**2. JA biosynthesis pathway and regulation**

#### **2.1. The scheme of JA biosynthesis pathway**

394 Lipid Metabolism

responses (Eckardt, 2008).

complex (Thines et al., 2007).

Yan et al, 2012).

enzymatically formed oxylipins, which play overlapped roles with OPDA in plant stress

JA biosynthesis and signaling pathways have been extensively investigated in dicotyledonous plants such as *Arabidopsis*, tobacco and tomato. In monocotyledonous species, only a scant number of JA biosynthetic enzymes have been described (Tani et al., 2008; Yan et al., 2012). Jasmonates are formed from the LOX-catalyzed peroxidation of trienoic fatty acids at carbon atom 13 to form 13-hydroperoxide, which is modified to an allene oxide fatty acid and subsequently cyclized to the compound 12-oxo-phytodienoic acid (OPDA). Jasmonic acid (JA) is synthesized from OPDA by the reduction of a double bond and three consecutive rounds of β-oxidation. The pathway can accept C18-PUFA (linolenic acid) as well as C16-PUFA (hexadecatrienoic acid), in the latter case the intermediate is the so-called dinor-OPDA that may also be metabolized to JA. JA can be further enzymatically converted into numerous derivatives or conjugates, some of which have well-described

JA signaling pathway, the transition process of JA-Ile as a chemical signal to biological signal, was elucidated in recent years. JA initiates signaling process upon formation of a SCFCOI1-JA-Ile-JAZ ternary complex (JAZ: jasmonate ZIM-domain protein; Sheard et al., 2010), in which the JAZ repressors are ubiquitinated and subsequently degraded to release transcription factors, e.g., MYC2, causing downstream transcription activation of defense responses or developmental regulation (Chini et al., 2007; Thines et al., 2007). The only jasmonate receptor identified to date has been the COI1 protein (Katsir et al., 2008; Yan et al., 2009), but interestingly, only JA-Ile was found as a ligand of the SCFCOI1 E3 ubiquitin ligase

Since discovered in the 1960s as secondary metabolites from the oils of jasmine flowers (Demole *et al.*, 1962), the biological roles of JA have received increased attention of researchers in the past decades. Jasmonates have gradually become realized as a defense and fertility hormone, and as such modulate numerable processes relating to development and stress responses. In *Arabidopsis* and tomato, JAs are directly involved in stamen and trichome development, vegetative growth, cell cycle regulation, senescence, anthocyanin biosynthesis regulation, and responses to various biotic and abiotic stresses (Creelman and Mullet, 1997; Wasternack, 2007; Howe and Jander, 2008; Browse, 2009; Avanci et al., 2010; Pauwels and Goossens, 2011). In monocots, much less is known about the role of JAs, however, it has been shown they are required for sex determination, reproductive bud initiation and elongation, leaf senescence, pigmentation of tissues and responses to the attack by pathogens and insects (Engelberth et al., 2004; Tani et al., 2008; Acosta et al., 2009;

In plants, the JA signal acts co-operatively with other plant hormones. A number of studies have already attracted attention to plant hormone cross-talk as it relates to defense responses. In *Arabidopsis,* JA was shown to interact synergistically with ethylene (Xu et al 1994), and, depending on particular stress, both synergistically and antagonistically with salicylic acid (Beckers and Spoel, 2006) and abscisic acid (ABA) (Anderson et al 2004) in

biological activity such as free JA, MeJA, *cis*-jasmone and JA–Ile.

In 1962, a floral scent compound, the methyl ester of jasmonic acid (MeJA) was isolated for the first time from the aromatic oil of *Jasminum grandiflorum* (Demole et al., 1962). However, the physiological effects of MeJA or its free acid (JA) were unknown until the 1980's when a senescence-promoting effect of JA (Ueda and Kato, 1980) and growth inhibition activity of MeJA to *Vicia faba* (Dathe, 1981) were observed. Now JA and derivatives (JAs) are the best characterized group of oxylipins in plants and are regarded as one of the the major hormones regulating both defense and development.

Biosynthesis of JAs originates from polyunsaturated fatty acids (PUFA) and is synthesized by one of the seven distinct branches of the lipoxygenase (LOX) pathway, the allene oxide synthase (AOS) branch (Feussner and Wasternack, 2002). The remaing six branches form other oxylipins including GLVs as well as epoxy-, hydroxy-, keto- or ether PUFA and epoxyhydroxy-PUFA (Feussner and Wasternack, 2002) (Figure 1). In the oxylipin biosynthesis (Figure 1), only 13-hydroperoxide from α-linolenic acid (18:3, α-LeA) can be utilized by the AOS branch for JA production. Other fatty acid hydroperoxides such as 9- 13- and 2-hydroperoxide, oxygenated by 9-LOX, 13-LOX and α-dioxygenase (α-DOX), respectively, or those whose substrates originate from α-LeA, hexadecatrienoic acid (16:3, HTA) or linoleic acid (18:2, LA) may be channeled to form other oxylipin subgroups. Biological functions of the majority of estimated 400-500 oxylipins is mostly unknown.

The biosynthesis of JA and MeJA was elucidated by Vick and Zimmerman (1983), and Hamberg and Hughes (1988). The original precursors PUFA are released from chloroplast membranes by the action of lipid hydrolyzing enzymes. Upon α-LeA liberation, a molecular oxygen is incorporated by a 13-LOX at carbon atom 13 of the substrate leading to the formation of a fatty acid hydroperoxide, 13-HPOT (13*S*-hydroperoxy-(*9Z*,11*E*,15) octadecatrienoic acid) (Figure 2). This intermediate compound can proceede to seven distinct enzymatic branches (Figure 1), one of which is dehydration by the allene oxide synthase (AOS) to an unstable allene oxide, 12,13-EOT ((9*Z*,13*S*,15*Z*)-12,13-oxido-9,11,15 octadecatrienoic acid) which can be cyclized to racemic 12-oxo-phytodienoic acid (OPDA). In the presence of an allene oxide cyclase (AOC), preferential product is the enantiomer, 9*S*,13*S/cis* (+)-OPDA (Figure 2). All the reactions from α-LeA to OPDA take place within a plastid. *cis* (+)-OPDA is subsequently transported into the peroxisome, where it is further converted into (+)-7-*iso*-JA by 12-oxo-phytodienoic acid reductase (OPR) and three beta oxidation steps involving three peroxisomal enzymatic functions (acyl-CoA oxidase, multifunctional protein, and l-3-ketoacyl-CoA thiolase) (Figure 2). (+)-7-*iso*-JA often epimerizes

#### 396 Lipid Metabolism

into a more stable *trans* configuration, (-)-JA or undergoes modifications to produce diverse JA derivatives including MeJA and (+)-7-*iso*-JA*-*Ile. The latter one is the bioactive form of JA produced by conjugation of JA to isoleucine by the enzyme encoded by the *JA resistant 1* (*JAR1*) gene (Staswick and Tiryaki, 2004).

Jasmonate Biosynthesis, Perception and Function in Plant Development and Stress Responses 397

**Figure 2.** Scheme of JA biosynthesis pathway in *A. thaliana* (Delker et al., 2006)

**2.2. The alternative routes of JA biosynthesis** 

considered as the alternative routes of JA biosynthesis.

jasmonoyl-1-amino-1-cyclopropane carboxylic acid, and MeJA for methyl jasmonates.

The enzymes and the intermediates are indicated as LOX2 for lipoxygenase 2, AOS for allene oxide synthase, AOC for allene oxide cyclase, and OPR3 for 12-oxophytodienoate reductase 3; 13-HPOT for 13*S*-hydroperoxy-9*Z*,11*E*,15*Z*-octadecatrienoic acid, 12,13-EOT for 12,13-epoxyoctadecatrienoic acid, OPDA for 12-oxophytodienoic acid, JA for jasmonates, JA-Ile for jasmonoyl-isoleucine, JA-ACC for

JA biosynthesis beginning with free linolenic acid (18:3) as the substrate is referred to as the Vick and Zimmermann pathway (Schaller et al., 2004) which was considered the major source of JA production in plants. However, there are several variations of this pathway

Linoleic acid (LA, 18:2) is a ubiquitous component of plant lipids. All seed oils of commercial importance including corn, sunflower and soybean oils usually contain over 50% of linoleate. Previously, LA was considered analogous to α-LeA for metabolism by the Vick and Zimmermann pathway (Schaller et al., 2004) yielding 9,10-dihydro-JA (DH-JA). This product was widely detected *in vivo* in some plant species (Miersch et al., 1999; Blechert

**Figure 1.** Overview of the oxylipin biosynthesis pathways in plants (Andreou et al., 2009) In the first reaction, free fatty acids (18:3 or 18:2) are oxidized by the addition of molecular oxygen yielding hydroperoxides (HPOT, hydroperoxy-octadecatrienoic acid; HOPD, hydroperoxyoctadecadienoic acids) through activity of oxygenases, lipoxygenase (LOX) or α-dioxygenase (α-DOX). Hydroperoxide products formed by LOXs are further metabolized by other enzymes: allene oxide synthase (AOS), hydroperoxide lyase (HPL), divinyl ether synthase (DES), peroxygenase (POX) and epoxyalcohol synthases (EAS).

**Figure 2.** Scheme of JA biosynthesis pathway in *A. thaliana* (Delker et al., 2006) The enzymes and the intermediates are indicated as LOX2 for lipoxygenase 2, AOS for allene oxide synthase, AOC for allene oxide cyclase, and OPR3 for 12-oxophytodienoate reductase 3; 13-HPOT for 13*S*-hydroperoxy-9*Z*,11*E*,15*Z*-octadecatrienoic acid, 12,13-EOT for 12,13-epoxyoctadecatrienoic acid, OPDA for 12-oxophytodienoic acid, JA for jasmonates, JA-Ile for jasmonoyl-isoleucine, JA-ACC for jasmonoyl-1-amino-1-cyclopropane carboxylic acid, and MeJA for methyl jasmonates.

#### **2.2. The alternative routes of JA biosynthesis**

396 Lipid Metabolism

(*JAR1*) gene (Staswick and Tiryaki, 2004).

into a more stable *trans* configuration, (-)-JA or undergoes modifications to produce diverse JA derivatives including MeJA and (+)-7-*iso*-JA*-*Ile. The latter one is the bioactive form of JA produced by conjugation of JA to isoleucine by the enzyme encoded by the *JA resistant 1*

**Figure 1.** Overview of the oxylipin biosynthesis pathways in plants (Andreou et al., 2009) In the first reaction, free fatty acids (18:3 or 18:2) are oxidized by the addition of molecular oxygen yielding hydroperoxides (HPOT, hydroperoxy-octadecatrienoic acid; HOPD, hydroperoxy-

epoxyalcohol synthases (EAS).

octadecadienoic acids) through activity of oxygenases, lipoxygenase (LOX) or α-dioxygenase (α-DOX). Hydroperoxide products formed by LOXs are further metabolized by other enzymes: allene oxide synthase (AOS), hydroperoxide lyase (HPL), divinyl ether synthase (DES), peroxygenase (POX) and

JA biosynthesis beginning with free linolenic acid (18:3) as the substrate is referred to as the Vick and Zimmermann pathway (Schaller et al., 2004) which was considered the major source of JA production in plants. However, there are several variations of this pathway considered as the alternative routes of JA biosynthesis.

Linoleic acid (LA, 18:2) is a ubiquitous component of plant lipids. All seed oils of commercial importance including corn, sunflower and soybean oils usually contain over 50% of linoleate. Previously, LA was considered analogous to α-LeA for metabolism by the Vick and Zimmermann pathway (Schaller et al., 2004) yielding 9,10-dihydro-JA (DH-JA). This product was widely detected *in vivo* in some plant species (Miersch et al., 1999; Blechert et al., 1995; Gundlach and Zenk, 1998), but not others, suggesting DH-JA biosynthesis from LA through Vick and Zimmermann pathway is not conserved (Gundlach and Zenk, 1998). Investigation by Gundlach and Zenk (1998) revealed that allene oxide cyclase (AOC), unlike most of the other enzymes of the Vick and Zimmerman pathway, discriminates between 18:3 and 18:2-derived pathway intermediates. This implies that AOC is the bottleneck for DH-JA production or alternatively, DH-OPDA (precursor of DH-JA) may result from the spontaneous cyclization of the 18:2-derived allene oxide (Gundlach and Zenk, 1998).

Jasmonate Biosynthesis, Perception and Function in Plant Development and Stress Responses 399

(MGDG) or digalactosyl-diacylglyceride (DGDG). Arabidopsides A, C, E and F have dino-OPDA at *sn2* position of glycerol backbone. Except for arabidopside F, all arabidopsides contain OPDA (Figure 4) (Hisamatsu et al., 2003; Hisamatsu 2005). At present, it is unclear whether the lipid-bound OPDA/dn-OPDA in the membranes is synthesized in situ from MGDG or DGDG or alternatively, OPDA/dn-OPDA is synthesized from free 18:3/16:3 and then incorporated into glycerol (Schaller et al., 2004). The latter possibility is supported by substantial amount of free 18:3 and 18:2 that were detected in tomato leaves after wounding (Conconi et al., 1996). However, Buseman et al. (2006) have shown that within the first 15 min after wounding, levels of OPDA-dnOPDA MGDG, OPDA-OPDA MGDG, and OPDA-OPDA DGDG increased 200 to 1000 folds. Yet in untreated leaves, the levels of these oxylipin-containing complex lipid species remained low, suggesting lipid-bound OPDA/dn-OPDA in wounding response synthesize on the esterified galactolipids rather than via the free fatty acids. Furthermore, OPDA and dn-OPDA sequestered in form of MGDG-O or DGDG-O may provide an abundant resource of OPDA/dn-OPDA, which may rapidly release under appropriate stress conditions for signaling or further metabolism (Schaller et

**Figure 4.** Arabidopsides of *Arabidopsis thaliana* (Hisamatsu et al., 2003, 2005)

**Figure 5.** The metabolites produced from JA in plants (Gfeller et al., 2010)

al., 2004).

Hexadecatrienoic acid (16:3) was proposed as an analog of linolenic acid for JA biosynthesis through the Vick and Zimmermann pathway (Weber et al., 1997), which is characterized by forming dinor-oxophytodienoic acid (dn-OPDA), a 16-carbon cyclopentanoic acid analog of *cis* (+)-OPDA. First identified in leaf extracts of *Arabidopsis* and potato plants (Weber et al., 1997), dn-OPDA dramatically accumulated upon wounding, suggesting an important role of this molecule in wounding response (Weber et al., 1997) (Figure 3). Although dn-OPDA forms after the first β-oxidation of *cis* (+)-OPDA (Figure 2), the detected dn-OPDA in wounded leaves believed to from 16:3 (Figure 3). Convincing genetic evidence for the role of 16:3 in JA biosynthesis came from the analysis ofthe *Arabidopsis* mutant *fad5* incapable of synthesizing 16:3 and JA (Weber et al., 1997).

OPDA and dn-OPDA are also constituents of arabidopsides (Figure 4), which are considered other alternative substrates for JA production in *Arabidopsis* (Gfeller et al., 2010). Arabidopsides are OPDA- and/or dn-OPDA-containing monogalactosyl-diacylglycerides

**Figure 3.** Alternative JA biosynthesis pathway (Schaller et al., 2004; Gfeller et al., 2010) See the abbreviations of the enzymes in the Fig. 2. dnOPDA indicated the intermediate dinoroxophytodienoic acid, OPC:8 is 3-oxo-2-(2'-[Z]-pentenyl)-cyclopentane-1-octanoicacid and OPC:6 is 3 oxo-2-(2'-pentenyl) cyclopentanehexanoic acid.

(MGDG) or digalactosyl-diacylglyceride (DGDG). Arabidopsides A, C, E and F have dino-OPDA at *sn2* position of glycerol backbone. Except for arabidopside F, all arabidopsides contain OPDA (Figure 4) (Hisamatsu et al., 2003; Hisamatsu 2005). At present, it is unclear whether the lipid-bound OPDA/dn-OPDA in the membranes is synthesized in situ from MGDG or DGDG or alternatively, OPDA/dn-OPDA is synthesized from free 18:3/16:3 and then incorporated into glycerol (Schaller et al., 2004). The latter possibility is supported by substantial amount of free 18:3 and 18:2 that were detected in tomato leaves after wounding (Conconi et al., 1996). However, Buseman et al. (2006) have shown that within the first 15 min after wounding, levels of OPDA-dnOPDA MGDG, OPDA-OPDA MGDG, and OPDA-OPDA DGDG increased 200 to 1000 folds. Yet in untreated leaves, the levels of these oxylipin-containing complex lipid species remained low, suggesting lipid-bound OPDA/dn-OPDA in wounding response synthesize on the esterified galactolipids rather than via the free fatty acids. Furthermore, OPDA and dn-OPDA sequestered in form of MGDG-O or DGDG-O may provide an abundant resource of OPDA/dn-OPDA, which may rapidly release under appropriate stress conditions for signaling or further metabolism (Schaller et al., 2004).

398 Lipid Metabolism

et al., 1995; Gundlach and Zenk, 1998), but not others, suggesting DH-JA biosynthesis from LA through Vick and Zimmermann pathway is not conserved (Gundlach and Zenk, 1998). Investigation by Gundlach and Zenk (1998) revealed that allene oxide cyclase (AOC), unlike most of the other enzymes of the Vick and Zimmerman pathway, discriminates between 18:3 and 18:2-derived pathway intermediates. This implies that AOC is the bottleneck for DH-JA production or alternatively, DH-OPDA (precursor of DH-JA) may result from the

Hexadecatrienoic acid (16:3) was proposed as an analog of linolenic acid for JA biosynthesis through the Vick and Zimmermann pathway (Weber et al., 1997), which is characterized by forming dinor-oxophytodienoic acid (dn-OPDA), a 16-carbon cyclopentanoic acid analog of *cis* (+)-OPDA. First identified in leaf extracts of *Arabidopsis* and potato plants (Weber et al., 1997), dn-OPDA dramatically accumulated upon wounding, suggesting an important role of this molecule in wounding response (Weber et al., 1997) (Figure 3). Although dn-OPDA forms after the first β-oxidation of *cis* (+)-OPDA (Figure 2), the detected dn-OPDA in wounded leaves believed to from 16:3 (Figure 3). Convincing genetic evidence for the role of 16:3 in JA biosynthesis came from the analysis ofthe *Arabidopsis* mutant *fad5* incapable of

OPDA and dn-OPDA are also constituents of arabidopsides (Figure 4), which are considered other alternative substrates for JA production in *Arabidopsis* (Gfeller et al., 2010). Arabidopsides are OPDA- and/or dn-OPDA-containing monogalactosyl-diacylglycerides

**Figure 3.** Alternative JA biosynthesis pathway (Schaller et al., 2004; Gfeller et al., 2010) See the abbreviations of the enzymes in the Fig. 2. dnOPDA indicated the intermediate dinoroxophytodienoic acid, OPC:8 is 3-oxo-2-(2'-[Z]-pentenyl)-cyclopentane-1-octanoicacid and OPC:6 is 3-

spontaneous cyclization of the 18:2-derived allene oxide (Gundlach and Zenk, 1998).

synthesizing 16:3 and JA (Weber et al., 1997).

oxo-2-(2'-pentenyl) cyclopentanehexanoic acid.

**Figure 4.** Arabidopsides of *Arabidopsis thaliana* (Hisamatsu et al., 2003, 2005)

**Figure 5.** The metabolites produced from JA in plants (Gfeller et al., 2010)
