Precursor 12-oxophytodienoic Acid Research Articles

Integrative analysis of multiple metabolomes and transcriptome revealed color expression mechanism in red skin root syndrome of Panax ginseng

Red skin root syndrome causes reduction of both production and quality of Panax ginseng (ginseng). However, its development process and key metabolites are still unknown. Multiple metabolomes including non-targeted metabolome, and anthocyanins-targeted and carotenoids-targeted metabolomes were performed on field grown red skin and healthy ginseng. The detected metabolites were analyzed in combination with previous transcriptome results. The total metabolite profiles revealed that primary metabolites especially citrate was decreased, but secondary metabolites mainly involved in phenylpropanoid pathway were increased in red skin ginseng compared to those in healthy ginseng. Targeted metabolome found that each gram of fresh red skin ginseng sample contained 8 μg β-carotene, 0.56 μg α-carotene, 0.017 μg rutin, 0.248 μg chalcone, and some anthocyanins including 0.023 μg cyanidin and 0.037 μg quercetin 3-O-glucoside, albeit in low concentrations but higher than those in healthy ginseng. Furthermore, integrative metabolome and transcriptome analysis revealed that changes of metabolites related to anthocyanins synthesis were correlated with the related gene expression patterns. Additionally, jasmonate (JA) and its precursor 12-oxophytodienoic acid increased their concentrations dramatically in red skin ginseng, suggesting the possible role of JA or JA signaling pathway in regulating red skin syndrome. Consistently, the expressions of 17 MYB transcription factors were differentially regulated in red skin ginseng. JA related cis-elements are widely distributed at their promoters. Taken together, our study revealed reduction of primary metabolism but increase of secondary metabolism in red skin ginseng. MYB transcription factors of JA signal pathway were supposed to mediate accumulation of anthocyanins and carotenoids that contributed to the development of red skin ginseng.

Increased tolerance to salt stress in OPDA-deficient rice ALLENE OXIDE CYCLASE mutants is linked to an increased ROS-scavenging activity.

Salinity stress represents a global constraint for rice, the most important staple food worldwide. Therefore the role of the central stress signal jasmonate for the salt response was analysed in rice comparing the responses to salt stress for two jasmonic acid (JA) biosynthesis rice mutants (cpm2 and hebiba) impaired in the function of ALLENE OXIDE CYCLASE (AOC) and their wild type. The aoc mutants were less sensitive to salt stress. Interestingly, both mutants accumulated smaller amounts of Na(+) ions in their leaves, and showed better scavenging of reactive oxygen species (ROS) under salt stress. Leaves of the wild type and JA mutants accumulated similar levels of abscisic acid (ABA) under stress conditions, and the levels of JA and its amino acid conjugate, JA-isoleucine (JA-Ile), showed only subtle alterations in the wild type. In contrast, the wild type responded to salt stress by strong induction of the JA precursor 12-oxophytodienoic acid (OPDA), which was not observed in the mutants. Transcript levels of representative salinity-induced genes were induced less in the JA mutants. The absence of 12-OPDA in the mutants correlated not only with a generally increased ROS-scavenging activity, but also with the higher activity of specific enzymes in the antioxidative pathway, such as glutathione S-transferase, and fewer symptoms of damage as, for example, indicated by lower levels of malondialdehyde. The data are interpreted in a model where the absence of OPDA enhanced the antioxidative power in mutant leaves.

The Novel Monocot-Specific 9-Lipoxygenase ZmLOX12 Is Required to Mount an Effective Jasmonate-Mediated Defense Against Fusarium verticillioides in Maize

Fusarium verticillioides is a major limiting factor for maize production due to ear and stalk rot and the contamination of seed with the carcinogenic mycotoxin fumonisin. While lipoxygenase (LOX)-derived oxylipins have been implicated in defense against diverse pathogens, their function in maize resistance against F. verticillioides is poorly understood. Here, we functionally characterized a novel maize 9-LOX gene, ZmLOX12. This gene is distantly related to known dicot LOX genes, with closest homologs found exclusively in other monocot species. ZmLOX12 is predominantly expressed in mesocotyls in which it is strongly induced in response to F. verticillioides infection. The Mutator transposon-insertional lox12-1 mutant is more susceptible to F. verticillioides colonization of mesocotyls, stalks, and kernels. The infected mutant kernels accumulate a significantly greater amount of the mycotoxin fumonisin. Reduced resistance to the pathogen is accompanied by diminished levels of the jasmonic acid (JA) precursor 12-oxo phytodienoic acid, JA-isoleucine, and expression of jasmonate-biosynthetic genes. Supporting the strong defense role of jasmonates, the JA-deficient opr7 opr8 double mutant displayed complete lack of immunity to F. verticillioides. Unexpectedly, the more susceptible lox12 mutant accumulated higher levels of kauralexins, suggesting that F. verticillioides is tolerant to this group of antimicrobial phytoalexins. This study demonstrates that this unique monocot-specific 9-LOX plays a key role in defense against F. verticillioides in diverse maize tissues and provides genetic evidence that JA is the major defense hormone against this pathogen.

Jasmonic acid and its precursor 12-oxophytodienoic acid control different aspects of constitutive and induced herbivore defenses in tomato.

The jasmonate family of growth regulators includes the isoleucine (Ile) conjugate of jasmonic acid (JA-Ile) and its biosynthetic precursor 12-oxophytodienoic acid (OPDA) as signaling molecules. To assess the relative contribution of JA/JA-Ile and OPDA to insect resistance in tomato (Solanum lycopersicum), we silenced the expression of OPDA reductase3 (OPR3) by RNA interference (RNAi). Consistent with a block in the biosynthetic pathway downstream of OPDA, OPR3-RNAi plants contained wild-type levels of OPDA but failed to accumulate JA or JA-Ile after wounding. JA/JA-Ile deficiency in OPR3-RNAi plants resulted in reduced trichome formation and impaired monoterpene and sesquiterpene production. The loss of these JA/JA-Ile -dependent defense traits rendered them more attractive to the specialist herbivore Manduca sexta with respect to feeding and oviposition. Oviposition preference resulted from reduced levels of repellant monoterpenes and sesquiterpenes. Feeding preference, on the other hand, was caused by increased production of cis-3-hexenal acting as a feeding stimulant for M. sexta larvae in OPR3-RNAi plants. Despite impaired constitutive defenses and increased palatability of OPR3-RNAi leaves, larval development was indistinguishable on OPR3-RNAi and wild-type plants, and was much delayed compared with development on the jasmonic acid-insensitive1 (jai1) mutant. Apparently, signaling through JAI1, the tomato ortholog of the ubiquitin ligase CORONATINE INSENSITIVE1 in Arabidopsis (Arabidopsis thaliana), is required for defense, whereas the conversion of OPDA to JA/JA-Ile is not. Comparing the signaling activities of OPDA and JA/JA-Ile, we found that OPDA can substitute for JA/JA-Ile in the local induction of defense gene expression, but the production of JA/JA-Ile is required for a systemic response.

Transcriptomic analysis of oxylipin biosynthesis genes and chemical profiling reveal an early induction of jasmonates in chickpea roots under drought stress

Drought is one of the major constraints in subtropical agriculture. Therefore improving water stress tolerance is of great importance to breed for drought tolerance in future. The first plant organ sensing dehydration is the root. Aim of the present work was to clarify the potential impact of the phyto-oxylipins pathway on drought tolerance of chickpea (Cicer arietinum), the third important legume crop worldwide. Therefore, we measured the expression of key genes involved in oxylipins metabolism by qPCR on samples from stressed and non-stressed roots of a drought-tolerant and a drought-sensitive chickpea variety using commercially available TaqMan assays. We demonstrate that the drought tolerant variety reacts to drought with sustained and earlier activation of a specific lipoxygenase (Mt-LOX 1) gene, two hydroperoxide lyases (Mt-HPL 1 and Mt-HPL 2), an allene oxide synthase (Mt-AOS), and an oxo-phytodienoate reductase (Mt-OPR). We further show that gene over-expression positively correlates with the levels of major oxylipin metabolites from the AOS branch of the pathway, which finally leads to the synthesis of jasmonates. Higher levels of jasmonic acid (JA), its precursor 12-oxophytodienoic acid (OPDA) and the active form JA-isoleucine (JA-Ile) were especially detected in the root tissues of the tolerant variety, prompting us to assume a role of jasmonates in the early signalling of drought stress in chickpea and its involvement in the tolerance mechanism of the drought-tolerant variety.

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.

Linolenate 9R‐Dioxygenase and Allene Oxide Synthase Activities of Lasiodiplodia theobromae

Jasmonic acid (JA) is synthesized from linolenic acid (18:3n-3) by sequential action of 13-lipoxygenase, allene oxide synthase (AOS), and allene oxide cyclase. The fungus Lasiodiplodia theobromae can produce large amounts of JA and was recently reported to form the JA precursor 12-oxophytodienoic acid. The objective of our study was to characterize the fatty acid dioxygenase activities of this fungus. Two strains of L. theobromae with low JA secretion (~0.2mg/L medium) oxygenated 18:3n-3 to 5,8-dihydroxy-9Z,12Z,15Z-octadecatrienoic acid as well as 9R-hydroperoxy-10E,12Z,15Z-octadecatrienoic acid, which was metabolized by an AOS activity into 9-hydroxy-10-oxo-12Z,15Z-octadecadienoic acid. Analogous conversions were observed with linoleic acid (18:2n-6). Studies using [11S-(2)H]18:2n-6 revealed that the putative 9R-dioxygenase catalyzed stereospecific removal of the 11R hydrogen followed by suprafacial attack of dioxygen at C-9. Mycelia from these strains of L. theobromae contained 18:2n-6 as the major polyunsaturated acid but lacked 18:3n-3. A third strain with a high secretion of JA (~200mg/L) contained 18:3n-3 as a major fatty acid and produced 5,8-dihydroxy-9Z,12Z,15Z-octadecatrienoic acid from added 18:3n-3. This strain also lacked the JA biosynthetic enzymes present in higher plants.

Spodoptera littoralis-Induced Lectin Expression in Tobacco

The induced defense response in plants towards herbivores is mainly regulated by jasmonates and leads to the accumulation of so-called jasmonate-induced proteins. Recently, a jasmonate (JA) inducible lectin called Nicotiana tabacum agglutinin or NICTABA was discovered in tobacco (N. tabacum cv Samsun) leaves. Tobacco plants also accumulate the lectin after insect attack by caterpillars. To study the functional role of NICTABA, the accumulation of the JA precursor 12-oxophytodienoic acid (OPDA), JA as well as different JA metabolites were analyzed in tobacco leaves after herbivory by larvae of the cotton leafworm (Spodoptera littoralis) and correlated with NICTABA accumulation. It was shown that OPDA, JA as well as its methyl ester can trigger NICTABA accumulation. However, hydroxylation of JA and its subsequent sulfation and glucosylation results in inactive compounds that have lost the capacity to induce NICTABA gene expression. The expression profile of NICTABA after caterpillar feeding was recorded in local as well as in systemic leaves, and compared to the expression of several genes encoding defense proteins, and genes encoding a tobacco systemin and the allene oxide cyclase, an enzyme in JA biosynthesis. Furthermore, the accumulation of NICTABA was quantified after S. littoralis herbivory and immunofluorescence microscopy was used to study the localization of NICTABA in the tobacco leaf.

Rapid Enzymatic Isomerization of 12‐Oxophytodienoic Acid in the Gut of Lepidopteran Larvae

In response to feeding larvae of the Mediterranean climbing cutworm (Spodoptera littoralis), leaves of the lima bean (Phaseolus lunatus) produce fatty acid-derived signaling compounds (oxylipins). The major products are the phytohormones jasmonic acid and its biosynthetic precursor 12-oxophytodienoic acid (OPDA), along with 13-hydroxy-12-oxooctadeca-9,15-dienoic acid, 9-hydroxy-12-oxooctadeca-10,15-dienoic acid (alpha- and gamma-ketol), as well as unsaturated aldehydes. Oxylipin production is highest at the feeding zone of the insect and decreases with distance from the damaged area. Accordingly, the feeding insect experiences high local concentrations of oxylipins, which are taken up into the alimentary canal and are finally excreted with the feces. In contrast to most other oxylipins, OPDA was not detectable in the insect's gut; instead the structurally related tetrahydrodicranenone B (iso-OPDA) was identified. Feeding experiments with deuterium-labeled OPDA proved that the isomerization is catalyzed by an enzyme from the insect's gut tissue. The phenomenon appears to be widespread among Lepidopteran larvae.

Changes in jasmonates and 12-oxophytodienoic acid contents of Medicago sativa L. during somatic embryogenesis

Jasmonic acid (JA), its methyl ester (MeJA) and the biosynthetic precursor 12-oxophytodienoic acid (OPDA) were detected quantitatively during somatic embryogenesis of Medicago sativa L. Using GC-MS analysis, these compounds were found in initial explants, in calli and in somatic embryos in the nanogram range per gram of fresh weight. In distinct stages of somatic embryogenesis, JA and 12-OPDA accumulated preferentially in cotyledonary embryos. Initial explants exhibited about five-fold higher JA content than OPDA content, whereas in other stages OPDA accumulated predominantly. These data suggest that also in embryogenic tissues OPDA and JA may have individual signalling properties.

Isolation and characterization of signal transduction mutants of Arabidopsis thaliana that constitutively activate the octadecanoid pathway and form necrotic microlesions.

Thionins are a group of antimicrobial polypeptides that form part of the plant's defense mechanism against pathogens. The Thi 2.1 thionin gene of Arabidopsis thaliana has been shown to be inducible by jasmonic acid (JA), an oxylipin-like hormone derived from oxygenated linolenic acid and synthesized via the octadecanoid pathway. The JA-dependent regulation of the Thi 2.1 gene has been exploited for setting up a genetic screen for the isolation of signal transduction mutants that constitutively express the Thi 2.1 gene. Ten cet-mutants have been isolated which showed a constitutive expression of the thionin gene. Allelism tests revealed that they represent at least five different loci. Some mutants are dominant, others recessive, but all cet mutations behaved as monogenic traits when backcrossed with Thi 2.1-GUS plants. Some of the mutants overproduce JA and its bioactive precursor 12-oxophytodienoic acid (OPDA) up to 40-fold while others have the same low levels as the control wildtype plants. Two of the mutants showed a strong induction of both the salicylic acid (SA)- and the JA-dependent signaling pathways, while the majority seems to be affected only in the octadecanoid pathway. The Thi 2.1 thionin gene and the Pdf 1.2 defensin gene are activated independently, though both are regulated by JA. The cet-mutants, except for one, also show a spontaneous leaf cell necrosis, a reaction often associated with the systemic acquired resistance (SAR) pathway.

The Arabidopsis male-sterile mutant, opr3, lacks the 12-oxophytodienoic acid reductase required for jasmonate synthesis.

Jasmonic acid (JA) and its precursor 12-oxophytodienoic acid (OPDA) act as plant growth regulators and mediate responses to environmental cues. To investigate the role of these oxylipins in anther and pollen development, we characterized a T-DNA-tagged, male-sterile mutant of Arabidopsis, opr3. The opr3 mutant plants are sterile but can be rendered fertile by exogenous JA but not by OPDA. Cloning of the mutant locus indicates that it encodes an isozyme of 12-oxophytodienoate reductase, designated OPR3. All of the defects in opr3 are alleviated by transformation of the mutant with an OPR3 cDNA. Our results indicate that JA and not OPDA is the signaling molecule that induces and coordinates the elongation of the anther filament, the opening of the stomium at anthesis, and the production of viable pollen. Just as importantly, our data demonstrate that OPR3 is the only isoform of OPR capable of reducing the correct stereoisomer of OPDA to produce JA required for male gametophyte development.

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.

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.

Induction of 12-Oxo-Phytodienoic Acid in Wounded Plants and Elicited Plant Cell Cultures

Jasmonic acid (JA) is rapidly biosynthesized from alpha-linolenic acid in plants upon contact with pathogens or wounding, and triggers gene activation, leading to the synthesis of defensive secondary metabolites and proteins. Despite the recent finding that its precursor, 12-oxo-phytodienoic acid (PDA), is a more powerful inducer of gene activation, interest has focused so far almost exclusively on JA. A validated negative chemical ionization-gas chromatography-mass spectrometry method has been developed that allows the simultaneous quantification of endogenous 12-oxo-PDA and JA in plant tissues. In six out of eight plant species tested maximal levels of 12-oxo-PDA exceeded peak levels of JA by approximately 3- to 5-fold after elicitation with a yeast cell wall preparation or when plants were wounded. These experiments support the hypothesis that 12-oxo-PDA acts as the predominant jasmonate signal in most plants, whereas JA remains an active metabolite of its precursor. Furthermore, JA but not 12-oxo-PDA was shown to be secreted into the medium from cultured plant cells, suggesting that JA may also act as an intercellular signal.

The octadecanoic pathway: signal molecules for the regulation of secondary pathways.

Plant defense against microbial pathogens and herbivores relies heavily on the induction of defense proteins and low molecular weight antibiotics. The signals between perception of the aggression, gene activation, and the subsequent biosynthesis of secondary compounds are assumed to be pentacylic oxylipin derivatives. The rapid, but transient, synthesis of cis-jasmonic acid was demonstrated after insect attack on a food plant and by microbial elicitor addition to plant suspension cultures. This effect is highly specific and not caused by a number of environmental stresses such as light, heavy metals, or cold or heat shock. Elicitation of Eschscholtzia cell cultures also led to a rapid alkalinization of the growth medium prior to jasmonate formation. Inhibition of this alkalinization process by the protein kinase inhibitor staurosporine also inhibited jasmonate formation. The induction of specific enzymes in the benzo[c]phenanthridine alkaloid pathway leading to the antimicrobial sanguinarine was induced to a qualitatively and quantitatively similar extent by fungal elicitor, methyl jasmonate, and its linolenic acid-derived precursor 12-oxophytodienoic acid. It is herein proposed that a second oxylipid cascade may exist in plants starting from linoleic acid via 15,16-dihydro-12-oxophytodienoic acid to 9,10-dihydrojasmonate. Experiments with synthetic trihomojasmonate demonstrated that beta-oxidation is not a prerequisite for biological activity and that 12-oxophytodienoic acid and derivatives are most likely fully active as signal transducers. Octadecanoic acid-derived compounds are essential elements in modulating the synthesis of antibiotic compounds and are thus integral to plant defense.