiology.Chapman and Hall, London, UK, pp. 82–106 (1992).

5 Sterling TM and Hall JC, Mechanism of action of natural auxins andthe auxinic herbicides, in Herbicide Activity: Toxicology, Biochemistryand Molecular Biology, ed. by Roe RM, Burton JD and Kuhr RJ. IOSPress, Amsterdam, The Netherlands, pp. 111–141 (1997).

6 Grossmann K, Mediation of herbicide effects by hormone interactions.J Plant Growth Regul 22:109–122 (2003).

7 Fedtke C and Duke SO, Herbicides, in Plant Toxicology, ed. by Hock Band Elstner EF. Marcel Dekker, New York, NY, pp. 247–330 (2005).

8 Dayan FE, Duke SO and Grossmann K, Herbicides as probes in plantbiology. Weed Sci (2009). in press.

9 Gianfagna TJ, Natural and synthetic growth regulators and their usein horticultural and agronomic crops, in Hormones. Physiology,Biochemistry and Molecular Biology, ed. by Davies PJ. KluwerAcademic Publishers, Dordrecht, The Netherlands, pp. 751–773(1995).

10 Krikorian AD, Kelly K and Smith DL, Hormones in tissue culture andmicropropagation, in Plant Hormones and their Role in Plant Growthand Development, ed. by Davies PJ. Martinus Nijhoff, Dordrecht, TheNetherlands, pp. 593–613 (1987).

11 Farrimond JA, Elliott MC and Clack DW, Charge separation as acomponent of the structural requirements for hormone activity.Nature 274:401–402 (1978).

12 Romano CP, Cooper ML and Klee HJ, Uncoupling auxin and ethyleneeffects in transgenic tobacco and Arabidopsis plants. Plant Cell5:181–189 (1993).

13 Gilbert FA, The status of plant-growth substances and herbicides in1945. Chem Rev 39:199–218 (1946).

14 Grossmann K, The mode of action of quinclorac: a case study of anew auxin-type herbicide, in Herbicides and their Mechanisms ofAction, ed. by Cobb AH and Kirkwood RC. Sheffield Academic Press,Sheffield, UK, pp. 181–214 (2000).

15 Kelley KB and Riechers DE, Recent developments in auxin biology andnew opportunities for auxinic herbicide research. Pestic BiochemPhysiol 89:1–11 (2007).

16 Dharmasiri N, Dharmasiri S and Estelle M, The F-box protein TIR1 is anauxin receptor. Nature 435:441–445 (2005).

17 Dharmasiri N, Dharmasiri S, Weijers D, Lechner E, Yamada M, Hobbie L,et al, Plant development is regulated by a family of auxin receptorF box proteins. Develop Cell 9:109–119 (2005).

18 Kepinski S and Leyser O, The Arabidopsis TIR1 protein is an auxinreceptor. Nature 435:446–451 (2005).


12

0


19 Tan X, Calderon-Villalobos LIA, Sharon M, Zheng C, Robinson CV,Estelle M, et al, Mechanism of auxin perception by the TIR1 ubiquitinligase. Nature 446:640–645 (2007).

20 Kraft M, Kuglitsch R, Kwiatkowski J, Frank M and Grossmann K,Indole-3-acetic acid and auxin herbicides up-regulate 9-cis-epoxycarotenoid dioxygenase gene expression and abscisicacid accumulation in cleavers (Galium aparine): interaction withethylene. J Exp Bot 58:1497–1503 (2007).

21 Hagen G and Guilfoyle T, Auxin-responsive gene expression: genes,promoters and regulatory factors. Plant Mol Biol 49:373–385 (2002).

22 Guilfoyle T, Sticking with auxin. Nature 446:621–622 (2007).23 Walsh TA, Neal R, Merlo AO, Honma M, Hicks GR, Wolff K, et al,

Mutations in an auxin receptor homolog AFB5 and in SGT1bconfer resistance to synthetic picolinate auxins and not to 2,4-dichlorophenoxyacetic acid or indole-3-acetic acid in Arabidopsis.Plant Physiol 142:542–552 (2006).

24 Badescu GO and Napier RM, Receptors for auxin: will it all end in TIRs?Trends Plant Sci 11:217–223 (2006).

25 Braun N, Wyrzykowska P, Muller P, David K, Couch D, Perrot-Rechenmann C, et al, Conditional repression of AUXIN BINDINGPROTEIN1 reveals that it coordinates cell division and cell expansionduring postembryonic shoot development in Arabidopsis andtobacco. The Plant Cell 20:2746–2762 (2008).

26 Argueso CT, Hansen M and Kieber JJ, Regulation of ethylenebiosynthesis. J Plant Growth Reg 26:92–105 (2007).

27 Grossmann K and Schmulling T, The effects of the herbicide quincloracon shoot growth in tomato is alleviated by inhibitors of ethylenebiosynthesis and by the presence of an antisense construct tothe 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene intransgenic plants. Plant Growth Reg 16:183–188 (1995).

28 Abeles FB, Morgan PW and Saltveit ME (eds), Ethylene in Plant Biology.Academic Press, San Diego, CA (1992).

29 Grossmann K, The mode of action of auxin herbicides: a new endingto a long, drawn out story. Trends Plant Sci 5:506–508 (2000).

30 Scheltrup F and Grossmann K, Abscisic acid is a causative factor inthe mode of action of the auxinic herbicide quinmerac in cleaver(Galium aparine L.). J Plant Physiol 147:118–126 (1995).

31 Grossmann K, Scheltrup F, Kwiatkowski J and Caspar G, Induction ofabscisic acid is a common effect of auxin herbicides in susceptibleplants. J Plant Physiol 149:475–478 (1996).

32 Hansen H and Grossmann K, Auxin-induced ethylene triggersabscisic acid biosynthesis and growth inhibition. Plant Physiol124:1437–1448 (2000).

33 Schwartz SH, Qin X and Zeevaart JAD, Elucidation of the indirectpathway of abscisic acid biosynthesis by mutants, genes, andenzymes. Plant Physiol 131:1591–1601 (2003).

34 Taylor IB, Sonneveld T, Bugg TDH and Thompson AJ, Regulation andmanipulation of the biosynthesis of abscisic acid, including thesupply of xanthophyll precursors. J Plant Growth Regul 24:253–273(2005).

35 Raghavan C, Ong EK, Dalling MJ and Stevenson TW, Regulation ofgenes associated with auxin, ethylene and ABA pathways by 2,4-dichlorophenoxy-acetic acid in Arabidopsis. Funct Integ Genomics6:60–70 (2006).

36 Grossmann K, Kwiatkowski J and Tresch S, Auxin herbicides induceH2O2 overproduction and tissue damage in cleavers (GaliumaparineL.). J Exp Bot 52:1811–1816 (2001).

37 Romero-Puertas MC, Mccarthy I, Gomez M, Sandalio LM, Corpas FJ, DelRio LA, et al, Reactive oxygen species-mediated enzymatic systemsinvolved in the oxidative action of 2,4-dichlorophenoxyacetic acid.Plant Cell Environ 27:1135–1148 (2004).

38 Dat J, Vandenabeele S, Vranova E, Van Montagu M, Inze D and VanBreusegem F, Dual action of the active oxygen species during plantstress responses. CMLS Cell Mol Life Sci 57:779–795 (2000).

39 Grossmann K and Hansen H, Ethylene-triggered abscisic acid: aprinciple in plant growth regulation? Physiol Plant 113:9–14 (2001).

40 Grossmann K, News from old compounds: the mode of action ofauxin herbicides, in Protection. Progress and Prospects in Science

and Regulation, ed. by Voss G and Ramos G. Wiley-VCH, Weinheim,Germany, pp. 131–142 (2003).

41 Tittle FL, Goudey JS and Spencer MS, Effect of 2,4-dichlorophenoxyacetic acid on endogenous cyanide, β-cyanoalanine synthase activity, and ethylene evolution in seedlingsof soybean and barley. Plant Physiol 94:1143–1148 (1990).

42 Miller JM and Conn EE, Metabolism of hydrogen cyanide by higherplants. Plant Physiol 65:1199–1202 (1980).

43 Solomanson LP, Cyanide as a metabolic inhibitor, in Cyanide in Biology,ed. by Vennesland EE, Conn EE, Knowles CJ, Westley J and Wissing F.Academic Press, London, UK, pp. 11–28 (1981).

44 Zagrobelny M, Bak S and Moller BL, Cyanogenesis in plants andarthropods. Phytochemistry 69:1457–1468 (2008).

45 Siegien I and Bogatec R, Cyanide action in plants – from toxic toregulatory. Acta Physiol Plant 28:483–497 (2006).

46 Peiser GD, Wang T-T, Hoffman NE, Yang SF, Liu H-W and Walsh CT,Formation of cyanide from carbon 1 of 1-aminocyclopropane-1-carboxylic acid during its conversion to ethylene. Proc Natl Acad SciUSA 81:3059–3063 (1984).

47 Grossmann K, A role for cyanide, derived from ethylene biosynthesis,in the development of stress symptoms. Physiol Plant 97:772–775(1996).

48 Yip W-K and Yang SF, Cyanide metabolism in relation to ethyleneproduction in plant tissue. Plant Physiol 88:473–476 (1988).

49 Manning K, Detoxification of cyanide by plants and hormone action,in Cyanide Compounds in Biology, ed. by Evered D and Garnety SF.Wiley, Ciba Foundation Symposium 140, Chichester, UK, pp. 92–110(1988).

50 McMahon JM, Smith S and Arteca RN, Molecular control of ethyleneproduction by cyanide in Arabidopsis thaliana. Physiol Plant109:180–187 (2000).

51 Ververidis P and Dilley DR, Catalytic and noncatalytic inactivation ofACC oxidase. PGRSA 23:81 (1995).

52 Liang W-S, Drought stress increases both cyanogenesis andβ-cyanoalanine synthase activity in tobacco. Plant Sci165:1109–1115 (2003).

53 Reinhardt D, Kende H and Boller T, Subcellular localization of1-aminocyclopropane-1-carboxylate oxidase in tomato cells. Planta195:142–146 (1994).

54 Wurtele ES, Nikolau BJ and Conn EE, Subcellular and developmentaldistribution of β-cyanoalanine synthase in barley leaves. PlantPhysiol 78:285–290 (1985).

55 Vahala J, Ruonala R, Keinanen M, Tuominen H and Kangasjarvi J,Ethylene insensitivity modulates ozone-induced cell death in birch.Plant Physiol 132:185–195 (2003).

56 Yip W-Y and Yang SF, Ethylene biosynthesis in relation to cyanidemetabolism. Bot Bull Acad Sin 39:1–7 (1998).

57 Grossmann K and Kwiatkowski J, Evidence for a causative role ofcyanide, derived from ethylene biosynthesis, in the herbicidalmode of action of quinclorac in barnyard grass. Pestic BiochemPhysiol 51:150–160 (1995).

58 Grossmann K and Scheltrup F, Selective induction of1-aminocyclopropane-1-carboxylic acid (ACC) synthase activ-ity is involved in the selectivity of the auxin herbicide quincloracbetween barnyard grass and rice. Pestic Biochem Physiol 58:145–153(1997).

59 Grossmann K and Kwiatkowski J, The mechanism of quincloracselectivity in grasses. Pestic Biochem Physiol 66:83–91 (2000).

60 Abdallah I, Fischer AJ, Elmore CL, Saltveit ME and Zaki M, Mechanismof resistance to quinclorac in smooth crabgrass (Digitariaischaemum). Pestic Biochem Physiol 84:38–48 (2006).

61 Sunohara Y and Matsumoto H, Quinclorac-induced cell death isaccompanied by generation of reactive oxygen species in maizeroot tissue. Phytochemistry 69:2312–2319 (2008).

62 Siefert F, Kwiatkowski J, Sarkar S and Grossmann K, Changes inendogenous cyanide and 1-aminocyclopropane-1-carboxylic acidlevels during the hypersensitive response of tobacco mosaic virus-infected tobacco leaves. Plant Growth Reg 17:109–113 (1995).


auxin herbicides: current status of mechanism and …passel.unl.edu/image/robles...

Documents