12-oxophytodienoic-acid and jasmonic-acid

Topics: alpha-Linolenic Acid; Bryopsida; Chloroplasts; Cyclopentanes; Fatty Acids; Fatty Acids, Unsaturated; Marchantia; Metabolic Networks and Pathways; Oxylipins; Peroxisomes; Species Specificity

Jasmonates (JAs) are lipid-derived signals mediating plant responses to biotic and abiotic stresses and in plant development. Following the elucidation of each step in their biosynthesis and the important components of perception and signaling, several activators, repressors and co-repressors have been identified which contribute to fine-tuning the regulation of JA-induced gene expression. Many of the metabolic reactions in which JA participates, such as conjugation with amino acids, glucosylation, hydroxylation, carboxylation, sulfation and methylation, lead to numerous compounds with different biological activities. These metabolites may be highly active, partially active in specific processes or inactive. Hydroxylation, carboxylation and sulfation inactivate JA signaling. The precursor of JA biosynthesis, 12-oxo-phytodienoic acid (OPDA), has been identified as a JA-independent signaling compound. An increasing number of OPDA-specific processes is being identified. To conclude, the numerous JA compounds and their different modes of action allow plants to respond specifically and flexibly to alterations in the environment.

Topics: Biotechnology; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Models, Biological; Oxylipins; Plant Growth Regulators; Plants; Signal Transduction; Stress, Physiological

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.

Topics: Cyclopentanes; Fatty Acids, Unsaturated; Flowers; Gene Expression Regulation, Plant; Isoleucine; Oxylipins; Plant Growth Regulators; Plant Leaves; Seeds; Signal Transduction; Solanum lycopersicum; Stress, Physiological

One of the most challenging questions in modern plant science is how plants regulate their morphological and developmental adaptation in response to changes in their biotic and abiotic environment. A comprehensive elucidation of the underlying mechanisms will help shed light on the extremely efficient strategies of plants in terms of survival and propagation. In recent years, a number of environmental stress conditions have been described as being mediated by signaling molecules of the oxylipin family. In this context, jasmonic acid, its biosynthetic precursor, 12-oxo-phytodienoic acid (OPDA), and also reactive electrophilic species such as phytoprostanes play pivotal roles. Although our understanding of jasmonic acid-dependent processes and jasmonic acid signal-transduction cascades has made considerable progress in recent years, knowledge of the regulation and mode of action of OPDA-dependent plant responses is just emerging. This minireview focuses on recent work concerned with the elucidation of OPDA-specific processes in plants. In this context, aspects such as the differential recruitment of OPDA, either by de novo biosynthesis or by release from cyclo-oxylipin-galactolipids, and the conjugation of free OPDA are discussed.

Topics: Cyclopentanes; Fatty Acids, Unsaturated; Galactolipids; Oxylipins; Plants; Signal Transduction

Jasmonates, a collective term combining both jasmonic acid (JA) and related derivatives, are ubiquitously distributed in the plant kingdom. They are characterized as lipid-derived signal molecules which mediate a plethora of physiological functions, in particular stress responses, male fertility, and a multitude of developmental processes. In the course of JA biosynthesis, the first oxylipin with signal character, cis-(+)-12-oxo-phytodienoic acid (OPDA), is produced in a cyclization reaction catalyzed by allene oxide cyclase (AOC). This enzyme-catalyzed ring closure is of particular importance, as it warrants the enantiomeric structure at the cyclopentenone ring which in the end results in the only bioactive JA enantiomer, cis-(+)-JA. In this review, we focus on the structural and molecular mechanisms underlying the above mentioned cyclization reaction. In this context, we will discuss the crystal structure of AOC2 of Arabidopsis thaliana with respect to putative binding sites of the instable substrate, 12,13-epoxy-9(Z),11,15(Z)-octadecatrienoic acid (12,13-EOT), as well as possible intermolecular rearrangements during the cyclization reaction.

Topics: Cyclization; Cyclopentanes; Fatty Acids, Unsaturated; Intramolecular Oxidoreductases; Linoleic Acid; Models, Chemical; Models, Molecular; Molecular Structure; Oxylipins; Plant Proteins; Protein Structure, Tertiary

Plant jasmonic acid (JA) and structurally similar animal prostaglandins play pivotal roles in regulating cellular responses against environmental cues, including the innate immune response(s). In plants, JA and its immediate precursor 12-oxo-phytodienoic acid (OPDA) are synthesized by the octadecanoid pathway, which employs at least five enzymes (lipase, lipoxygenase, allene oxide synthase and cyclase, and OPDA reductase), in addition to the enzymes involved in the beta-oxidation steps. Genetic, molecular, and biochemical analyses have led to the identification of almost all the genes of the octadecanoid pathway in Arabidopsis--a model dicotyledonous plant. In this regard, rice (Oryza sativa L.)--an important socio-economic monocotyledonous model research plant--remains poorly characterized. Until now, no gene has been specifically associated with this pathway. It is therefore of utmost importance to identify, characterize, and assign the pathway specific genes in rice. In this review, we have surveyed the rice genome, extracted a large number of putative genes of the octadecanoid pathway, and discussed their relationship with the known pathway genes from other plant species. Moreover, the achievements made so far on the rice octadecanoid pathway have also been summarized to reflect the contribution of rice towards extending our knowledge on this critical pathway in plants.

Topics: Animals; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Intramolecular Oxidoreductases; Methyltransferases; Oryza; Oxylipins; Phospholipases; Phylogeny; Plant Proteins

It is known that octadecanoids, i.e. jasmonic acid and related compounds are involved in plant defence reactions against (1) microbial pathogens, (2) herbivores and (3) damage by UV-B or UV-C light as well as (4) senescence and (5) mechanotransduction. Jasmonic acid is likely to occur ubiquitously in the plant kingdom, and it has also been found in some fungi. The pathway of octadecanoid biosynthesis was elucidated in the early 80s by Vick and Zimmerman. This review summarizes recent progress in the identification and characterization of octadecanoid biosynthetic enzymes and in the understanding of the regulation of octadecanoid biosynthesis.

Topics: alpha-Linolenic Acid; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Intramolecular Oxidoreductases; Lipid Peroxidation; Lipoxygenase; Membrane Lipids; Oxylipins; Phylogeny; Plant Physiological Phenomena; Signal Transduction; Stearic Acids

Plants respond to situations requiring the initiation of inducible defence reactions with a complex array of signalling events that ultimately result in the activation of sets of defence genes. Among the chemical signals involved in the induction of defence reactions are cyclic oxylipins derived from C18- or C16-unsaturated fatty acids, the octadecanoids and the hexadecanoids. Key to understanding octadecanoid biology are the C18-metabolite 12-oxophytodienoic acid (OPDA) and the C12-compound jasmonic acid which is biosynthetically derived from 12-oxophytodienoic acid. Different octadecanoids likely have different biological functions. The bouquet of signalling compounds, rather than any single compound, is probably decisive for the biological response that results. This means that the processes regulating the pool sizes of different octadecanoids and their distribution within the plant are key to understanding octadecanoid biology. Recent results, including the cloning of several enzymes of the octadecanoid biosynthetic pathway, have provided first insights into these processes and into how the octadecanoid system is linked to other defence-related signalling pathways of the plant cell.

Topics: Cyclopentanes; Fatty Acids, Unsaturated; Models, Chemical; Oxylipins; Plant Diseases; Signal Transduction

Accumulating experimental data have shown that endogenous hormones play important roles in regulating seed dormancy and germination.

Topics: Abscisic Acid; Amino Acids; Germination; Seeds; Zanthoxylum

Twelve-oxo-phytodienoic acid (OPDA), the cyclopentenone precursor of jasmonic acid (JA), is required for the wounding response of plants. OPDA is derived from plastid-localized α-linolenic acid (α-LeA; 18:3) via the octadecanoid pathway, and is further exported from plastids to the cytosol for JA biosynthesis. However, the mechanism of OPDA transport from plastids has yet to be elucidated. In the current study, a plastid inner envelope-localized protein, designated 12-oxo-Phtyodienoic Acid Transporter 1 (OPDAT1), was identified and shown to potentially be involved in OPDA export from plastids, in Populus trichocarpa. Torr. OPDAT1 is expressed predominantly in young leaves of P. trichocarpa. Functional expression of OPDAT1 in yeast cells revealed that OPDAT1 is involved in OPDA transport. Loss-of-function of OPDAT1 in poplar resulted in increased accumulation of OPDA in the extracted plastids and a reduction in JA concentration, whereas an OPDAT1-overexpressing line showed a reverse tendency in OPDA accumulation and JA biosynthesis. OPDAT1 transcripts were rapidly induced by mechanical wounding of leaves, and an opdat1 mutant transgenic plant displayed increased susceptibility to spider mite (Tetranychus urticae) infestation. Collectively, these data suggest that OPDAT1 is an inner envelope transporter for OPDA, and this has potential implications for JA biosynthesis in poplar under environmental stresses.

Topics: Cyclopentanes; Fatty Acids, Unsaturated; Oxylipins; Plastids; Populus

Despite the importance of volatile organic compounds (VOCs) for plants, control mechanisms for their basal and stress-induced biosynthesis and release remain unclear. We sampled and characterized headspace and internal leaf volatile pools in rice (Oryza sativa), after a simulated herbivory treatment, which triggers an endogenous jasmonate burst. Certain volatiles, such as linalool, were strongly upregulated by simulated herbivory stress. In contrast, other volatiles, such as β-caryophyllene, were constitutively emitted and fluctuated according to time of day. Transcripts of the linalool synthase gene transiently increased 1-3 h after exposure of rice to simulated herbivory, whereas transcripts of caryophyllene synthase peaked independently at dawn. Unexpectedly, although emission and accumulation patterns of rice inducible and constitutive VOCs were substantially different, both groups of volatiles were compromised in jasmonate-deficient hebiba mutants, which lack the allene oxide cyclase (AOC) gene. This suggests that rice employs at least two distinct oxylipin-dependent mechanisms downstream of AOC to control production of constitutive and herbivore-induced volatiles. Levels of the JA precursor, 12-oxo-phytodienoic acid (OPDA), were correlated with constitutive volatile levels suggesting that OPDA or its derivatives could be involved in control of volatile emission in rice.

Topics: Acyclic Monoterpenes; Animals; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Herbivory; Intramolecular Oxidoreductases; Mutation; Oryza; Oxylipins; Plant Growth Regulators; Plant Leaves; Plant Proteins; Sesquiterpenes; Volatile Organic Compounds

The evolution of adaptive interactions with beneficial, neutral and detrimental microbes was one of the key features enabling plant terrestrialization. Extensive studies have revealed conserved and unique molecular mechanisms underlying plant-microbe interactions across different plant species; however, most insights gleaned to date have been limited to seed plants. The liverwort Marchantia polymorpha, a descendant of early diverging land plants, is gaining in popularity as an advantageous model system to understand land plant evolution. However, studying evolutionary molecular plant-microbe interactions in this model is hampered by the small number of pathogens known to infect M. polymorpha. Here, we describe four pathogenic fungal strains, Irpex lacteus Marchantia-infectious (MI)1, Phaeophlebiopsis peniophoroides MI2, Bjerkandera adusta MI3 and B. adusta MI4, isolated from diseased M. polymorpha. We demonstrate that salicylic acid (SA) treatment of M. polymorpha promotes infection of the I. lacteus MI1 that is likely to adopt a necrotrophic lifestyle, while this effect is suppressed by co-treatment with the bioactive jasmonate in M. polymorpha, dinor-cis-12-oxo-phytodienoic acid (dn-OPDA), suggesting that antagonistic interactions between SA and oxylipin pathways during plant-fungus interactions are ancient and were established already in liverworts.

Topics: Cyclopentanes; Drug Antagonism; Evolution, Molecular; Fatty Acids, Unsaturated; Fungi; Gene Expression Regulation, Plant; Host-Pathogen Interactions; Marchantia; Oxylipins; Plant Diseases; Salicylic Acid

Multiple long-distance signals have been identified for pathogen-induced systemic acquired resistance, but mobile signals for symbiont-induced systemic resistance (ISR) are less well understood. We used ISR-positive and -negative mutants of maize (

Topics: Cyclopentanes; Disease Resistance; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Isomerism; Lipoxygenase; Oxylipins; Plant Diseases; Trichoderma; Xylem; Zea mays

Wounding is a serious environmental stress in plants. Oxylipins such as jasmonic acid play an important role in defense against wounding. Mechanisms to adapt to wounding have been investigated in vascular plants; however, those mechanisms in nonvascular plants remain elusive. To examine the response to wounding in

Topics: Amino Acids; Bryopsida; Chromatography, Liquid; Citric Acid Cycle; Cyclopentanes; Energy Metabolism; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Glycolysis; Intramolecular Oxidoreductases; Oxylipins; Photosynthetic Reaction Center Complex Proteins; Plant Proteins; Plants, Genetically Modified; Protein Biosynthesis; Protein Folding; Proteome; Proteomics; Reactive Oxygen Species; Stress, Physiological; Tandem Mass Spectrometry

In the field, plants experience high light (HL) intensities that are often accompanied by elevated temperatures. Such conditions are a serious threat to agriculture production, because photosynthesis is highly sensitive to both HL intensities and high-temperature stress. One of the potential cellular targets of HL and heat stress (HS) combination is PSII because its degree of photoinhibition depends on the balance between the rate of PSII damage (induced by light stress), and the rate of PSII repair (impaired under HS). Here, we studied the responses of Arabidopsis (

Topics: Acclimatization; Arabidopsis; Chloroplasts; Cyclopentanes; Fatty Acids, Unsaturated; Heat-Shock Response; Hydrogen Peroxide; Light; Oxylipins; Photosynthesis; Photosystem II Protein Complex; Plant Growth Regulators; Plant Stomata; RNA, Messenger; Transcriptome

Jasmonates are vital plant hormones that not only act in the stress response to biotic and abiotic influences, such as wounding, pathogen attack, and cold acclimation, but also drive developmental processes in cooperation with other plant hormones. The biogenesis of jasmonates starts in the chloroplast, where several enzymatic steps produce the jasmonate precursor 12-oxophytodienoic acid (OPDA) from α-linolenic acid. OPDA in turn is exported into the cytosol for further conversion into active jasmonates, which subsequently induces the expression of multiple genes in the nucleus. Despite its obvious importance, the export of OPDA across the chloroplast membranes has remained elusive. In this study, we characterized a protein residing in the chloroplast outer membrane, JASSY, which has proven indispensable for the export of OPDA from the chloroplast. We provide evidence that JASSY has channel-like properties and propose that it thereby facilitates OPDA transport. Consequently, a lack of JASSY in

Topics: Acclimatization; Arabidopsis; Chloroplast Proteins; Chloroplasts; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Oxylipins; Plant Immunity

To determine whether drought-induced root jasmonate [jasmonic acid (JA) and jasmonic acid-isoleucine (JA-Ile)] accumulation affected shoot responses to drying soil, near-isogenic wild-type (WT) tomato (Solanum lycopersicum cv. Castlemart) and the def-1 mutant (which fails to accumulate jasmonates during water deficit) were self- and reciprocally grafted. Rootstock hydraulic conductance was entirely rootstock dependent and significantly lower in def-1, yet def-1 scions maintained a higher leaf water potential as the soil dried due to their lower stomatal conductance (gs). Stomatal sensitivity to drying soil (the slope of gsversus soil water content) was low in def-1 self-grafts but was normalized by grafting onto WT rootstocks. Although soil drying increased 12-oxo-phytodienoic acid (OPDA; a JA precursor and putative antitranspirant) concentrations in def-1 scions, foliar JA accumulation was negligible and foliar ABA accumulation reduced compared with WT scions. A WT rootstock increased drought-induced ABA and JA accumulation in def-1 scions, but decreased OPDA accumulation. Xylem-borne jasmonates were biologically active, since supplying exogenous JA via the transpiration stream to detached leaves decreased transpiration of WT seedlings but had the opposite effect in def-1. Thus foliar accumulation of both ABA and JA at WT levels is required for both maximum (well-watered) gs and stomatal sensitivity to drying soil.

Topics: Cyclopentanes; Fatty Acids, Unsaturated; Mutation; Oxylipins; Plant Proteins; Plant Shoots; Plant Stomata; Soil; Solanum lycopersicum; Water; Xylem

Reactive electrophile species (RES), including prostaglandins, phytoprostanes and 12-oxo phytodienoic acid (OPDA), activate detoxification responses in plants and animals. However, the pathways leading to the activation of defense reactions related to abiotic or biotic stress as a function of RES formation, accumulation or treatment are poorly understood in plants. Here, the thiol-modification of proteins, including the RES-activated basic region/leucine zipper transcription factor TGA2, was studied. TGA2 contains a single cysteine residue (Cys186) that was covalently modified by reactive cyclopentenones but not required for induction of detoxification genes in response to OPDA or prostaglandin A1. Activation of the glutathione-S-transferase 6 (GST6) promoter was responsive to cyclopentenones but not to unreactive cyclopentanones, including jasmonic acid suggesting that thiol reactivity of RES is important to activate the TGA2-dependent signaling pathway resulting in GST6 activation We show that RES modify thiols in numerous proteins in vivo, however, thiol reactivity alone appears not to be sufficient for biological activity as demonstrated by the failure of several membrane permeable thiol reactive reagents to activate the GST6 promoter.

Topics: Amino Acids; Arabidopsis; Arabidopsis Proteins; Basic-Leucine Zipper Transcription Factors; Cyclopentanes; Cysteine; Escherichia coli; Fatty Acids, Unsaturated; Glutathione Transferase; Nuclear Proteins; Oxylipins; Pipecolic Acids; Plant Leaves; Plants, Genetically Modified; Promoter Regions, Genetic; Prostaglandins A; Recombinant Proteins; Seedlings; Signal Transduction

In cultivated tomato (Solanum lycopersicum), increases in photosynthetically active radiation (PAR) induce type VI leaf glandular trichomes, which are important defensive structures against arthropod herbivores. Yet, how PAR affects the type VI trichome-associated leaf chemistry and its biological significance with respect to other photomorphogenic responses in this agronomically important plant species is unknown. We used the type VI trichome-deficient tomato mutant odorless-2 (od-2) and its wild type to investigate the influence of PAR on trichome-associated chemical defenses against thrips (Frankliniella occidentalis). High PAR increased thrips resistance in wild-type plants, but not in od-2. Furthermore, under high PAR, thrips preferred od-2 over the wild type. Both genotypes increased type VI trichome densities under high PAR. Wild-type plants, however, produced more trichome-associated allelochemicals, i.e. terpenes and phenolics, these being undetectable or barely altered in od-2. High PAR increased leaf number and thickness, and induced profound but similar metabolomic changes in wild-type and od-2 leaves. Enhanced PAR also increased levels of ABA in wild-type and od-2 plants, and of auxin in od-2, while the salicylic acid and jasmonate concentrations were unaltered. However, in both genotypes, high PAR induced the expression of jasmonic acid-responsive defense-related genes. Taken together, our results demonstrate that high PAR-mediated induction of trichome-associated chemical defenses plays a prominent role in tomato-thrips interactions.

Topics: Abscisic Acid; Animals; Cyclopentanes; Disease Resistance; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Genotype; Indoleacetic Acids; Light; Metabolomics; Mutation; Oxylipins; Pheromones; Plant Diseases; Plant Leaves; RNA, Messenger; Salicylic Acid; Solanum lycopersicum; Thysanoptera; Trichomes; Volatile Organic Compounds

Jasmonic acid (JA) is involved in a variety of physiological responses in seed plants. However, the detection and role of JA in lycophytes, a group of seedless vascular plants, have remained elusive until recently. This study provides the first evidence of 12-oxo-phytodienoic acid (OPDA), JA and jasmonoyl-isoleucine (JA-Ile) in the model lycophyte Selaginella moellendorffii. Mechanical wounding stimulated the accumulation of OPDA, JA and JA-Ile. These data were corroborated by the detection of enzymatically active allene oxide synthase (AOS), allene oxide cyclase (AOC), 12-oxo-phytodienoic acid reductase 3 (OPR3) and JA-Ile synthase (JAR1) in S. moellendorffii. SmAOS2 is involved in the first committed step of JA biosynthesis. SmAOC1 is a crucial enzyme for generating the basic structure of jasmonates and is actively involved in the formation of OPDA. SmOPR5, a functionally active OPR3-like enzyme, is also vital for the reduction of (+)-cis-OPDA, the only isomer of the JA precursor. The conjugation of JA to Ile by SmJAR1 demonstrates that S. moellendorffii produces JA-Ile. Thus, the four active enzymes have characteristics similar to those in seed plants. Wounding and JA treatment induced the expression of SmAOC1 and SmOPR5. Furthermore, JA inhibited the growth of shoots in S. moellendorffii, which suggests that JA functions as a signaling molecule in S. moellendorffii. This study proposes that JA evolved as a plant hormone for stress adaptation, beginning with the emergence of vascular plants.

Topics: Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Intramolecular Oxidoreductases; Isoleucine; Ligases; Oxidoreductases; Oxylipins; Plant Proteins; Plant Shoots; Selaginellaceae

Fungi can produce jasmonic acid (JA) and its isoleucine conjugate in large quantities, but little is known about the biosynthesis. Plants form JA from 18:3

Topics: Alkenes; Cyclopentanes; Fatty Acids, Unsaturated; Fusarium; Mycelium; Oxidation-Reduction; Oxides; Oxylipins

In plants, cis-jasmone (CJ) is synthesized from α-linolenic acid (LA) via two biosynthetic pathways using jasmonic acid (JA) and iso-12-oxo-phytodienoic acid (iso-OPDA) as key intermediates. However, there have been no reports documenting CJ production by microorganisms. In the present study, the production of fungal-derived CJ by Lasiodiplodia theobromae was observed for the first time, although this production was not observed for Botrytis cinerea, Verticillium longisporum, Fusarium oxysporum, Gibberella fujikuroi, and Cochliobolus heterostrophus. To investigate the biosynthetic pathway of CJ in L. theobromae, administration experiments using [18,18,18-

Topics: Acetates; Ascomycota; Biosynthetic Pathways; Cyclopentanes; Deuterium; Fatty Acids, Unsaturated; Metabolome; Oxylipins

Jasmonates (JAs) are plant hormones that integrate external stress stimuli with physiological responses. (+)-7-iso-JA-L-Ile is the natural JA ligand of COI1, a component of a known JA receptor. The upstream JA biosynthetic precursor cis-(+)-12-oxo-phytodienoic acid (cis-(+)-OPDA) has been reported to act independently of COI1 as an essential signal in several stress-induced and developmental processes. Wound-induced increases in the endogenous levels of JA/JA-Ile are accompanied by two to tenfold increases in the concentration of OPDA, but its means of perception and metabolism are unknown. To screen for putative OPDA metabolites, vegetative tissues of flowering Arabidopsis thaliana were extracted with 25% aqueous methanol (v/v), purified by single-step reversed-phase polymer-based solid-phase extraction, and analyzed by high throughput mass spectrometry. This enabled the detection and quantitation of a low abundant OPDA analog of the biologically active (+)-7-iso-JA-L-Ile in plant tissue samples. Levels of the newly identified compound and the related phytohormones JA, JA-Ile and cis-(+)-OPDA were monitored in wounded leaves of flowering Arabidopsis lines (Col-0 and Ws) and compared to the levels observed in Arabidopsis mutants deficient in the biosynthesis of JA (dde2-2, opr3) and JA-Ile (jar1). The observed cis-(+)-OPDA-Ile levels varied widely, raising questions concerning its role in Arabidopsis stress responses.

Topics: Arabidopsis; Cyclopentanes; Diazonium Compounds; Fatty Acids, Unsaturated; Flowers; Isoleucine; Oxylipins; Plant Growth Regulators; Plant Leaves; Pyridines; Stereoisomerism

Under oxidative stress conditions the lipid constituents of cells can undergo oxidation whose frequent consequence is the production of highly reactive α,β-unsaturated carbonyls. These molecules are toxic because they can add to biomolecules (such as proteins and nucleic acids) and several enzyme activities cooperate to eliminate these reactive electrophile species. CeQORH (chloroplast envelope Quinone Oxidoreductase Homolog, At4g13010) is associated with the inner membrane of the chloroplast envelope and imported into the organelle by an alternative import pathway. In the present study, we show that the recombinant ceQORH exhibits the activity of a NADPH-dependent α,β-unsaturated oxoene reductase reducing the double bond of medium-chain (C⩾9) to long-chain (18 carbon atoms) reactive electrophile species deriving from poly-unsaturated fatty acid peroxides. The best substrates of ceQORH are 13-lipoxygenase-derived γ-ketols. γ-Ketols are spontaneously produced in the chloroplast from the unstable allene oxide formed in the biochemical pathway leading to 12-oxo-phytodienoic acid, a precursor of the defense hormone jasmonate. In chloroplasts, ceQORH could detoxify 13-lipoxygenase-derived γ-ketols at their production sites in the membranes. This finding opens new routes toward the understanding of γ-ketols role and detoxification.

Topics: Arabidopsis; Arabidopsis Proteins; Chloroplasts; Cyclopentanes; Fatty Acids, Unsaturated; Lipoxygenase; Membrane Lipids; Membrane Proteins; Oxidation-Reduction; Oxylipins; Quinone Reductases; Quinones

The exquisite harmony between hormones and their corresponding signaling pathways is central to prioritizing plant responses to simultaneous and/or successive environmental trepidations. The crosstalk between jasmonic acid (JA) and salicylic acid (SA) is an established effective mechanism that optimizes and tailors plant adaptive responses. However, the underlying regulatory modules of this crosstalk are largely unknown. Global transcriptomic analyses of mutant plants (ceh1) with elevated levels of the stress-induced plastidial retrograde signaling metabolite 2-C-methyl-D-erythritol cyclopyrophosphate (MEcPP) revealed robustly induced JA marker genes, expected to be suppressed by the presence of constitutively high SA levels in the mutant background. Analyses of a range of genotypes with varying SA and MEcPP levels established the selective role of MEcPP-mediated signal(s) in induction of JA-responsive genes in the presence of elevated SA. Metabolic profiling revealed the presence of high levels of the JA precursor 12-oxo-phytodienoic acid (OPDA), but near wild type levels of JA in the ceh1 mutant plants. Analyses of coronatine-insensitive 1 (coi1)/ceh1 double mutant plants confirmed that the MEcPP-mediated induction is JA receptor COI1 dependent, potentially through elevated OPDA. These findings identify MEcPP as a previously unrecognized central regulatory module that induces JA-responsive genes in the presence of high SA, thereby staging a multifaceted plant response within the environmental context.

Topics: Arabidopsis; Arabidopsis Proteins; Cyclopentanes; Erythritol; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Genes, Plant; Models, Biological; Mutation; Oxylipins; Plastids; Salicylic Acid; Signal Transduction

Understanding how plants allocate their resources to growth or defence is of long-term importance to the development of new and improved varieties of different crops. Using molecular genetics, plant physiology, hormone analysis and Next-Generation Sequencing (NGS)-based transcript profiling, we have isolated and characterized the rice (Oryza sativa) LESION AND LAMINA BENDING (LLB) gene that encodes a chloroplast-targeted putative leucine carboxyl methyltransferase. Loss of LLB function results in reduced growth and yield, hypersensitive response (HR)-like lesions, accumulation of the antimicrobial compounds momilactones and phytocassanes, and constitutive expression of pathogenesis-related genes. Consistent with these defence-associated responses, llb shows enhanced resistance to rice blast (Magnaporthe oryzae) and bacterial blight (Xanthomonas oryzae pv. oryzae). The lesion and resistance phenotypes are likely to be caused by the over-accumulation of jasmonates (JAs) in the llb mutant including the JA precursor 12-oxo-phytodienoic acid. Additionally, llb shows an increased lamina inclination and enhanced early seedling growth due to elevated brassinosteroid (BR) synthesis and/or signalling. These findings show that LLB functions in the chloroplast to either directly or indirectly repress both JA- and BR-mediated responses, revealing a possible mechanism for controlling how plants allocate resources for defence and growth.

Topics: Amino Acid Sequence; Chloroplasts; Cyclopentanes; Disease Resistance; Fatty Acids, Unsaturated; Genes, Reporter; Magnaporthe; Mutation; Oryza; Oxylipins; Phenotype; Plant Diseases; Plant Growth Regulators; Plant Leaves; Seedlings; Xanthomonas

Oxylipins of the jasmonate family are active as signals in plant responses to biotic and abiotic stresses as well as in development. Jasmonic acid (JA), its precursor cis-12-oxo-phytodienoic acid (OPDA) and the isoleucine conjugate of JA (JA-Ile) are the most prominent members. OPDA and JA-Ile have individual signalling properties in several processes and differ in their pattern of gene expression. JA-Ile, but not OPDA, is perceived by the SCFCOI1-JAZ co-receptor complex. There are, however, numerous processes and genes specifically induced by OPDA. The recently identified OPDA-Ile suggests that OPDA specific responses might be mediated upon formation of OPDA-Ile. Here, we tested OPDA-Ile-induced gene expression in wild type and JA-deficient, JA-insensitive and JA-Ile-deficient mutant background. Tests on putative conversion of OPDA-Ile during treatments revealed only negligible conversion. Expression of two OPDA-inducible genes, GRX480 and ZAT10, by OPDA-Ile could be detected in a JA-independent manner in Arabidopsis seedlings but less in flowering plants. The data suggest a bioactivity in planta of OPDA-Ile.

Topics: Arabidopsis; Cyclopentanes; Ecotype; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Isoleucine; Oxylipins; Plant Leaves; RNA, Messenger

Expression takes place for most of the jasmonic acid (JA)-induced genes in a COI1-dependent manner via perception of its conjugate JA-Ile in the SCF

Topics: Arabidopsis; Arabidopsis Proteins; Cyclopentanes; Fatty Acids, Unsaturated; Isoleucine; Oxylipins; Plant Growth Regulators; Signal Transduction

The pericarp anatomy and the effects of storage after harvest, storage temperature and early cypsela imbibition on phytohormone profiles were studied in inbred sunflower lines B123 and B91. On day 0, germination of B123 cypselas was near 0%, indicating dormancy, whereas that of B91 cypselas was near 100%, indicating non-dormancy. The germination of B123 and B91 on day 33 at room temperature (25 °C) storage was similar. Cell wall thickness and sclerification of the pericarp were higher in B123 than B91, suggesting that structural characteristics may contribute to physical dormancy in B123. Jasmonates (JAs), salicylic acid (SA) and abscisic acid (ABA) were measured in dry and imbibed pericarps. SA content of dry pericarp was higher on day 33 than day 0. SA content during imbibition on day 33 was similar for room and low (-20 °C) storage temperatures. ABA content after 12 h imbibition was similar on days 0 and 33 at low temperature, but it increased on day 33 at room temperature for B123. 12-Oxo-phytodienoic acid (OPDA) was maximal on day 0 for B123, but peaked at day 33 at low temperature for B91. JA was higher on days 0 and 33 at room temperature as compared with low temperature. Our findings indicate that pericarp hormone profiles are affected in the two lines with different dormancy degree depending on storage conditions and imbibition processes.

Topics: Abscisic Acid; Cell Wall; Cyclopentanes; Fatty Acids, Unsaturated; Germination; Helianthus; Oxylipins; Plant Dormancy; Plant Growth Regulators; Salicylic Acid; Seeds; Temperature

Resistance of tomato (Solanum Lycopersicum) to the fungal pathogen Botrytis cinerea requires complex interplay between hormonal signalling. In this study, we explored the involvement of new oxylipins in the tomato basal and induced response to this necrotroph through the functional analysis of the tomato α-dioxygenase2 (α-DOX2)-deficient mutant divaricata. We also investigated the role of SA in the defence response against this necrotrophic fungus using SA-deficient tomato nahG plants. The plants lacking dioxigenase α-DOX2, which catalyses oxylipins production from fatty acids, were more susceptible to Botrytis, and hexanoic acid-induced resistance (Hx-IR) was impaired; hence α-DOX2 is required for both tomato defence and the enhanced protection conferred by natural inducer hexanoic acid (Hx) against B. cinerea. The divaricata plants accumulated less pathogen-induced callose and presented lower levels of jasmonic acid (JA) and 12-oxo-phytodienoic acid (OPDA) upon infection if compared to the wild type. Glutathion-S-transferase (GST) gene expression decreased and ROS production significantly increased in Botrytis-infected divaricata plants. These results indicate that absence of α-DOX2 influences the hormonal changes, oxidative burst and callose deposition that occur upon Botrytis infection in tomato. The study of SA-deficient nahG tomato plants showed that the plants with low SA levels displayed increased resistance to Botrytis, but were unable to display Hx-IR. This supports the involvement of SA in Hx-IR. NaghG plants displayed reduced callose and ROS accumulation upon infection and an increased GST expression. This reflects a positive relationship between SA and these defensive mechanisms in tomato. Finally, Hx boosted the pathogen-induced callose in nahG plants, suggesting that this priming mechanism is SA-independent. Our results support the involvement of the oxylipins pathway and SA in tomato response to Botrytis, probably through complex crosstalk of the hormonal balance with callose and ROS accumulation, and reinforce the role of the oxidative stress in the outcome of the plant-Botrytis interaction.

Topics: Botrytis; Caproates; Cyclopentanes; Dioxygenases; Disease Resistance; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Glucans; Oxylipins; Plant Diseases; Plant Growth Regulators; Plant Proteins; Reactive Oxygen Species; Salicylic Acid; Solanum lycopersicum

The citrus rootstocks sour orange and Cleopatra mandarin display differential resistance against Tetranychus urticae. Sour orange plants support reduced oviposition, growth rates and damage compared with Cleopatra mandarin plants. Jasmonic acid signalling and flavonoid accumulation have been revealed as key mechanisms for the enhanced resistance of sour orange plants. In this study, we observed that the release of T. urticae herbivore-induced plant volatiles (HIPVs) from sour orange plants has a marked repellent effect on conspecific mites associated with the production of the terpenes α-ocimene, α-farnesene, pinene and d-limonene, and the green leaf volatile 4-hydroxy-4-methyl-2-pentanone. By contrast, T. urticae HIPVs from Cleopatra mandarin plants promote conspecific mite attraction associated with an increase in (2-butoxyethoxy) ethanol, benzaldehyde and methyl salicylate levels. HIPVs released from sour orange plants following T. urticae infestation induce resistance in Cleopatra mandarin plants, thereby reducing oviposition rates and stimulating the oxylipin biosynthetic gene lipoxygenase2 (LOX2). Cleopatra HIPVs do not affect the response to T. urticae of these rootstocks. We conclude that sour orange plants promote herbivore-induced resistance in Cleopatra mandarin plants and, despite the weak basal resistance of these rootstocks, herbivore resistance can be induced through the combination of HIPVs, such as α-ocimene and d-limonene.

Topics: Animals; Chromatography, High Pressure Liquid; Citrus; Cyclopentanes; Disease Resistance; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Genotype; Herbivory; Insect Repellents; Metabolomics; Oxylipins; Plant Diseases; Plant Proteins; Salicylic Acid; Smell; Tetranychidae; Volatilization

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.

Topics: Abscisic Acid; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Glutathione Transferase; Intramolecular Oxidoreductases; Isoleucine; Lipid Peroxidation; Oryza; Oxylipins; Plant Growth Regulators; Plant Leaves; Plant Proteins; Plant Roots; Reactive Oxygen Species; Seedlings; Sodium Chloride; Stress, Physiological

12-Oxo-phytodienoic acid (OPDA) is an intermediate in jasmonic acid (JA) biosynthesis. OPDA exerts JA-dependent and JA-independent biological effects; therefore, it is considered a signaling molecule in flowering plants. OPDA is induced by bacterial infection and wounding and inhibits growth in the moss Physcomitrella patens. The functions of OPDA and allene oxide cyclase (AOC) in the liverwort Marchantia polymorpha were explored, which represents the most basal lineage of extant land plants. The analysis of OPDA showed that it is present in M. polymorpha and is increased by wounding. OPDA has been suggested to be involved in the response to environmental stresses. Moreover, OPDA showed growth inhibitory activity in M. polymorpha. Nonetheless JA in M. polymorpha was not found in this study. AOC synthesizes OPDA from an unstable allene oxide. A database search of the M. polymorpha genome identified only a putative gene encoding allene oxide cyclase (MpAOC). Recombinant MpAOC showed AOC activity similar to that in flowering plants. MpAOC was localized to chloroplasts, as in flowering plants. Expression of MpAOC was induced by wounding and OPDA treatment, and positive feedback regulation of OPDA was demonstrated in M. polymorpha. Overexpression of MpAOC increased the endogenous OPDA level and suppressed growth in M. polymorpha. These results indicate the role of OPDA as a signaling molecule regulating growth and the response to wounding in the liverwort M. polymorpha.

Topics: Bryopsida; Cyclopentanes; Fatty Acids, Unsaturated; Intramolecular Oxidoreductases; Marchantia; Molecular Structure; Oxylipins

Plant oxylipins derive from oxygenation of polyunsaturated fatty acids in thylakoid membranes and oxylipins such as jasmonic acid (JA) and 12-oxo-phytodienoic acid (OPDA) play important roles in adaptation to photo-oxidative stress. OPDA functions both as a JA precursor and as a biologically active signaling molecule that induces expression of a specific set of genes. These genes can be induced by OPDA in the JA-insensitive coronatine insensitive1 (coi1) mutant, suggesting that there is an alternative pathway for OPDA signaling, independent of COI1-dependent JA signaling. However, little is known about OPDA signaling in photo-oxidative stress responses. In this study, we isolated Arabidopsis mutants with constitutively enhanced expression from the OPDA-responsive HsfA2 promoter. We used deletion mapping and complementation analysis to identify one responsible gene as CATALASE2. Our results thus indicate that ROS-producing cellular metabolism links to OPDA signaling.

Topics: Adaptation, Physiological; Arabidopsis; Arabidopsis Proteins; Cyclopentanes; DNA-Binding Proteins; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Genetic Complementation Test; Heat Shock Transcription Factors; Heat-Shock Proteins; Heat-Shock Response; Light; Oxidation-Reduction; Oxidative Stress; Oxylipins; Plant Growth Regulators; Plant Proteins; Plants, Genetically Modified; Promoter Regions, Genetic; Reactive Oxygen Species; Sequence Deletion; Signal Transduction; Thylakoids; Transcription Factors

Membranes are primary sites of perception of environmental stimuli. Polyunsaturated fatty acids are major structural constituents of membranes that also function as modulators of a multitude of signal transduction pathways evoked by environmental stimuli. Different stresses induce production of a distinct blend of oxygenated polyunsaturated fatty acids, "oxylipins." We employed three Arabidopsis (Arabidopsis thaliana) ecotypes to examine the oxylipin signature in response to specific stresses and determined that wounding and drought differentially alter oxylipin profiles, particularly the allene oxide synthase branch of the oxylipin pathway, responsible for production of jasmonic acid (JA) and its precursor 12-oxo-phytodienoic acid (12-OPDA). Specifically, wounding induced both 12-OPDA and JA levels, whereas drought induced only the precursor 12-OPDA. Levels of the classical stress phytohormone abscisic acid (ABA) were also mainly enhanced by drought and little by wounding. To explore the role of 12-OPDA in plant drought responses, we generated a range of transgenic lines and exploited the existing mutant plants that differ in their levels of stress-inducible 12-OPDA but display similar ABA levels. The plants producing higher 12-OPDA levels exhibited enhanced drought tolerance and reduced stomatal aperture. Furthermore, exogenously applied ABA and 12-OPDA, individually or combined, promote stomatal closure of ABA and allene oxide synthase biosynthetic mutants, albeit most effectively when combined. Using tomato (Solanum lycopersicum) and Brassica napus verified the potency of this combination in inducing stomatal closure in plants other than Arabidopsis. These data have identified drought as a stress signal that uncouples the conversion of 12-OPDA to JA and have revealed 12-OPDA as a drought-responsive regulator of stomatal closure functioning most effectively together with ABA.

Topics: Abscisic Acid; Adaptation, Physiological; Arabidopsis; Brassica napus; Cyclopentanes; Droughts; Fatty Acids, Unsaturated; Lyases; Oxylipins; Plant Stomata; Plants, Genetically Modified; Solanum lycopersicum; Stress, Physiological; Subcellular Fractions

Through evolution, plants have developed a myriad of strategies to adapt to environmental perturbations. Using 3 Arabidopsis ecotypes in conjunction with various transgenic and mutant lines, we provide evidence that wounding and drought differentially alter the metabolic signatures derived from the 2 main competing oxylipin-pathway branches, namely the JA and its precursor 12-OPDA produced by Allene oxide synthase (AOS) branch, and aldehydes and corresponding alcohols generated by Hydroperoxide lyase (HPL) branch. Specifically, we show that wounding induces production of both HPL and AOS-derived metabolites whereas, drought stress only elicits production of hexenal but suppresses hexenol, and further uncouples the conversion of 12-OPDA to JA. This finding led to uncovering of 12-OPDA as a functional convergence point of oxylipin and ABA pathways to control stomatal aperture in plant adaptive responses to drought. In addition, using transgenic lines overexpressing plastidial and extraplastidial HPL enzyme establish the strong interdependence of AOS- and HPL-branch pathways, and the importance of this linkage in tailoring plant adaptive responses to the nature of perturbations.

Topics: Adaptation, Physiological; Aldehyde-Lyases; Arabidopsis; Cyclopentanes; Cytochrome P-450 Enzyme System; Droughts; Fatty Acids, Unsaturated; Intramolecular Oxidoreductases; Oxylipins; Plant Stomata; Stress, Physiological; Water

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.

Topics: Aldehydes; Animals; Cyclopentanes; Fatty Acids, Unsaturated; Food Preferences; Gene Expression Regulation, Plant; Herbivory; Larva; Manduca; Oviposition; Oxylipins; RNA Interference; Secondary Metabolism; Solanum lycopersicum; Terpenes; Trichomes

The brown planthopper (BPH, Nilaparvata lugens) is a destructive, monophagous, piercing-sucking insect pest of rice. Previous studies indicated that jasmonic acid (JA) positively regulates rice defense against chewing insect pests but negatively regulates it against the piercing-sucking insect of BPH. We here demonstrated that overexpression of allene oxide cyclase (AOC) but not OPR3 (cis-12-oxo-phytodienoic acid (OPDA) reductase 3, an enzyme adjacent to AOC in the JA synthetic pathway) significantly increased rice resistance to BPH, mainly by reducing the feeding activity and survival rate. Further analysis revealed that plant response to BPH under AOC overexpression was independent of the JA pathway and that significantly higher OPDA levels stimulated rice resistance to BPH. Microarray analysis identified multiple candidate resistance-related genes under AOC overexpression. OPDA treatment stimulated the resistance of radish seedlings to green peach aphid Myzus persicae, another piercing-sucking insect. These results imply that rice resistance to chewing insects and to sucking insects can be enhanced simultaneously through AOC-mediated increases of JA and OPDA and provide direct evidence of the potential application of OPDA in stimulating plant defense responses to piercing-sucking insect pests in agriculture.

Topics: Animals; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Hemiptera; Herbivory; Intramolecular Oxidoreductases; Oryza; Oxidoreductases Acting on CH-CH Group Donors; Oxylipins; Plant Growth Regulators; Plant Proteins; Plants, Genetically Modified

Peroxisomes are vital eukaryotic organelles that house a variety of metabolic functions. To fully define the proteome of plant peroxisomes, we recently performed a proteomic analysis of peroxisomes from etiolated Arabidopsis seedlings, verified the peroxisomal localization of candidate proteins by in vivo targeting analysis of fluorescent proteins, and subjected the T-DNA mutants of the newly confirmed proteins to a series of phenotypic analysis. Our reverse genetics approach revealed the role of a cysteine protease - Response to Drought 21A-like Cysteine Protease1 (RDL1) - in seed germination, indole-3-butyric acid (IBA) β-oxidation and stress response. Here, we developed a quick assay aimed at identifying peroxisomal proteins involved in the metabolism of 12-oxo-phytodienoic acid (OPDA), which is converted to jasmonic acid in the peroxisome through β-oxidation. We performed a survey of the same mutants analyzed in our previous reverse genetics study with this new assay by measuring the response of mutants to OPDA's inhibitory effect on root elongation. Mutants of RDL1 and SERINE CARBOXYPEPTIDASE-LIKE20 (SCPL20) exhibited statistically significant hypersensitivity to OPDA, indicating the potential involvement of these proteins in OPDA metabolism. This convenient assay may be used in the future to rapidly screen for mutants defective in OPDA metabolism or signaling.

Topics: Arabidopsis; Arabidopsis Proteins; Cyclopentanes; DNA, Bacterial; Fatty Acids, Unsaturated; Genes, Plant; Mutation; Oxylipins; Peroxisomes; Phenotype; Plant Roots; Proteome; Proteomics; Seedlings

Jasmonates are oxylipin signals that play important roles in the development of fertile flowers and in defense against pathogens and herbivores in leaves. The aim of this work was to understand the synthesis and function of jasmonates in roots. Grafting experiments with a jasmonate-deficient mutant demonstrated that roots produce jasmonates independently of leaves, despite low expression of biosynthetic enzymes. Levels of 12-oxo-phytodienoic acid, jasmonic acid, and its isoleucine derivative increased in roots upon osmotic and drought stress. Wounding resulted in a decrease of preformed 12-oxo-phytodienoic acid concomitant with an increase of jasmonic acid and jasmonoyl-isoleucine. 13-Lipoxygenases catalyze the first step of lipid oxidation leading to jasmonate production. Analysis of 13-lipoxygenase-deficient mutant lines showed that only one of the four 13-lipoxygenases, LOX6, is responsible and essential for stress-induced jasmonate accumulation in roots. In addition, LOX6 was required for production of basal 12-oxo-phytodienoic acid in leaves and roots. Loss-of-function mutants of LOX6 were more attractive to a detritivorous crustacean and more sensitive to drought, indicating that LOX6-derived oxylipins are important for the responses to abiotic and biotic factors.

Topics: Animals; Arabidopsis; Arabidopsis Proteins; Crustacea; Cyclopentanes; Droughts; Fatty Acids, Unsaturated; Feeding Behavior; Lipoxygenase; Mutation; Osmosis; Oxylipins; Phenotype; Plant Leaves; Plant Roots; Plant Shoots; Signal Transduction; Stress, Physiological

The jasmonate family of phytohormones plays central roles in plant development and stress acclimation. However, the architecture of their signaling circuits remains largely unknown. Here we describe a jasmonate family binding protein, cyclophilin 20-3 (CYP20-3), which regulates stress-responsive cellular redox homeostasis. (+)-12-Oxo-phytodienoic acid (OPDA) binding promotes CYP20-3 to form a complex with serine acetyltransferase 1, which triggers the formation of a hetero-oligomeric cysteine synthase complex with O-acetylserine(thiol)lyase B in chloroplasts. The cysteine synthase complex formation then activates sulfur assimilation that leads to increased levels of thiol metabolites and the buildup of cellular reduction potential. The enhanced redox capacity in turn coordinates the expression of a subset of OPDA-responsive genes. Thus, we conclude that CYP20-3 is a key effector protein that links OPDA signaling to amino acid biosynthesis and cellular redox homeostasis in stress responses.

Topics: Amino Acids; Arabidopsis; Chloroplasts; Chromatography, Affinity; Cyclopentanes; Cyclophilins; Fatty Acids, Unsaturated; Homeostasis; Oxidation-Reduction; Oxidative Stress; Oxylipins; Protein Interaction Maps; Serine O-Acetyltransferase; Signal Transduction

The importance of phytohormone balance is increasingly recognized as central to the outcome of plant-pathogen interactions. Next to their well-known developmental role, brassinosteroids (BR) were recently found to be involved in plant innate immunity. In this study, we examined the role of BR in rice (Oryza sativa) innate immunity during infection with the root-knot nematode Meloidogyne graminicola, and we studied the inter-relationship with the jasmonate (JA) pathway. Exogenous epibrassinolide (BL) supply at low concentrations induced susceptibility in the roots whereas high concentrations of BL enforced systemic defense against this nematode. Upon high exogenous BL supply on the shoot, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) confirmed a strong feedback inhibitory effect, leading to reduced BR biosynthesis in the root. Moreover, we demonstrate that the immune suppressive effect of BR is at least partly due to negative cross-talk with the JA pathway. Mutants in the BR biosynthesis or signaling pathway accumulate slightly higher levels of the immediate JA-precursor 12-oxo-phytodienoic acid, and qRT-PCR data showed that the BR and JA pathway are mutually antagonistic in rice roots. Collectively, these results suggest that the balance between the BR and JA pathway is an effective regulator of the outcome of the rice-M. graminicola interaction.

Topics: Animals; Brassinosteroids; Cyclopentanes; Dose-Response Relationship, Drug; Fatty Acids, Unsaturated; Feedback, Physiological; Gene Expression Regulation, Plant; Models, Biological; Mutation; Oryza; Oxylipins; Plant Diseases; Plant Growth Regulators; Plant Immunity; Plant Roots; RNA, Plant; Signal Transduction; Tylenchoidea

Calcium-dependent protein kinases (CDPKs) modulate plant development and growth and are important regulators of biotic and abiotic stress responses. Recently it was found that simultaneously silencing Nicotiana attenuata NaCDPK4 and NaCDPK5 (IRcdpk4/5 plants) results in accumulation of exceptionally high JA levels after wounding or simulated herbivory treatments, which in turn induced high levels of defense metabolites that slowed the growth of Manduca sexta, a specialist insect herbivore. To investigate the mechanism by which NaCDPK4 and NaCDPK5 regulate JA accumulation, we analyzed the transcript levels of all important enzymes involved in JA biosynthesis, but these genes showed no differences between wild-type and IRcdpk4/5 plants. Moreover, the dynamics of JA were similar between these plants, excluding the possibility of decreased degradation rates in IRcdpk4/5 plants. To gain insight into the mechanism by which NaCDPK4 and NaCDPK5 regulate JA biosynthesis, free fatty acids, including C18:3, and (9S,13S)-12-oxo-phytodienoic acid (OPDA), two important precursors of JA were quantified at different times before and after wounding and simulated herbivore feeding treatments. We show that after these treatments, IRcdpk4/5 plants have decreased levels of C18:3, but have enhanced OPDA and JA levels, suggesting that NaCDPK4 and NaCDPK5 have a role in the early steps of JA biosynthesis. The possible role of NaCDPK4 and NaCDPK5 regulating AOS and AOC enzymatic activity is discussed.

Topics: Cyclopentanes; Fatty Acids, Unsaturated; Nicotiana; Oxylipins; Plant Growth Regulators; Plant Proteins; Protein Kinases

Jasmonates and phytoprostanes are oxylipins that regulate stress responses and diverse physiological and developmental processes. 12-Oxo-phytodienoic acid (OPDA) and phytoprostanes are structurally related electrophilic cyclopentenones, which activate similar gene expression profiles that are for the most part different from the action of the cyclopentanone jasmonic acid (JA) and its biologically active amino acid conjugates. Whereas JA-isoleucine signals through binding to COI1, the bZIP transcription factors TGA2, TGA5, and TGA6 are involved in regulation of gene expression in response to phytoprostanes. Here root growth inhibition and target gene expression were compared after treatment with JA, OPDA, or phytoprostanes in mutants of the COI1/MYC2 pathway and in different TGA factor mutants. Inhibition of root growth by phytoprostanes was dependent on COI1 but independent of jasmonate biosynthesis. In contrast, phytoprostane-responsive gene expression was strongly dependent on TGA2, TGA5, and TGA6, but not dependent on COI1, MYC2, TGA1, and TGA4. Different mutant and overexpressing lines were used to determine individual contributions of TGA factors to cyclopentenone-responsive gene expression. Whereas OPDA-induced expression of the cytochrome P450 gene CYP81D11 was primarily regulated by TGA2 and TGA5, the glutathione S-transferase gene GST25 and the OPDA reductase gene OPR1 were regulated by TGA5 and TGA6, but less so by TGA2. These results support the model that phytoprostanes and OPDA regulate differently (i) growth responses, which are COI1 dependent but jasmonate independent; and (ii) lipid stress responses, which are strongly dependent on TGA2, TGA5, and TGA6. Identification of molecular components in cyclopentenone signalling provides an insight into novel oxylipin signal transduction pathways.

Topics: Arabidopsis; Arabidopsis Proteins; Basic-Leucine Zipper Transcription Factors; Cyclopentanes; Cytochrome P-450 Enzyme System; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Genes, Plant; Isoleucine; Nuclear Proteins; Oxylipins; Plant Roots; Plants, Genetically Modified; Prostaglandins A; Signal Transduction; Stress, Physiological; Transcription, Genetic; Transcriptome

Oxylipins including jasmonates are signaling compounds in plant growth, development, and responses to biotic and abiotic stresses. In Arabidopsis (Arabidopsis thaliana) most mutants affected in jasmonic acid (JA) biosynthesis and signaling are male sterile, whereas the JA-insensitive tomato (Solanum lycopersicum) mutant jai1 is female sterile. The diminished seed formation in jai1 together with the ovule-specific accumulation of the JA biosynthesis enzyme allene oxide cyclase (AOC), which correlates with elevated levels of JAs, suggest a role of oxylipins in tomato flower/seed development. Here, we show that 35S::SlAOC-RNAi lines with strongly reduced AOC in ovules exhibited reduced seed set similarly to the jai1 plants. Investigation of embryo development of wild-type tomato plants showed preferential occurrence of AOC promoter activity and AOC protein accumulation in the developing seed coat and the embryo, whereas 12-oxo-phytodienoic acid (OPDA) was the dominant oxylipin occurring nearly exclusively in the seed coat tissues. The OPDA- and JA-deficient mutant spr2 was delayed in embryo development and showed an increased programmed cell death in the developing seed coat and endosperm. In contrast, the mutant acx1a, which accumulates preferentially OPDA and residual amount of JA, developed embryos similar to the wild type, suggesting a role of OPDA in embryo development. Activity of the residual amount of JA in the acx1a mutant is highly improbable since the known reproductive phenotype of the JA-insensitive mutant jai1 could be rescued by wound-induced formation of OPDA. These data suggest a role of OPDA or an OPDA-related compound for proper embryo development possibly by regulating carbohydrate supply and detoxification.

Topics: Apoptosis; Cyclopentanes; Endosperm; Fatty Acids, Unsaturated; Fruit; Gene Expression Regulation, Plant; Intramolecular Oxidoreductases; Mutation; Organ Specificity; Ovule; Oxylipins; Phenotype; Plant Proteins; Promoter Regions, Genetic; RNA Interference; Seeds; Solanum lycopersicum

Vesicle fusion processes in plants are important for both development and stress responses. Transgenic potato plants with reduced expression of SYNTAXIN-RELATED1 (StSYR1), a gene encoding the potato homolog of Arabidopsis PENETRATION1 (AtPEN1), display spontaneous necrosis and chlorosis at later stages of development. In accordance with this developmental defect, tuber number, weight and overall yield are significantly reduced in StSYR1-RNAi lines. Enhanced resistance of StSYR1-RNAi plants to Phytophthora infestans, the causal agent of late blight disease of potato, correlates with enhanced levels of salicylic acid, whereas levels of 12-oxophytodienoic acid and jasmonic acid are unaltered. Cultured cells of StSYR1-RNAi lines secrete at least two compounds which are not detectable in the supernatant of control cells, suggesting an involvement of StSYR1 in secretion processes to the apoplast.

Topics: Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression; Genes, Plant; Oxylipins; Phytophthora infestans; Plant Development; Plant Diseases; Plant Immunity; Plant Proteins; Plant Tubers; Plants, Genetically Modified; Qa-SNARE Proteins; RNA Interference; Salicylic Acid; Solanum tuberosum

Little is known regarding production and function of endogenous jasmonates (JAs) in root nodules of soybean plants inoculated with Bradyrhizobium japonicum. We investigated (1) production of jasmonic acid (JA) and 12-oxophytodienoic acid (OPDA) in roots of control and inoculated plants and in isolated nodules; (2) correlations between JAs levels, nodule number, and plant growth during the symbiotic process; and (3) effects of exogenous JA and OPDA on nodule cell number and size. In roots of control plants, JA and OPDA levels reached a maximum at day 18 after inoculation; OPDA level was 1.24 times that of JA. In roots of inoculated plants, OPDA peaked at day 15, whereas JA level did not change appreciably. Shoot dry matter of inoculated plants was higher than that of control at day 21. Chlorophyll a decreased more abruptly in control plants than in inoculated plants, whereas b decreased gradually in both cases. Exogenous JA or OPDA changed number and size of nodule central cells and peripheral cells. Findings from this and previous studies suggest that increased levels of JA and OPDA in control plants are related to senescence induced by nutritional stress. OPDA accumulation in nodulated roots suggests its involvement in "autoregulation of nodulation."

Topics: Adult; Bradyrhizobium; Chlorophyll; Chlorophyll A; Cyclopentanes; Fatty Acids, Unsaturated; Glycine max; Humans; Oxylipins; Plant Growth Regulators; Plant Roots; Plant Shoots; Root Nodules, Plant; Symbiosis

The moss Physcomitrella patens is an evolutionarily basal model system suitable for the analysis of plant defence responses activated after pathogen assault. Upon infection with the necrotroph Botrytis cinerea, several defence mechanisms are induced in P. patens, including the fortification of the plant cell wall by the incorporation of phenolic compounds and the induced expression of related genes. Botrytis cinerea infection also activates the accumulation of reactive oxygen species and cell death with hallmarks of programmed cell death in moss tissues. Salicylic acid (SA) levels also increase after fungal infection, and treatment with SA enhances transcript accumulation of the defence gene phenylalanine ammonia-lyase (PAL) in P. patens colonies. The expression levels of the genes involved in 12-oxo-phytodienoic acid (OPDA) synthesis, including lipoxygenase (LOX) and allene oxide synthase (AOS), increase in P. patens gametophytes after pathogen assault, together with a rise in free linolenic acid and OPDA concentrations. However, jasmonic acid (JA) could not be detected in healthy or infected tissues of this plant. Our results suggest that, although conserved defence signals, such as SA and OPDA, are synthesized and are probably involved in the defence response of P. patens against B. cinerea infection, JA production appears to be missing. Interestingly, P. patens responds to OPDA and methyl jasmonate by reducing moss colony growth and rhizoid length, suggesting that jasmonate perception is present in mosses. Thus, P. patens can provide clues with regard to the evolution of different defence pathways in plants, including signalling and perception of OPDA and jasmonates in nonflowering and flowering plants.

Topics: Apoptosis; Biological Evolution; Botrytis; Bryopsida; Cell Death; Cell Wall; Cyclopentanes; Fatty Acids, Unsaturated; Oxylipins; Reactive Oxygen Species; Salicylic Acid; Signal Transduction

Hessian fly (HF) is a biotrophic insect that interacts with wheat on a gene-for-gene basis. We profiled changes in membrane lipids in two isogenic wheat lines: a susceptible line and its backcrossed offspring containing the resistance gene H13. Our results revealed a 32 to 45% reduction in total concentrations of 129 lipid species in resistant plants during incompatible interactions within 24 h after HF attack. A smaller and delayed response was observed in susceptible plants during compatible interactions. Microarray and real-time polymerase chain reaction analyses of 168 lipid-metabolism-related transcripts revealed that the abundance of many of these transcripts increased rapidly in resistant plants after HF attack but did not change in susceptible plants. In association with the rapid mobilization of membrane lipids, the concentrations of some fatty acids and 12-oxo-phytodienoic acid (OPDA) increased specifically in resistant plants. Exogenous application of OPDA increased mortality of HF larvae significantly. Collectively, our data, along with previously published results, indicate that the lipids were mobilized through lipolysis, producing free fatty acids, which were likely further converted into oxylipins and other defense molecules. Our results suggest that rapid mobilization of membrane lipids constitutes an important step for wheat to defend against HF attack.

Topics: Animals; Cyclopentanes; Diptera; Fatty Acids; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Genes, Plant; Host-Parasite Interactions; Larva; Membrane Lipids; Models, Biological; Oligonucleotide Array Sequence Analysis; Oxylipins; Plant Diseases; Plant Epidermis; Plant Growth Regulators; Plant Immunity; Plant Leaves; RNA, Plant; Salicylic Acid; Seedlings; Time Factors; Triticum

Jasmonates (JAs) are ubiquitously occurring signaling compounds in plants formed in response to biotic and abiotic stress as well as in development. (+)-7-iso-jasmonoyl isoleucine, the bioactive JA, is involved in most JA-dependent processes mediated by the F-box protein COI1 in a proteasome-dependent manner. However, there is an increasing number of examples, where the precursor of JA biosynthesis, cis-(+)-12-oxophytodienoic acid (OPDA) is active in a JA/COI1-independent manner. Here, we discuss those OPDA-dependent processes, thereby giving emphasis on tomato embryo development. Recent data on seed coat-generated OPDA and its role in embryo development is discussed based on biochemical and genetic evidences.

Topics: Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Isoleucine; Models, Biological; Mutation; Oxylipins; Plant Proteins; Seeds; Signal Transduction; Solanum lycopersicum

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.

Topics: Adaptation, Physiological; Cicer; Cyclopentanes; Droughts; Fatty Acids, Unsaturated; Genes, Plant; Isoleucine; Lipoxygenase; Oxylipins; Plant Proteins; Plant Roots; Polymerase Chain Reaction; Stress, Physiological; Transcriptome; Water

Root-to-shoot communication plays an important role in the adaptation to environmental stress. In this study, we established a model system for root-to-shoot signalling to observe global gene expression in Arabidopsis thaliana. The roots of Arabidopsis seedlings were wounded and the expression in the shoots of 68 and 5 genes was up-regulated threefold at 30 min and 6 h post-injury, respectively. These genes were designated early and late Root-to-Shoot responsive (RtS) genes, respectively. Many of the early RtS genes were found to encode transcription factors such as AtERFs, whereas others were associated with jasmonic acid (JA) and ethylene (ET). Some of the late RtS genes were shown to be regulated by 12-oxo-phytodienoic acid (OPDA). In fact, elevated levels of JA and OPDA were detected in the shoots of seedlings 30 min and 6 h, respectively, after wounding of the roots. A mutant analysis revealed that JA and ET are involved in the expression of the early RtS genes. Thus, root-to-shoot communication for many RtS genes is associated with the systemic production of JA, OPDA and possibly ET.

Topics: Arabidopsis; Cyclopentanes; Dehydration; Ethylenes; Fatty Acids, Unsaturated; Gene Expression Profiling; Gene Expression Regulation, Plant; Genes, Plant; Mutation; Oligonucleotide Array Sequence Analysis; Oxylipins; Plant Roots; Plant Shoots; RNA, Plant; Signal Transduction; Stress, Physiological; Transcription Factors

Arabidopsis thaliana COMATOSE (CTS) encodes an ABC transporter involved in peroxisomal import of substrates for β-oxidation. Various cts alleles and mutants disrupted in steps of peroxisomal β-oxidation have previously been reported to exhibit a severe block on seed germination. Oxylipin analysis on cts, acyl CoA oxidase1 acyl CoA oxidase2 (acx1 acx2), and keto acyl thiolase2 dry seeds revealed that they contain elevated levels of 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA), and JA-Ile. Oxylipin and transcriptomic analysis showed that accumulation of these oxylipins occurs during late seed maturation in cts. Analysis of double mutants generated by crossing cts with mutants in the JA biosynthesis pathway indicate that OPDA, rather than JA or JA-Ile, contributes to the block on germination in cts seeds. We found that OPDA was more effective at inhibiting wild-type germination than was JA and that this effect was independent of CORONATINE INSENSITIVE1 but was synergistic with abscisic acid (ABA). Consistent with this, OPDA treatment increased ABA INSENSITIVE5 protein abundance in a manner that parallels the inhibitory effect of OPDA and OPDA+ABA on seed germination. These results demonstrate that OPDA acts along with ABA to regulate seed germination in Arabidopsis.

Topics: Abscisic Acid; Arabidopsis; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Developmental; Gene Expression Regulation, Plant; Germination; Mutation; Oxylipins; Seeds

Jasmonic acid and its derived metabolites (JAs) orchestrate plant defense against insects and fungi. 12-Oxo-phytodienoic acid (OPDA), a JA precursor, has also been implicated in plant defense. We sought to define JAs and OPDA functions through comparative defense susceptibility characteristics of three Arabidopsis (Arabidopsis thaliana) genotypes: aos, lacking JAs and OPDA; opda reductase3 (opr3), deficient in JA production but can accumulate OPDA; and transgenics that overexpress OPR3. opr3, like aos, is susceptible to cabbage loopers (Trichoplusia ni) but, relative to aos, opr3 has enhanced resistance to a necrotrophic fungus. Gas chromatography-mass spectrometry reveals that opr3 produces OPDA but no detectable JAs following wounding and looper infestation; unexpectedly, substantial levels of JAs accumulate in opr3 upon fungal infection. Full-length OPR3 transcripts accumulate in fungal-infected opr3, potentially through splicing of the T-DNA containing intron. Fungal resistance correlates with levels of JAs not OPDA; therefore, opr3 resistance to some pests is likely due to JA accumulation, and signaling activities ascribed to OPDA should be reassessed because opr3 can produce JAs. Together these data (1) reinforce the primary role JAs play in plant defense against insects and necrotrophic fungi, (2) argue for a reassessment of signaling activities ascribed to OPDA, and (3) provide evidence that mutants with intron insertions can retain gene function.

Topics: Animals; Arabidopsis; Arabidopsis Proteins; Botrytis; Brassica; Cyclopentanes; DNA, Bacterial; Fatty Acids, Unsaturated; Fertility; Gene Expression Regulation, Plant; Immunity, Innate; Indoles; Introns; Molecular Sequence Data; Moths; Mutagenesis, Insertional; Mutation; Oxidoreductases; Oxylipins; Plant Diseases; Thiazoles

How plants perceive herbivory is not yet well understood. We investigated early responses of the model plant Arabidopsis (Arabidopsis thaliana) to attack from the generalist grasshopper herbivore, Schistocerca gregaria (Caelifera). When compared with wounding alone, S. gregaria attack and the application of grasshopper oral secretions (GS) to puncture wounds elicited a rapid accumulation of various oxylipins, including 13-hydroperoxy octadecatrienoic acid, 12-oxo-phytodienoic acid (OPDA), jasmonic acid, and jasmonic acid-isoleucine. Additionally, GS increased cytosolic calcium levels, mitogen-activated protein kinase (MPK3 and MPK6) activity, and ethylene emission but not the accumulation of hydrogen peroxide. Although GS contain caeliferin A16:0, a putative elicitor of caeliferan herbivores, treatment with pure, synthetic caeliferin A16:0 did not induce any of the observed responses. With mutant plants, we demonstrate that the observed changes in oxylipin levels are independent of MPK3 and MPK6 activity but that MPK6 is important for the GS-induced ethylene release. Biochemical and pharmacological analyses revealed that the lipase activity of GS plays a central role in the GS-induced accumulation of oxylipins, especially OPDA, which could be fully mimicked by treating puncture wounds only with a lipase from Rhizopus arrhizus. GS elicitation increased the levels of OPDA-responsive transcripts. Because the oral secretions of most insects used to study herbivory-induced responses in Arabidopsis rapidly elicit similar accumulations of OPDA, we suggest that lipids containing OPDA (arabidopsides) play an important role in the activation of herbivory-induced responses.

Topics: Animals; Arabidopsis; Arabidopsis Proteins; Cyclopentanes; Ethylenes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Grasshoppers; Insecta; Lipase; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Mouth; Oxylipins; Reactive Oxygen Species; RNA, Messenger

Jasmonates are well-characterized signals in the development of plants and their response to abiotic and biotic stresses, such as touch and wounding by herbivores. A gap in our knowledge on jasmonate-induced processes, however, is the cellular localization of jasmonates. Here, a novel antibody-based approach was developed to visualize jasmonates in cross-sections of plant material. Antibodies raised in rabbits against BSA-coupled jasmonic acid (JA) are specific for JA, its methyl ester and isoleucine conjugate. They do not bind to 12-oxophytodienoic acid, 12-hydoxy-JA or coronatine. These antibodies were used in combination with newly established fixation and embedding methods. Jasmonates were rapidly and uniformly distributed within all cells near the site of damage of a mechanically wounded tomato (Solanum lycopersicum) leaf. Leaf tissue distally located to the wound site exhibited identical distribution, but had a lower signal intensity. The occurrence of jasmonates in all cell types of a wounded leaf was accompanied by transcript accumulation of early JA-induced genes visualized by in situ hybridization. With these new antibodies, a powerful tool is available to detect cell-specifically the occurrence of jasmonates in any jasmonate-dependent stress response or developmental process of plants.

Topics: Antibodies; Arabidopsis; Cyclopentanes; Fatty Acids, Unsaturated; Immunohistochemistry; Oxylipins; Plant Growth Regulators; Plant Leaves; Solanum lycopersicum

It is known that wounding systemically activates the expression of various defense-related genes in plants. However, most studies of wound-induced systemic response are concerned with a leaf-to-leaf response. We have recently reported that the long distance signaling was also observed in the shoots of Arabidopsis seedling with wounded roots. We identified early and late root-to-shoot responsive (RtS) genes that were upregulated in the shoots of root-wounded seedlings at 30 min and 6 h post-injury, respectively. It is likely that the primary signals were rapidly transfered from injured roots to shoots, and then these signals were converted into chemical signals. In fact, increase of JA and OPDA content activated the expression of early and late RtS genes in shoots, respectively. In addition, we visualized wound-induced root-to-shoot response by using RtS promoter-luciferase (Luc) transgenic plants. Analysis of the AtERF13 promoter::Luc transgenic plants clearly shows that the wound-induced root-to-shoot signaling was rapidly activated via the vascular systems.

Topics: Arabidopsis; Arabidopsis Proteins; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Oxylipins; Plant Roots; Plant Shoots; Stress, Physiological; Time Factors

Venus flytrap's leaves can catch an insect in a fraction of a second. Since the time of Charles Darwin, scientists have struggled to understand the sensory biology and biomechanics of this plant, Dionaea muscipula. Here we show that insect-capture of Dionaea traps is modulated by the phytohormone abscisic acid (ABA) and jasmonates. Water-stressed Dionaea, as well as those exposed to the drought-stress hormone ABA, are less sensitive to mechanical stimulation. In contrast, application of 12-oxo-phytodienoic acid (OPDA), a precursor of the phytohormone jasmonic acid (JA), the methyl ester of JA (Me-JA), and coronatine (COR), the molecular mimic of the isoleucine conjugate of JA (JA-Ile), triggers secretion of digestive enzymes without any preceding mechanical stimulus. Such secretion is accompanied by slow trap closure. Under physiological conditions, insect-capture is associated with Ca(2+) signaling and a rise in OPDA, Apparently, jasmonates bypass hapto-electric processes associated with trap closure. However, ABA does not affect OPDA-dependent gland activity. Therefore, signals for trap movement and secretion seem to involve separate pathways. Jasmonates are systemically active because application to a single trap induces secretion and slow closure not only in the given trap but also in all others. Furthermore, formerly touch-insensitive trap sectors are converted into mechanosensitive ones. These findings demonstrate that prey-catching Dionaea combines plant-specific signaling pathways, involving OPDA and ABA with a rapidly acting trigger, which uses ion channels, action potentials, and Ca(2+) signals.

Topics: Abscisic Acid; Action Potentials; Amino Acids; Animals; Cyclopentanes; Droseraceae; Fatty Acids, Unsaturated; Indenes; Insecta; Oxylipins; Plant Growth Regulators; Plant Leaves; Predatory Behavior; Stress, Mechanical; Time Factors

Ecological performance is all about timing and the endogenous clock that allows the entrainment of rhythms and anticipation of fitness-determining events is being rapidly characterized. How plants anticipate daily abiotic stresses, such as cold in early mornings and drought at noon, as well as biotic stresses, such as the timing of pathogen infections, is being explored, but little is known about the clock's role in regulating responses to insect herbivores and mutualists, whose behaviors are known to be strongly diurnally regulated and whose attack is known to reconfigure plant metabolomes. We developed a liquid chromatography-mass spectrometry procedure and analyzed its output with model-based peak picking algorithms to identify metabolites with diurnal accumulation patterns in sink/source leaves and roots in an unbiased manner. The response of metabolites with strong diurnal patterns to simulated attack from the specialist herbivore, Manduca sexta larvae was analyzed and annotated with in-house and public databases. Roots and leaves had largely different rhythms and only 10 ions of 182 oscillating ions in leaves and 179 oscillating ions in roots were rhythmic in both tissues: root metabolites mainly peaked at dusk or night, while leaf metabolites peaked during the day. Many oscillating metabolites showed tissue-specific regulation by simulated herbivory of which systemic responses in unattacked tissues were particularly pronounced. Diurnal and herbivory-elicited accumulation patterns of disaccharide, phenylalanine, tyrosine, lyciumoside I, coumaroyl tyramine, 12-oxophytodienoic acid and jasmonic acid and those of their related biosynthetic transcripts were examined in detail. We conclude that oscillating metabolites of N. attenuata accumulate in a highly tissue-specific manner and the patterns reveal pronounced diurnal rhythms in the generalized and specialized metabolism that mediates the plant's responses to herbivores and mutualists. We propose that diurnal regulation will prove to an important element in orchestrating a plant's responses to herbivore attack.

Topics: Animals; Circadian Rhythm; Cyclopentanes; Disaccharides; Fatty Acids, Unsaturated; Genes, Plant; Glycosides; Herbivory; Larva; Manduca; Mechanical Phenomena; Nicotiana; Organ Specificity; Oxylipins; Phenylalanine; Plant Leaves; Plant Roots; Polyamines; RNA, Messenger; Tyrosine

This study was conducted to determine if constitutive levels of jasmonic acid (JA) and other octadecanoid compounds were elevated prior to herbivory in a maize genotype with documented resistance to fall armyworm (Spodoptera frugiperda) and other lepidopteran pests. The resistant inbred Mp708 had approximately 3-fold higher levels of jasmonic acid (JA) prior to herbivore feeding than the susceptible inbred Tx601. Constitutive levels of cis-12-oxo-phytodienoic acid (OPDA) also were higher in Mp708 than Tx601. In addition, the constitutive expression of JA-inducible genes, including those in the JA biosynthetic pathway, was higher in Mp708 than Tx601. In response to herbivory, Mp708 generated comparatively higher levels of hydrogen peroxide, and had a greater abundance of NADPH oxidase transcripts before and after caterpillar feeding. Before herbivore feeding, low levels of transcripts encoding the maize insect resistance cysteine protease (Mir1-CP) and the Mir1-CP protein were detected consistently. Thus, Mp708 appears to have a portion of its defense pathway primed, which results in constitutive defenses and the ability to mount a stronger defense when caterpillars attack. Although the molecular mechanisms that regulate the constitutive accumulation of JA in Mp708 are unknown, it might account for its enhanced resistance to lepidopteran pests. This genotype could be valuable in studying the signaling pathways that maize uses to response to insect herbivores.

Topics: Animals; Breeding; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Hydrogen Peroxide; Lepidoptera; NADPH Oxidases; Oxidoreductases Acting on CH-CH Group Donors; Oxylipins; Plant Proteins; RNA, Messenger; RNA, Plant; Signal Transduction; Zea mays

Lipases are involved in the generation of jasmonates, which regulate responses to biotic and abiotic stresses. Two sn-1-specific acyl hydrolases, DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1) and DONGLE (DGL), have been reported to be localized in plastids and to be essential and sufficient for jasmonate biosynthesis in Arabidopsis (Arabidopsis thaliana) leaves. Here, we show that levels of 12-oxo-phytodienoic acid (OPDA) and jasmonic acid in three different DGL RNA interference lines and the dad1 mutant were similar to wild-type levels during the early wound response as well as after Pseudomonas infection. Due to the lack of sn-2 substrate specificity, synthesis of dinor OPDA was not expected and also not found to be affected in DGL knockdown and DGL-overexpressing lines. As reported, DAD1 participates in jasmonate formation only in the late wound response. In addition, DGL protein was found to be localized in lipid bodies and not in plastids. Furthermore, jasmonate levels in 16 additional mutants defective in the expression of lipases with predicted chloroplast localization did not show strong differences from wild-type levels after wounding, except for a phospholipase A (PLA) PLA-Igamma1 (At1g06800) mutant line that displayed diminished wound-induced dinor OPDA, OPDA, and jasmonic acid levels. A quadruple mutant defective in four DAD1-like lipases displayed similar jasmonate levels as the mutant line of PLA-Igamma1 after wounding. Hence, we identify PLA-Igamma1 as a novel target gene to manipulate jasmonate biosynthesis. Our results suggest that, in addition to DAD1 and PLA-Igamma1, still unidentified enzymes with sn-1 and sn-2 hydrolase activity are involved in wound- and pathogen-induced jasmonate formation, indicating functional redundancy within the lipase family.

Topics: Arabidopsis; Arabidopsis Proteins; Cyclopentanes; Fatty Acids, Unsaturated; Mutation; Oxylipins; Phospholipases A; Phospholipases A1; Plant Diseases; Plants, Genetically Modified; Pseudomonas syringae

Jasmonate (JA) activates plant defense, promotes pollen maturation, and suppresses plant growth. An emerging theme in JA biology is its involvement in light responses; here, we examine the interdependence of the JA- and light-signaling pathways in Arabidopsis thaliana. We demonstrate that mutants deficient in JA biosynthesis and signaling are deficient in a subset of high irradiance responses in far-red (FR) light. These mutants display exaggerated shade responses to low, but not high, R/FR ratio light, suggesting a role for JA in phytochrome A (phyA) signaling. Additionally, we demonstrate that the FR light-induced expression of transcription factor genes is dependent on CORONATINE INSENSITIVE1 (COI1), a central component of JA signaling, and is suppressed by JA. phyA mutants had reduced JA-regulated growth inhibition and VSP expression and increased content of cis-(+)-12-oxophytodienoic acid, an intermediate in JA biosynthesis. Significantly, COI1-mediated degradation of JASMONATE ZIM DOMAIN1-beta-glucuronidase (JAZ1-GUS) in response to mechanical wounding and JA treatment required phyA, and ectopic expression of JAZ1-GUS resulted in exaggerated shade responses. Together, these results indicate that JA and phyA signaling are integrated through degradation of the JAZ1 protein, and both are required for plant responses to light and stress.

Topics: Anthocyanins; Arabidopsis; Arabidopsis Proteins; Chlorophyll; Cyclopentanes; Fatty Acids, Unsaturated; Flowers; Gene Expression Regulation, Plant; Light; Mutation; Oxylipins; Phytochrome A; Plant Growth Regulators; RNA, Plant; Signal Transduction

epi-Jasmonic acid (epi-JA) and tuberonic acid (TA) were synthesized from the key aldehyde, all cis-2-(2-hydroxy-5-vinylcyclopentyl)acetaldehyde (14), which was in turn prepared stereoselectively from the (1R)-acetate of 4-cyclopentene-1,3-diol (10) through S(N)2-type allylic substitution with CH(2)[double bond, length as m-dash]CHMgBr followed by Mitsunobu inversion, Eschenmoser-Claisen rearrangement, and regioselective Swern oxidation of the corresponding bis-TES ether (13). Wittig reaction of the aldehyde 14 with [Ph(3)P(CH(2))Me](+)Br(-) followed by oxidation afforded epi-JA (3) stereoselectivity over the trans isomer. Similarly, TA (5) was synthesized. Furthermore, the above findings were applied successfully to improve the total efficiency of the previous synthesis of 12-oxo-PDA (1).

Topics: Acetates; Aldehydes; alpha-Linolenic Acid; Cyclopentanes; Fatty Acids, Unsaturated; Lactones; Oxylipins; Stereoisomerism

Jasmonic acid (JA) is a plant hormone that plays an important role in a wide variety of plant physiological processes. The plant pathogenic fungus, Lasiodiplodia theobromae also produces JA; however, its biosynthesis in this fungus has yet to be explored. Administration of [1-(13)C] and [2-(13)C] NaOAc into L. theobromae established that JA in this fungus originates from a fatty acid synthetic pathway. The methyl ester of 12-oxo-phytodienoic acid (OPDA) was detected in the culture extracts of L. theobromae by GC-MS analysis. This finding indicates the presence of OPDA (a known intermediate of JA biosynthesis in plants) in L. theobromae. (2)H NMR spectroscopic data of JA produced by L. theobromae with the incorporation of [9,10,12,13,15,16-(2)H(6)] linolenic acid showed that five deuterium atoms remained intact. In plants, this is speculated to arise from JA being produced by the octadecanoid pathway. However, the observed stereoselectivity of the cyclopentenone olefin reduction in L. theobromae was opposite to that observed in plants. These data suggest that JA biosynthesis in L. theobromae is similar to that in plants, but differing in the facial selectivity of the enone reduction.

Topics: alpha-Linolenic Acid; Ascomycota; Cyclization; Cyclopentanes; Fatty Acids, Unsaturated; Gas Chromatography-Mass Spectrometry; Molecular Structure; Nuclear Magnetic Resonance, Biomolecular; Oxylipins; Plant Growth Regulators; Plants

Adventitious root formation (ARF) in the model plant Petunia hybrida cv. Mitchell has been analysed in terms of anatomy, gene expression, enzymatic activities and levels of metabolites. This study focuses on the involvement of wound response and primary metabolism. Microscopic techniques were complemented with targeted transcript, enzyme and metabolite profiling using real time polymerase chain reaction (PCR), Northern blot, enzymatic assays, chromatography and mass spectrometry. Three days after severance from the stock plants, first meristematic cells appeared which further developed into root primordia and finally adventitious roots. Excision of cuttings led to a fast and transient increase in the wound-hormone jasmonic acid, followed by the expression of jasmonate-regulated genes such as cell wall invertase. Analysis of soluble and insoluble carbohydrates showed a continuous accumulation during ARF. A broad metabolite profiling revealed a strong increase in organic acids and resynthesis of essential amino acids. Substantial changes in enzyme activities and metabolite levels indicate that specific enzymes and metabolites might play a crucial role during ARF. Three metabolic phases could be defined: (i) sink establishment phase characterized by apoplastic unloading of sucrose and being probably mediated by jasmonates; (ii) recovery phase; and (iii) maintenance phase, in which a symplastic unloading occurs.

Topics: Amino Acids; Carbohydrate Metabolism; Cell Respiration; Citric Acid Cycle; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Genes, Plant; Glycolysis; Oxylipins; Petunia; Plant Proteins; Plant Roots; RNA, Messenger

* The cpr5-1 Arabidopsis thaliana mutant exhibits constitutive activation of salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signalling pathways and displays enhanced tolerance of heat stress (HS). * cpr5-1 crossed with jar1-1 (a JA-amino acid synthetase) was compromised in basal thermotolerance, as were the mutants opr3 (mutated in OPDA reductase3) and coi1-1 (affected in an E3 ubiquitin ligase F-box; a key JA-signalling component). In addition, heating wild-type Arabidopsis led to the accumulation of a range of jasmonates: JA, 12-oxophytodienoic acid (OPDA) and a JA-isoleucine (JA-Ile) conjugate. Exogenous application of methyl jasmonate protected wild-type Arabidopsis from HS. * Ethylene was rapidly produced during HS, with levels being modulated by both JA and SA. By contrast, the ethylene mutant ein2-1 conferred greater thermotolerance. * These data suggest that JA acts with SA, conferring basal thermotolerance while ET may act to promote cell death.

Topics: Adaptation, Physiological; Arabidopsis; Arabidopsis Proteins; Cyclopentanes; Ethylenes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Heat-Shock Response; Membrane Proteins; Mutation; Nucleotidyltransferases; Oxylipins; Phenotype; RNA, Messenger; Salicylic Acid; Signal Transduction; Temperature

Gall-inducing insects are accomplished plant parasites that can profoundly influence host-plant physiology. We recently reported that the caterpillar Gnorimoschema gallaesolidaginis failed to significantly alter emissions of host-plant volatiles that often recruit natural enemies of insect herbivores, and demonstrated that a caterpillar species feeding on linolenate-deficient plant tissues avoids inducing some of the indirect defenses of its host plant. Here, we investigate whether absence of volatile responses to the galler G. gallaesolidaginis could similarly be explained by a lack of linolenate in galls. We screened interior and exterior tissue of galls and control stems of Solidago altissima for free linolenate, linoleate, 12-oxo-phytodienoate, jasmonate, and salicylate. We found, unexpectedly, that G. gallaesolidaginis strongly increased amounts of linolenic and linoleic acids inside galls without associated increases in two downstream products, 12-oxo-phytodienoic or jasmonic acid. In contrast, the generalist caterpillar Heliothis virescens induced elevated levels of linolenic, linoleic, 12-oxo-phytodienoic, and jasmonic acids in S. altissima. Moreover, these two fatty acids and 12-oxo-phytodienoate were significantly and positively associated with jasmonic acid, suggesting that increased levels of these precursors can lead directly to greater amounts of jasmonic acid. Taken together, these findings suggest that gall insects may be able to nutritionally enhance their food source without inducing concomitant increases in phytohormones and associated defense responses.

Topics: alpha-Linolenic Acid; Animals; Cyclopentanes; Fatty Acids; Fatty Acids, Unsaturated; Host-Parasite Interactions; Linoleic Acids; Moths; Oxylipins; Plant Growth Regulators; Plant Tumors; Solidago; Species Specificity

The antagonistic action between jasmonic acid (JA) and salicylic acid (SA) in plant defence responses has been well documented. However, their relationship in secondary metabolite production is largely unknown. Here, we report that PB90, a protein elicitor from Phytophthora boehmeriae, triggers JA generation, SA accumulation and flavonol glycoside production of Ginkgo biloba cells. JA inhibitors suppress not only PB90-triggered JA generation, but also the elicitor-induced flavonol glycoside production. However, the elicitor can still enhance flavonol glycoside production even though the JA generation is totally inhibited. Over-expression of SA hydrolase gene NahG not only abolishes SA accumulation, but also suppresses the elicitor-induced flavonol glycoside production when JA signalling is inhibited. Interestingly, expression of NahG does not inhibit the elicitor-induced flavonol glycoside accumulation in the absence of JA inhibitors. Moreover, JA levels are significantly enhanced when SA accumulation is impaired in the transgenic cells. Together, the data suggest that both JA and SA are involved in PB90-induced flavonol glycoside production. Furthermore, we demonstrate that JA signalling might be enhanced to substitute for SA to mediate the elicitor-induced flavonol glycoside accumulation when SA signalling is impaired, which reveals an unusual complementary relationship between JA and SA in mediating plant secondary metabolite production.

Topics: Cyclopentanes; Fatty Acids, Unsaturated; Flavonols; Fungal Proteins; Gene Expression Regulation, Plant; Genes, Plant; Ginkgo biloba; Glycosides; Mixed Function Oxygenases; Oxylipins; Phytophthora; Plants, Genetically Modified; Salicylic Acid; Signal Transduction

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.

Topics: Animals; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Profiling; Gene Expression Regulation, Plant; Nicotiana; Oxylipins; Plant Growth Regulators; Plant Leaves; Plant Lectins; RNA, Plant; Spodoptera

Jasmonic acid (JA) and its biologically active derivatives (bioactive JAs) perform a critical role in regulating plant responses to wound stress. The perception of bioactive JAs by the F-box protein COI1 triggers the SCF(COI1)/ubiquitin-dependent degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins that repress the expression of JA-response genes. JA is required for many wound-inducible systemic defense responses, but little is known about the role of the hormone in long-distance signal relay between damaged and undamaged leaves. Here, we show that the wounding of Arabidopsis thaliana leaves results in the rapid (<5 min) accumulation of jasmonoyl-l-isoleucine (JA-Ile), the bioactive form of JA, in leaves distal to the wound site. The rapid systemic increase in JA-Ile preceded the onset of early transcriptional responses, and was associated with JAZ degradation. Wound-induced systemic production of JA-Ile required the JA biosynthetic enzyme 12-oxo-phytodienoic acid (OPDA) reductase 3 (OPR3) in undamaged responding leaves, but not in wounded leaves, and was largely dependent on the JA-conjugating enzyme JAR1. Interestingly, the wound-induced synthesis of JA/JA-Ile in systemic leaves was correlated with a rapid decline in OPDA levels. These results are consistent with a model in which a rapidly transmitted wound signal triggers the systemic synthesis of JA, which, upon conversion to JA-Ile, activates the expression of early response genes by the SCF(COI1)/JAZ pathway.

Topics: Adaptation, Physiological; Arabidopsis; Arabidopsis Proteins; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Isoleucine; Nucleotidyltransferases; Oxidoreductases; Oxylipins; Plant Leaves; Plants, Genetically Modified; RNA, Plant; Signal Transduction

Topics: Cyclopentanes; Fatty Acids, Unsaturated; Oxylipins; Plant Growth Regulators; Plants; Signal Transduction

The oxylipin pathway is commonly involved in induced plant defenses, and is the main signal-transduction pathway induced by insect folivory. Herbivory induces the production of several oxylipins, and consequently alters the so-called 'oxylipin signature' in the plant. Jasmonic acid (JA), as well as pathway intermediates are known to induce plant defenses. Indirect defense against herbivorous insects comprises the production of herbivore-induced plant volatiles (HIPVs). To unravel the precise oxylipin signal-transduction underlying the production of HIPVs in Arabidopsis thaliana and the resulting attraction of parasitoid wasps, we used a multidisciplinary approach that includes molecular genetics, metabolite analysis, and behavioral analysis. Mutant plants affected in the jasmonate pathway (18:0 and/or 16:0 -oxylipin routes; mutants dde2-2, fad5, opr3) were studied to assess the effects of JA and its oxylipin intermediates 12-oxo-phytodienoate (OPDA) and dinor-OPDA (dnOPDA) on HIPV emission and parasitoid (Diadegma semiclausum) attraction. Interference with the production of the oxylipins JA and OPDA altered the emission of HIPVs, in particular terpenoids and the phenylpropanoid methyl salicylate, which affected parasitoid attraction. Our data show that the herbivore-induced attraction of parasitoid wasps to Arabidopsis plants depends on HIPVs that are induced through the 18:0 oxylipin-derivative JA. Furthermore, our study shows that the 16:0-oxylipin route towards dnOPDA does not play a role in HIPV induction, and that the role of 18:0 derived oxylipin-intermediates, such as OPDA, is either absent or limited.

Topics: Animals; Arabidopsis; Behavior, Animal; Cyclopentanes; Fatty Acids, Unsaturated; Genotype; Host-Parasite Interactions; Oxylipins; Plants, Genetically Modified; Signal Transduction; Wasps

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 omega-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.

Topics: Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression; Genes, Plant; Intramolecular Oxidoreductases; Magnaporthe; Oryza; Oxidoreductases Acting on CH-CH Group Donors; Oxylipins; Plant Diseases; Plants, Genetically Modified

Both enzymatically and non-enzymatically generated oxylipins fulfill signalling functions in plant responses to biotic and oxidative stress on the cellular level. We studied the impact of two different exogenously applied cyclopentenone-oxylipins on the proteome of Arabidopsis thaliana leaves: the enzymatically formed 12-oxo-phytodienoic-acid, a member of the jasmonate family of mediators, and A(1)-phytoprostane generated by a free-radical mechanism upon biotic and oxidative stress. Infiltration of leaves with these oxylipins led to induction of classical stress proteins like chaperones as well as enzymes connected to the cellular redox and detoxification systems. A large proportion of the regulated proteins are localized in chloroplasts where these oxylipins are formed. Furthermore, we show that cyclopentenone-oxylipins spontaneously react with several proteins and glutathione in vitro and in vivo. Conjugation to the glutathione sulfhydryl group is a reversible process that is also catalysed by glutathione-S-transferases. In vitro, an oxidative stress inducible glutathione-S-transferase, GST6, localized both in plastids and the cytosol can be covalently modified and partially inactivated by several cyclopentenone-oxylipins.

Topics: Arabidopsis; Arabidopsis Proteins; Chloroplasts; Cyclopentanes; Fatty Acids, Unsaturated; Glutathione Transferase; Molecular Chaperones; Oxidation-Reduction; Oxidative Stress; Oxylipins; Plant Leaves; Proteome; Signal Transduction

Root-knot nematodes (RKN) are severe pests of maize. Although lipoxygenase (LOX) pathways and their oxylipin products have been implicated in plant-nematode interactions, prior to this report there was no conclusive genetic evidence for the function of any plant LOX gene in such interactions. We showed that expression of a maize 9-LOX gene, ZmLOX3, increased steadily and peaked at 7 days after inoculation with Meloidogyne incognita RKN. Mu-insertional lox3-4 mutants displayed increased attractiveness to RKN and an increased number of juveniles and eggs. A set of jasmonic acid (JA)- and ethylene (ET)-responsive and biosynthetic genes as well as salicylic acid (SA)-dependent genes were overexpressed specifically in the roots of lox3-4 mutants. Consistent with this, levels of JA, SA, and ET were elevated in lox3-4 mutant roots, but not in leaves. Unlike wild types, in lox3-4 mutant roots, a phenylalanine ammonia lyase (PAL) gene was not RKN-inducible, suggesting a role for PAL-mediated metabolism in nematode resistance. In addition to these alterations in the defense status of roots, lox3-4 knockout mutants displayed precocious senescence and reduced root length and plant height compared with the wild type, suggesting that ZmLOX3 is required for normal plant development. Taken together, our data indicate that the ZmLOX3-mediated pathway may act as a root-specific suppressor of all three major defense signaling pathways to channel plant energy into growth processes, but is required for normal levels of resistance against nematodes.

Topics: Aldehyde-Lyases; Animals; Cyclopentanes; Cytochrome P-450 Enzyme System; Disease Susceptibility; Ethylenes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Genes, Plant; Immunity, Innate; Intramolecular Oxidoreductases; Lipoxygenase; Models, Biological; Mutation; Nematoda; Organ Specificity; Oxylipins; Plant Diseases; Plant Roots; RNA, Messenger; Salicylic Acid; Zea mays

The conditional flu mutant of Arabidopsis accumulates excess amounts of protochlorophyllide within plastid membranes in the dark and generates singlet oxygen upon light exposure. By varying the length of the dark period, the level of the photosensitizer protochlorophyllide may be modulated, and conditions have been established that either endorse the cytotoxicity of (1)O(2) or reveal its signaling role. Two criteria have been used to distinguish between these two modes of activity of (1)O(2): the impact of the EXECUTER1 mutation and the prevalence of either non-enzymatic or enzymatic lipid peroxidation. During illumination of etiolated flu seedlings, toxic effects of (1)O(2) prevail and non-enzymatic lipid peroxidation proceeds rapidly. In contrast, in light-grown flu plants that were subjected to an 8 h dark/light shift, lipid peroxidation occurred almost exclusively enzymatically. The resulting oxidation product, 13-hydroperoxy octadecatrienoic acid (13-HPOT), serves as a substrate for synthesis of 12-oxo phytodienoic acid (OPDA) and jasmonic acid (JA), both of which are known to control various metabolic and developmental processes in plants. Inactivation of the EXECUTER1 protein abrogates not only (1)O(2)-mediated cell death and growth inhibition of flu plants, but also enzymatic lipid peroxidation. However, inactivation of jasmonate biosynthesis in the aos/flu double mutant does not affect (1)O(2)-mediated growth inhibition and cell death. Hence, JA and OPDA do not act as second messengers during (1)O(2) signaling, but form an integral part of a stress-related signaling cascade activated by (1)O(2) that encompasses several signaling pathways known to be activated by abiotic and biotic stressors.

Topics: Arabidopsis; Arabidopsis Proteins; Cell Death; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Profiling; Gene Expression Regulation, Plant; Lipid Peroxidation; Mutation; Oxidation-Reduction; Oxidative Stress; Oxylipins; Singlet Oxygen; Time Factors

12-oxo-phytodienoic acid and several phytoprostanes are cyclopentenone oxylipins that are formed via the enzymatic jasmonate pathway and a nonenzymatic, free radical-catalyzed pathway, respectively. Both types of cyclopentenone oxylipins induce the expression of genes related to detoxification, stress responses, and secondary metabolism, a profile clearly distinct from that of the cyclopentanone jasmonic acid. Microarray analyses revealed that 60% of the induction by phytoprostanes and 30% of the induction by 12-oxo-phytodienoic acid was dependent on the TGA transcription factors TGA2, TGA5, and TGA6. Moreover, treatment with phytoprostanes and 12-oxo-phytodienoic acid inhibited cell division and root growth, a property also shared by jasmonic acid. Besides being potent signals, cyclopentenones and other lipid peroxidation products are reactive electrophiles that can covalently bind to and damage proteins. To this end, we show that at least two of the induced detoxification enzymes efficiently metabolize cyclopentenones in vitro. Accumulation of two of these metabolites was detectable during Pseudomonas infection. The cyclopentenone oxylipin gene induction profile resembles the defense response induced by a variety of lipophilic xenobiotics. Hence, oxidized lipids may activate chemosensory mechanisms of a general broad-spectrum detoxification network involving TGA transcription factors.

Topics: Arabidopsis; Arabidopsis Proteins; Basic-Leucine Zipper Transcription Factors; Cell Cycle; Cell Division; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Oxylipins; Plant Roots; Pseudomonas; Reverse Transcriptase Polymerase Chain Reaction; Transcriptional Activation

Jasmonates are derived from oxygenated fatty acids (oxylipins) via the octadecanoid pathway and are characterized by a pentacyclic ring structure. They have regulatory functions as signaling molecules in plant development and adaptation to environmental stress. Recently, we solved the structure of allene oxide cyclase 2 (AOC2) of Arabidopsis thaliana, which is, together with the other three AOCs, a key enzyme in the biosynthesis of jasmonates, in that it releases the first cyclic and biologically active metabolite -- 12-oxo-phytodienoic acid (OPDA). On the basis of models for the bound substrate, 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid, and the product, OPDA, we proposed that a conserved Glu promotes the reaction by anchimeric assistance. According to this hypothesis, the transition state with a pentadienyl carbocation and an oxyanion is stabilized by a strongly bound water molecule and favorable pi-pi interactions with aromatic residues in the cavity. Stereoselectivity results from steric restrictions to the necessary substrate isomerizations imposed by the protein environment. Here, site-directed mutagenesis was used to explore and verify the proposed reaction mechanism. In a comparative analysis of the AOC family from A. thaliana involving enzymatic characterization, in vitro import, and transient expression of AOC-enhanced green fluorescent protein fusion proteins for analysis of subcellular targeting, we demonstrate that all four AOC isoenzymes may contribute to jasmonate biosynthesis, as they are all located in chloroplasts and, in concert with the allene oxide synthase, they are all able to convert 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid into enantiomerically pure cis(+)-OPDA.

Topics: Animals; Arabidopsis; Arabidopsis Proteins; Binding Sites; Cyclization; Cyclopentanes; Fatty Acids, Unsaturated; Intramolecular Oxidoreductases; Isoenzymes; Molecular Structure; Oxylipins; Point Mutation; Protein Conformation; Recombinant Fusion Proteins; Substrate Specificity; Tissue Distribution; Zea mays

Expression of AtPHO1;H10, a member of the Arabidopsis (Arabidopsis thaliana) PHO1 gene family, is strongly induced following numerous abiotic and biotic stresses, including wounding, dehydration, cold, salt, and pathogen attack. AtPHO1;H10 expression by wounding was localized to the cells in the close vicinity of the wound site. AtPHO1;H10 expression was increased by application of the jasmonic acid (JA) precursor 12-oxo-phytodienoic acid (OPDA), but not by JA or coronatine. Surprisingly, induction of AtPHO1;H10 by OPDA was dependent on the presence of CORONATINE INSENSITIVE1 (COI1). The induction of AtPHO1;H10 expression by wounding and dehydration was dependent on COI1 and was comparable in both the wild type and the OPDA reductase 3-deficient (opr3) mutant. In contrast, induction of AtPHO1;H10 expression by exogenous abscisic acid (ABA) was independent of the presence of either OPDA or COI1, but was strongly decreased in the ABA-insensitive mutant abi1-1. The involvement of the ABA pathway in regulating AtPHO1;H10 was distinct between wounding and dehydration, with induction of AtPHO1;H10 by wounding being comparable to wild type in the ABA-deficient mutant aba1-3 and abi1-1, whereas a strong reduction in AtPHO1;H10 expression occurred in aba1-3 and abi1-1 following dehydration. Together, these results reveal that OPDA can modulate gene expression via COI1 in a manner distinct from JA, and independently from ABA. Furthermore, the implication of the ABA pathway in coregulating AtPHO1;H10 expression is dependent on the abiotic stress applied, being weak under wounding but strong upon dehydration.

Topics: Arabidopsis; Arabidopsis Proteins; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Genes, Plant; Oxylipins; Signal Transduction

The pathway of jasmonic acid (JA) biosynthesis was established in the 1980s by Vick and Zimmerman but, until now, the preparative biosynthesis of the jasmonic acid precursors 12-oxo-phytodienoic acid (OPDA) and 3-oxo-2-[2'-pentenyl]-cyclopentan-1-octanoic acid (OPC-8:0) in their endogenous and biologically relevant cis(+)-configuration was only possible in small amounts and had to put up with high costs. This was mainly due to the lack of high amounts of pure and enzymatically active allene oxide cyclase (AOC), which is a key enzyme in the biosynthesis of jasmonates in that it releases, in a coupled reaction with allene oxide synthase (AOS), the first cyclic and biological active metabolite - OPDA. We describe here the expression and purification of AOS and AOC and their subsequent coupling to solid matrices to produce an enantioselective, reusable bioreactor for octadecanoid production. With the method described here it is possible to produce optically pure enantiomers of octadecanoids in high amounts in a cost- and time-efficient manner. Furthermore, it could be demonstrated that a physical interaction of AOS and AOC, hitherto postulated to be required for substrate channeling from AOS to AOC, is not necessary for the in vitro cyclization of the unstable epoxide generated by the AOS reaction.

Topics: Cyclopentanes; Enzymes, Immobilized; Fatty Acids, Unsaturated; Intramolecular Oxidoreductases; Oxylipins; Protein Binding; Stereoisomerism

Pathogen infection leads to defence induction as well as to changes in carbohydrate metabolism of plants. Salicylic acid and oxylipins are involved in the induction of defence, but it is not known if these signalling molecules also mediate changes in carbohydrate metabolism. In this study, the effect of application of salicylic acid and the oxylipins 12-oxo-phytodienoic acid (OPDA) and jasmonic acid on photosynthesis was investigated by kinetic chlorophyll fluorescence imaging and compared with the effects of infection by virulent and avirulent strains of Pseudomonas syringae. Both pathogen strains and OPDA caused a similar change in fluorescence parameters of leaves of Arabidopsis thaliana. The response to OPDA appeared faster compared with that to the pathogens and persisted only for a short time. Infiltration with jasmonic acid or salicylic acid did not lead to a localized and distinct fluorescence response of the plant. To capture the faint early symptoms of the plant response, a novel algorithm was applied identifying the unique fluorescence signature-the set of images that, when combined, yield the highest contrast between control and infected leaf segments. Unlike conventional fluorescence parameters, this non-biased approach indeed detected the infection as early as 6 h after inoculation with bacteria. It was posssible to identify distinct fluorescence signatures characterizing the early and late phases of the infection. Fluorescence signatures of both infection phases were found in leaves infiltrated with OPDA.

Topics: Arabidopsis; Chlorophyll; Cyclopentanes; Fatty Acids, Unsaturated; Fluorescence; Host-Parasite Interactions; Oxylipins; Plant Leaves; Pseudomonas syringae; Virulence

Topics: Calcium; Calcium Signaling; Cell Compartmentation; Cell Nucleus; Cells, Cultured; Cyclopentanes; Cytosol; Fatty Acids, Unsaturated; Inflammation Mediators; Nicotiana; Oxylipins; Plant Growth Regulators

Jasmonic acid (JA) is an important regulator of plant development and stress responses. Several enzymes involved in the biosynthesis of JA from alpha-linolenic acid have been characterized. The final biosynthesis steps are the beta-oxidation of 12-oxo-phytoenoic acid. We analyzed JA biosynthesis in the Arabidopsis mutants pex6, affected in peroxisome biogenesis, and aim1, disrupted in fatty acid beta-oxidation. Upon wounding, these mutants exhibit reduced JA levels compared to wild type. pex6 accumulated the precursor OPDA. Feeding experiments with deuterated OPDA substantiate this accumulation pattern, suggesting the mutants are impaired in the beta-oxidation of JA biosynthesis at different steps. Decreased expression of JA-responsive genes, such as VSP1, VSP2, AtJRG21 and LOX2, following wounding in the mutants compared to the wild type reflects the reduced JA levels of the mutants. By use of these additional mutants in combination with feeding experiments, the necessity of functional peroxisomes for JA-biosynthesis is confirmed. Furthermore an essential function of one of the two multifunctional proteins of fatty acid beta-oxidation (AIM1) for wound-induced JA formation is demonstrated for the first time. These data confirm that JA biosynthesis occurs via peroxisomal fatty acid beta-oxidation machinery.

Topics: Adenosine Triphosphatases; Arabidopsis; Arabidopsis Proteins; ATPases Associated with Diverse Cellular Activities; Cyclopentanes; Deuterium; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Multienzyme Complexes; Mutation; Oxidation-Reduction; Oxylipins

The induction of jasmonic acid (JA) is one of the major signaling events in plants in response to insect herbivore damage and leads to the activation of direct and indirect defensive measures. Green leafy volatiles, which constitute a major portion of volatile organic compounds, often are released in response to insect herbivore attack and have been shown to significantly activate JA production in exposed corn (Zea mays) seedlings, thereby priming these plants specifically against subsequent herbivore attack. To explore the factors determining the specificity of the octadecanoid signaling pathway in corn, we analyzed qualitative and quantitative changes in major octadecanoids. The time course and the amount of induced JA and 12-oxophytodienoic acid levels in corn seedlings were strikingly different after wounding, application of caterpillar regurgitant, or treatment with cis-3-hexenyl acetate (Z-3-6:AC). Exposure to Z-3-6:AC induced accumulation of transcripts encoded by three putative 12-oxophytodienoate10,11-reductase genes (ZmOPR1/2, ZmOPR5, and ZmOPR8). Although changes in ZmOPR5 RNAs were detected only after exposure to Z-3-6:AC, ZmOPR1/2 RNAs and ZmOPR8 RNAs also were abundant after treatment with crude regurgitant elicitor or mechanical damage. The physiological implications of these findings in the context of plant-insect interactions are discussed.

Topics: Acetates; Animals; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Genes, Plant; Insecta; Oxidoreductases; Oxylipins; Plant Extracts; Plant Leaves; RNA, Messenger; Signal Transduction; Time Factors; Up-Regulation; Volatilization; Zea mays

Conifers produce terpenoid-based oleoresins as constitutive and inducible defenses against herbivores and pathogens. Much information is available about the genes and enzymes of the late steps of oleoresin terpenoid biosynthesis in conifers, but almost nothing is known about the early steps which proceed via the methylerythritol phosphate (MEP) pathway. Here we report the cDNA cloning and functional identification of three Norway spruce (Picea abies) genes encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS), which catalyzes the first step of the MEP pathway, and their differential expression in the stems of young saplings. Among them are representatives of both types of plant DXS genes. A single type I DXS gene is constitutively expressed in bark tissue and not affected by wounding or fungal application. In contrast, two distinct type II DXS genes, PaDXS2A and PaDXS2B, showed increased transcript abundance after these treatments as did two other genes of the MEP pathway tested, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and 4-hydroxyl 3-methylbutenyl diphosphate reductase (HDR). We also measured gene expression in a Norway spruce cell suspension culture system that, like intact trees, accumulates monoterpenes after treatment with methyl jasmonate. These cell cultures were characterized by an up-regulation of monoterpene synthase gene transcripts and enzyme activity after elicitor treatment, as well as induced formation of octadecanoids, including jasmonic acid and 12-oxophytodienoic acid. Among the Type II DXS genes in cell cultures, PaDXS2A was induced by treatment with chitosan, methyl salicylate, and Ceratocystis polonica (a bark beetle-associated, blue-staining fungal pathogen of Norway spruce). However, PaDXS2B was induced by treatment with methyl jasmonate and chitosan, but was not affected by methyl salicylate or C. polonica. Our results suggest distinct functions of the three DXS genes in primary and defensive terpenoid metabolism in Norway spruce.

Topics: Acetates; Amino Acid Sequence; Cells, Cultured; Cloning, Molecular; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Gene Library; Intramolecular Lyases; Molecular Sequence Data; Oxylipins; Picea; Resins, Plant; Sequence Homology, Amino Acid; Terpenes; Transcription, Genetic; Transferases

Cis-jasmone is a highly appreciated fragrance and plant-derived signal molecule that controls pollination, attracts parasitoids of attacking herbivores, and serves as an intra- and interspecific signal that controls gene expression. cis-Jasmone is produced from linolenic acid along the jasmonic acid cascade. In addition to the conversion of jasmonic acid into cis-jasmone, a novel pathway might exist that converts cis-oxophytodienoic acid (OPDA), an early precursor of jasmonic acid, into iso-OPDA. The planar iso-OPDA is degraded by beta-oxidation to 3,7-didehydrojasmonic acid, which yields cis-jasmone by spontaneous decarboxylation. The degradation of iso-OPDA to cis-jasmone is demonstrated for many plant species and the yeast Saccharomyces cerevisiae.

Topics: Cyclopentanes; Fatty Acids, Unsaturated; Molecular Structure; Oxidation-Reduction; Oxylipins; Plant Growth Regulators; Protein Precursors; Signal Transduction

A GC-MS-based method for the simultaneous quantification of common oxylipins along with labile and highly reactive compounds based on in situ derivatization with pentafluorobenzyl hydroxylamine to the corresponding O-2,3,4,5,6-pentafluorobenzyl oximes (PFB oximes) is presented. The approach covers oxo derivatives such as jasmonic acid (JA), 12-oxophytodienoic acid (OPDA), certain phytoprostanes, unsaturated oxo-acids, oxo-hydroxy acids, and aldehyde fragments from the polar head of fatty acids. In the positive electron impact-MS mode, the PFB oximes display characteristic fragment ions that greatly facilitate the identification of oxylipins in complex matrices. In addition, the fluorinated derivatives allow a highly selective and low-background analysis by negative chemical ionization. Besides showing the general value of the method for the identification of a broad range of oxylipins (18 examples), we also demonstrate sensitivity, linearity, and reproducibility for the quantification of JA, OPDA, 11-oxo-9-undecenoic acid, and 13-oxo-9,11-tridecadienoic acid. The efficiency of the method is demonstrated by differential profiling of these four oxylipins in lima bean leaves after mechanical wounding and feeding by the herbivore Spodoptera littoralis. Caterpillar feeding induced several oxylipins, whereas after wounding only the level of JA increased. The rapid in situ derivatization prevents the isomerization of cis-JA to trans-JA. The resting level of JA in lima beans showed an isomer ratio of 80:20 for trans/cis-JA. After wounding, de novo synthesis of JA alters the ratio to 20:80 in favor of the cis isomer.

Topics: Animals; Cyclopentanes; Fatty Acids, Monounsaturated; Fatty Acids, Unsaturated; Gas Chromatography-Mass Spectrometry; Hydroxylamines; Oximes; Oxylipins; Phaseolus; Plant Leaves; Plants; Spodoptera

A highly sensitive and accurate multiplex gas chromatography-mass spectrometry (GC-MS/MS)-technique is reported for the quantitative analysis of acidic phytohormones in Arabidopsis thaliana and other plant species. The optimized setup allows the routine processing and analysis of between 20 mg and 5 g of tissue. The protocol was designed and the equipment used was chosen to facilitate implementation of the method into other laboratories and to provide access to state-of-the-art analytical tools for the acidic phytohormones and related signaling molecules.

Topics: Abscisic Acid; Arabidopsis; Cyclopentanes; Diazomethane; Fatty Acids, Unsaturated; Gas Chromatography-Mass Spectrometry; Indoleacetic Acids; Oxylipins; Plant Growth Regulators; Plant Proteins; Salicylic Acid

Upon a dark/light shift the conditional flu mutant of Arabidopsis starts to generate singlet oxygen (1O2) that is restricted to the plastid compartment. Distinct sets of genes are activated that are different from those induced by hydrogen peroxide/superoxide. One of the genes that is rapidly upregulated is EDS1 (enhanced disease susceptibility). The EDS1 protein has been shown to be required for the resistance to biotrophic pathogens and the accumulation of salicylic acid (SA) that enhances the defenses of a plant by inducing the synthesis of pathogen-related (PR) proteins. Because of the similarity of its N-terminal portion to the catalytic site of lipases, EDS1 has also been implicated with the release of polyunsaturated fatty acids and the subsequent formation of various oxylipins. The release of singlet oxygen in the flu mutant triggers a drastic increase in the concentration of free SA and activates the expression of PR1 and PR5 genes. These changes depend on the activity of EDS1 and are suppressed in flu/eds1 double mutants. Soon after the beginning of singlet oxygen production, the synthesis of oxylipins such as jasmonic acid (JA) and 12-oxophytodienoic acid (OPDA) also start and plants stop growing and induce a cell-death response. The inactivation of EDS1 does not affect oxylipin synthesis, growth inhibition and the initiation of cell death, but it does allow plants to recover much faster from singlet oxygen-mediated growth inhibition and it also suppresses the spread of necrotic lesions in leaves. Hence, singlet oxygen activates a complex stress-response program with EDS1 playing a key role in initiating and modulating several steps of it. This program includes not only responses to oxidative stress, but also responses known to be activated during plant-pathogen interactions and wounding.

Topics: Apoptosis; Arabidopsis; Arabidopsis Proteins; Circadian Rhythm; Cyclopentanes; DNA-Binding Proteins; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Light; Mutation; Oxylipins; RNA, Messenger; Salicylic Acid; Singlet Oxygen

Tocopherols (vitamin E) are lipophilic antioxidants that are synthesized by all plants and are particularly abundant in seeds. Two tocopherol-deficient mutant loci in Arabidopsis thaliana were used to examine the functions of tocopherols in seedlings: vitamin e1 (vte1), which accumulates the pathway intermediate 2,3-dimethyl-5-phytyl-1,4-benzoquinone (DMPBQ); and vte2, which lacks all tocopherols and pathway intermediates. Only vte2 displayed severe seedling growth defects, which corresponded with massively increased levels of the major classes of nonenzymatic lipid peroxidation products: hydroxy fatty acids, malondialdehyde, and phytoprostanes. In the absence of pathogens, the phytoalexin camalexin accumulated in vte2 seedlings to levels 100-fold higher than in wild-type or vte1 seedlings. Similarly, gene expression profiling in wild-type, vte1, and vte2 seedlings indicated that increased levels of nonenzymatic lipid peroxidation in vte2 corresponded to increased expression of many defense-related genes, which were not induced in vte1. Both biochemical and transcriptional analyses of vte2 seedlings indicate that nonenzymatic lipid peroxidation plays a significant role in modulating plant defense responses. Together, these results establish that tocopherols in wild-type plants or DMPBQ in vte1 plants limit nonenzymatic lipid peroxidation during germination and early seedling development, thereby preventing the inappropriate activation of transcriptional and biochemical defense responses.

Topics: Arabidopsis; Arabidopsis Proteins; Biomarkers; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Profiling; Gene Expression Regulation, Plant; Germination; Immunity, Innate; Indoles; Lipid Peroxidation; Malondialdehyde; Mutation; Oxylipins; Plant Diseases; RNA, Messenger; Seedlings; Thiazoles; Tocopherols; Up-Regulation

Plants are constantly exposed to environmental changes and need to integrate multiple external stress cues. Calcium-dependent protein kinases (CDPKs) are implicated as major primary Ca2+ sensors in plants. CDPK activation, like activation of mitogen-activated protein kinases (MAPKs), is triggered by biotic and abiotic stresses, although distinct stimulus-specific stress responses are induced. To investigate whether CDPKs are part of an underlying mechanism to guarantee response specificity, we identified CDPK-controlled signaling pathways. A truncated form of Nicotiana tabacum CDPK2 lacking its regulatory autoinhibitor and calcium-binding domains was ectopically expressed in Nicotiana benthamiana. Infiltrated leaves responded to an abiotic stress stimulus with the activation of biotic stress reactions. These responses included synthesis of reactive oxygen species, defense gene induction, and SGT1-dependent cell death. Furthermore, N-terminal CDPK2 signaling triggered enhanced levels of the phytohormones jasmonic acid, 12-oxo-phytodienoic acid, and ethylene but not salicylic acid. These responses, commonly only observed after challenge with a strong biotic stimulus, were prevented when the CDPK's intrinsic autoinhibitory peptide was coexpressed. Remarkably, elevated CDPK signaling compromised stress-induced MAPK activation, and this inhibition required ethylene synthesis and perception. These data indicate that CDPK and MAPK pathways do not function independently and that a concerted activation of both pathways controls response specificity to biotic and abiotic stress.

Topics: Apoptosis; Blotting, Northern; Calcium; Cyclopentanes; Ethylenes; Fatty Acids, Unsaturated; Green Fluorescent Proteins; Immunoblotting; Mitogen-Activated Protein Kinases; Mutagenesis, Site-Directed; Nicotiana; Oxylipins; Plant Lectins; Plasmids; Protein Kinases; Reactive Oxygen Species; Receptor Cross-Talk; Signal Transduction

Jasmonic acid (JA) and methyl jasmonate (MeJA), collectively known as JAs, regulate diverse physiological processes in plants, including the response to wounding. Recent reports suggest that a cyclopentenone precursor of JA, 12-oxo-phytodienoic acid (OPDA), can also induce gene expression. However, little is known about the physiological significance of OPDA-dependent gene expression. We used microarray analysis of approximately 21,500 Arabidopsis (Arabidopsis thaliana) genes to compare responses to JA, MeJA, and OPDA treatment. Although many genes responded identically to both OPDA and JAs, we identified a set of genes (OPDA-specific response genes [ORGs]) that specifically responded to OPDA but not to JAs. ORGs primarily encoded signaling components, transcription factors, and stress response-related genes. One-half of the ORGs were induced by wounding. Analysis using mutants deficient in the biosynthesis of JAs revealed that OPDA functions as a signaling molecule in the wounding response. Unlike signaling via JAs, OPDA signaling was CORONATINE INSENSITIVE 1 independent. These results indicate that an OPDA signaling pathway functions independently of JA/MeJA signaling and is required for the wounding response in Arabidopsis.

Topics: Acrolein; Arabidopsis; Blotting, Northern; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Profiling; Gene Expression Regulation, Plant; Genes, Plant; Mutation; Oligonucleotide Array Sequence Analysis; Oxylipins; Plant Diseases; Plant Leaves; Reproducibility of Results; RNA, Messenger; Signal Transduction; Time Factors

The naturally occurring enantiomer of the various octadecanoids and jasmonates is established in a biosynthetic step catalyzed by the allene oxide cyclase (AOC). The AOC converts an allene oxide formed by an allene oxide synthase (AOS). Here, we show cloning and characterization of cDNAs encoding the AOC and a third AOS, respectively, in addition to the two AOSs previously published (Plant J. 21, 199-213, 2000). The ORF of the AOC-cDNA of 717 bp codes for a protein of 238 amino acid residues carrying a putative chloroplast target sequence. Overexpression without chloroplast target sequence revealed AOC activity. The AOC was found to be a single copy gene which mapped on chromosome 6H. AOC mRNA accumulation appeared in leaf segments upon treatment with various jasmonates, octadecanoids and ABA or during stress such as treatment with sorbitol or glucose solutions. Infection with powdery mildew activated AOC expression in susceptible and resistant lines of barley which correlated with PR1b expression. Among different tissues of barley seedlings, the scutellar node and leaf base accumulated AOC mRNA preferentially which correlated with accumulation of mRNAs for other biosynthetic enzymes (lipoxygenases, AOSs). AOC mRNA accumulation appeared also abundantly in parts of the root containing the tip and correlated with elevated levels of jasmonates. The data suggest a link of AOC expression and JA formation and support role of JA in stress responses and development of barley.

Topics: Amino Acid Sequence; Chromosome Mapping; Cloning, Molecular; Cyclopentanes; DNA, Plant; Escherichia coli; Fatty Acids, Unsaturated; Gene Expression; Genome, Plant; Hordeum; Intramolecular Oxidoreductases; Isoenzymes; Lipoxygenase; Molecular Sequence Data; Oxylipins; Plant Structures; Recombinant Proteins; RNA, Messenger; Sequence Alignment; Tissue Distribution

The allene oxide cyclase (AOC), an enzyme in jasmonate biosynthesis, occurs in vascular bundles and ovules of tomato flowers which exhibit a tissue-specific oxylipin signature (Plant J. 24, 113-126, 2000). Constitutive overexpression of the AOC did not led to altered levels of jasmonates in leaves, but these levels increased upon wounding or other stresses suggesting regulation of jasmonate biosynthesis by substrate availability (Plant J. 33, 577-589, 2003). Here, we show dramatic changes in levels of jasmonic acid (JA), of 12-oxo-phytodienoic acid (OPDA), their methyl esters (JAME, OPDAME), and of dinor-OPDA in most flower organs upon constitutive overexpression of AOC. Beside a dominant occurrence of OPDAME and JA in most flower organs, the ratio among the various compounds was altered differentially in the organs of transgenic flowers, e.g. OPDAME increased up to 53-fold in stamen, and JA increased about 51-fold in buds and 7.5-fold in sepals. The increase in jasmonates and octadecanoids was accompanied by decreased levels of free lipid hydro(per)oxy compounds. Except for 16:2, the AOC overexpression led to a significant increase in free but not esterified polyunsaturated fatty acids in all flower organs. The data suggest different regulation of JA biosynthesis in leaves and flowers of tomato.

Topics: Cyclopentanes; Fatty Acids; Fatty Acids, Nonesterified; Fatty Acids, Unsaturated; Flowers; Gene Expression; Immunohistochemistry; Intramolecular Oxidoreductases; Oxylipins; Plant Leaves; Plants, Genetically Modified; RNA, Messenger; Solanum lycopersicum; Tissue Distribution

In suspension-cultured rice ( Oryza sativaL.) cells, jasmonic acid (JA) functions as a signal transducer in elicitor N-acetylchitoheptaose-induced phytoalexin production. Differential screening of a cDNA library constructed using poly(A)(+) RNA from suspension-cultured rice cells treated with JA (10(-4) M) for 2 h yielded a cDNA for a gene that responded to exogenous JA by an increase in mRNA level. Nucleotide sequence analysis indicated that the cDNA encodes an homologue of the yeast Old Yellow Enzyme. The deduced amino acid sequence was very similar to the sequences of 12-oxophytodienoic acid reductases (OPR) 1 and 2 from Arabidopsis thaliana(AtOPR1 and AtOPR2) and OPR1 from tomato ( Lycopersicon esculentum) (LeOPR1). The cDNA-encoded protein purified from recombinant Escherichia coli cells as a hexahistidine-tagged fusion protein exhibited OPR activity similar to that of AtOPR1, AtOPR2, and LeOPR1, which catalyze reduction of (-)- cis-12-oxophytodienoic acid (OPDA) preferentially over (+)- cis-OPDA, a natural precursor of JA. Thus the rice enzyme was termed OsOPR1. The physiological roles of OsOPR1 are discussed. This is the first report of the cloning of an OPR gene from a monocot plant.

Topics: Cells, Cultured; Cloning, Molecular; Cycloheximide; Cyclopentanes; DNA, Complementary; Fatty Acids, Unsaturated; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Molecular Sequence Data; Oryza; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Oxylipins; Phylogeny; Plant Growth Regulators; Plant Proteins; RNA, Messenger; Sequence Analysis, DNA

Oxylipins of the jasmonate pathway and synthetic functional analogs have been analyzed for their elicitor-like activities in an assay based on the induced accumulation of glyceollins, the phytoalexins of soybean (Glycine max L.), in cell suspension cultures of this plant. Jasmonic acid (JA) and its methyl ester showed weak phytoalexin-inducing activity when compared to an early jasmonate biosynthetic precursor, 12-oxo-phytodienoic acid (OPDA), as well as to the bacterial phytotoxin coronatine and certain 6-substituted indanoyl-L-isoleucine methyl esters, which all were highly active. Interestingly, different octadecanoids and indanoyl conjugates induced the accumulation of transcripts of various defense-related genes to different degrees, indicating distinct induction competencies. Therefore, these signaling compounds and mimics were further analyzed for their effects on signal transduction elements, such as the transient enhancement of the cytosolic Ca2+ concentration and MAP kinase activation, which are known to be initiated by a soybean pathogen-derived beta-glucan elicitor. In contrast to the beta-glucan elicitor, none of the other compounds tested triggered these early signaling elements. Moreover, endogenous levels of OPDA and JA in soybean cells were shown to be unaffected after treatment with beta-glucans. Thus, OPDA and JA, which are functionally mimicked by coronatine and a variety of 6-substituted derivatives of indanoyl-L-isoleucine methyl ester, represent highly efficient signaling compounds of a lipid-based pathway not deployed in the beta-glucan elicitor-initiated signal transduction.

Topics: Acetates; Amino Acids; Benzopyrans; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Glycine max; Indenes; Mitogen-Activated Protein Kinases; Molecular Mimicry; Oxylipins; Plant Growth Regulators; Pterocarpans; RNA, Messenger; Signal Transduction

Allene oxide synthase (AOS) catalyzes the entrance reaction in the biosynthesis of the octadecanoids 12-oxophytodienoic acid (OPDA) and jasmonic acid (JA). The enzyme is feedback-regulated by JA and thus a target of the JA-signalling pathway. A fusion genetic approach was used to isolate mutants in this signalling pathway. Seeds from transgenic Arabidopsis thaliana plants expressing the Escherichia coli uidA gene encoding beta-glucuronidase (GUS) under the control of the AOS promoter were mutagenized with ethylmethane sulfonate and the progeny was screened for individuals exhibiting constitutive expression of uidA in the absence of an added octadecanoid. From 21,000 mutagenized plants, 8 lines showing constitutive AOS expression were obtained. The mutant lines were characterized further and fell into four classes, I to IV. All showed signs of growth inhibition encompassing both shoot and root systems, and accumulated higher than normal levels of OPDA. Mutants belonging to classes I and IV failed to set seeds due to defects in flower development which prevented self-pollination. One mutant, designated cas1, was characterized in more detail and showed, in addition to elevated levels of AOS mRNA, AOS polypeptide, OPDA, and JA, constitutive expression of JA-responsive genes ( VSP2, PDF1.2). The cas1 mutation is recessive and affects a single locus. Using cleaved amplified polymorphic sequences (CAPS) and simple sequence length polymorphisms (SSLP), the mutated gene was mapped to chromosome IV next to the SSLP marker CIW7.

Topics: Arabidopsis; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Glucuronidase; Intramolecular Oxidoreductases; Mutation; Oxylipins; Phenotype; Plants, Genetically Modified; Seeds; Signal Transduction; Stearates

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.

Topics: Cyclopentanes; Fatty Acids, Unsaturated; Flowers; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Immunohistochemistry; Intramolecular Oxidoreductases; Lipoxygenase; Microscopy, Electron; Oxylipins; Plant Stems; Plastids; RNA, Messenger; Signal Transduction; Solanum lycopersicum; Wound Healing

Although the supply of 12-oxo-PDA (1) and OPC-8:0 (2), the metabolites in the linolenic acid cascade leading to epi-jasmonic acid, is in demand for biological investigations, the previous syntheses of these metabolites suffer from low efficiency. Recently, we established a reaction to install an alkyl group onto the ring of cyclopentene monoacetate 4 by using a reagent system consisting of RMgCl (3 equiv) and CuCN (cat). The reaction was applied to ClMg(CH2)8OTBDPS (11) with modification by which the quantity of 11 could be reduced to 2 equiv without decreasing efficiency. The product 12 obtained in 88% yield with 92% regioselectivity was successfully transformed into the key iodo-lactone 17 in good yield, from which 12-oxo-PDA (1) and OPC-8:0 (2) were synthesized as described in Schemes 3 and 5 through construction of the cis side chain by Wittig reaction. Note that the Wittig reaction proceeded with high cis selectivity of >95%, which is higher than in similar cases reported previously. Synthesis of the 13-isomers of 1 and 2 was also accomplished. With these compounds in hand, the epimerization speed of 1 and 2 was investigated to rule out overestimation of the finding in the literature that 1 and 2 change to the 13-epimers easily. Instead, we observed that the compounds are quite stable at room temperature for an extended period of days under slightly acidic and neutral conditions.

Topics: Alkylation; alpha-Linolenic Acid; Caprylates; Cyclopentanes; Fatty Acids, Unsaturated; Oxylipins; Stereoisomerism

Occurrence of an essential enzyme in jasmonate (JA) biosynthesis, the allene oxide cyclase, (AOC) was analyzed in different developmental stages and various organs of Arabidopsis thaliana plants by immuno blot analysis and immunocytological approaches. Levels of AOC and of the two preceding enzymes in JA biosynthesis increased during seedling development accompanied by increased levels of JA and 12-oxophytodienoic acid levels after 4 and 8 weeks. Most tissues including all vascular bundles and that of flower buds contain AOC protein. Flowers shortly before opening, however, contain AOC protein preferentially in ovules, stigma cells and vascular bundles, whereas in anthers and pollen AOC could not be detected. The putative roles of AOC and JA in development are discussed.

Topics: Arabidopsis; Blotting, Northern; Cyclopentanes; Fatty Acids, Unsaturated; Immunohistochemistry; Intramolecular Oxidoreductases; Lipoxygenase; Oxylipins; Plant Structures; Tissue Distribution

Colonization of barley (Hordeum vulgare cv Salome) roots by an arbuscular mycorrhizal fungus, Glomus intraradices Schenck & Smith, leads to elevated levels of endogenous jasmonic acid (JA) and its amino acid conjugate JA-isoleucine, whereas the level of the JA precursor, oxophytodienoic acid, remains constant. The rise in jasmonates is accompanied by the expression of genes coding for an enzyme of JA biosynthesis (allene oxide synthase) and of a jasmonate-induced protein (JIP23). In situ hybridization and immunocytochemical analysis revealed that expression of these genes occurred cell specifically within arbuscule-containing root cortex cells. The concomitant gene expression indicates that jasmonates are generated and act within arbuscule-containing cells. By use of a near-synchronous mycorrhization, analysis of temporal expression patterns showed the occurrence of transcript accumulation 4 to 6 d after the appearance of the first arbuscules. This suggests that the endogenous rise in jasmonates might be related to the fully established symbiosis rather than to the recognition of interacting partners or to the onset of interaction. Because the plant supplies the fungus with carbohydrates, a model is proposed in which the induction of JA biosynthesis in colonized roots is linked to the stronger sink function of mycorrhizal roots compared with nonmycorrhizal roots.

Topics: Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Hordeum; Immunohistochemistry; In Situ Hybridization; Intramolecular Oxidoreductases; Mycorrhizae; Oxylipins; Plant Proteins; Plant Roots; Reactive Oxygen Species; Symbiosis

A highly sensitive and accurate multiplex gas chromatography-tandem mass spectrometry (GC-MS/MS) technique is reported for indole-3-acetic acid, abscisic acid, jasmonic acid, 12-oxo-phytodienoic acid and salicylic acid. The optimized setup allows the routine processing and analysis of up to 60 plant samples of between 20 and 200 mg of fresh weight per day. The protocol was designed and the equipment used was chosen to facilitate implementation of the method into other laboratories and to provide access to state-of-the-art analytical tools for the acidic phytohormones and related signalling molecules. Whole-plant organ-distribution maps for indole-3-acetic acid, abscisic acid, jasmonic acid, 12-oxo-phytodienoic acid and salicylic acid were generated for Arabidopsis thaliana (L.) Heynh. For leaves of A. thaliana, a spatial resolution of hormone quantitation down to approximately 2 mm(2) was achieved.

Topics: Abscisic Acid; Algorithms; Arabidopsis; Biological Transport; Cyclopentanes; Fatty Acids, Unsaturated; Gas Chromatography-Mass Spectrometry; Indoleacetic Acids; Molecular Structure; Oxylipins; Plant Growth Regulators; Salicylic Acid; Signal Transduction

The jasmonic acid (JA)-dependent regulation of the Thi2.1 gene had previously been exploited for setting up a genetic screen for the isolation of signal transduction mutants of Arabidopsis thaliana (L.) Heynh. that constitutively express the thionin gene. Several cet mutants had been isolated which showed a Constitutive Expression of the Thionin gene. These cet mutants, except for one, also showed spontaneous leaf cell necrosis and were up-regulated in the expression of the PR1 gene, reactions often associated with the systemic acquired resistance (SAR) pathway. Four of these cet mutants, cet1, cet2, cet3 and cet4.1 were crossed with the fad triple and coi1 mutants that are blocked at two steps within the JA-dependent signaling pathway, and with transgenic NahG plants that are deficient in salicylic acid (SA) and are unable to activate SAR. Analysis of the various double-mutant lines revealed that the four cet genes act within a signaling cascade at or prior to branch points from which not only JA-dependent signals but also SA-dependent signaling and cell death pathways diverge.

Topics: Apoptosis; Arabidopsis; Arabidopsis Proteins; Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Immunity, Innate; Mutation; Oxylipins; Plant Proteins; Plants, Genetically Modified; Salicylic Acid; Signal Transduction

Coronatine-inducible tyrosine aminotransferase (TAT), which catalyses the transamination from tyrosine to p-hydroxyphenylpyruvate, is the first enzyme of a pathway leading via homogentisic acid to plastoquinone and tocopherols, the latter of which are known to be radical scavengers in plants. TAT can be also induced by the octadecanoids methyl jasmonate (MeJA) and methyl-12-oxophytodienoic acid (MeOPDA), as well as by wounding, high light, UV light and the herbicide oxyfluorfen. In order to elucidate the role of octadecanoids in the process of TAT induction in Arabidopsis thaliana (L.) Heynh., the jasmonate-deficient mutant delayed dehiscence (dde1) was used, in which the gene for 12-oxophytodienoic acid reductase 3 is disrupted. The amount of immunodetectable TAT was low. The enzyme was still fully induced by coronatine as well as by MeJA although induction by the latter was to a lesser extent and later than in the wild type. Treatment with MeOPDA, wounding and UV light, however, had hardly any effects. Tocopherol levels that showed considerable increases in the wild type after some treatments were much less affected in the mutant. However, starting levels of tocopherol were higher in non-induced dde1 than in the wild type. We conclude that jasmonate plays an important role in the signal transduction pathway regulating TAT activity and the biosynthesis of its product tocopherol.

Topics: Acetates; Arabidopsis; Cyclopentanes; Enzyme Induction; Fatty Acids, Unsaturated; Halogenated Diphenyl Ethers; Herbicides; Light; Mutation; Oxylipins; Phenyl Ethers; Phenylpyruvic Acids; Stress, Mechanical; Tocopherols; Tyrosine; Tyrosine Transaminase; Ultraviolet Rays

Topics: Alternaria; Animals; Arabidopsis; Arabidopsis Proteins; Cyclopentanes; Diptera; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Oxidoreductases; Oxylipins; Plant Diseases; Plant Growth Regulators; Plant Proteins; Signal Transduction

In this work, the role of jasmonic acid (JA) in leaf senescence is examined. Exogenous application of JA caused premature senescence in attached and detached leaves in wild-type Arabidopsis but failed to induce precocious senescence of JA-insensitive mutant coi1 plants, suggesting that the JA-signaling pathway is required for JA to promote leaf senescence. JA levels in senescing leaves are 4-fold higher than in non-senescing ones. Concurrent with the increase in JA level in senescing leaves, genes encoding the enzymes that catalyze most of the reactions of the JA biosynthetic pathway are differentially activated during leaf senescence in Arabidopsis, except for allene oxide synthase, which is constitutively and highly expressed throughout leaf development. Arabidopsis lipoxygenase 1 (cytoplasmic) expression is greatly increased but lipoxygenase 2 (plastidial) expression is sharply reduced during leaf senescence. Similarly, AOC1 (allene oxide cyclase 1), AOC2, and AOC3 are all up-regulated, whereas AOC4 is down-regulated with the progression of leaf senescence. The transcript levels of 12-oxo-PDA reductase 1 and 12-oxo-PDA reductase 3 also increase in senescing leaves, as does PED1 (encoding a 3-keto-acyl-thiolase for beta-oxidation). This represents the first report, to our knowledge, of an increase in JA levels and expression of oxylipin genes during leaf senescence, and indicates that JA may play a role in the senescence program.

Topics: Acetyl-CoA C-Acetyltransferase; Arabidopsis; Chlorophyll; Cyclopentanes; Defensins; Fatty Acids, Unsaturated; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Intramolecular Oxidoreductases; Lipoxygenase; Mutation; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Oxylipins; Plant Leaves; Plant Proteins; Signal Transduction; Up-Regulation

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.

Topics: Antimicrobial Cationic Peptides; Arabidopsis; Arabidopsis Proteins; Chromosome Segregation; Crosses, Genetic; Cyclopentanes; Fatty Acids, Unsaturated; Fusarium; Lectins; Mutation; Oxylipins; Plant Diseases; Plant Growth Regulators; Plant Lectins; Plant Proteins; Salicylic Acid; Signal Transduction; Stearic Acids

To investigate the signaling pathways through which defense responses are activated following pathogen infection, we have isolated and characterized the cpr22 mutant. This plant carries a semidominant, conditional lethal mutation that confers constitutive expression of the pathogenesis-related (PR) genes PR-1, PR-2, PR-5 and the defensin gene PDF1.2. cpr22 plants also display spontaneous lesion formation, elevated levels of salicylic acid (SA) and heightened resistance to Peronospora parasitica Emco5. The cpr22 locus was mapped to chromosome 2, approximately 2 cM telomeric to the AthB102 marker. By analyzing the progeny of crosses between cpr22 plants and either NahG transgenic plants or npr1 mutants, all of the cpr22-associated phenotypes except PDF1.2 expression were found to be SA dependent. However, the SA signal transducer NPR1 was required only for constitutive PR-1 expression. A cross between cpr22 and ndr1-1 mutants revealed that enhanced resistance to P. parasitica is mediated by an NDR1-dependent pathway, while the other cpr22-induced defenses are not. Crosses between either coi1-1 or etr1-1 mutants further demonstrated that constitutive PDF1.2 expression is mediated by a JA- and ethylene-dependent pathway. Based on these results, the cpr22 mutation appears to induce its associated phenotypes by activating NPR1-dependent and NPR1-independent branches of the SA pathway, as well as an ethylene/JA signaling pathway. Interestingly, the SA-dependent phenotypes, but not the SA-independent phenotypes, are suppressed when cpr22 mutants are grown under high humidity.

Topics: Arabidopsis; Arabidopsis Proteins; Chromosome Segregation; Cyclopentanes; Defensins; Ethylenes; Fatty Acids, Unsaturated; Genes, Plant; Humidity; Mixed Function Oxygenases; Models, Biological; Mutation; Oomycetes; Oxylipins; Phenotype; Plant Diseases; Plant Growth Regulators; Plant Leaves; Plant Proteins; Salicylic Acid; Signal Transduction; Thiadiazoles; Transcription Factors

The Arabidopsis opr3 mutant is defective in the isoform of 12-oxo-phytodienoate (OPDA) reductase required for jasmonic acid (JA) biosynthesis. Oxylipin signatures of wounded opr3 leaves revealed the absence of detectable 3R,7S-JA as well as altered levels of its cyclopentenone precursors OPDA and dinor OPDA. In contrast to JA-insensitive coi1 plants and to the fad3 fad7 fad8 mutant lacking the fatty acid precursors of JA synthesis, opr3 plants exhibited strong resistance to the dipteran Bradysia impatiens and the fungus Alternaria brassicicola. Analysis of transcript profiles in opr3 showed the wound induction of genes previously known to be JA-dependent, suggesting that cyclopentenones could fulfill some JA roles in vivo. Treating opr3 plants with exogenous OPDA powerfully up-regulated several genes and disclosed two distinct downstream signal pathways, one through COI1, the other via an electrophile effect of the cyclopentenones. We conclude that the jasmonate family cyclopentenone OPDA (most likely together with dinor OPDA) regulates gene expression in concert with JA to fine-tune the expression of defense genes. More generally, resistance to insect and fungal attack can be observed in the absence of JA.

Topics: Alternaria; Animals; Arabidopsis; Arabidopsis Proteins; Cyclopentanes; Diptera; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Oxidoreductases; Oxylipins; Plant Diseases; Plant Proteins

Topics: Arabidopsis; Arabidopsis Proteins; Cyclopentanes; Fatty Acids, Unsaturated; Oxidoreductases; Oxylipins; Plant Diseases; Plant Proteins

Recent evidence suggests that oxidized lipid-derived molecules play significant roles in inducible plant defence responses against microbial pathogens, either by directly deterring parasite multiplication, or as signals involved in the induction of sets of defence genes. The synthesis of these oxylipins was hypothesized to be initiated by the phospholipase A2-mediated release of unsaturated fatty acids from membrane lipids. Here, we demonstrate that, in tobacco leaves reacting hypersensitively to tobacco mosaic virus, a strong increase in soluble phospholipase A2 (PLA2) activity occurs at the onset of necrotic lesion appearance. This rapid PLA2 activation occurred before the accumulation of 12-oxophytodienoic and jasmonic acids, two fatty acid-derived defence signals. Three PLA2 isoforms were separated and the most active enzyme was partially purified, its N-terminal sequence displaying similarity with patatin, the major storage protein in potato tubers. Three related tobacco patatin-like cDNAs, called NtPat1, NtPat2 and NtPat3, were cloned, with NtPat2 encoding the PLA2 isolated from infected leaves. RT-PCR experiments showed a rapid transcriptional activation of the three NtPat genes in virus-infected leaves, preceding the increase in PLA2 activity. Recombinant NtPat1 and NtPat3 enzymes were active in an assay using labelled bacterial membranes, and also displayed high bona fide PLA2 activity on phosphatidylcholine substrate. These results point to a possible new role of patatin-like phospholipases in inducible plant defence responses. The induction kinetics together with the enzymatic activity data indicate that the NtPat proteins may provide precursors for oxylipin synthesis during the hypersensitive response to pathogens.

Topics: Adaptation, Physiological; Amino Acid Sequence; Carboxylic Ester Hydrolases; Cyclopentanes; Enzyme Induction; Fatty Acids, Unsaturated; Molecular Sequence Data; Nicotiana; Oxylipins; Phospholipases A; Phospholipases A2; Plant Growth Regulators; Plant Leaves; Plant Proteins; Plants, Toxic; Reverse Transcriptase Polymerase Chain Reaction; Sequence Alignment; Tobacco Mosaic Virus

A crucial step in the biosynthesis of jasmonic acid (JA) is the formation of its correct stereoisomeric precursor, cis(+)12-oxophytodienoic acid (OPDA). This step is catalysed by allene oxide cyclase (AOC), which has been recently cloned from tomato. In stems, young leaves and young flowers, AOC mRNA accumulates to a low level, contrasting with a high accumulation in flower buds, flower stalks and roots. The high levels of AOC mRNA and AOC protein in distinct flower organs correlate with high AOC activity, and with elevated levels of JA, OPDA and JA isoleucine conjugate. These compounds accumulate in flowers to levels of about 20 nmol g-1 fresh weight, which is two orders of magnitude higher than in leaves. In pistils, the level of OPDA is much higher than that of JA, whereas in flower stalks, the level of JA exceeds that of OPDA. In other flower tissues, the ratios among JA, OPDA and JA isoleucine conjugate differ remarkably, suggesting a tissue-specific oxylipin signature. Immunocytochemical analysis revealed the specific occurrence of the AOC protein in ovules, the transmission tissue of the style and in vascular bundles of receptacles, flower stalks, stems, petioles and roots. Based on the tissue-specific AOC expression and formation of JA, OPDA and JA amino acid conjugates, a possible role for these compounds in flower development is discussed in terms of their effect on sink-source relationships and plant defence reactions. Furthermore, the AOC expression in vascular bundles might play a role in the systemin-mediated wound response of tomato.

Topics: Cyclopentanes; Fatty Acids, Unsaturated; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Intramolecular Oxidoreductases; Oxylipins; Plant Leaves; Plant Roots; Plant Stems; RNA, Messenger; Solanum lycopersicum; Stereoisomerism; Transcription, Genetic

Plants are able to respond to herbivore damage with de novo biosynthesis of an herbivore-characteristic blend of volatiles. The signal transduction initiating volatile biosynthesis may involve the activation of the octadecanoid pathway, as exemplified by the transient increase of endogenous jasmonic acid (JA) in leaves of lima bean (Phaseolus lunatus) after treatment with the macromolecular elicitor cellulysin. Within this pathway lima bean possesses at least two different biologically active signals that trigger different biosynthetic activities. Early intermediates of the pathway, especially 12-oxo-phytodienoic acid (PDA), are able to induce the biosynthesis of the diterpenoid-derived 4,8, 12-trimethyltrideca-1,3,7,11-tetraene. High concentrations of PDA result in more complex patterns of additional volatiles. JA, the last compound in the sequence, lacks the ability to induce diterpenoid-derived compounds, but is highly effective at triggering the biosynthesis of other volatiles. The phytotoxin coronatine and amino acid conjugates of linolenic acid (e.g. linolenoyl-L-glutamine) mimic the action of PDA, but coronatine does not increase the level of endogenous JA. The structural analog of coronatine, the isoleucine conjugate of 1-oxo-indanoyl-4-carboxylic acid, effectively mimics the action of JA, but does not increase the level of endogenous JA. The differential induction of volatiles resembles previous findings on signal transduction in mechanically stimulated tendrils of Bryonia dioica.

Topics: alpha-Linolenic Acid; Amino Acids; Cellulase; Cucurbitaceae; Cyclopentanes; Fabaceae; Fatty Acids, Unsaturated; Indenes; Mevalonic Acid; Oils, Volatile; Oxylipins; Physical Stimulation; Plant Growth Regulators; Plant Leaves; Plants, Medicinal; Signal Transduction; Stearic Acids; Terpenes; Time Factors; Volatilization

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.

Topics: Abscisic Acid; Arabidopsis; Cyclopentanes; Enzyme Induction; Fatty Acids, Unsaturated; Intramolecular Oxidoreductases; Oxylipins; Plant Growth Regulators; RNA, Messenger; Salicylic Acid; Signal Transduction

Octadecanoids, potent cyclic plant signaling molecules derived from alpha-linolenic acid, are involved in the regulation of a multitude of physiological processes such as senescence, herbivore and pathogen defense, mechanoperception and morphogenesis. The first cyclic intermediate in the Vick-Zimmerman pathway of octadecanoid biosynthesis is 12-oxo-phytodienoic acid. Its conversion to the end product of the pathway, jasmonic acid, a C12 compound, first proceeds through reduction to 3-oxo-2-(pent-2'-enyl)-cyclopentane-1-octanoic acid, which is then converted to jasmonic acid by three cycles of beta-oxidation. The first of these conversions is a decisive point in the biosynthetic sequence, in that it channels the octadecanoid into the pathway of beta-oxidation. 12-Oxo-phytodienoate reductase was purified to apparent homogeneity from a cell suspension culture of Corydalis sempervirens. The enzyme is soluble and a monomer of apparent molecular mass 41 kDa which prefers NADPH over NADH to reduce the 10,11-double bond of 12-oxo-phytodienoic acid. The structure of the reaction product was proved by derivatization, GC/MS and NMR analysis. The enzyme accepts both the cis and the trans isomer of 12-oxo-phydodienoic acid, with a preference for the cis-isomer (6:1). 12-Oxo-phytodienoate reductase will also convert the synthetic substrate 2-cyclohexenone to cyclohexanone, but the enzyme did not reduce some other cyclic alpha,beta-unsaturated ketones tested (the plant hormone abscisic acid or the steroids testosterone and progesterone). Characteristic parameters of 12-oxo-phytodienoate reductase were determined.

Topics: Cells, Cultured; Chromatography, Gel; Cyclohexanones; Cyclopentanes; Electrophoresis, Polyacrylamide Gel; Fatty Acids, Unsaturated; Gas Chromatography-Mass Spectrometry; Isomerism; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Oxylipins; Plants; Stearic Acids

Jasmonic acid and its precursors are potent regulatory molecules in plants. We devised a method for the simultaneous extraction of these compounds from plant leaves to quantitate changes in the levels of jasmonate family members during health and on wounding. During our study, we identified a novel 16-carbon cyclopentenoic acid in leaf extracts from Arabidopsis and potato. The new compound, a member of the jasmonate family of signals, was named dinor-oxo-phytodienoic acid. Dinor-oxo-phytodienoic acid was not detected in the Arabidopsis mutant fad5, which is incapable of synthesizing 7Z,10Z, 13Z-hexadecatrienoic acid (16:3), suggesting that the metabolite is derived directly from plastid 16:3 rather than by beta-oxidation of the 18-carbon 12-oxo-phytodienoic acid. Simultaneous quantitation of jasmonate family members in healthy leaves of Arabidopsis and potato suggest that different plant species have different relative levels of jasmonic acid, oxo-phytodienoic acid, and dinor-oxo-phytodienoic acid. We term these profiles "oxylipin signatures." Dinor-oxo-phytodienoic acid levels increased dramatically in Arabidopsis and potato leaves on wounding, suggesting roles in wound signaling. Treatment of Arabidopsis with micromolar levels of dinor-oxo-phytodienoic acid increased the ability of leaf extracts to transform linoleic acid into the alpha-ketol 13-hydroxy-12-oxo-9(Z) octadecenoic acid indicating that the compound can regulate part of its own biosynthetic pathway. Tightly regulated changes in the relative levels of biologically active jasmonates may permit sensitive control over metabolic, developmental, and defensive processes in plants.

Topics: Arabidopsis; Cyclopentanes; Fatty Acids, Unsaturated; Mass Spectrometry; Oxylipins; Plant Growth Regulators; Signal Transduction

The phytotoxic principle, coronatine, which is present in several pathovars of the plant pathogen, Pseudomonas syringae was shown to be highly active in completely different, jasmonate-selective bioassays. At nanomolar to micromolar concentrations, coronatine induced the accumulation of defense-related secondary metabolites in several plant cell cultures, induced transcript accumulation of the elicitor-responsive gene encoding the berberine bridge enzyme of Eschscholtzia californica, as well as the coiling response of Bryonia dioica tendrils. Biological activity critically depended upon the structure of coronatine, and slight modifications, such as methylation of the carboxyl moiety or reduction of the carbonyl group, rendered the molecules almost inactive. Coronafacic acid, obtained by hydrolysis of coronatine, was also nearly inactive. Coronatine did not elicit the accumulation of endogenous jasmonic acid in the systems analyzed. While coronafacic acid is similar in structure to jasmonic acid, we found coronatine to be a close structural analogue of the cyclic C18-precursor of jasmonic acid, 12-oxo-phytodienoic acid. The phytotoxic symptoms produced by coronatine can now be understood on the basis of the toxin's action as a mimic of the octadecanoid signalling molecules of higher plants.

Topics: Amino Acids; Bacterial Toxins; Berberine; Biological Assay; Cells, Cultured; Cyclopentanes; Dose-Response Relationship, Drug; Fatty Acids, Unsaturated; Indenes; Oxylipins; Plants; Pseudomonas; RNA, Messenger; Signal Transduction; Structure-Activity Relationship