PESTICIDE

Biochemistry & Physiology

Pesticide Biochemistry and Physiology 89 (2007) 111 www.elsevier.com/locate/ypest

Recent developments in auxin biology and new opportunities for auxinic herbicide research
Kevin B. Kelley 1, Dean E. Riechers
Received 16 October 2006; accepted 11 April 2007 Available online 20 April 2007

University of Illinois, Department of Crop Sciences, 1102 S. Goodwin Avenue, Urbana, IL 61801, USA

Abstract Auxinic herbicides mimic the eects of natural auxin. However, in spite of decades of research, the site(s) of action of auxinic herbicides has remained unknown and many physiological aspects of their function are unclear. Recent advances in auxin biology provide new opportunities for research into the mode of action of auxinic herbicides. Of considerable interest is the discovery of auxin receptors (TIR1 and possibly ABP1) that may lead to the discovery of auxinic herbicide site(s) of action. Knowledge of auxin-conjugating enzymes and auxin signal transduction components may shed new light on herbicide activity, selectivity in dicots, and mechanisms leading to phytotoxicity in sensitive plants. Analysis of genes induced in response to auxin may provide a novel approach for detection of o-target herbicide injury in crops. For example, the auxin-responsive gene GH3 is highly and specically induced in response to auxinic herbicides in soybean, and may oer a novel method for diagnosing auxinic herbicide injury. Advances in our understanding of auxin biology will provide many new avenues and opportunities for auxinic herbicide research in the future. 2007 Elsevier Inc. All rights reserved.
Keywords: Auxin biosynthesis; Auxinic herbicide; Auxin homeostasis; Auxin receptor; Herbicide detection; Herbicide resistance; Phytohormone; Phytotoxicity; Plant growth regulator; Signal transduction; TIR1; AFB; ABP1

1. Introduction Auxin was the rst plant hormone discovered and has been extensively researched for many decades [1]. Since its discovery, our understanding of auxin function has greatly increased. However, auxin biology is very complex, and many aspects of auxin action have proven dicult to elucidate. Soon after the discovery of the chemical structure of auxin, synthetic compounds with similar structure were tested for auxinic activity, leading to the discovery of auxinic herbicides, the rst selective synthetic herbicides [2]. Auxinic herbicides have been successfully used to control broadleaf weeds for over 60 years with minimal development of auxinic herbicide-resistant weeds [3]. However, the mode of action of these herbicides is as complex as
*

Corresponding author. Fax: +1 217 333 5299. E-mail address: riechers@uiuc.edu (D.E. Riechers). Present address: USDA-ARS, Aberdeen, ID 83210, USA.

the action of natural auxin, and in spite of decades of research, much remains unknown about the mechanism of action of these herbicides. The herbicidal eect of auxinic herbicides has been attributed to an overinduction of the auxin response in susceptible plants [2,4]. While the concentration of natural auxin and its eects are tightly controlled under most circumstances, auxinic herbicides overcome the natural regulatory mechanisms of susceptible plants to cause an uncontrolled auxin response. At low doses, auxinic herbicides possess similar hormonal properties to natural auxin. However, as rates increase, they cause various growth abnormalities in sensitive dicots, ranging from leaf epinasty and/or cupping and stem twisting, to thickening of stems and roots, and ultimately chlorosis and necrosis [2,5]. Lack of phytotoxicity in grasses has been attributed to several factors, including anatomical dierences in vascular structure and dierences in ability to metabolize the herbicide [2].

0048-3575/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2007.04.002

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Auxinic herbicides have an aromatic ring and a carboxylic acid moiety, as does indole-3-acetic acid (IAA), the most commonly found natural auxin, and are commonly divided into three classes: phenoxyacetic acids (e.g., 2,4-D and MCPA), benzoic acids (e.g., dicamba), and pyridine acids (e.g., picloram, clopyralid) [2]. The distinction between these classes depends on the type of aromatic ring they possess and the location of a carboxylic acid moiety in relation to the aromatic ring. However, recent research indicates that the carboxyl group may not be requisite for auxin activity, as was originally presumed. By using a computational approach in concert with bioassays of many molecules considered auxin-like, it was determined that the chemical condition of the ring system is the determining factor for the auxin activity of molecules; this was demonstrated by the discovery of a non-carboxylated molecule with auxin activity (2,6-dibromo-phenol) [6]. A newer herbicide family, the quinolinecarboxylic acids (e.g., quinclorac, quinmerac), has been developed that also has auxin-like activity [7]. However, unlike the above-mentioned herbicide groups, these compounds also have herbicidal activity on some grasses, suggesting a potentially distinct but related mechanism of action. Research in the last several years has shed a great deal of light on auxin regulation and signal transduction. The regulation of the auxin signal is very complex, involving several pathways for auxin biosynthesis [8,9] and several ways in which auxin can be stored and converted quickly to an active form, or targeted for degradation [10,11]. Furthermore, many molecular components are involved in perceiving and transmitting the auxin signal resulting in plant developmental changes [11,12]. The discovery of soluble auxin receptors that are responsible for induced gene expression should be of particular interest to herbicide physiology research [1315]. This review examines what is currently known about the auxin hormonal response, how it is regulated, and what new opportunities this knowledge provides for future research into auxinic herbicide mechanism of action, crop selectivity, and weed resistance. 2. Auxinic herbicide resistance

not believed to be the cause for resistance in biotypes of yellow starthistle (Centaurea solstitialis) [17], wild mustard (Sinapis arvensis L.) [18], or kochia (Kochia scoparia) [19], indicating that these resistance mechanisms may include an altered site of action. In the case of wild mustard, several studies characterizing physiological, biochemical, and genetic dierences between resistant and susceptible biotypes support this conclusion [2027]. Genetic research indicates that the resistance mechanisms to escape phytotoxicity may potentially be dierent among these three biotypes. Inheritance studies have shown that auxinic herbicide resistance in yellow starthistle is controlled by a single recessive nuclear allele [28], while resistance in wild mustard is controlled by a single dominant nuclear allele [21,29,30], and resistance in kochia appears to be a quantitative trait controlled by multiple genes [19,31]. Each of these biotypes may provide unique opportunities to study auxin biology and auxinic herbicide mode of action. If resistance is due to altered signal perception or signal transduction, this may result in gene expression differences between resistant and susceptible biotypes in response to auxin and/or auxinic herbicides. As expected, resistant biotypes of both kochia and wild mustard display dierent gene expression patterns in response to the auxinic herbicide dicamba [20,32]. These dierences in expression may help to elucidate the complex auxin signaling pathways as well as reveal more information about the mechanisms of auxinic herbicide resistance in these biotypes. Recent developments in the understanding of auxin biology may also help elucidate the resistance mechanisms utilized by these plants. For example, resistance to auxinic herbicides in wild mustard is believed to be due to an altered auxin-binding protein (ABP). These plants may have altered auxin binding activity compared to susceptible plants [21], and ABP expression studies support this as a possible mechanism of resistance [22]. An ABP has been identied as a potential auxin receptor (see Section 3.2). 3. Auxin regulation and action

Auxinic herbicide resistance is less common than for several other herbicide classes. For example, the acetolactate synthase inhibitor and triazine herbicides each have over 150 resistant weed biotypes, but less than 40 weed biotypes have been found with resistance to auxinic herbicides in spite of the extensive use of auxinic herbicides throughout several decades [2,3]. Auxinic herbicides are considered to have a lower risk of resistance development than most herbicide classes [16]. However, a few weed biotypes have developed resistance to auxinic herbicides. It has proven dicult to elucidate the resistance mechanism(s) for these biotypes. The mechanism of resistance to auxinic herbicides has been under investigation for several of these biotypes. Altered metabolism, translocation, and absorption are

The phytohormone auxin is involved in many plant developmental processes, including the dierentiation of vascular tissues, the formation of lateral and adventitious roots, the control of apical dominance, and tropic responses such as leaves and stems growing toward light [12,3336]. Auxin inuences plant development by causing changes at the cellular level, which aect cell turgor and elongation, as well as cell division and dierentiation [37 40]. The most common and most studied form of natural auxin is IAA [11]. Though a comprehensive picture of auxin biology has yet to be completed, a great deal has been learned about how IAA is synthesized and regulated, as well as how the auxin signal leads to biochemical changes necessary for plant development.

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3.1. Auxin biosynthesis, storage, and degradation Auxin levels are tightly controlled to regulate their activity on plant development. IAA biosynthesis can take place through both tryptophan (Trp)-dependent and Trp-independent routes, although these pathways are not fully understood [11]. Trp-dependent biosynthesis of IAA involves the action of cytochrome P-450 monooxygenases [41], and there appears to be more than one potential pathway leading from Trp to IAA [8]. Less is known about Trpindependent biosynthesis of IAA. Analyses of Arabidopsis thaliana mutants defective in Trp biosynthetic enzymes indicate that IAA can be synthesized from the Trp precursor indole-3-glycerol phosphate in a pathway that does not include Trp [9]. The presence of multiple pathways indicates that auxin may potentially be synthesized through alternate pathways in dierent tissues, and these dierences may be signicant in regulating developmental processes. Dierences in developmental and tissue-specic auxin biosynthesis may also indicate dierences in sensitivity to auxinic herbicides. As a result, research aimed towards identifying these potential dierences in sensitivity may lead to new weed management strategies. IAA is frequently found in inactive forms conjugated via ester linkages to sugars or amide linkages to peptides or amino acids [11]. These inactive forms can serve as auxin storage, which can be readily hydrolyzed to release active auxin or as the rst step in auxin degradation. Arabidopsis maintains approximately 90% of IAA in amide linkages, about 10% as ester linkages, and about 1% as free IAA [11]. Conjugates between IAA and small peptides have also been discovered in Arabidopsis seeds and may be an important seed-storage form of auxin to provide developing plants with a source of auxin [42]. Proteins with IAA glucosyl-transferase activity have been identied, and an Arabidopsis transgenic line that overexpresses one of these proteins is less sensitive to exogenous IAA when assayed by measuring root elongation. This line shows a phenotype consistent with depleted auxin levels, including reduced vascular development and impaired gravitropism [43], indicating that IAAglucose conjugates may play a role in IAA inactivation and are not merely a storage form of auxin. However, the auxinic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was not a substrate for IAA glucosyl-transferase, and transgenic overexpression of this enzyme did not reduce sensitivity to 2,4-D [43]. Sugar conjugates of 2,4-D have been detected in vivo, however, and conjugation between auxinic herbicides and sugars and/or amino acids is a common detoxication process [2]. The principal amino acid conjugates of IAA in plants include alanine (Ala), leucine (Leu), aspartate (Asp), and glutamate (Glu) [10,11]. IAA conjugates of Ala and Leu are eective substrates for IAA-conjugate hydrolysis and cause auxin-related symptoms when exogenously applied [44], indicating they can readily be converted to active IAA and are likely storage forms of auxin. However,

IAA conjugates of Asp and Glu are not substrates for conjugate hydrolysis; oxidation of IAAAsp has been detected [10,45], indicating that conjugation of IAA to Asp or Glu may play a role in inactivation of IAA. Six IAAamino acid conjugating enzymes have been isolated from Arabidopsis, and all six conjugate IAA to multiple amino acids in vitro [10] (rst identied as GH3 in soybean; refer to Sections 4 and 5). The expression of these enzymes is induced in response to exogenous auxin [10], suggesting they may play an important role in inactivating excess auxin to help the plant maintain auxin homeostasis. Mutant analysis in Arabidopsis supports this putative role for GH3 enzymes. Overexpression of these enzymes results in phenotypes consistent with decreased auxin levels, including reduced shoot elongation and inhibited lateral root growth [46,47]. However, inactivation results in an increase in sensitivity to exogenous auxin [10]. Auxin substrates for GH3 enzymes include IAA, indole-3-butyric acid (IBA), indole-3-pyruvic acid (IPA), phenylacetic acid (PAA), and a-naphthaleneacetic acid (NAA). Trp and the active halogenated auxin analogs 4-chloroindole-3-acetic acid (4-Cl-IAA), 2,4-D, and dicamba are not substrates for GH3 enzymes [10]. If these amino acid-conjugating enzymes play a major role in auxin regulation, then this may partially explain why 2,4-D and dicamba are eective growth regulator herbicides (Fig. 1). As more enzymes involved in auxin regulation are identied and characterized, their eect on and their response to auxinic herbicides can be determined. This will undoubtedly broaden our

Fig. 1. GH3 regulation of auxin vs. auxinic herbicides. (a) Indole-3-acetic acid (IAA) induces gene expression as part of its hormonal activity. Many genes are induced (see also Fig. 2), including GH3. The GH3 enzyme conjugates IAA to amino acids, converting it to an inactive form. This process is part of a regulated physiological response to natural auxin by removing excess IAA. (b) Exogenously applied dicamba also induces a hormonal response, mimicking the eect of IAA. GH3 is also induced in response to dicamba. However, dicamba is not a substrate for the GH3 enzymes conjugation activity. As a result, dicamba is not inactivated, thus representing one of several plausible reasons that dicamba and other auxinic herbicides are able to overstimulate the auxin response, resulting in phytotoxicity.

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understanding of auxinic herbicide detoxication and selectivity as well as auxin regulation. 3.2. Auxin signaling Following decades of research on auxin molecular biology, several elements of the auxin signaling pathway have been elucidated. These include two apparent types of receptors that perceive the auxin signal and factors inuenced by these receptors, resulting in induced gene expression and also possibly cell wall modications important for cell expansion. These two types of receptors include ABP1 (auxin-binding protein 1), a membrane-bound protein thought to initiate cell wall expansion [48,49], and more recently TIR1 (Transport Inhibitor Response 1) and related auxin signaling F-box (AFB) proteins, soluble proteins that initiate the induction of gene expression [1315]. Auxin receptor research is of particular interest to auxinic herbicide researchers because the specic site(s) of action for auxinic herbicides (which are likely auxin receptors) have yet to be elucidated. ABP1 is auxin specic and confers an auxin-dependent response [48]. ABP1 is mainly found in the endoplasmic reticulum (ER) where the pH is too high for auxin binding, but some ABP1 is also found at the plasma membrane. Experiments with ABP1 antisera indicated that extracellular ABP1 may be necessary for auxin-mediated cell expansion [50] possibly by activating H+-ATPase activity [49]. No known function has been demonstrated at the ER. The Arabidopsis genome has only one ABP1 gene. A homozygous null mutation of this gene results in embryo lethality, and antisense suppression of ABP1 in tobacco (Nicotiana tabacum) cells eliminates cell elongation and reduces cell division [51]. These results suggest that ABP1 is essential for plant development and indicate that ABP1 may be an important auxin receptor. Auxinic herbicide resistance in wild mustard may be due to an altered ABP1 (refer to Section 2). Transfer of this ABP transgene to the Arabidopsis null mutant could potentially result in functional complementation and concomitant resistance to auxinic herbicides. If successful, this could provide further evidence for ABP1 as an auxin receptor, as well as for an altered ABP1 as the molecular basis for resistance in wild mustard. The fastest reported response to exogenous auxin is the hyperpolarization of the plasma membrane, which can occur within seconds after exogenous application and presumably enables cell expansion. This may take place through the binding of auxin to ABP1 resulting in Ca2+dependent protein kinase mediated phosphorylation [49,52], thereby rapidly activating proton-pumping ATPases on the plasma membrane that actively pump H+ ions into the apoplast [39]. The electrochemical gradient created by the increased concentration of H+ ions is oset by an accompanying antiport that pumps K+ ions into cells [53]. Other research suggests that in addition to acidication caused by proton pumping, the displacement of Ca2+

ions further destabilizes the cell wall [54] allowing cell expansion to take place. In addition, calcium may play an important role in auxinic herbicide resistance in wild mustard. In both isolated protoplasts and intact seedlings, the inhibitory eects of auxinic herbicides on a sensitive biotype could be reduced by pretreatment with a calcium ionophore, while pretreatment with a calcium channel blocker increases the sensitivity of the resistant biotype to auxinic herbicides [23,25,26]. The role of ABP1 in auxin signaling remains unclear, and there is no evidence showing that auxin binding to ABP1 results in auxin-induced gene expression. However, auxin-induced gene expression was detected in a cellular extract free of membranes [55] indicating that, in addition to ABP1, a soluble receptor is necessary to mediate the auxin response. This led to the discovery of TIR1 as an auxin receptor (discussed below). The discovery of a soluble auxin receptor (TIR1) was preceded by the identication of regulatory elements in the promoter of the auxin-responsive gene GH3 [56] (refer to Section 5.1). Auxin-response elements (AuxREs) are short cis-elements in the promoters of auxin-responsive genes [57,58]. AuxREs have a core TGTCTC element [57], and putative AuxREs have been found in many genes throughout the Arabidopsis genome [11]. By using a highly active palindromic repeat of the TGTCTC element in a yeast one-hybrid system, the rst auxin-response factor (ARF) was identied in Arabidopsis [59]. Subsequently, 23 ARF genes were identied in the Arabidopsis genome [60]. All contain an amino-terminal DNA binding domain that binds to AuxREs, a middle region that is not highly conserved but is likely a transcriptional activator or repressor domain, and a carboxy-terminal dimerization domain [61]. ARFs bind to AuxREs as monomers, homodimers, or heterodimers to up- or down-regulate gene expression of auxin-responsive genes [11]. The function of ARFs can be inuenced by microRNAs, small RNA molecules that target the degradation of other RNAs. MicroRNAs can target the degradation of several ARFs, inhibiting their ability to activate or repress the expression of auxin-responsive genes [11,62,63]. Recent research also implicates microRNAs in the regulation of TIR1 and related F-box protein receptors (see below) [64]. The exact function of microRNAs in auxin signaling remains unclear, but they appear to be important factors in regulating the auxin response. Another family of proteins, called Aux/IAA proteins, also can form heterodimers with ARFs to repress expression of auxin-responsive genes. There have been 29 Aux/IAA genes identied in the Arabidopsis genome, and they generally have four domains [65]. Domain I is an active repression domain, domain II controls protein stability, and domains III and IV are dimerization domains that have sequences that are highly conserved with ARF dimerization domains [11,66]. These dimerization domains allow Aux/IAA proteins to dimerize with ARFs, inhibiting their ability to activate expression of auxin-responsive genes [67] (Fig. 2a).

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Fig. 2. Expression of auxin-responsive genes. (a) Auxin-response elements (AuxREs) are promoter elements in auxin-responsive genes. Auxin-response factors (ARFs) bind to AuxREs to induce gene expression. ARFs consist of a DNA binding domain (DBD) and three other domains (labeled II, III, and IV). Aux/IAA proteins also have four domains. Domains III and IV have sequence similarity to ARFs, and in the absence of auxin form heterodimers with ARFs, thus inhibiting their ability to activate expression of auxin-responsive genes. (b) Auxin binding to the TIR1 factor causes the SCFTIR1 complex to covalently bind ubiquitin to Aux/IAA, targeting it for degradation by the 26S proteosome. Following the removal of Aux/IAA, ARFs are free to activate expression of auxin-responsive genes, either as monomers or homodimers. *Aux/IAAs are among the genes induced by this process. Their expression appears to ensure a rapid reduction in auxin-induced gene expression following removal of the auxin stimulus.

The high number of Aux/IAA and ARF proteins encoded by genes in the Arabidopsis genome could lead to a diverse collection of dimer combinations. This diversity could result in temporal and spatial dierences in gene expression that may be important for regulation of the various growth and developmental processes controlled by auxin. Mutational analysis of individual members of these gene families supports their distinct roles in development, but some mutants missing single members of these families only show subtle phenotypic dierences [11,35,68], indicating that some level of functional redundancy likely exists as well. Induction of primary auxin-responsive genes involves the degradation of the Aux/IAA repressor proteins by a SCFTIR1 (Skp1-Cullin-F-box, Transport Inhibitor Response 1) ubiquitin ligase protein complex [11,69]. The SCFTIR1 complex targets Aux/IAA proteins for degradation by the 26S proteosome [70,71]. Degraded Aux/IAAs no longer bind to ARFs to repress expression, thereby allowing ARFs to promote auxin-induced gene expression [70] (Fig. 2b). Degradation of Aux/IAA proteins via the SCFTIR1 complex is activated by auxin, which can take place in a soluble membrane-free cell extract [55], suggesting that the auxin receptor initiating this response is cytoplasmic. The TIR1 factor in the SCFTIR1 complex has been identied as this receptor (Fig. 2b); TIR1 binds auxin directly, and transgenic expression of TIR1 in animal cells can independently result in an auxin-regulated response [13,14]. The TIR1 factor has an F-box motif and 16 leucine-rich repeats [69] that

bind to domain II of Aux/IAA proteins, leading to ubiquitination by the SCFTIR1 complex [71]. Three other F-box proteins, named AFB1, 2, and 3, have also been shown to act as auxin receptors and cause a similar response. While loss of one of these proteins causes only a modest phenotypic response, loss of all four results in auxin insensitivity [15]. The discovery that TIR1 and related AFB proteins are the auxin receptors through which this process is initiated identies a new class of receptor [14]. There are also two additional homologs of TIR1 in Arabidopsis; one homolog, AFB5, may be important as a site of action specic for pyridine acid herbicides such as picloram [72]. An Arabidopsis mutant decient in AFB5 displayed a high degree of resistance to picloram, but negligible resistance to 2,4-D and a slight increase in sensitivity to IAA. Complementation of this mutant line with the wild-type AFB5 resulted in a restoration of sensitivity to picloram [72]. This indicates that receptor interactions with 2,4-D or IAA may not necessarily apply to other auxinic herbicides. The interaction of AFB5 with auxinic herbicides of dierent chemistries is an example of how the discovery of these F-box auxin receptors provide new opportunities to research their role in the mechanism of action of auxinic herbicides. 4. Auxin-induced gene expression Several classes of genes are induced in response to the auxin stimulus through the TIR1 receptor. The proteins

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encoded by several of these genes play key roles in auxin signaling and regulation [4,10,67], while the function of others remains unknown. These genes may also play a role in the phytotoxicity and selectivity of auxinic herbicides. This role may be elucidated as the functions of these genes are fully characterized and the eects of auxinic herbicides on their expression in both monocot and dicot species are determined. As with factors involved in other aspects of auxin biology (e.g., biosynthesis and signal transduction), Arabidopsis mutants with up- or down-regulated expression of these genes may assist in determining their role in herbicide phytotoxicity. 4.1. Families of primary auxin-responsive genes Three families of primary auxin-responsive genes have been characterized from several plant species, including the Aux/IAA family, the GH3 family, and the SAUR family [12]. Due to the fact that Aux/IAAs repress expression of early-responsive genes, and that auxin stimulates the degradation of Aux/IAA repressor proteins, it is surprising that Aux/IAAs are among the primary auxin-responsive genes; in eect, repressing their own expression (Fig. 2a). However, it appears that the regulation of these genes helps to ensure a transient response to the auxin stimulus [11]. Increases in auxin concentration result in degradation of Aux/IAA repressor proteins (Fig. 2b), allowing for increased expression of other auxin-responsive genes important for essential developmental responses, but also result in increased expression of Aux/IAA repressor proteins that help to quickly reduce the expression of auxinresponsive genes once the auxin stimulus is removed via degradation, inactivation, reduced biosynthesis, and/or sequestration [11]. GH3 was rst isolated from soybean [73], and related genes have been found in a number of dierent plant species [12], including 19 closely-related genes in Arabidopsis [74]. Six of the encoded Arabidopsis proteins specically adenylate auxin, while other members of this protein family adenylated other substrates, including jasmonic acid [74]. Arabidopsis GH3 proteins that adenylated auxin were later determined to conjugate auxin to amino acids and are presumably involved in maintaining auxin homeostasis (Fig. 1a) (refer to Section 3.1) [10]. Soybean GH3 has a similar enzymatic activity [10], and GH3 is strongly and specically induced by auxinic herbicides in soybean leaves [75,76]. Furthermore, 2,4-D and dicamba are not substrates for inactivation by GH3 (Fig. 1b) [10]. GH3 may therefore play a major role in maintaining auxin homeostasis under normal conditions, but is unable to attenuate the unregulated eects of auxinic herbicides (Fig. 1b). SAURs (small auxin up RNAs) are small genes (transcripts less than 500 bp) that are induced within 5 min of auxin application. They were initially found as a cluster of ve genes in the soybean genome [77], and have since been found in several other plant species [12]. SAURs are expressed in the epidermis and cortex, and exogenous

auxin increases their expression in these tissues but not in other tissues [78]. They are short-lived mRNAs and appear to encode short-lived proteins as well. A SAUR gene was characterized from maize, and the protein was determined to have a half-life of about seven minutes [79]. The short half-lives of the mRNAs appear to be due to an element in the 3 0 untranslated region [11]. SAURs bind calmodulin (CaM) and may be regulated by calcium/CaM at the posttranslational level [79,80] (which incidentally also provides evidence that calcium/CaM are involved in auxin-mediated signal transduction). Other research indicates that calcium plays a vital role in the auxin-induced growth response [81] and also an important role in the resistance mechanism of wild mustard to auxinic herbicides [23,25,26]. The precise function that SAURs have in auxin signal transduction remains to be determined. 4.2. Other primary auxin-responsive genes Other primary auxin-responsive genes have also been found, and several may play a role in the ecacy or selectivity of auxinic herbicides. Many genes encoding glutathione S-transferases (GSTs) are induced by auxin [12,73]. GST enzymes conjugate various pesticide substrates to reduced glutathione, resulting in detoxication [82,83]. Besides auxin, they are induced by other diverse stimuli including abiotic and biotic stresses, heavy metals, and other plant hormones [12,73]. Additionally, 1-aminocyclopropane-1-carboxylate (ACC) synthase expression is induced by auxin [4]. ACC is the precursor to ethylene, and ACC synthase catalyzes the rate-limiting step in ethylene production, which is stimulated by auxin [84]. Typically, induction of ACC synthase and stimulation of ethylene production are key processes in the phytotoxic eect of auxinic herbicides, coupled with a massive stimulation of abscisic acid (ABA) biosynthesis [4]; in contrast, however, some species are unresponsive to exogenous ethylene treatment [85]. ACC synthase is also induced by other stresses that are not auxin related [84]. Other auxin-responsive genes have been identied (e.g., transcription factors involved in various biosynthetic pathways, including ABA and ethylene biosynthesis), although our understanding of the role that some of them play in the auxin response is limited [20,32,8689]. As more is learned about the function of these genes and their response to auxinic herbicides, it may be determined what role, if any, they play in promoting auxinic herbicide phytotoxicity and/or selectivity. 5. Identifying markers for auxinic herbicide injury Another application of auxin biology relevant to herbicide physiology research involves the use of auxin-responsive genes as markers specic to auxinic herbicide injury. This represents a new and novel method for herbicide detection in plants because most herbicides block metabolic pathways (e.g., fatty acid or amino acid biosynthesis) rather than induce gene expression. Several dicot crops are

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very sensitive to auxinic herbicides, and o-target auxinic herbicide injury is a recurring problem. For example, in the Midwestern United States, soybean elds are often in close proximity to cereal crop elds to which auxinic herbicides are commonly applied. This results in frequent reports of soybean symptoms resembling injury caused by o-target movement of auxinic herbicides [90,91]. However, the causal agent of these symptoms is often dicult to identify because auxinic herbicides can move o-target to injure soybean in several ways that may not be readily identiable. Furthermore, other stresses such as virus infection and herbicides with other modes of action cause symptoms that may be mistaken for auxinic herbicide injury [92]. Current diagnostic tools are limited in their ability to identify or rule out auxinic herbicides as the cause of injury symptoms observed in a growers eld. Herbicide residue analysis is a commonly used method for detection of herbicide exposure [93]. Auxinic herbicide residues can be detected in injured plants, but they can also cause injury at very low doses well below the detection threshold of analytical equipment [94,95]. However, gene expression dierences specic to auxinic herbicides may be able to exclude or implicate auxinic herbicides as the cause of injury symptoms observed in growers elds [75,76]. Gene expression dierences are part of the injury response [75] and may be more useful at exposure doses below the detection limits of residue analysis. This novel approach to herbicide detection is especially promising because it is much easier to measure and quantify expression of a highly-induced gene than it is to detect down-regulation of a constitutivelyexpressed gene, or to measure the depletion of the product(s) of an enzymatic reaction or biochemical pathway. 5.1. GH3 expression characteristics Among the known auxin-inducible genes, several characteristics of the soybean gene GH3 indicate that it has potential to be a valuable molecular marker for auxinic herbicide injury in soybean. Its low constitutive expression, coupled with its response to auxins and lack of response to other stimuli, suggest that GH3 may be strongly and specifically induced by auxinic herbicides. Constitutive GH3 expression is very limited. Expression correlates strongly with developmental events presumably mediated by auxin [96]. GH3 transcripts and protein are detected in developing tissues, including the inner cortex of roots and oral tissues [78,97]. Neither GH3 transcripts nor protein were detected in leaf tissue in the absence of exogenous auxin [96,97]. This indicates that GH3 expression is highly correlated with developmental events inuenced by auxin, and that GH3 is not expressed in mature soybean leaves in the absence of exogenous auxin. GH3 expression is highly induced at the transcript and protein levels by active auxins (including IAA, NAA, and 2,4-D), but expression is unaected by other hormones such as cytokinin, ethylene (applied as Ethephon), or gibberellic acid (GA), or by fusiccoccin, inactive auxin analogs, or

cold or heat shock [73,97]. The specic response of GH3 expression to several auxins (but not to other plant hormones or stress treatments), taken together with the tissue-specic endogenous expression of GH3, indicate that GH3 expression is specically regulated by active auxins. GH3 expression is induced in a dose-dependent manner within minutes of exogenous auxin application in most if not all soybean tissues, including leaves [78,96]. In excised soybean plumules, incubation in a 2,4-D solution resulted in a rapid, dose-dependent induction of GH3 expression at both the transcript and protein levels [73,97,98]. GH3 expression is rapidly and strongly induced by natural and synthetic auxins in a dose-dependent manner; however, the doseresponse and induction kinetics of GH3 expression in leaves of whole plants treated with auxinic herbicides may be dierent from excised plant organs incubated in solution. In addition to soybean, GH3 homologs may have potential as molecular markers in other auxinic herbicide-sensitive crops as well. Auxin-inducible genes with high sequence similarity to GH3 have been isolated from several other plant species [10,99102]. Furthermore, by using antisera raised against soybean GH3, proteins of a similar size were detected from several monocot and dicot species [97]. As determined by immunoblot analysis, these proteins (presumably GH3 homologs) were strongly induced in dicot species in response to 2,4-D, but very weakly induced if at all in monocot species [97], consistent with the sensitivity of dicots and the tolerance of monocots to 2,4-D [2]. In contrast to GH3, SAURs, and Aux/IAA genes may have higher expression levels in untreated tissues and are induced by auxin in a limited number of tissues [96]. GSTs and ACC synthase respond to numerous other stimuli besides auxin [12,84]. However, constitutive GH3 expression is low in tissues with correspondingly low endogenous auxin levels, including leaves [73,78,96,97]. GH3 expression is specically and rapidly induced by active auxins to high levels in a dose-dependent manner in most if not all tissues, but is not aected by inactive auxin analogs, other plant hormones, protein synthesis inhibitors, or heat or cold shock [73,97,98]. All of these factors provide compelling evidence that GH3 would be strongly and specically induced by auxinic herbicides in injured soybean leaves. 5.2. Auxinic herbicide eects on GH3 expression Based on the characteristics indicating that GH3 would be suitable for use in a diagnostic assay, GH3 expression at both the transcript and protein levels were measured in response to auxinic herbicide injury in soybean leaves [75,76]. The expression of GH3 in auxinic herbicide-injured soybean leaves was similar to expression in response to exogenous auxin. Expression of GH3 at the transcript level was strongly induced in soybean in a dose-dependent fashion within eight hours in response to dicamba, clopyralid and 2,4-D and expression was detectable up to 17 days after treatment (DAT) [75,76]. GH3 protein levels

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responded similarly, although higher doses were required to detect a response 8 h after treatment. Protein expression levels peaked one to three DAT, but dropped below detection by seven DAT in surviving plants [76]. However, since a colorimetric detection assay was used, more sensitive immunodetection methods (i.e., chemilluminescent detection) may have detected expression at later time points and/or in response to reduced herbicide doses [76]. Evaluation of GH3 expression at both the transcript and protein levels in response to other stresses potentially present in a soybean eld revealed that GH3 expression is specic to auxinic herbicides. Expression was evaluated in response to the non-auxinic herbicides glyphosate, imazethapyr, and fomesafen, as well as heat, salt, and drought stress, and infection with Soybean mosaic virus or Bean pod mottle virus [75,76]. Expression of GH3 at the transcript level in response to these stresses was minimal (equivalent to a low constitutive level), while GH3 protein expression was not detected in response to any of these stresses. These results indicate that GH3 expression has excellent potential for use in a novel diagnostic assay for auxinic herbicide injury. The assay may not be eective for extended periods of time after exposure, but likely long enough for samples to be collected once the injury has been identied (several days to a week or more depending on the detection method employed [75,76]). A protein-based immunoassay may not detect injury from lower auxinic herbicide doses, although more sensitive immunodetection methods (e.g., chemilluminescence) may alleviate this problem. However, GH3 expression is very specic for auxinic herbicides, and detection of elevated expression levels in soybean leaves would provide strong evidence for involvement of an auxinic herbicide in causing any injury reported in the eld. 6. Research opportunities As a more complete picture of auxin biology is assembled, new opportunities for auxinic herbicide research are also created. Identifying the site(s) of action for auxinic herbicides has eluded researchers for many years [4]. However, potential solutions to this uncertainty may be attainable in the near future. For example, the identication of the soluble auxin receptors TIR1 and related AFBs [13 15] provides new opportunities for herbicide mechanism of action research into the possibility that TIR1 is a major site of action for auxinic herbicides [72]. In addition, further characterization of ABP1 may help to elucidate its role in signaling and determine its potential as a site of action for auxin and auxinic herbicides [22]. These recent developments may nally lead to identifying the site(s) to which auxinic herbicides bind to initiate their phytotoxic eect. In addition to identication of auxin receptors, several other recent developments in auxin biology may provide unique opportunities for auxinic herbicide research. Several genes involved in auxin inactivation via conjugation to amino acids or glucose have been identied and are currently being characterized. Some auxinic herbicides are not

substrates for several of these enzymes [10,43, Fig. 1], though conjugation to sugars and/or amino acids is a signicant mechanism for auxinic herbicide detoxication [2]. Research conducted in parallel with the auxin inactivation studies may help to identify enzymes involved in auxinic herbicide detoxication and selectivity between monocots and dicots. In addition, dierent pathways for auxin biosynthesis have been identied [8,9,41], which may prove signicant at dierent developmental stages and among species. These distinctions may help to explain dierences in auxinic herbicide selectivity as well as herbicide ecacy at various developmental stages. Auxin-responsive genes provide another important resource for investigating auxin biology and auxinic herbicide mechanism of action. By evaluating the response of these genes to auxinic herbicides (via genomic or proteomic methods) and determining their function in auxin biology, it may be revealed if these genes also play signicant roles in mediating auxinic herbicide phytotoxicity and/or selectivity. For example, ACC synthase is an auxin-responsive gene that converts ACC to ethylene [12,84]. It is known that for some plant species the induction of ACC synthase and subsequent ethylene production are rate-limiting steps in promoting auxinic herbicide phytotoxicity [2,4,85]. Other known (and yet to be discovered) auxin-responsive genes may also play important roles in mediating auxinic herbicide phytotoxicity and/or selectivity. Auxin-responsive genes may also have use in diagnosing o-target auxinic herbicide injury due to their distinct expression patterns. GH3 expression is strongly and specifically induced in soybean leaves in response to auxinic herbicides [75,76]; consequently, detecting elevated expression levels in injured leaves would provide strong evidence for an auxinic herbicide as the cause of the injury. In addition, this application may have use in resistant weed management; an assay may be developed to rapidly determine whether weeds are resistant to an auxinic herbicide based on the presence or absence of GH3 expression [76]. Recent advances in auxin molecular biology have created many new opportunities to realize a more complete picture of auxinic herbicide mechanism of action. In addition, recent discoveries in auxin biology provide new opportunities to elucidate weed resistance mechanisms, develop innovative applications for herbicide injury diagnosis [2,17,20,32,75,76], determine selectivity mechanisms between dicots and monocots, and better understand the basis for tolerance of certain dicots towards specic auxinic herbicides [2]. Auxinic herbicide phytotoxicity involves a massive accumulation of ethylene, ABA, and possibly cyanide as well [4,103]. Advances in our understanding of plant hormones will likely clarify their respective roles, both individually and in combination, towards promoting phytotoxicity. Auxinic herbicides have been very eective at controlling broadleaf weeds in cereal crops for many decades, and will likely be instrumental for weed management for decades to come. Likewise, auxinic herbicides have both puzzled and intrigued herbicide physiologists

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for many decades, and may continue to perplex researchers for years to come. However, it appears that more pieces to the puzzle are nally falling into place. Acknowledgments Funding for research in the laboratory of D.E.R. involving the eects of auxinic herbicides on soybean injury and GH3 expression in soybean was provided by the Illinois Soybean Association and Dow AgroSciences. Thanks and appreciation are extended to Loyd Wax, Ray Zielinski, Leslie Domier, Aaron Hager, Kris Lambert, and Qin Zhang for helpful discussions and research assistance. References
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