Molecular players regulating the jasmonate signalling network

Molecular players regulating the jasmonate signalling network Oscar Lorenzo and Roberto Solano Many plant developmental and stress responses require the coordinated interaction of the jasmonate and other signalling pathways, such as those for ethylene, salicylic acid and abscisic acid. Recent research in Arabidopsis has uncovered several key players that regulate crosstalk between these signalling pathways and that shed light on the molecular mechanisms modulating this coordinated interaction. Genes that are involved in the regulation of protein stability through the ubiquitin-proteasome pathway (COI1, AXR1 and SGT1b), signalling proteins (MPK4) and transcription factors (AtMYC2, ERF1, NPR1 and WRKY70) form a regulatory network that allows the plant to fine-tune specific responses to different stimuli. Addresses Departamento de Gene´tica Molecular de Plantas, Centro Nacional de Biotecnologı´a-CSIC, Campus Universidad Auto´noma, 28049 Madrid, Spain  Present address: Dpto. de Fisiologı´a Vegetal, Centro de Investigaciones Agrarias Luso-Espan˜ol, Facultad de Biologı´a, Universidad de Salamanca, Plaza de los Doctores de la Reina s/n, 37007 Salamanca, Spain. Corresponding author: Solano, Roberto ([email protected])

Current Opinion in Plant Biology 2005, 8:532–540 This review comes from a themed issue on Cell signalling and gene regulation Edited by George Coupland and Salome Prat Monguio Available online 21st July 2005 1369-5266/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2005.07.003

Introduction

is no doubt that jasmonates (JAs) are essential plant hormones that regulate defence responses and developmental processes. These signal molecules are involved in the regulation of many physiological and developmental processes, including root growth, tuberization, fruit ripening, senescence, tendril coiling and pollen development. They are also important regulators of plant responses to environmental stresses, such as ozone exposure, wounding, water deficit, and attack by pathogens and pests [2–14]. Several genetic screens worldwide have identified mutants that are affected in their response to JAs, and the molecular cloning of the corresponding mutated genes has identified some components of the JA signalling pathway. A brief description of the Arabidopsis JA signalling mutants identified to date is presented in Table 1. In addition, gain-of-function experiments in transgenic plants have implied the involvement of ethylene response factor (ERF) transcription factors (i.e. the octadecanoid-derivative responsive Catharanthus AP2domain protein ORCA3 and ERF1 [15,16]) in the regulation of JA-inducible gene expression. This review focuses on the most recent genes to be identified, and on how these genes shed light on the molecular interconnection between the JA and other signalling pathways in the activation of developmental and stress responses.

The proteasome connection: JA crosstalk with auxin, pathogens and light Recent research in plant hormonal signalling has uncovered ubiquitin-mediated protein degradation as a central regulatory mechanism that is involved in many different hormonal pathways [17,18]. JA signalling is a good example of one such hormone pathway as three JA-signalling genes, CORONATINE INSENSITIVE 1 (COI1), SGT1b/ JAI4 and AUXIN RESISTANT 1 (AXR1) (described in Table 1), are part of the ubiquitin-proteasome pathway.

Plant responses to exogenous or endogenous stimuli do not result from the activation of a single signalling pathway but are the consequence of a complex network of interactions among different modulation routes. Different stimuli promote an asymmetric activation of these complex signalling networks, and the final balance of interactions determines the specific response to the initial stimulus. Thus, understanding plant responses to stress requires not only the discovery of the pathways involved but also knowledge of how these pathways are combined by the cell, and of which molecular players determine these interactions.

COI1 has a central role in JA signalling and is required for all JA-dependent responses tested to date [19,20]. COI1 has been found to encode an F-box protein, providing the first indication that ubiquitin-mediated protein degradation is involved in JA signalling [20]. This hypothesis has been further supported by the demonstration that COI1 is present in a functional E3-type ubiquitin ligase complex. Moreover, plants that are deficient in other components of SCF (skip–cullin–F-box) complexes also show impaired JA responses [21–23].

Forty-three years after the discovery of methyl jasmonate as a major lipid constituent of the jasmine scent [1], there

The existence of a COI1 function that is conserved in species other than Arabidopsis has been recently demon-

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Jasmonate signalling network Lorenzo and Solano 533

Table 1 Mutants and genes modulating the jasmonate-signalling pathway in Arabidopsis. Mutant a

Characteristics (screen/phenotype)

Reduced sensitivity to JA coi1 Reduced root growth inhibition and anthocyanin accumulation by coronatine (and JA). Male-sterile. Enhanced sensitivity to A. brassicicola, Pythium irregulare and Bradysia impatiens. jar1/jin4/jai2 Reduced root growth inhibition by MeJA. Enhanced sensitivity to Pythium irregulare. jin1/jai1 Reduced root growth inhibition by JA. AtMYC2 is a nuclear-localised bHLHzip transcription factor. jai3 Reduced root growth inhibition by JA in ein3 background. mpk4 Alteration in the expression of JA- and SA-response genes. Dwarf phenotype. jai4/sgt1b Reduced root growth inhibition by JA in the ein3 background. axr1 Reduced root growth inhibition by MeJA. jue1–3 oji1

a

Reference(s)

F-box–leucine-rich repeat (LRR) (At2 g39940)

[19,20]

JA–amino synthetase (At2 g46370) AtMYC2 (At1 g32640)

[52,61,76] [52,76]

AtMPK4 (At4 g01370)

[52] [69]

SGT1b (At4 g11260) RUB-activating enzyme E1 (At1 g05180)

Reduced expression of pLOX2::LUC. Enhanced sensitivity to ozone. Reduced root growth inhibition by MeJA. Reduced expression of VSP.

Suppressor of JA-related defects in coi1, such as root growth, senescence and defence.

[52] [22,28] [77] [78]

Constitutive JA response cet1–9 Constitutive expression of thionin (Thi2.1:BAR). High constitutive levels of JA and 12-oxo-phytodienoic acid (OPDA). Spontaneous microlesions. cex1 Constitutive root growth inhibition and expression of JA-inducible genes VSP, Thi2.1 and PDF1.2. CEX1 is a negative regulator of JA signalling downstream of COI. cev1 Constitutive expression of pVSP1::LUC. CEV1 acts as negative regulator of JA and ET pathways upstream of COI1 and ETR1. joe1 Increased expression of pLOX2::LUC and accumulation of anthocyanins. joe2 Increased expression of pLOX2::LUC and reduced inhibition of root growth. cas1 Constitutive expression of allene oxide synthase (AOS1). Others cos1

Gene

[79]

[80]

AtCeSA3 (At5 g05170)

[56,57]

[77] [77] [81] Lumazine synthase (At2 g44050)

[25]

All mutants for which the corresponding genes have been identified are highlighted in bold.

strated by the identification of a COI1 homologue in tomato (LeCOI1). Interestingly, mutations in LeCOI1 show that, at least in tomato, JA is involved in developmental processes, such as ovule development, that are not impaired in Arabidopsis coi1 mutants [24], suggesting that other COI1-like proteins might regulate these processes in Arabidopsis. Putative targets of COI1-dependent proteasome degradation have been identified and their functional analysis will be instrumental to furthering our understanding of the molecular mechanisms that regulate JA responses [21,25]. Using a two-hybrid strategy, Devoto et al. [21] identified (among other proteins) RPD3b, a histone deacetylase, as a COI1 target. Because histone deacetylation is believed to decrease the accessibility of chromatin to www.sciencedirect.com

the transcription machinery [26], COI1-dependent proteasome degradation of RPD3b would be a probable mechanism for derepression of JA-dependent transcription. However, constitutive expression of an RPD3brelated histone deacetylase in Arabidopsis (i.e. RPD3a/ HD19) has the opposite effect, increasing transcription of the JA-induced ERF1 transcription factor and ERF1 targets [27]. Therefore, the regulation of JA signalling by histone deacetylation is complex and awaits further characterisation. Another putative target of COI1 is COS1. cos1 has been identified as a suppressor of coi1, restoring some JAregulated responses, such as root growth, senescence and defence, in the coi1 mutant [25]. COS1 encodes lumazine synthase, a key component of the riboflavin Current Opinion in Plant Biology 2005, 8:532–540

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pathway, which suggests the involvement of this pathway in the modulation of JA signalling. COI1 is a component that is specific to the JA pathway, whereas SGT1b/JAI4 and AXR1 are shared by other signalling pathways [22,28,29]. AXR1 is a positive regulator of auxin responses, and it modulates the activity of SCFTIR1 by modifying cullin through the addition of the ubiquitin-like protein Nedd8/Rub1 [30,31]. Because cullin is a common component of SCF complexes, the pleiotropic effect of axr1 mutations suggests that this protein also regulates SCFCOI1. The jasmonic-acid-insensitive4 (jai4) mutation was identified in a screen for JA-insensitive mutants in an ethyleneinsensitive (ein3) background. JAI4 has been recently identified by map-based cloning and found to encode SGT1b (O Lorenzo, R Solano, unpublished). The yeast SGT1 homologue interacts with components of SCF complexes, suggesting that it has a role in the regulation of the ubiquitin-proteasome pathway [32]. The conserved function of SGT1 in mediating SCF activity in plants is supported by the complementation of the yeast sgt1 mutations by the two highly related Arabidopsis SGT1 genes, and by the observation that the SGT1 proteins Hv SGT1 and NbSGT1 co-immunoprecipitate with core SCF subunits in barley and Nicotiana benthamiana [33,34]. Like mutations in AXR1, mutations in SGT1b have pleiotropic effects that impair plant responses not only to JA but also to auxin and pathogens, suggesting that both SGT1b and AXR1 are regulators of SCF complexes and are involved in several different signalling pathways [29,33,35]. Regulation of SCF function also explains interactions between light and JA pathways. Genetic and molecular analysis have suggested the involvement of the COP9 signalosome (CSN) together with COP1 (ubiquitin-ligase E3) and COP10 (E2) in the regulation of photomorphogenesis through the light-dependent degradation of HY5 and HYH transcription factors [36,37]. The CSN interacts not only with COP1 but also with SCF-type E3 ligases such as SCFTIR1 and SCFCOI1 [23,30]. Transgenic plants in which the functionalities of components of the CSN are reduced show a reduced response to JA with regard to the inhibition of root growth and the induction of JAresponsive genes such as VSP (VEGETATIVE STORAGE PROTEIN), which suggests the involvement of CSN in the regulation of JA signalling [23]. AtMYC2 constitutes an additional point of crosstalk between the JA and light pathways. This transcription factor, previously shown to be a key component of the JA pathway (see below), has recently been shown to function as a negative regulator of blue-light-mediated photomorphogenic growth [38]. This surprising discovery suggests that AtMYC2 acts as a general integrator of different environmental stresses. Current Opinion in Plant Biology 2005, 8:532–540

JA and ethylene: cooperation and antagonism JA and ethylene (ET) can either cooperate or act as antagonists in the regulation of different stress responses (i.e. to pathogen attack, wounding and ozone exposure) and developmental processes (i.e. exaggerated apical hook development) [11–14]. The selection of the specific response that is appropriate for each stress stimulus seems to be determined by the type of interaction established between the JA and ET signalling pathways. Thus, in the case of pathogens, both hormones cooperate or synergise in the activation of defence gene expression [16,39,40]. Mutants that are affected in the synthesis or perception of either JA or ET are impaired in the induction of defences and show an increased susceptibility to pathogens [7,41– 44]. However, in the case of the wound response in Arabidopsis, an antagonistic interaction between JA and ET has been described in the activation of local responses in the damaged tissues [14,45]. Two transcription factors, ERF1 and AtMYC2/JIN1 (JASMONATE INSENSITIVE 1) have been shown to participate in the regulation of these interactions in Arabidopsis. ERF1 plays a key role in the integration of JA and ET signals in the activation of plant defences. ERF1 expression is induced upon necrotrophic pathogen infection and regulates ‘in vivo’ the expression of defence-related genes, such as PDF1.2 and b-CHI. Constitutive expression of ERF1 in transgenic plants is sufficient to enhance resistance to several necrotrophic fungi [46,47]. In addition to being regulated by pathogens, the expression of ERF1 (and ERF1-targets) is induced by ET and JA, and this induction depends on the simultaneous action of the JA and ET signalling pathways. In fact, mutations that block either of these signalling pathways (i.e. ein2 or coi1) block the induction of ERF1 expression and its targets [16,40,48]. The simultaneous requirement of both signals for ERF1 expression explains, at the molecular level, the cooperation of both pathways in the activation of pathogen defence genes. In addition to ERF1, several other ERFs are also induced by JA and ET and are possibly involved in the regulation of defences against pathogens, which suggests that these genes might share partially redundant functions with ERF1 [49–51]. JIN1 encodes a nuclear-localised bHLHzip-type transcription factor (AtMYC2) that differentially regulates two branches in the JA-signalling pathway [52]. One of these branches, which is negatively regulated by AtMYC2, is required for the expression of pathogen defence genes. Consistently, jin1 mutants show increased resistance to pathogens [52,53,54]. The other branch, which is positively regulated by AtMYC2, induces the expression of genes that are involved in the response to wounding (either mechanical or biotic). Interestingly, www.sciencedirect.com

Jasmonate signalling network Lorenzo and Solano 535

these two branches are also differentially regulated by ERF1, but the effects of ERF1 are opposite to those of AtMYC2. ERF1 positively regulates expression of pathogen-response genes, and prevents the JA-mediated induction of wound-response genes such as VSP2 [52]. Therefore, the interplay between AtMYC2 and ERF1 can explain how plants select the correct response to two different stresses (i.e. pathogen attack and wounding) that share the same signals (ET and JA) for their activation. Two JA-regulated MYC transcription factors (JAMYC2 and JAMYC10) have been identified in tomato, using a ‘one-hybrid’ strategy, and found to specifically recognise a T/G box in the promoter of the JA-induced LEUCINE AMINOPEPTIDASE (LAP) gene [55]. When constitutively expressed in a jin1/atmyc2 mutant background, both JAMYC2 and JAMYC10 complemented the phenotypic defects of this mutant in terms of root-growth inhibition by JA and the expression of JA-induced genes such as VSP2 and PDF1.2, indicating that the JIN1/AtMYC2 function is conserved in dicotyledonous plants. Modulation of the synthesis or availability of JA and ET might be another regulatory level in the crosstalk between these hormones, as suggested by the mutant phenotypes of the constitutive response mutant cev1 and the JA-insensitive mutant jar1. The cev1 mutant has a stunted phenotype, resulting from an increased production of JA and ET, which can be suppressed by mutations that block the JA and ET signalling pathways (i.e. coi1 and etr1). CEV1 encodes a cellulose synthase, indicating that the cell wall might be involved in stress signalling and that CEV1 might coordinate the simultaneous synthesis of both hormones in response to stress [56,57]. JAR1 encodes an enzyme that has JA adenylation activity and that is able to form conjugates between JA and several amino acids, mainly with isoleucine [58,59]. In addition to amino acids, JAR1 is able to conjugate JA with 1-aminocyclopropane-1-carboxylic acid (ACC), the ET precursor, which can then be detected in Arabidopsis leaves. JA–ACC is a jasmonate derivative that is poorly effective in inhibiting root growth, suggesting that JA–ACC conjugation might provide a mechanism that regulates the availability of both JA and ET.

JA and abscisic acid interconnections Several physiological and developmental processes are influenced by crosstalk between JA and abscisic acid (ABA), including seed germination and plant defences against pathogens and wounding. Exogenous application of JA inhibits germination in several species and shows a synergistic effect with ABA in Arabidopsis [60–62]. Nevertheless, the effects of the exogenous application of hormones might not reliably reflect endogenous hormonal function. For example, two JA-insensitive mutants, coi116 and jar1, are hypersensitive to ABA during seed www.sciencedirect.com

germination, which indicates that JA antagonises ABAmediated inhibition of germination [61,62]. Antagonistic interactions between ABA and JA pathways have also been observed to modulate pathogen defence responses [53], whereas positive interactions between ABA and JA have been reported in responses to wounding. ABA perception is required for the induction of the PROTEINASE INHIBITOR (PIN) gene in response to wounding [63], and induction of JA synthesis (and signalling) by ABA has been widely documented [64–66]. JIN1/ AtMYC2 might be a key regulator of these interactions between ABA and JA. In addition to JA, AtMYC2 expression is also activated by ABA in a COI1-dependent manner, which suggests that ABA precedes JA in the activation of AtMYC2-mediated wound responses [52,67,68]. In responses to pathogen attack, the negative effect of ABA on the expression of JA-regulated defence genes [53] cannot be explained solely by the activation of AtMYC2 by ABA, which prevents the induction of pathogen-related (PR) genes, because this repression by ABA also occurs in an atmyc2 mutant background [52,53,55]. This indicates that ABA acts independently of JA in the regulation of pathogen defences, and that AtMYC2 should not be the only point of confluence.

Antagonistic interactions between JA and salicylic acid The crosstalk interactions between JA and salicylic acid (SA) are probably the most intensively studied interactions involving JA and have been extensively summarised in several excellent reviews [11,13,14]. Here, we focus only on recently identified players that regulate this crosstalk. Although the above-mentioned reviews show many examples of cooperative interactions between JA and SA, in general, these two hormones interact in a mutually antagonistic fashion. MPK4 (encoding MAP-kinase 4) was the first gene to be identified as a regulator of the negative crosstalk between JA and SA in the activation of defences. MPK4 is required for the induction of some JA-regulated defence genes such as PDF1.2 and THI2.1. mpk4 loss-of-function mutants constitutively express SA-regulated PR genes, probably as a result of their elevated SA levels [69]. This indicates that the MAP kinase cascade that involves MPK4 regulates JA–SA crosstalk by simultaneously repressing SA biosynthesis and promoting the perception of or response to JA. Similarly, constitutive ERF1 overexpression that promotes the activation of JA- or ETdependent defences, reduces tolerance of Pseudomonas syringae, suggesting a negative crosstalk between the JA/ ET and the SA signalling pathways [46]. It has also been shown that the induction of SA after Pseudomonas infection prevents JA accumulation, with this negative interaction being modulated through Current Opinion in Plant Biology 2005, 8:532–540

536 Cell signalling and gene regulation

NPR1 [70,71]. This protein, activated by SA, interacts with members of the TGA family of transcription factors and activates SA-dependent expression of PR genes. Surprisingly, the negative effect of NPR1 on JA signalling does not require its nuclear localisation, but seems to be exerted through a novel NPR1 function in the cytosol [70]. NPR1 might not work alone because an NPR1-like gene (NPR4) has recently been shown to be required for full expression of the JA-induced PDF1.2 gene [72].

In summary, JA–SA crosstalk constitutes an excellent example of the complex regulatory networks that allow the plant to fine-tune specific responses to different sets of pathogens.

Conclusions Recent research in plant hormone signalling has uncovered two major concepts: first, hormone signalling pathways are not isolated but are connected in complex regulatory networks; and second, ubiquitin-mediated protein degradation is a central regulatory mechanism involving many different hormonal pathways. In the case of JA-signalling, both concepts apply (Figure 1). The ubiquitin-proteasome pathway plays a central role in JA signalling and crosstalk with other pathways. In most JAregulated processes, the precise plant response is not activated by JA alone but is the result of a network of interactions between different signalling pathways. Key players that modulate these regulatory networks have been identified, and determining how these genes interact has extended our understanding of the molecular

The transcription factor WRKY70 has recently been shown to be a key regulator of JA–SA crosstalk. WRKY70 expression is positively regulated by SA and negatively regulated by JA. Constitutive ectopic expression of WRKY70 enhances resistance to virulent pathogens and results in constitutive expression of SA-responsive PR genes. Conversely, antisense suppression of WRKY70 activates the expression of JA-induced genes, which suggests that this transcription factor might function as a switch between the SA and JA signalling pathways [73].

Figure 1

Biotic/abiotic stress

ABA JAR1 ABA

ET

MPK4 SA

JA

AXR1 CSN SGT1b

NPR1

SCFCOI1

ERF1

AtMYC2

WRKY70

PDF1.2, b-CHI, HEL

VSP, Lox, Thi2.1

PR1 Current Opinion in Plant Biology

Stress-responsive network involving the JA, ET, SA and ABA signalling pathways. Different types of biotic or abiotic stress, such as pathogen infection or wounding, induce the synthesis and subsequent activation of several hormonal pathways (i.e. JA, ET, SA and ABA). JA and JA–isoleucine (JA–Ile) (which is synthesized by JAR1) activates, via SCFCOI1, the expression of AtMYC2, which upregulates wound-response genes (i.e. VSP, Lox and Thi2.1) and represses pathogen-response genes (i.e. PDF1.2, b-CHI and HEL). The cooperation of the ET and JA signals through the transcriptional induction of ERF1 has the opposite effect, activating the expression of pathogen-response genes and repressing wounding-response genes. ABA has a negative effect on the transcriptional activation of JA/ET-regulated defence genes. In the response to wounding, ABA’s activity seems to precede JA synthesis. SCFCOI1 is a central regulator of all JA-dependent responses, the activity of which is presumably modulated by several ubiquitin-proteasome pathway genes (e.g. AXR1, SGT1b and CSN) that are also involved in the modulation of other SCF complexes. The negative interactions of the JA and SA pathways are exemplified by the negative effect on JA signalling of NPR1 and WRKY70, which are positive regulators of SA-dependent responses. MPK4 has the opposite role, negatively influencing SA biosynthesis but positively regulating JA signalling. COI1-dependent and AtMYC2-dependent PR1 repression represent two other examples of the antagonism between these signalling pathways. In summary, this complex network of interactions allows the plant to select the correct set of genes in response to each particular stress. Arrows indicate induction or positive interaction, whereas dashed lines indicate repression or negative interaction. Thicker arrows represent the main JA pathway. Current Opinion in Plant Biology 2005, 8:532–540

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Jasmonate signalling network Lorenzo and Solano 537

mechanisms that underlie JA-dependent responses. However, the identification of additional components of the JAregulated network is necessary to complete this understanding. A major step in the JA signalling pathway remains to be uncovered: the JA receptor. In the case of auxin signalling, it has just been demonstrated that the F-box protein TIR1 is the auxin receptor [74,75]. By analogy, COI1, the closest F-box protein to TIR1 in the Arabidopsis genome, could well be the JA receptor. Certainly, further research on JA signalling and JA-regulated responses will clarify this point and will extend our understanding of the molecular mechanisms that underlie the JA-regulated network.

Acknowledgements We thank the members of the Solano laboratory for comments on the manuscript. Work in this laboratory that is described in this review was financed by Grants 07G/0048/2000 and 07B/0044/2002 from the Comunidad de Madrid and BIO2001-0567 from the Spanish Ministerio de Ciencia y Tecnologı´a. OL is supported by a ‘Ramon y Cajal’ research contract.

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11. Turner JG, Ellis C, Devoto A: The jasmonate signal pathway. Plant Cell 2002, 14:S153-S164. 12. Farmer EE, Almeras E, Krishnamurthy V: Jasmonates and related oxylipins in plant responses to pathogenesis and herbivory. Curr Opin Plant Biol 2003, 6:372-378. 13. Devoto A, Turner JG: Regulation of jasmonate-mediated plant responses in Arabidopsis. Ann Bot (Lond) 2003, 92:329-337. 14. Rojo E, Solano R, Sanchez-Serrano JJ: Interactions between signaling compounds involved in plant defense. J Plant Growth Regul 2003, 22:82-98. 15. van der Fits L, Memelink J: ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science 2000, 289:295-297. 16. Lorenzo O, Piqueras R, Sanchez-Serrano JJ, Solano R: ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense. Plant Cell 2003, 15:165-178. 17. Devoto A, Muskett PR, Shirasu K: Role of ubiquitination in the regulation of plant defence against pathogens. Curr Opin Plant Biol 2003, 6:307-311. 18. Moon J, Parry G, Estelle M: The ubiquitin-proteasome pathway and plant development. Plant Cell 2004, 16:3181-3195. 19. Feys B, Benedetti CE, Penfold CN, Turner JG: Arabidopsis mutants selected for resistance to the phytotoxin coronatine are male sterile, insensitive to methyl jasmonate, and resistant to a bacterial pathogen. Plant Cell 1994, 6:751-759. 20. Xie DX, Feys BF, James S, Nieto-Rostro M, Turner JG: COI1: an Arabidopsis gene required for jasmonate-regulated defense and fertility. Science 1998, 280:1091-1094. 21. Devoto A, Nieto-Rostro M, Xie D, Ellis C, Harmston R, Patrick E, Davis J, Sherratt L, Coleman M, Turner JG: COI1 links jasmonate signalling and fertility to the SCF ubiquitin-ligase complex in Arabidopsis. Plant J 2002, 32:457-466. 22. Xu L, Liu F, Lechner E, Genschik P, Crosby WL, Ma H, Peng W, Huang D, Xie D: The SCF(COI1) ubiquitin-ligase complexes are required for jasmonate response in Arabidopsis. Plant Cell 2002, 14:1919-1935. 23. Feng S, Ma L, Wang X, Xie D, Dinesh-Kumar SP, Wei N, Deng XW: The COP9 signalosome interacts physically with SCF COI1 and modulates jasmonate responses. Plant Cell 2003, 15:1083-1094. 24. Li L, Zhao Y, McCaig BC, Wingerd BA, Wang J, Whalon ME,  Pichersky E, Howe GA: The tomato homolog of CORONATINEINSENSITIVE1 is required for the maternal control of seed maturation, jasmonate-signaled defense responses, and glandular trichome development. Plant Cell 2004, 16:126-143. This paper describes the phenotypic and molecular characterisation of the tomato sterile mutant jai1, which is defective in the tomato homologue of COI1. Importantly, the effects of the jai1 mutation include sterility caused by a defect in the maternal control of seed maturation. This defect is associated with the loss of accumulation of jasmonate (JA)regulated proteinase inhibitor proteins in reproductive tissues. Other jai1 phenotypes that are related to defence are susceptibility to two-spotted spider mites and modulation of mite behaviour, the abnormal development of glandular trichomes and the absence of JA-responsive gene expression. COI1 plays a central role in the expression of the JA-regulated transcriptome in tomato. Comparison of the JA/COI1 signalling pathway in tomato and Arabidopsis indicates that this pathway regulates distinct developmental processes, and suggests a role for JA in the promotion of glandular-trichome-based defences. 25. Xiao S, Dai L, Liu F, Wang Z, Peng W, Xie D: COS1: an Arabidopsis  coronatine insensitive1 suppressor essential for regulation of jasmonate-mediated plant defense and senescence. Plant Cell 2004, 16:1132-1142. The authors identify the cos1 mutant in a screen for suppressors of coi1. The cos1 mutation restores the coi1-deficient phenotypes related to root elongation, senescence, plant defence responses, and jasmonate-inducible gene expression. This suppression seems to be COI-specific because cos1 does not affect the phenotype of other F-box mutants such as tir1. The COS1 gene is found to encode lumazine synthase, Current Opinion in Plant Biology 2005, 8:532–540

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supporting the involvement of the riboflavin pathway downstream of COI1 in jasmonate signalling. 26. Lusser A, Kolle D, Loidl P: Histone acetylation: lessons from the plant kingdom. Trends Plant Sci 2001, 6:59-65. 27. Zhou C, Zhang L, Duan J, Miki B, Wu K: HISTONE  DEACETYLASE19 is involved in jasmonic acid and ethylene signaling of pathogen response in Arabidopsis. Plant Cell 2005, 17:1196-1204. After the identification of HISTONE DEACETYLASE19 (AtRPD3A) in Arabidopsis, the authors reported its expression pattern and induction by wounding, by infection by the fungal pathogen Alternaria brassicicola, and by JA and ET. By means of transgenic analysis, the authors demonstrate a role for HDA19 in the regulation of JA/ET gene expression and pathogen response in Arabidopsis. Constitutive overexpression of HDA19 decreased histone acetylation levels, and increased expression of JA- and ET-regulated genes (e.g. ERF1 and PRs) and resistance to the A. brassicicola. Opposite effects were found in HDA19-RNAi lines. 28. Tiryaki I, Staswick PE: An Arabidopsis mutant defective in jasmonate response is allelic to the auxin-signaling mutant axr1. Plant Physiol 2002, 130:887-894. 29. Gray WM, Muskett PR, Chuang H-W, Parker JE: Arabidopsis SGT1b is required for SCF TIR1-mediated auxin response. Plant Cell 2003, 15:1310-1319. 30. Schwechheimer C, Serino G, Deng XW: Multiple ubiquitin ligase-mediated processes require COP9 signalosome and AXR1 function. Plant Cell 2002, 14:2553-2563. 31. del Pozo JC, Dharmasiri S, Hellmann H, Walker L, Gray WM, Estelle M: AXR1-ECR1-dependent conjugation of RUB1 to the Arabidopsis Cullin AtCUL1 is required for auxin response. Plant Cell 2002, 14:421-433. 32. Kitagawa K, Skowyra D, Elledge SJ, Harper JW, Hieter P: SGT1 encodes an essential component of the yeast kinetochore assembly pathway and a novel subunit of the SCF ubiquitin ligase complex. Mol Cell 1999, 4:21-33. 33. Azevedo C, Sadanandom A, Kitagawa K, Freialdenhoven A, Shirasu K, Schulze-Lefert P: The RAR1 interactor SGT1, an essential component of R gene-triggered disease resistance. Science 2002, 295:2073-2076. 34. Liu Y, Schiff M, Serino G, Deng XW, Dinesh-Kumar SP: Role of SCF ubiquitin-ligase and the COP9 signalosome in the N genemediated resistance response to Tobacco mosaic virus. Plant Cell 2002, 14:1483-1496. 35. Austin MJ, Muskett P, Kahn K, Feys BJ, Jones JDG, Parker JE: Regulatory role of SGT1 in early R gene-mediated plant defenses. Science 2002, 295:2077-2080. 36. Osterlund MT, Hardtke CS, Wei N, Deng XW: Targeted destabilization of HY5 during light-regulated development of Arabidopsis. Nature 2000, 405:462-466. 37. Holm M, Ma LG, Qu LJ, Deng XW: Two interacting bZIP proteins are direct targets of COP1-mediated control of lightdependent gene expression in Arabidopsis. Genes Dev 2002, 16:1247-1259. 38. Yadav V, Mallappa C, Gangappa SN, Bhatia S, Chattopadhyay S:  A basic helix-loop-helix transcription factor in Arabidopsis, MYC2, acts as a repressor of blue light-mediated photomorphogenic growth. Plant Cell 2005, 17:1953-1966. The authors report a newly discovered role for AtMYC2, which had previously been shown to mediate ABA- and JA-responses, in blue-light signalling. They show that the AtMYC2 transcription factor is expressed in seedlings grown under various light conditions, and interacts with the Zbox and G-box light-responsive elements of minimal light-regulated promoters. Phenotypic and genetic analyses of atmyc2 mutants demonstrate that AtMYC2 acts as a negative regulator of blue-light-mediated photomorphogenic growth and blue- and far-red-light-regulated gene expression. In addition, the authors also show that AtMYC2 functions as a positive regulator of lateral root formation. Taken together, these results demonstrate that AtMYC2 is a key integrator of different signalling pathways in Arabidopsis. 39. Xu Y, Chang PF, Liu D, Narasimhan ML, Raghothama KG, Hasegawa PM, Bressan RA: Plant defense genes are synergically induced by ethylene and methyl jasmonate. Plant Cell 1994, 6:1077-1085. Current Opinion in Plant Biology 2005, 8:532–540

40. Penninckx IAMA, Thomma BPHJ, Buchala A, Metraux J-P, Broekaert WF: Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin. Plant Cell 1998, 10:2103-2113. 41. Knoester M, van Loon LC, van den Heuvel J, Henning J, Bol JF, Linthorst HJM: Ethylene-insensitive tobacco lacks nonhost resistance against soil-borne fungi. Proc Natl Acad Sci USA 1998, 95:1933-1937. 42. Vijayan P, Shockey J, Levesque CA, Cook RJ, Browse J: A role for jasmonate in pathogen defense of Arabidopsis. Proc Natl Acad Sci USA 1998, 95:7209-7214. 43. Thomma BPHJ, Eggermont K, Penninckx IAMA, Mauch-Mani B, Vogelsang R, Cammue BPA, Broekaert WF: Separate jasmonate-dependent and salicylate-dependent defenseresponse pathways in Arabidopsis are essential for resistance to distinct microbial pathogens. Proc Natl Acad Sci USA 1998, 95:15107-15111. 44. Thomma BPHJ, Eggermont K, Tierens KFM-J, Broekaert WF: Requirement of functional Ethylene-Insensitive2 gene for efficient resistance of Arabidopsis to infection by Botrytis cinerea. Plant Physiol 1999, 121:1093-1101. 45. Rojo E, Leon J, Sanchez-Serrano JJ: Cross-talk between wound signalling pathways determines local versus systemic gene expression in Arabidopsis thaliana. Plant J 1999, 20:135-142. 46. Berrocal-Lobo M, Molina A, Solano R: Constitutive expression of ETHYLENE-RESPONSE-FACTOR1 in Arabidopsis confers resistance to several necrotrophic fungi. Plant J 2002, 29:23-32. 47. Berrocal-Lobo M, Molina A: Ethylene response factor 1  mediates Arabidopsis resistance to the soilborne fungus Fusarium oxysporum. Mol Plant Microbe Interact 2004, 17:763-770. Using mutants that are impaired in the ET, JA, and SA signalling pathways, the authors explored the signal transduction network that controls resistance to the soil-borne fungus Fusarium oxysporum. They showed that all three hormonal signals and NPR1 function are required for resistance. Expression of ERF1 was upregulated after inoculation with the fungus, but this upregulation was blocked in ein2-5 and coi1-1 mutants. In addition, constitutive ERF1-overexpression in Arabidopsis also confers enhanced resistance to this pathogen. These results extend previous observations from work on other pathogens [46] and support a wide role for ERF1 in the activation of defence responses. 48. Solano R, Stepanova A, Chao QM, Ecker JR: Nuclear events in ethylene signaling: a transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSEFACTOR1. Genes Dev 1998, 12:3703-3714. 49. Chen W, Provart NJ, Glazebrook J, Katagiri F, Chang HS, Eulgem T, Mauch F, Luan S, Zou G, Whitham SA: Expression profile matrix of Arabidopsis transcription factor genes suggests their putative functions in response to environmental stresses. Plant Cell 2002, 14:559-574. 50. Onate-Sanchez L, Singh KB: Identification of Arabidopsis ethylene-responsive element binding factors with distinct induction kinetics after pathogen infection. Plant Physiol 2002, 128:1313-1322. 51. Brown RL, Kazan K, McGrath KC, Maclean DJ, Manners JM: A role for the GCC-box in jasmonate-mediated activation of the PDF1.2 gene of Arabidopsis. Plant Physiol 2003, 132:1020-1032. 52. Lorenzo O, Chico JM, Sanchez-Serrano JJ, Solano R:  Jasmonate-insensitive1 encodes a MYC transcription factor essential to discriminate between different jasmonateregulated defence responses in Arabidopsis. Plant Cell 2004, 16:1938-1950. The authors exploit the negative crosstalk between ethylene (ET) and jasmonate (JA) to design a screening for JA-insensitive mutants using an ET-insensitive (ein3) background. They isolated five independent loci and further characterised jai1/jin1. JIN1 encodes the bHLH-leucine-zipper transcription factor AtMYC2, which is nuclear-localised and rapidly upregulated by JA and abscisic acid in a COI1-dependent manner. AtMYC2 differentially regulates the expression of two groups of JAinduced genes. The first group includes genes that are involved in defense responses against pathogens and is repressed by AtMYC2. Consistently, jin1 mutants show increased resistance to necrotrophic www.sciencedirect.com

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pathogens. The second group, which are integrated by genes that are involved in JA-mediated systemic responses to wounding, are activated by AtMYC2. Gain-of-function experiments confirmed the relevance of AtMYC2 in the activation of JA signalling. Finally, the authors also present data that indicate that the two AtMYC2-regulated branches of the JA signalling pathway are antagonistically regulated by ERF1. 53. Anderson JP, Badruzsaufari E, Schenk PM, Manners JM,  Desmond OJ, Ehlert C, Maclean DJ, Ebert PR, Kazan K: Antagonistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene expression and disease resistance in Arabidopsis. Plant Cell 2004, 16:3460-3479. The authors investigate the suggested role of abscisic acid (ABA) in enhancing disease susceptibility and provide evidence that ABA application suppresses the basal and induced transcription levels of JA- and ETregulated defence genes. Mutations that cause ABA deficiencies (i.e. aba1 and aba2) and disruption of the AtMYC2 transcription factor increased the expression of JA–ET-regulated defence genes. Consistently, aba1, aba2 and atmyc2 mutants showed increased resistance to the necrotrophic fungus Fusarium oxysporum. Furthermore, overexpression of AtMYC2 confirmed the negative regulatory effect of this protein on defence gene expression in Arabidopsis. Finally, analysis of JA-, ET- and ABA-insensitive mutants led the authors to propose a model in which the ABA and JA/ET signalling pathways are mutually antagonistic, and modulate defence and stress-responsive gene expression in response to biotic and abiotic stresses. 54. Nickstadt A, Thomma BPHJ, Feussner I, Kangasja¨ rvi J, Zeier J, Loeffler C, Scheel D, Berger S: The jasmonate-insensitive mutant jin1 shows increased resistance to biotrophic as well as necrotrophic pathogens. Mol Plant Pathol 2004, 5:425-434. 55. Boter M, Ruiz-Rivero O, Abdeen A, Prat S: Conserved MYC  transcription factors play a key role in jasmonate signaling both in tomato and Arabidopsis. Genes Dev 2004, 18:1577-1591. Using yeast one-hybrid screening, the authors identified two bHLH-leu zipper proteins, JAMYC2 and JAMYC10, that specifically recognise a T/ G-box motif present in the tomato leucine aminopeptidase (LAP) promoter. Site-directed mutagenesis of this G-element resulted in reduced jasmonate (JA) activation. Other motifs were found in this LAP promoter region by in vivo footprinting and mutation of these motifs abolished the JA response. The authors reported that JAMYC2 and JAMYC10 are induced by JA preceding LAP and PIN2 transcript accumulation and that overexpression of JAMYC enhanced JA-induced expression of these genes. Knockout mutants in the AtMYC2 homologue were identified and these showed JA-insensitivity and differential regulation of defencerelated genes. JAMYCs were able to complement these phenotypes, pointing to a key role for MYC transcription factors in JA-induced defence gene activation and a conservation of this function in dicotyledonous plants. 56. Ellis C, Turner JG: The Arabidopsis mutant cev1 has constitutively active jasmonate and ethylene signal pathways and enhanced resistance to pathogens. Plant Cell 2001, 13:1025-1033. 57. Ellis C, Karafyllidis I, Wasternack C, Turner JG: The Arabidopsis mutant cev1 links cell wall signaling to jasmonate and ethylene responses. Plant Cell 2002, 14:1557-1566. 58. Staswick PE, Tiryaki I, Rowe ML: Jasmonate response locus JAR1 and several related Arabidopsis genes encode enzymes of the firefly luciferase superfamily that show activity on jasmonic, salicylic, and indole-3-acetic acids in an assay for adenylation. Plant Cell 2002, 14:1405-1415. 59. Staswick PE, Tiryaki I: The oxylipin signal jasmonic acid is  activated by an enzyme that conjugates it to isoleucine in Arabidopsis. Plant Cell 2004, 16:2117-2127. This work demonstrates that JAR1 is a JA–amino synthetase that is required to activate JA signalling in Arabidopsis. Therefore, activation of JA by conjugation with amino acids (mainly isoleucine) is required for full JA responses. Furthermore, the authors also found 1-aminocyclopropane-1-carboxylic acid (ACC), the ethylene precursor, conjugated to JA in plant tissues, providing a mechanism to co-regulate the availability of JA and ACC for conversion to the active hormones JA–isoleucine and ethylene, respectively. 60. Wilen RW, van Rooijen GJ, Pearce DW, Pharis RP, Holbrook IA, Moloney MM: Effects of jasmonic acid on embryo specific processes in Brassica and Linum oilseeds. Plant Physiol 1991, 95:399-405.

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61. Staswick PE, Su W, Howell SH: Methyl jasmonate inhibition of root growth and induction of a leaf protein are decreased in an Arabidopsis thaliana mutant. Proc Natl Acad Sci USA 1992, 89:6837-6840. 62. Ellis C, Turner JG: A conditionally fertile coi1 allele indicates cross-talk between plant hormone signalling pathways in Arabidopsis thaliana seeds and young seedlings. Planta 2002, 215:549-556. 63. Carrera E, Prat S: Expression of the Arabidopsis abi1-1 mutant allele inhibits proteinase inhibitor wound-induction in tomato. Plant J 1998, 15:765-771. 64. Hildmann T, Ebneth M, Pena-Cortes H, Sanchez-Serrano JJ, Willmitzer L, Prat S: General roles of abscisic and jasmonic acids in gene activation as a result of mechanical wounding. Plant Cell 1992, 4:1157-1170. 65. Pena-Cortes H, Fisahn J, Willmitzer L: Signals involved in woundinduced proteinase inhibitor II gene expression in tomato and potato plants. Proc Natl Acad Sci USA 1995, 92:4106-4113. 66. Leon J, Rojo E, Sanchez-Serrano JJ: Wound signalling in plants. J Exp Bot 2001, 52:1-9. 67. Abe H, Yamaguchi-Shinozaki K, Urao T, Iwasaki T, Hosokawa D, Shinozaki K: Role of Arabidopsis MYC and MYB homologs in drought- and abscisic acid-regulated gene expression. Plant Cell 1997, 9:1859-1868. 68. Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K: Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell 2003, 15:63-78. 69. Petersen M, Brodersen P, Naested H, Andreasson E, Lindhart U, Johansen B, Nielsen HB, Lacy M, Austin MJ, Parker JE: Arabidopsis Map kinase 4 negatively regulates systemic acquired resistance. Cell 2000, 103:1111-1120. 70. Spoel SH, Koornneef A, Claessens SM, Korzelius JP, Van Pelt JA, Mueller MJ, Buchala AJ, Metraux JP, Brown R, Kazan K et al.: NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathways through a novel function in the cytosol. Plant Cell 2003, 15:760-770. 71. Dong X: NPR1, all things considered. Curr Opin Plant Biol 2004,  7:547-552. Several recent papers have shown the essential role of the NPR1 protein in SA-mediated systemic acquired resistance (SAR), in induced systemic resistance (ISR) and in crosstalk inhibition of JA-mediated defence responses. This review summarises the molecular characterisation of NPR1 and TGA transcription factors. The author also reviews the roles of the AtWhy1 and WRKY70 transcription factors in plant defence, which are involved in SA-mediated defence and SA–JA crosstalk, respectively. 72. Liu G, Holub EB, Alonso JM, Ecker JR, Fobert PR: An Arabidopsis NPR1-like gene, NPR4, is required for disease resistance. Plant J 2005, 41:304-318. 73. Li J, Brader G, Palva ET: The WRKY70 transcription factor: a  node of convergence for jasmonate-mediated and salicylatemediated signals in plant defense. Plant Cell 2004, 16:319-331. Expression analysis of the Arabidopsis transcription factor WRKY70 reveals that it is activated by salicylic acid (SA) and elicitors from Erwinia carotovora but repressed by jasmonate (JA). The authors use an overexpression approach to show that WRKY70 acts as a constitutive activator of SA-induced NPR-1-independent expression of PR1 and as a repressor of JA-responsive genes in an NPR1-dependent fashion. Consistently, WRKY70 antisense suppression activated the JA-responsive genes and diminished the induction of PR genes. This differential regulation of defence-related genes is supported by transcriptomic profiling and suggests that WRKY70 integrates signals from these mutually antagonistic pathways in Arabidopsis. 74. Dharmasiri N, Dharmasiri S, Estelle M: The F-box protein TIR1 is  an auxin receptor. Nature 2005, 435:441-445. This report and [75] confirm the perception of auxin by the TRANSPORT INHIBITOR RESPONSE 1 (TIR1) protein. The finding that this F-box is an auxin receptor is sustained by in vitro pull-down assays in which auxin binds directly to SCFTIR, thereby promoting the Aux/IAA-SCFTIR1 interaction. Moreover, the authors of these papers also use heterologous nonauxin-responsive systems (i.e. Xenopus embryos and insect cells) to show that auxin specifically interacts with TIR1. Three closely related TIR1 family members also function as auxin receptors since the quadCurrent Opinion in Plant Biology 2005, 8:532–540

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ruple mutant lacks the ability to bind IAA. These results define a new type of plant receptors and a novel mode of hormone action in regulating the direct-binding interaction between the SCF-target and the F-box protein. 75. Kepinski S, Leyser O: The Arabidopsis F-box protein TIR1 is an  auxin receptor. Nature 2005, 435:446-451. See annotation for [74]. 76. Berger S, Bell E, Mullet JE: Two methyl jasmonate-insensitive mutants show altered expression of AtVsp in response to methyl jasmonate and wounding. Plant Physiol 1996, 111:525-531. 77. Jensen AB, Raventos D, Mundy J: Fusion genetic analysis of jasmonate-signalling mutants in Arabidopsis. Plant J 2002, 29:595-606. 78. Kanna M, Tamaoki M, Kubo A, Nakajima N, Rakwal R, Agrawal GK, Tamogami S, Ioki M, Ogawa D, Saji H, Aono M:

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Isolation of an ozone-sensitive and jasmonate-semiinsensitive Arabidopsis mutant (oji1). Plant Cell Physiol 2003, 44:1301-1310. 79. Hilpert B, Bohlmann H, op den Camp RO, Przybyla D, Miersch O, Buchala A, Apel K: Isolation and characterization of signal transduction mutants of Arabidopsis thaliana that constitutively activate the octadecanoid pathway and form necrotic microlesions. Plant J 2001, 26:435-446. 80. Xu L, Liu F, Wang Z, Peng W, Huang R, Huang D, Xie D: An Arabidopsis mutant cex1 exhibits constant accumulation of jasmonate-regulated AtVSP Thi2.1 and PDF1.2. FEBS Lett 2001, 494:161-164. 81. Kubigsteltig II, Weiler EW: Arabidopsis mutants affected in the transcriptional control of allene oxide synthase, the enzyme catalyzing the entrance step in octadecanoid biosynthesis. Planta 2003, 217:748-757.

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