Identification of a peroxisomal acyl-activating enzyme involved in the biosynthesis of jasmonic acid in Arabidopsis

Jasmonic acid (JA) is a lipid-derived signal that regulates a wide variety of developmental and defense-related processes in higher plants. JA is synthesized from linolenic acid via an enzymatic pathway that initiates in the plastid and terminates in peroxisomes. The C18 JA precursor 12-oxo-phytodienoic acid (OPDA) is converted in the peroxisome to 3-oxo-2-(2'-[Z]-pentenyl)cyclopentane-1-octanoic acid (OPC-8:0), which subsequently undergoes three rounds of beta-oxidation to yield JA. Although most JA biosynthetic enzymes have been identified, several key steps in the pathway remain to be elucidated. To address this knowledge gap, we employed co-expression analysis to identify genes that are coordinately regulated with known JA biosynthetic components in Arabidopsis. Among the candidate genes uncovered by this approach was a 4-coumarate-CoA ligase-like member of the acyl-activating enzyme (AAE) gene family, which we have named OPC-8:0 CoA Ligase1 (OPCL1). In response to wounding, opcl1 null mutants exhibited reduced levels of JA and hyperaccumulation of OPC-8:0. Recombinant OPCL1 was active against both OPDA and OPC-8:0, as well as medium-to-long straight-chain fatty acids. Subcellular localization studies with green fluorescent protein-tagged OPCL1 showed that the protein is targeted to peroxisomes. These findings establish a physiological role for OPCL1 in the activation of JA biosynthetic precursors in leaf peroxisomes, and further indicate that OPC-8:0 is a physiological substrate for the activation step. The results also demonstrate the utility of co-expression analysis for identification of factors that contribute to jasmonate homeostasis.     

Octadecanoid signaling component "burst" in rice (Oryza sativa L.) seedling leaves upon wounding by cut and treatment with fungal elicitor chitosan

Octadecanoid pathway components, 12-oxo-phytodieonic acid (OPDA) and jasmonic acid (JA), are key biologically active regulators of plant self-defense response(s). However, to date these compounds have been studied mostly in dicots, and used large (1-10 g fresh weight, FW) samples for quantification, even when examined in mature rice plants, which is a drawback considering their rapid responsiveness to stress. Focusing on rice--a monocot cereal crop research model--this work describes an efficient and simultaneous quantification of both OPDA and JA using a minimum amount of 200mg FW seedling leaf tissue upon wounding (by cut) and treatment with fungal elicitor, chitosan (CT) by high-pressure liquid chromatography-turboionspray tandem mass spectrometry. Transient OPDA/JA "burst" was consistently and reproducibly detected within 3 min in wounded and CT treated leaves. OPDA peaked dramatically around 5 min and returned to its basal level within 15 min, whereas JA induction upon wounding and CT treatment were in parallel to OPDA production, peaking at 30 and 60 min, respectively. Present results mark a major advance in our understanding of key inducible octadecanoid pathway components in rice, and strongly suggest a role for the octadecanoid pathway downstream of perception of at least these two fundamentally different extracellular stimuli.     

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

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

Jasmonate biosynthesis in Arabidopsis thaliana requires peroxisomal beta-oxidation enzymes--additional proof by properties of pex6 and aim1

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.     

Jasmonate biosynthesis and the allene oxide cyclase family of Arabidopsis thaliana

In biosynthesis of octadecanoids and jasmonate (JA), the naturally occurring enantiomer is established in a step catalysed by the gene cloned recently from tomato as a single-copy gene (Ziegler et al., 2000). Based on sequence homology, four full-length cDNAs were isolated from Arabidopsis thaliana ecotype Columbia coding for proteins with AOC activity. The expression of AOCgenes was transiently and differentially up-regulated upon wounding both locally and systemically and was induced by JA treatment. In contrast, AOC protein appeared at constitutively high basal levels and was slightly increased by the treatments. Immunohistochemical analyses revealed abundant occurrence of AOC protein as well as of the preceding enzymes in octadecanoid biosynthesis, lipoxygenase (LOX) and allene oxide synthase (AOS), in fully developed tissues, but much less so in 7-day old leaf tissues. Metabolic profiling data of free and esterified polyunsaturated fatty acids and lipid peroxidation products including JA and octadecanoids in wild-type leaves and the jasmonate-deficient mutant OPDA reductase 3 (opr3) revealed preferential activity of the AOS branch within the LOX pathway. 13-LOX products occurred predominantly as esterified derivatives, and all 13-hydroperoxy derivatives were below the detection limits. There was a constitutive high level of free 12-oxo-phytodienoic acid (OPDA) in untreated wild-type and opr3 leaves, but an undetectable expression of AOC. Upon wounding opr3 leaves exhibited only low expression of AOC, wounded wild-type leaves, however, accumulated JA and AOC mRNA. These and further data suggest regulation of JA biosynthesis by OPDA compartmentalization and a positive feedback by JA during leaf development.     

Concurrent activation of cell death-regulating signaling pathways by singlet oxygen in Arabidopsis thaliana

Upon a dark/light shift the conditional flu mutant of Arabidopsis starts to generate singlet oxygen ((1)O(2)), a non-radical reactive oxygen species that is restricted to the plastid compartment. Immediately after the shift, plants stop growing and develop necrotic lesions. We have established a protoplast system, which allows detection and characterization of the death response in flu induced by the release of (1)O(2). Vitamin B6 that quenches (1)O(2) in fungi was able to protect flu protoplasts from cell death. Blocking ethylene production was sufficient to partially inhibit the death reaction. Similarly, flu mutant seedlings expressing transgenic NahG were partially protected from the death provoked by the release of (1)O(2), indicating a requirement for salicylic acid (SA) in this process, whereas in cells depleted of both, ethylene and SA, the extent of cell death was reduced to the wild-type level. The flu mutant was also crossed with the jasmonic acid (JA)-depleted mutant opr3, and with the JA, OPDA and dinor OPDA (dnOPDA)-depleted dde2-2 mutant. Analysis of the resulting double mutants revealed that in contrast to the JA-induced suppression of H(2)O(2)/superoxide-dependent cell death reported earlier, JA promotes singlet oxygen-mediated cell death in flu, whereas other oxylipins such as OPDA and dnOPDA antagonize this death-inducing activity of JA.     

Induction of the Arabidopsis PHO1;H10 gene by 12-oxo-phytodienoic acid but not jasmonic acid via a CORONATINE INSENSITIVE1-dependent pathway

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.     

Identification of the OsOPR7 gene encoding 12-oxophytodienoate reductase involved in the biosynthesis of jasmonic acid in rice

Enzyme 12-oxophytodienoate (OPDA) reductase (EC1.3.1.42), which is involved in the biosynthesis of jasmonic acid (JA), catalyses the reduction of 10, 11-double bonds of OPDA to yield 3-oxo-2-(2′-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0). The rice OsOPR1 gene encodes OPDA reductase (OPR) converting (−)-cis-OPDA preferentially, rather than (+)-cis-OPDA, a natural precursor of JA. Here, we provide evidence that an OPR family gene in rice chromosome 8, designated OsOPR7, encodes the enzyme involved in the JA biosynthesis. Recombinant OsOPR7-His protein efficiently catalysed the reduction of both enantiomers of cis-OPDA, similar to the OPR3 protein in Arabidopsis thaliana (L.) Heynh. The expression of OsOPR7 mRNA was induced and reached maximum levels within 0.5 h of mechanical wounding and drought stress, and the endogenous JA level started to increase in accordance with the increase in OsOPR7 expression. The GFP-OsOPR7 fusion protein was detected exclusively in peroxisomes in onion epidermal cells. Furthermore, complementation analysis using an Arabidopsis opr3 mutant indicated that the OsOPR7 gene, but not OsOPR1, was able to complement the phenotypes of male sterility in the mutant caused by JA deficiency, and that JA production in the opr3 mutant was also restored by the expression of the OsOPR7 gene. We conclude that the OsOPR7 gene encodes the enzyme catalysing the reduction of natural (+)-cis-OPDA for the JA biosynthesis in rice. Tomoyuki Tani and Hiroyuki Sobajima have equally contributed to this work.     

The role of EDS1 (enhanced disease susceptibility) during singlet oxygen-mediated stress responses of Arabidopsis

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.     

Role of cis-12-oxo-phytodienoic acid in tomato embryo development

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.     

Methods for the analysis of oxylipins in plants

Plant oxylipins comprise a highly diverse and complex class of molecules that are derived from lipid oxidation. The initial oxidation of unsaturated fatty acids may either occur by enzymatic or chemical reactions. A large variety of oxylipin classes are generated by an array of alternative reactions further converting hydroperoxy fatty acids. The structural diversity of oxylipins is further increased by their occurrence either as free fatty acid derivatives or as esters in complex lipids. Lipid peroxidation is common to all biological systems, appearing in developmentally regulated processes and as a response to environmental changes. The oxylipins formed may perform various biological roles; some of them have signaling functions. In order to elucidate the roles of oxylipins in a given biological context, comprehensive analytical assays are available for determining the oxylipin profiles of plant tissues. This review summarizes indirect methods to estimate the general peroxidation state of a sample and more sophisticated techniques for the identification, structure determination and quantification of oxylipins.