Fidelity in plant hormone modifications catalyzed by Arabidopsis GH3 acyl acid amido synthetases.

GRETCHEN HAGEN 3 (GH3) acyl acid amido synthetases conjugate amino acids to acyl acid hormones to either activate or inactivate the hormone molecule. The largest subgroup of GH3 proteins modify the growth-promoting hormone auxin (indole-3-acetic acid; IAA) with the second largest class activating the defense hormone jasmonic acid (JA). The two-step reaction mechanism of GH3 proteins provides a potential proofreading mechanism to ensure fidelity of hormone modification. Examining pyrophosphate release in the first-half reaction of Arabidopsis GH3 proteins that modify IAA (AtGH3.2/YDK2, AtGH3.5/WES1, AtGH3.17/VAS2), JA (AtGH3.11/JAR1), and other acyl acids (AtGH3.7, AtGH3.12/PBS3) indicates that acyl acid-AMP intermediates are hydrolyzed into acyl acid and AMP in the absence of the amino acid, a typical feature of pre-transfer editing mechanisms. Single-turnover kinetic analysis of AtGH3.2/YDK2 and AtGH3.5/WES1 shows that non-cognate acyl acid-adenylate intermediates are more rapidly hydrolyzed than the cognate IAA-adenylate. In contrast, AtGH3.11/JAR1 only adenylates JA, not IAA. While some of the auxin-conjugating GH3 proteins in Arabidopsis (i.e., AtGH3.5/WES1) accept multiple acyl acid substrates, others, like AtGH3.2/YDK2, are specific for IAA; however, both these proteins share similar active site residues. Biochemical analysis of chimeric variants of AtGH3.2/YDK2 and AtGH3.5/WES1 indicates that the C-terminal domain contributes to selection of cognate acyl acid substrates. These findings suggest that the hydrolysis of non-cognate acyl acid-adenylate intermediates, or proofreading, proceeds via a slowed structural switch that provides a checkpoint for fidelity before the full reaction proceeds.

Structure-activity relationship study of nitrogen signaling factors.

We have synthesized 10 analogs of oxylipins, which are nitrogen signaling factors (NSFs) that mediate cell-to-cell communication in the fission yeast Schizosaccharomyces pombe, and evaluated their structure-activity relationships with the aim of developing molecular probes for NSFs. We found that the OH or OAc group at C10 could be replaced with a compact amide (17) or carbamate (19). Introducing an alkyne as a detection tag at C10 led to decreased, though still sufficient, activity. Introducing an alkyne at the C18 position showed a similar trend, suggesting tolerance is relatively low even for compact functional groups such as alkynes. Although introduction of a diazirine moiety as a photoreactive group at the C5 position decreased the activity, we found that introducing diazirine at the C13 position was acceptable, and compound 38 exhibited potent NSF activity. These findings will be helpful in the development of molecular probes for NSFs.

Isolation and Structure Determination of cis-OPDA-α-Monoglyceride from Arabidopsis thaliana.

cis-12-oxo-Phytodieneoic acid-α-monoglyceride (1) was isolated from Arabidopsis thaliana. The chemical structure of 1 was elucidated based on exhaustive 1D and 2D NMR spectroscopic measurements and supported by FDMS and HRFDMS data. The absolute configuration of the cis-OPDA moiety in 1 was determined by comparison of 1H NMR spectra and ECD measurements. With respect to the absolute configuration of the ß-position of the glycerol backbone, the 2:3 ratio of (S) to (R) was determined by making ester-bonded derivatives with (R)-(+)-α-methoxy-α-trifluoromethylphenylacetyl chloride and comparing 1H NMR spectra. Wounding stress did not increase endogenous levels of 1, and it was revealed 1 had an inhibitory effect of A. thaliana post germination growth. Notably, the endogenous amount of 1 was higher than the amounts of (+)-7-iso-jasmonic acid and (+)-cis-OPDA in intact plants. 1 also showed antimicrobial activity against Gram-positive bacteria, but jasmonic acid did not. It was also found that α-linolenic acid-α-monoglyceride was converted into 1 in the A. thaliana plant, which implied α-linolenic acid-α-monoglyceride was a biosynthetic intermediate of 1.

Simulated Herbivory Affects the Volatile Emissions of Oak Saplings, while Neighbourhood Affects Flavan-3-ols Content of Their Leaves.

To what extent particular plant defences against herbivorous insects are constitutive or inducible will depend on the costs and benefits in their neighbourhood. Some defensive chemicals in leaves are thought to be costly and hard to produce rapidly, while others, including volatile organic compounds that attract natural enemies, might be cheaper and can be released rapidly. When surrounding tree species are more closely related, trees can face an increased abundance of both specialist herbivores and their parasitoids, potentially increasing the benefits of constitutive and inducible defences. To test if oaks (Quercus robur) respond more to herbivore attacks with volatile emission than with changes in leaf phenolic chemistry and carbon to nitrogen ratio (C: N), and whether oaks respond to the neighbouring tree species, we performed an experiment in a forest in Poland. Oak saplings were placed in neighbourhoods dominated by oak, beech, or pine trees, and half of them were treated with the phytohormone methyl jasmonate (elicitor of anti-herbivore responses). Oaks responded to the treatment by emitting a different volatile blend within 24 h, while leaf phenolic chemistry and C: N remained largely unaffected after 16 days and multiple treatments. Leaf phenolics were subtly affected by the neighbouring trees with elevated flavan-3-ols concentrations in pine-dominated plots. Our results suggest that these oaks rely on phenols as a constitutive defence and when attacked emit volatiles to attract natural enemies. Further studies might determine if the small effect of the neighbourhood on leaf phenolics is a response to different levels of shading, or if oaks use volatile cues to assess the composition of their neighbourhood.

Identification of "modified OPDA (mo-OPDA)" as a Michael adduct of cis-OPDA.

cis-(+)-12-Oxo-phytodienoic acid (cis-OPDA) is a significant plant oxylipin, known as a biosynthetic precursor of the plant hormone jasmonoyl-l-isoleucine (JA-Ile), and a bioactive substance in plant environmental stresses. A recent study showed that a plant dioxygenase, Jasmonate Induced Dioxygenase 1 (JID1), converts cis-OPDA into an unidentified metabolite termed "modified-OPDA (mo-OPDA)" in Arabidopsis thaliana. Here, using ultra-performance liquid chromatography coupled with triple quad mass spectrometry (UPLC-MS/MS) experiment, the chemical identity of "mo-OPDA" was demonstrated and identified as a conjugate between cis-OPDA and 2-mercaptoethanol (cis-OPDA-2ME), an artifact produced by Michael addition during the JID1 digestion of cis-OPDA. However, previous reports demonstrated a decreased accumulation of cis-OPDA in the JID1-OE line, suggesting the existence of an unknown JID1-mediated mechanism regulating the level of cis-OPDA in A. thaliana.

Oxylipin profile of human milk and human milk-derived extracellular vesicles.

BACKGROUND: Small Extracellular Vesicles (sEVs) are nano-sized vesicles that are present in all biofluids including human milk (HM) playing a crucial role in cell-to-cell communication and the stimulation of the neonatal immune system. Oxylipins, which are bioactive lipids formed from polyunsaturated fatty acids, have gained considerable attention due to their potential role in mitigating disease progression and modulating the inflammatory status of breastfed infants. This study aims at an in-depth characterization of the oxylipin profiles of HM and, for the first time, of HM-derived sEVs (HMEVs) employing an ad-hoc developed and validated ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method. RESULTS: The UPLC-MS/MS method covered a panel of 13 oxylipins for quantitation and 93 oxylipins for semi-quantitation. In 200 µL of HM and HMEV isolates of 15 individuals, 42 out of 106 oxylipins were detected in either HM or HMEVs, with 38 oxylipins being detected in both matrices. Oxylipins presented distinct profiles in HM and HMEVs, suggesting specific mechanisms responsible for the encapsulation of target molecules in HMEVs. Ten and eight oxylipins were quantified with ranges between 0.03 - 73 nM and 0.30 pM-0.07 nM in HM and HMEVs, respectively. The most abundant oxylipins found in HMEVs were docosahexaenoic acid derivatives (17-HDHA and 14-HDHA) with known anti-inflammatory properties, and linoleic acid derivatives (9-10-DiHOME and 12,13-DiHOME) in HM samples. SIGNIFICANCE AND NOVELTY: This is the first time a selective, relative enrichment of anti-inflammatory oxylipins in HMEVs has been described. Future studies will focus on the anti-inflammatory and pro-healing capacity of oxylipins encapsulated in HMEVs, with potential clinical applications in the field of preterm infant care, specifically the prevention of severe intestinal complications including necrotizing enterocolitis.

Oxylipin biosynthesis via an unprecedented 16-hydroperoxide lyase pathway in green tissues of cucumber (Cucumis sativus L.) plants.

The plant lipoxygenase cascade is a source of various regulatory oxylipins that play a role in cell signalling, stress adaptation, and immune response. Recently, we detected an unprecedented 16(S)-lipoxygenase, CsLOX3, in the leaves and fruit pericarp of cucumber (Cucumis sativus L.). In the present work, an array of products biosynthesized through the conversions of α-linolenic acid 16-hydroperoxide (16-HPOT) was detected. Firstly, a prominent 15-hydroxy-9,12-pentadecadienoic acid (Me/TMS) was detected, the product of hydroperoxide lyase (HPL) chain cleavage of 16-HPOT and further reduction of aldehyde 15-oxo-9,12-pentadecadienoic acid to alcohol. Besides, the presence of dicarboxylic acid, 3,6-pentadecadiene-1,15-dioic acid, was deduced from the detection of its catalytic hydrogenation product, pentadecane-1,15-dioic acid. Finally, 12,15-dihydroxypentadecanoic acid (Me/TMS) was detected amongst the hydrogenated products, thus indicating the presence of the parent 12,15-dihydroxy-9,13-pentadecadienoic acid. To confirm the proposed HPL chain cleavage, the 16(S)-HPOT was prepared and incubated with the recombinant cucumber HPL CYP74B6 enzyme. The CYP74B6 possessed high activity towards 16-HPOT. Chain cleavage yields the (9Z,12Z)-15-oxo-9,12-pentadecadienoic acid, undergoing a spontaneous isomerization into (9Z,13E)-15-oxo-9,13-pentadecadienoic acid. Thus, the cucumber plants as well as the recombinant cucumber HPL CYP74B6 possessed unprecedented 16-HPL activity, cleaving 16-HPOT into a C15 fragment, 15-oxo-9,12-pentadecadienoic acid, and a complementary volatile C3 fragment, propionic aldehyde. The 16-LOX/16-HPL route of oxylipin biosynthesis presents a novel facet of the plant LOX pathway.

Sensing nature's alarm: SnO2/MXene gas sensor unveils methyl jasmonate signatures of plant insect stress.

The incorporation of artificial intelligence into agriculture presents challenges, particularly due to hardware limitations, especially in sensors. Currently, pest detection relies heavily on manual scouting by humans. Therefore, the objective of this study is to create a chemoresistive sensor that enables early identification of the characteristic volatile compound, viz., methyl jasmonate, released during pest infestations. Given the lower reactivity of esters, we have fine-tuned a composite consisting of SnO2 nanoparticles and 2D-MXene sheets to enhance adsorption and selective oxidation, resulting in heightened sensitivity. The optimized composite demonstrated a notable response even at concentrations as low as 120 ppb, successfully confirming pest infestations in tomato crops.

The versatile CYP74 clan enzyme CYP440A19 from the European lancelet Branchiostoma lanceolatum biosynthesizes novel macrolactone, epoxydiene, and related oxylipins.

The present work reports the detection and cloning of a new CYP74 clan gene of the European lancelet (Branchiostoma lanceolatum) and the biochemical characterization of the recombinant protein CYP440A19. CYP440A19 possessed epoxyalcohol synthase (EAS) activity towards the 13-hydroperoxides of linoleic and α-linolenic acids, which were converted into oxiranylcarbinols, i.e., (11S,12R,13S)-11-hydroxy-12,13-epoxy derivatives. The conversion of 9-hydroperoxides produced distinct products. Linoleic acid 9(S)-hydroperoxide (9-HPOD) was mainly converted into 9,14-diol (10E,12E)-9,14-dihydroxy-10,12-octadecadienoic acid and macrolactone 9(S),10(R)-epoxy-11(E)-octadecen-13(S)-olide. In addition, (8Z)-colneleic acid was formed. Brief incubations of the enzyme with 9-HPOD in a biphasic system of hexane-water enabled the isolation of the short-lived 9,10-epoxydiene (9S,10R,11E,13E)-9,10-epoxy-11,13-octadecadienoic acid. The structure and stereochemistry of the epoxyalcohols, macrolactone, (8Z)-colneleic acid (Me), and 9,10-epoxydiene (Me) were confirmed by 1H-NMR, 1H-1H-COSY, 1H-13C-HSQC, and 1H-13C-HMBC spectroscopy. Macrolactone and cis-9,10-epoxydiene are novel products. The 9-hydroperoxide of α-linolenic acid was mainly converted into macrolactone 9(S),10(R)-epoxy-11(E),15(Z)-octadecadiene-13(S)-olide and a minority of divinyl ethers, particularly (8Z)-colnelenic acid. The versatility of enzyme catalysis, as well as the diversity of CYP74s and other enzymes involved in oxylipin biosynthesis, demonstrates the complexity of the lipoxygenase pathway in lancelets.

Non-enantioselective, enantioselective, and two-dimensional liquid chromatography coupled with tandem mass spectrometry for the study of stereochemical disposition of oxylipins in cGMP-regulated hemin-treated platelets.

Oxylipins are important low abundant signaling molecules in living organisms. In platelets they play a primary role in platelet activation and aggregation in the course of thrombotic events. In vivo, they are enzymatically synthesized by cyclooxygenases, lipoxygenases, or cytochrome P450 isoenzmes, resulting in diverse polyunsaturated fatty acid (FA) metabolites including hydroxy-, epoxy-, oxo-FAs, and endoperoxides with pro-thrombotic or anti-thrombotic effects. In a recent study, it was reported that hemin induces platelet death which was accompanied by enhanced reactive oxygen species (ROS) production (measured by flow cytometry) and lipid peroxidation (as determined by proxy using flow cytometry with BODIPY-C11 as sensor). Lipidomic studies further indicated significant changes of the platelet lipidome upon ex vivo hemin treatment, amongst others oxylipins were increased. The effect could be (at least partly) reversed by riociguat/diethylamine NONOate diethylammonium salt (DEA/NO) which modulates the soluble guanylate cyclase(sGC)-cGMP-cGMP-dependent protein kinase I(cGKI) signaling axis. In the original work, oxylipins were measured by a non-enantioselective UHPLC-tandem-MS assay which may not give the full picture whether oxylipin elevation is due to ROS or by enzymatic processes. We present here the study of the stereochemical disposition of hemin-induced platelet lipidome alterations using Chiralpak IA-U column with amylose tris(3,5-dimethylphenylcarbamate) chiral selector immobilized on 1.6 µm silica particles. It was found that the major platelet oxylipins 12-HETE, 12-HEPE and 14-HDoHE (from 12-LOX) and 12-HHT (from COX-1) were present in S-configuration indicating their enzymatic formation. On the other hand, both R and S enantiomers of 9- and 13-HODE, 11- and 15-HETE were detected, possibly due to enzyme promiscuity rather than non-specific oxidation (by ROS or autoxidation), as confirmed by multi-loop based two-dimensional LC-MS using selective comprehensive mode with achiral RPLC in the 1st dimension and chiral LC in the 2nd using a multiple heart-cutting interface. For 12-HETrE, a peak at the retention time of the R-enantiomer was ruled out as isobaric interference by 2D-LC-MS. In particular, arachidonic acid derivates 12(S)-HHT, 11(R)-HETE and 15(S)-HETE were found to be sensitive to hemin and cGMP modulation.

Anti-inflammatory discovery of sesquiterpenoids and a jasmonic acid derivative from Artemisia stolonifera.

Eleven previously undescribed sesquiterpenoids (8-18), one undescribed jasmonic acid derivative (35) and 28 known compounds were isolated from the leaves of Artemisia stolonifera. Undescribed compounds with their absolute configurations were determined by extensive spectroscopic analysis, single-crystal X-ray diffraction and ECD calculation. Compound 8 was identified as a rare sesquiterpenoid featuring a rearranged 5/8 bicyclic ring system, whereas compound 17 was found to be an unprecedented monocyclic sesquiterpenoid with methyl rearrangement. Evaluation of biological activity showed that compounds 1-5 and 7 displayed cytotoxicity against six tumor cells. In the meantime, compounds 11, 12, 18 and 35 exhibited inhibitory effects against LPS-stimulated NO production in RAW 264.7 macrophage cells and reduced the transcription of IL-6 and IL-1ß in a dose-dependent manner at 25, 50 and 100 µM. Moreover, the anti-inflammatory-based network pharmacology and molecular docking analyses revealed potential target proteins of 11, 12, 18 and 35.

An oriental melon 9-lipoxygenase gene CmLOX09 response to stresses, hormones, and signal substances / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

In plants, lipoxygenases (LOXs) play a crucial role in biotic and abiotic stresses. In our previous study, five 13-LOX genes of oriental melon were regulated by abiotic stress but it is unclear whether the 9-LOX is involved in biotic and abiotic stresses. The promoter analysis revealed that CmLOX09 (type of 9-LOX) has hormone elements, signal substances, and stress elements. We analyzed the expression of CmLOX09 and its downstream genes-CmHPL and CmAOS-in the leaves of four-leaf stage seedlings of the oriental melon cultivar "Yumeiren" under wound, hormone, and signal substances. CmLOX09, CmHPL, and CmAOS were all induced by wounding. CmLOX09 was induced by auxin (indole acetic acid, IAA) and gibberellins (GA3); however, CmHPL and CmAOS showed differential responses to IAA and GA3. CmLOX09, CmHPL, and CmAOS were all induced by hydrogen peroxide (H2O2) and methyl jasmonate (MeJA), while being inhibited by abscisic acid (ABA) and salicylic acid (SA). CmLOX09, CmHPL, and CmAOS were all induced by the powdery mildew pathogen Podosphaera xanthii. The content of 2-hexynol and 2-hexenal in leaves after MeJA treatment was significantly higher than that in the control. After infection with P. xanthii, the diseased leaves of the oriental melon were divided into four levels-levels 1, 2, 3, and 4. The content of jasmonic acid (JA) in the leaves of levels 1 and 3 was significantly higher than that in the level 0 leaves. In summary, the results suggested that CmLOX09 might play a positive role in the response to MeJA through the hydroperoxide lyase (HPL) pathway to produce C6 alcohols and aldehydes, and in the response to P. xanthii through the allene oxide synthase (AOS) pathway to form JA.