BIOSYNTHESIS AND ACTION OF JASMONATES IN PLANTS

CREELMAN & MULLET BIOSYNTHESIS AND ACTION OF JASMONATES Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997. 48:355–81 Copyright © 1997 by Annual Reviews Inc. All rights reserved

BIOSYNTHESIS AND ACTION OF JASMONATES IN PLANTS Robert A. Creelman and John E. Mullet Department of Biochemistry and Biophysics, Crop Biotechnology Center, Texas A&M University, College Station, Texas 77843 KEY WORDS: jasmonic acid, chemistry, gene expression, insect and disease resistance

ABSTRACT Jasmonic acid and its derivatives can modulate aspects of fruit ripening, production of viable pollen, root growth, tendril coiling, and plant resistance to insects and pathogens. Jasmonate activates genes involved in pathogen and insect resistance, and genes encoding vegetative storage proteins, but represses genes encoding proteins involved in photosynthesis. Jasmonic acid is derived from linolenic acid, and most of the enzymes in the biosynthetic pathway have been extensively characterized. Modulation of lipoxygenase and allene oxide synthase gene expression in transgenic plants raises new questions about the compartmentation of the biosynthetic pathway and its regulation. The activation of jasmonic acid biosynthesis by cell wall elicitors, the peptide systemin, and other compounds will be related to the function of jasmonates in plants. Jasmonate modulates gene expression at the level of translation, RNA processing, and transcription. Promoter elements that mediate responses to jasmonate have been isolated. This review covers recent advances in our understanding of how jasmonate biosynthesis is regulated and relates this information to knowledge of jasmonate modulated gene expression.

CONTENTS INTRODUCTION..................................................................................................................... CHEMISTRY AND QUANTITATION................................................................................... JA ACCUMULATION AND DISTRIBUTION ...................................................................... BIOSYNTHETIC PATHWAY AND REGULATION ............................................................ Linolenic Acid and Lipases ................................................................................................. Lipoxygenase (LOX)............................................................................................................

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356 CREELMAN & MULLET Allene Oxide Synthase and Other Steps in JA Biosynthesis ................................................ JASMONATE SIGNAL TRANSDUCTION .......................................................................... JA FUNCTION AND RESPONSIVE GENES ........................................................................ Seed Germination and Growth............................................................................................ Vegetative Sinks and Storage Proteins................................................................................ Photosynthesis, Senescence, and Abiotic Stress.................................................................. Flower and Fruit Development ........................................................................................... Insect and Disease Resistance............................................................................................. JA’s Dual Role in Development and Defense ..................................................................... CONCLUDING REMARKS ....................................................................................................

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INTRODUCTION Jasmonic acid (JA), and its methyl ester (methyl jasmonate, MeJA) are linolenic acid (LA)-derived cyclopentanone-based compounds of wide distribution in the plant kingdom. MeJA was first identified as a component of the essential oil of several plant species, while JA was first obtained from a fungal culture filtrate. Early studies showed that exogenous JA or MeJA can promote senescence and act as a growth regulator. Subsequent research revealed that JA specifically alters gene expression and that wounding and elicitors could cause JA/MeJA accumulation in plants. These results implied a role for jasmonate in plant defense that has recently been confirmed. Other research described roles for jasmonates in vegetative development, fruit development, and pollen viability. The dual role of JA in plant development and defense is examined in this review. This review emphasizes new information in this field since the last review in this series (103). Other excellent reviews are available that cover JA/MeJA with respect to herbivory (7, 13), signaling (40, 106), chemistry and biochemistry (51, 56), gene expression (89), and chromatography (117). For the purposes of this review, (3R, 7RS)-JA/MeJA will be referred to collectively as jasmonates unless it is necessary to identify specific isomers.

CHEMISTRY AND QUANTITATION The jasmonate molecule (Figure 1) contains two chiral centers located at C3 and C7 generating four possible stereoisomers, since either chiral center can have an R or S absolute configuration. The mirror image isomers, (3R, 7S) and (3S, 7R), have their side chains in a cis orientation. These isomers are known as (+)− and (−)-7-iso-JA or (+)− and (−)-epi-JA. The enantiomers, (3R, 7R)and (3S, 7S)-JA or (−)-JA and (+)-JA, have their side chains in the trans configuration. Because of increased steric hindrance, the cis orientation is less stable and will epimerize to the more stable trans configuration. This occurs via a keto-enol tautomerization involving the C6 ketone and the C7 proton to

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Figure 1 Chemical structures of various isomers of jasmonic acid and coronatine. The 3S, 7S isomer is the final product of the jasmonic acid biosynthetic pathway and is easily converted to its diastereomer 3R, 7S isomer during extraction. The corresponding enantiomers are found in synthetic material. Coronatine is a bacterial phytotoxin with biological activities similar to jasmonic acid.

form the corresponding diastereomers. During extraction or in the presence of acids or bases, (+)-7-iso-JA, thought to be the initial jasmonate formed in plants, is believed to epimerize to an equilibrium mixture of approximately 9:1 (−)-JA:(+)-7-iso-JA (80). The actual equilibrium concentration in planta is unknown. Consequently, analysis of jasmonates isolated from plants should indicate which isomers are being analyzed. Commercially available synthetic MeJA used in many experiments is composed of a 9:1 ratio (±)-MeJA:(±)-7iso-MeJA. The methyl esters may be converted to the free acids with either basic hydrolysis or incubation with commercially available esterases.

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A monoclonal antibody for the analysis of (−)-JA (analyzed as the methyl ester) has been described (1). Assuming that (−)-MeJA has a cross reaction of 100%, (+)-7-iso-MeJA had a cross reactivity of 86%. Albrecht et al (1) suggested that, when using this monoclonal antibody, it is advisable to allow endogenous jasmonates to reach their stable equilibrium before quantitation. Furthermore, as is the case with any antibody-based technique, procedures must be developed to estimate losses during extraction. In addition, antibody methods must be validated using a physical method such as GC-MS to check for possible errors due to interfering compounds present in extracts. Synthetic JA or jasmonate analogs containing deuterium or 13C have been used to quantify endogenous jasmonates by GC-MS selected ion monitoring. The jasmonate diastereomers can be resolved by gas chromatography using capillary columns enabling their separate quantitation. Furthermore, mass spectra of endogenous compounds can be to used to unequivocally identify jasmonates based on comparison with published spectra. Mueller and Brodschelm (80) derivatized jasmonates to the pentafluorobenzyl esters for quantitation by GC-MS-NICI and reported a limit of detection of ∼500 fg. The presence of the fluorine atoms accounts for the increased sensitivity of this method. Creelman et al (29) used (13C,2H3)-MeJA, whereas Gundlach et al (53) used 9,10 dihydrojasmonic acid to estimate JA levels. However, use of compounds that are structurally similar to the compound being measured may under- or overestimate endogenous levels unless the recovery efficiencies are identical. To circumvent this problem, Creelman & Mullet (28) synthesized (2-13C)-JA and used it to measure JA in soybean tissue. Use of (2-13C)-JA in calculating endogenous levels of JA must take into account the m/e 225 (M+1) in isotope dilution calculations because of the natural abundance of heavy isotopes. A significant improvement was the synthesis of (1,2-13C)-JA with a molecular weight two mass units higher than endogenous JA (Z-P Zhang, ES McCloud & IT Baldwin, personal communication). The positive and negative ion electrospray mass spectra of several jasmonate amino acid conjugates were determined by combined HPLC-MS (102).

JA ACCUMULATION AND DISTRIBUTION The level of JA in plants varies as a function of tissue and cell type, developmental stage, and in response to several different environmental stimuli. JA levels are low in soybean seeds but increase to 2 µg/g fresh weight in developing axes within 12 h of imbibition. In soybean seedlings, levels of JA are higher in the hypocotyl hook, a zone of cell division, and young plumules compared to the zone of cell elongation and more mature regions of the stem,


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older leaves, and roots. High levels of JA are also found in flowers and pericarp tissues of developing reproductive structures (28, 71). In mature soybean leaves, high levels of jasmonate responsive gene expression are observed in paraveinal mesophyll cells and bundle sheath cells that surround veins and to a lesser extent in epidermal cells (50, 61). Little expression of jasmonate responsive genes is observed in palisade parenchyma cells unless leaves are treated with jasmonate (61). This suggests that JA levels are lower or compartmentalized differently in palisade cells or that these cells are less sensitive to JA. In illuminated plants, JA will accumulate in chloroplasts because it is a weak acid, assuming it becomes distributed in cellular compartments like ABA (58). Because palisade cells are filled with chloroplasts, this may lower the level of JA in the cytoplasm of these cells below the threshold needed to activate gene expression. The accumulation of JA in chloroplasts may help explain why exogenous JA activates gene expression but increasing endogenous levels of JA by overexpression of allene oxide synthase does not (57). Exogenous JA will cause transient and large changes in the concentration of JA in most plant cells before reaching internal equilibrium in tissues. In contrast, over-expression of allene oxide synthase in chloroplasts provides cells with elevated rates of JA synthesis over the life of the plants. JA is synthesized in these plants in cells containing chloroplasts where it can be sequestered as it is produced. This may keep the level of JA from rising in other regions of the cell where JA receptors that modulate gene expression are presumably located. Jasmonate levels are rapidly and transiently increased by mechanical perturbations such as those causing tendril coiling (39, 123) and turgor reduction induced by water deficit (28). Mechanical impedance during root growth might also induce JA accumulation, causing inhibition of root growth (113). Other mechanical perturbations, involving wind or touch, induce changes in growth that may be mediated in part by JA. In Arabidopsis thaliana, the TCH genes are upregulated in response to mechanical perturbation (19). The TCH genes encode calmodulin and two other calcium-binding proteins. Expression of these genes increases when cytoplasmic calcium levels rise (4). In animal cells, calcium stimulates eicosanoid biosynthesis by activating phospholipase and lipoxygenase. In plant cells, similar enzymes are also involved in JA biosynthesis, suggesting that a similar signal transduction pathway involving calcium may activate synthesis of JA in response to touch and turgor reduction. Jasmonate accumulates in response to plant wounding (29). Localized wound-induced JA accumulation in injured cells could result from the mixing of compartments that contain lipases, membranes rich in LA, the precursor of


JA, lipoxygenase and the other enzymes that are involved in JA biosynthesis (described below). However, the situation is more complex because JA accumulation can be induced in cell cultures and plants by oligosaccharides derived from plant cell walls, by elicitors such as chitosans derived from fungal cell walls, and by peptide inducers such as systemin (37, 53, 82). These compounds are thought to stimulate JA biosynthesis via receptor-mediated processes. Elicitor-induced phosphorylation of a plasmalemma protein (42) and inhibition of elicitor-mediated JA accumulation by the protein kinase inhibitor staurosporin (16) are consistent with this idea. Furthermore, wound-induced accumulation of JA requires a MAP kinase (104). In some plants, ABA appears to be needed for wound, elicitor, or systemin-mediated JA accumulation (89). JA and JA responsive genes also accumulate systemically in plants in response to localized wounding (84). The systemic signal is apparently released at the wound site and migrates through the phloem to other parts of the plant. Systemin, an 18–amino acid peptide, has been shown to move in the phloem and to induce JA and JA-responsive genes throughout the apical portion of plants (84). Systemin may be released from the wound site upon hydrolysis of a precursor polypeptide (77). Electrical signals have also been proposed to mediate systemic induction of JA in response to wounding (124). In addition, systemic accumulation of JA as well as transfer of JA among plants could occur via the vapor phase in the form of MeJA (43, 49), although the significance of this latter pathway under physiological conditions is not clear.

BIOSYNTHETIC PATHWAY AND REGULATION The biosynthesis of jasmonates begins with LA (Figure 2). This fatty acid is converted to 13-hydroperoxylinolenic acid by lipoxygenase. 13-hydroperoxylinolenic acid is a substrate for allene oxide synthase [also known as hydroperoxide dehydratase or hydroperoxide dehydrase; see Simpson & Gardner (105)] and allene oxide cyclase resulting in the formation of 12-oxo-phytodienoic acid (12-oxo-PDA). Following reduction and three steps of beta oxidation, (−)-7-iso-JA is formed. Jasmonic acid can be catabolized to form MeJA and numerous conjugates and catabolites that may have biological activity (56). The accumulation of JA in plants in response to wounding, or treatment with elicitors and systemin, can be blocked using inhibitors of lipoxygenase (8, 36, 41, 86). Therefore, increases in JA level mediated by these inducers results from de novo synthesis rather than release from JA conjugates.


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Figure 2 Biosynthetic pathway of jasmonic acid. It is postulated that signals (such as elicitors) interact with a membrane receptor, which causes the eventual production of 13-hydroperoxylinolenic acid. Production of 13-hydroperoxylinolenic acid is believed to occur with the release of linolenic acid via either a phospholipase or lipase followed by oxidation by lipoxygenase (LOX), but a preliminary oxidation of linolenic acid while still esterified to a phospholipid and subsequent release by a lipase cannot be ruled out. 13-hydroperoxylinolenic acid can then be catabolized by hydroperoxy lyase (HL), eventually forming volatile aldehydes and traumatic acid, or via peroxygenase pathway to cutin monomers. Jasmonic acid arises from 13-hydroperoxylinolenic acid via an allene oxide synthase (AOS) and an allene oxide cyclase (AOC)-dependent pathway with 12-oxophytodienoic acid (12-oxo-PDA) as an intermediate. Jasmonic acid then acts to modulate gene expression or can be further catabolized.

Linolenic Acid and Lipases The A. thaliana fad3-2 fad7-2 fad8 mutant has very low levels of linolenic acid and is unable to accumulate JA in response to wounding (76; RA Creelman, M McConn, J Browse & JE Mullet, unpublished data). Application of LA to plants results in accumulation of JA (44). This indicates that the level, distribution, or availability of LA could determine the rate of JA biosynthesis. In one study, the level of free linolenic acid measured before wounding was many


times higher than the maximum JA accumulated after wounding (27). Free LA levels doubled within 1 h after wounding, while JA levels rose 10-fold (27). Therefore, the wound-induced increase in JA level could have resulted from release of linolenic acid from phospholipids, or the utilization of LA present before wounding for JA biosynthesis. Plant membranes, especially chloroplast membranes, are a rich source of LA esterified in glycerolipids and phospholipids. This has led to the suggestion that increases in JA could result from the activation of phospholipases that release LA from membranes (44). Plant extracts also contain highly active acyl hydroxylases that can release fatty acids from lipids. In animal systems, phospholipase A2, activated by micromolar levels of calcium, releases arachidonic acid used in the biosynthesis of eicosanoids. The eicosanoids, leukotrienes, and prostaglandins have chemical structures similar to jasmonates. These compounds mediate localized stress and inflammatory responses in animal cells. Direct evidence for the role of a specific phospholipase in JA synthesis is lacking. However, analysis of phospholipid changes and phospholipase A activity was done in tobacco cells treated with elicitors derived from Phytophthora parasitica var. nicotianae (97). Time course studies showed that the amount of phosphatidylcholine was reduced in response to elicitors and that a concomitant increase of phospholipase A activity occurred. In soybean cell culture, harpin and an extract from the pathogenic fungus Verticillium dahliae promoted rapid increases in phospholipase A activity (23). Upon wounding of potato tuber tissue, JA levels rose about 100-fold in 4 h; however, inhibitors of animal phospholipase A2 (manoalide and quinacrine) did not inhibit the accumulation of JA (69). Phospholipase D, which cleaves the head group from phospholipids, could also trigger release of LA and stimulate JA biosynthesis. Plant phospholipase D has been identified in plants and is proposed to play a role in plant defense (121). In some instances, fatty acids may be oxidized before release from lipids for JA biosynthesis. Fuessner et al (46) describe the presence of lipoxygenase (LOX) in lipid bodies which oxidizes fatty acids before further metabolism. Phospholipases preferring oxygenated fatty acids have been observed in several plant species (6, 9). Therefore, the oxidation of fatty acids during high irradiance, exposure to ozone, or as a consequence of the oxidative burst associated with plant defense (20) may stimulate the activity of phospholipases or nonspecific acyl hydrolases resulting in release of oxidized fatty acids for JA biosynthesis. In animal cells, arachidonic acid is delivered to cells for eicosanoid biosynthesis via low density lipoproteins (54). In plants, transfer of linolenic acid among cells could be carried out by lipid transfer proteins that are localized in


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the extracellular space (114). Current models postulate that jasmonate biosynthesis is regulated by pathogens or herbivory through the production of elicitors or systemic signaling molecules that interact with receptors present on the plasma membrane. The observation that the enzymes of the JA biosynthetic pathway are primarily localized in plastids (10, 17) suggests that mechanisms must exist to shuttle LA released from the plasma membrane to the plastid. Alternatively, signal perception could be transmitted to plastids for subsequent release of free LA in that organelle.

Lipoxygenase (LOX) Treatment of plants with LOX inhibitors (8, 86) and transgenic plants with reduced LOX activity (10) have reduced ability to synthesize JA. Therefore, LOX mediates an essential step in JA biosynthesis. In animals, eicosanoid biosynthesis is regulated in part through calcium and protein modulated interaction of LOX with membranes and its substrate (35, 79). In plants, the role of LOX in the regulation of JA biosynthesis has been difficult to analyze because most plants have numerous genes encoding LOX, different isoforms of LOX have different enzyme specificity, and LOX is present in more than one compartment in plant cells (i.e. 45, 64, 83, 100). Plant lipoxygenases (EC 1.13.11.12) oxygenate linolenic acid at the 9 or 13 position to give 9- or 13-hydroperoxylinolenic acid. The role of 9-hydroperoxides and their catabolites in plants is unclear (119). The role of LOX isozymes in the production of hydroperoxide isomers also needs further investigation. LOX from tendrils of Bryonia dioica consists of a major isoform with pI = 6.5 and minor constituents with pI = 6.7 and 7.3 (38). These LOX isoforms primarily produce 13-hydroperoxylinolenic acid, whereas a preparation from cell cultures contains at least seven LOX isoforms in the pI range from 6.3–6.7 and 7.3–7.5 with the major reaction product 9-hydroperoxylinolenic acid (38). LOX isozymes are found in the plasmalemma/microsomes of cucumber cotyledons (Cucumis sativus L.) (81). In soybean leaves, LOX accumulates in the vacuoles of paraveinal mesophyll cells (52). Elsewhere, LOX is associated with epidermal and cortical cells and is present in vacuoles and plastids. In plastids, a methyl jasmonate–induced LOX is sequestered into protein inclusion bodies (52). This may be important in restricting the interaction of LOX with fatty acids. Similarly, in barley, jasmonate–induced LOX isozymes are localized in plastids (45). Changes in the distribution and abundance of LOX during development and in different tissues and compartments is due in part to the expression of different members of the Lox gene family. For example, two different LOX


genes, AtLox1 (78) and AtLox2 (11), have been identified in A. thaliana. AtLox1 is expressed in leaves, roots, inflorescences, and young seedlings, with the highest expression found in roots and young seedlings. Because AtLox1 lacks obvious targeting sequences, this enzyme is most likely localized in the cytoplasm. In contrast, AtLox2 is localized in chloroplasts (10). The presence of a plastid transit sequence suggests that a rice LOX that catalyzes the exclusive formation of 13-hydroperoxylinolenate is also localized in chloroplasts (90). AtLox2 mRNA levels are high in leaves and inflorescences but low in seeds, roots, and stems. The physiological role of this chloroplast lipoxygenase was analyzed by reducing LOX2 accumulation in transgenic plants (10). The reduction of AtLox2 expression caused no obvious changes in plant growth. However, the wound-induced accumulation of JA observed in control plants was absent in leaves of transgenic plants lacking LOX2. Therefore, plastid localized LOX2 is required for wound-induced synthesis of jasmonates in Arabidopsis leaves.

Allene Oxide Synthase and Other Steps in JA Biosynthesis The fate of 13-hydroperoxylinolenate produced by lipoxygenase is another key branchpoint in the jasmonate biosynthetic pathway (Figure 2). Hydroperoxide lyase will cleave 13-hydroperoxylinolenate to form volatile six carbon aldehydes and 12-oxo-dodecenoic acid (119). 13-hydroperoxylinolenate can also be used by peroxygenase to produce precursors of cutin molecules (18). In contrast, production of jasmonates requires that 13-hydroperoxylinolenate be metabolized to allene oxide by allene oxide synthase (AOS; IUBMB name hydroperoxide dehydratase, EC 4.2.1.92). Other names previously used for AOS include hydroperoxide isomerase, hydroperoxide cyclase, and fatty acid hydroperoxide dehydrase. Flax and Arabidopsis AOS have been cloned and characterized (21, 107, 108; E Bell, RA Creelman & JE Mullet, unpublished data). Flax AOS is a 55-kDa hemoprotein with the spectral characteristics of a cytochrome P450 and a turnover rate of 1000 min−1. The primary structure of AOS deduced from its cDNA reveals a protein of 536 amino acids containing a C-terminal domain homologous to a region in many cytochrome P450s that contain a heme-binding cysteine. The flax cDNA encodes a 58–amino acid N-terminal sequence characteristic of chloroplast transit peptides. This is consistent with localization of AOS activity in chloroplasts (120). The Arabidopsis AOS (available from the Ohio State University Arabidopsis Biological Resource Center as EST 94J16T7, GenBank Accession T20864) shares a high degree of homology with flax AOS and also contains a putative N-terminal plastid targeting sequence (RA Creelman, E Bell & JE Mullet, unpublished


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data). The Arabidopsis AOS gene exists as a single copy, based on analysis of total DNA Southern blots (E Bell, RA Creelman & JE Mullet, unpublished data). Allene oxide synthase activity has been localized to the plastid outer envelope in spinach (17), but given its putative plastid targeting sequence and high turnover number further localization studies are warranted. Over-expression of flax AOS in transgenic potato plants increased JA levels (57), indicating that the amount of AOS protein limits JA biosynthesis. The high turnover rate of flax AOS (∼1000 min−1) may help this enzyme compete for substrate also used by hydroperoxy lyase and peroxygenase depending on the localization of these enzymes and their substrate accessibility. 13-hydroperoxylinolenate produced by a plastid localized LOX may be more accessible to AOS localized to plastids. Recent studies have shown that AOS activity was higher in tissues with elevated JA, suggesting that differences in JA that occur during plant development may be caused by variation in AOS abundance or activity (105). Similar analysis of AOS level and activity needs to be carried out in plants exposed to elicitors and systemin, or after wounding. Nonenzymatic cyclization of allene oxide will yield a racemic mixture of 12-oxo-PDA. However, allene oxide cyclase (55) catalyzes the stereospecific formation of the 9S, 13S enantiomer of 12-oxo-PDA. The enzymes catalyzing reduction of 12-oxo-PDA and β-oxidation leading to JA have been demonstrated in vitro but have not been extensively characterized (119). The tomato mutant, JL5, is inhibited in the conversion of 13-hydroperoxylinolenate to 12-oxo-PDA (60). This mutant, which could be altered in AOS or AOC activity, was identified by screening plants for reduced levels of woundinduced Pin2 expression. Diethyldithiocarbamic acid (DIECA), an inhibitor of JA biosynthesis, was shown to efficiently reduce 13-hydroperoxylinolenate to 13-hydroxylinolenic acid (37, 41). This suggests that DIECA inhibits JA biosynthesis by reducing the precursor pool leading to allene oxide. Salicylic acid (SA), a mediator of some plant defense responses, inhibits the conversion of 13-S-hydroperoxy linolenic acid to 12-oxo-PDA (37, 41, 87, 124).

JASMONATE SIGNAL TRANSDUCTION The JA signal transduction pathway is mainly unknown. It is presumed that jasmonate interacts with receptors in the cell that activate a signaling pathway resulting in changes in transcription, translation, and other responses mediated by JA. Lack of high specific activity JA and the lipophilic and volatile nature of JA and MeJA will make direct analysis of JA receptors difficult. Jasmonate receptors and other components of the signal transduction pathway are more likely to be discovered through analysis of mutants that are insensitive or


altered in their response to JA. To date, up to four different classes of JA-insensitive mutants have been identified; coi1, jar1, jin1, and jin4 (12, 14, 113). Genetic studies were unable to determine whether jin4 and jar1 were allelic (14). The mutants jar1, jin1, and jin4 were recovered using a root growth screen (wild type A. thaliana root growth is inhibited by 1–10 mM JA). In contrast, coi1 was identified because plants were resistant to coronatine (47). Coronatine is a chlorosis-inducing toxin that has a chemical structure (Figure 1) and biological activity similar to JA. The coi1 mutant also shows a MeJA–insensitive root growth phenotype. Application of jasmonate to plants causes large changes in translation, transcription, and mRNA populations (103). Treatment of barley leaves with jasmonate reduced synthesis of the large and small subunits of Rubisco and other proteins (122). Decreased translation of the large subunit in chloroplasts was correlated with a site-specific cleavage in the 5′-untranslated portion of the rbcL mRNA (94, 96). The altered rbcL mRNA 5′-end presumably reduces access to the ribosome–binding site located near the site of translation initiation. Reduced synthesis of Rubisco small subunit and other cytoplasmic proteins occurred through supression of translation initiation and reduction of mRNA levels (95). The promoters of two jasmonate–inducible genes, Pin2 and VspB, have been studied in some detail (67, 74). A 50–bp domain was identified in the promoters of both genes that conferred JA responsiveness on truncated reporter gene constructs. The VspB 50–bp domain did not confer JA responsiveness on a truncated −46 CaMV promoter, but induction was observed when this DNA region was added to a −90 CaMV truncated promoter. This indicates that factors binding to the −90 CaMV promoter were required to observe JA–stimulated transcription. Both JA–responsive domains contain a G-box sequence (CACGTG), which in other promoters has been shown to bind bZIP transcription factors (126). Because bZIP protein binding sites are found in numerous promoters that are not responsive to JA, it is likely that this factor, if it plays a role in JA mediated responses, interacts with other trans factors to modulate transcription. Mutation of the G-box in the Pin2 promoter did not prevent JA-mediated induction of Pin2 (72). Jasmonate induced accumulation of VspB and Pin2 mRNA is blocked by cycloheximide. Bestatin, an inhibitor of aminopeptidases in plants and animals, induces Pin2 in the absence of JA (101). This observation suggests that induction of Pin2, and perhaps other jasmonate modulated plant genes, is normally prevented by the action of an aminopeptidase. Induction of JA–responsive genes could therefore be mediated by inactivation of the protease or stabilization of the target protein.


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JA FUNCTION AND RESPONSIVE GENES Jasmonate modulates the expression of numerous genes and influences specific aspects of plant growth, development, and responses to abiotic and biotic stresses (see Table 1). Many of these responses were identified by application of jasmonate to plants, sometimes at nonphysiological levels. Interactions between JA and other plant growth regulators make assignment of physiological roles for JA even more complicated. In the section below, proposed actions of JA in plants are related to the level of JA in plant tissues, the activity of JA responsive genes, and insights provided by JA insensitive and JA deficient plants.

Seed Germination and Growth JA and MeJA inhibit the germination of nondormant seeds and stimulate the germination of dormant seeds. JA, MeJA, ABA, and ethylene inhibit germination of the recalcitrant seeds of Quercus robur (48). When these dessicationsensitive seeds were dried, the concentration of MeJA and JA increased before Table 1 Jasmonate functions and responsive genes Physiological Function

JA Responsive Genes

References

Seed germination and growth (-) seed, pollen germination

???

47, 48, 76

(-) root growth

???

12, 14, 113

(+) tendril coiling

???

38, 39, 123

Photosynthesis/vegetative sinks (-) photosynthetic apparatus

(-) rbcL, rbcS

24, 94–96

(+) vegetative protein storage

(+) Vsp, Lox

11, 15, 22, 45, 50, 52, 61, 65, 73, 75, 87

(+) tuberization

(+) Pin2

63, 85, 87, 88, 93

???

47, 76

Flower and fruit development (+) pollen viability (+) fruit ripening, pigments

(+) EFE

30, 71

(+) seed development

(+) cruciferin, napin, Vsps

110, 125

(+) insect resistance

(+) Pin2, other genes

36, 37, 41, 43, 44, 49, 62

(+) disease resistance

(+) osmotin, thionin, RIP60

9a, 129

(+) Chs, Pal, HMGR, PPO, pdf, ???

25, 26, 29, 49, 82, 91

Insect and disease resistance


the loss in seed viability. The increase in jasmonate was correlated with lipid peroxidation, which suggests that the production of jasmonate may not be regulating germination but rather is a consequence of membrane damage. In apple, jasmonate stimulated the germination of dormant embryos and increased alkaline lipase activity (92). Lipase activation may stimulate the mobilization of lipid reserves to provide sugars for seedling growth. Inhibitors of lipoxygenase-inhibited embryo germination and JA partially reversed inhibitor action. The level of jasmonate in soybean seeds 12 days post-anthesis is low (∼0.1 µg/g fresh weight), whereas in older seeds JA levels are higher (0.5 ng/g fresh weight) (28). 12 h after imbibition, the level of JA increased fivefold to 2 µg/g fresh weight in axes. JA levels declined with further seedling development. The observed increase in JA levels following imbibition is correlated with seed reserve mobilization and therefore may be a consequence rather than a trigger of germination. The jasmonate-insensitive mutants, jin4 and jar1, show increased sensitivity to ABA inhibition of germination (14, 113). Therefore, JA may stimulate seed germination by decreasing sensitivity to ABA. Alternatively, JA-mediated growth inhibition could block seed germination. JA strongly inhibits root growth by a mechanism not mediated by ethylene (14). JA also inhibits IAA-stimulated coleoptile elongation possibly by blocking incorporation of glucose into cell wall polysaccharides (118). Furthermore, JA activates the differential growth involved in tendril coiling, a response that does not directly involve ethylene or IAA (39). Further work is needed to define the role of JA in growth processes.

Vegetative Sinks and Storage Proteins Plants have the capacity to accumulate large amounts of carbon and nitrogen in specific cells and tissues and to mobilize these materials for use in other parts of the plant. This capacity is used during seed formation when nutrients are moved from the vegetative plant to developing seeds, and during seed germination when carbon and nitrogen are mobilized for seedling development. Transient storage and mobilization of nutrients also occur during vegetative growth. For example, carbon often accumulates in chloroplasts during the day and is mobilized at night to other parts of the plant. Carbon and nitrogen may also accumulate in cells located in meristematic regions for use during rapid cell growth. For osmotic reasons, cells are only able to accumulate a limited amount of sucrose and amino acids. Therefore, large amounts of carbon and nitrogen accumulate as polymers in the form of starch, fructans, and proteins. A role for jasmonic acid in protein storage in plants was suggested, in part, because jasmonate levels are high in vegetative sinks. As noted above, jasmon-


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ate levels are higher in soybean axes, plumules, and the hypocotyl hook relative to the hypocotyl zone of cell elongation and the nonelongating portion of stems and roots (28). In six-week-old soybean seedlings, JA levels are higher in young growing leaves that are importing carbon and nitrogen than in older fully expanded leaves (28). High levels of JA are present in developing reproductive structures, especially pods, with lower levels in seeds. Jasmonate or a derivative of jasmonate, tuberonic acid, has been proposed to play a role in the formation of tubers, a special type of vegetative sink (68, 85, 93). A second reason to suggest that jasmonate plays an important role in protein storage during plant development derives from the discovery that genes encoding vegetative storage proteins (VSPs) (111) are regulated by jasmonate (2). Vegetative storage proteins were first described in soybean (127, 128). The VSPs accumulate in the vacuoles of paraveinal mesophyll and bundle sheath cells that surround veins in soybean leaves (50). If pods are continuously removed from plants, the VSPs accumulate and can account for as much as 45% of the soluble protein in leaves (128). Other studies showed that the VSPs accumulate in pods and other parts of the developing reproductive structure but not in seeds (109). This led to the suggestion that the VSPs represent temporary deposits of amino acids derived from disassembly of Rubisco and other leaf proteins that were being mobilized for seed development. The observation that jasmonate regulates VSP accumulation was made by Anderson (2, 3) when treating soybean cell cultures with this compound. Later studies showed that the soybean VSPs accumulate in soybean axes, hypocotyl hooks, and young developing leaves and that VspB expression is correlated with endogenous levels of JA in plants (28, 73, 75). VspB expression also increases in young leaves when plants are exposed to water deficit (75). This is most likely due to inhibition of leaf growth but continued import and storage of carbon and nitrogen in the leaf. The soybean VSPs include two proteins having low acid phosphatase activity (Vspa, Vspb) (33) and lipoxygenase (65, 115). The genes encoding these proteins are regulated in a complex way by JA, sugars, phosphate, nitrogen, and auxin (34, 73, 98, 112).

Photosynthesis, Senescence, and Abiotic Stress Application of JA to leaves decreases expression of nuclear and chloroplast genes involved in photosynthesis (22, 122). JA treatments also cause a loss of chlorophyll from leaves or cell cultures (122). Jasmonate’s ability to cause chlorosis led to the suggestion that this compound plays a role in plant senescence (116). However, this suggestion is difficult to reconcile with the finding of high JA levels in zones of cell division, young leaves, and reproductive


structures. Unfortunately, a complete analysis of JA levels in senescing leaves has not been carried out. A limited study of this question in soybean revealed only small changes in JA level in soybean leaves during pod fill (22). Thus although JA can induce senescence-like symptoms, the role of this hormone in mediating senescence is at present unclear. If jasmonate-induced chlorosis and inhibition of genes encoding proteins involved in photosynthesis are not involved in senescence, then what is the physiological role of this JA activity? JA may inhibit the synthesis of chloroplast proteins during an early phase of leaf formation where cell division and import of nutrients are very active. This is consistent with higher levels of JA in young leaves compared with older leaves of soybean. In monocot leaves, meristematic cells are localized to the leaf base. This region of the leaf contains little chlorophyll, and expression of genes involved in photosynthesis is limited. Plastids in this developmental stage often contain starch grains, and these cells may accumulate vegetative storage proteins before cell enlargement and chloroplast development. Once cells stop dividing and begin to elongate, chloroplast development is initiated. If developing monocot leaves are similar to soybean hypocotyls, higher jasmonate concentrations will be present in the meristematic cells of the leaf base than in expanding and mature cells nearer the leaf apex. If this is the case, JA could act to inhibit premature accumulation of the photosynthetic apparatus in meristematic cells of the leaf base while stimulating accumulation of carbon and nitrogen reserves needed for later cell development. We speculate that treatment of excised mature leaves with JA may be recapitulating this earlier phase of development. Application of JA to mature leaves may drive the developmental program in reverse by inhibiting expression of chloroplast genes and stimulating accumulation of vegetative storage and other proteins. The expression of many genes involved in photosynthesis is higher during plant illumination compared to darkness. JA’s ability to inhibit expression of genes involved in photosynthesis could lower expression of these genes in darkness. If JA is distributed like ABA in plant cells, this compound will accumulate in chloroplasts in illuminated plants because of the increase in pH in this compartment. At night, JA accumulated in chloroplasts during the day will be released into the cytoplasm, where it could inhibit expression of genes involved in photosynthesis. The ability of JA to inhibit expression of genes involved in photosynthesis suggests that jasmonate could help reduce the plant’s capacity for carbon assimilation under conditions of excess light or carbon. The photosynthetic apparatus may absorb more light energy than can be used for photosynthesis under conditions where carbon fixation exceeds the capacity of cells to export


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or store carbon. Inhibition of genes encoding the photosynthetic apparatus under these conditions would eventually balance energy absorbing and using capacities. In the short term, JA-mediated induction of vegetative storage protein synthesis under conditions of high sugar accumulation creates a sink for carbon and nitrogen and releases phosphate from sugar phosphate pools for further carbon fixation. Excess light absorption also occurs in plants exposed to water deficit, which induces stomatal closure, making plants deficient in CO2. Under these conditions, the products of photosynthetic electron transport can no longer be used for carbon fixation, and the energy harvested by the chlorophyll antennae must be dissipated rather than used for the formation of ATP and reducing power. Some of the excess energy can be dissipated via the xanthophyll cycle or through other energy-quenching mechanisms (31). However, once the capacity of these systems is exceeded, membrane damage will occur. Lipoxygenase and other enzymes that metabolize fatty acids may protect membranes from damage by removing oxidized fatty acids. The lipoxygenase-mediated generation of JA could, in turn, induce changes in the cell that ameliorate further photochemical damage. For example, jasmonate-induced loss of chlorophyll would decrease the amount of energy absorbed by the photosynthetic apparatus. The accumulation of anthocyanins that is stimulated by JA in illuminated plants (49) could also provide some protection from excess radiation.

Flower and Fruit Development Jasmonate might be expected to play a role in formation of flowers, fruit, and seed because of the relatively high levels of this compound in developing plant reproductive tissues. The presence of jasmonate and related volatile fatty acid derivatives may be involved in insect attraction related to pollen dispersal. Other aspects of flower, fruit, and seed development that can be modulated by jasmonate include fruit ripening, fruit carotenoid composition, and expression of genes encoding seed and vegetative storage proteins. Jasmonate-stimulated tomato and apple fruit ripening most likely occurs through activation of EFE and production of ethylene (30). It is possible that jasmonate levels gradually increase in developing fruit leading to enhanced synthesis of ethylene and subsequent fruit ripening. Application of JA to tomato fruit inhibited the accumulation of lycopene and stimulated accumulation of β-carotene (99). The biochemical basis and physiological role of this JA-mediated change needs further investigation. Soybean Vsps and A. thaliana AtVsp show high expression in flowers and developing fruit (15, 110). The vegetative storage proteins may provide tem-


porary storage of carbon and nitrogen arriving at the reproductive apex for use during rapid synthesis of seed storage proteins. The AtVSP proteins were missing in flowers of coi1 mutants that are insensitive to JA but could be induced by treatment of plants with JA (12). The JA-deficient LA mutant of A. thaliana also does not express AtVsp unless plants are provided with exogenous JA (RA Creelman, M McConn, E Bell, J Browse & JE Mullet, unpublished data). Ovules of the LA-deficient mutant were viable, indicating that JA and expression of the AtVsp were not essential for seed formation. Therefore, although JA may modulate expression of genes encoding seed storage proteins (125), JA is not essential for production of viable ovules in A. thaliana. However, fatty acid mutants of A. thaliana that lack LA and JA and the coi1 JA-insensitive mutant fail to produce viable pollen unless supplied with JA (76).

Insect and Disease Resistance JA plays an important role in plant insect and disease resistance. Several lines of evidence support this conclusion. First, JA accumulates in wounded plants (29) and in plants or cell cultures treated with elicitors of pathogen defense (53). Second, JA activates genes encoding protease inhibitors that help protect plants from insect damage (62). JA also activates expression of genes encoding antifungal proteins such as thionin (9a), osmotin (129), PDF (91), and the ribosome-inactivating protein RIP60 (24). JA modulates expression of cell wall proteins such as PRP (29) that may be involved in synthesis of barriers to infection. Furthermore, JA induces genes involved in phytoalexin biosynthesis (Chs, Pal, HMGR) (25, 29) and phenolics (polyphenol oxidase; 37) that are involved in plant defense. The oxylipin pathway that leads to JA is also the source of other volatile aldehydes and alcohols that function in plant defense and wound healing. For example, the C6-aldehyde 2-hexenal completely inhibited growth of Pseudomonas syringae and E. coli (32), and C6-aldehydes and alcohols reduced aphid fecundity (59). These compounds are synthesized from 13-hydroperoxylinolenic acid via the action of hydroperoxy lyase (Figure 2). Jasmonate, wounding, and elicitors increase the expression of lipoxygenase and stimulate hydroperoxy lyase activity (5, 52). This response enhances the ability of plants to produce the six carbon compounds that contribute to plant protection. A third type of evidence for JA’s role in pest resistance comes from analysis of plants having modified levels of JA. For example, treatment of potato with JA increases resistance to Phytophthora infestans (26). The tomato mutant, JL5, which is inhibited in the conversion of 13-hydroperoxylinolenic acid


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to 12-oxo-PDA is more susceptible to damage by Manduca sexta (60). An A. thaliana mutant deficient in LA contains negligible amounts of JA and neither accumulates JA nor induces JA-responsive genes when wounded. These mutants are very susceptible to fungal gnats (M McConn, RA Creelman, E Bell, JE Mullet & J Browse, unpublished data). Treatment of the mutants with JA restored fungal gnat resistance, demonstrating an essential role for JA in resistance. Although a general role for JA in plant defense is now well established, the specific way that JA is deployed relative to other defense mechanisms in response to insects and pathogens needs further investigation. The complexity of plant defense responses and JA’s role was demonstrated in a recent study of two genes (Pdf1.1, Pdf1.2) that are involved in fungal resistance in A. thaliana. SA, an inducer of many genes involved in responses to pathogens, was able to induce PR-1 but not Pdf (91). Pdf expression was induced by JA, ethylene, and oxygen radical generators such as paraquat and rose bengal. The A. thaliana mutants, ein2 and coi1, which are blocked in their responses to ethylene and MeJA, respectively, were not altered in pathogen-mediated induction of PR-1 but blocked in accumulation of PDF. These results have several important implications. First, the oxidative burst that often accompanies plant responses to pathogens may induce JA by producing oxidized fatty acids. The oxidative burst has been linked to programmed cell death (70) responses and represents one line of plant pathogen defense. JA released from injured cells could then activate further defense responses including systemic ones. Second, this study shows that pathogens such as A. brassicola can trigger at least two defense pathways, one involving SA and one involving JA and ethylene. This observation is consistent with reports that JA and ethylene synergistically induce genes that are involved in plant defense (129). Furthermore, SA is an inhibitor of JA biosynthesis and action (36, 86). This interaction may allow the plant to modulate the relative amount of SA- and JA-inducible defenses as a function of time after attack or in response to specific pathogens or herbivores.

JA’s Dual Role in Development and Defense In this final section, we attempt to provide a rationale for JA’s dual role in plant development and defense. This discussion starts with the following questions: Why are genes encoding vegetative storage proteins and genes such as Pin2 that are involved in insect defense regulated similarly? Why are jasmonate-inducible genes involved in plant defense also regulated by sugars, phosphate, and auxin? Why are JA levels high in young apical sinks and especially reproductive structures but inducible by wounding or elicitation in older parts of the plant?


The regulation of expression of VspB, which encodes a soybean vegetative storage protein, and Pin2, which encodes a tomato protease inhibitor involved in plant defense, is remarkably similar. Both genes are highly expressed in apical regions of vegetative plants and in flowers and reproductive tissues. Both genes are induced upon wounding and application of JA. Expression of these genes is much higher when JA/wounding treatment occurs in illuminated plants and induction of gene expression by JA is synergistically stimulated by sugars and inhibited by phosphate and auxin (34, 66). The products of these two genes are targeted to vacuoles, and large amounts of the two proteins often accumulate in plant cells. The similarity of expression of these two genes and localization of their gene products suggest that they play nearly identical roles in plants, except for the activity of the encoded proteins. For many reasons it is not surprising that some proteins involved in plant defense also function as vegetative storage proteins. Unlike seed storage proteins that have to be stored at high density in nearly dehydrated seeds, vegetative storage proteins accumulate in fully hydrated plants containing large vacuoles. Therefore, the constraints on proteins that serve as VSPs are fewer than for seed storage proteins. Plants need to accumulate large amounts of vegetative storage proteins and proteins involved in defense without disrupting metabolism. Sequestering these proteins in vacuoles may help accomplish this. Moreover, both types of proteins are mobilized to recover amino acids when the need for storage or defense is gone. Therefore many proteins involved in plant defense, in particular those that are sequestered in vacuoles for action when ingested, are ideally suited for a role as vegetative storage proteins. Because VSPs that can also aid in plant defense have additional value for the plant, perhaps most VSPs will eventually be found to serve a role in plant defense. Elevated expression of JA-responsive genes such as Pin2 in vegetative apices, young leaves, and reproductive structures may simply be the consequence of their role as vegetative storage proteins. However, the accumulation of defensive proteins in these tissues also provides the plant with a preformed deterrent to herbivory and disease in regions of the plant critical to survival and reproduction. The differential accumulation of compounds involved in defense in plant tissues of high value is consistent with the “optimal defense theory” described by researchers working in the area of chemical ecology (7). This theory predicts that defense should be allocated to plant parts that contribute significantly to a plant’s fitness and have a high probability of attack. A second class of chemical defense theory, the “C/N theory,” emphasizes the fact that allocation of carbon and nitrogen to defense may occur in competition with the use of these resources for growth and development (7). This theory


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rationalizes why plants may induce carbon-rich defenses (i.e. tannins, phenols) vs nitrogen-rich defenses (i.e. proteins) depending on nutrient availability. The inducible or activated defense system mediated by JA is consistent with the C/N theory of chemical defense in several ways. In older leaves, JA levels and JA-inducible defense is activated in response to wounding or elicitors, thus limiting the allocation of nutrients to defense to situations where this is required. In addition, expression of Pin2 is regulated not only by the presence of JA but also by sugars (63, 88). Dual regulation by sugars and JA is also observed in other genes such as Chs that are involved in plant defense. Furthermore, Vsp expression and perhaps genes such as Pin2 is inhibited when plants are grown in limiting nitrogen conditions (112). This type of regulation would minimize the allocation of carbon and nitrogen to proteins and secondary metabolites involved in plant defense under conditions where nutrients are limiting.

CONCLUDING REMARKS The volume of publications and reviews on jasmonate over the past decade documents the increasing interest in this compound and its role in plants. Research on this topic has solidified our understanding of the chemistry and biosynthetic pathway of jasmonates. However, additional research is needed into the mechanisms that regulate the synthesis of JA in plants during development and in response to wounding and oligosaccharides and peptides that modulate JA biosynthesis. Transgenic plants containing sense/antisense constructs of genes in the biosynthetic pathway and mutants deficient in JA will help provide definitive information. Our understanding of the JA signal transduction pathway will rapidly advance as genes identified through analysis of JA-insensitive mutants. Recent direct evidence for JA’s role in plant defense confirms this role for JA originally suggested from studies of soybean cell cultures. The deployment of JA-inducible genes as part of the complex plant defense system will be a topic of intense future study. Insight gained from these studies should lead to better design of durable plant defense and improved utilization of proteins and genes from nonplant sources for plant protection. ACKNOWLEDGMENTS

We thank our colleagues in this field for their insightful comments and for sending recent publications on this topic. Because of length restrictions and the availability of other reviews, we apologize in advance for being unable to cite all of the excellent publications on this topic. The authors’ research on jasmon-


ate has been supported by the USDA National Research Initiative, the National Science Foundation, and the Texas Agricultural Experiment Station. Visit the Annual Reviews home page at http://www.annurev.org

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