12-oxophytodienoic Acid Reductase Research Articles

Loop 6 and the β-hairpin flap are structural hotspots that determine cofactor specificity in the FMN-dependent family of ene-reductases.

Flavin mononucleotide (FMN)-dependent ene-reductases constitute a large family of oxidoreductases that catalyze the enantiospecific reduction of carbon-carbon double bonds. The reducing equivalents required for substrate reduction are obtained from reduced nicotinamide by hydride transfer. Most ene-reductases significantly prefer, or exclusively accept, either NADPH or NADH. Despite their usefulness in biocatalytic applications, the structural determinants for cofactor preference remain elusive. We employed the NADPH-preferring 12-oxophytodienoic acid reductase 3 from Solanum lycopersicum (SlOPR3) as a model enzyme of the ene-reductase family and applied computational and structural methods to investigate the binding specificity of the reducing coenzymes. Initial docking results indicated that the arginine triad R283, R343, and R366 residing on and close to a critical loop at the active site (loop 6) are the main contributors to NADPH binding. In contrast, NADH binds unfavorably in the opposite direction toward the β-hairpin flap within a largely hydrophobic region. Notably, the crystal structures of SlOPR3 in complex witheither NADPH4 or NADH4 corroborated these different binding modes. Molecular dynamics simulations confirmed NADH binding near the β-hairpin flap and provided structural explanations for the low binding affinity of NADH to SlOPR3. We postulate that cofactor specificity is determined by the arginine triad/loop 6 and the residue(s) controlling access to a hydrophobic cleft formed by the β-hairpin flap. Thus, NADPH preference depends on a properly positioned arginine triad, whereas granting access to the hydrophobic cleft at the β-hairpin flap favors NADH binding.

Jasmonates, Their Biosynthesis, Metabolism and Signalling Pathway in Plant Organisms

Jasmonates, derivatives of jasmonic acid, are lipid-derived oxylipins with cyclopentanone ring, produced as a result of dioxygenation mediated by lipoxygenases. The biosynthesis of jasmonic acid is catalysed by a series of different enzymes, starting from lipoxygenase, allene oxide synthase, allene oxide cyclase and 12-oxophytodienoic acid reductase to the final three rounds of β-oxidation. Consequently, a few metabolites known as jasmonates are formed from the resulting jasmonic acid. Some of these metabolites are active and able to work on the jasmonate receptor and thus start the jasmonate signalling pathway. Products of gene expression resulting from this pathway have a great significance in plant growth, development, reproduction, senescence and also in defence and adaptation to various adverse environmental conditions.

Identification of Genes and Metabolic Pathways Involved in Resin Yield in Masson Pine by Integrative Analysis of Transcriptome, Proteome and Biochemical Characteristics.

Masson pine (Pinus massoniana L.) is one of the most important resin-producing tree species in southern China. However, the molecular regulatory mechanisms of resin yield are still unclear in masson pine. In this study, an integrated analysis of transcriptome, proteome, and biochemical characteristics from needles of masson pine with the high and common resin yield was investigated. The results showed that chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophyll (Chl C), carotenoids (Car), glucose (Glu), gibberellin A9 (GA9), gibberellin A15 (GA15), and gibberellin A53 (GA53) were significantly increased, whereas fructose (Fru), jasmonic acid (JA), jasmonoyl-L-isoleucine (JA-ILE), gibberellin A1 (GA1), gibberellin A3 (GA3), gibberellin A19 (GA19), and gibberellin A24 (GA24) were significantly decreased in the high resin yield in comparison with those in the common one. The integrated analysis of transcriptome and proteome showed that chlorophyll synthase (chlG), hexokinase (HXK), sucrose synthase (SUS), phosphoglycerate kinase (PGK), dihydrolipoamide dehydrogenase (PDH), dihydrolipoamide succinyltransferase (DLST), 12-oxophytodienoic acid reductase (OPR), and jasmonate O-methyltransferases (JMT) were consistent at the transcriptomic, proteomic, and biochemical levels. The pathways of carbohydrate metabolism, terpenoid biosynthesis, photosynthesis, and hormone biosynthesis may play crucial roles in the regulation of resin yield, and some key genes involved in these pathways may be candidates that influence the resin yield. These results provide insights into the molecular regulatory mechanisms of resin yield and also provide candidate genes that can be applied for the molecular-assisted selection and breeding of high resin-yielding masson pine.

Specific Changes in Arabidopsis thaliana Rosette Lipids during Freezing Can Be Associated with Freezing Tolerance.

While the roles of a few specific lipids in plant freezing tolerance are understood, the effect of many plant lipids remains to be determined. Acclimation of plants to non-freezing cold before exposure to freezing temperatures improves the outcome of plants, compared to plants exposed to freezing without acclimation. Arabidopsis thaliana plants were subjected to one of three treatments: (1) “control”, i.e., growth at 21 °C, (2) “non-acclimated”, i.e., 3 days at 21 °C, 2 h at −8 °C, and 24 h recovery at 21 °C, and (3) “acclimated”, i.e., 3 days at 4 °C, 2 h at −8 °C, and 24 h recovery at 21 °C. Plants were harvested at seven time points during the treatments, and lipid levels were measured by direct-infusion electrospray ionization tandem mass spectrometry. Ion leakage was measured at the same time points. To examine the function of lipid species in relation to freezing tolerance, the lipid levels in plants immediately following the freezing treatment were correlated with the outcome, i.e., ion leakage 24-h post-freezing. Based on the correlations, hypotheses about the functions of specific lipids were generated. Additionally, analysis of the lipid levels in plants with mutations in genes encoding patatin-like phospholipases, lipoxygenases, and 12-oxophytodienoic acid reductase 3 (opr3), under the same treatments as the wild-type plants, identified only the opr3-2 mutant as having major lipid compositional differences compared to wild-type plants.

12-Oxophytodienoic Acid Reductase 3 (OPR3) Functions as NADPH-Dependent α,β-Ketoalkene Reductase in Detoxification and Monodehydroascorbate Reductase in Redox Homeostasis

12-Oxophytodienoic Acid Reductase 3 (OPR3) Functions as NADPH-Dependent α,β-Ketoalkene Reductase in Detoxification and Monodehydroascorbate Reductase in Redox Homeostasis

12-Oxophytodienoic Acid Reductase 3 (OPR3) Functions as NADPH-Dependent α,β-Ketoalkene Reductase in Detoxification and Monodehydroascorbate Reductase in Redox Homeostasis

Arabidopsis (Arabidopsis thaliana) 12-oxophytodienoic acid reductase isoform 3 (OPR3) is involved in the synthesis of jasmonic acid (JA) by reducing the α,β-unsaturated double bond of the cyclopentenone moiety in 12-oxophytodienoic acid (12-OPDA). Recent research revealed that JA synthesis is not strictly dependent on the peroxisomal OPR3. The ability of OPR3 to reduce trinitrotoluene suggests that the old yellow enzyme homolog OPR3 has additional functions. Here, we show that OPR3 catalyzes the reduction of a wide spectrum of electrophilic species that share a reactivity toward the major redox buffers glutathione (GSH) and ascorbate (ASC). Furthermore, we show that 12-OPDA reacts with ASC to form an ASC-12-OPDA adduct, but in addition OPR3 has the ability to regenerate ASC from monodehydroascorbate. The presented data characterize OPR3 as a bifunctional enzyme with NADPH-dependent α,β-ketoalkene double-bond reductase and monodehydroascorbate reductase activities (MDHAR). opr3 mutants showed a slightly less-reduced ASC pool in leaves in line with the MDHAR activity of OPR3 in vitro. These functions link redox homeostasis as mediated by ASC and GSH with OPR3 activity and metabolism of reactive electrophilic species.

One-pot synthesis of bioactive cyclopentenones from α-linolenic acid and docosahexaenoic acid

Oxidation products of the poly-unsaturated fatty acids (PUFAs) arachidonic acid, α-linolenic acid and docosahexaenoic acid are bioactive in plants and animals as shown for the cyclopentenones prostaglandin 15d-PGJ2 and PGA2, cis-(+)-12-oxophytodienoic acid (12-OPDA), and 14-A-4 neuroprostane. In this study an inexpensive and simple enzymatic multi-step one-pot synthesis is presented for 12-OPDA, which is derived from α-linolenic acid, and the analogous docosahexaenoic acid (DHA)-derived cyclopentenone [(4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl]-cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid, OCPD]. The three enzymes utilized in this multi-step cascade were crude soybean lipoxygenase or a recombinant lipoxygenase, allene oxide synthase and allene oxide cyclase from Arabidopsis thaliana. The DHA-derived 12-OPDA analog OCPD is predicted to have medicinal potential and signaling properties in planta. With OCPD in hand, it is shown that this compound interacts with chloroplast cyclophilin 20-3 and can be metabolized by 12-oxophytodienoic acid reductase (OPR3) which is an enzyme relevant for substrate bioactivity modulation in planta.

Characterization of 12-Oxophytodienoic Acid Reductases from Rose-scented Geranium (Pelargonium graveolens)

Pelargonium graveolens L'Hér, also referred to as rose geranium, is a popular herbal plant with typical rosy fragrance largely based on the blend of monoterpenoid constituents. Among them, citronellol, which is biosynthesized from geraniol via double bond reduction, is the most abundant scent compound. In this study, three 12-oxophytodienoic acid reductases (PgOPRl-3) hive been cloned from P. graveolens, as -possible candidates for the double-bond reductase involved in citronellol biosynthesis. The bacterially expressed recombinant PgOPRs did not reduce geraniol to citronellol, but stereoselectively converted citral into (S)-citronellal in the presence of NADPH. Thus, the a,-unsaturated carbonyl moiety in the substrate is essential for the catalytic activity of PgOPRs; as reported for OPRs from other plants and structurally related yeast old yellow enzymes. PgOPRs promiscuously accepted linear and cyclic α,β- uisaturated carbonyl substrates, including methacrolein, a typical reactive carbonyl compound. The possible biotechnological applications for PgOPRs in plant metabolic'engineering, based on their catalytic properties, are discussed herein.

Homologs of old yellow enzyme in plants

Old Yellow Enzyme (OYE) is a flavin mononucleotide-dependent oxidoreductase. 12-oxophytodienoic acid reductases (OPRs) are OYE homologs and are represented by multigene families in plants. OPRs catalyze the reduction of double bonds in α,β-unsaturated aldehydes or ketones. They belong to the octadecanoid pathway, which converts linolenic acid to jasmonic acid (JA). Individual OPR family members may have distinct functions due to their different substrate specificity, subcellular localization, tissue distribution, and differential regulation of their expression in response to specific environmental cues. Based on their differential preferences for 12-oxophytodienoate (OPDA) stereoisomers, these enzymes are classified into two subgroups: OPRI and OPRII. OPRI enzymes preferentially catalyze the reduction of cis-(−)OPDA over the JA precursor cis-(+)OPDA , and therefore, they are not involved in JA biosynthesis. Although a significant progress has been made to understand the exact physiological roles of OPR enzymes, the function of OPRI subgroup members in plants remains largely unknown. Enzymes belonging to this subgroup are possibly involved in defense responses and signaling. The members of the OPRII subgroup are required for JA biosynthesis because they catalyze the reduction of the JA precursor cis-(+)OPDA. This review will highlight some characteristics of this family in Arabidopsis and other species and discuss the physiological role of OPR family members in plants.

Production of 3-Oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic Acid in the Fungus Aspergillus oryzae: A Step Towards Heterologous Production of Pyrethrins in Fungi.

Pyrethrins are natural insecticides, which accumulate to high concentrations in pyrethrum (Chrysanthemum cinerariaefolium) flowers. Synthetic pyrethroids are more stable, more efficacious and cheaper, but contemporary requirements for safe and environmentally friendly pesticides encourage a return to the use of natural pyrethrins, and this would be favoured by development of an efficient route to their production by microbial fermentation. The biosynthesis of pyrethrins involves ester linkage between an acid moiety (chrysanthemoyl or pyrethroyl, synthesised via the mevalonic acid pathway from glucose), and an alcohol (pyrethrolone). Pyrethrolone is generated from 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid, which originates from α-linolenic acid via the jasmonic acid biosynthetic cascade. The first four genes in this cascade, encoding lipoxygenase 2, allene-oxide synthase, allene-oxide cyclase 2 and 12-oxophytodienoic acid reductase 3, were amplified from an Arabidopsis thaliana cDNA library, cloned in a purpose-built fungal multigene expression vector and expressed in Aspergillus oryzae. HPLC-MS analysis of the transgenic fungus homogenate gave good evidence for the presence of 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid.

Identification and characterization of genes involved in the jasmonate biosynthetic and signaling pathways in mulberry (Morus notabilis)

Jasmonate (JA) is an important phytohormone regulating growth, development, and environmental response in plants, particularly defense response against herbivorous insects. Recently, completion of the draft genome of the mulberry (Morus notabilis) in conjunction with genome sequencing of silkworm (Bombyx mori) provides an opportunity to study this unique plant-herbivore interaction. Here, we identified genes involved in JA biosynthetic and signaling pathways in the genome of mulberry for the first time, with the majority of samples showing a tissue-biased expression pattern. The analysis of the representative genes 12-oxophytodienoic acid reductase (OPRs) and jasmonate ZIM-domain (JAZs) was performed and the results indicated that the mulberry genome contains a relatively small number of JA biosynthetic and signaling pathway genes. A gene encoding an important repressor, MnNINJA, was identified as an alternative splicing variant lacking an ethylene-responsive element binding factor-associated amphiphilic repression motif. Having this fundamental information will facilitate future functional study of JA-related genes pertaining to mulberry-silkworm interactions.

Cloning and Functional Characterization of Key Enzymes in Putative Octadecanoid Pathway of <i>Physcomitrella patens</i>

Jasmonic acid and its metabolites are ubiquitously occurring lipid-derived signaling compounds that regulate growth, development and defense processes in flowering plants. However, their functions in lower land plants have not been well characterized yet. The model moss, <span style="text-decoration: underline;"><em>Physcomitrella patens</em></span> is a member of bryophytes and represents a key evolutionary position between green algae and flowering plants. According to the proposed land plant evolution, jasmonic acid signaling pathway has evolved after the evolutionary split of bryophytes and vascular plants, indicating its absence in bryophytes. However, the putative key homologous genes involved in jasmonic acid biosynthesis and its signaling pathway are available in <span style="text-decoration: underline;"><em>P. patens</em></span> genome. Allene oxide synthase catalyzes the first committed step of jasmonic acid biosynthesis in flowering plants. A putative allene oxide synthase gene of<span style="text-decoration: underline;"><em> P. patens</em></span> was cloned and shown to have the same in vitro function as observed in flowering plants. Moreover, based on substrate specificity, plant 12-oxophytodienoic acid reductases can be classified into two groups, group I and group II, of which only group II isozymes involve in jasmonic acid biosynthesis. Six putative 12-oxophytodienoic acid reductase genes, among which only one represents group II, were identified in<span style="text-decoration: underline;"><em> P. patens genome</em></span>. Two 12-oxo-phytodienoic acid reductase genes, which represent group I and group II, were cloned and characterized. Striking results were obtained since the 12-oxo-phytodienoic acid reductase, which represents group II, exhibited group I type activity. This unusual functional property clearly explains why<span style="text-decoration: underline;"> <em>P. patens</em></span>, probably all the bryophytes, does not contain jasmonic and its signaling pathway though it contains the corresponding homologous genes. Moreover, the ancestral plant 12-oxophytodienoic acid reductases might have possessed group I type activity and early precursors of jasmonic acid might have functioned as signaling molecules in ancestral land plants instead of jasmonic acid. <em>Tropical Agricultural Research Vol. 23 (2): 160-167 (2012)</em> DOI: <span style="text-decoration: underline;"><a href="http://dx.doi.org/10.4038/tar.v23i2.4647">http://dx.doi.org/10.4038/tar.v23i2.4647</a></span>

Oxylipins are not required for R gene-mediated resistance in potato

The role of 9- and 13-lipoxygenase-derived oxylipins for race-cultivar-specific resistance in potato was analyzed by expressing RNA interference constructs against oxylipin biosynthetic genes in transgenic potato plants carrying the resistance gene R1 against Phytophthora infestans. Down-regulation of 9-lipoxygenase expression resulted in highly reduced levels of 9-hydroxyoctadecatrienoic acid after treatment with the pathogen-associated molecular pattern Pep-13. However, neither 9-lipoxygenase nor 9-divinyl ether synthase RNAi plants exhibited alterations in their resistance to P. infestans. Similarly, successful down-regulation of transcript accumulation of the 13-lipoxygenase pathway genes encoding allene oxide cyclase, 12-oxophytodienoic acid reductase 3 and the jasmonic acid receptor coronatine-insensitive 1 resulted in highly reduced levels of jasmonic acid after Pep-13 treatment. Race-cultivar-specific resistance, however, was not lost in these plants. Our results suggest that neither 9-lipoxygenase-derived oxylipins nor jasmonic acid are required for R-gene-mediated resistance in potato. Importantly, in tobacco, the silencing of 9-lipoxygenase expression was previously demonstrated to suppress race-cultivar-specific resistance. Thus, we conclude a differential requirement of oxylipins for R-gene-mediated resistance in different solanaceous plants.

Bioreduction of α-methylcinnamaldehyde derivatives: chemo-enzymatic asymmetric synthesis of Lilial™ and Helional™

Nonracemic aryl-substituted alpha-methyldihydrocinnamaldehyde derivatives employed as olfactory principles in perfumes (Lilial, Helional) were obtained via enzymatic reduction of the corresponding cinnamaldehyde precursors using cloned and overexpressed ene-reductases. (R)-Enantiomers were obtained using the old-yellow-enzyme (OYE) homolog YqjM from Bacillus subtilis and 12-oxophytodienoic acid reductase isoenzyme OPR1 from tomato (e.e.(max) 53%), and (S)-aldehydes were furnished in up to 97% e.e. using isoenzyme OPR3, nicotinamide 2-cyclohexene-1-one reductase NCR from Zymomonas mobilis and yeast OYE isoenzymes 1-3 under optimised reaction conditions in the presence of t-butyl methyl ether as the co-solvent. The stereochemical outcome of the reduction of alpha-methylcinnamaldehyde using NCR and OYEs 1-3 [previously reported to be (R)] was unambiguously corrected to be (S).

PAMP‐induced defense responses in potato require both salicylic acid and jasmonic acid

To elucidate the molecular mechanisms underlying pathogen-associated molecular pattern (PAMP)-induced defense responses in potato (Solanum tuberosum), the role of the signaling compounds salicylic acid (SA) and jasmonic acid (JA) was analyzed. Pep-13, a PAMP from Phytophthora, induces the accumulation of SA, JA and hydrogen peroxide, as well as the activation of defense genes and hypersensitive-like cell death. We have previously shown that SA is required for Pep-13-induced defense responses. To assess the importance of JA, RNA interference constructs targeted at the JA biosynthetic genes, allene oxide cyclase and 12-oxophytodienoic acid reductase, were expressed in transgenic potato plants. In addition, expression of the F-box protein COI1 was reduced by RNA interference. Plants expressing the RNA interference constructs failed to accumulate the respective transcripts in response to wounding or Pep-13 treatment, neither did they contain significant amounts of JA after elicitation. In response to infiltration of Pep-13, the transgenic plants exhibited a highly reduced accumulation of reactive oxygen species as well as reduced hypersensitive cell death. The ability of the JA-deficient plants to accumulate SA suggests that SA accumulation is independent or upstream of JA accumulation. These data show that PAMP responses in potato require both SA and JA and that, in contrast to Arabidopsis, these compounds act in the same signal transduction pathway. Despite their inability to fully respond to PAMP treatment, the transgenic RNA interference plants are not altered in their basal defense against Phytophthora infestans.

Identification of a Jasmonic Acid-Responsive Region in the Promoter of the Rice 12-Oxophytodienoic Acid Reductase 1 GeneOsOPR1

The rice 12-oxophytodienoic acid reductase 1 gene (OsOPR1), isolated as a jasmonic acid (JA)-responsive gene, has been suggested to be involved in defense responses in rice. We identified a 19-base pair region that is essential to the JA-responsiveness of OsOPR1 by deletion and mutation analysis of the promoter by dual luciferase assay. This region contains possible recognition sites for basic leucine zipper transcription factors.

Insect Elicitors and Exposure to Green Leafy Volatiles Differentially Upregulate Major Octadecanoids and Transcripts of 12-Oxo Phytodienoic Acid Reductases in Zea mays

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.

Cloning and characterization of a putative 12-oxophytodienoic acid reductase cDNA induced by osmotic stress in roots of foxtail millet

Foxtail millet is a gramineous crop with low water requirement. Cloning of osmotic responses-related genes from foxtail millet is a key step for understanding the mechanism of its tolerance to drought. Here we reported the cloning and characterization of a cDNA (SiOPR1) encoding a putative 12-oxophytodienoic acid reductase 1 from foxtail millet by using RACE methods. Sequence analysis showed that SiOPR1 encoded a polypeptide of 374 amino acids with a predicted molecular mass of 41.9 kDa and pI of 5.14. Multiple alignment result showed that OPR1 protein was very conservative among gramineous crops. RNA gel blot analysis results indicated that SiOPR1 was up-regulated by osmotic stress, and its expression was limited in the roots of foxtail millet. However, SiOPR1 expression was not affected by ABA, NaCl and MeJA treatments both in roots and shoots. Therefore, it is suggested that SiOPR1 gene play an important role in response to drought stress.

15. Rice 12-oxophytodienoic acid reductase 8 (OsOPR8) is the isozyme involved in biosynthesis of jasmonic acid

15. Rice 12-oxophytodienoic acid reductase 8 (OsOPR8) is the isozyme involved in biosynthesis of jasmonic acid

43. Identification of the gene encoding 12-oxophytodienoic acid (OPDA) reductase involved in biosynthesis of jasmonic acid in rice

43. Identification of the gene encoding 12-oxophytodienoic acid (OPDA) reductase involved in biosynthesis of jasmonic acid in rice