The power of mutants for investigating jasmonate biosynthesis and signaling

Mutant analysis includes approaches that range from traditional screening of mutant populations (forward genetics), to identifying mutations in known genes (reverse genetics), to examining the effects of site-specific mutations that encode modified proteins. All these methodologies have been applied to study jasmonate synthesis and signaling, and their use has led to important discoveries. The fad3 fad7 fad8 mutant of Arabidopsis, and other mutants defective in jasmonate synthesis, revealed the roles of jasmonate in flower development and plant defense against necrotrophic fungal pathogens. The coi1 mutant identified the F-box protein that is now known to be the receptor for jasmonoyl-isoleucine, the active form of jasmonate hormone. Investigations of how JASMONATE-ZIM DOMAIN (JAZ) proteins bind to COI1 and facilitate jasmonate perception have relied on the jai3 mutant, on JAZΔJas constructs, and on site-specific mutations in the Jas and ZIM domains of these proteins.     

Role of beta-oxidation in jasmonate biosynthesis and systemic wound signaling in tomato

Jasmonic acid (JA) is a lipid-derived signal that regulates plant defense responses to biotic stress. Here, we report the characterization of a JA-deficient mutant of tomato (Lycopersicon esculentum) that lacks local and systemic expression of defensive proteinase inhibitors (PIs) in response to wounding. Map-based cloning studies demonstrated that this phenotype results from loss of function of an acyl-CoA oxidase (ACX1A) that catalyzes the first step in the peroxisomal beta-oxidation stage of JA biosynthesis. Recombinant ACX1A exhibited a preference for C12 and C14 straight-chain acyl-CoAs and also was active in the metabolism of C18 cyclopentanoid-CoA precursors of JA. The overall growth, development, and reproduction of acx1 plants were similar to wild-type plants. However, the mutant was compromised in its defense against tobacco hornworm (Manduca sexta) attack. Grafting experiments showed that loss of ACX1A function disrupts the production of the transmissible signal for wound-induced PI expression but does not affect the recognition of this signal in undamaged responding leaves. We conclude that ACX1A is essential for the beta-oxidation stage of JA biosynthesis and that JA or its derivatives is required both for antiherbivore resistance and the production of the systemic wound signal. These findings support a role for peroxisomes in the production of lipid-based signaling molecules that promote systemic defense responses.     

Octadecanoid and jasmonate signaling in tomato (Lycopersicon esculentum Mill.) leaves: endogenous jasmonates do not induce jasmonate biosynthesis

Jasmonates and their precursors, the octadecanoids, are signals in stress-induced alteration of gene expression. Several mRNAs coding for enzymes of jasmonic acid (JA) biosynthesis are up-regulated upon JA treatment or endogenous increase of the JA level. Here we investigated the positive feedback of endogenous JA on JA formation, as well as its beta-oxidation steps. JA-responsive gene expression was recorded in terms of proteinase inhibitor2 (pin2) mRNA accumulation. JA formed upon treatment of tomato (Lycopersicon esculentum cv. Moneymaker) leaves with JA derivatives carrying different lengths of the carboxylic acid side chain was quantified by gas chromatography-mass spectrometry (GC-MS). The data revealed that beta-oxidation of the side chain occurs up to a butyric acid moiety. The amount of JA formed from side-chain modified JA derivatives correlated with pin2-mRNA accumulation. JA derivatives with a carboxylic side chain of 3, 5 or 7 carbon atoms were unable to form JA and to express on pin2, whereas even-numbered derivatives were active. After treatment of tomato leaves with (10-(2)H)-(-)-12-oxophytoenoic acid, (4-(2)H)-(-)-JA and its methyl ester were formed and could be quantified separately from the endogenously nonlabeled JA pool by GC-MS analysis via isotopic discrimination. The level of 8 nmol per g fresh weight JA and its methyl ester originated exclusively from labeled 12-oxophytoenic acid. This and further data indicate that endogenous synthesis of the JA precursor 12-oxophytodienoic acid, as well as of JA and its methyl ester, are not induced in tomato leaves, suggesting that positive feedback in JA biosynthesis does not function in vivo.     

Chemical genetic approaches for the elucidation of signaling pathways

New chemical methods that use small molecules to perturb cellular function in ways analogous to genetics have recently been developed. These approaches include both synthetic methods for discovering small molecules capable of acting like genetic mutations, and techniques that combine the advantages of genetics and chemistry to optimize the potency and specificity of small-molecule inhibitors. Both approaches have been used to study protein function in vivo and have provided insights into complex signaling cascades.     

Methyl jasmonate signaling and signal crosstalk between methyl jasmonate and abscisic acid in guard cells

Plants tightly control stomatal aperture in response to various environmental changes. A drought-inducible phytohormone, abscisic acid (ABA), triggers stomatal closure and ABA signaling pathway in guard cells has been well studied. Similar to ABA, methyl jasmonate (MeJA) induces stomatal closure in various plant species but MeJA signaling pathway is still far from clear. Recently we found that Arabidopsis calcium dependent protein kinase CPK6 functions as a positive regulator in guard cell MeJA signaling and provided new insights into cytosolic Ca²⁺-dependent MeJA signaling. Here we discuss the MeJA signaling and also signal crosstalk between MeJA and ABA pathways in guard cells.Key words: methyl jasmonate, abscisic acid, guard cell, reactive oxygen species, nitric oxide, calciumStomata, which are formed by pairs of specialized cells called guard cells, control gas exchanges and transpirational water loss. Guard cells can shrink and swell in response to various physiological stimuli, resulting in stomatal closing and opening.^1,2 To optimize growth under various environmental conditions, plants have developed fine-tuned signal pathway in guard cells. Abscisic acid (ABA) is synthesized under drought stress and induces stomatal closure to reduce transpirational water loss.² ABA signal transduction in guard cells has been widely studied. ABA induces increases of various second messengers such as cytosolic Ca²⁺, reactive oxygen species (ROS) and nitric oxide (NO) in guard cells. These early signal components finally evoke ion efflux through plasma membrane ion channels, resulting in reduction of guard cell turgor pressure.Jasmonates are plant hormones synthesized via the octadecanoid pathway and regulate various physiological processes in plants such as pollen maturation, tendril coiling, senescence and responses to wounding and pathogen attacks.³ Similar to ABA, jasmonates also trigger stomatal closure and the response is conserved among various plant species including Arabidopsis thaliana,⁴ Hordeum vulgare,⁵ Commelina benghalensis,⁶ Vicia faba,⁷ Nicotiana glauca,⁸ Paphiopedilum Supersuk⁹ and Paphiopedilum tonsum.⁹ A volatile methyl ester of jasmonic acid (JA), methy jasmonate (MeJA), has been widely used for studying jasmonate signaling pathway. To date, pharmacological and reverse genetic approaches have revealed many important signal components involved in MeJA-induced stomatal closure and suggest a signal crosstalk between MeJA and ABA in guard cells. In this review, we mainly focus on the three important second messengers, ROS, NO and cytosolic Ca²⁺ and discuss recent advance about MeJA signaling and signal interaction between MeJA and ABA in guard cells.     

Transient pulse formation in jasmonate signaling pathway

The jasmonate (JA) signaling pathway in plants is activated as defense response to a number of stresses like attacks by pests or pathogens and wounding by animals. Some recent experiments provide significant new knowledge on the molecular detail and connectivity of the pathway. The pathway has two major components in the form of feedback loops, one negative and the other positive. We construct a minimal mathematical model, incorporating the feedback loops, to study the dynamics of the JA signaling pathway. The model exhibits transient gene expression activity in the form of JA pulses in agreement with experimental observations. The dependence of the pulse amplitude, duration and peak time on the key parameters of the model is determined computationally. The deterministic and stochastic aspects of the pathway dynamics are investigated using both the full mathematical model and a reduced version of it. We also compare the mechanism of pulse formation with the known mechanisms of pulse generation in some bacterial and viral systems.     

Enzymes of jasmonate biosynthesis occur in tomato sieve elements

The allene oxide cyclase (AOC) is a plastid-located enzyme in the biosynthesis of the signaling compound jasmonic acid (JA). In tomato, AOC occurs specifically in ovules and vascular bundles [Hause et al. (2000) Plant J. 24; 113]. Immunocytological analysis of longitudinal sections of petioles and flower stalks revealed the occurrence of AOC in companion cells (CC) and sieve elements (SE). Electron microscopic analysis led to the conclusion that the AOC-containing structures of SE are plastids. AOC was not detected in SE of 35S::AOCantisense plants. The enzymes preceding AOC in JA biosynthesis, the allene oxide synthase (AOS) and the lipoxygenase, were also detected in SE. In situ hybridization showed that the SE are free of AOC-mRNA suggesting AOC protein traffic from CC to SE via plasmodesmata. A control by in situ hybridization of AOS mRNA coding for a protein with a size above the exclusion limit of plasmodesmata indicated mRNA in CC and SE. The data suggest that SE carry the capacity to form 12-oxo-phytodienoic acid, the unique precursor of JA. Together with preferential generation of JA in vascular bundles [Stenzel et al. (2003) Plant J. 33: 577], the data support a role of JA in systemic wound signaling.     

Chemical genetic blockade of transformation reveals dependence on aberrant oncogenic signaling

BACKGROUND: Our understanding of protein kinase inhibition in the treatment of cancer is clearly limited by the lack of inhibitors that selectively block a single kinase implicated in neoplastic transformation. One approach to developing specific inhibitors is to engineer in protein kinases silent mutations that allow selective inhibition while retaining kinase activity. Because it is implicated in a large number of malignancies, EGFR provides an attractive target for such selective kinase inhibition.RESULTS: We generated an inhibitor-sensitized allele of the transforming receptor tyrosine kinase v-erbB. Transformation of immortalized rodent fibroblasts by sensitized versions of v-erbB (v-erbB-as1) was blocked by 1-napthyl PP1 (NaPP1), a cell-permeable ATP-competitive inhibitor. NaPP1 also reversed morphological transformation by v-erbB-as1. Signaling through MAP kinase and PI(3) kinase was initially blocked by inhibitor treatment and then recovered to levels comparable to those in nontransformed cells. Surprisingly, NaPP1-treated v-erbB-as1 cells failed to re-enter the cell cycle, showed decreased levels of D- and A-type cyclins, and showed increased levels of p27. To extend this result, we showed that NaPP1 treatment of v-Src-as1 cells also led to cell cycle arrest. Arrested cells could be rescued with a conditional allele of Raf or by transduction of a constitutive allele of cyclin D1.CONCLUSIONS: These data suggest that mammalian cells can become dependent on aberrant oncogenic signaling; this dependency renders them incapable of returning to a normal, proliferative phenotype.     

Impact of methyl jasmonate on squalene biosynthesis in microalga Schizochytrium mangrovei

Squalene is an effective antioxidant and a potential chemopreventive agent. In this work, the effect of methyl jasmonate (MJA) on squalene biosynthesis in microalga Schizochytrium mangrovei was investigated. The maximum squalene content (1.17 ± 0.06 mg/g cell dry weight, DW) reached during the next 3 h after MJA treatment (0.1 mM) at 48 h of cultivation, which was 60% higher than that of control. The activity of squalene synthase (SS) increased 2-fold over control at this point. The maximum cholesterol content of 0.45 ± 0.03 mg/g DW was reached at hour 51 when MJA concentration was 0.4 mM, whereas the squalene content was lower at this point. The observations suggested that the increased squalene content was resulted from an increased activity of SS. MJA could be used to regulate the key enzymes in squalene biosynthetic pathway for the increased production of this compound in thraustochytrids. This research also provided novel information on the stimulation effect of methyl jasmonate on the biosynthesis of essential intermediate involved in the primary metabolism in microorganism.     

Catalases negatively regulate methyl jasmonate signaling in guard cells

Methyl jasmonate (MeJA)-induced stomatal closure is accompanied by the accumulation of hydrogen peroxide (H?O?) in guard cells. In this study, we investigated the roles of catalases (CATs) in MeJA-induced stomatal closure using cat mutants cat2, cat3-1 and cat1 cat3, and the CAT inhibitor, 3-aminotriazole (AT). When assessed with 2',7'-dichlorodihydrofluorescein, the reduction of catalase activity by means of mutations and the inhibitor accumulated higher basal levels of H?O? in guard cells whereas they did not affect stomatal aperture in the absence of MeJA. In contrast, the cat mutations and the treatment with AT potentiated MeJA-induced stomatal closure and MeJA-induced H?O? production. These results indicate that CATs negatively regulate H?O? accumulation in guard cells and suggest that inducible H?O? production rather than constitutive elevation modulates stomatal apertures in Arabidopsis.     

Ion channel-forming alamethicin is a potent elicitor of volatile biosynthesis and tendril coiling. Cross talk between jasmonate and salicylate signaling in lima bean

Alamethicin (ALA), a voltage-gated, ion channel-forming peptide mixture from Trichoderma viride, is a potent elicitor of the biosynthesis of volatile compounds in lima bean (Phaseolus lunatus). Unlike elicitation with jasmonic acid or herbivore damage, the blend of substances emitted comprises only the two homoterpenes, 4,11-dimethylnona-1,3,7-triene and 4,8,12-trimethyltrideca-1,3,7,11-tetraene, and methyl salicylate. Inhibition of octadecanoid signaling by aristolochic acid and phenidone as well as mass spectrometric analysis of endogenous jasmonate demonstrate that ALA induces the biosynthesis of volatile compounds principally via the octadecanoid-signaling pathway (20-fold increase of jasmonic acid). ALA also up-regulates salicylate biosynthesis, and the time course of the production of endogenous salicylate correlates well with the appearance of the methyl ester in the gas phase. The massive up-regulation of the SA-pathway (90-fold) interferes with steps in the biosynthetic pathway downstream of 12-oxophytodienoic acid and thereby reduces the pattern of emitted volatiles to compounds previously shown to be induced by early octadecanoids. ALA also induces tendril coiling in various species like Pisum, Lathyrus, and Bryonia, but the response appears to be independent from octadecanoid biosynthesis, because inhibitors of lipoxygenase and phospholipase A(2) do not prevent the coiling reaction.     

Cooperation and functional diversification of two closely related galactolipase genes for jasmonate biosynthesis

Jasmonic acid (JA) plays pivotal roles in diverse plant biological processes, including wound response. Chloroplast lipid hydrolysis is a critical step for JA biosynthesis, but the mechanism of this process remains elusive. We report here that DONGLE (DGL), a homolog of DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1), encodes a chloroplast-targeted lipase with strong galactolipase and weak phospholipase A(1) activity. DGL is expressed in the leaves and has a specific role in maintaining basal JA content under normal conditions, and this expression regulates vegetative growth and is required for a rapid JA burst after wounding. During wounding, DGL and DAD1 have partially redundant functions for JA production, but they show different induction kinetics, indicating temporally separated roles: DGL plays a role in the early phase of JA production, and DAD1 plays a role in the late phase of JA production. Whereas DGL and DAD1 are necessary and sufficient for JA production, phospholipase D appears to modulate wound response by stimulating DGL and DAD1 expression.     

Omega hydroxylated JA-Ile is an endogenous bioactive jasmonate that signals through the canonical jasmonate signaling pathway

Jasmonates are fatty acid derivatives that control several plant processes including growth, development and defense. Despite the chemical diversity of jasmonates, only jasmonoyl-L-isoleucine (JA-Ile) has been clearly characterized as the endogenous ligand of the jasmonate co-receptors (COI1-JAZs) in higher plants. Currently, it is accepted that ω-hydroxylation of JA-Ile leads to inactivation of the molecule. This study shows that ω-hydroxylated JA-Ile (12-OH-JA-Ile) retains bioactivity and signals through the canonical JA-pathway. The results suggest that 12-OH-JA-Ile differentially activates a subset of JA-Ile co-receptors that may control and/or modulate particular jasmonate dependent responses. It is proposed that after a strong immune response mediated by JA-Ile, the ω-hydroxylated form modulates JA-Ile activated processes thereby improving plant resilience.