Occurrence of hydroxylated jasmonic acids in leaflets of Solanum demissum plants grown under long- and short-day conditions

Under short-day (SD) conditions both 11-OH-jasmonic acid (11-OH-JA) and a smaller quantity of 12-OH-JA occurred in leaflets of Solanum demissum Lindl. Plants which had formed tubers. This is the first time that 11-OH-JA has been detected as a native substance in higher plants. Under long-day (LD) conditions no tubers were formed and none of these compounds were detectable. A positive correlation was found between the occurrence of 11-OH-JA and 12-OH-JA in leaflets of S. demissum and tuber formation, but a causal relation has yet to be proved. The (-)-JA content in leaflets was not significantly different under short and long days. Mild stress applied to detached SD and LD leaflets caused a rapid accumulation of JA in these leaflets. Upon this treatment an increase in the levels of hydroxylated JAs was detected in SD leaflets only.
JA was a potent promotor of tuber formation in vitro in S. demissum explants. Lipoxygenase (LOX: EC 1.13.11.12) is involved in the biosynthesis of JA. Under SD conditions, application of salicylhydroxamic acid (SHAM), an inhibitor of LOX activity, to the roots did not prevent tuber formation in vivo. It is suggested that daylength controls the hydroxylation of JA. The enzyme(s), responsible for the hydroxylation of JA, would only be effective under SD conditions.     

Facile preparation of optically active jasmonates and their biological activities in rice

A facile and efficient method has been developed for the optical resolution of racemic jasmonic acid (JA) on a relatively large scale and was successfully utilized for the preparation of optically pure (+)-JA and (?)-JA. We indicated that (+)-JA has lower growth inhibitory activity than (?)-JA in the rice seedling growth test and confirmed in line with an earlier observation that their respective biologically-active forms, (+)-JA-Ile and (?)-JA-Ile, show comparable inhibitory activities. We compared the metabolism of (+)-JA and (?)-JA into (+)-JA-Ile and (?)-JA-Ile, respectively, in the JA-deficient rice cpm2, and found that the exogenously applied (+)-JA was metabolized to the corresponding Ile conjugate less efficiently as compared with (?)-JA. Such metabolic rate difference may cause a discrepancy between biological potencies of (+)-JA and (?)-JA in rice.

Abbreviations: FW: fresh weight; Ile: isoleucine; JA: jasmonic acid; JA-Ile: jasmonoyl-l-isoleucine; LC-ESI-MS/MS: liquid chromatography and electrospray ionization tandem mass spectrometry; MeJA: methyl jasmonate; OPDA: 12-oxophytodienoic acid     

Octadecanoid-derived alteration of gene expression and the "oxylipin signature" in stressed barley leaves. Implications for different signaling pathways

Stress-induced gene expression in barley (Hordeum vulgare cv Salome) leaves has been correlated with temporally changing levels of octadecanoids and jasmonates, quantified by means of gas chromatography/mass spectrometry-single ion monitoring. Application of sorbitol-induced stress led to a low and transient rise of jasmonic acid (JA), its precursor 12-oxophytodienoic acid (OPDA), and the methyl esters JAME and OPDAME, respectively, followed by a large increase in their levels. JA and JAME peaked between 12 and 16 h, about 4 h before OPDA and OPDAME. However, OPDA accumulated up to a 2.5-fold higher level than the other compounds. Dihomo-JA and 9, 13-didehydro-OPDA were identified as minor components. Kinetic analyses revealed that a transient threshold of jasmonates or octadecanoids is necessary and sufficient to initiate JA-responsive gene expression. Although OPDA and OPDAME applied exogenously were metabolized to JA in considerable amounts, both of them can induce gene expression, as evidenced by those genes that did not respond to endogenously formed JA. Also, coronatine induces JA-responsive genes independently from endogenous JA. Application of deuterated JA showed that endogenous synthesis of JA is not induced by JA treatment. The data are discussed in terms of distinct signaling pathways.     

Jasmonates in flower and seed development

Jasmonates are ubiquitously occurring lipid-derived signaling compounds active in plant development and plant responses to biotic and abiotic stresses. Upon environmental stimuli jasmonates are formed and accumulate transiently. During flower and seed development, jasmonic acid (JA) and a remarkable number of different metabolites accumulate organ- and tissue specifically. The accumulation is accompanied with expression of jasmonate-inducible genes. Among these genes there are defense genes and developmentally regulated genes. The profile of jasmonate compounds in flowers and seeds covers active signaling molecules such as JA, its precursor 12-oxophytodienoic acid (OPDA) and amino acid conjugates such as JA-Ile, but also inactive signaling molecules occur such as 12-hydroxy-JA and its sulfated derivative. These latter compounds can occur at several orders of magnitude higher level than JA. Metabolic conversion of JA and JA-Ile to hydroxylated compounds seems to inactivate JA signaling, but also specific functions of jasmonates in flower and seed development were detected. In tomato OPDA is involved in embryo development. Occurrence of jasmonates, expression of JA-inducible genes and JA-dependent processes in flower and seed development will be discussed.     

Kinetics of the Accumulation of Jasmonic Acid and Its Derivatives in Systemic Leaves of Tobacco (Nicotiana tabacum cv. Xanthi nc) and Translocation of Deuterium-Labeled Jasmonic Acid from the Wounding Site to the Systemic Site

In plants, the mobile signal needed for wound-induced systemic acquired resistance (WSR) has been elusive. The signal compound involved in WSR is supposed to be JA or its derivatives. On the basis of kinetic study of the accumulation of JA or its derivatives, it was discovered that JA, JA-Ile, tuberonic acid (TA, 12-OH epi-JA), and tuberonic acid glucoside (TAG) accumulated in systemic tissues in response to mechanical wounding stress in the tobacco plant (Nicotiana tabacum). Attempts to recover deuterium-labeled JA in systemic leaves after feeding the wounded leaves with deuterium-labeled JA were successfully done. It was also found that the translocated deuterium-labeled JA was metabolized to TA in systemic leaves under feeding of deuterium-labeled JA to the wounding leaves.     

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.     

A defect in glyoxysomal fatty acid beta-oxidation reduces jasmonic acid accumulation in Arabidopsis.

The final steps of jasmonic acid (JA) biosynthesis are thought to involve peroxisomal beta-oxidation, but this has not been directly demonstrated. The last and key step in fatty acid beta-oxidation is catalyzed by 3-ketoacyl-CoA thiolase (KAT) (EC 2.3.1.16). A mutant of Arabidopsis thaliana ecotype Landsberg erecta, which lacks a functional KAT protein and is defective in glyoxysomal fatty acid beta-oxidation has been reported. In this study, the mutant was found to accumulate reduced level of JA in both its wounded cotyledons and leaves, while only the cotyledons accumulate 3-oxo-2-(pent-2'-enyl)-cyclopentane-1-octanoic acid (OPC-8:0). This indicates that a defect in one of the thiolase isoenzymes impairs beta-oxidation of OPC-8:0 to JA. The mutant had sufficient thiolase activity for the synthesis of JA in the unwounded but not in the wounded tissues. Activities of the enzymes in the JA pathway that catalyze the steps, which precede beta-oxidation were not altered by the mutation in a thiolase protein. Thus, reduced levels of JA in the wounded tissues of the mutant were attributed to the defect in a thiolase protein.     

Allene oxide synthase: a major control point in Arabidopsis thaliana octadecanoid signalling

The analysis of allene oxide synthase (AOS) mRNA levels, of AOS polypeptide levels and specific enzymatic activities, as well as the quantitative determination of the levels of the octadecanoids cis-12-oxophytodienoic acid (cis-OPDA) and JA following a number of treatments, has shown that AOS is a regulatory site in octadecanoid biosynthesis in A. thaliana. AOS activity, mRNA and polypeptide levels are increased in wounded leaves locally and systemically. The methyl esters of OPDA or JA (OPDAME, JAME) and coronatine, are strong inducers of AOS mRNA, polypeptide and enzymatic activity. Ethephon also induces AOS activity. Salicylic acid (SA) was an inducer of AOS activity while abscisic acid (ABA) had no effect. At the level of the octadecanoids, the consequences of induction of AOS by the different inducers were distinctly different, depending on the nature of the inducer. Wounding led to a strong, bi-phasic accumulation of JA in wounded leaves and to a less pronounced increase in JA-levels in systemic leaves. Levels of OPDA changed very little in wounded leaves and remained constant or even declined in systemic leaves. Ethephon treatment resulted in a strong, transient increase in JA-levels kinetically coinciding with the second, more pronounced peak in wound-induced JA. In SA-treated leaves, the level of cis-OPDA increased throughout the experimental period while there was no effect on JA levels during the first 24 h following treatment and only a slight accumulation after 48 h. Clearly, mechanisms in addition to regulating substrate (LA) availability and the regulation of AOS accumulation control the output of the octadecanoid pathway.     

Expression of a Flax Allene Oxide Synthase cDNA Leads to Increased Endogenous Jasmonic Acid (JA) Levels in Transgenic Potato Plants but Not to a Corresponding Activation of JA-Responding Genes

Both jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are thought to be significant components of the signaling pathway regulating the expression of plant defense genes in response to various stresses. JA and MeJA are plant lipid derivatives synthesized from [alpha]-linolenic acid by a lipoxygenase-mediated oxygenation leading to 13-hydroperoxylinolenic acid, which is subsequently transformed by the action of allene oxide synthase (AOS) and additional modification steps. AOS converts lipoxygenase-derived fatty acid hydroperoxide to allene epoxide, which is the precursor for JA formation. Overexpression of flax AOS cDNA under the regulation of the cauliflower mosaic virus 35S promoter in transgenic potato plants led to an increase in the endogenous level of JA. Transgenic plants had six- to 12-fold higher levels of JA than the nontransformed plants. Increased levels of JA have been observed when potato and tomato plants are mechanically wounded. Under these conditions, the proteinase inhibitor II (pin2) genes are expressed in the leaves. Despite the fact that the transgenic plants had levels of JA similar to those found in nontransgenic wounded plants, pin2 genes were not constitutively expressed in the leaves of these plants. Transgenic plants with increased levels of JA did not show changes in water state or in the expression of water stress-responsive genes. Furthermore, the transgenic plants overexpressing the flax AOS gene, and containing elevated levels of JA, responded to wounding or water stress by a further increase in JA and by activating the expression of either wound- or water stress-inducible genes. Protein gel blot analysis demonstrated that the flax-derived AOS protein accumulated in the chloroplasts of the transgenic plants.     

Substrate specificity and products of side-reactions catalyzed by jasmonate:amino acid synthetase (JAR1)

Jasmonate:amino acid synthetase (JAR1) is involved in the function of jasmonic acid (JA) as a plant hormone. It catalyzes the synthesis of several JA-amido conjugates, the most important of which appears to be JA-Ile. Structurally, JAR1 is a member of the firefly luciferase superfamily that comprises enzymes that adenylate various organic acids. This study analyzed the substrate specificity of recombinant JAR1 and determined whether it catalyzes the synthesis of mono- and dinucleoside polyphosphates, which are side-reaction products of many enzymes forming acyl approximately adenylates. Among different oxylipins tested as mixed stereoisomers for substrate activity with JAR1, the highest rate of conversion to Ile-conjugates was observed for (+/-)-JA and 9,10-dihydro-JA, while the rate of conjugation with 12-hydroxy-JA and OPC-4 (3-oxo-2-(2Z-pentenyl)cyclopentane-1-butyric acid) was only about 1-2% that for (+/-)-JA. Of the two stereoisomers of JA, (-)-JA and (+)-JA, rate of synthesis of the former was about 100-fold faster than for (+)-JA. Finally, we have demonstrated that (1) in the presence of ATP, Mg(2+), (-)-JA and tripolyphosphate the ligase produces adenosine 5'-tetraphosphate (p(4)A); (2) addition of isoleucine to that mixture halts the p(4)A synthesis; (3) the enzyme produces neither diadenosine triphosphate (Ap(3)A) nor diadenosine tetraphosphate (Ap(4)A) and (4) Ap(4)A cannot substitute ATP as a source of adenylate in the complete reaction that yields JA-Ile.     

Resistance to Magnaporthe grisea in transgenic rice with suppressed expression of genes encoding allene oxide cyclase and phytodienoic acid reductase

Linolenic acid (18:3) and its derivative jasmonic acid (JA) are important molecules in disease resistance in many dicotyledonous plants. We have previously used 18:3- and JA-deficient rice (F78Ri) to investigate the roles of fatty acids and their derivatives in resistance to the blast fungus Magnaporthe grisea [A. Yara, T. Yaeno, J.-L. Montillet, M. Hasegawa, S. Seo, K. Kusumi, K. Iba, Enhancement of disease resistance to Magnaporthe grisea in rice by accumulation of hydroxy linoleic acid, Biochem. Biophys. Res. Commun. 370 (2008) 344-347; A. Yara, T. Yaeno, M. Hasegawa, H. Seto, J.-L. Montillet, K. Kusumi, S. Seo, K. Iba, Disease resistance against Magnaporthe grisea is enhanced in transgenic rice with suppression of ω-3 fatty acid desaturases, Plant Cell Physiol. 48 (2007) 1263-1274]. However, because F78Ri plants are suppressed in the first step of the JA biosynthetic pathway, we could not confirm the specific contribution of JA to disease resistance. In this paper, we generated two JA-deficient rice lines (AOCRi and OPRRi) with suppressed expression of the genes encoding allene oxide cyclase (AOC) and 12-oxo-phytodienoic acid reductase (OPR), which catalyze late steps in the JA biosynthetic pathway. The levels of disease resistance in the AOCRi and OPRRi lines were equal to that in wild-type plants. Our data suggest that resistance to M. grisea is not dependent on JA synthesis.     

Effects of theobroxide, a natural product, on the level of endogenous jasmonoids

The natural potato microtuber inducing substance, theobroxide, strongly induces the formation of tuber of potato (Solanum tuberosum L.) and flower bud of morning glory (Pharbitis nil) plants under non-inducing conditions (long days) (Yoshihara et al., 2000). In the present study, theobroxide was evaluated for its effect on the level of endogenous jasmonoids in different tissues of such two plants. An in vitro bioassay using cultures of single-node segments of potato stems was performed with the supplement of theobroxide in the medium. The endogenous jasmonic acid (JA) and its analogue tuberonic acid (TA, 12-hydroxyjasmonic acid) in segments and microtubers were quantitatively analyzed. The increase in the endogenous JA level caused by theobroxide was observed in both segments and microtubers. Endogenous TA was only detected in segments, and the content increased with the concentration of theobroxide. As for morning glory, the whole plant was sprayed with theobroxide for 1 approximately 5 weeks under different photoperiods and endogenous JA in the leaves was quantitatively analyzed. Theobroxide spraying increased the level of endogenous JA in the leaves of the plants grown under both long and short days.     

12-hydroxyjasmonic acid glucoside is a COI1-JAZ-independent activator of leaf-closing movement in Samanea saman

Jasmonates are ubiquitously occurring plant growth regulators with high structural diversity that mediate numerous developmental processes and stress responses. We have recently identified 12-O-β-D-glucopyranosyljasmonic acid as the bioactive metabolite, leaf-closing factor (LCF), which induced nyctinastic leaf closure of Samanea saman. We demonstrate that leaf closure of isolated Samanea pinnae is induced upon stereospecific recognition of (-)-LCF, but not by its enantiomer, (+)-ent-LCF, and that the nonglucosylated derivative, (-)-12-hydroxyjasmonic acid also displays weak activity. Similarly, rapid and cell type-specific shrinkage of extensor motor cell protoplasts was selectively initiated upon treatment with (-)-LCF, whereas flexor motor cell protoplasts did not respond. In these bioassays related to leaf movement, all other jasmonates tested were inactive, including jasmonic acid (JA) and the potent derivates JA-isoleucine and coronatine. By contrast, (-)-LCF and (-)-12-hydroxyjasmonic acid were completely inactive with respect to activation of typical JA responses, such as induction of JA-responsive genes LOX2 and OPCL1 in Arabidopsis (Arabidopsis thaliana) or accumulation of plant volatile organic compounds in S. saman and lima bean (Phaseolus lunatus), generally considered to be mediated by JA-isoleucine in a COI1-dependent fashion. Furthermore, application of selective inhibitors indicated that leaf movement in S. saman is mediated by rapid potassium fluxes initiated by opening of potassium-permeable channels. Collectively, our data point to the existence of at least two separate JA signaling pathways in S. saman and that 12-O-β-D-glucopyranosyljasmonic acid exerts its leaf-closing activity through a mechanism independent of the COI1-JAZ module.     

The jasmonate-induced expression of the Nicotiana tabacum leaf lectin

Previous experiments with tobacco (Nicotiana tabacum L. cv Samsun NN) plants revealed that jasmonic acid methyl ester (JAME) induces the expression of a cytoplasmic/nuclear lectin in leaf cells and provided the first evidence that jasmonates affect the expression of carbohydrate-binding proteins in plant cells. To corroborate the induced accumulation of relatively large amounts of a cytoplasmic/nuclear lectin, a detailed study was performed on the induction of the lectin in both intact tobacco plants and excised leaves. Experiments with different stress factors demonstrated that the lectin is exclusively induced by exogeneously applied jasmonic acid and JAME, and to a lesser extent by insect herbivory. The lectin concentration depends on leaf age and the position of the tissue in the leaf. JAME acts systemically in intact plants but very locally in excised leaves. Kinetic analyses indicated that the lectin is synthesized within 12 h exposure time to JAME, reaching a maximum after 60 h. After removal of JAME, the lectin progressively disappears from the leaf tissue. The JAME-induced accumulation of an abundant nuclear/cytoplasmic lectin is discussed in view of the possible role of this lectin in the plant.     

Involvement of Jasmonic Acid in Elicitor-Induced Phytoalexin Production in Suspension-Cultured Rice Cells

It has been suggested that jasmonic acid (JA) could be an integral part of a general signal transduction system regulating inducible defense genes in plants. It was reported that treatment with an elicitor (N-acetylchitoheptaose) induced production of phytoalexin in suspension-cultured rice (Oryza sativa L.) cells. In this study, the role of JA in the induction of phytoalexin production by N-acetylchitoheptaose was investigated. Exogenously applied ([plus or minus])-JA (10-4 M) clearly induced the production of momilactone A, a major phytoalexin, in suspension-cultured rice cells. On the other hand, in rice cells treated with N-acetylchitoheptaose, endogenous JA was rapidly and transiently accumulated prior to accumulation of momilactone A. Treatment with ibuprofen, an inhibitor of JA biosynthesis, reduced production of momilactone A in the cells treated with N-acetylchitoheptaose, but the addition of ([plus or minus])-JA increased production of momilactone A to levels higher than those in the elicited rice cells. These results strongly suggest that JA functions as a signal transducer in the induction of biosynthesis of momilactone A by N-acetylchitoheptaose in suspension-cultured rice cells.     

Jasmonate-Inducible Genes Are Activated in Rice by Pathogen Attack without a Concomitant Increase in Endogenous Jasmonic Acid Levels

The possible role of the octadecanoid signaling pathway with jasmonic acid (JA) as the central component in defense-gene regulation of pathogen-attacked rice was studied. Rice (Oryza sativa L.) seedlings were treated with JA or inoculated with the rice blast fungus Magnaporthe grisea (Hebert) Barr., and gene-expression patterns were compared between the two treatments. JA application induced the accumulation of a number of pathogenesis-related (PR) gene products at the mRNA and protein levels, but pathogen attack did not enhance the levels of (-)-JA during the time required for PR gene expression. Pathogen-induced accumulation of PR1-like proteins was reduced in plants treated with tetcyclacis, a novel inhibitor of jasmonate biosynthesis. There was an additive and negative interaction between JA and an elicitor from M. grisea with respect to induction of PR1-like proteins and of an abundant JA-and wound-induced protein of 26 kD, respectively. Finally, activation of the octadecanoid signaling pathway and induction of a number of PR genes by exogenous application of JA did not confer local acquired resistance to rice. The data suggest that accumulation of nonconjugated (-)-JA is not necessary for induction of PR genes and that JA does not orchestrate localized defense responses in pathogen-attacked rice. Instead, JA appears to be embedded in a signaling network with another pathogen-induced pathway(s) and may be required at a certain minimal level for induction of some PR genes.     

Involvement of phospholipase D in wound-induced accumulation of jasmonic acid in arabidopsis

Multiple forms of phospholipase D (PLD) were activated in response to wounding, and the expressions of PLDalpha, PLDbeta, and PLDgamma differed in wounded Arabidopsis leaves. Antisense abrogation of the common plant PLD, PLDalpha, decreased the wound induction of phosphatidic acid, jasmonic acid (JA), and a JA-regulated gene for vegetative storage protein. Examination of the genes involved in the initial steps of oxylipin synthesis revealed that abrogation of the PLDalpha attenuated the wound-induced expression of lipoxygenase 2 (LOX2) but had no effect on allene oxide synthase (AOS) or hydroperoxide lyase in wounded leaves. The systemic induction of LOX2, AOS, and vegetative storage protein was lower in the PLDalpha-suppressed plants than in wild-type plants, with AOS exhibiting a distinct pattern. These results indicate that activation of PLD mediates wound induction of JA and that LOX2 is probably a downstream target through which PLD promotes the production of JA.     

Systemic induction of H2O2 in pea seedlings pretreated by wounding and exogenous jasmonic acid

Mechanical stress was one of stresses with whichplants often met. With the development of fruit andvegetable finish machining in food industry, artificialinjury also appeared. As response to other stresses,plants have evolved with some adaptive mechanismsto cope with wounding[1]. Jasmonic acid (JA) andmethyl jasmonate (MeJA), as important signal mole-cules in plant response to wounding, have attracted agreat deal of attention. The studies on some crops, suchas potato[2], rice[3], and tomato[…