**3.8. JA-Signaling regulates Male Fertility via Transcription Factors MYB21 and MYB24**

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

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

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

#### 418 Lipid Metabolism

repression" model has been proposed; in which DELLAs compete with MYC2 for binding to JAZ1 in *Arabidopsis*. Without GA, stabilized DELLA proteins bind to JAZ1 and release MYC2 to promote JA signaling. GA triggers degradation of DELLAs, which releases free JAZ1 to bind to MYC2 and, thus, attenuates JA signaling (Hou et al., 2010). Furthermore, GA significantly suppresses JA-activation of JA-responsive genes, whereas, GA alone does not significantly affect the expression of JA-responsive genes (Hou et al., 2010). This study suggested GA negatively regulates JA signaling. However, GA was found to mobilize the expression of DAD1, a key enzyme of JA biosynthesis in flowers. This is consistent with the observation that the JA content in the young flower buds of the GA-deficient quadruple mutant *ga1-3 gai-t6 rgat2 rgl1-1* is much lower than that in the WT. The conclusion of these observervations suggests that GA promotes JA biosynthesis to control the expression of MYB21, MYB24, and MYB57, which are essential for male anther development (Cheng et al., 2009).

Jasmonate Biosynthesis, Perception and Function in Plant Development and Stress Responses 419

INSENSITIVE3 (EIN3) and EIN3-LIKE1 (EIL1), two central positive transcription factors for the ET responses (Zhu et al., 2011). At least JAZ1, JAZ3, and JAZ9 can bind EIN3 and EIL1. Therefore, the JAZ proteins can repress the function of EIN3/EIL1, possibly by suppressing the DNA binding of EIN3 (Zhu et al., 2011). In the current model, ET is needed for EIN3/EIL1 stabilization and JA for EIN3/EIL1 release from the JAZ protein repression due to ubiquitin-mediated proteolysis, providing a reasonable explanation for the synergy in many ET/JA-regulated processes (Zhu et al., 2011). As we described above, MYC2, MYC3, and MYC4 are the key signaling players for JA signaling downstream JAZs are also required for ET signaling (Lorenzo et al., 2004). On the other hand, Overexpression of ET-responsive transcription factors ERF1 and ORA59 significantly activates JA responses (Lorenzo et al.,

Very limited information for the interaction between JA and ABA is available so far. ABA and MeJA were reported to induce stomatal closure, most likely by triggering the production of reactive oxygen species (ROS) in stomatal guard cells (Munemasa et al., 2007). The *coi1* mutation suppresses only MeJA-mediated ROS production without influencing ABAmediated ROS production, suggesting that *COI1*-dependent JA signaling acts through ABA pathway for stomatal closure (Munemasa et al., 2007). Anderson et al. (2004) showed that interaction between ABA and ethylene signaling is mutually antagonistic in vegetative tissues. Exogenous ABA suppressed both basal and JA/ethylene–activated transcription of defense genes. By contrast, ABA deficiency as conditioned by mutations in the *ABA1* and *ABA2* genes, which encode enzymes involved in ABA biosynthesis, resulted in up-regulation of basal and induced transcription from JA-ethylene responsive defense genes (Anderson et al., 2004).

**4. THE physiological roles of JA in plant development and defense** 

JA is one of the major defense hormones in plants (Browse, 2009), and it provides a major mechanism of induced defenses against insects herbivores and a wide spectrum of pathogen species, especially necrotrophic fungi. The defense property of jasmonates to various insect herbivores has been extensively studied in the past decades. To the best of our knowledge, there is no report supporting a negative role of jasmonates in defense against insect species. This topic has been covered by a number of excellent reviews (Farmer et al., 2003; Felton and Tumlinson, 2008; Browse, 2009; Howe and Jander, 2008). Here, we describe the major lines of evidence that point to the evolution, action and significance of JA as a defense hormone *in planta*. (1) Mechanical wounding or damage caused by herbivore feeding results in rapid accumulation of JAs at the site of wounding (Glauser et al., 2008). Successive feeding on the leaves causes steady increase of JA content throughout the entire plant (Reymond et al., 2004). (2) Wounding or exogenous application of JA/MeJA generally up-regulates the genes involved in JA biosynthesis (Mueller, 1997; Leon and Sanchez-Serrano, 1999). Most JA biosynthesis genes such as *LOX2*, *LOX*3, *LOX4*, *AOS*, *OPR3*, and *AOC3* and signaling genes such as *MYC2*, *JAZ1*, *JAZ2*, *JAZ5*, *JAZ6*, *JAZ7*, *JAZ8*, *JAZ9,* and *JAZ10* were found highly inducible in response to wounding, MeJA treatment, and herbivore feeding (Chung et al.,

**4.1. JA is an essential signal for plant defense against insect herbivory** 

2003; Pré et al., 2008).

The mutually antagonistic interactions between SA and JA pathways were shown by analysis of SA- and JA-marker gene expression in SA and JA signaling mutants of *Arabidopsis*. JAsignaling mutant *coi1* displayed enhanced basal and inducible expression of SA marker gene *PR1*, while SA signaling mutant *npr1* (*non-repressor of pr genes 1*) showed concomitant increases in basal or induced levels of JA marker gene *PDF1.2* (Mur et al., 2006). Interestingly, exogenous SA promotes JA-dependent induction of defense gene *PDF1.2* when applied at low concentrations. However, at higher SA concentrations, JA-induced induction of *PDF1.2* is suppressed, suggesting the interaction between these pathways may be dose dependent (Mur et al., 2006). The antagonistic interaction between SA and JA is mediated by NPR1, the centrral regulator of SA signaling (Spoel et al., 2003). WRKY70 is a versatile transcription factor with roles in multiple signaling pathways and physiological processes. It regulates the antagonistic interactions between SA and JA pathways. Overexpression of WRKY70 leads to the constitutive expression of the SA-responsive *PR* genes and increased resistance to SA-sensitive pathogens but reduces resistance to JA-sensitive pathogens. In contrast, suppression of WRKY70 leads to increased expression of JA-responsive genes and increased resistance to a pathogen sensitive to JA-dependent defenses (Li et al., 2004). Another important negative regulator of SA signaling is MPK4. The *Arabidopsis mpk4* mutant exhibits increased SA levels, constitutive expression of *PR1*, and increased resistance to *P. syringae* in the absence of pathogen attack. In contrast, the JA-dependent induction of the *PDF1.2* gene was abolished in the *mpk4* mutant (Petersen et al., 2000).

A number of studies provide evidence for positive interactions between the JA and ET signaling pathways. For example, both JA and ET signaling are required for the expression of the defense-related gene *PDF1.2* in response to infection by *Alternaria brassicicola*  (Penninckx et al., 1998). Evidence that JA and ET coordinatively regulate many other defense-related genes was obtained in an *A. thaliana* microarray experiment, which showed nearly half of the genes that were induced by ET were also induced by JA treatment (Schenk et al., 2000). Some evidence suggest also antagonistic interactions between the JA and ET defense pathways although a number of JA-specific or ET-specific genes were found in wounding and defense responses (Lorenzo et al., 2003). Crosstalk between JA and ET was found mediated through the physical interaction of JAZ proteins with ETHYLENE INSENSITIVE3 (EIN3) and EIN3-LIKE1 (EIL1), two central positive transcription factors for the ET responses (Zhu et al., 2011). At least JAZ1, JAZ3, and JAZ9 can bind EIN3 and EIL1. Therefore, the JAZ proteins can repress the function of EIN3/EIL1, possibly by suppressing the DNA binding of EIN3 (Zhu et al., 2011). In the current model, ET is needed for EIN3/EIL1 stabilization and JA for EIN3/EIL1 release from the JAZ protein repression due to ubiquitin-mediated proteolysis, providing a reasonable explanation for the synergy in many ET/JA-regulated processes (Zhu et al., 2011). As we described above, MYC2, MYC3, and MYC4 are the key signaling players for JA signaling downstream JAZs are also required for ET signaling (Lorenzo et al., 2004). On the other hand, Overexpression of ET-responsive transcription factors ERF1 and ORA59 significantly activates JA responses (Lorenzo et al., 2003; Pré et al., 2008).

418 Lipid Metabolism

repression" model has been proposed; in which DELLAs compete with MYC2 for binding to JAZ1 in *Arabidopsis*. Without GA, stabilized DELLA proteins bind to JAZ1 and release MYC2 to promote JA signaling. GA triggers degradation of DELLAs, which releases free JAZ1 to bind to MYC2 and, thus, attenuates JA signaling (Hou et al., 2010). Furthermore, GA significantly suppresses JA-activation of JA-responsive genes, whereas, GA alone does not significantly affect the expression of JA-responsive genes (Hou et al., 2010). This study suggested GA negatively regulates JA signaling. However, GA was found to mobilize the expression of DAD1, a key enzyme of JA biosynthesis in flowers. This is consistent with the observation that the JA content in the young flower buds of the GA-deficient quadruple mutant *ga1-3 gai-t6 rgat2 rgl1-1* is much lower than that in the WT. The conclusion of these observervations suggests that GA promotes JA biosynthesis to control the expression of MYB21, MYB24, and MYB57,

The mutually antagonistic interactions between SA and JA pathways were shown by analysis of SA- and JA-marker gene expression in SA and JA signaling mutants of *Arabidopsis*. JAsignaling mutant *coi1* displayed enhanced basal and inducible expression of SA marker gene *PR1*, while SA signaling mutant *npr1* (*non-repressor of pr genes 1*) showed concomitant increases in basal or induced levels of JA marker gene *PDF1.2* (Mur et al., 2006). Interestingly, exogenous SA promotes JA-dependent induction of defense gene *PDF1.2* when applied at low concentrations. However, at higher SA concentrations, JA-induced induction of *PDF1.2* is suppressed, suggesting the interaction between these pathways may be dose dependent (Mur et al., 2006). The antagonistic interaction between SA and JA is mediated by NPR1, the centrral regulator of SA signaling (Spoel et al., 2003). WRKY70 is a versatile transcription factor with roles in multiple signaling pathways and physiological processes. It regulates the antagonistic interactions between SA and JA pathways. Overexpression of WRKY70 leads to the constitutive expression of the SA-responsive *PR* genes and increased resistance to SA-sensitive pathogens but reduces resistance to JA-sensitive pathogens. In contrast, suppression of WRKY70 leads to increased expression of JA-responsive genes and increased resistance to a pathogen sensitive to JA-dependent defenses (Li et al., 2004). Another important negative regulator of SA signaling is MPK4. The *Arabidopsis mpk4* mutant exhibits increased SA levels, constitutive expression of *PR1*, and increased resistance to *P. syringae* in the absence of pathogen attack. In contrast, the JA-dependent induction of the *PDF1.2* gene was abolished in

A number of studies provide evidence for positive interactions between the JA and ET signaling pathways. For example, both JA and ET signaling are required for the expression of the defense-related gene *PDF1.2* in response to infection by *Alternaria brassicicola*  (Penninckx et al., 1998). Evidence that JA and ET coordinatively regulate many other defense-related genes was obtained in an *A. thaliana* microarray experiment, which showed nearly half of the genes that were induced by ET were also induced by JA treatment (Schenk et al., 2000). Some evidence suggest also antagonistic interactions between the JA and ET defense pathways although a number of JA-specific or ET-specific genes were found in wounding and defense responses (Lorenzo et al., 2003). Crosstalk between JA and ET was found mediated through the physical interaction of JAZ proteins with ETHYLENE

which are essential for male anther development (Cheng et al., 2009).

the *mpk4* mutant (Petersen et al., 2000).

Very limited information for the interaction between JA and ABA is available so far. ABA and MeJA were reported to induce stomatal closure, most likely by triggering the production of reactive oxygen species (ROS) in stomatal guard cells (Munemasa et al., 2007). The *coi1* mutation suppresses only MeJA-mediated ROS production without influencing ABAmediated ROS production, suggesting that *COI1*-dependent JA signaling acts through ABA pathway for stomatal closure (Munemasa et al., 2007). Anderson et al. (2004) showed that interaction between ABA and ethylene signaling is mutually antagonistic in vegetative tissues. Exogenous ABA suppressed both basal and JA/ethylene–activated transcription of defense genes. By contrast, ABA deficiency as conditioned by mutations in the *ABA1* and *ABA2* genes, which encode enzymes involved in ABA biosynthesis, resulted in up-regulation of basal and induced transcription from JA-ethylene responsive defense genes (Anderson et al., 2004).

#### **4. THE physiological roles of JA in plant development and defense**

#### **4.1. JA is an essential signal for plant defense against insect herbivory**

JA is one of the major defense hormones in plants (Browse, 2009), and it provides a major mechanism of induced defenses against insects herbivores and a wide spectrum of pathogen species, especially necrotrophic fungi. The defense property of jasmonates to various insect herbivores has been extensively studied in the past decades. To the best of our knowledge, there is no report supporting a negative role of jasmonates in defense against insect species. This topic has been covered by a number of excellent reviews (Farmer et al., 2003; Felton and Tumlinson, 2008; Browse, 2009; Howe and Jander, 2008). Here, we describe the major lines of evidence that point to the evolution, action and significance of JA as a defense hormone *in planta*. (1) Mechanical wounding or damage caused by herbivore feeding results in rapid accumulation of JAs at the site of wounding (Glauser et al., 2008). Successive feeding on the leaves causes steady increase of JA content throughout the entire plant (Reymond et al., 2004). (2) Wounding or exogenous application of JA/MeJA generally up-regulates the genes involved in JA biosynthesis (Mueller, 1997; Leon and Sanchez-Serrano, 1999). Most JA biosynthesis genes such as *LOX2*, *LOX*3, *LOX4*, *AOS*, *OPR3*, and *AOC3* and signaling genes such as *MYC2*, *JAZ1*, *JAZ2*, *JAZ5*, *JAZ6*, *JAZ7*, *JAZ8*, *JAZ9,* and *JAZ10* were found highly inducible in response to wounding, MeJA treatment, and herbivore feeding (Chung et al.,

2008). (3) Insect feeding or wounding induces hundreds of defense-related genes in JAdependent manner, including genes involved in pathogenesis, indole glucosinolate metabolism, and detoxification (Reymond et al., 2004). (4) Insect feeding, wounding, or MeJA treatment activates synthesis of anti-insect substance, e.g., proteinase inhibitors (PIs) in *Arabidopsis* (Farmer et al., 1992), nicotine in tobacco, papain inhibitor(s) in tomato (Bolter, 1993), vinblastine in rose periwinkle (*Catharanthus roseus*), artemisinin in annual wormwood (*Artemisia annua*) (De Geyter et al., 2012), and poisonous secondary metabolites such as glucosinates and camalexin in *Arabidopsis*. (5) JA biosynthesis or perception mutants of *Arabidopsis* such as, *fad3-2 fad7-2 fad8*, *aos*, *opr3*, *jar1,* and *coi1,* as well as those from other species such as tomato *jar1*, and maize *opr7 opr8* are highly susceptible to insect attack (McConn et al. 1997; Laudert and Weiler, 1998; Stintzi et al., 2001; Staswick et al., 1998; Xie et al., 1998; Li et al., 2004; Yan et al., 2012). These JA mutants are shown to be compromised in resistance to a wide range of arthropod herbivores including caterpillars (*Lepidoptera*), beetles (*Coleoptera*), thrips (*Thysanoptera*), leafhoppers (*Homoptera*), spider mites (*Acari*), fungal gnats (*Diptera*), and mirid bugs (*Heteroptera*) (Howe and Jander, 2008). On the other hand, JA-pathway overexpression mutants such as *cev1*, cex1, and *fou2* are highly resistant to insect and pathogen attacks (Ellis and Turner, 2001; Xu et al., 2001; Bonaventure et al., 2007). (6) Exogenous application of JA or MeJA can elevate resistance of a number of plant species to insects attack (Avdiushko et al., 1997). The JA precursor OPDA also contributes to plant defense against insect attacks (Stintzi et al., 2001). (7) When attacted by herbivores, plants can rapidly release volatile organic compounds (VOC, consisting mainly of fatty acidderived products and terpenes) and green leafy volatiles (GLV, including mainly of (*Z*)*-3* hexenal, (*Z*)-3-hexenol, and (*Z*)-3-hexenyl acetate). These can effectively induce direct defense response — activation of JA biosynthesis pathway in the attacked and neighboring plants, and indirect defense response — attraction of insect enemies that parasitize or prey on feeding insects (Paré and Tumlinson, 1999; Engelberth et al., 2004).

Jasmonate Biosynthesis, Perception and Function in Plant Development and Stress Responses 421

application of JA and ET can activate expression of genes in both JA biosynthesis and signaling pathway (Chung et al., 2008; Pré et al., 2008). As to SA interaction with JA/ET, either to biotrophic or necrotrophic pathogens, SA was suggested to act mutually

Considering only the role of JA, it has been frequently demonstrated to be an indispensible phytohormone signal for resistance/susceptibility to several diseases caused by fungal,

1. **JA mediates resistance of plant to necrotrophic pathogens such as** *Botrytis cenerea* **and** *Alternaria brassicicola*. JA perception mutant *coi1* display enhanced susceptibility to *B. cenerea* and *A. brassicicola* (Thomma et al., 1998) and JA biosynthesis mutant *aos* as well as signaling mutant *coi1* is also highly susceptible to *B. cenerea* (Rowe et al., 2010). Interestingly, there are exceptions that JA negatively regulates resistance to necrotrophic fungi. For example, JA signaling mutant *jin1/jai1*, which acts downstream of *COI1* in JA signaling pathway, showed higher resistance to necrotrophic fungi such as *B. cinerea* and *Plectosphaerella cucumerina* (Lorenzo et al., 2004). JA signaling mutant *coi1* displayed enhanced resistance to necrotrophic fungus *Fusarium oxysporium*

2. **JA biosynthesis mutants show extremely high susceptibility to soil-borne oomycete**  *Pythium* **spp.** *Arabidopsis* fatty acid desaturase triple mutant *fad3-2 fad7-2 fad8*, deficient in biosynthesis of the JA precursor linolenic acid, is more susceptible to the root pathogen *Pythium mastophorum*; 90% of the triple mutant plants did not survive the infection as compared to only 10% of wild-type plants (Vijayan et al., 1998). Exogenously applied MeJA reduced death rate of *fad3-2 fad7-2 fad8*. Another *Arabidopsis*  JA mutany *jar1* (*jasmonic-acid resistant 1*), shows reduced sensitivity to jasmonates and deficient JA signaling (Staswick et al., 1998). Both *fad3-2 fad7-2 fad8* and *jar1* plants exhibit enhanced susceptibility to *Pythium irregular*e (Staswick et al., 1998). In maize, the double mutant *opr7 opr8*, deficient in JA biosynthesis, showed extreme susceptibility to *Pythium aristosporium* (Yan et al., 2012). When tarnsfered to a field from sterile soil *opr7 opr8* plants displayed "wilting" phenotype due to root rots 6 d after the transfer (Figure 10H and 10I), and 11 days after transplanting, all the *opr7 opr8* plants died, while wild-

3. **JA signaling promotes pathogenesis of biotrophic pathogens**. Resistance of host plants against fungal and bacterial biotrophic pathogens is associated with activation of SA-dependent signaling and SAR. As SA and JA/ET signaling tend to be mutually inhibitory, JA/ET signaling is expected to have negative effects on resistance to these pathogens. Results from studies of *Peronospora parasitica*, *Erysiphe* spp., and P*seudomonas syringae* support the idea that SA signaling is important for resistance against biotrophs. In the cases of *P. parasitica* and *Erisyphe* spp., JA/ET-dependent responses do no seem to play a major role because infection does not induce JA/ET pathways. However, JA/ET signaling may also be effective if activated artificially (Glazebrook, 2005). In the case of *bacterial biotroph P.syringae,* SA-dependent defense responses clearly play an important role in limiting *P.syringae* growth. Mutants with defects in SA signaling, including *eds1*, *pad4*, *eds5*, *sid2*, and *npr1*, show enhanced susceptibility to virulent strains and in some

type plants continued to display normal growth (Yan et al., 2012).

antagonistically with JA/ET pathways (Lorenzo and Solano, 2005).

bacterial, and viral pathogens.

(Thatcher et al., 2009).

### **4.2. The roles of JA in induced systemic resistance (ISR) against microbial pathogens**

JA is an essential phytohormone for defense response against a wide spectrum of pathogens, alone or in combination with other hormones, such as ET, SA, and ABA (Browse, 2009; Adie et al., 2007). Although all plant hormones including GA, auxin (IAA), and brassinosteroids (BR) may be involved in plant defense responses against pathogens (Smith et al., 2009), numerous studies have shown that SA, JA, and ET are the major players in induced resistance of plants (Dong, 1998; Kunkel and Brooks, 2002). The SA-mediated pathway is typically activated in response to pathogens and mediates the initiation of a hypersensitive response (HR) and induction of pathogenesis-related proteins (PRs) that confer systemic acquired resistance (SAR) against a broad array of pathogens (Smith et al., 2009). Rhizobacteria-mediated induced systemic resistance (ISR) in plants primarily depends on JA and ET (Pieterse et al., 1998). Regarding the relationship of JA with ET, the widely held belief is that ET acts synergistically with JA in the activation of responses to pathogens (Lorenzo and Solano, 2005). Several defense-related genes including *PR1*, *PR3*, *PR4*, *PR5,* and *PDF1.2* are synergistically induced by JA and ET (Lorenzo et al., 2003). Exogenous application of JA and ET can activate expression of genes in both JA biosynthesis and signaling pathway (Chung et al., 2008; Pré et al., 2008). As to SA interaction with JA/ET, either to biotrophic or necrotrophic pathogens, SA was suggested to act mutually antagonistically with JA/ET pathways (Lorenzo and Solano, 2005).

420 Lipid Metabolism

**pathogens**

2008). (3) Insect feeding or wounding induces hundreds of defense-related genes in JAdependent manner, including genes involved in pathogenesis, indole glucosinolate metabolism, and detoxification (Reymond et al., 2004). (4) Insect feeding, wounding, or MeJA treatment activates synthesis of anti-insect substance, e.g., proteinase inhibitors (PIs) in *Arabidopsis* (Farmer et al., 1992), nicotine in tobacco, papain inhibitor(s) in tomato (Bolter, 1993), vinblastine in rose periwinkle (*Catharanthus roseus*), artemisinin in annual wormwood (*Artemisia annua*) (De Geyter et al., 2012), and poisonous secondary metabolites such as glucosinates and camalexin in *Arabidopsis*. (5) JA biosynthesis or perception mutants of *Arabidopsis* such as, *fad3-2 fad7-2 fad8*, *aos*, *opr3*, *jar1,* and *coi1,* as well as those from other species such as tomato *jar1*, and maize *opr7 opr8* are highly susceptible to insect attack (McConn et al. 1997; Laudert and Weiler, 1998; Stintzi et al., 2001; Staswick et al., 1998; Xie et al., 1998; Li et al., 2004; Yan et al., 2012). These JA mutants are shown to be compromised in resistance to a wide range of arthropod herbivores including caterpillars (*Lepidoptera*), beetles (*Coleoptera*), thrips (*Thysanoptera*), leafhoppers (*Homoptera*), spider mites (*Acari*), fungal gnats (*Diptera*), and mirid bugs (*Heteroptera*) (Howe and Jander, 2008). On the other hand, JA-pathway overexpression mutants such as *cev1*, cex1, and *fou2* are highly resistant to insect and pathogen attacks (Ellis and Turner, 2001; Xu et al., 2001; Bonaventure et al., 2007). (6) Exogenous application of JA or MeJA can elevate resistance of a number of plant species to insects attack (Avdiushko et al., 1997). The JA precursor OPDA also contributes to plant defense against insect attacks (Stintzi et al., 2001). (7) When attacted by herbivores, plants can rapidly release volatile organic compounds (VOC, consisting mainly of fatty acidderived products and terpenes) and green leafy volatiles (GLV, including mainly of (*Z*)*-3* hexenal, (*Z*)-3-hexenol, and (*Z*)-3-hexenyl acetate). These can effectively induce direct defense response — activation of JA biosynthesis pathway in the attacked and neighboring plants, and indirect defense response — attraction of insect enemies that parasitize or prey

on feeding insects (Paré and Tumlinson, 1999; Engelberth et al., 2004).

**4.2. The roles of JA in induced systemic resistance (ISR) against microbial** 

JA is an essential phytohormone for defense response against a wide spectrum of pathogens, alone or in combination with other hormones, such as ET, SA, and ABA (Browse, 2009; Adie et al., 2007). Although all plant hormones including GA, auxin (IAA), and brassinosteroids (BR) may be involved in plant defense responses against pathogens (Smith et al., 2009), numerous studies have shown that SA, JA, and ET are the major players in induced resistance of plants (Dong, 1998; Kunkel and Brooks, 2002). The SA-mediated pathway is typically activated in response to pathogens and mediates the initiation of a hypersensitive response (HR) and induction of pathogenesis-related proteins (PRs) that confer systemic acquired resistance (SAR) against a broad array of pathogens (Smith et al., 2009). Rhizobacteria-mediated induced systemic resistance (ISR) in plants primarily depends on JA and ET (Pieterse et al., 1998). Regarding the relationship of JA with ET, the widely held belief is that ET acts synergistically with JA in the activation of responses to pathogens (Lorenzo and Solano, 2005). Several defense-related genes including *PR1*, *PR3*, *PR4*, *PR5,* and *PDF1.2* are synergistically induced by JA and ET (Lorenzo et al., 2003). Exogenous Considering only the role of JA, it has been frequently demonstrated to be an indispensible phytohormone signal for resistance/susceptibility to several diseases caused by fungal, bacterial, and viral pathogens.


cases, avirulent strains. *P. syringae* DC3000 inhibits SA signaling by producing a toxin called coronatine, which imitates JA-Ile (a bioactive JA-amino acid conjugate). The coronamic acid moiety of coronatine structurally resembles ACC (the ET precursor, aminocyclopropane carboxylic acid). Resistance of JA insensitive mutant *coi1* to *P. syringae* DC3000 is associated with elevated levels of SA and enhanced expression of SA-regulated genes, suggesting that coronatine contributes to virulence by activating JA signaling, thereby repressing SA-dependent defense mechanisms that limit *P. syringae*  growth (Glazebrook, 2005).

Jasmonate Biosynthesis, Perception and Function in Plant Development and Stress Responses 423

grapevine, exogenous jasmonate can rescue growth in the salt-sensitive cell line and salt stress response is modulated by JA-signaling components such as JAZ proteins (Ismail et al.,

JA plays import role of plant resistance against ozone (O3) stress. JA biosynthesis mutant *fad3/7/8* and JA-signaling mutant *jar1* have greater sensitivity to O3 (Rao et al., 2000). Furthermore, MeJA pretreatment decreased O3-induced H2O2 content and SA concentrations

In addition to defense function, jasmonate was frequently shown to serve essential roles in reproductive processes of plants. In *Arabidopsis*, JA biosynthesis mutants such as *fad3/7/8, aos, opr3,* and JA signaling mutant *coi1* are male sterile, strongly supporting JA as an essential signal for development of male organ of bisexual flowers (Browse, 2009). This JAdependent male sterility phenotype consists of three characteristics: 1) the anthers of mutants lost dehiscence to shed pollen at flowering time; 2) the pollen grains in the anthers are predominantly (>97%) inviable; 3) the stamen filaments are substantially shorter in mutant, that is, the anthers do not elongate sufficiently to the stigma level (Browse, 2009). The fertility defective phenotype of JA biosynthetic mutants is rescued by exogenous JA treatment; however, the signaling mutant *coi1* cannot be rescued by JA application (Browse, 2009). Some JA signaling components have also been implicated in stamen development. *MYB21* and *MYB24* are JA-responsive transcription factors in *opr3* stamens and were isolated as JA-dependent transcription factors for flower development (Mandaokar et al., 2006). A *myb21* mutants exhibited shorter anther filaments, delayed anther dehiscence, and greatly reduced male fertility. A *myb24* mutants was phenotypically wild type, but creation of a *myb21myb24* double mutant indicated that introduction of the *myb21* mutation exacerbated all three aspects of the *myb24* phenotype. Exogenous jasmonate could not restore fertility to *myb21* or *myb21myb24* mutant plants. All these results indicate that *MYB21* and *MYB24* are JA signaling components mediating JA response during stamen

In addition, overexpression of Jas domain-defective JAZ proteins such as JAZ1-ΔJas and JAZ10.4 which are resistant to SCFCOI1/26S proteolysis complex resultes male sterile phenotypes, further demonstrating that JA is essential for male fertility in *Arabidopsis* (Thines et al., 2007; Chung and Howe, 2009). However, some JA signaling mutants are male fertile instead of sterile. For example, *myc2* mutant does not show a male-sterile phenotype (Lorenzo et al., 2004); *jar1* mutant is male fertile (Staswick and Tiryaki, 2004). In these mutants, it may be possible that an alternative component or ligand for JA signaling exists. Surprisingly, in contrast to *Arabidopsis*, JA perception mutant *jar1* of tomato (*Solanum lycopersicum*) are male-fertile but female-sterile (Li et al., 2004), suggesting that JA roles in

Like *Arabidopsis*, the monocotyledonous plant rice bears bisexual flowers. The JA-deficient mutant of rice *hebiba* showed male sterility (Riemann et al., 2003), supporting that JA is

reproductive differentiation of plants largely depend on the species.

**4.4. JAs are required signals for pollen development in dicots and for sex** 

and completely abolished O3-induced cell death (Rao et al., 2000).

2012).

**determination in monocots** 

development (Mandaokar et al., 2006).


#### **4.3. JAs serve an important role against abiotic stresses**

Salinity is one better studied abiotic stress in plants. Foliar application of MeJA can effectively alleviate salinity stress symptoms in soybean seedlings (Yoon et al., 2009). In grapevine, exogenous jasmonate can rescue growth in the salt-sensitive cell line and salt stress response is modulated by JA-signaling components such as JAZ proteins (Ismail et al., 2012).

422 Lipid Metabolism

growth (Glazebrook, 2005).

positive effects to resistance against RNA viruses.

12 hr later and remained elevated 5 d later (Runyon et al., 2010).

**4.3. JAs serve an important role against abiotic stresses** 

cases, avirulent strains. *P. syringae* DC3000 inhibits SA signaling by producing a toxin called coronatine, which imitates JA-Ile (a bioactive JA-amino acid conjugate). The coronamic acid moiety of coronatine structurally resembles ACC (the ET precursor, aminocyclopropane carboxylic acid). Resistance of JA insensitive mutant *coi1* to *P. syringae* DC3000 is associated with elevated levels of SA and enhanced expression of SA-regulated genes, suggesting that coronatine contributes to virulence by activating JA signaling, thereby repressing SA-dependent defense mechanisms that limit *P. syringae* 

4. **JAs may have positive roles in plant resistance against viruses.** Members of the geminivirus family are plant viruses with circular, single-stranded DNA genomes that infect a wide range of plant species and cause extensive yield losses in important crops such as tomato, maize, and cotton. In *Arabidopsis*, exogenous application of jasmonates reduces susceptibility to geminivirus infection (Lozano-Durán et al., 2011). In a case of turnip crinkle virus (TCV), SA, but not JA/ET, is required for the development of hypersensitive reaction (HR) and systemic resistance in *Arabidopsis* (Kachroo et al., 2000). However, applying 60 μM JA and then 100 μM SA 24 h later, enhanced resistance to *Cucumber mosaic virus* (CMV), *Tobacco mosaic virus* (TMV), and TCV in *Arabidopsis*, tobacco, tomato, and hot pepper (Shang et al., 2011), indicating JA and SA have additive

5. **JA positively regulates resistance against parasitic plants.** In plant-parasitic plants interaction, both JA and SA were found to positively regulate host-plant defense responses to parasitic plants (Bar-Nun et al., 2008; Runyon et al., 2010). The holoparasitic plant, *Orobanche aegyptiaca*, is capable of infecting many host plants, including *Arabidopsis thaliana*. Low dose exposure to MeJA or methyl salicylic acid (MeSA) effectively induced resistance of *Arabidopsis* seedlings to *O. aegyptiaca* (Bar-Nun et al., 2008). Runyon et al. (2010) reported that the parasitic plant *Cuscuta pentagona* grew larger on mutant tomato plants, in which the SA (*NahG*) or JA (*jin1*) pathways were disrupted, suggesting that these hormones can act independently to reduce parasite growth. Large increases of both JA and SA were detected in host plant tomato after parasitism was established (*i.e.,* haustoria formation) (Runyon et al., 2010). Host production of JA was transitory and reached a maximum at 36 hr, whereas SA peaked

6. **JA is a key player in induced systemic resistance against root knot nematodes (RKN)**. Foliar application of JA induces a systemic defense response that reduces avirulent nematode reproduction on susceptible tomato plants. JA enhances *Mi*-mediated resistance (*Mi* is a resistant gene in tomato) of resistant lines at high temperature (Cooper et al., 2005). However, using JA-signaling mutant *jar1* (equal to *coi1* of *Arabidopsis*) and JA biosynthesis mutant *def1*, it was found that endogenous JA signaling

pathway is required for tomato susceptibility to RKNs (Bhattarai et al., 2008).

Salinity is one better studied abiotic stress in plants. Foliar application of MeJA can effectively alleviate salinity stress symptoms in soybean seedlings (Yoon et al., 2009). In JA plays import role of plant resistance against ozone (O3) stress. JA biosynthesis mutant *fad3/7/8* and JA-signaling mutant *jar1* have greater sensitivity to O3 (Rao et al., 2000). Furthermore, MeJA pretreatment decreased O3-induced H2O2 content and SA concentrations and completely abolished O3-induced cell death (Rao et al., 2000).

### **4.4. JAs are required signals for pollen development in dicots and for sex determination in monocots**

In addition to defense function, jasmonate was frequently shown to serve essential roles in reproductive processes of plants. In *Arabidopsis*, JA biosynthesis mutants such as *fad3/7/8, aos, opr3,* and JA signaling mutant *coi1* are male sterile, strongly supporting JA as an essential signal for development of male organ of bisexual flowers (Browse, 2009). This JAdependent male sterility phenotype consists of three characteristics: 1) the anthers of mutants lost dehiscence to shed pollen at flowering time; 2) the pollen grains in the anthers are predominantly (>97%) inviable; 3) the stamen filaments are substantially shorter in mutant, that is, the anthers do not elongate sufficiently to the stigma level (Browse, 2009). The fertility defective phenotype of JA biosynthetic mutants is rescued by exogenous JA treatment; however, the signaling mutant *coi1* cannot be rescued by JA application (Browse, 2009). Some JA signaling components have also been implicated in stamen development. *MYB21* and *MYB24* are JA-responsive transcription factors in *opr3* stamens and were isolated as JA-dependent transcription factors for flower development (Mandaokar et al., 2006). A *myb21* mutants exhibited shorter anther filaments, delayed anther dehiscence, and greatly reduced male fertility. A *myb24* mutants was phenotypically wild type, but creation of a *myb21myb24* double mutant indicated that introduction of the *myb21* mutation exacerbated all three aspects of the *myb24* phenotype. Exogenous jasmonate could not restore fertility to *myb21* or *myb21myb24* mutant plants. All these results indicate that *MYB21* and *MYB24* are JA signaling components mediating JA response during stamen development (Mandaokar et al., 2006).

In addition, overexpression of Jas domain-defective JAZ proteins such as JAZ1-ΔJas and JAZ10.4 which are resistant to SCFCOI1/26S proteolysis complex resultes male sterile phenotypes, further demonstrating that JA is essential for male fertility in *Arabidopsis* (Thines et al., 2007; Chung and Howe, 2009). However, some JA signaling mutants are male fertile instead of sterile. For example, *myc2* mutant does not show a male-sterile phenotype (Lorenzo et al., 2004); *jar1* mutant is male fertile (Staswick and Tiryaki, 2004). In these mutants, it may be possible that an alternative component or ligand for JA signaling exists. Surprisingly, in contrast to *Arabidopsis*, JA perception mutant *jar1* of tomato (*Solanum lycopersicum*) are male-fertile but female-sterile (Li et al., 2004), suggesting that JA roles in reproductive differentiation of plants largely depend on the species.

Like *Arabidopsis*, the monocotyledonous plant rice bears bisexual flowers. The JA-deficient mutant of rice *hebiba* showed male sterility (Riemann et al., 2003), supporting that JA is required for male organ formation of bisexual flower plants. Maize is another monocot and belongs to monoecious plants, which bears distinct male inflorescence (called tassel) and female inflorescence (called ear) on the same plant. The monosexual florets in the tassel or the ears develop from bisexual floret primordia of top or axillary meristems though a sex determination program mediated by a number of sex-determining genes (Bortiri and Hake, 2007)*.* Recent study on *ts1* (*tasselseed1*), a mutant in which male inflorescence (tassel) becomes female-fertile structure that can be pollinated to bear seeds, showed that jasmonates is an essential phytohormone that initiates sex determination program of tassel (Acosta et al., 2009). In our recent study, the JA-deficient mutant *opr7 opr8* showed 100% feminized tassel, strongly supporting the JA signal requirement for tassel formation in maize (Figure 10A and 10B) (Yan et al., 2012). *TS1* encodes a 13-lipoxygenase (i.e. *LOX8*), disruption of which causes JA-deficiency locally in the tassel meristem. *opr7 opr8* is a double mutant of OPR isoforms required for JA biosynthesis, mutation of which results in JA depletion systemically in the plant. Several studies have showed that gibberellin (GA) is involved in ear formation. GA biosynthesis mutants such as *an1*, *d1*, *d2*, *d3,* and *d5* and GA perception mutants *D8* and *D9*, all showed dwarfism and masculinized ears (i.e. male florets are produced in ears), indicating GA is another important phytohormone for sex determination in maize (Chuck, 2010). Putting the studies of JA and GA together, we may hypothesize that JA and GA act antagonistically in male and female flowers, respectively, in maize sex determination process.

Jasmonate Biosynthesis, Perception and Function in Plant Development and Stress Responses 425

integrates other plant hormones including CK (cytokinins), GA, IAA, ABA, and ET to regulate growth processes and defense responses (Sano et al., 1996; Cheng et al., 2009;

Trichomes are branching structures or hair-like appendages differentiated from epidermal cells in the aerial part of plant, which function as barriers to protect plants against herbivores, insects, abiotic damage, UV irradiation, and excessive transpiration (Ishida et al., 2008). Trichome formation is initiated by various environmental cues, such as wounding and insect attack (Yoshida et al., 2009), and by different endogenous developmental signals, including phytohormones, such as jasmonate (Traw and Bergelson, 2003; Li et al., 2004; Yoshida et al., 2009), gibberellin (Perazza et al., 1998), ethylene (Plett et al., 2009), and salicylic acid (Traw and Bergelson, 2003). Tomato JA perception mutant *jar1* has no trichomes on the surface of young fruit and significantly less on the leaf and stem surfaces (Li et al., 2004). JA biosynthesis mutant *aos* and perception mutant *coi1* produced fewer trichomes than the wild type and MeJA treatment increases trichome density in *aos* but not *coi1*, indicating JA signal is a positive regulator of trichome development in *Arabidopsis* (Yoshida et al., 2009). JA signal controls trichome patterning in *Arabidopsis* via a key transcription factor GRABRA3 of which JA treatment enhanced expression prior to trichome initiation (Yoshida et al., 2009). GRABRA3 interacts with other transcription factors such as TRANSPARENT TESTA GLABRA1 (TTG1) and GLABRA1 (GL1) to control trichome initiation (Yoshida et al., 2009). Furthermore, a recent study showed that JAZ proteins interact with these transcription factors to regulate trichome development (Qi et al., 2011).

JA perception mutant *jar1* of tomato bears much smaller fruits compared with wild type, and the young seeds of the mutant fruits suffer from high rate of seed abortion (>99%), indicating that JA signal is an essential signal during the early stage of fruit development and seed maturation in tomato (Li et al., 2004). In apples (*Malus sylvestris*) and sweet cherries (*Prunus avium*), endogenous JA accumulated in the early ripening stage of the fruit and seeds, also indicating that JA plays an important role in fruit/seed development (Kondo et al., 2000).

Leaf senescence involves senescence-associated cell death (PCD), which is controlled by age under the influence of endogenous and environmental factors (Lim et al., 2007). Several phytohormones including JA, cytokinins, ethylene, ABA, and SA were implicated in leaf senescence program (Lim et al., 2007). Regarding the role of JA in leaf senescence, most studies support that JA positively regulates leaf senescence process (Ueda and Kato, 1980; He et al., 2002; Schenk et al., 2000; Castillo and León, 2008). Senescence-like phenotypes are induced by exogenous application of MeJA or JA in *Artemisia absinthium* or *Arabidopsis*  (Ueda and Kato, 1980; He et al., 2002); and some senescence-up-regulated genes such as

**4.9. JAs act as an internal signal facilitating leaf senescence** 

Nagpal et al., 2005; Anderson et al., 2004; Lorenzo et al., 2003).

**4.7. JA involved in trichome development** 

**4.8. JAs promote fruit/seed ripening** 

#### **4.5. JAs has a role in female organgenesis in some plant species**

In tomato, JA signaling mutant *jar1* (the ortholog of *coi1*) showed seed-bearing sterility: 1) the size and mass of mature ripened *jai1-1* fruit were significantly less than those of mature wild-type fruit; 2) vast majority (>99%) of fertilize ova of the mutant fruit were not viable during the fruit development and only a few viable seeds were recovered from the fruit. It is estimated that the number of viable seeds produced by *jai1-1* plants was <0.1% of the viable seed yield from wild-type plants grown under identical conditions (Li et al., 2004). In maize, JA-deficient mutant *opr7 opr8* showed outgrowth of multiple female reproductive buds and extreme elongation of ear shanks, indicating JA is a crucial signal for female organ growth (Figure 10C and 10D) (Yan et al., 2012).

#### **4.6. JAs regulate vegetative growth**

Activation of JA defense signaling against biotic and abiotic stresses depletes available resources and severely restricts plant growth. It is well known that JAs act in plant as growth inhibitors in root and shoots (Staswick et al., 1992). Wound-induced accumulation of endogenous JAs strongly suppresses plant growth of roots and shoots by inhibiting cell mitosis (Zhang and Turner, 2008). The inhibition role of JAs depends on JA signaling pathway. JA perception mutant *coi1* relieved JA inhibition to roots and shoots (Xie et al., 1998). JA-signaling mutants such as *jin1/myc2*, *jin4/jar1,* and *jai3* have largely reduced growth inhibition to roots and leaves by JA application (Lorenzo et al., 2004). JA signal integrates other plant hormones including CK (cytokinins), GA, IAA, ABA, and ET to regulate growth processes and defense responses (Sano et al., 1996; Cheng et al., 2009; Nagpal et al., 2005; Anderson et al., 2004; Lorenzo et al., 2003).

#### **4.7. JA involved in trichome development**

424 Lipid Metabolism

maize sex determination process.

(Figure 10C and 10D) (Yan et al., 2012).

**4.6. JAs regulate vegetative growth** 

**4.5. JAs has a role in female organgenesis in some plant species** 

In tomato, JA signaling mutant *jar1* (the ortholog of *coi1*) showed seed-bearing sterility: 1) the size and mass of mature ripened *jai1-1* fruit were significantly less than those of mature wild-type fruit; 2) vast majority (>99%) of fertilize ova of the mutant fruit were not viable during the fruit development and only a few viable seeds were recovered from the fruit. It is estimated that the number of viable seeds produced by *jai1-1* plants was <0.1% of the viable seed yield from wild-type plants grown under identical conditions (Li et al., 2004). In maize, JA-deficient mutant *opr7 opr8* showed outgrowth of multiple female reproductive buds and extreme elongation of ear shanks, indicating JA is a crucial signal for female organ growth

Activation of JA defense signaling against biotic and abiotic stresses depletes available resources and severely restricts plant growth. It is well known that JAs act in plant as growth inhibitors in root and shoots (Staswick et al., 1992). Wound-induced accumulation of endogenous JAs strongly suppresses plant growth of roots and shoots by inhibiting cell mitosis (Zhang and Turner, 2008). The inhibition role of JAs depends on JA signaling pathway. JA perception mutant *coi1* relieved JA inhibition to roots and shoots (Xie et al., 1998). JA-signaling mutants such as *jin1/myc2*, *jin4/jar1,* and *jai3* have largely reduced growth inhibition to roots and leaves by JA application (Lorenzo et al., 2004). JA signal

required for male organ formation of bisexual flower plants. Maize is another monocot and belongs to monoecious plants, which bears distinct male inflorescence (called tassel) and female inflorescence (called ear) on the same plant. The monosexual florets in the tassel or the ears develop from bisexual floret primordia of top or axillary meristems though a sex determination program mediated by a number of sex-determining genes (Bortiri and Hake, 2007)*.* Recent study on *ts1* (*tasselseed1*), a mutant in which male inflorescence (tassel) becomes female-fertile structure that can be pollinated to bear seeds, showed that jasmonates is an essential phytohormone that initiates sex determination program of tassel (Acosta et al., 2009). In our recent study, the JA-deficient mutant *opr7 opr8* showed 100% feminized tassel, strongly supporting the JA signal requirement for tassel formation in maize (Figure 10A and 10B) (Yan et al., 2012). *TS1* encodes a 13-lipoxygenase (i.e. *LOX8*), disruption of which causes JA-deficiency locally in the tassel meristem. *opr7 opr8* is a double mutant of OPR isoforms required for JA biosynthesis, mutation of which results in JA depletion systemically in the plant. Several studies have showed that gibberellin (GA) is involved in ear formation. GA biosynthesis mutants such as *an1*, *d1*, *d2*, *d3,* and *d5* and GA perception mutants *D8* and *D9*, all showed dwarfism and masculinized ears (i.e. male florets are produced in ears), indicating GA is another important phytohormone for sex determination in maize (Chuck, 2010). Putting the studies of JA and GA together, we may hypothesize that JA and GA act antagonistically in male and female flowers, respectively, in

Trichomes are branching structures or hair-like appendages differentiated from epidermal cells in the aerial part of plant, which function as barriers to protect plants against herbivores, insects, abiotic damage, UV irradiation, and excessive transpiration (Ishida et al., 2008). Trichome formation is initiated by various environmental cues, such as wounding and insect attack (Yoshida et al., 2009), and by different endogenous developmental signals, including phytohormones, such as jasmonate (Traw and Bergelson, 2003; Li et al., 2004; Yoshida et al., 2009), gibberellin (Perazza et al., 1998), ethylene (Plett et al., 2009), and salicylic acid (Traw and Bergelson, 2003). Tomato JA perception mutant *jar1* has no trichomes on the surface of young fruit and significantly less on the leaf and stem surfaces (Li et al., 2004). JA biosynthesis mutant *aos* and perception mutant *coi1* produced fewer trichomes than the wild type and MeJA treatment increases trichome density in *aos* but not *coi1*, indicating JA signal is a positive regulator of trichome development in *Arabidopsis* (Yoshida et al., 2009). JA signal controls trichome patterning in *Arabidopsis* via a key transcription factor GRABRA3 of which JA treatment enhanced expression prior to trichome initiation (Yoshida et al., 2009). GRABRA3 interacts with other transcription factors such as TRANSPARENT TESTA GLABRA1 (TTG1) and GLABRA1 (GL1) to control trichome initiation (Yoshida et al., 2009). Furthermore, a recent study showed that JAZ proteins interact with these transcription factors to regulate trichome development (Qi et al., 2011).

#### **4.8. JAs promote fruit/seed ripening**

JA perception mutant *jar1* of tomato bears much smaller fruits compared with wild type, and the young seeds of the mutant fruits suffer from high rate of seed abortion (>99%), indicating that JA signal is an essential signal during the early stage of fruit development and seed maturation in tomato (Li et al., 2004). In apples (*Malus sylvestris*) and sweet cherries (*Prunus avium*), endogenous JA accumulated in the early ripening stage of the fruit and seeds, also indicating that JA plays an important role in fruit/seed development (Kondo et al., 2000).

#### **4.9. JAs act as an internal signal facilitating leaf senescence**

Leaf senescence involves senescence-associated cell death (PCD), which is controlled by age under the influence of endogenous and environmental factors (Lim et al., 2007). Several phytohormones including JA, cytokinins, ethylene, ABA, and SA were implicated in leaf senescence program (Lim et al., 2007). Regarding the role of JA in leaf senescence, most studies support that JA positively regulates leaf senescence process (Ueda and Kato, 1980; He et al., 2002; Schenk et al., 2000; Castillo and León, 2008). Senescence-like phenotypes are induced by exogenous application of MeJA or JA in *Artemisia absinthium* or *Arabidopsis*  (Ueda and Kato, 1980; He et al., 2002); and some senescence-up-regulated genes such as

#### 426 Lipid Metabolism

*SEN1*, *SEN4*, *SEN5*, *SAG12*, *SAG14*, and *SAG15* are responsive to JA treatment (He et al., 2002; Schenk et al., 2000). Delayed yellowing phenotype during natural senescence and upon dark incubation of detached leaves was observed in JA biosynthesis mutant *kat2* and signaling mutant *coi1* (Castillo and León, 2008). Casting doubt about the role of JA in senescence, JA-defective mutants *aos* and *opr3* senesced similar ro wild type under natural senescence conditions or upon dark treatment (He et al., 2002; Schommer et al., 2008). In maize, strong genetic evidence was obtained for JA involvement in the leaf senescence (Yan et al., 2012). The leaves of JA-deficient mutant *opr7 opr8* displayed senesced substantially later than wild type (Figure 10G).

Jasmonate Biosynthesis, Perception and Function in Plant Development and Stress Responses 427

2011). In *Arabidopsis*, camalexin (3-thiazol-2'yl-indole) is the main phytoalexin induced by a variety of microorganisms including bacteria, fungi, and oomycetes. JA signaling is required for the activation of camalexin synthesis in response to infection by *P. syringae* pv. *maculicola* ES4326 (Zhou et al., 1999). Glucosinolates are a group of thioglucosides found in all cruciferous plants such as *Arabidopsis* and *Brassica napus*. The hydrolysed products of glucosinolates contribute to plant defense against microorganisms. MeJA treatment increases total glucosinolate content in leaves of *B. napus* up to 20 fold (Doughty et al., 1995). In *Arabidopsis*, accumulation of camalexin and indole glucosinolates can be trigged by elicitors from the plant pathogen *Erwinia carotovora,* and this induction effect is *COI1* dependent (Brader et al., 2001). There are also a number of examples that jasmonates effectively activate secondary metabolites in medicinal plant species such as artemisinin synthesis in *Artemisia annua* and vinblastine (an alkaloid) in *Catharanthus roseus* (De Geyter et al., 2012). All pathways of the above metabolites, including nicotine, camalexin, glucosinolates, artemisinin, and vinblastine belong to JA-elicited plant secondary metabolism, which is regulated by JA-signaling components such as *COI1*, *MYC2*, *ERF1* and

*JAZs* (De Geyter et al., 2012)*.*

**Figure 10.** Genetic morphological phenotypes of JA-deficient mutant in maize

*Pythium aristosporum* compared with wild type (left).

(A) Tassel of wild type. (B) Feminized tassel structure of JA deficient mutant *opr7 opr8*. (C) The ear of wild type. (D) Multiple elongated ears of *opr7 opr8*.(E) Anthocyanins accumulation in the brace roots of wildtype. (F) Lack anthocyanins pigmentation of *opr7 opr8* brace roots. (G) The senescence phenotype of third leaves of wild type (left) and *opr7 opr8* (right). (H) & (I) *opr7 opr8* (right) is highly susceptible to

#### **4.10. JAs activate secondary metabolism beneficial to development and defense**

Secondary metabolites play diverse roles in plants. For example, flowers synthesize and accumulate anthocyanin pigments in petals to attract pollinating insects. In addition, anthocyanins absorb visible as well as UV radiation and are effective antioxidants and scavengers of reactive oxygen species, protecting plant tissues from the effects of excess incidental visible or UV-B radiation and oxidative stress (Quina et al., 2009). Other secondary metabolites such as polyamines, quinones, terpenoids, alkaloids, phenylpropanoids, and glucosinolates act as phytoalexins to protect plants against microorganisms or herbivores (Chen et al., 2006).

Gaseous MeJA enhance production of anthocyanins in soybean seedlings (Franceschi and Grimes, 1991). Wounding or MeJA treatment activates rapidly the expression of anthocyanin biosynthesis genes and increase anthocyanin level in the detached corolla of *Petunia hybrida* (Moalem-Beno *et al.*, 1997). In *Arabidopsis*, JA or MeJA treatment strongly enhances anthocyanin accumulation in the shoots, especially in the petiole of the seedling (Lorenzo et al., 2004) and this JAs-activating anthocyanin accumulation depends on COI1 mediated JA signaling pathway (Shan et al., 2009). JA induces anthocyanin biosynthesis via up-regulation of the 'late' anthocyanin biosynthetic genes *DFR*, *LDOX*, and *UF3GT* (Shan et al., 2009). JA coincidently activates anthocyanin biosynthetic regulators such as transcription factors *PAP1*, *PAP2*, and *GL3* (Shan et al., 2009). Either these biosynthetic genes or transcription factors are *COI1*-dependent (Shan et al., 2009). In the monocot plant maize, *opr7opr8* double mutant lack anthocyanin pigmentation in brace roots and auricles (Figure 10E and 10F), but not in leaf blade or sheath, indicating that endogenous JA controls anthocyanins pigmentation in specific tissues of maize (Yan et al., 2012).

JAs also effectively activate defensive metabolites against insects or pathogens. Early studies concluded that MeJA application strongly induced anti-insect protein accumulation such as proteinase inhibitors I and II (PI-I, II) (Farmer et al., 1992) and vegetative storage protein (VSP) (Liu et al., 2005). Nicotine, an alkaloid toxic to most insects by interfering with the transmitter substance between nerves and muscles, widely exists in tobacco (*Nicotiana tabacum*) and related species. Exogenous application of JA or wounding of leaves activate nicotine biosynthesis in a *COI1*- and *MYC2*-dependent manner, indicating JA signal is required in tobacco to control nicotine metabolism (Shoji et al., 2008; Shoji and Hashimoto, 2011). In *Arabidopsis*, camalexin (3-thiazol-2'yl-indole) is the main phytoalexin induced by a variety of microorganisms including bacteria, fungi, and oomycetes. JA signaling is required for the activation of camalexin synthesis in response to infection by *P. syringae* pv. *maculicola* ES4326 (Zhou et al., 1999). Glucosinolates are a group of thioglucosides found in all cruciferous plants such as *Arabidopsis* and *Brassica napus*. The hydrolysed products of glucosinolates contribute to plant defense against microorganisms. MeJA treatment increases total glucosinolate content in leaves of *B. napus* up to 20 fold (Doughty et al., 1995). In *Arabidopsis*, accumulation of camalexin and indole glucosinolates can be trigged by elicitors from the plant pathogen *Erwinia carotovora,* and this induction effect is *COI1* dependent (Brader et al., 2001). There are also a number of examples that jasmonates effectively activate secondary metabolites in medicinal plant species such as artemisinin synthesis in *Artemisia annua* and vinblastine (an alkaloid) in *Catharanthus roseus* (De Geyter et al., 2012). All pathways of the above metabolites, including nicotine, camalexin, glucosinolates, artemisinin, and vinblastine belong to JA-elicited plant secondary metabolism, which is regulated by JA-signaling components such as *COI1*, *MYC2*, *ERF1* and *JAZs* (De Geyter et al., 2012)*.*

426 Lipid Metabolism

later than wild type (Figure 10G).

microorganisms or herbivores (Chen et al., 2006).

*SEN1*, *SEN4*, *SEN5*, *SAG12*, *SAG14*, and *SAG15* are responsive to JA treatment (He et al., 2002; Schenk et al., 2000). Delayed yellowing phenotype during natural senescence and upon dark incubation of detached leaves was observed in JA biosynthesis mutant *kat2* and signaling mutant *coi1* (Castillo and León, 2008). Casting doubt about the role of JA in senescence, JA-defective mutants *aos* and *opr3* senesced similar ro wild type under natural senescence conditions or upon dark treatment (He et al., 2002; Schommer et al., 2008). In maize, strong genetic evidence was obtained for JA involvement in the leaf senescence (Yan et al., 2012). The leaves of JA-deficient mutant *opr7 opr8* displayed senesced substantially

**4.10. JAs activate secondary metabolism beneficial to development and defense** 

Secondary metabolites play diverse roles in plants. For example, flowers synthesize and accumulate anthocyanin pigments in petals to attract pollinating insects. In addition, anthocyanins absorb visible as well as UV radiation and are effective antioxidants and scavengers of reactive oxygen species, protecting plant tissues from the effects of excess incidental visible or UV-B radiation and oxidative stress (Quina et al., 2009). Other secondary metabolites such as polyamines, quinones, terpenoids, alkaloids, phenylpropanoids, and glucosinolates act as phytoalexins to protect plants against

Gaseous MeJA enhance production of anthocyanins in soybean seedlings (Franceschi and Grimes, 1991). Wounding or MeJA treatment activates rapidly the expression of anthocyanin biosynthesis genes and increase anthocyanin level in the detached corolla of *Petunia hybrida* (Moalem-Beno *et al.*, 1997). In *Arabidopsis*, JA or MeJA treatment strongly enhances anthocyanin accumulation in the shoots, especially in the petiole of the seedling (Lorenzo et al., 2004) and this JAs-activating anthocyanin accumulation depends on COI1 mediated JA signaling pathway (Shan et al., 2009). JA induces anthocyanin biosynthesis via up-regulation of the 'late' anthocyanin biosynthetic genes *DFR*, *LDOX*, and *UF3GT* (Shan et al., 2009). JA coincidently activates anthocyanin biosynthetic regulators such as transcription factors *PAP1*, *PAP2*, and *GL3* (Shan et al., 2009). Either these biosynthetic genes or transcription factors are *COI1*-dependent (Shan et al., 2009). In the monocot plant maize, *opr7opr8* double mutant lack anthocyanin pigmentation in brace roots and auricles (Figure 10E and 10F), but not in leaf blade or sheath, indicating that endogenous JA controls

JAs also effectively activate defensive metabolites against insects or pathogens. Early studies concluded that MeJA application strongly induced anti-insect protein accumulation such as proteinase inhibitors I and II (PI-I, II) (Farmer et al., 1992) and vegetative storage protein (VSP) (Liu et al., 2005). Nicotine, an alkaloid toxic to most insects by interfering with the transmitter substance between nerves and muscles, widely exists in tobacco (*Nicotiana tabacum*) and related species. Exogenous application of JA or wounding of leaves activate nicotine biosynthesis in a *COI1*- and *MYC2*-dependent manner, indicating JA signal is required in tobacco to control nicotine metabolism (Shoji et al., 2008; Shoji and Hashimoto,

anthocyanins pigmentation in specific tissues of maize (Yan et al., 2012).

**Figure 10.** Genetic morphological phenotypes of JA-deficient mutant in maize

(A) Tassel of wild type. (B) Feminized tassel structure of JA deficient mutant *opr7 opr8*. (C) The ear of wild type. (D) Multiple elongated ears of *opr7 opr8*.(E) Anthocyanins accumulation in the brace roots of wildtype. (F) Lack anthocyanins pigmentation of *opr7 opr8* brace roots. (G) The senescence phenotype of third leaves of wild type (left) and *opr7 opr8* (right). (H) & (I) *opr7 opr8* (right) is highly susceptible to *Pythium aristosporum* compared with wild type (left).

#### **5. Conclusion**

Our understanding of the biosynthesis, regulation, and signaling mechanisms of jasmonates has increased substantially in the last few years. JA biosynthesis enzymes showed 'selfactivation', in which the final product, JA, positively regulate the enzyme activity of this pathway. Currently, only (+)-7-iso-jasmonoyl-L-Ile has been conclusively shown to function as the bioactive ligand to JA signaling machinery SCFCOI1/JAZs complex. The molecular mechanism of JA signal perception and transduction was found to mimic many aspects of the auxin signaling process. In the presence of low levels of JA, JAZ proteins repress the expression of JA-responsive genes by interacting directly with the bHLH (basic helix-loophelix) transcription factors MYC2, MYC3, and MYC4, which are positive regulators of JA responses. When JA levels increased, the bioactive form of ligand JA-Ile promotes binding of JAZs to SCFCOI1 to form SCFCOI1-JAZ-JA-Ile reception complex and subsequent degradation of JAZ repressors via the ubiquitin/26S proteasome pathway, resulting in derepression of primary response genes. A number of recent studies found a wide spectrum of JA functions in plant including the regulation of developmental and defense processes, such as, resistance against insects and pathogens, root growth, fruit/seed maturation, leaf senescence, anthocyanin pigmentation, sex determination (of monoecious plant), female or reproductive organ formation.

Jasmonate Biosynthesis, Perception and Function in Plant Development and Stress Responses 429

Anderson JP, Badruzsaufari E, Schenk PM, Manners JM, Desmond OJ, Ehlert C, Maclean DJ, Ebert PR, and Kazan K (2004) Antagonistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene expression and disease

Andersson MX, Hamberg M, Kourtchenko O, Brunnstrom A, McPhail KL, Gerwick WH, Gobel C, Feussner I, and Ellerstrom M (2006) Oxylipin profiling of the hypersensitive response in Arabidopsis thaliana. Formation of a novel oxo-phytodienoic acid-

Andreou A, Brodhun F, and Feussner I (2009) Biosynthesis of oxylipins in non-mammals.

Antico CJ (2012) Insights into the role of jasmonic acid-mediated defenses against

Avanci NC, Luche DD, Goldman GH, and Goldman MH (2010) Jasmonates are phytohormones with multiple functions, including plant defense and reproduction.

Avdiushko SA, Brown GC, Dahlman DL, and Hildebrand DF (1997) Methyl jasmonate exposure induces insect resistance in cabbage and tobacco. Environmental Entomology

Bannenberg G, Martinez M, Hamberg M, and Castresana C (2009) Diversity of the enzymatic activity in the lipoxygenase gene family of Arabidopsis thaliana. Lipids

Bar-Nun N, Sachs T, andMayer AM (2008) A role for IAA in the infection of Arabidopsis

Beckers GJ, and Spoel SH (2006) Fine-tuning plant defence signalling: salicylate versus

Bell E, Creelman RA, and Mullet JE (1995) A chloroplast lipoxygenase is required for wound-induced jasmonic acid accumulation in Arabidopsis. Proc Natl Acad Sci U S A

Berger S, Bell E, and Mullet JE (1996) Two methyl jasmonate-insensitive mutants show altered expression of AtVsp in response to methyl jasmonate and wounding. Plant

Bhattarai KK, Xie QG, Mantelin S, Bishnoi U, Girke T, Navarre DA, and Kaloshian I (2008) Tomato susceptibility to root-knot nematodes requires an intact jasmonic acid signaling

Blechert S, Brodschelm W, Holder S, Kammerer L, Kutchan TM, Mueller MJ, Xia ZQ, and Zenk MH (1995) The octadecanoic pathway: signal molecules for the regulation of

Bolter CJ (1993) Methyl Jasmonate Induces Papain Inhibitor(s) in Tomato Leaves. Plant

Bonaventure G, Gfeller A, Proebsting WM, Hortensteiner S, Chetelat A, Martinoia E, and Farmer EE (2007) A gain-of-function allele of TPC1 activates oxylipin biogenesis after

Bortiri E, and Hake S (2007) Flowering and determinacy in maize. J Exp Bot 58:909-916.

containing galactolipid, arabidopside E. J Biol Chem 281:31528-31537.

necrotrophic and biotrophic fungal pathogens. Frontiers in Biology 7:48.

resistance in Arabidopsis. Plant Cell 16:3460-3479.

thaliana by Orobanche aegyptiaca Ann Bot 101:261-265.

pathway. Mol Plant Microbe Interact 21:1205-1214.

leaf wounding in Arabidopsis. Plant J 49:889-898.

secondary pathways. Proc Natl Acad Sci U S A 92:4099-4105.

jasmonate. Plant Biol (Stuttg) 8:1-10.

Prog Lipid Res 48:148-170.

Genet Mol Res 9:484-505.

26:642-654.

44:85-95.

92:8675-8679.

Physiology 111:525-531.

Physiol 103:1347-1353.
