1. Jasmonate biosynthetic pathway
Jasmonates are oxylipins (oxygenated fatty acids) that originate from linolenic acid released from chloroplast membranes by lipase enzymes and subsequently oxygenated by lipoxygenases (LOXs) to hydroperoxide derivatives (Fig. 1; for a detailed discussion of the different branches of the LOX pathway refer to Wasternack, 2006, 2007). The initial step in the biosynthesis of JAs via the octadecanoid pathway (Fig. 1a) is catalysed by an allene oxide synthase (AOS) that converts 13-hydroperoxy-linolenic acid (13-HPOT) to an unstable allene oxide intermediate, which is in turn modified by an allene oxide cyclase (AOC) to form 12-oxo-phytodienoic acid (OPDA) (Vick & Zimmerman, 1984). All the reactions leading to the formation of OPDA occur in the plastids, whereas the subsequent steps take place in the peroxisome. Reduction of OPDA by OPDA reductase 3 (OPR3) is followed by three rounds of β-oxidation to finally yield jasmonic acid ((+)-7-iso-JA in Fig. 1; Cruz Castillo et al., 2004; Li et al., 2005; Theodoulou et al., 2005). For detailed reviews on JA biosynthesis refer to Delker et al. (2006); Schaller et al., 2005; Wasternack (2006), (2007).
Figure 1. (a) Schematic diagram of the jasmonate (JA) biosynthetic pathway. Biosynthesis of JA from α-linolenic acid requires the enzymes 13-lipoxygenase (13-LOX), allene oxide synthase (AOS) and allene oxide cyclase (AOC) in the chloroplast, and the enzymatic activity of 12-oxo-phytodienoic acid reductase3 (OPR3), acyl-CoA oxidase (ACX), multifunctional protein (MFP) and 3-ketoacyl-CoA thiolase (KAT) in the peroxisome. Intermediates (square boxes): 13-HPOT, 13-hydroperoxylinolenic acid; OPC-8:0, 3-oxo-2- (2′-pentenyl)cyclopentane-1-octanoic acid; OPDA, 12-oxo-phytodienoic acid; JA, (+)-7-iso-jasmonic acid. (b) Metabolism of jasmonates. JA may be metabolized to different compounds depending on the chemical modification of the carboxylic acid group, the pentenyl side chain or the pentanone ring. JA metabolites: MeJA, methyl jasmonate; JA-ACC, conjugate with 1-aminocyclopropane-1-carboxylic acid; JA–Ile, conjugate with the amino acid isoleucine; 12-HSO4-JA, sulfonated derivative of the 12-hydroxy-jasmonic acid; 12-O-GlcJA, glucosylated derivative of the 12-hydroxy-jasmonic acid; JA-O-Glc, glucosylated derivative of JA (adapted from Wasternack, 2007).
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Mutants have been isolated for virtually every step of the jasmonate biosynthetic pathway. Several characterized Arabidopsis JA biosynthetic mutants are male sterile (McConn & Browse, 1996; Sanders et al., 2000; Stintzi & Browse, 2000; Ishiguro et al., 2001; Park et al., 2002; von Malek et al., 2002). For example, the Arabidopsis dad1 (defective in anther dehiscence 1) mutant displays defects in anther dehiscence, pollen maturation, filament elongation and flower opening. This male-sterile phenotype can be rescued by the exogenous application of JA or linolenic acid. The DAD1 gene encodes a chloroplastic phospholipase A1 (Ishiguro et al., 2001). Application of JA also rescues the severe male sterility present in the mutants dde1 (delayed dehiscence 1) and opr3 (oxo-phytodienoic acid reductase 3). Cloning of these mutant loci indicated that they encode the JA biosynthetic enzyme OPR3 (Sanders et al., 2000; Stintzi & Browse, 2000).
Recent analysis of acyl-coenzyme A oxidase (ACX) mutants in Arabidopsis has shown that the JA synthesis mediated by ACX1/5, one of the three key enzymes involved in β-oxidation, is essential for male reproductive development and defence against chewing insects such as Trichoplusia ni (Schilmiller et al., 2007). Additionally, analysis of the mutants aim1 (abnormal inflorescence meristem 1) and pex6 (peroxin 6), which are defective in fatty acid β-oxidation and peroxisome biogenesis, respectively, as well as feeding experiments, have provided further proof of the importance of the peroxisomal β-oxidation step in the octadecanoid biosynthetic pathway (Delker et al., 2006). Previous to the action of the enzymes ACX1/5, MFP (multifunctional protein) and KAT (L-3-ketoacyl CoA thiolase), which are responsible for carrying out the fatty acid β-oxidation in the peroxisome, an activation step of OPDA or its products 3-oxo-2(2′-[Z]-pentenyl) cyclopentane-1-octanoic acid (OPC-8:0) and 3-oxo-2(2′-[Z]-pentenyl)cyclopentane-1-hexanoic acid (OPC-6 : 0) may occur. This mechanism has been suggested by the identification of three 4-coumarate : CoA ligase- (4CL)-like proteins in Arabidopsis, which displayed in vitro specific activation of OPDA and its derivatives as well as peroxisomal localization (Schneider et al., 2005; Koo et al., 2006).
Jasmonic acid can be metabolized to several derivatives (Fig. 1b), with MeJA (a volatile active methyl ester, derivative of jasmonic acid) being one of the best characterized. Among the JA amino acid conjugates, a role in pathogen resistance has been reported for JA–Ile (jasmonoyl–isoleucine). A recent study on N. attenuata transgenic plants impaired in the production of JA–Ile by silenced threonine deaminase indicates that this conjugated derivative of JA is mediating plant defence mechanisms against insects such as Manduca sexta. Also, silencing of JASMONATE RESISTANT 4 (JAR4), the N. attenuata homologue of the Arabidopsis JA-amino synthetase JAR1 (JASMONATE RESISTANT 1) gene, confirmed the role of JA–Ile in activating defence responses (Kang et al., 2006). JAR1 is required in Arabidopsis to activate JA signalling (Staswick & Tiryaki, 2004) and to confer resistance against the soil fungus Pythium irregulare (Staswick et al., 1998). The identification of JAR1 uncovered an enzyme that conjugates JA to isoleucine and clearly revealed that in addition to the well known methylated forms (Falkenstein et al., 1991; Feys et al., 1994; for a recent review see Wasternack, 2007), amino acid conjugates of JA are also bioactive. Similarly, the JA precursor OPDA is able to induce plant defence mechanisms in the absence of jasmonic acid (Stintzi et al., 2001). The involvement of OPDA during wound-induced gene expression has been explored in a recent DNA microarray study, where it has been shown that OPDA triggers the expression of a subset of genes distinct from those induced by JA. These OPDA-specific response genes responded to OPDA but not to JAs (Taki et al., 2005).
2. Jasmonate signalling pathway
Several mutants involved in JA signal perception and transduction have been isolated, the best characterized of which is coi1 (coronatine insensitive 1; for review see Turner et al., 2002; Devoto & Turner, 2005; Lorenzo & Solano, 2005). Mutations of COI1 display resistance to JA inhibition of root growth, various degrees of male sterility, accumulation of anthocyanins, altered senescence and increased susceptibility to several necrotrophic pathogens, biotrophic fungi and pests (Penninckx et al., 1996; Thomma et al., 1998; Norman-Setterblad et al., 2000; Ellis & Turner, 2002; Ellis et al., 2002; Reymond et al., 2004; Zarate et al., 2007). It has been shown that COI1 is required for all the JA-mediated responses analysed so far. Interestingly, characterization of the tomato homologue of COI1 indicates that JAs might have different regulatory functions during reproductive development in different species. In tomato, in addition to jasmonate-related defence responses (a characteristic shared with the Arabidopsis coi1 mutant), COI1 is required for the maternal control of seed maturation and glandular trichome development (Li et al., 2004b). Microarray studies have searched for wound-, jasmonate- and COI1-regulated genes revealing that genes induced by both wounding and MeJA generally required COI1 in Arabidopsis (Devoto et al., 2005). In particular, this study also highlighted the complexity of the signal transduction network mediated by JAs, wounding and COI1. The expression of several JA-induced genes does not appear to require COI1, whereas the induction of several others by wounding does not appear to require jasmonate. In addition, the involvement of OPDA-induced genes during the wound response in Arabidopsis is accomplished via a COI1-independent pathway (Taki et al., 2005). COI1 encodes an F-box protein that is part of an E3 ubiquitin ligase complex involved in ubiquitin-mediated protein degradation. In Arabidopsis the COI1 protein associates with CUL1 (Cullin1), RBX1 (RING-box protein 1) and the Skp1-like proteins ASK1 (Arabidopsis S-PHASE KINASE-ASSOCIATED PROTEIN 1) and ASK2 to form the SCFCOI1 (SKP1, CDC53p/CUL1, F-box) ubiquitin–ligase protein degradation complex (Devoto et al., 2002; Xu et al., 2002). Importantly, JA-related phenotypes have been identified in plants with mutations in components of the ubiquitin proteasome system (Tiryaki & Staswick, 2002; Xu et al., 2002; Lorenzo & Solano, 2005; Moon et al., 2007) linking JAs signalling with ubiquitin-mediated protein degradation.
The Arabidopsis COI1 is closely related to the auxin signalling TIR1 (TRANSPORT INHIBITOR RESPONSE 1) gene (Ruegger et al., 1998), which encodes the first plant F-box protein to be identified as part of an SCF complex (Gray et al., 1999). More recently, TIR1 was demonstrated to act as an auxin receptor (Dharmasiri et al., 2005; Kepinski & Leyser, 2005). AXR1 is a positive regulator of auxin response that modulates SCFTIR1. activity (Leyser et al., 1993) and is additionally considered to be a connection node regulating the activity of different SCF complexes. As a result of the similarities in the mode of action of COI1 and TIR1, it is tempting to speculate that jasmonate and auxin might use analogous signalling mechanisms sharing common components such as AXR1. Interestingly, the auxin-resistant mutant axr1-24 (AUXIN RESISTANT 1) has been isolated in a screen for decreased sensitivity to JA. This allele of axr1 also displays reduced inhibition of root growth by ACC. AXR1 was also found to be necessary for resistance to P. irregulare in Arabidopsis (Tiryaki & Staswick, 2002). This work provided initial evidence for the involvement of auxin signalling components in defence. For a recent review on the role of the ubiquitin/proteasome system in regulating hormonal signalling pathways and responses to biotic stresses, see Dreher & Callis (2007).
Plants use an extremely complex network of synergistic defensive tactics to guard themselves from different pathogens. Fundamental to inducible basal resistance is the ability to recognize and respond to small molecular motifs named pathogen-associated molecular patterns (PAMPs; Nürnberger et al., 2004), recently renamed microbial associated molecular patterns (MAMPs; Mackey & McFall, 2006). Significantly, a recent study has shown that bacterial PAMPS such as bacterial flagellin (flg22) downregulate auxin signalling in Arabidopsis by targeting auxin receptor transcripts (Navarro et al., 2006). Specific suppression of auxin receptors such as TIR1 was shown to occur via post-transcriptional gene silencing mediated by the microRNA miR393. Repression of auxin signalling results in reduced Pseudomonas syringae growth, associating auxin with disease susceptibility via miRNA-mediated suppression of auxin signalling (Navarro et al., 2006). Whether JAs utilize small RNA-mediated sequence-specific mRNA degradation to regulate expression of SCFCOI1 targets remains to be demonstrated.
Potential targets for COI1-mediated ubiquitination have been identified via yeast two-hybrid screen and immuno-assays (Devoto et al., 2002), as well as gene silencing (Xu et al., 2002). Among these putative targets of the SCFCOI1 E3 ligase complex, the histone deacetylase RPD3b/HDAC6 constitutes a particularly interesting substrate, considering its known function as transcriptional repressor (Devoto et al., 2002). Incidentally, constitutive overexpression of histone deacetylase 19 (RPD3A/HDAC19), the closest homologue of the COI1-interacting partner RPD3b in Arabidopsis, decreased histone acetylation levels and enhanced expression of jasmonic acid-responsive PR (pathogenesis-related) genes (Zhou et al., 2005). It has not been shown whether or not RPD3A can also interact with COI1.
Since TIR1 was identified as an auxin receptor (Dharmasiri et al., 2005; Kepinski & Leyser, 2005), the sequence similarity between COI1 and TIR1 has led to hypothesize that the SCFCOI1 might be the jasmonate receptor (Chini et al., 2007; Thines et al., 2007). Recently, members of the jasmonate ZIM-domain (JAZ) protein family have been identified by Thines et al. (2007) as direct targets of SCFCOI1 ubiquitin ligase and the 26S proteasome. The JAZ1 protein acts to repress transcription of jasmonate-responsive genes. Importantly, the JAs (see above), but not other biologically active JA derivatives such as OPDA or MeJA, promotes physical interaction between COI1 and JAZ1 in yeast. At the same time, an independent study identified an Arabidopsis JAZ gene (JAI3/JAZ3) as a repressor of JA signalling (Chini et al., 2007). Collectively, these results suggest that the COI1–JAZ complex might be a site of perception for JA–Ile. Moreover, it was demonstrated that JAI3 interacts with MYC2, and that JAI3 is a negative regulator of MYC2 function. Therefore SCFCOI1-dependent proteasome degradation of JAZ proteins releases MYC2, leading to transcriptional activation of jasmonate responses (Chini et al., 2007). Whether COI1 acts as a receptor only for JA–Ile, and the degree to which other JAZs and JAZ-like proteins contribute to jasmonate signalling, remain to be elucidated. In addition, it is possible that other JAs mediate the interaction between COI1 and other members of the JAZ family, or with SCFCOI1 substrates that await identification.
JASMONATE ASSOCIATED 1 (JAS1), a novel mediator of wounding and JA-regulated growth inhibition, was selected in a MeJA-based growth inhibition screen for transgenic plants expressing candidate JA signalling genes obtained from microarray experiments (Yan et al., 2007). The demonstration that JAS1.1 and JAS1.3 are targeted to the nucleus, and that the expression of JAS1.3 affects transcript levels for at least three other genes, suggests that this protein might act as a transcriptional regulator. Intriguingly, JAS1 corresponds to JAZ10, one of the members of the ZIM domain-containing JAZ protein family. It remains to be demonstrated whether JAS1/JAZ10 also acts as a repressor of JA signalling via COI1, as has been shown for JAZ1 and JAZ3 (Chini et al., 2007; Thines et al., 2007).
So far, only one coi1 suppressor, cos1, has been identified in Arabidopsis. The gene encodes a lumazine synthase, which catalyses the penultimate step in the riboflavin (vitamin B2) biosynthetic pathway. The mutation reverts the coi1 phenotype in terms of JA-induced root growth, gene expression, senescence and defence responses (Xiao et al., 2004).
Constitutive JA-signalling mutants such as cet (constitutive expressor of THI2.1; Hilpert et al., 2001) and cev1 (constitutive expressor of VSP1; Ellis et al., 2002) have been isolated in Arabidopsis. Nine of the 10 cet mutants identified in a genetic screen for constitutive expression of the THI2.1 (THIONIN 2.1) JA-regulated gene display spontaneous leaf cell necrosis, and some display upregulation of the PR1 (pathogenesis-related 1) gene, a common feature of the systemic acquired resistance (SAR) response (Hilpert et al., 2001). SAR is an inducible plant-defence response involving a cascade of transcriptional events mediated by SA. This defence mechanism, often induced after a local infection, provides increased resistance in organs distant from the site of primary infection against subsequent pathogen infections (Durrant & Dong, 2004). Analysis of multiple mutant lines between cet and the fad3-2, fad7-2, fad8 triple mutant (a JA-biosynthetic mutant), coi1 (a JA-signalling mutant) and NahG (transgenic plant expressing salicylate hydroxylase and thus a SA-deficient plant) suggest that at least some of the CET genes act in the cell death stress response in a JA- and SA-dependent manner (Nibbe et al., 2002). The molecular components responsible for the phenotype of the cet mutants remain to be identified.
The mutant cev1 displays constitutive activation of JA signalling, showing increased defence responses against fungal and bacterial pathogens as well as aphids (Ellis et al., 2002). Recently, it has been shown that cev1 is also more resistant to infestation by silverleaf whitefly (an obligate phloem-feeding pest with a different feeding habit from that of aphids; Zarate et al., 2007). This mutant overproduces not only JA, but also OPDA and ethylene, along with constitutive expression of the biotic stress-responsive genes VSP (VEGETATIVE STORAGE PROTEIN), PDF1.2 (PLANT DEFENSIN), Thi2.1 (THIONIN) and CHI-B (CHITINASE B) (Ellis & Turner, 2001; Ellis et al., 2002). It has been found that CEV1 encodes the cellulose synthase CeSA3, providing a link between cell wall signalling and JA and ethylene-regulated defence responses (Ellis et al., 2002).
In Arabidopsis, Lorenzo et al. (2004) have identified five jai1/jin1 (jasmonate insensitive 1) mutant alleles, and have cloned MYC2, a nuclear localized basic helix–loop–helix-leucine zipper (bHLHzip)-type transcription factor that antagonistically regulates two branches of the JA-signalling pathway in a COI1-dependent manner. Although MYC2 was originally isolated as an ABA-response mutant (Abe et al., 2003), it is a positive regulator of a subset of JA-inducible genes and is essential for JA-dependent developmental processes in Arabidopsis. However, the molecular mechanisms underlying the control of MYC2 expression remain largely unknown. Other alleles of jai have been identified and found to encode COI1 (JAI5), JAR1(JIN4/JAI2) and SGT1b (SUPPRESSOR OF THE G2 ALLELE OF skp1-4; JAI4), a yeast regulator of the activity of SCF complexes (Kitagawa et al., 1999). For further information on allelic relationships, refer to reviews by Devoto & Turner (2005); Lorenzo & Solano (2005).
Signalling JA mutants share some common phenotypes, such as reduced root growth inhibition by JA or MeJA, anthocyanin accumulation, enhanced sensitivity to pathogens, and altered expression of JA-responsive genes. Analyses of JA mutants have mostly focused on altered JA-inducible gene expression and defence responses, whereas a detailed analysis of the causes underlying the stunted growth that characterizes some of them has seldom been performed.