**2.3. Derivatives and Metabolites of JA**

The JA biosynthetic pathway from linolenic acid yields (+)-7-*iso*-JA (3*R*,7*S*-JA) as the final product (Sembdner and Parthier, 1993). However, this molecule readily isomerizes to the thermodynamically favored stereoisomer (-)-JA (3*R*,7*R*-JA) (Figure 2) resulting in a molar equilibrium of about 9: 1 ((-)-JA : (+)-7-*iso*-JA) under normal conditions (Sembdner and Parthier, 1993). In addition to isomerization, JA undergoes a series of molecular modifications to form a variety of metabolites in plants (Figure 5).

Jasmonate Biosynthesis, Perception and Function in Plant Development and Stress Responses 401

(senescence-associated gene) 101-like acyl hyrolase. The free hexadecatrienoic acid (16:3) liberated by a phospholipase A2 from the *sn*-*2* position of MGDG or DGDG can also become substrate for JA biosynthesis to form dn-OPDA (Figure 3). Alternatively, 13-LOX may oxygenate 18:3 and 16:3 esterified in galactolipids (Buseman et al., 2006; Kourtchenko et al., 2007). The resulting hydroperoxy galactolipids may then be substrates for AOS and AOC, yielding arabidopsides, which belong to galactolipid species containing esterified OPDA and dnOPDA in plastidic membranes. These lipid species may serve as storage lipids that may allow the rapid release of OPDA and dnOPDA, the JA biosynthetic intermediates. Conclusively, lipase activity is essential to supply the JA biosynthesis pathway with either

The first reported lipase involved in JA biosynthesis was DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1), a chloroplastic glycerolipid lipase. DAD1 belongs to phospholipase A1 (PLA1) family. *Arabidopsis dad1* T-DNA insertion mutants are male sterile from decreased JA accumulation required for reproduction (Ishiguro et al., 2001), but remain capable of synthesizing JA, indicating that other lipases contribute to JA production.

A homolog of DAD1, the DONGLE protein was characterized as an essential lipase involved in the early wound response in *Arabidopsis* leaves. A DGL-overexpressing mutant *dgl-D* displayed dwarfism with small round leaves, extremely high basal JA accumulation, increased expression of JA-responsive genes, and increased resistance to the necrotrophic

*Arabidopsis* PLA1 family comprises seven PLA1 with predicted plastidic transit signaling peptides: DGL (At1g05800; PLA-Iα1), DAD1 (At2g44810; PLA-Iβ1), At2g31690 (PLA-Iα2), At4g16820 (PLA-Iβ2), At1g06800 (PLA-Iγ1), At2g30550 (PLA-Iγ2), and At1g51440 (PLA-Iγ3) (Ryu, 2004). In addition to these seven PLA1 lipases (DAD1-like lipases), Ellinger et al (2010), identified 14 additional putative lipases with predicted plastid transit peptides suggesting, up to 21 lipases may contribute to JA production in *Arabidopsis*. Mutant lines of 18 different lipases, including DGL and DAD1, have been assessed for wound-induced jasmonate levels. However, none of the single lipase mutants or the quadruple mutant line (*pla-Iβ2 / Iγ1 / Iγ2 / Iγ3*) were completely abolished in JA formation under basal and wound-induced conditions (Ellinger et al., 2010), indicating that multiple lipases with both *sn-1* and *sn-2* (or dual *sn-*

Lipoxygenases (LOXs) are non-heme iron-containing dioxygenases widely distributed in yeast, algae, fungus, plant, and animal species (Shin et al, 2008). LOX is the first synthesis enzyme of Vick and Zimmermann pathway. LOX isozymes catalyze the incorporation of molecular oxygen at either position 9 or 13 of polyunsaturated fatty acids (PUFA) such as linoleic (LA,18:2) and α-linolenic (LeA,18:3) acid to produce PUFA hydroperoxides (HPOT and HPOD), which can be further converted to different oxidized fatty acids (oxylipins) through the action of enzymes participating in seven LOX-pathway branches (Figure 1).

*1*/*sn-2*) galactolipid substrate specificity participate in JA formation in plant.

free PUFA (18:3 and 16:3) or OPDA/dnOPDA as precursors.

fungus *Alternaria brassicicola* (Hyun et al., 2008).

**2.6. Lipoxygenase (LOX)** 


With the above reactions, (+)-7-*iso*-JA can be converted into more than 30 distinct jasmonates which were found to be widespread in *Angiospermae*, *Gymnospermae*, *Pteridophyta*, *Algae* such as *Euglena*, *Spirulina*, and *Chiarella,* and the red alga *Gelidium*  (Sembdner and Parthier, 1993). However, just several jasmonates *e.g.,* free JA, *cis*-jasmone, MeJA and JA-Ile, are considered to be the major bioactive JA forms in plants (Fonseca et al., 2009). Other jasmonate derivatives or conjugates have been viewed as clearance metabolites playing important roles in hormone homeostasis (Sembdner and Parthier, 1993), in which JA biosynthesis (and deconjugation) and JA degradation (or conjugation) are balanced to control the actual active JA level for fine-tuning developemental and defensive events.

#### **2.4. The Enzymes for JA biosynthesis and derivation**

The enzymes of JA biosynthesis and metabolism have been extensively investigated in *Arabidopsis* and several articles have reviewed structures, biochemical activities, and functional regulation (Schaller and Stintzi, 2009; Schaller, 2001; Delker et al., 2006). Here we focus on the genes encoding the enzymes for JA biosynthesis and metabolism.

#### **2.5. Phosholipase A (PLA)**

According to the classical Vick and Zimmerman pathway (Vick and Zimmerman, 1983), JA biosynthesis initiates by release of 18:3 from chloroplast membrane galactolipids by a lipase. The lipase belongs to one of the following five enzyme (Wasternack, 2007; Delker et al., 2006): (1) phospholipase A1 (PLA1), which cleaves the acyl group of phospholipid and glycerolipids at the *sn*-1 position; (2) phospholipase A2 (PLA2), which cleaves the acyl group in *sn*-2 position; (3) patatin-like acyl hydrolases, which has little *sn-1*/*sn-2* specificity and is homologous to animal Ca2+-independent PLA2; (4) DAD-like lipase with activity of phospholipid and galactolipid acyl hydrolase that may have *sn-1* or *sn-2* specificity; (5) SAG (senescence-associated gene) 101-like acyl hyrolase. The free hexadecatrienoic acid (16:3) liberated by a phospholipase A2 from the *sn*-*2* position of MGDG or DGDG can also become substrate for JA biosynthesis to form dn-OPDA (Figure 3). Alternatively, 13-LOX may oxygenate 18:3 and 16:3 esterified in galactolipids (Buseman et al., 2006; Kourtchenko et al., 2007). The resulting hydroperoxy galactolipids may then be substrates for AOS and AOC, yielding arabidopsides, which belong to galactolipid species containing esterified OPDA and dnOPDA in plastidic membranes. These lipid species may serve as storage lipids that may allow the rapid release of OPDA and dnOPDA, the JA biosynthetic intermediates. Conclusively, lipase activity is essential to supply the JA biosynthesis pathway with either free PUFA (18:3 and 16:3) or OPDA/dnOPDA as precursors.

The first reported lipase involved in JA biosynthesis was DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1), a chloroplastic glycerolipid lipase. DAD1 belongs to phospholipase A1 (PLA1) family. *Arabidopsis dad1* T-DNA insertion mutants are male sterile from decreased JA accumulation required for reproduction (Ishiguro et al., 2001), but remain capable of synthesizing JA, indicating that other lipases contribute to JA production.

A homolog of DAD1, the DONGLE protein was characterized as an essential lipase involved in the early wound response in *Arabidopsis* leaves. A DGL-overexpressing mutant *dgl-D* displayed dwarfism with small round leaves, extremely high basal JA accumulation, increased expression of JA-responsive genes, and increased resistance to the necrotrophic fungus *Alternaria brassicicola* (Hyun et al., 2008).

*Arabidopsis* PLA1 family comprises seven PLA1 with predicted plastidic transit signaling peptides: DGL (At1g05800; PLA-Iα1), DAD1 (At2g44810; PLA-Iβ1), At2g31690 (PLA-Iα2), At4g16820 (PLA-Iβ2), At1g06800 (PLA-Iγ1), At2g30550 (PLA-Iγ2), and At1g51440 (PLA-Iγ3) (Ryu, 2004). In addition to these seven PLA1 lipases (DAD1-like lipases), Ellinger et al (2010), identified 14 additional putative lipases with predicted plastid transit peptides suggesting, up to 21 lipases may contribute to JA production in *Arabidopsis*. Mutant lines of 18 different lipases, including DGL and DAD1, have been assessed for wound-induced jasmonate levels. However, none of the single lipase mutants or the quadruple mutant line (*pla-Iβ2 / Iγ1 / Iγ2 / Iγ3*) were completely abolished in JA formation under basal and wound-induced conditions (Ellinger et al., 2010), indicating that multiple lipases with both *sn-1* and *sn-2* (or dual *sn-1*/*sn-2*) galactolipid substrate specificity participate in JA formation in plant.

#### **2.6. Lipoxygenase (LOX)**

400 Lipid Metabolism

defensive events.

**2.5. Phosholipase A (PLA)** 

**2.3. Derivatives and Metabolites of JA** 

modifications to form a variety of metabolites in plants (Figure 5).

1-Aminocyclopropane-1-carboxylic acid (ACC).

**2.4. The Enzymes for JA biosynthesis and derivation** 

2. C1 carboxyl group can be decarboxylated. 3. Glycosylation of C1 carboxyl group. 4. Reduction of C6 carbonyl group. 5. Reduction of C9,10 double bond. 6. Hydroxylation of carbon at C11 or C12.

The JA biosynthetic pathway from linolenic acid yields (+)-7-*iso*-JA (3*R*,7*S*-JA) as the final product (Sembdner and Parthier, 1993). However, this molecule readily isomerizes to the thermodynamically favored stereoisomer (-)-JA (3*R*,7*R*-JA) (Figure 2) resulting in a molar equilibrium of about 9: 1 ((-)-JA : (+)-7-*iso*-JA) under normal conditions (Sembdner and Parthier, 1993). In addition to isomerization, JA undergoes a series of molecular

1. C1 carboxyl group can be methyl-esterified or conjugated with amino acids or with

With the above reactions, (+)-7-*iso*-JA can be converted into more than 30 distinct jasmonates which were found to be widespread in *Angiospermae*, *Gymnospermae*, *Pteridophyta*, *Algae* such as *Euglena*, *Spirulina*, and *Chiarella,* and the red alga *Gelidium*  (Sembdner and Parthier, 1993). However, just several jasmonates *e.g.,* free JA, *cis*-jasmone, MeJA and JA-Ile, are considered to be the major bioactive JA forms in plants (Fonseca et al., 2009). Other jasmonate derivatives or conjugates have been viewed as clearance metabolites playing important roles in hormone homeostasis (Sembdner and Parthier, 1993), in which JA biosynthesis (and deconjugation) and JA degradation (or conjugation) are balanced to control the actual active JA level for fine-tuning developemental and

The enzymes of JA biosynthesis and metabolism have been extensively investigated in *Arabidopsis* and several articles have reviewed structures, biochemical activities, and functional regulation (Schaller and Stintzi, 2009; Schaller, 2001; Delker et al., 2006). Here we

According to the classical Vick and Zimmerman pathway (Vick and Zimmerman, 1983), JA biosynthesis initiates by release of 18:3 from chloroplast membrane galactolipids by a lipase. The lipase belongs to one of the following five enzyme (Wasternack, 2007; Delker et al., 2006): (1) phospholipase A1 (PLA1), which cleaves the acyl group of phospholipid and glycerolipids at the *sn*-1 position; (2) phospholipase A2 (PLA2), which cleaves the acyl group in *sn*-2 position; (3) patatin-like acyl hydrolases, which has little *sn-1*/*sn-2* specificity and is homologous to animal Ca2+-independent PLA2; (4) DAD-like lipase with activity of phospholipid and galactolipid acyl hydrolase that may have *sn-1* or *sn-2* specificity; (5) SAG

focus on the genes encoding the enzymes for JA biosynthesis and metabolism.

Lipoxygenases (LOXs) are non-heme iron-containing dioxygenases widely distributed in yeast, algae, fungus, plant, and animal species (Shin et al, 2008). LOX is the first synthesis enzyme of Vick and Zimmermann pathway. LOX isozymes catalyze the incorporation of molecular oxygen at either position 9 or 13 of polyunsaturated fatty acids (PUFA) such as linoleic (LA,18:2) and α-linolenic (LeA,18:3) acid to produce PUFA hydroperoxides (HPOT and HPOD), which can be further converted to different oxidized fatty acids (oxylipins) through the action of enzymes participating in seven LOX-pathway branches (Figure 1). Plant LOXs are classified with respect to their positional specificity of LA dioxygenation. LOXs adding O2 to C-9 or C-13 of the hydrocarbon backbone of LA are designated as 9-LOX or 13-LOX (Feussner and Wasternack, 2002). However, in plants some LOXs have dual positional specificity to C-9 and C-13 of LA and produce both 9- and 13-hydroperoxides of linoleic acid (Hughes *et al*. 2001, Kim *et al*. 2002, Garbe *et al*. 2006). Plants LOXs were found in several subcellular compartments including chloroplast, vacuole and cytosol. According to their localization and sequence similarity, plant LOXs can be classified into type 1- and type 2-LOXs. Type 1-LOXs harbor no chloroplast-transit peptide but the members of this group share a high similarity (>75%) of amino acid sequence to one another. Type 2-LOXs carry a putative chloroplast transit peptide and show only a moderate overall similarity ( 35%) of amino acid sequence to one another. To date, these LOX forms all belong to the subfamily of 13-LOXs (Feussner and Wasternack, 2002). After LOX activity and fluxing through the lipoxygenase pathway branches, PUFA (mainly LA and LeA) can be converted to hundreds of oxylipin species which physiological roles are largely unclear in plants. However, the jasmonates, a small group of oxylipins, whose members are well known signal molecules mediating defense responses against pathogens and insects. JA alone does not completely describe the effects of lipoxygenase activity, but the other hundreds of oxylipin compounds posses biochemical roles in determing a wide spectrum of responses. Plant LOXs have been correlated with seed germination, vegetative and reproductive growth, fruit maturation, plant senescence, and responses to pathogen attacks and insect wounding (Porta and Rocha-Sosa, 2002).

Jasmonate Biosynthesis, Perception and Function in Plant Development and Stress Responses 403

9-/13-HPOT, the product of LOXs can serve as substrate for several enzymes (Figure 1). Allene oxide synthase (AOS) catalyzes 9-/13-HPOT to the unstable epoxide, either 9,10- EOT (9,10-Epoxyoctadecatrienoic acid) or 12,13-EOT (12,13-Epoxyoctadecatrienoic acid). The unstable epoxide can be either hydrolysed non-enzymatically to *α*- and *γ* -ketols or cyclized to 12-oxo-phytodienoic acid (OPDA). Only 12, 13-EOT provides substrate for the following enzyme in JA biosynthesis, allene oxide cyclase (AOC) (Figure 1). AOS and other HPOT-utilizing enzymes such as HPL (hydroperoxide lyase) and DES (divinyl ether synthase) belong to the family of CYP74 enzymes, which are independent from molecular oxygen and NADPH, exhibit low affinity to CO, and use an acyl hydroperoxide as the substrate and the oxygen donor (Stumpe and Feussner, 2006). CYP74 enzymes have been phylogenetically classified into CYP74A, CYP74B, CYP74C, and CYP74D (Stumpe and Feussner, 2006). With some exceptions, plant AOS enzymes belong to CYP74A (Stumpe and Feussner, 2006). According to the specificity of AOS to the substrates, 9-/13 hydroperoxides, AOS enzymes specialize into 9- or 13-AOS, which use either 9- or 13 hydroperoxide, respectively, as substrate. AOS enzymes from barley and rice show no substrate specificity for either (9*S*)-hydroperoxides or (13*S*)-hydroperoxides, and designated 9/13-AOS (Stumpe and Feussner, 2006). Like LOXs, only 13-AOS functions in JA biosynthesis. All 13-AOS carry a plastid-transit peptide except AOS from guayule (Pan et al., 1995) and barley (Maucher at al., 2000), indicating that during JA biosynthesis, AOS localizes to chloroplast. Interestingly, barley AOS, which lacks plastid-transit peptide, was also found localized in plastid (Maucher at al., 2000). Plant species may contain one or multiple AOS genes. For example, *Arabidopsis* jus has a single copy of AOS gene while rice may have four AOS genes (Agrawal et al., 2004). AOS genes from plant species such as flax (Song *et al*., 1993), guayule (Pan *et al*., 1995), *Arabidopsis* (Laudert *et al*., 1996), tomato (Howe *et al*., 2000), barley (Maucher *et al*., 2000), rice (Ha et al., 2002; Agrawal et al., 2004) and corn (Utsunomiya *et al*., 2000) have been cloned or purified so far. The diagram of oxylipin biosynthesis (Figure 1) clearly showed AOS branch competes with other HPOTusing branches for substrate, indicating AOS activity is crucial to control influx of HPOT into JA biosynthesis (Figure 1 & 2). Overexpression of flax AOS in transgenic potato plants led to 6-12 folds increased of basal JA level (Harms et al., 1995). However, overexpression of *Arabidopsis* AOS in either *Arabidopsis* or tobacco did not alter the basal level of JA (Laudert et al., 2000), indicating that the basal expression level of AOS varied in plant species which may be the bottleneck or not for JA production in rest plants. One important property of *AOS* genes in plants is that they are strongly induced by wounding and JA- / MeJA-, and OPDA- treatment in many plant species (Harms et al., 1995; Laudert and weiler, 1998). Other plant hormones such and Ethylene and abscisic acid (ABA) can also induce AOS *Arabidopsis* (Laudert and weiler, 1998). *Arabidopsis* JA-deficient mutant *aos* (Park et al., 2002) or *dde2* (*delayed-dehiscence2*) (von Malek et al., 2002) showed malesterile phenotype and no JA induction in wounding response, demonstrating AOS

**2.7. Allene oxide synthase (AOS)** 

enzyme is essential for JA biosynthesis pathway in plants.

Every plant species harbors several LOX isozymes, encoded by a LOX gene family. For example, the *Arabidopsis* genome contains six LOX genes (Bannenberg et al., 2009), while rice and maize have 14 (Umate, 2011) and 13 LOX genes (Nemchenko et al. 2006), respectively. All LOX isoforms may contribute to oxylipin production, but only 13-LOXs with chloroplast-transit peptide participate in Vick and Zimmerman pathway for JA production. Additionally, several LOX genes may function for JA biosynthesis, e.g., LOX2, LOX3, LOX4 and LOX6 in *Arabidopsis* contain chloroplast signaling peptides and show 13S-lipoxygenase activity, both required for JA biosynthesis (Bannenberg et al., 2009). However, no LOX gene in *Arabidopsis* shows dual 9-/13-LOX activity (Bannenberg et al., 2009). Compelling evidence establishes LOXs involvment in JA biosynthesis in plants. LOX2 of *Arabidopsis* localizes to chloroplasts (Bell et al., 1995), and transgenic plants lacking LOX2 no longer produced JA as observed in control plants, indicating requirement of LOX2 for wound-induced accumulation of jasmonates in leaves (Bell et al., 1995). *lox3 lox4* double mutant is male sterilie, revealing redundant role of LOX3 and LOX4 in florescence JA biosynthesis (Caldelari et al., 2011). In maize strong evidence establishes *TS1* encoding ZmLOX8, as indispensible for JA biosynthesis in tassel (Acosta et al., 2009). In the *ts1* mutant, the male sex determination process – abortion of pistil primordia in bisexual floral meristem, fails from deficient lipoxygenase activity and subsequent low endogenous JA concentrations (Acosta et al., 2009). In addition to peroxidation of JA percurors, LOXs may indirectly regulate JA biosynthesis in plants. For example, the maize disruption mutant, *lox10,* is devoid of green leaf volatiles (GLV) and reduced JA production (Christensen et al., 2012).

#### **2.7. Allene oxide synthase (AOS)**

402 Lipid Metabolism

wounding (Porta and Rocha-Sosa, 2002).

Plant LOXs are classified with respect to their positional specificity of LA dioxygenation. LOXs adding O2 to C-9 or C-13 of the hydrocarbon backbone of LA are designated as 9-LOX or 13-LOX (Feussner and Wasternack, 2002). However, in plants some LOXs have dual positional specificity to C-9 and C-13 of LA and produce both 9- and 13-hydroperoxides of linoleic acid (Hughes *et al*. 2001, Kim *et al*. 2002, Garbe *et al*. 2006). Plants LOXs were found in several subcellular compartments including chloroplast, vacuole and cytosol. According to their localization and sequence similarity, plant LOXs can be classified into type 1- and type 2-LOXs. Type 1-LOXs harbor no chloroplast-transit peptide but the members of this group share a high similarity (>75%) of amino acid sequence to one another. Type 2-LOXs carry a putative chloroplast transit peptide and show only a moderate overall similarity ( 35%) of amino acid sequence to one another. To date, these LOX forms all belong to the subfamily of 13-LOXs (Feussner and Wasternack, 2002). After LOX activity and fluxing through the lipoxygenase pathway branches, PUFA (mainly LA and LeA) can be converted to hundreds of oxylipin species which physiological roles are largely unclear in plants. However, the jasmonates, a small group of oxylipins, whose members are well known signal molecules mediating defense responses against pathogens and insects. JA alone does not completely describe the effects of lipoxygenase activity, but the other hundreds of oxylipin compounds posses biochemical roles in determing a wide spectrum of responses. Plant LOXs have been correlated with seed germination, vegetative and reproductive growth, fruit maturation, plant senescence, and responses to pathogen attacks and insect

Every plant species harbors several LOX isozymes, encoded by a LOX gene family. For example, the *Arabidopsis* genome contains six LOX genes (Bannenberg et al., 2009), while rice and maize have 14 (Umate, 2011) and 13 LOX genes (Nemchenko et al. 2006), respectively. All LOX isoforms may contribute to oxylipin production, but only 13-LOXs with chloroplast-transit peptide participate in Vick and Zimmerman pathway for JA production. Additionally, several LOX genes may function for JA biosynthesis, e.g., LOX2, LOX3, LOX4 and LOX6 in *Arabidopsis* contain chloroplast signaling peptides and show 13S-lipoxygenase activity, both required for JA biosynthesis (Bannenberg et al., 2009). However, no LOX gene in *Arabidopsis* shows dual 9-/13-LOX activity (Bannenberg et al., 2009). Compelling evidence establishes LOXs involvment in JA biosynthesis in plants. LOX2 of *Arabidopsis* localizes to chloroplasts (Bell et al., 1995), and transgenic plants lacking LOX2 no longer produced JA as observed in control plants, indicating requirement of LOX2 for wound-induced accumulation of jasmonates in leaves (Bell et al., 1995). *lox3 lox4* double mutant is male sterilie, revealing redundant role of LOX3 and LOX4 in florescence JA biosynthesis (Caldelari et al., 2011). In maize strong evidence establishes *TS1* encoding ZmLOX8, as indispensible for JA biosynthesis in tassel (Acosta et al., 2009). In the *ts1* mutant, the male sex determination process – abortion of pistil primordia in bisexual floral meristem, fails from deficient lipoxygenase activity and subsequent low endogenous JA concentrations (Acosta et al., 2009). In addition to peroxidation of JA percurors, LOXs may indirectly regulate JA biosynthesis in plants. For example, the maize disruption mutant, *lox10,* is devoid of green leaf volatiles (GLV) and reduced JA production (Christensen et al., 2012).

9-/13-HPOT, the product of LOXs can serve as substrate for several enzymes (Figure 1). Allene oxide synthase (AOS) catalyzes 9-/13-HPOT to the unstable epoxide, either 9,10- EOT (9,10-Epoxyoctadecatrienoic acid) or 12,13-EOT (12,13-Epoxyoctadecatrienoic acid). The unstable epoxide can be either hydrolysed non-enzymatically to *α*- and *γ* -ketols or cyclized to 12-oxo-phytodienoic acid (OPDA). Only 12, 13-EOT provides substrate for the following enzyme in JA biosynthesis, allene oxide cyclase (AOC) (Figure 1). AOS and other HPOT-utilizing enzymes such as HPL (hydroperoxide lyase) and DES (divinyl ether synthase) belong to the family of CYP74 enzymes, which are independent from molecular oxygen and NADPH, exhibit low affinity to CO, and use an acyl hydroperoxide as the substrate and the oxygen donor (Stumpe and Feussner, 2006). CYP74 enzymes have been phylogenetically classified into CYP74A, CYP74B, CYP74C, and CYP74D (Stumpe and Feussner, 2006). With some exceptions, plant AOS enzymes belong to CYP74A (Stumpe and Feussner, 2006). According to the specificity of AOS to the substrates, 9-/13 hydroperoxides, AOS enzymes specialize into 9- or 13-AOS, which use either 9- or 13 hydroperoxide, respectively, as substrate. AOS enzymes from barley and rice show no substrate specificity for either (9*S*)-hydroperoxides or (13*S*)-hydroperoxides, and designated 9/13-AOS (Stumpe and Feussner, 2006). Like LOXs, only 13-AOS functions in JA biosynthesis. All 13-AOS carry a plastid-transit peptide except AOS from guayule (Pan et al., 1995) and barley (Maucher at al., 2000), indicating that during JA biosynthesis, AOS localizes to chloroplast. Interestingly, barley AOS, which lacks plastid-transit peptide, was also found localized in plastid (Maucher at al., 2000). Plant species may contain one or multiple AOS genes. For example, *Arabidopsis* jus has a single copy of AOS gene while rice may have four AOS genes (Agrawal et al., 2004). AOS genes from plant species such as flax (Song *et al*., 1993), guayule (Pan *et al*., 1995), *Arabidopsis* (Laudert *et al*., 1996), tomato (Howe *et al*., 2000), barley (Maucher *et al*., 2000), rice (Ha et al., 2002; Agrawal et al., 2004) and corn (Utsunomiya *et al*., 2000) have been cloned or purified so far. The diagram of oxylipin biosynthesis (Figure 1) clearly showed AOS branch competes with other HPOTusing branches for substrate, indicating AOS activity is crucial to control influx of HPOT into JA biosynthesis (Figure 1 & 2). Overexpression of flax AOS in transgenic potato plants led to 6-12 folds increased of basal JA level (Harms et al., 1995). However, overexpression of *Arabidopsis* AOS in either *Arabidopsis* or tobacco did not alter the basal level of JA (Laudert et al., 2000), indicating that the basal expression level of AOS varied in plant species which may be the bottleneck or not for JA production in rest plants. One important property of *AOS* genes in plants is that they are strongly induced by wounding and JA- / MeJA-, and OPDA- treatment in many plant species (Harms et al., 1995; Laudert and weiler, 1998). Other plant hormones such and Ethylene and abscisic acid (ABA) can also induce AOS *Arabidopsis* (Laudert and weiler, 1998). *Arabidopsis* JA-deficient mutant *aos* (Park et al., 2002) or *dde2* (*delayed-dehiscence2*) (von Malek et al., 2002) showed malesterile phenotype and no JA induction in wounding response, demonstrating AOS enzyme is essential for JA biosynthesis pathway in plants.

#### **2.8. Allene oxide cyclase (AOC)**

Allene oxide cyclase (AOC) catalyzes the stereospecific cyclization of the unstable allene oxide, the product of AOS into the cis-(+) enantiomer OPDA, the precursor of JA (Figure 2). The unstable allene oxide is either 9,10-EOT (9,10-Epoxyoctadecatrienoic acid) or 12,13-EOT (12,13-Epoxyoctadecatrienoic acid), corresponding to 9-/13-HPOT, the substrates of AOS. These unstable substrates of AOC, 9,10-EOT and 12,13-EOT can spontaneously and rapidly hydrolyze to a mixture of α- and γ- ketols (t1/2 < 30 minutes in water) (Schaller et al., 2004). However, *in vivo* α- and γ- ketols are not detectable (Schaller et al., 2004), suggesting tight coupling of AOS and AOC reactions, which effectively convert HPOT into OPDA. AOC was firstly purified as a 47kDa dimer from maize kernals (Ziegler et al., 1997) and was found to accepted only 12,13-EOT (12,13(*S*)-epoxylinolenic acid) but not 12,13- EOD (12,13(*S*) epoxylinoleic acid) as a substrate (Ziegler et al., 1999). This is in contrast to AOS, which produces both allene oxides using 13(*S*)-hydroperoxy 18:3 and 18:2. Thus, it appears AOC provides additional specificity to the octadecanoid pathway for JA production in plants (Schaller et al., 2004). To date, one *AOC* gene from tomato (Ziegler et al., 2000), one from barley (Maucher et al., 2004) and four from *Arabidopsis* (Stenzel et al., 2003) have been cloned. Monocot *AOC* genes are less stuied, but at least two exisist in the rice genome (Agrawal et al., 2004). *Arabidopsis* AOCs are enzymatically active and form *cis*-(+)-OPDA, with AOC2 having greatest activity. The N-terminal of cloned *AOC* genes revealed the presence of chloroplast-transit peptide and localization in chloroplast was confirmed immunohistochemically (Ziegler et al., 2000; Stenzel et al., 2003), supporting OPDA production of JA biosynthesis is localized in chloroplast. *Arabidopsis* and rice AOC genes, in particular AOC2 and AOC1, respectively are differentially regulated upon wounding, JAtreatment, and environmental stresses (Agrawal et al., 2004).

Jasmonate Biosynthesis, Perception and Function in Plant Development and Stress Responses 405

remains unknown (Schaller, 2001). A number of compounds containing an olefinic bond of α,β-unsaturated ketones and aldehydes may be substrates of OYE (Schaller, 2001). Several OYE homologues have been identified in prokaryotic and eukaryotic organisms (Vaz et al., 1995; Kohli and Massey, 1998; Xu et al., 1999). The first identified OYE in higher plant is OPR1 of *Arabidopsis* (Schaller and Weiler, 1997). Plant OPR isomers are encoded by small gene families identified in across broad plant genre, (Schaller et al., 2004). Better-studied OPR families include five *OPRs* (three of which are characterized) in *Arabidopsis* (Sanders et

**Figure 6.** β-Oxidation Scheme of JA Biosynthesis (Li et al., 2005)

Abbreviations: ACS (acyl-CoA synthetase), ACX (acyl-CoA oxidase), MFP (multifunctional protein), KAT (3-ketoacyl-CoA thiolase), and OPC8:0 (3-oxo-2(2'[*Z*]-pentenyl)-cyclopentane-1-octanoic acid)

al., 2000), six in pea (Matsui et al., 2004), three in tomato (Strassner et al., 2002), 13 in rice (Agrawal et al., 2004), and eight in maize (Zhang et al., 2005). All theses OPRs can catalyze the reduction of α,β-unsaturated carbonyls (conjugated enones) in a wide spectrum of substrates in including four stereoisomers of OPDA(Sanders et al., 2000). Earlier studies on the enzymatic activity of OPRs in *Arabidopsis* and tomato revealed that different OPR isomers have distinct substrate preferences to warrant classification into separate groups, group I and II, depending on their substrate specificity to OPDA stereoisomers (Schaller et al., 1998). OPR group I enzymes preferentially catalyze the reduction of (9*R*,13*R*)-12-oxo-

#### **2.9. Oxo-phytodienoic acid reductase (OPR)**

The second half of the JA biosynthesis pathway, beginning with *cis*-(+)-OPDA, occurs in the peroxisome, requiring OPDA or its CoA ester to transport from the chloroplast into the peroxisome. An OPDA-specific transporter is not yet known, however a peroxisomal ABC transporter protein COMATOSE (CTS, Footitt et al., 2002), also known as PXA1 (Zolman et al., 2001) or PED3 (Hayashi et al., 2002), may mediate transportation of OPDA into peroxisome. While *cts* mutants are JA-deficient, suggesting involvement of CTS with JAproduction, substantial residual JA implicates CTS-independent OPDA transport, possiblely by ion trapping of OPDA (Theodoulou et al., 2005).

The first step of peroxisomal JA biosynthesis is the conversion of OPDA, a cyclopentenone to cyclopentanone (3-oxo-2-(2'(*Z*)-pentenyl)-cyclopentane-1-octanoic acid, OPC-8:0) catalyzed by OPDA reductase (OPR). OPR enzymes belong to Old Yellow Enzyme (OYE) (EC 1.6.99.1), initially isolated from brewer's bottom yeast and shown to possess a flavin cofactor. Despite extensive biochemical and spectroscopic characterization, the physiological role of the enzyme remained obscure. OYE has been described as a diaphorase catalyzing the oxidation of NADPH in presence of molecular oxygen, but the physiological oxidant remains unknown (Schaller, 2001). A number of compounds containing an olefinic bond of α,β-unsaturated ketones and aldehydes may be substrates of OYE (Schaller, 2001). Several OYE homologues have been identified in prokaryotic and eukaryotic organisms (Vaz et al., 1995; Kohli and Massey, 1998; Xu et al., 1999). The first identified OYE in higher plant is OPR1 of *Arabidopsis* (Schaller and Weiler, 1997). Plant OPR isomers are encoded by small gene families identified in across broad plant genre, (Schaller et al., 2004). Better-studied OPR families include five *OPRs* (three of which are characterized) in *Arabidopsis* (Sanders et

404 Lipid Metabolism

**2.8. Allene oxide cyclase (AOC)** 

treatment, and environmental stresses (Agrawal et al., 2004).

**2.9. Oxo-phytodienoic acid reductase (OPR)** 

by ion trapping of OPDA (Theodoulou et al., 2005).

Allene oxide cyclase (AOC) catalyzes the stereospecific cyclization of the unstable allene oxide, the product of AOS into the cis-(+) enantiomer OPDA, the precursor of JA (Figure 2). The unstable allene oxide is either 9,10-EOT (9,10-Epoxyoctadecatrienoic acid) or 12,13-EOT (12,13-Epoxyoctadecatrienoic acid), corresponding to 9-/13-HPOT, the substrates of AOS. These unstable substrates of AOC, 9,10-EOT and 12,13-EOT can spontaneously and rapidly hydrolyze to a mixture of α- and γ- ketols (t1/2 < 30 minutes in water) (Schaller et al., 2004). However, *in vivo* α- and γ- ketols are not detectable (Schaller et al., 2004), suggesting tight coupling of AOS and AOC reactions, which effectively convert HPOT into OPDA. AOC was firstly purified as a 47kDa dimer from maize kernals (Ziegler et al., 1997) and was found to accepted only 12,13-EOT (12,13(*S*)-epoxylinolenic acid) but not 12,13- EOD (12,13(*S*) epoxylinoleic acid) as a substrate (Ziegler et al., 1999). This is in contrast to AOS, which produces both allene oxides using 13(*S*)-hydroperoxy 18:3 and 18:2. Thus, it appears AOC provides additional specificity to the octadecanoid pathway for JA production in plants (Schaller et al., 2004). To date, one *AOC* gene from tomato (Ziegler et al., 2000), one from barley (Maucher et al., 2004) and four from *Arabidopsis* (Stenzel et al., 2003) have been cloned. Monocot *AOC* genes are less stuied, but at least two exisist in the rice genome (Agrawal et al., 2004). *Arabidopsis* AOCs are enzymatically active and form *cis*-(+)-OPDA, with AOC2 having greatest activity. The N-terminal of cloned *AOC* genes revealed the presence of chloroplast-transit peptide and localization in chloroplast was confirmed immunohistochemically (Ziegler et al., 2000; Stenzel et al., 2003), supporting OPDA production of JA biosynthesis is localized in chloroplast. *Arabidopsis* and rice AOC genes, in particular AOC2 and AOC1, respectively are differentially regulated upon wounding, JA-

The second half of the JA biosynthesis pathway, beginning with *cis*-(+)-OPDA, occurs in the peroxisome, requiring OPDA or its CoA ester to transport from the chloroplast into the peroxisome. An OPDA-specific transporter is not yet known, however a peroxisomal ABC transporter protein COMATOSE (CTS, Footitt et al., 2002), also known as PXA1 (Zolman et al., 2001) or PED3 (Hayashi et al., 2002), may mediate transportation of OPDA into peroxisome. While *cts* mutants are JA-deficient, suggesting involvement of CTS with JAproduction, substantial residual JA implicates CTS-independent OPDA transport, possiblely

The first step of peroxisomal JA biosynthesis is the conversion of OPDA, a cyclopentenone to cyclopentanone (3-oxo-2-(2'(*Z*)-pentenyl)-cyclopentane-1-octanoic acid, OPC-8:0) catalyzed by OPDA reductase (OPR). OPR enzymes belong to Old Yellow Enzyme (OYE) (EC 1.6.99.1), initially isolated from brewer's bottom yeast and shown to possess a flavin cofactor. Despite extensive biochemical and spectroscopic characterization, the physiological role of the enzyme remained obscure. OYE has been described as a diaphorase catalyzing the oxidation of NADPH in presence of molecular oxygen, but the physiological oxidant

**Figure 6.** β-Oxidation Scheme of JA Biosynthesis (Li et al., 2005) Abbreviations: ACS (acyl-CoA synthetase), ACX (acyl-CoA oxidase), MFP (multifunctional protein), KAT (3-ketoacyl-CoA thiolase), and OPC8:0 (3-oxo-2(2'[*Z*]-pentenyl)-cyclopentane-1-octanoic acid)

al., 2000), six in pea (Matsui et al., 2004), three in tomato (Strassner et al., 2002), 13 in rice (Agrawal et al., 2004), and eight in maize (Zhang et al., 2005). All theses OPRs can catalyze the reduction of α,β-unsaturated carbonyls (conjugated enones) in a wide spectrum of substrates in including four stereoisomers of OPDA(Sanders et al., 2000). Earlier studies on the enzymatic activity of OPRs in *Arabidopsis* and tomato revealed that different OPR isomers have distinct substrate preferences to warrant classification into separate groups, group I and II, depending on their substrate specificity to OPDA stereoisomers (Schaller et al., 1998). OPR group I enzymes preferentially catalyze the reduction of (9*R*,13*R*)-12-oxo-

10,15(*Z*)-octadecatrienoic acid (9*R*,13*R*-OPDA), while OPR group II enzymes preferentially catalyze (9*S*,13*S*)-12-oxo-10,15(*Z*)-octadecatrienoic acid (9*S*,13*S*-OPDA), a intermediate biosynthetic precursor in JA biosynthesis (Schaller et al., 1998). OPR3 of *Arabidopsis* and tomato, belonging to group II, have been shown to efficiently reduce the natural isomer 9*S*,13*S*-OPDA to OPC 8:0, the precursor of JA (Schaller et al., 1998). In contrast, OPR group I enzymes such as OPR1/2 of *Arabidopsis* and tomato have very low affinity for 9*S*,13*S*-OPDA (Schaller et al., 1998) and unlikely to be involved in JA biosynthesis but instead function in other yet unknown biochemical processes. In addition, to uncover the molecular determinants of substrate specificity between OPR group I and II, crystal structural comparison and mutational analysis of tomato OPR1/3 in complex with OPDA enantiomers revealed that two active-site residues, i.e., Tyr78 and Tyr246 in OPR1 and Phe74 and His244 in OPR3 of tomato, are critical for substrate specificity (Breithaupt et al. 2009). Thus, the biochemical studies conclude OPR3 rather than OPR1 and OPR2, is responsible for JA production in *Arabidopsis* and tomato.

Jasmonate Biosynthesis, Perception and Function in Plant Development and Stress Responses 407

confirmed as an OPC-activating enzyme, designated OPCL1 (OPC-8:CoA ligase 1) (Koo et al., 2006). Loss-of-function mutants for *OPCL1* hyper-accumulate OPC-8:0, OPC-6:0, and OPC-4:0, suggesting a metabolic block in OPC-CoA ester formation. The mutants are also compromised in wound-induced JA accumulation. However, about 50% of wild-type levels remain in the mutants indicating that OPCL1 is responsible for only part of the woundinduced JA production, and that additional acyl-CoA synthetases may be involved in OPC-8:0-activation (Koo et al., 2006). Another two 4CL-like proteins At4g05160 and At5g63380, were selected as acyl-CoA synthetases for JA biosynthesis. The recombinant At4g05160 protein showed, *in vitro,* a distinct activity with broad substrate specificity including, medium-chain fatty acids, long-chain fatty acids, as well as, OPDA and OPC-8:0. The closest paralogue of At4g05160, At5g63380, showed high activity with long-chain fatty acids and OPDA (Schneider et al., 2005), suggesting OPDA-CoA could function as substrate for OPR3

Peroxisomal β-oxidation (Figure 6) of JA biosynthesis is catalyzed by three proteins (1) acyl-CoA oxidase (ACX), (2) the multifunctional protein (MFP) which exhibits 2-*trans*-enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, D-3-hydroxyacyl-CoA epimerase and Δ3, Δ2-enoyl-CoA isomerase activities, and (3) L-3-ketoacyl-CoA thiolase (KAT) (Schaller et al., 2004). The first *ACX* gene, named *ACX1A* was isolated from tomato. ACX1A was shown to catalyze the first step in the peroxisomal beta-oxidation stage of JA biosynthesis (Li et al., 2005). Recombinant ACX1A exhibited a preference for C12 and C14 straight-chain acyl-CoAs and also was active in the metabolism of cyclopentanoid-CoA precursors of JA (Li et al., 2005). *acx1* tomato mutant produced very little JA in wounded leaves (for 1-hour wound, 5% of wild type) and impaired in wound-induced defense gene activation and insect resistance (Li et al., 2005). *Arabidopsis* genome contains six *ACX* genes, designated *ACX1 to ACX6* (Rylott et al., 2003). *acx1* mutant of Arabidopsis produced 20% of JA production in wild type while *acx1/5* double mutant showed severe JA deficiency symptoms including impaired male fertility and susceptible to leaf-chewing insect (Schilmiller et al., 2007). For MFP roles in JA biosynthesis Arabidopsis *aim1* mutant, in which one of two MFP i.e. MFP2 is disrupted, showed impairment in wound-induced JA accumulation and defensive gene expression (Delker et al., 2007). In tobacco a similar result was obtained, that is, a stressresponsive MFP orthologous to *Arabidopsis* AIM1 are involved in β-oxidation (Ohya et al., 2008). Among five *KAT* genes in *Arabidopsis*, *KAT2* was shown to play a major role in driving wound-activated responses by participating in the biosynthesis of JA in wounded leaves (Castillo et al., 2004). The final step of JA biosynthesis is that jasmonyl-CoA releases free acid by jasmonyl-thioesterase. There is no report of cloning of jasmonyl-thioesterase in plant so far except that in *Arabidopsis* two peroxyisomal acyl-thioesterases, ACH1 and ACH2 have showed thioesterase activity of hydrolyzing both medium and long-chain fatty acyl-

The floral scent methyl jasmonate (MeJA) has been identified as a vital cellular regulator that mediates diverse developmental processes and defense responses against biotic and

to form OPC-CoA, the substrate of β-oxidation.

CoAs but not jasmonyl-CoA (Tilton et al., 2004).

**2.11. Carboxyl methyltransferase (JMT)** 

Numerous genetic studies identifed JA biosynthetic OPR enzymes across several plant species. A knockout *Arabidopsis* mutant, *dde1/opr3,* displayed male sterilility and compromised defense responses resulting from JA deficiency, indicating OPR3 is essential for the JA biosynthesis pathway and other OPR isomers such as OPR1/2 can not substitute for OPR3 in JA production (Sanders et al., 2000; Stintzi and Browse, 2000). One orthologue of OPR3, i.e., OsOPR7, was identified as a JA biosynthetic OPR in rice (Tani et al., 2008). *OPR7* and *OPR8* in maize, orthologous to *OPR3* of *Arabidopsis*, are segmentally duplicated genes, sharing 94.5% identity in amino acid sequence to each other and responsible for JA biosynthesis in maize (Yan et al., 2012). *opr7 opr8* double mutant showed a number of genetic phenotypes such as tasselseed and susceptibility to insect and pathogen, reflecting JA essential functions in monocotyledonous plants (Yan et al., 2012). Thus in plants, the physiological role of OPR group II enzymes in plants is primarily for production of the jasmonates, which mediate many development and defense-related processes (Yan et al., 2012). However, the biological significance of plants with multiple OPR group I enzymes is not clearly understood so far.

#### **2.10. β-Oxidation enzymes**

β-oxidation in lipid metabolism was believed to be located in the peroxisomes of all higher plants (Masterson and Wood, 2001) but also detected in mitochondria in a non-oilseed plant (Masterson and Wood, 2001). The terminal steps of peroxisomal JA biosynthesis are three βoxidation reactions, which shorten the carboxyl side chain from the intermediates OPC-8:0 or OPC-6:0 produced from OPDA or dn-OPDA (Vick and Zimmernan, 1983). Prior to entry into the β-oxidation reactions, the carboxylic group of OPC-8:0 or OPC-6:0 must activate as a CoA ester. *Arabidopsis* possesses an acyl-activating superfamily containing 63 different genes, whose proteins are potential acyl-activating enzymes (AAEs) (Shockey et al., 2003). Within this superfamily, a subgroup, called the 4-coumarate:CoA ligase (4CL)-like family, contains 13 members shown to possess peroxisomal acyl-activating activity involved in the biosynthesis of jasmonic acid (Koo et al., 2006). One of these 13 genes, At1g20510 was confirmed as an OPC-activating enzyme, designated OPCL1 (OPC-8:CoA ligase 1) (Koo et al., 2006). Loss-of-function mutants for *OPCL1* hyper-accumulate OPC-8:0, OPC-6:0, and OPC-4:0, suggesting a metabolic block in OPC-CoA ester formation. The mutants are also compromised in wound-induced JA accumulation. However, about 50% of wild-type levels remain in the mutants indicating that OPCL1 is responsible for only part of the woundinduced JA production, and that additional acyl-CoA synthetases may be involved in OPC-8:0-activation (Koo et al., 2006). Another two 4CL-like proteins At4g05160 and At5g63380, were selected as acyl-CoA synthetases for JA biosynthesis. The recombinant At4g05160 protein showed, *in vitro,* a distinct activity with broad substrate specificity including, medium-chain fatty acids, long-chain fatty acids, as well as, OPDA and OPC-8:0. The closest paralogue of At4g05160, At5g63380, showed high activity with long-chain fatty acids and OPDA (Schneider et al., 2005), suggesting OPDA-CoA could function as substrate for OPR3 to form OPC-CoA, the substrate of β-oxidation.

Peroxisomal β-oxidation (Figure 6) of JA biosynthesis is catalyzed by three proteins (1) acyl-CoA oxidase (ACX), (2) the multifunctional protein (MFP) which exhibits 2-*trans*-enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, D-3-hydroxyacyl-CoA epimerase and Δ3, Δ2-enoyl-CoA isomerase activities, and (3) L-3-ketoacyl-CoA thiolase (KAT) (Schaller et al., 2004). The first *ACX* gene, named *ACX1A* was isolated from tomato. ACX1A was shown to catalyze the first step in the peroxisomal beta-oxidation stage of JA biosynthesis (Li et al., 2005). Recombinant ACX1A exhibited a preference for C12 and C14 straight-chain acyl-CoAs and also was active in the metabolism of cyclopentanoid-CoA precursors of JA (Li et al., 2005). *acx1* tomato mutant produced very little JA in wounded leaves (for 1-hour wound, 5% of wild type) and impaired in wound-induced defense gene activation and insect resistance (Li et al., 2005). *Arabidopsis* genome contains six *ACX* genes, designated *ACX1 to ACX6* (Rylott et al., 2003). *acx1* mutant of Arabidopsis produced 20% of JA production in wild type while *acx1/5* double mutant showed severe JA deficiency symptoms including impaired male fertility and susceptible to leaf-chewing insect (Schilmiller et al., 2007). For MFP roles in JA biosynthesis Arabidopsis *aim1* mutant, in which one of two MFP i.e. MFP2 is disrupted, showed impairment in wound-induced JA accumulation and defensive gene expression (Delker et al., 2007). In tobacco a similar result was obtained, that is, a stressresponsive MFP orthologous to *Arabidopsis* AIM1 are involved in β-oxidation (Ohya et al., 2008). Among five *KAT* genes in *Arabidopsis*, *KAT2* was shown to play a major role in driving wound-activated responses by participating in the biosynthesis of JA in wounded leaves (Castillo et al., 2004). The final step of JA biosynthesis is that jasmonyl-CoA releases free acid by jasmonyl-thioesterase. There is no report of cloning of jasmonyl-thioesterase in plant so far except that in *Arabidopsis* two peroxyisomal acyl-thioesterases, ACH1 and ACH2 have showed thioesterase activity of hydrolyzing both medium and long-chain fatty acyl-CoAs but not jasmonyl-CoA (Tilton et al., 2004).

#### **2.11. Carboxyl methyltransferase (JMT)**

406 Lipid Metabolism

production in *Arabidopsis* and tomato.

not clearly understood so far.

**2.10. β-Oxidation enzymes** 

10,15(*Z*)-octadecatrienoic acid (9*R*,13*R*-OPDA), while OPR group II enzymes preferentially catalyze (9*S*,13*S*)-12-oxo-10,15(*Z*)-octadecatrienoic acid (9*S*,13*S*-OPDA), a intermediate biosynthetic precursor in JA biosynthesis (Schaller et al., 1998). OPR3 of *Arabidopsis* and tomato, belonging to group II, have been shown to efficiently reduce the natural isomer 9*S*,13*S*-OPDA to OPC 8:0, the precursor of JA (Schaller et al., 1998). In contrast, OPR group I enzymes such as OPR1/2 of *Arabidopsis* and tomato have very low affinity for 9*S*,13*S*-OPDA (Schaller et al., 1998) and unlikely to be involved in JA biosynthesis but instead function in other yet unknown biochemical processes. In addition, to uncover the molecular determinants of substrate specificity between OPR group I and II, crystal structural comparison and mutational analysis of tomato OPR1/3 in complex with OPDA enantiomers revealed that two active-site residues, i.e., Tyr78 and Tyr246 in OPR1 and Phe74 and His244 in OPR3 of tomato, are critical for substrate specificity (Breithaupt et al. 2009). Thus, the biochemical studies conclude OPR3 rather than OPR1 and OPR2, is responsible for JA

Numerous genetic studies identifed JA biosynthetic OPR enzymes across several plant species. A knockout *Arabidopsis* mutant, *dde1/opr3,* displayed male sterilility and compromised defense responses resulting from JA deficiency, indicating OPR3 is essential for the JA biosynthesis pathway and other OPR isomers such as OPR1/2 can not substitute for OPR3 in JA production (Sanders et al., 2000; Stintzi and Browse, 2000). One orthologue of OPR3, i.e., OsOPR7, was identified as a JA biosynthetic OPR in rice (Tani et al., 2008). *OPR7* and *OPR8* in maize, orthologous to *OPR3* of *Arabidopsis*, are segmentally duplicated genes, sharing 94.5% identity in amino acid sequence to each other and responsible for JA biosynthesis in maize (Yan et al., 2012). *opr7 opr8* double mutant showed a number of genetic phenotypes such as tasselseed and susceptibility to insect and pathogen, reflecting JA essential functions in monocotyledonous plants (Yan et al., 2012). Thus in plants, the physiological role of OPR group II enzymes in plants is primarily for production of the jasmonates, which mediate many development and defense-related processes (Yan et al., 2012). However, the biological significance of plants with multiple OPR group I enzymes is

β-oxidation in lipid metabolism was believed to be located in the peroxisomes of all higher plants (Masterson and Wood, 2001) but also detected in mitochondria in a non-oilseed plant (Masterson and Wood, 2001). The terminal steps of peroxisomal JA biosynthesis are three βoxidation reactions, which shorten the carboxyl side chain from the intermediates OPC-8:0 or OPC-6:0 produced from OPDA or dn-OPDA (Vick and Zimmernan, 1983). Prior to entry into the β-oxidation reactions, the carboxylic group of OPC-8:0 or OPC-6:0 must activate as a CoA ester. *Arabidopsis* possesses an acyl-activating superfamily containing 63 different genes, whose proteins are potential acyl-activating enzymes (AAEs) (Shockey et al., 2003). Within this superfamily, a subgroup, called the 4-coumarate:CoA ligase (4CL)-like family, contains 13 members shown to possess peroxisomal acyl-activating activity involved in the biosynthesis of jasmonic acid (Koo et al., 2006). One of these 13 genes, At1g20510 was

The floral scent methyl jasmonate (MeJA) has been identified as a vital cellular regulator that mediates diverse developmental processes and defense responses against biotic and abiotic stresses (Cheong and Choi, 2003). The enzyme converting JA to methyl jasmonates (MeJA) is JA carboxyl methyltransferase (JMT), which was first cloned and characterized from *Arabidopsis* (Seo et al., 2001). As JMT does not carry any transit signal peptides, it is presumably a cytoplasmic enzyme (Seo et al., 2001). JMT is constitutively expressed in almost all the organs of mature plants, but not in young seedling (Seo et al., 2001), indicating young plant avoids to produce MeJA. However, JMT can be induced by both wounding and MeJA treatment (Seo et al., 2001). Transgenic *Arabidopsis* overexpressing *JMT* exhibited constitutive expression of jasmonate-responsive genes, including *VSP* and *PDF1.2*, and enhanced level of resistance against the virulent fungus *Botrytis cinerea* (Seo et al., 2001), indicating JMT is a key enzyme for airborne-jasmonate-regulated plant responses.

Jasmonate Biosynthesis, Perception and Function in Plant Development and Stress Responses 409

enzymes in JA biosynthesis pathway. Many studies show that most enzyme genes for JA biosynthesis such as *LOX*, *AOS*, *AOC*, *OPR3*, *JMT* and *JRA1* are induced by JA treatment (Wasternack, 2007) or wounding (Schaller, 2001), implying that JA biosynthesis is a JAdependent process in plants. Monitoring of MeJA-responsive genes in *Arabidopsis* by cDNA microarrays also concluded that JA biosynthesis is regulated by a positive feedback loop (Sasaki et al., 2001). Further evidence for this conclusion has come from mutants with constitutively up-regulated JA levels such as *cev1* and *fou2.* These mutants showed typical phenotypes associated with exogenous JA treatment such as roots/shoots growth inhibition, anthocyanin accumulation in the leaves, and over-expression of JA-dependent genes (Ellis and Turner, 2001; Bonaventure et al., 2007). However, the expression levels of JA biosynthetic enzymes do not determine the actual output of JA biosynthesis in limited substrate conditions. The second strategy for JA biosynthesis regulation is controlling the substrate availability. For example, the fully expanded leaves of *Arabidopsis* carry LOX, AOS, and AOC proteins abundantly; however, JA formation is at a substantially low level. JA production rapidly occurs only upon strong external stimuli such as wounding, which largely induces JA biosynthesis enzymes and releases the substrate LA from the membranes (Stenzel et al., 2003). Furthermore, wound induction of JA is transient and appears before the expression induction of *LOX*, *AOS,* and *AOC* genes (Howe et al., 2000). These data clearly show that JA biosynthesis is regulated by enzyme activities and substrate availability. The third strategy of JA biosynthesis regulation is to store or reuse intermediates or conjugates of jasmonate in case of over-produced or quick release release is required for healing in situation of insect or pathogen attacks. Recently, esterified OPDA was found in galactolipids (monogalactosyldiacylglycerol, MGDG and digalactosyldiacylglycerol, DGDG) (Stelmach et al., 2001). A novel oxylipin category, socalled arabidopsides A, B, C, D, E, F, G and F (Figure 4) were found containing OPDA and/or dino-OPDA (Hisamatsu et al., 2003; Hisamatsu, 2005). These compounds could accumulate to 7-8% of total lipids in plants if challenged by pathogens (Anderson et al., 2006). In addition, JA derivatives including jasmonyl-amino acids such as JA-Ile (jasmonoylisoleucine), JA-Leu (jasmonoyl-leucine), and JA-Val (jasmonoyl-valine) and jasmonyl-ACC (1-amino- cyclopropane-1-carboxylic acid) conjugates are present and all, except JA-Ile, are

considered storage forms of jasmonates in plants (Staswick et al., 2002).

The initial product (+)-7-*iso*-JA, synthesized in peroxisomes epimerizes simultaneously to a more thermo-stable *trans* configuration, (-)-JA, which is generally known as jasmonic acid (JA) (Wasternack, 2007; Creelman and Mullet, 1997). Both (-)-JA and (+)-7-iso-JA are bioactive but the former is more active (Wasternack, 2007). JA can convert into a number of derivatives and conjugates (Figure 5). The JA precursor OPDA, the free acid and methyl ester of JA, i.e., JA and MeJA, and conjugates JA-Ile and JA-trp (jasmonyl-L-tryptophan) are assumed the most active JA forms in plants (Fonseca ea al., 2009). However, in COI1-JAZ (Coronatine Insensitive 1 and Jasmonate ZIM-domain-containing protein, respectively)

**3. JA perception and signaling pathway** 

**3.1. Bioactive JA forms and JA signaling ligand** 

#### **2.12. JA-amino acid synthetase (JAR1)**

Since jasmonoyl-L-isoleucine (JA-Ile) is the only one bioactive ligand, known so far, involving in JA signaling, JAR1 (JASMONATE RESISTANT 1), a JA amino acid synthetase that conjugates isoleucine to JA (Staswick and Tiryaki, 2004), was assumed a most important JA derivation enzyme in plants. Recent studies indicate JA-Ile promotes binding of the JAZ proteins to SCFCOI1 complexes and results in subsequent degradation of JAZ by the ubiquitination/26S-proteasomes (Thines et al., 2007). *JAR1* is one of 19 closely related *Arabidopsis* genes that are similar to the auxin-induced soybean *GH3* gene family (Staswick et al., 2002). Analysis of fold-predictions for this protein family suggested that JAR1 might belong to the acyl adenylate-forming firefly luciferase superfamily. These enzymes activate the carboxyl groups of a variety of substrates including JA, indole-3-acetic acid (IAA) and salicylic acid (SA) for their subsequent biochemical modification (Staswick et al., 2002), thereby regulating hormone activity. The first *jar1* mutant was identified that affected signaling in the jasmonate pathway. *jar1* plants have reduced sensitivity to root growth inhibition in the presence of exogenous JA ( Staswick *et al*., 2002). *jar1* was shown to be susceptible to soil oomycete (Staswick et al., 1998) and necrotrophic pathogens (Antico et al., 2012). In wounding *JAR1* transcript was found increased dramatically in wounded tissue and JA–Ile accumulated mostly near the wound site with a minor increase in unwounded tissue (Suza and Staswick, 2008). However, the reduced accumulation of JA–Ile had little or no effect on several jasmonate-dependent wound-induced genes such as *VSP2,* for *LOX2*, *PDF1.2*, *WRKY33*, *TAT3* and *CORI3*. Morphologically, *jar1* mutation is male fertile while JA biosynthesis and signaling mutants are male sterile (Suza and Staswick, 2008).

#### **2.13. JA Biosynthesis Regulation**

The levels of jasmonic acid in plants vary with developmental stage, organs, and are variable in response to different environmental stimuli (Creelman and Mullet, 1995). High levels of jasmonates are found in flowers, pericarp tissues of developing fruit, and in the chloroplasts of illuminated plants; Jasmonate levels increase rapidly in response to mechanical perturbations such as tendril coiling and when plants suffer wounding (Creelman and Mullet, 1995). There are several strategies applicable for plants to regulate generation or activities of jasmontes. The first strategy is to regulate the expression of the enzymes in JA biosynthesis pathway. Many studies show that most enzyme genes for JA biosynthesis such as *LOX*, *AOS*, *AOC*, *OPR3*, *JMT* and *JRA1* are induced by JA treatment (Wasternack, 2007) or wounding (Schaller, 2001), implying that JA biosynthesis is a JAdependent process in plants. Monitoring of MeJA-responsive genes in *Arabidopsis* by cDNA microarrays also concluded that JA biosynthesis is regulated by a positive feedback loop (Sasaki et al., 2001). Further evidence for this conclusion has come from mutants with constitutively up-regulated JA levels such as *cev1* and *fou2.* These mutants showed typical phenotypes associated with exogenous JA treatment such as roots/shoots growth inhibition, anthocyanin accumulation in the leaves, and over-expression of JA-dependent genes (Ellis and Turner, 2001; Bonaventure et al., 2007). However, the expression levels of JA biosynthetic enzymes do not determine the actual output of JA biosynthesis in limited substrate conditions. The second strategy for JA biosynthesis regulation is controlling the substrate availability. For example, the fully expanded leaves of *Arabidopsis* carry LOX, AOS, and AOC proteins abundantly; however, JA formation is at a substantially low level. JA production rapidly occurs only upon strong external stimuli such as wounding, which largely induces JA biosynthesis enzymes and releases the substrate LA from the membranes (Stenzel et al., 2003). Furthermore, wound induction of JA is transient and appears before the expression induction of *LOX*, *AOS,* and *AOC* genes (Howe et al., 2000). These data clearly show that JA biosynthesis is regulated by enzyme activities and substrate availability. The third strategy of JA biosynthesis regulation is to store or reuse intermediates or conjugates of jasmonate in case of over-produced or quick release release is required for healing in situation of insect or pathogen attacks. Recently, esterified OPDA was found in galactolipids (monogalactosyldiacylglycerol, MGDG and digalactosyldiacylglycerol, DGDG) (Stelmach et al., 2001). A novel oxylipin category, socalled arabidopsides A, B, C, D, E, F, G and F (Figure 4) were found containing OPDA and/or dino-OPDA (Hisamatsu et al., 2003; Hisamatsu, 2005). These compounds could accumulate to 7-8% of total lipids in plants if challenged by pathogens (Anderson et al., 2006). In addition, JA derivatives including jasmonyl-amino acids such as JA-Ile (jasmonoylisoleucine), JA-Leu (jasmonoyl-leucine), and JA-Val (jasmonoyl-valine) and jasmonyl-ACC (1-amino- cyclopropane-1-carboxylic acid) conjugates are present and all, except JA-Ile, are considered storage forms of jasmonates in plants (Staswick et al., 2002).

#### **3. JA perception and signaling pathway**

408 Lipid Metabolism

abiotic stresses (Cheong and Choi, 2003). The enzyme converting JA to methyl jasmonates (MeJA) is JA carboxyl methyltransferase (JMT), which was first cloned and characterized from *Arabidopsis* (Seo et al., 2001). As JMT does not carry any transit signal peptides, it is presumably a cytoplasmic enzyme (Seo et al., 2001). JMT is constitutively expressed in almost all the organs of mature plants, but not in young seedling (Seo et al., 2001), indicating young plant avoids to produce MeJA. However, JMT can be induced by both wounding and MeJA treatment (Seo et al., 2001). Transgenic *Arabidopsis* overexpressing *JMT* exhibited constitutive expression of jasmonate-responsive genes, including *VSP* and *PDF1.2*, and enhanced level of resistance against the virulent fungus *Botrytis cinerea* (Seo et al., 2001),

indicating JMT is a key enzyme for airborne-jasmonate-regulated plant responses.

biosynthesis and signaling mutants are male sterile (Suza and Staswick, 2008).

The levels of jasmonic acid in plants vary with developmental stage, organs, and are variable in response to different environmental stimuli (Creelman and Mullet, 1995). High levels of jasmonates are found in flowers, pericarp tissues of developing fruit, and in the chloroplasts of illuminated plants; Jasmonate levels increase rapidly in response to mechanical perturbations such as tendril coiling and when plants suffer wounding (Creelman and Mullet, 1995). There are several strategies applicable for plants to regulate generation or activities of jasmontes. The first strategy is to regulate the expression of the

Since jasmonoyl-L-isoleucine (JA-Ile) is the only one bioactive ligand, known so far, involving in JA signaling, JAR1 (JASMONATE RESISTANT 1), a JA amino acid synthetase that conjugates isoleucine to JA (Staswick and Tiryaki, 2004), was assumed a most important JA derivation enzyme in plants. Recent studies indicate JA-Ile promotes binding of the JAZ proteins to SCFCOI1 complexes and results in subsequent degradation of JAZ by the ubiquitination/26S-proteasomes (Thines et al., 2007). *JAR1* is one of 19 closely related *Arabidopsis* genes that are similar to the auxin-induced soybean *GH3* gene family (Staswick et al., 2002). Analysis of fold-predictions for this protein family suggested that JAR1 might belong to the acyl adenylate-forming firefly luciferase superfamily. These enzymes activate the carboxyl groups of a variety of substrates including JA, indole-3-acetic acid (IAA) and salicylic acid (SA) for their subsequent biochemical modification (Staswick et al., 2002), thereby regulating hormone activity. The first *jar1* mutant was identified that affected signaling in the jasmonate pathway. *jar1* plants have reduced sensitivity to root growth inhibition in the presence of exogenous JA ( Staswick *et al*., 2002). *jar1* was shown to be susceptible to soil oomycete (Staswick et al., 1998) and necrotrophic pathogens (Antico et al., 2012). In wounding *JAR1* transcript was found increased dramatically in wounded tissue and JA–Ile accumulated mostly near the wound site with a minor increase in unwounded tissue (Suza and Staswick, 2008). However, the reduced accumulation of JA–Ile had little or no effect on several jasmonate-dependent wound-induced genes such as *VSP2,* for *LOX2*, *PDF1.2*, *WRKY33*, *TAT3* and *CORI3*. Morphologically, *jar1* mutation is male fertile while JA

**2.12. JA-amino acid synthetase (JAR1)** 

**2.13. JA Biosynthesis Regulation** 

#### **3.1. Bioactive JA forms and JA signaling ligand**

The initial product (+)-7-*iso*-JA, synthesized in peroxisomes epimerizes simultaneously to a more thermo-stable *trans* configuration, (-)-JA, which is generally known as jasmonic acid (JA) (Wasternack, 2007; Creelman and Mullet, 1997). Both (-)-JA and (+)-7-iso-JA are bioactive but the former is more active (Wasternack, 2007). JA can convert into a number of derivatives and conjugates (Figure 5). The JA precursor OPDA, the free acid and methyl ester of JA, i.e., JA and MeJA, and conjugates JA-Ile and JA-trp (jasmonyl-L-tryptophan) are assumed the most active JA forms in plants (Fonseca ea al., 2009). However, in COI1-JAZ (Coronatine Insensitive 1 and Jasmonate ZIM-domain-containing protein, respectively) binding experiments JA-Ile rather than OPDA, JA, and MeJA can promote COI1-JAZ binding, indicating only JA-Ile is the direct JA signaling ligand in plants (Thines et al, 2007). The trans configuration, (-)-JA-L-isoleucine was demonstrated to be the active molecule form of JA for COI1-JAZ binding (Thines et al, 2007). However, the recent study showed that (+)-7-iso-JA-L-Ile, which is also structurally more similar to coronatine, is highly active. The previously proposed active form (-)-JA-L-Ile, which contains a small amount of the C7 epimer (+)-7-iso-JA-L-Ile, if purified, is inactive (Fonseca ea al., 2009). In summary, currently (+)-7-iso-JA-L-Ile is the only proven ligand for JA signaling in plants.

Jasmonate Biosynthesis, Perception and Function in Plant Development and Stress Responses 411

and JA-Ile were tested for affinity in COI1-JAZ1 binding. Surprisingly, only JA-Ile functioned as ligand for COI1-JAZ interaction (Thines et al., 2007). JA-Ile is a conjugate product of JA with isoluecine by JAR1, a JA-amino acid conjugation enzyme similar to auxin-responsive GH3 family proteins of soybean (Staswick and Tiryaki, 2004). This provided biochemical explination as to why *jar1* mutant showed the phenotype 'JA-RESISTANT' and demonstrating JAR1 as a provider of a JA signal rather than a component of JA perception machinery in plant. More recently, inositol pentakisphosphate (IP5) was found as an existing cofactor of COI1 crystal structure and COI1 protein lacking IP5 lost ligand-binding acivity (Sheard et al., 2010). Based on the information available so far, the true jasmonates receptor is a co-repressor complex, consisting of the SCFCOI1 E3 ubiquitin ligase complex, JAZ degrons (JAZ1 to JAZ12), and a newly discovered third component,

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

*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.

inositol pentakisphosphate (IP5) (Sheard et al., 2010).

**Proteins** 

#### **3.2. Ubiquitination-based JA receptor: COI1-JAZ complex**

Critical to comprehending hormonal control of development and defense events is the understanding of hormone perception. In the past decades, several mechanisms of plant hormone perception have been elucidated (Spartz and Gray, 2008; Chow and MeCourt, 2006). Cytokinins (CK) and ethylene were found to be perceived by two-component-based hormone receptors while brassinosteroids (BR) by leucine-rich repeat (LRR)-based hormone receptors (Chow and MeCourt, 2006) and ABA (abscisic acid) by nuclear RCAR/PYR1/PYL– PP2C complexes (Raghavendra et al, 2010). More recently, auxin, JA, GA (gibberellic acid), and SA (salicylic acid) are found to be perceived by nuclear SCFTIR1, SCFCOI1, SCFDELLA, and SCFNPR complexes respectively (Chow and MeCourt, 2006; Lumba et al., 2010; Fu et al. 2012).

Early researchers believed that screening for Arabidopsis mutants insensitive to growth inhibition by bacterial coronatine, which is structurally analogous to JA and MeJA, would result in discovering JA receptor protein(s) in plants. Exhaustive screens identified only the alleles of *coronatine insensitive 1 (coi1)* and *jasmonates resistant 1 (jar1),* suggesting COI1 and JAR1 function in JA perception in plant. However, cloning of COI1 and JAR1 showed that COI1 encodes an F-box protein (Xie et al., 1998) and JAR1 an auxin-induced GH3 protein (Staswick and Tiryaki, 2004), and neither protein shows homology to known plant receptor proteins. The investigators reasoned that COI1, rather than JAR1, is a potential JA-receptor or a component of a receptor complex from two lines of evidence. First, *coi1* mutant displays severe JA signal-phenotypes such as male sterility, defective responses to JA-treatment and wounding, and high susceptiblity to insect and necrotrophic pathogens whereas *jar1* is fertile and only partially defective to JA-treatment and wounding. Secondly, *COI1* locus encodes an F-box protein which is known to associate with SKP1, Cullin, and Rbx proteins to form an E3 ubiquitin ligase, known as the SCF complex. Several SCF complexes in plant have been implicated in a number of important processes, for example, SCFTIR1 complex is an auxin receptor, implying SCFCOI1 may function as analog of SCFTIR1 for JA signaling. The components of SCFCOI1 complex were demonstrated to exist in Arabidopsis and mutations in the components of SCF resulted in reduced JA-dependent responses (Xu et al., 2002). Now the question becomes: what is the substrate(s) of SCFCOI1 E3 ubiquitin ligase complex? This substrate was anticipated to function as a key negative regulator in JA signaling (Turner et al., 2002; Browse, 2005). Later, three laboratories simultaneously found the substrates of SCFCOI1 complex, which were called JAZ proteins consisting of 12 members in Arabidopsis (Chini et al., 2007; Thines et al., 2007; Yan et al., 2007). Bioactive JA forms JA, OPDA, MeJA, and JA-Ile were tested for affinity in COI1-JAZ1 binding. Surprisingly, only JA-Ile functioned as ligand for COI1-JAZ interaction (Thines et al., 2007). JA-Ile is a conjugate product of JA with isoluecine by JAR1, a JA-amino acid conjugation enzyme similar to auxin-responsive GH3 family proteins of soybean (Staswick and Tiryaki, 2004). This provided biochemical explination as to why *jar1* mutant showed the phenotype 'JA-RESISTANT' and demonstrating JAR1 as a provider of a JA signal rather than a component of JA perception machinery in plant. More recently, inositol pentakisphosphate (IP5) was found as an existing cofactor of COI1 crystal structure and COI1 protein lacking IP5 lost ligand-binding acivity (Sheard et al., 2010). Based on the information available so far, the true jasmonates receptor is a co-repressor complex, consisting of the SCFCOI1 E3 ubiquitin ligase complex, JAZ degrons (JAZ1 to JAZ12), and a newly discovered third component, inositol pentakisphosphate (IP5) (Sheard et al., 2010).

410 Lipid Metabolism

binding experiments JA-Ile rather than OPDA, JA, and MeJA can promote COI1-JAZ binding, indicating only JA-Ile is the direct JA signaling ligand in plants (Thines et al, 2007). The trans configuration, (-)-JA-L-isoleucine was demonstrated to be the active molecule form of JA for COI1-JAZ binding (Thines et al, 2007). However, the recent study showed that (+)-7-iso-JA-L-Ile, which is also structurally more similar to coronatine, is highly active. The previously proposed active form (-)-JA-L-Ile, which contains a small amount of the C7 epimer (+)-7-iso-JA-L-Ile, if purified, is inactive (Fonseca ea al., 2009). In summary, currently

Critical to comprehending hormonal control of development and defense events is the understanding of hormone perception. In the past decades, several mechanisms of plant hormone perception have been elucidated (Spartz and Gray, 2008; Chow and MeCourt, 2006). Cytokinins (CK) and ethylene were found to be perceived by two-component-based hormone receptors while brassinosteroids (BR) by leucine-rich repeat (LRR)-based hormone receptors (Chow and MeCourt, 2006) and ABA (abscisic acid) by nuclear RCAR/PYR1/PYL– PP2C complexes (Raghavendra et al, 2010). More recently, auxin, JA, GA (gibberellic acid), and SA (salicylic acid) are found to be perceived by nuclear SCFTIR1, SCFCOI1, SCFDELLA, and SCFNPR complexes respectively (Chow and MeCourt, 2006; Lumba et al., 2010; Fu et al. 2012). Early researchers believed that screening for Arabidopsis mutants insensitive to growth inhibition by bacterial coronatine, which is structurally analogous to JA and MeJA, would result in discovering JA receptor protein(s) in plants. Exhaustive screens identified only the alleles of *coronatine insensitive 1 (coi1)* and *jasmonates resistant 1 (jar1),* suggesting COI1 and JAR1 function in JA perception in plant. However, cloning of COI1 and JAR1 showed that COI1 encodes an F-box protein (Xie et al., 1998) and JAR1 an auxin-induced GH3 protein (Staswick and Tiryaki, 2004), and neither protein shows homology to known plant receptor proteins. The investigators reasoned that COI1, rather than JAR1, is a potential JA-receptor or a component of a receptor complex from two lines of evidence. First, *coi1* mutant displays severe JA signal-phenotypes such as male sterility, defective responses to JA-treatment and wounding, and high susceptiblity to insect and necrotrophic pathogens whereas *jar1* is fertile and only partially defective to JA-treatment and wounding. Secondly, *COI1* locus encodes an F-box protein which is known to associate with SKP1, Cullin, and Rbx proteins to form an E3 ubiquitin ligase, known as the SCF complex. Several SCF complexes in plant have been implicated in a number of important processes, for example, SCFTIR1 complex is an auxin receptor, implying SCFCOI1 may function as analog of SCFTIR1 for JA signaling. The components of SCFCOI1 complex were demonstrated to exist in Arabidopsis and mutations in the components of SCF resulted in reduced JA-dependent responses (Xu et al., 2002). Now the question becomes: what is the substrate(s) of SCFCOI1 E3 ubiquitin ligase complex? This substrate was anticipated to function as a key negative regulator in JA signaling (Turner et al., 2002; Browse, 2005). Later, three laboratories simultaneously found the substrates of SCFCOI1 complex, which were called JAZ proteins consisting of 12 members in Arabidopsis (Chini et al., 2007; Thines et al., 2007; Yan et al., 2007). Bioactive JA forms JA, OPDA, MeJA,

(+)-7-iso-JA-L-Ile is the only proven ligand for JA signaling in plants.

**3.2. Ubiquitination-based JA receptor: COI1-JAZ complex** 
