**3.3. JA Signaling Model: SCFCOI1/JAZ Proteins Imitates SCFTIR1/AUX/IAA Proteins**

*coi1* is a completely insensitive mutant to JA/coronatine. COI1 was map-cloned and revealed as an F-box protein that functions as the substrate-recruiting element of the Skp1–Cul1–Fbox protein (SCF) ubiquitin E3 ligase complex. As described above, JAZ family proteins are transcriptional repressors and SCFCOI1 substrate targets, which associate with COI1 in a hormone-dependent manner*.* Recent research established JA signaling model (Figure 7) (Chini et al, 2007; Thines et al., 2007; Sheard et al., 2010). In the absence or low level of hormone signal, JAZ repressor complex, including JAZ proteins, adaptor protein NINJA, and co-repressor TPL, actively repress the activity of JA-responsive transcription factors

**Figure 7.** Model of JA signaling in *Arabidopsis* (Browse and Howe, 2008). (A) At low intracellular levels of JA signal (JA-Ile), SCFCOI1 complex has no essential activity of E3 ubiquitin ligase, resulting in accumulation of JAZ proteins which repress the activity of transcription factors such as MYC2 that positively regulate JA-responsive genes. (B) At high level of JA signal such as upon wounding, rapid accumulation of bioactive JA-Ile promotes SCFCOI1-mediated ubiquitination and subsequent degradation of JAZ proteins via the 26S proteasome. JA-induced removal of JAZ proteins causes derepression of transcription factors and the activation of JA-responsive genes.

(e.g., MYC2), which bind to *cis*-acting elements of jasmonate-response genes, preventing transcription activity. In response to stimuli such as wounding JA-Ile stimulates the specific binding of JAZ proteins to COI1, leading to poly-ubiquitination and subsequent degradation of JAZs by the 26S proteasome. JAZ degradation relieves repression of MYC2 and other transcription factors, permitting the expression of jasmonate-responsive genes such as *PDF1.2*. The role of COI1-mediated JAZ degradation in jasmonate signaling is analogous to auxin signaling through the receptor SCFTIR1 complex, which degrades the AUX/IAA transcriptional repressors in hormone-dependent manner (Gray et al., 2001; Kepinski and Leyser, 2005). Supported by its sequence homology and functional similarity to TIR1, COI1 is recognized for a critical role in the direct perception of the jasmonate signal (Xie et al., 1998; Katsir et al., 2008; Sheard et al., 2010). Mimicking AUX/IAA proteins which are induced specifically in response to auxin (Gray et al., 2001), JAZ proteins are highly inducible by JA/MeJA treatment or wounding (Thines et al., 2007; Chung et al., 2008).

Jasmonate Biosynthesis, Perception and Function in Plant Development and Stress Responses 413

proteins is also known as TIFY family (Vanholme et al., 2007). 12 of 18 TIFY proteins are JAZs. The functions of the remaining TIFYs are unknown, except PPD1 and PPD2, which showed, to additively repress the proliferation of dispersed meristematic cells (DMCs) in

The first prominent characteristic of JAZ proteins is that they possess two domains, ZIM and Jas domains, and both are important for JAZs function (Thines et al., 2007). The former may have two biochemical roles *in vivo*. 1) JAZ1ΔJas and JAZ3ΔJas can not be degraded by SCFCOI1 complex plus 26S proteasome but still can suppress JA signaling, indicating JAZ may interact with MYC2 via the first conserved domain ZIM (Chini et al., 2007; Thines et al., 2007). 2) ZIM domains are responsible for homo- and hetero dimerization (Chini et al., 2009; Chung and Howe, 2009). All JAZs except JAZ7 are prone to form homo-/hetero-dimers (Pauwels and Goossens, 2011). The biological meaning of JAZ dimerization *in vivo* remains elusive but extensive dimerization of JAZs or JAZ with other proteins such as MYC2 and NINJA may help to establish insensitivity to SCFCOI1 E3 ubiquitin ligase in the situation of low JA signal. The second prominent feature of JAZs is "logically-paradoxical"; most *JAZ*  genes are highly induced upon JA treatment or by wounding in JA-dependent manner (Chini et al., 2007; Thines et al., 2007; Yan et al., 2007) but, on the other hand, JAZ proteins are degraded during activated signaling (Chini et al., 2007; Thines et al., 2007). Similarly, AUX/IAA proteins have the same paradoxical feature, that is, AUX/IAA genes are induced by auxin but the proteins disappear rapidly (Abel et al., 1994). The third important feature of JAZ genes is that several splice-variant transcripts of single *JAZ* gene exist naturally in plants (Figure 9). For example, *JAZ10* has four alternative splice variants and three of them, *At5g13220.2, At5g13220.3,* and *At5g13220.4* encode stable JAZΔJas isomers, which attenuate

**Figure 8.** The structures and phylogeny of *Arabidopsis* ZIM-Domain containing proteins JAZs. ZIM

domain and Jas domain are shown in yellow and pink bars, respectively (Browse, 2009).

leaves (Pauwels and Goossens, 2011).

JA signal output (Yan et al., 2007 Chung and Howe, 2009).

#### **3.4. Characterization of JA Signaling Repressors: JAZs, NINJA, and TPL**

JAZs proteins were designated as JAZs because they were annotated as ZIM-domain containing proteins (ZIM: Zinc-finger protein expressed in Inflorescence Meristem) and their expression depends on jasmonates (Chini et al, 2007; Thines et al., 2007). Thines et al., (2007) in their study found that eight ZIM-domain containing unknown proteins (JAZs) were significantly induced in stamens and seedlings of *opr3* mutant after JA application. JAZ1 protein, one out of the eight JAZs that were tested for activity as a substrate of SCFCOI1 complex, acts to repress transcription of jasmonate-responsive genes. Jasmonate treatment causes JAZ1 degradation and JA–Ile promotes physical interaction between COI1 and JAZ1 proteins in the absence of other plant proteins (Thines et al., 2007). Additionally, *JAZ1Δ3A*, a mutant with distruption of conserved domain 3A (i.e., Jas domain in Yan et al., 2007) shows typical JA-signaling phenotypes such as male sterility and root growth insensitivity to JA (Thines et al., 2007). In a separate study, Chini et al., (2007) characterized the mutant *jasmonate-insensitive3-1* (*jai3-1*) they identified in a genetic screen for JA-insensitivity. Positional cloning of the *jai3-1* mutation revealed that a base substitution in JAI3 results in a truncated protein that causes a jasmonate-insensitive phenotype and impaired transcriptional responses to jasmonate (Chini et al., 2007). *jai3-1* is a dominant mutant with a missing conserved CT/Jas domain of JAI3, which encodes JAZ3.,This mutant showed some JA-deficiency phenotypes such as root growth insensitivity to JA treatment (Chini et al., 2007). In the third independent study, Yan et al. (2007) profiled the transriptome depletion of *aos* mutant compared to wild type and identified 35 JA-dependent genes responsible for JA signaling depletion in *aos* mutant. Three of these genes encode ZIM-domain containing proteins. Overexpression of a predicted alternatively spliced transcript At5g13220.3, called *Jasmonate-Associated 1* (*JAS1,* identical to *JAZ10*), resulted in reduced sensitivity to MeJA and elevated growth of roots and shoots under MeJA treatment (Yan et al., 2007). In total, 12 *JAZ* genes (Figure 8) have been identified so far in *Arabidopsis* (Chini et al., 2007; Yan et al., 2007; Browse, 2009). All are believed to have redundant function in JA signaling pathway as transcription repressors (Chini et al., 2007). The gene family of ZIM-domain containing proteins is also known as TIFY family (Vanholme et al., 2007). 12 of 18 TIFY proteins are JAZs. The functions of the remaining TIFYs are unknown, except PPD1 and PPD2, which showed, to additively repress the proliferation of dispersed meristematic cells (DMCs) in leaves (Pauwels and Goossens, 2011).

412 Lipid Metabolism

(e.g., MYC2), which bind to *cis*-acting elements of jasmonate-response genes, preventing transcription activity. In response to stimuli such as wounding JA-Ile stimulates the specific binding of JAZ proteins to COI1, leading to poly-ubiquitination and subsequent degradation of JAZs by the 26S proteasome. JAZ degradation relieves repression of MYC2 and other transcription factors, permitting the expression of jasmonate-responsive genes such as *PDF1.2*. The role of COI1-mediated JAZ degradation in jasmonate signaling is analogous to auxin signaling through the receptor SCFTIR1 complex, which degrades the AUX/IAA transcriptional repressors in hormone-dependent manner (Gray et al., 2001; Kepinski and Leyser, 2005). Supported by its sequence homology and functional similarity to TIR1, COI1 is recognized for a critical role in the direct perception of the jasmonate signal (Xie et al., 1998; Katsir et al., 2008; Sheard et al., 2010). Mimicking AUX/IAA proteins which are induced specifically in response to auxin (Gray et al., 2001), JAZ proteins are highly

inducible by JA/MeJA treatment or wounding (Thines et al., 2007; Chung et al., 2008).

JAZs proteins were designated as JAZs because they were annotated as ZIM-domain containing proteins (ZIM: Zinc-finger protein expressed in Inflorescence Meristem) and their expression depends on jasmonates (Chini et al, 2007; Thines et al., 2007). Thines et al., (2007) in their study found that eight ZIM-domain containing unknown proteins (JAZs) were significantly induced in stamens and seedlings of *opr3* mutant after JA application. JAZ1 protein, one out of the eight JAZs that were tested for activity as a substrate of SCFCOI1 complex, acts to repress transcription of jasmonate-responsive genes. Jasmonate treatment causes JAZ1 degradation and JA–Ile promotes physical interaction between COI1 and JAZ1 proteins in the absence of other plant proteins (Thines et al., 2007). Additionally, *JAZ1Δ3A*, a mutant with distruption of conserved domain 3A (i.e., Jas domain in Yan et al., 2007) shows typical JA-signaling phenotypes such as male sterility and root growth insensitivity to JA (Thines et al., 2007). In a separate study, Chini et al., (2007) characterized the mutant *jasmonate-insensitive3-1* (*jai3-1*) they identified in a genetic screen for JA-insensitivity. Positional cloning of the *jai3-1* mutation revealed that a base substitution in JAI3 results in a truncated protein that causes a jasmonate-insensitive phenotype and impaired transcriptional responses to jasmonate (Chini et al., 2007). *jai3-1* is a dominant mutant with a missing conserved CT/Jas domain of JAI3, which encodes JAZ3.,This mutant showed some JA-deficiency phenotypes such as root growth insensitivity to JA treatment (Chini et al., 2007). In the third independent study, Yan et al. (2007) profiled the transriptome depletion of *aos* mutant compared to wild type and identified 35 JA-dependent genes responsible for JA signaling depletion in *aos* mutant. Three of these genes encode ZIM-domain containing proteins. Overexpression of a predicted alternatively spliced transcript At5g13220.3, called *Jasmonate-Associated 1* (*JAS1,* identical to *JAZ10*), resulted in reduced sensitivity to MeJA and elevated growth of roots and shoots under MeJA treatment (Yan et al., 2007). In total, 12 *JAZ* genes (Figure 8) have been identified so far in *Arabidopsis* (Chini et al., 2007; Yan et al., 2007; Browse, 2009). All are believed to have redundant function in JA signaling pathway as transcription repressors (Chini et al., 2007). The gene family of ZIM-domain containing

**3.4. Characterization of JA Signaling Repressors: JAZs, NINJA, and TPL** 

The first prominent characteristic of JAZ proteins is that they possess two domains, ZIM and Jas domains, and both are important for JAZs function (Thines et al., 2007). The former may have two biochemical roles *in vivo*. 1) JAZ1ΔJas and JAZ3ΔJas can not be degraded by SCFCOI1 complex plus 26S proteasome but still can suppress JA signaling, indicating JAZ may interact with MYC2 via the first conserved domain ZIM (Chini et al., 2007; Thines et al., 2007). 2) ZIM domains are responsible for homo- and hetero dimerization (Chini et al., 2009; Chung and Howe, 2009). All JAZs except JAZ7 are prone to form homo-/hetero-dimers (Pauwels and Goossens, 2011). The biological meaning of JAZ dimerization *in vivo* remains elusive but extensive dimerization of JAZs or JAZ with other proteins such as MYC2 and NINJA may help to establish insensitivity to SCFCOI1 E3 ubiquitin ligase in the situation of low JA signal. The second prominent feature of JAZs is "logically-paradoxical"; most *JAZ*  genes are highly induced upon JA treatment or by wounding in JA-dependent manner (Chini et al., 2007; Thines et al., 2007; Yan et al., 2007) but, on the other hand, JAZ proteins are degraded during activated signaling (Chini et al., 2007; Thines et al., 2007). Similarly, AUX/IAA proteins have the same paradoxical feature, that is, AUX/IAA genes are induced by auxin but the proteins disappear rapidly (Abel et al., 1994). The third important feature of JAZ genes is that several splice-variant transcripts of single *JAZ* gene exist naturally in plants (Figure 9). For example, *JAZ10* has four alternative splice variants and three of them, *At5g13220.2, At5g13220.3,* and *At5g13220.4* encode stable JAZΔJas isomers, which attenuate JA signal output (Yan et al., 2007 Chung and Howe, 2009).

**Figure 8.** The structures and phylogeny of *Arabidopsis* ZIM-Domain containing proteins JAZs. ZIM domain and Jas domain are shown in yellow and pink bars, respectively (Browse, 2009).

#### 414 Lipid Metabolism

The molecular mechanism by which JAZ proteins repress downstream gene expression is unknown. Pauwels et al. (2010) reported a mechanism that JAZ proteins co-repress JA signal by recruiting the Groucho/Tup1-type co-repressor TOPLESS (TPL) and TPL-related proteins (TPRs) through a newly characterized adaptor protein, designated Novel Interactor of JAZ (NINJA). NINJA acts as a transcriptional repressor whose activity is mediated by a functional TPL-binding motif EAR (ERF-associated amphiphilic repression). Accordingly, both NINJA and TPL proteins function as negative regulators of JA response (Pauwels et al., 2010).

Jasmonate Biosynthesis, Perception and Function in Plant Development and Stress Responses 415

2007; Chung and Howe, 2009). MYC2 was identified as a key regulator of JA signaling, acting as a basic helix-loop-helix (bHLH, also called MYC) transcription factor for JA signal transduction. MYC2 was map-cloned from *jai1* (*jasmonate-insensitive 1*) mutant mutant (Lorenzo et al., 2004), which is allelic to the previously characterized mutant *jin1* (*methyl jasmonate-insensitive 1*) (Berger et al., 1996). MYC2 differentially regulates two branches of JA-mediated responses. That is, it positively regulates a wound-responsive gene set, including *VSP2*, *LOX3*, and *TAT*, but represses the expression of a pathogenresponsive gene set such as *PR4*, *PR1*, and *PDF1.2* (Lorenzo et al., 2004). Interestingly, the ethylene-responsive transcription factor ERF1 also co-regulated these two gene sets, but in opposite direction, i.e., ERF1 activated pathogen-responsive genes but represses wound-

In Arabidopsis, there are 133 bHLH genes, constituting one of the largest families of transcription factors (Heim et al., 2003). Based on the amino acid sequence similarity of both the entire protein and of the bHLH domain, Arabidopsis bHLH proteins are divided into 12 major groups and 25 subgroups (Heim et al., 2003). MYC2 is a member of the subgroup IIIe, along with MYC3 (At5g46760), MYC4 (At4g17880), and MYC5 (At5g46830) (Heim et al., 2003). In contrast to severe JA-synthesis and JA-perception mutants such as *aos* and *coi1*, *myc2* plants are male-fertile and only partially defense-compromised (Lorenzo et al., 2004). This indicates that other JAZ-interacting transcription factors activate the expression of early JA-responsive genes following JA-mediated ubiquitination-proteasomal removal of JAZ repressors. The close paralogues MYC3 and MYC4, but not MYC5, showed to interact with JAZ1, JAZ3, and JAZ9 proteins in both pull-down and yeast two-hybrid assays. Although *myc3* and *myc4* loss-of-function mutants did not show an evident JA-related phenotype, the triple mutant *myc2 myc3 myc4* is as impaired *as coi1-1* in the activation of several, but not all, JA-mediated responses such as the defense against bacterial pathogens and insect herbivory. Moreover, overexpression of cDNAs encoding MYC3 and MYC4 proteins resulted in anthocyanin accumulation and higher transcript levels of JA-responsive genes compared to wild type. In addition, similar to plants overexpressing MYC2, MYC3 overexpression plants were hypersensitive to JA-mediated root growth inhibition. Based on these results, it is concluded that in addition to previously characterized MYC2, MYC3 and MYC4 are also JAZ-interacting transcription factors that activate JA-responses through SCFCOI1 complex plus 26S proteasome (Niu et al., 2011; Brown et al., 2003; Cheng et al., 2011; Fernández-

**3.6. AP2/ERF Transcription Factors Involve in JA Signaling Network** 

AP2/ERF transcription factors are considered the second important group of transcription factors that belong to a large plant-specific APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) superfamily, containing at least 122 members in *Arabidopsis* (Nakano et al., 2006). Many ERF genes have been shown to be regulated by a variety of stress related stimuli, such as wounding, JA, ethylene, salicylic acid, or infection by different types of pathogens (Pré et al., 2008). Four transcription factors, ERF1, AtERF2, AtERF14, and ORA59, were suggested to function as positive regulators involving in JA signing pathway while AtERF4 was

responsive genes (Lorenzo et al., 2004).

Calvo et al., 2011).


**Figure 9.** The sequences of ZIM and Jas domains in the transcripts of JAZs (Yan et al., 2007)

#### **3.5. JAZ proteins control the MYC-type transcription factors activity**

Transcription factors dependent on JA signal are supposed to be the key components for JA signaling pathway. Up to date, MYC2 is the only transcription factor known to interact directly with JAZ proteins. According to the current model of JA signaling (Figure 7), MYC2 is the most important transcription factor to activate transcription of the early JAresponsive genes including downstream transcription factors (such as WRKYs, MYBs, and AP2/ERFs), JA biosynthesis genes, and JAZ proteins (Lorenzo et al., 2004; Chini et al., 2007; Chung and Howe, 2009). MYC2 was identified as a key regulator of JA signaling, acting as a basic helix-loop-helix (bHLH, also called MYC) transcription factor for JA signal transduction. MYC2 was map-cloned from *jai1* (*jasmonate-insensitive 1*) mutant mutant (Lorenzo et al., 2004), which is allelic to the previously characterized mutant *jin1* (*methyl jasmonate-insensitive 1*) (Berger et al., 1996). MYC2 differentially regulates two branches of JA-mediated responses. That is, it positively regulates a wound-responsive gene set, including *VSP2*, *LOX3*, and *TAT*, but represses the expression of a pathogenresponsive gene set such as *PR4*, *PR1*, and *PDF1.2* (Lorenzo et al., 2004). Interestingly, the ethylene-responsive transcription factor ERF1 also co-regulated these two gene sets, but in opposite direction, i.e., ERF1 activated pathogen-responsive genes but represses woundresponsive genes (Lorenzo et al., 2004).

414 Lipid Metabolism

2010).

The molecular mechanism by which JAZ proteins repress downstream gene expression is unknown. Pauwels et al. (2010) reported a mechanism that JAZ proteins co-repress JA signal by recruiting the Groucho/Tup1-type co-repressor TOPLESS (TPL) and TPL-related proteins (TPRs) through a newly characterized adaptor protein, designated Novel Interactor of JAZ (NINJA). NINJA acts as a transcriptional repressor whose activity is mediated by a functional TPL-binding motif EAR (ERF-associated amphiphilic repression). Accordingly, both NINJA and TPL proteins function as negative regulators of JA response (Pauwels et al.,

Protein Name Protein # Sequence of ZIM domain Sequence of Jas domain

JAZ1 At1g19180.1 PLTIFYAGQVIVFNDFSAEKAKEVINLA PIARRASLHRFLEKRKDRVTSKAPY

JAZ2 At1g74950.1 PLTIFYGGRVMVFDDFSAEKAKEVIDLA PIARRASLHRFLEKRKDRITSKAPY JAZ3 At3g17860.1 QLTIFYAGSVCVYDDISPEKAKAIMLLA PLARKASLARFLEKRKERVTSVSPY

JAZ4 At1g48500.1 QLTIFYAGSVLVYQDIAPEKAQAIMLLA PQTRKASLARFLEKRKERVINVSPY At1g48500.2 QLTIFYAGSVLVYQDIAPEKAQAIMLLA PQTRKASLARFLEKRKERY\* At1g48500.3 QLTIFYAGSVLVYQDIAPEKAQAIMLLA PQTRKASLARFLEKRKERY\* JAZ5 At1g17380.1 LTIFFGGKVLVYNEFPVDKAKEIMEVA RIARRASLHRFFAKRKDRAVARAPY JAZ6 At1g72450.1 QLTIFFGGKVMVFNEFPEDKAKEIMEVA RIARRASLHRFFAKRKDRAVARAPY JAZ7 At2g34600.1 ILTIFYNGHMCVSSDLTHLEANAILSLA KASMKRSLHSFLQKRSLRIQATSPY JAZ8 At1g30135.1 RITIFYNGKMCFSSDVTHLQARSIISIA KASMKKSLQSFLQKRKIRIQATSPY JAZ9 At1g70700.1 QLTIFYGGTISVFNDISPDKAQAIMLCA PQARKASLARFLEKRKERLMSAMPY

JAZ10 At5g13220.1 MTIFYNGSVSVFQVSRNKAGEIMKVA PIARRKSLQRFLEKRKERLVSTSPY At5g13220.2 MTIFYNGSVSVFQVSRNKAGEIMKVA PIARRKSLQRFLEKRKER\* At5g13220.3 MTIFYNGSVSVFQVSRNKAGEIMKVA PIARRKSLQRFLEKRKER\*

JAZ11 At3g43440.1 QLTIIFGGSFSVFDGIPAEKVQEILHIA PIARRRSLQRFFEKRRHRFVHTKPY

JAZ12 At5g20900.1 QLTIFFGGSVTVFDGLPSEKVQEILRIA PIARRHSLQRFLEKRRDRLVNKNPY

Transcription factors dependent on JA signal are supposed to be the key components for JA signaling pathway. Up to date, MYC2 is the only transcription factor known to interact directly with JAZ proteins. According to the current model of JA signaling (Figure 7), MYC2 is the most important transcription factor to activate transcription of the early JAresponsive genes including downstream transcription factors (such as WRKYs, MYBs, and AP2/ERFs), JA biosynthesis genes, and JAZ proteins (Lorenzo et al., 2004; Chini et al.,

**Figure 9.** The sequences of ZIM and Jas domains in the transcripts of JAZs (Yan et al., 2007)

**3.5. JAZ proteins control the MYC-type transcription factors activity** 

At5g13220.4 MTIFYNGSVSVFQVSRNKAGEIMKVA \*

At1g19180.2 PLTIFYAGQVIVFNDFSAEKAKEVINLA PIARRASLHRFLEKRKDRVTSKAPY

At3g17860.2 QLTIFYAGSVCVYDDISPEKAKAIMLLA PLARKASLARFLEKRKERVTSVSPY At3g17860.3 QLTIFYAGSVCVYDDISPEKAKAIMLLA PLARKASLARFLEKRKERVTSVSPY

At1g70700.2 QLTIFYGGTISVFNDISPDKAQAIMLCA PQARKASLARFLEKRKERLMSAMPY

At3g43440.2 -------------------GVPAQKVQEILHIA PIARRRSLQRFFEKRRHRFVHTKPY

At5g20900.2 QLTIIFGGSCRVFNGVPAQKVQEILHIA PIARRRSLQRFFEKRRHRFVHTKPY

In Arabidopsis, there are 133 bHLH genes, constituting one of the largest families of transcription factors (Heim et al., 2003). Based on the amino acid sequence similarity of both the entire protein and of the bHLH domain, Arabidopsis bHLH proteins are divided into 12 major groups and 25 subgroups (Heim et al., 2003). MYC2 is a member of the subgroup IIIe, along with MYC3 (At5g46760), MYC4 (At4g17880), and MYC5 (At5g46830) (Heim et al., 2003). In contrast to severe JA-synthesis and JA-perception mutants such as *aos* and *coi1*, *myc2* plants are male-fertile and only partially defense-compromised (Lorenzo et al., 2004). This indicates that other JAZ-interacting transcription factors activate the expression of early JA-responsive genes following JA-mediated ubiquitination-proteasomal removal of JAZ repressors. The close paralogues MYC3 and MYC4, but not MYC5, showed to interact with JAZ1, JAZ3, and JAZ9 proteins in both pull-down and yeast two-hybrid assays. Although *myc3* and *myc4* loss-of-function mutants did not show an evident JA-related phenotype, the triple mutant *myc2 myc3 myc4* is as impaired *as coi1-1* in the activation of several, but not all, JA-mediated responses such as the defense against bacterial pathogens and insect herbivory. Moreover, overexpression of cDNAs encoding MYC3 and MYC4 proteins resulted in anthocyanin accumulation and higher transcript levels of JA-responsive genes compared to wild type. In addition, similar to plants overexpressing MYC2, MYC3 overexpression plants were hypersensitive to JA-mediated root growth inhibition. Based on these results, it is concluded that in addition to previously characterized MYC2, MYC3 and MYC4 are also JAZ-interacting transcription factors that activate JA-responses through SCFCOI1 complex plus 26S proteasome (Niu et al., 2011; Brown et al., 2003; Cheng et al., 2011; Fernández-Calvo et al., 2011).

#### **3.6. AP2/ERF Transcription Factors Involve in JA Signaling Network**

AP2/ERF transcription factors are considered the second important group of transcription factors that belong to a large plant-specific APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) superfamily, containing at least 122 members in *Arabidopsis* (Nakano et al., 2006). Many ERF genes have been shown to be regulated by a variety of stress related stimuli, such as wounding, JA, ethylene, salicylic acid, or infection by different types of pathogens (Pré et al., 2008). Four transcription factors, ERF1, AtERF2, AtERF14, and ORA59, were suggested to function as positive regulators involving in JA signing pathway while AtERF4 was

#### 416 Lipid Metabolism

identified as a negative regulator for this process (Lorenzo et al., 2003; Oñate-Sánchez et al., 2007; Pré et al., 2008; McGrath et al., 2005).

Jasmonate Biosynthesis, Perception and Function in Plant Development and Stress Responses 417

WD40 protein TTG1 (TRANSPARENT TEXTA GLABRA 1) and R2R3-MYB proteins such as MYB75 and GL1 (GLABRA 1). Apart from anthocyanin biosynthesis and trichome formation, GL3/EGL3/TT8 complex may be involved in many other processes including root hair formation, flavonoid biosynthesis, stomata patterning, and seed coat mucilage production (Pauwels and Goossens, 2011). JAZ-interacting domain (JID) was found in a number of bHLH transcription factors including MYC2/MYC3/MYC4, indicating that JAZs may have much wider function spectrum than currently known. Indeed, JID domain is present in GL3, EGL3 and TT8, and interaction of these proteins with eight different JAZs

**3.8. JA-Signaling regulates Male Fertility via Transcription Factors MYB21 and** 

JA was repeatedly shown to be essential for male fertility in *Arabidopsis*. Many JA biosynthesis and signaling mutants such as *dad1*, *fad3/7/8*, *lox3/4*, *aos*, *opr3,* and *coi1* are male sterile because of a combination of defective anther dehiscence, insufficient filament elongation, and severely reduced pollen viability (Browse, 2009). Transcriptome analysis of JA-treated stamens in *opr3* and wild type identified two R2R3 MYB proteins, MYB21 and MYB24, as key regulators of the stamen maturation processes triggered by JA (Mandaokar et al., 2006). Overexpression of *MYB21* in the *coi1-1* or *opr3* mutants could partially restore male fertility (Cheng et al., 2009), whereas the *myb21-1* knockout mutant had strong reduction of fertility that could not be rescued by exogenous JA (Mandaokar et al., 2006).

On the other hand, as the major JA-signaling components, JAZ proteins were found directly involved in stamen development. Overexpression of JAZ1ΔJas (Thines et al., 2007) and JAZ10.4 (Chung and Howe, 2009), both of which lack the full Jas domain and are resistant to degradation by SCFCOI1/26S proteasome, results in male sterility. However, the JAZ3 splice acceptor mutant *jai3-1*, which expresses *JAZ3* without the Jas domain (Chini et al., 2007) and JAZ10.3 (Yan et al., 2007), which lost a portion of Jas domain, are still fertile. This suggests a threshold level of JA signaling determines fertility. This notion was also strongly supported by the findings that COI1 leaky mutant allele coi1-16 is only partially male-sterile (Xiao et al., 2004). Interestingly, JAZ proteins were shown to regulate MYB21/MYB24, the transcription factors responsible for stamen and pollen maturation. A select set of JAZ proteins (JAZ1, JAZ8, and JAZ11) interact directly with MYB21 and MYB24, revealing a mechanism in which JA triggers COI1-dependent JAZ degradation to control MYB21 and MYB24 levels and thereby stamen development (Song et al., 2011). In addition, GA was found to promote JA biosynthesis in flower to control the expression of MYB21, MYB24, and

As an important signal for plant development and defense, JA does not act independently but cooperatively with other phytohormonal signaling pathways including GA (gibberellin), SA (salicylic acid), Ethylene, and ABA (abscisic acid). For JA-GA interaction, a "relief of

MYB57 in the filament of the flower (Cheng et al., 2009).

**3.9. JA Signal Interaction with GA, SA, Ethylene and ABA** 

has been detected (Qi et al., 2011).

**MYB24** 

The expression of *ERF1* can be activated rapidly by ethylene or jasmonate in wild-type plant but not in JA or ethylene (ET) signaling mutants *coi1* or *ein2* (*ethylene insensitive2*), suggesting *ERF1* expression depends on JA and/or ethylene signal. Constitutive overexpression of *ERF1* activates the expression of several defense-related genes, including *PLANT DEFENSIN1.2* (*PDF1.2*) and *BASIC CHITINASE* (*ChiB*) (Lorenzo et al., 2003), and was shown to confer resistance to necrotrophic fungi such as *Botrytis cinerea* and *Plectosphaerella cucumerina*  (Lorenzo et al., 2003)*.* All these results suggest that ERF1 acts downstream of the intersection between ethylene and jasmonate pathways and suggest that this transcription factor is a key element in the integration of both signals for the regulation of defense response genes (Lorenzo et al., 2003). *ORA59*, a close paralogue of *ERF1* in *Arabidopsis*, has also been shown to integrate JA and ET signals in defense responses against *B. cinerea*. Overexpression line of ORA59 showed a severe dwarf phenotype under normal growth conditions, similar to plant overexpressing *ERF1* (Pré et al., 2008). RNAi-silencing of *ORA59* compromises JA- and ETinduced expression of several defense-related genes such as *PDF1.2*, *HEL,* and *ChiB* (Pré et al., 2008). Two more *ERF1*-like genes, *AtERF2* and *AtERF14* have shown to behave similarly as *ERF1* and *ORA59*. Constitutive overexpression of *AtERF2* or AtERF14 causes high levels of *PDF1.2* and *ChiB* gene expression in transgenic *Arabidopsis* plants (Brown et al., 2003; Oñate-Sánchez et al., 2007). In contrast to *ERF1*, *ORA59*, *AtERF2,* and *AtERF14*, AtERF4 (At3g15210) negatively regulates the expression of *PDF1.2* (McGrath et al., 2005). Loss-offunction mutants of *AtERF4* showed impaired induction of defense genes following exogenous ET treatment and increased susceptibility to *Fusarium oxysporum*. Moreover, the expression of other *ERF* genes such as *ERF1* and *AtERF2* depends on *AtERF14* expression (McGrath et al., 2005). Collectively, several of members of the ERF family negatively and positively control the expression of a number of defense genes mediated by jasmonates.

### **3.7. JA-signaling controlling anthocyanin accumulation and trichome development via transcription factors WD40/bHLH/R2R3-MYB complex**

Current genetic and physiological evidence shows that JA regulates the activity of "WDrepeat/bHLH/MYB complex", which mediates anthocyanin accumulation and trichome initiation in a *COI1*-dependent manner. Overexpression of the MYB transcription factor MYB75 and bHLH factors such as GL3 (GLABRA 3) and EGL3 (ENHANCER OF GLABRA 3) restored anthocyanin accumulation and trichome initiation in the *coi1* mutant, respectively (Qi et al., 2011). Anthocyanin biosynthesis and trichome initiation are both inducible by JA (Maes et al., 2008; Qi et al., 2011). This induction requires both the JA receptor component COI1 and the GL3/EGL3/TT8-type bHLH proteins (Maes et al., 2008; Qi et al., 2011). Interestingly, the major JA signaling players, MYC2/MYC3/MYC4 are also involved in the JA-mediated anthocyanin accumulation (Lorenzo et al., 2004; Niu et al., 2011), but may not be required for trichome induction. bHLH factors GL3, EGL3 and TT8 (TRANSPARENT TESTA 8) function in complexes in which they interact directly with the WD40 protein TTG1 (TRANSPARENT TEXTA GLABRA 1) and R2R3-MYB proteins such as MYB75 and GL1 (GLABRA 1). Apart from anthocyanin biosynthesis and trichome formation, GL3/EGL3/TT8 complex may be involved in many other processes including root hair formation, flavonoid biosynthesis, stomata patterning, and seed coat mucilage production (Pauwels and Goossens, 2011). JAZ-interacting domain (JID) was found in a number of bHLH transcription factors including MYC2/MYC3/MYC4, indicating that JAZs may have much wider function spectrum than currently known. Indeed, JID domain is present in GL3, EGL3 and TT8, and interaction of these proteins with eight different JAZs has been detected (Qi et al., 2011).

416 Lipid Metabolism

2007; Pré et al., 2008; McGrath et al., 2005).

identified as a negative regulator for this process (Lorenzo et al., 2003; Oñate-Sánchez et al.,

The expression of *ERF1* can be activated rapidly by ethylene or jasmonate in wild-type plant but not in JA or ethylene (ET) signaling mutants *coi1* or *ein2* (*ethylene insensitive2*), suggesting *ERF1* expression depends on JA and/or ethylene signal. Constitutive overexpression of *ERF1* activates the expression of several defense-related genes, including *PLANT DEFENSIN1.2* (*PDF1.2*) and *BASIC CHITINASE* (*ChiB*) (Lorenzo et al., 2003), and was shown to confer resistance to necrotrophic fungi such as *Botrytis cinerea* and *Plectosphaerella cucumerina*  (Lorenzo et al., 2003)*.* All these results suggest that ERF1 acts downstream of the intersection between ethylene and jasmonate pathways and suggest that this transcription factor is a key element in the integration of both signals for the regulation of defense response genes (Lorenzo et al., 2003). *ORA59*, a close paralogue of *ERF1* in *Arabidopsis*, has also been shown to integrate JA and ET signals in defense responses against *B. cinerea*. Overexpression line of ORA59 showed a severe dwarf phenotype under normal growth conditions, similar to plant overexpressing *ERF1* (Pré et al., 2008). RNAi-silencing of *ORA59* compromises JA- and ETinduced expression of several defense-related genes such as *PDF1.2*, *HEL,* and *ChiB* (Pré et al., 2008). Two more *ERF1*-like genes, *AtERF2* and *AtERF14* have shown to behave similarly as *ERF1* and *ORA59*. Constitutive overexpression of *AtERF2* or AtERF14 causes high levels of *PDF1.2* and *ChiB* gene expression in transgenic *Arabidopsis* plants (Brown et al., 2003; Oñate-Sánchez et al., 2007). In contrast to *ERF1*, *ORA59*, *AtERF2,* and *AtERF14*, AtERF4 (At3g15210) negatively regulates the expression of *PDF1.2* (McGrath et al., 2005). Loss-offunction mutants of *AtERF4* showed impaired induction of defense genes following exogenous ET treatment and increased susceptibility to *Fusarium oxysporum*. Moreover, the expression of other *ERF* genes such as *ERF1* and *AtERF2* depends on *AtERF14* expression (McGrath et al., 2005). Collectively, several of members of the ERF family negatively and positively control the expression of a number of defense genes mediated by jasmonates.

**3.7. JA-signaling controlling anthocyanin accumulation and trichome development via transcription factors WD40/bHLH/R2R3-MYB complex** 

Current genetic and physiological evidence shows that JA regulates the activity of "WDrepeat/bHLH/MYB complex", which mediates anthocyanin accumulation and trichome initiation in a *COI1*-dependent manner. Overexpression of the MYB transcription factor MYB75 and bHLH factors such as GL3 (GLABRA 3) and EGL3 (ENHANCER OF GLABRA 3) restored anthocyanin accumulation and trichome initiation in the *coi1* mutant, respectively (Qi et al., 2011). Anthocyanin biosynthesis and trichome initiation are both inducible by JA (Maes et al., 2008; Qi et al., 2011). This induction requires both the JA receptor component COI1 and the GL3/EGL3/TT8-type bHLH proteins (Maes et al., 2008; Qi et al., 2011). Interestingly, the major JA signaling players, MYC2/MYC3/MYC4 are also involved in the JA-mediated anthocyanin accumulation (Lorenzo et al., 2004; Niu et al., 2011), but may not be required for trichome induction. bHLH factors GL3, EGL3 and TT8 (TRANSPARENT TESTA 8) function in complexes in which they interact directly with the
