12-oxophytodienoic Acid Research Articles

Impact of phytohormones on wheat resistance to Hessian fly under heat stress

The Hessian fly (HF, Mayetiola destructor) is one of the most destructive pests of wheat and wheat-related cereals. Wheat resistance and/or susceptibility to HF are often affected by the levels of phytohormones in plants. In this study, we tested the impact of phytohormones on Molly wheat resistance to HF biotype GP by externally applying phytohormones, including salicylic acid (SA), jasmonic acid (JA), 12-oxophytodienoic acid (OPDA), and auxin (indole acetic acid, IAA) to wheat seedlings under heat conditions. Our results indicated that the impact of externally applied phytohormones on wheat resistance to HF depends on the timing of phytohormone application and/or HF larval density at HF feeding sites in the plants. The early application of SA, OPDA, and IAA enhanced wheat resistance to HF under heat stress at low larval density, while the delayed application of SA, OPDA, and IAA did not affect wheat resistance to HF at high larval density.

Molecular cloning and characterization of heat-responsive LcOPR1, a gene encoding oxophytodienoic acid reductase in lentil.

Improving crop plants using biotechnological implications is a promising and modern approach compared to traditional methods. High-temperature exposure to the reproductive stage induces flower abortion and declines grain filling performance, leading to smaller grain production and low yield in lentil and other legumes. Thus, cloning effective candidate genes and their implication in temperature stress tolerance in lentil (Lens culinaris Medik.) using biotechnological tools is highly demandable. The 12-oxophytodienoic acid reductases (OPRs) are flavin mononucleotide-dependent oxidoreductases with vital roles in plants. They are members of the old yellow enzyme (OYE) family. These enzymes are involved in the octadecanoid pathway, which contributes to jasmonic acid biosynthesis and is essential in plant stress responses. Lentil is one of the vital legume crops affected by the temperature fluctuations caused by global warming. Therefore, in this study, the LcOPR1 gene was successfully cloned and isolated from lentils using RT-PCR to evaluate its functional responses in lentil under heat stress. The bioinformatics analysis revealed that the full-length cDNA of LcOPR1 was 1303 bp, containing an 1134 bp open reading frames (ORFs), encoding 377 amino acids with a predicted molecular weight of 41.63 and a theoretical isoelectric point of 5.61. Bioinformatics analyses revealed that the deduced LcOPR1 possesses considerable homology with other plant 12-oxophytodienoic acid reductases (OPRs). Phylogenetic tree analysis showed that LcOPR1 has an evolutionary relationship with other OPRs in different plant species of subgroup I, containing enzymes that are not required for jasmonic acid biosynthesis. The expression analysis of LcOPR1 indicated that this gene is upregulated in response to the heat-stress condition and during recovery in lentil. This study finding might be helpful to plant breeders and biotechnologists in LcOPR1 engineering and/or plant breeding programs in revealing the biological functions of LcOPR1 in lentils and the possibility of enhancing heat stress tolerance by overexpressing LcOPR1 in lentil and other legume plants under high temperature.

Inducible defense phytohormones in annual bluegrass (Poa annua) and creeping bentgrass (Agrostis stolonifera) in response to annual bluegrass weevil (Listronotus maculicollis) infestation.

The annual bluegrass weevil (Listronotus maculicollis) is the most damaging insect pest of short-mown turfgrass on golf courses in eastern North America. Listronotus maculicollis larvae cause limited visible damage as stem-borers (L1-3), compared to the crown-feeding (L4-5) developmental instars. Prolonged larval feeding results in discoloration and formation of irregular patches of dead turf, exposing soil on high-value playing surfaces (fairways, collars, tee boxes, and putting greens). Annual bluegrass (Poa annua) is highly susceptible to L. maculicollis compared to a tolerant alternate host plant, creeping bentgrass (Agrostis stolonifera). This study explored whether defense signaling phytohormones contribute to A. stolonifera tolerance in response to L. maculicollis. Concentrations (ng/g) of salicylic acid (SA), jasmonic acid (JA), jasmonic-isoleucine (JA-Ile), 12-oxophytodienoic acid (OPDA), and abscisic acid (ABA) were extracted from turfgrass (leaf, stem, and root) tissue samples as mean larval age reached 2nd (L2), 3rd (L3), and 4th (L4) instar. Poa annua infested with L. maculicollis larvae (L2-4) possessed significantly greater SA in above-ground tissues than A. stolonifera. Levels of constitutive JA, JA-Ile, OPDA, and ABA were significantly higher within non-infested A. stolonifera aboveground tissues compared to P. annua. Inducible defense phytohormones may play a role in P. annua susceptibility to L. maculicollis but are unlikely to provide tolerance in A. stolonifera. Additional studies in turfgrass breeding, particularly focusing on cultivar selection for increased constitutive JA content, could provide a non-chemical alternative management strategy for L. maculicollis for turfgrass managers.

Deciphering Aphanomyces euteiches-pea-biocontrol bacterium interactions through untargeted metabolomics

Aphanomyces euteiches causes root rot in pea, leading to significant yield losses. However, the metabolites involved in this pathosystem have not been thoroughly studied. This study aimed to fill this gap and explore mechanisms of bacterial suppression of A. euteiches via untargeted metabolomics using pea grown in a controlled environment. Chemical isotope labeling (CIL), followed by liquid chromatography-mass spectrometry (LC–MS), was used for metabolite separation and detection. Univariate and multivariate analyses showed clear separation of metabolites from pathogen-treated pea roots and roots from other treatments. A three-tier approach positively or putatively identified 5249 peak pairs or metabolites. Of these, 403 were positively identified in tier 1; 940 were putatively identified with high confidence in tier 2. There were substantial changes in amino acid pool, and fatty acid and phenylpropanoid pathway products. More metabolites, including salicylic and jasmonic acids, were upregulated than downregulated in A. euteiches-infected roots. 1-aminocyclopropane-1-carboxylic acid and 12-oxophytodienoic acid were upregulated in A. euteiches + bacterium-treated roots compared to A. euteiches-infected roots. A great number of metabolites were up- or down-regulated in response to A. euteiches infection compared with the control and A. euteiches + bacterium-treated plants. The results of this study could facilitate improved disease management.

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.

Nitrogen Deficiency Enhances Eggplant Defense against Western Flower Thrips via the Induction of the Jasmonate Pathway.

Plant nutrition is connected to defense against insect herbivores, but the exact mechanism underlying the effect of the nitrogen (N) supply on the anti-herbivore capacity of eggplants (Solanum melongena) has not been studied in detail. Therefore, we examined the impact of low (LN, 0.5 mM) and high (HN, 5 mM) nitrate levels on eggplant resistance against the western flower thrips Frankliniella occidentalis (WFT), a major destructive eggplant pest. Our results showed that LN plants displayed enhanced defense responses to WFT compared to HN plants. This included increased transcript levels of key genes in the jasmonic acid (JA) pathway, the accumulation of JA-amido conjugates (jasmonoyl-isoleucine, jasmonoyl-phenylalanine, and jasmonoyl-valine), JA precursor (12-oxophytodienoic acid), and methyl jasmonate, higher transcript levels of defense marker genes (MPK3, MPK7, and WRKY53), and increased activities of polyphenol oxidase and peroxidase upon a WFT attack. Our findings suggest that N deficiency can prime JA-mediated defense responses in eggplants, resulting in increased anti-herbivore resistance.

Exploring the interplay of phytohormones and polyamines in drought-stressed Cress (<i>Lepidium sativum</i> L.) leaves

The study investigated prolonged drought stress effects on phytohormone and polyamine levels in cress (Lepidium sativum L.) leaves, elucidating plant responses to harsh environments. Phytohormones, 12-oxophytodienoic acid (OPDA), Jasmonic acid (JA), Jasmonyl-Isoleucine (JA-Ile), Abscisic acid (ABA), and salicylic acid (SA) increased, especially ABA that rose consistently. Indole-3-acetic acid (IAA) responded akin to ABA and SA. Cytokinins such as zeatin (ZT) and isopentenyl adenine (iP) diversified drought tolerance, ZT fell sharply, and iP remained stable. Gibberellic acid (GA3) declined, while brassinolide (BL) surged with prolonged drought. Polyamines reduced, indicating sensitivity to severe drought. Findings uncovered intricate regulatory mechanisms for plant adaptation to water scarcity. Interaction of OPDA, JA, JA-Ile, ABA, SA, IAA, ZT, iP, GA3, BL, and polyamines contributes to drought coping and physiological balance. Insights into cress leaf response and drought tolerance mechanisms contribute significantly to plant drought adaptation understanding.

Establishment of new convenient two-line system for hybrid production by targeting mutation of OPR3 in allopolyploid Brassica napus

Abstract The two-line pollination control system, which usually depends on the utilization of thermosensitive or photoperiod genic male-sterile lines, has been widely used in various crops. However, this system is susceptible to instability issues caused by uncontrollable weather fluctuations. A stable and handy two-line pollination control system is highly desirable in many crop species for heterosis exploitation. Oxophytodienoic acid reductase 3 (OPR3) was proven to be involved in jasmonate biosynthesis. In the present study, CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat) was utilized to mutate two OPR3 homologs in Brassica napus. After two OPR3 homologs were simultaneously mutated, mutants exhibited complete male sterility, and fertility could be easily restored by exogenous MeJA treatment. Hybrids produced from crosses between the opr3 sterile lines and normal varieties exhibited heterosis. This new two-line system based on OPR3 mutation provides higher stability and convenience than traditional systems. By using exogenous MeJA treatment to restore fertility, the system enables more precise control of male fertility transition, which has great potential to significantly contribute to the maneuverable production of hybrid seeds in rapeseed as well as other Brassica species crops.

Metabolomic Analysis ofthe Desert Moss Syntrichia caninervis Provides Insights into Plant Dehydration andRehydration Response.

Desiccation-tolerant (DT) plants can survive extreme dehydration and tolerate the loss of up to 95% of their water content, making them ideal systems to determine the mechanism behind extreme drought stress and identify potential approaches for developing drought-tolerant crops. The desert moss Syntrichia caninervis is an emerging model for extreme desiccation tolerance that has benefited from high-throughput sequencing analyses, allowing identification of stress-tolerant genes; however, its metabolic response to desiccation is unknown. A liquid chromatography-mass spectrometry analysis of S. caninervis at six dehydration-rehydration stages revealed 912 differentially abundant compounds, belonging to 93 metabolic classes. Many (256) metabolites accumulated during rehydration in S. caninervis, whereas only 71 accumulated during the dehydration period, in contrast to the pattern observed in vascular DT plants. During dehydration, nitrogenous amino acids (l-glutamic acid and cysteinylglycine), alkaloids (vinleurosine) and steroids (physalin D) accumulated, whereas glucose 6-phosphate decreased. During rehydration, γ-aminobutyric acid, glucose 6-phosphate and flavonoids (karanjin and aromadendrin) accumulated, as did the plant hormones 12-oxo phytodienoic acid (12-OPDA) and trans-zeatin riboside. The contents ofl-arginine, maltose, turanose, lactulose and sucrose remained high throughout dehydration-rehydration. Syntrichia caninervis thus accumulates antioxidants to scavenge reactive oxygen species, accumulating nitrogenous amino acids and cytoprotective metabolites and decreasing energy metabolism to enter a protective state from dehydration-induced damage. During subsequent rehydration, many metabolites rapidly accumulated to prevent oxidative stress and restore physiological activities while repairing cells, representing a more elaborate rehydration repair mechanism than vascular DT plants, with a faster and greater accumulation of metabolites. This metabolic kinetics analysis in S. caninervis deepens our understanding of its dehydration mechanisms and provides new insights into the different strategies of plant responses to dehydration and rehydration.

Palliative Effects of Kaolin on Citrus Plants Under Controlled Stress Conditions of High Temperature and High Light Intensity

Under field conditions high temperatures are usually associated to high light intensity for periods of time that are getting longer because of global warming caused by climate change. These adverse conditions lead to significant reductions in yield and fruit quality in crops of great economic relevance such as citrus. In this work, the effect of high temperatures and high light intensity occurring alone or simultaneously has been studied in Carrizo citrange plants, a citrus genotype widely used as a rootstock, and the mitigating effect of kaolin (KL) evaluated. The combined stress conditions affected the plants in a unique manner at both, physiological and hormonal regulation levels, different to the effects of each individual stress. KL alleviated the deleterious effect of combined stress in different ways: (i) reducing leaf damage and abscission, (ii) improving physiological and gas exchange parameters, (iii) decreasing foliar proline content, (iv) increasing chlorophyll content, (v) preventing carotenoid degradation, and (vi) modulating levels of hormones and their precursors involved in plant responses to stress [abscisic acid (ABA), salicylic acid (SA), cinnamic acid (CA), indole-3-acetic acid (IAA), jasmonic acid (JA) and 12-oxophytodienoic acid (OPDA)].

Dynamic changes in the levels of metabolites and endogenous hormones during the germination of Zanthoxylum nitidum (Roxb.) DC. Seeds

ABSTRACT Accumulating experimental data have shown that endogenous hormones play important roles in regulating seed dormancy and germination. Zanthoxylum nitidum is a medicinal plant that propagates via seeds, which require a long dormancy period for normal germination, and complex changes in metabolites occur during the germination process. However, the regulatory network of endogenous hormones and metabolites during the germination of Z. nitidum seeds remains unclear. This study investigated the dynamic changes in the levels of metabolites and endogenous hormones during the germination of Z. nitidum seeds. The results revealed an increase in the levels of gibberellin 3 (GA3), 12-oxophytodienoic acid (OPDA), 1-aminocyclopropane-1-carboxylic acid (ACC) and trans-zeatin (TZ) and decrease in the levels of abscisic acid (ABA), jasmonic acid (JA), N-[(-)-jasmonoyl]-(S)-isoleucine (JA-Ile) and trans-zeatin riboside (TZR). Overall, 112 differential metabolites (DAMs) were screened from 3 seed samples (Sa, Sb and Sc), most of which are related to primary metabolism. A total of 16 DAMs (including 3 monosaccharides, 3 phosphate lipids, 3 carboxylic acids, 1 amino acid, 2 pyrimidines, and 4 nucleotides) were identified in the three sample comparison pairs (Sa vs Sb, Sa vs Sc, and Sb vs Sc); these DAMs were significantly enriched in purine metabolism; glycerophospholipid metabolism, citrate cycle (TCA cycle), alanine, aspartate and glutamate metabolism and pyruvate metabolism. OPDA, ACC and GAs were significantly positively correlated with upregulated metabolites, whereas ABA and JA were significantly positively correlated with downregulated metabolites. Finally, a hypothetical metabolic network of endogenous hormones that regulate seed germination was constructed. This study deepens our understanding of the importance of endogenous hormonal profiles that mediate seed germination.

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.

A non-JA producing oxophytodienoate reductase functions in salicylic acid-mediated antagonism with jasmonic acid during pathogen attack.

Peroxisome-localized oxo-phytodienoic acid (OPDA) reductases (OPR) are enzymes converting 12-OPDA into jasmonic acid (JA). However, the biochemical and physiological functions of the cytoplasmic non-JA producing OPRs remain largely unknown. Here, we generated Mutator-insertional mutants of the maize OPR2 gene and tested its role in resistance to pathogens with distinct lifestyles. Functional analyses showed that the opr2 mutants were more susceptible to the (hemi)biotrophic pathogens Colletotrichum graminicola and Ustilago maydis, but were more resistant to the necrotrophic fungus Cochliobolus heterostrophus. Hormone profiling revealed that increased susceptibility to C.graminicola was associated with decreased salicylic acid (SA) but increased JA levels. Mutation of the JA-producing lipoxygenase 10 (LOX10) reversed this phenotype in the opr2 mutant background, corroborating the notion that JA promotes susceptibility to this pathogen. Exogenous SA did not rescue normal resistance levels in opr2 mutants, suggesting that this SA-inducible gene is the key downstream component of the SA-mediated defences against C.graminicola. Disease assays of the single and double opr2 and lox10 mutants and the JA-deficient opr7opr8 mutants showed that OPR2 negatively regulates JA biosynthesis, and that JA is required for resistance against C.heterostrophus. Overall, this study uncovers a novel function of a non-JA producing OPR as a major negative regulator of JA biosynthesis during pathogen infection, a function that leads to its contrasting contribution to either resistance or susceptibility depending on pathogen lifestyle.

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.

Mycorrhizal Symbiosis Triggers Local Resistance in Citrus Plants Against Spider Mites.

Citrus plants are a highly mycotrophic species with high levels of fungal colonization. Citrus aurantium rootstocks typically show abundant root colonization by Rhizophagus irregularis three weeks after inoculation. Mycorrhizal symbiosis protects plants against multiple biotic stressors, however, such protection against spider mites remains controversial. We examined mycorrhiza-induced resistance (MIR) in citrus against the two-spotted spider mite Tetranychus urticae. Mycorrhized C. aurantium displayed reduced levels of damage in leaves and lower mite oviposition rates, compared to non-mycorrhized controls. Mycorrhization did not affect host choice of mites in Y-tube assays; of note, C. aurantium has innate strong antixenotic resistance against this mite. Analysis of metabolism pathways in mycorrhized citrus plants showed upregulated expression of the oxylipin-related genes LOX-2 and PR-3 early after infestation. Accordingly, jasmonic acid (JA), 12-oxo phytodienoic acid (OPDA), and JA-Ile concentrations were increased by mycorrhization. Non-targeted metabolomic analysis revealed the amino acid, oxocarboxylic acid, and phenylpropanoid metabolism as the three major pathways with more hits at 24 h post infection (hpi) in mycorrhized plants. Interestingly, there was a transition to a priming profile of these pathways at 48 hpi following infestation. Three flavonoids (i.e., malic acid, coumaric acid, and diconiferyl alcohol) were among the priming compounds. A mixture containing all these compounds provided efficient protection against the mite. Unexpectedly, systemic resistance did not improve after 72 h of primary infestation, probably due to the innate strong systemic resistance of C. aurantium. This is the first study to show that MIR is functional against T. urticae in locally infested citrus leaves, which is mediated by a complex pool of secondary metabolites and is likely coordinated by priming of JA-dependent responses.

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.

AtGH3.10 is another jasmonic acid-amido synthetase in Arabidopsis thaliana.

Jasmonoyl-isoleucine (JA-Ile) is a key signaling molecule that activates jasmonate-regulated flower development and the wound stress response. For years, JASMONATE RESISTANT1 (JAR1) has been the sole jasmonoyl-amino acid synthetase known to conjugate jasmonic acid (JA) to isoleucine, and the source of persisting JA-Ile in jar1 knockout mutants has remained elusive until now. Here we demonstrate through recombinant enzyme assays and loss-of-function mutant analyses that AtGH3.10 functions as a JA-amido synthetase. Recombinant AtGH3.10 could conjugate JA to isoleucine, alanine, leucine, methionine, and valine. The JA-Ile accumulation in the gh3.10-2 jar1-11 double mutant was nearly eliminated in the leaves and flower buds while its catabolism derivative 12OH-JA-Ile was undetected in the flower buds and unwounded leaves. Residual levels of JA-Ile, JA-Ala, and JA-Val were nonetheless detected in gh3.10-2 jar1-11, suggesting the activities of similar promiscuous enzymes. Upon wounding, the accumulation of JA-Ile and 12OH-JA-Ile and the expression of JA-responsive genes OXOPHYTODIENOIC ACID REDUCTASE3 and JASMONATE ZIM-DOMAIN1 observed in WT, gh3.10-1, and jar1-11 leaves were effectively abolished in gh3.10-2 jar1-11. Additionally, an increased proportion of undeveloped siliques associated with retarded stamen development was observed in gh3.10-2 jar1-11. These findings conclusively show that AtGH3.10 contributes to JA-amino acid biosynthesis and functions partially redundantly with AtJAR1 in sustaining flower development and the wound stress response in Arabidopsis.

Molecular cloning, characterization and expression analysis of two 12-oxophytodienoate reductases (NtOPR1 and NtOPR2) from Nicotiana tabacum.

12-oxophytodienoic acid (OPDA) is a signaling molecule involved in defense and stress responses in plants. 12-oxophytodienoate reductase (OPR) is involved in the biosynthesis of jasmonic acid and trigger the conversion of OPDA into 3-oxo-2(2'[Z]-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0). Sequence analysis revealed that Nicotiana tabacum 12-oxophytodienoate reductase 1 (OPR1) and OPR2 encoded polypeptides of 375 and 349 amino acids with molecular masses of 41.67 and 39.04 kilodaltons (kDa), respectively, while the deduced protein sequences of NtOPR1 and NtOPR2 showed high homology with other 12-oxophytodienoate reductases. BLAST (Basic local alignment search tool) analysis revealed that both NtOPRs belong to the family of Old Yellow Enzymes (OYE), and analysis of genomic DNA structure indicated that both genes include 5 exons and 4 introns. Phylogenetic analysis using MEGA X showed that NtOPR1 and NtOPR2 shared a close evolutionary relationship with Nicotiana attenuata 12-oxophytodienoate reductases. In silico analysis of subcellular localization indicated the probable locations of NtOPR1 and NtOPR2 to be the cytoplasm and the peroxisome, respectively. Tissue-specific expression assays via qRT-PCR revealed that NtOPR1 and NtOPR2 genes were highly expressed in Nicotiana tabacum roots, temperately expressed in leaves and flowers, while low expression was observed in stem tissue. Presently, two 12-oxophytodienoate reductase genes (NtOPR1 and NtOPR2) were cloned and comprehensively characterized. Our findings provide comprehensive analyses that may guide future deep molecular studies of 12-oxophytodienoate reductases in Nicotiana tabacum.

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

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

The In Vitro Interaction of 12-Oxophytodienoic Acid and Related Conjugated Carbonyl Compounds with Thiol Antioxidants.

α,β-unsaturated carbonyls interfere with numerous plant physiological processes. One mechanism of action is their reactivity toward thiols of metabolites like cysteine and glutathione (GSH). This work aimed at better understanding these interactions. Both 12-oxophytodienoic acid (12-OPDA) and abscisic acid (ABA) conjugated with cysteine. It was found that the reactivity of α,β-unsaturated carbonyls with GSH followed the sequence trans-2-hexenal < 12-OPDA ≈ 12-OPDA-ethylester < 2-cyclopentenone << methyl vinylketone (MVK). Interestingly, GSH, but not ascorbate (vitamin C), supplementation ameliorated the phytotoxic potential of MVK. In addition, 12-OPDA and 12-OPDA-related conjugated carbonyl compounds interacted with proteins, e.g., with members of the thioredoxin (TRX)-fold family. 12-OPDA modified two cysteinyl residues of chloroplast TRX-f1. The OPDAylated TRX-f1 lost its activity to activate the Calvin–Benson-cycle enzyme fructose-1,6-bisphosphatase (FBPase). Finally, we show that 12-OPDA interacts with cyclophilin 20-3 (Cyp20-3) non-covalently and affects its peptidyl-prolyl-cis/trans isomerase activity. The results demonstrate the high potential of 12-OPDA as a diverse interactor and cellular regulator and suggest that OPDAylation may occur in plant cells and should be investigated as novel regulatory mechanism.