Integrative effects of phytohormones in the phenolic acids production in Salvia verticillata L. under multi-walled carbon nanotubes and methyl jasmonate elicitation.

Salvia verticillata L. is a well-known herb rich in rosmarinic acid (RA) and with therapeutic values. To better understand the possible roles of phytohormones in the production of phenolic acids in S. verticillata, in this work, we investigated some physiological and biochemical responses of the species to methyl jasmonate (MJ) and multi-walled carbon nanotubes (MWCNTs) as two effective elicitors. The leaves were sprayed with aqueous solutions containing 100 mg L-1 MWCNTs and 100 µM MJ and then harvested during interval times of exposure up to 96 h. The level of abscisic acid, as the first effective phytohormone, was altered in the leaves in response to MJ and MWCNTs elicitation (2.26- and 3.06-fold more than the control, respectively), followed by significant increases (P ˂ 0.05) detected in jasmonic acid and salicylic acid contents up to 8 h after exposure. Obtained data revealed that simultaneously with changes in phytohormone profiles, significant (P ˂ 0.05) rises were observed in the content of H2O2 (8.85- and 9.74-folds of control), and the amount of lipid peroxidation (10.18- and 17.01-folds of control) during the initial times after exposure to MJ and MWCNTs, respectively. Later, the content of phenolic acids increased in the elicited leaves due to changes in the transcription levels of key enzymes involved in their biosynthesis pathways, so 2.71- and 11.52-fold enhances observed in the RA content of the leaves after exposure to MJ and MWCNTs, respectively. It is reasonable to conclude that putative linkages between changes in some phytohormone pools lead to the accumulation of phenolic acids in the leaves of S. verticillata under elicitation. Overall, the current findings help us improve our understanding of the signal transduction pathways of the applied stimuli that led to enhanced secondary metabolite production in medicinal plants.

Knocking down a DNA demethylase gene affects potato plant defense against a specialist insect herbivore.

DNA demethylase (DML) is involved in plant development and responses to biotic and abiotic stresses; however, its role in plant-herbivore interaction remains elusive. Here, we found that herbivory by the potato tuber moth, Phthorimaea operculella, rapidly induced the genome-wide DNA methylation and accumulation of DML gene transcripts in potato plants. Herbivory induction of DML transcripts was suppressed in jasmonate-deficient plants, whereas exogenous application of methyl jasmonate (MeJA) improved DML transcripts, indicating that the induction of DML transcripts by herbivory is associated with jasmonate signaling. Moreover, P. operculella larvae grew heavier on DML gene (StDML2) knockdown plants than on wild-type plants, and the decreased biosynthesis of jasmonates in the former may be responsible for this difference, since the larvae feeding on these two genotypes supplemented with MeJA showed similar growth. In addition, P. operculella adult moths preferred to oviposit on StDML2 knockdown plants than on wild-type plants, which was associated with the reduced emission of ß-caryophyllene in the former. In addition, supplementing ß-caryophyllene to these two genotypes further disrupted moths' oviposit choice preference for them. Interestingly, in StDML2 knockdown plants, hypermethylation was found at the promoter regions for the key genes StAOS and StAOC in the jasmonate biosynthetic pathway, as well as for the key gene StTPS12 in ß-caryophyllene production. Our findings suggest that knocking down StDML2 can affect herbivore defense via jasmonate signaling and defense compound production in potato plants.

Transcription factor SlWRKY50 enhances cold tolerance in tomato by activating the jasmonic acid signaling.

Tomato (Solanum lycopersicum) is a cold-sensitive crop but frequently experiences low-temperature stimuli. However, tomato responses to cold stress are still poorly understood. Our previous studies have shown that using wild tomato (Solanum habrochaites) as rootstock can significantly enhance the cold resistance of grafted seedlings, in which a high concentration of jasmonic acids (JAs) in scions exerts an important role, but the mechanism of JA accumulation remains unclear. Herein, we discovered that tomato SlWRKY50, a Group II WRKY transcription factor that is cold inducible, responds to cold stimuli and plays a key role in JA biosynthesis. SlWRKY50 directly bound to the promoter of tomato allene oxide synthase gene (SlAOS), and overexpressing SlWRKY50 improved tomato chilling resistance, which led to higher levels of Fv/Fm, antioxidative enzymes, SlAOS expression, and JA accumulation. SlWRKY50-silenced plants, however, exhibited an opposite trend. Moreover, diethyldithiocarbamate acid (a JA biosynthesis inhibitor) foliar treatment drastically reduced the cold tolerance of SlWRKY50-overexpression plants to wild-type levels. Importantly, SlMYC2, the key regulator of the JA signaling pathway, can control SlWRKY50 expression. Overall, our research indicates that SlWRKY50 promotes cold tolerance by controlling JA biosynthesis and that JA signaling mediates SlWRKY50 expression via transcriptional activation by SlMYC2. Thus, this contributes to the genetic knowledge necessary for developing cold-resistant tomato varieties.

Crystalline silica-induced proinflammatory eicosanoid storm in novel alveolar macrophage model quelled by docosahexaenoic acid supplementation.

Introduction: Phagocytosis of inhaled crystalline silica (cSiO2) particles by tissue-resident alveolar macrophages (AMs) initiates generation of proinflammatory eicosanoids derived from the ω-6 polyunsaturated fatty acid (PUFA) arachidonic acid (ARA) that contribute to chronic inflammatory disease in the lung. While supplementation with the ω-3 PUFA docosahexaenoic acid (DHA) may influence injurious cSiO2-triggered oxylipin responses, in vitro investigation of this hypothesis in physiologically relevant AMs is challenging due to their short-lived nature and low recovery numbers from mouse lungs. To overcome these challenges, we employed fetal liver-derived alveolar-like macrophages (FLAMs), a self-renewing surrogate that is phenotypically representative of primary lung AMs, to discern how DHA influences cSiO2-induced eicosanoids. Methods: We first compared how delivery of 25 µM DHA as ethanolic suspensions or as bovine serum albumin (BSA) complexes to C57BL/6 FLAMs impacts phospholipid fatty acid content. We subsequently treated FLAMs with 25 µM ethanolic DHA or ethanol vehicle (VEH) for 24 h, with or without LPS priming for 2 h, and with or without cSiO2 for 1.5 or 4 h and then measured oxylipin production by LC-MS lipidomics targeting for 156 oxylipins. Results were further related to concurrent proinflammatory cytokine production and cell death induction. Results: DHA delivery as ethanolic suspensions or BSA complexes were similarly effective at increasing ω-3 PUFA content of phospholipids while decreasing the ω-6 PUFA arachidonic acid (ARA) and the ω-9 monounsaturated fatty acid oleic acid. cSiO2 time-dependently elicited myriad ARA-derived eicosanoids consisting of prostaglandins, leukotrienes, thromboxanes, and hydroxyeicosatetraenoic acids in unprimed and LPS-primed FLAMs. This cSiO2-induced eicosanoid storm was dramatically suppressed in DHA-supplemented FLAMs which instead produced potentially pro-resolving DHA-derived docosanoids. cSiO2 elicited marked IL-1α, IL-1ß, and TNF-α release after 1.5 and 4 h of cSiO2 exposure in LPS-primed FLAMs which was significantly inhibited by DHA. DHA did not affect cSiO2-triggered death induction in unprimed FLAMs but modestly enhanced it in LPS-primed FLAMs. Discussion: FLAMs are amenable to lipidome modulation by DHA which suppresses cSiO2-triggered production of ARA-derived eicosanoids and proinflammatory cytokines. FLAMs are a potential in vitro alternative to primary AMs for investigating interventions against early toxicant-triggered inflammation in the lung.

The regulatory roles of MYC TFs in plant stamen development.

The stamen, as the male reproductive organ of flowering plants, plays a critical role in completing the life cycle of plants. MYC transcription factors are members of the bHLH IIIE subgroup and participate in a number of plant biological processes. In recent decades, a number of studies have confirmed that MYC transcription factors actively participate in the regulation of stamen development and have a critical impact on plant fertility. In this review, we summarized how MYC transcription factors play a role in regulating secondary thickening of the anther endothecium, the development and degradation of the tapetum, stomatal differentiation, and the dehydration of the anther epidermis. With regard to anther physiological metabolism, MYC transcription factors control dehydrin synthesis, ion and water transport, and carbohydrate metabolism to influence pollen viability. Additionally, MYCs participate in the JA signal transduction pathway, where they directly or indirectly control the development of stamens through the ET-JA, GA-JA, and ABA-JA pathways. By identifying the functions of MYCs during plant stamen development, it will help us to obtain a more comprehensive understanding not only on the molecular functions of this TF family but also the mechanisms underlying stamen development.

The Role of α-Linolenic Acid and Its Oxylipins in Human Cardiovascular Diseases.

α-linolenic acid (ALA) is an essential C-18 n-3 polyunsaturated fatty acid (PUFA), which can be elongated to longer n-3 PUFAs, such as eicosapentaenoic acid (EPA). These long-chain n-3 PUFAs have anti-inflammatory and pro-resolution effects either directly or through their oxylipin metabolites. However, there is evidence that the conversion of ALA to the long-chain PUFAs is limited. On the other hand, there is evidence in humans that supplementation of ALA in the diet is associated with an improved lipid profile, a reduction in the inflammatory biomarker C-reactive protein (CRP) and a reduction in cardiovascular diseases (CVDs) and all-cause mortality. Studies investigating the cellular mechanism for these beneficial effects showed that ALA is metabolized to oxylipins through the Lipoxygenase (LOX), the Cyclooxygenase (COX) and the Cytochrome P450 (CYP450) pathways, leading to hydroperoxy-, epoxy-, mono- and dihydroxylated oxylipins. In several mouse and cell models, it has been shown that ALA and some of its oxylipins, including 9- and 13-hydroxy-octadecatrienoic acids (9-HOTrE and 13-HOTrE), have immunomodulating effects. Taken together, the current literature suggests a beneficial role for diets rich in ALA in human CVDs, however, it is not always clear whether the described effects are attributable to ALA, its oxylipins or other substances present in the supplemented diets.

Bioactive Oxylipins Profile in Marine Microalgae.

Microalgae are photosynthetic microscopic organisms that serve as the primary food source in aquatic environments. Microalgae can synthesize a wide variety of molecules, such as polyunsaturated fatty acids (PUFAs) of the omega-3 and omega-6 series. Oxidative degradation of PUFA due to radical and/or enzymatic conversion leads to the formation of oxylipins, which are compounds known for their bioactive properties. In the present study, we aim to profile oxylipins from five microalgae species grown in 10-L photo-bioreactors under optimal conditions. During their exponential phase, microalgae were harvested, extracted and analyzed by LC-MS/MS to determine the qualitative and quantitative profile of oxylipins for each species. The five different selected microalgae revealed a high diversity of metabolites, up to 33 non-enzymatic and 24 enzymatic oxylipins present in different concentrations. Taken together, these findings highlight an interesting role of marine microalgae as a source of bioactive lipids mediators, which we hypothesize have an important function in preventive health measures such as amelioration of inflammation. The rich mixture of oxylipins may display advantages to biological organisms, especially by providing for human health benefits including antioxidant, anti-inflammatory, neuroprotective or immunomodulator activities. Some oxylipins are also well known for their cardiovascular properties.

High omega-6/omega-3 fatty acid and oxylipin ratio in plasma is linked to an adverse cardiometabolic profile in middle-aged adults.

Omega-6 and omega-3 oxylipins may be surrogate markers of systemic inflammation, which is one of the triggers for the development of cardiometabolic disorders. In the current study, we investigated the relationship between plasma levels of omega-6 and omega-3 oxylipins with body composition and cardiometabolic risk factors in middle-aged adults. Seventy-two 72 middle-aged adults (39 women; 53.6±5.1 years old; 26.7±3.8 kg/m2) were included in this cross-sectional study. Plasma levels of omega-6 and omega-3 fatty acids and oxylipins were determined using targeted lipidomic. Body composition, dietary intake, and cardiometabolic risk factors were assessed with standard methods. The plasma levels of the omega-6 fatty acids and derived oxylipins, the hydroxyeicosatetraenoic acids (HETEs; arachidonic acid (AA)-derived oxylipins) and dihydroxy-eicosatrienoic acids (DiHETrEs; AA-derived oxylipins), were positively associated with glucose metabolism parameters (i.e., insulin levels and homeostatic model assessment of insulin resistance index (HOMA); all r≥0.21, P<.05). In contrast, plasma levels of omega-3 fatty acids and derived oxylipins, specifically hydroxyeicosapentaenoic acids (HEPEs; eicosapentaenoic acid-derived oxylipins), as well as series-3 prostaglandins, were negatively associated with plasma glucose metabolism parameters (i.e., insulin levels, HOMA; all r≤0.20, P<.05). The plasma levels of omega-6 fatty acids and derived oxylipins, HETEs and DiHETrEs were also positively correlated with liver function parameters (i.e., glutamic pyruvic transaminase, gamma-glutamyl transferase (GGT), and fatty liver index; all r≥0.22 and P<.05). In addition, individuals with higher omega-6/omega-3 fatty acid and oxylipin ratio showed higher levels of HOMA, total cholesterol, low-density lipoprotein-cholesterol, triglycerides, and GGT (on average +36%), as well as lower levels of high-density lipoprotein cholesterol (-13%) (all P<.05). In conclusion, the omega-6/omega-3 fatty acid and oxylipin ratio, as well as specific omega-6 and omega-3 oxylipins plasma levels, reflect an adverse cardiometabolic profile in terms of higher insulin resistance and impaired liver function in middle-aged adults.

Factors affecting variability in free oxylipins in mammalian tissues.

PURPOSE OF THE REVIEW: Along with the growing interest in oxylipins is an increasing awareness of multiple sources of variability in oxylipin data. This review summarizes recent findings that highlight the experimental and biological sources of variation in free oxylipins. RECENT FINDINGS: Experimental factors that affect oxylipin variability include different methods of euthanasia, postmortem changes, cell culture reagents, tissue processing conditions and timing, storage losses, freeze-thaw cycles, sample preparation techniques, ion suppression, matrix effects, use and availability of oxylipin standards, and postanalysis procedures. Biological factors include dietary lipids, fasting, supplemental selenium, vitamin A deficiency, dietary antioxidants and the microbiome. Overt, but also more subtle differences in health affect oxylipin levels, including during resolution of inflammation and long-term recovery from disease. Sex, genetic variation, exposure to air pollution and chemicals found in food packaging and household and personal care products, as well as many pharmaceuticals used to treat health conditions also affect oxylipin levels. SUMMARY: Experimental sources of oxylipin variability can be minimized with proper analytical procedures and protocol standardization. Fully characterizing study parameters will help delineate biological factors of variability, which are rich sources of information that can be used to probe oxylipin mechanisms of action and to investigate their roles in health.

Dissecting the biochemical and molecular-genetic regulation of diverse metabolic pathways governing aroma formation in indigenous aromatic indica rice varieties.

BACKGROUND: Aromatic rice is characterized by its distinct flavor and fragrance, imparted by more than 200 volatile organic compounds. The desirable trait of aroma relies on the type of the variety, with some varieties exhibiting considerably higher aroma content. This prompted us to undergo an exhaustive study of the aroma-associated biochemical pathways and expression of related genes, encoding the enzymes involved in those pathways in indigenous aromatic rice cultivars. METHODS AND RESULTS: The higher aroma level in aromatic rice varieties was attributed to higher transcript levels of PDH, P5CS, OAT, ODC, DAO and TPI, but lower P5CDH and BADH2, as revealed by comparative expression profiling of genes in 11 aromatic and four non-aromatic varieties. Some of the high-aroma containing varieties exhibited lower expression of SPDS and SPMS genes, concomitant with higher PAO expression. Protein immunoblot analyses showed lesser BADH2 protein accumulation in the aromatic varieties. The involvement of shikimate pathway in aroma formation was justified by higher levels of shikimic and ferulic acids due to higher expression of SK1, SK2 and ARD genes. The aromatic varieties exhibited higher expression of LOX3 and HPL, with higher corresponding enzyme activity, accompanied with lower accumulation of lipid hydroperoxides and higher level of total terpenoids, signifying the role of oxylipin pathway and terpene-related volatiles in aroma formation. The pattern of transcript level and metabolite accumulation followed the same trend in both vegetative (seedling) and reproductive (seed) tissues. Sequence analyses revealed several mutations in the upstream region and different exons and introns of BADH2 in the examined aromatic varieties. The allele specific marker system acted as fingerprint to distinguish the selected aromatic rice varieties. The cleaved amplified polymorphic sequence marker established the absence of any mutation in the 14th exon of BADH2 in the aromatic varieties. CONCLUSION: The present work clearly highlighted the biochemical and molecular-genetic mechanism of differential aroma levels which could be attributed to differential regulation of metabolites and genes, belonging to 2-acetyl-1-pyrroline, shikimate, oxylipin and terpenoid metabolic pathways in the indigenous aromatic rice varieties.

Human CYP2B6 produces oxylipins from polyunsaturated fatty acids and reduces diet-induced obesity.

Multiple factors in addition to over consumption lead to obesity and non-alcoholic fatty liver disease (NAFLD) in the United States and worldwide. CYP2B6 is the only human detoxification CYP whose loss is associated with obesity, and Cyp2b-null mice show greater diet-induced obesity with increased steatosis than wildtype mice. However, a putative mechanism has not been determined. LC-MS/MS revealed that CYP2B6 metabolizes PUFAs, with a preference for metabolism of ALA to 9-HOTrE and to a lesser extent 13-HOTrE with a preference for metabolism of PUFAs at the 9- and 13-positions. To further study the role of CYP2B6 in vivo, humanized-CYP2B6-transgenic (hCYP2B6-Tg) and Cyp2b-null mice were fed a 60% high-fat diet for 16 weeks. Compared to Cyp2b-null mice, hCYP2B6-Tg mice showed reduced weight gain and metabolic disease as measured by glucose tolerance tests, however hCYP2B6-Tg male mice showed increased liver triglycerides. Serum and liver oxylipin metabolite concentrations increased in male hCYP2B6-Tg mice, while only serum oxylipins increased in female hCYP2B6-Tg mice with the greatest increases in LA oxylipins metabolized at the 9 and 13-positions. Several of these oxylipins, specifically 9-HODE, 9-HOTrE, and 13-oxoODE, are PPAR agonists. RNA-seq data also demonstrated sexually dimorphic changes in gene expression related to nuclear receptor signaling, especially CAR > PPAR with qPCR suggesting PPARγ signaling is more likely than PPARα signaling in male mice. Overall, our data indicates that CYP2B6 is an anti-obesity enzyme, but probably to a lesser extent than murine Cyp2b's. Therefore, the inhibition of CYP2B6 by xenobiotics or dietary fats can exacerbate obesity and metabolic disease potentially through disrupted PUFA metabolism and the production of key lipid metabolites.

Comparative Proteomics of Potato Cultivars with a Variable Dormancy Period.

The control of the duration of the dormancy phase is a significant challenge in the potato industry and for seed producers. However, the proteome landscape involved in the regulation of the length of the dormancy period over potato cultivars remains largely unexplored. In this study, we performed for the first time a comparative proteome profiling of potato cultivars with differential duration of tuber dormancy. More specifically, the proteome profiling of Agata, Kennebec and Agria commercial potato varieties with short, medium and medium-long dormancy, respectively, was assessed at the endodormancy stage using high-resolution two-dimensional electrophoresis (2-DE) coupled to reversed-phase liquid chromatography-tandem mass spectrometry (LC-TripleTOF MS/MS). A total of 11 proteins/isoforms with statistically significant differential abundance among cultivars were detected on 2-DE gels and confidently identified by LC-TripleTOF MS/MS. Identified proteins have known functions related to tuber development, sprouting and the oxylipins biosynthesis pathway. Fructokinase, a mitochondrial ADP/ATP carrier, catalase isozyme 2 and heat shock 70 kDa were the proteins with the strongest response to dormancy variations. To the best of our knowledge, this study reports the first candidate proteins underlying variable dormancy length in potato cultivars.

Obesity reprograms the pulmonary polyunsaturated fatty acid-derived lipidome, transcriptome, and gene-oxylipin networks.

Obesity exacerbates inflammation upon lung injury; however, the mechanisms by which obesity primes pulmonary dysregulation prior to external injury are not well studied. Herein, we tested the hypothesis that obesity dysregulates pulmonary PUFA metabolism that is central to inflammation initiation and resolution. We first show that a high-fat diet (HFD) administered to C57BL/6J mice increased the relative abundance of pulmonary PUFA-containing triglycerides and the concentration of PUFA-derived oxylipins (particularly prostaglandins and hydroxyeicosatetraenoic acids), independent of an increase in total pulmonary PUFAs, prior to onset of pulmonary inflammation. Experiments with a genetic model of obesity (ob/ob) generally recapitulated the effects of the HFD on the pulmonary oxylipin signature. Subsequent pulmonary next-generation RNA sequencing identified complex and unique transcriptional regulation with the HFD. We found the HFD increased pathways related to glycerophospholipid metabolism and immunity, including a unique elevation in B cell differentiation and signaling. Furthermore, we conducted computational integration of lipidomic with transcriptomic data. These analyses identified novel HFD-driven networks between glycerophospholipid metabolism and B cell receptor signaling with specific PUFA-derived pulmonary oxylipins. Finally, we confirmed the hypothesis by demonstrating that the concentration of pulmonary oxylipins, in addition to inflammatory markers, were generally increased in mice consuming a HFD upon ozone-induced acute lung injury. Collectively, these data show that a HFD dysregulates pulmonary PUFA metabolism prior to external lung injury, which may be a mechanism by which obesity primes the lungs to respond poorly to infectious and/or inflammatory challenges.

Identification and Characterization of Jasmonic Acid Biosynthetic Genes in Salvia miltiorrhiza Bunge.

Jasmonic acid (JA) is a vital plant hormone that performs a variety of critical functions for plants. Salvia miltiorrhiza Bunge (S. miltiorrhiza), also known as Danshen, is a renowned traditional Chinese medicinal herb. However, no thorough and systematic analysis of JA biosynthesis genes in S. miltiorrhiza exists. Through genome-wide prediction and molecular cloning, 23 candidate genes related to JA biosynthesis were identified in S. miltiorrhiza. These genes belong to four families that encode lipoxygenase (LOX), allene oxide synthase (AOS), allene oxide cyclase (AOC), and 12-OPDA reductase3 (OPR3). It was discovered that the candidate genes for JA synthesis of S. miltiorrhiza were distinct and conserved, in contrast to related genes in other plants, by evaluating their genetic structures, protein characteristics, and phylogenetic trees. These genes displayed tissue-specific expression patterns concerning to methyl jasmonate (MeJA) and wound tests. Overall, the results of this study provide valuable information for elucidating the JA biosynthesis pathway in S. miltiorrhiza by comprehensive and methodical examination.

Dynamic calcium signals mediate the feeding response of the carnivorous sundew plant.

Some of the most spectacular examples of botanical carnivory-in which predator plants catch and digest animals presumably to supplement the nutrient-poor soils in which they grow-occur within the Droseraceae family. For example, sundews of the genus Drosera have evolved leaf movements and enzyme secretion to facilitate prey digestion. The molecular underpinnings of this behavior remain largely unknown; however, evidence suggests that prey-induced electrical impulses are correlated with movement and production of the defense hormone jasmonic acid (JA), which may alter gene expression. In noncarnivorous plants, JA is linked to electrical activity via changes in cytoplasmic Ca2+. Here, we find that dynamic Ca2+ changes also occur in sundew (Drosera spatulata) leaves responding to prey-associated mechanical and chemical stimuli. Furthermore, inhibition of these Ca2+ changes reduced expression of JA target genes and leaf movements following chemical feeding. Our results are consistent with the presence of a conserved Ca2+-dependent JA signaling pathway in the sundew feeding response and provide further credence to the defensive origin of plant carnivory.

Variation on a theme: the structures and biosynthesis of specialized fatty acid natural products in plants.

Plants are able to construct lineage-specific natural products from a wide array of their core metabolic pathways. Considerable progress has been made toward documenting and understanding, for example, phenylpropanoid natural products derived from phosphoenolpyruvate via the shikimate pathway, terpenoid compounds built using isopentyl pyrophosphate, and alkaloids generated by the extensive modification of amino acids. By comparison, natural products derived from fatty acids have received little attention, except for unusual fatty acids in seed oils and jasmonate-like oxylipins. However, scattered but numerous reports show that plants are able to generate many structurally diverse compounds from fatty acids, including some with highly elaborate and unique structural features that have novel bioproduct functionalities. Furthermore, although recent work has shed light on multiple new fatty acid natural product biosynthesis pathways and products in diverse plant species, these discoveries have not been reviewed. The aims of this work, therefore, are to (i) review and systematize our current knowledge of the structures and biosynthesis of fatty acid-derived natural products that are not seed oils or jasmonate-type oxylipins, specifically, polyacetylenic, very-long-chain, and aromatic fatty acid-derived natural products, and (ii) suggest priorities for future investigative steps that will bring our knowledge of fatty acid-derived natural products closer to the levels of knowledge that we have attained for other phytochemical classes.

Omega-6 Polyunsaturated Fatty Acids Enhance Tumor Aggressiveness in Experimental Lung Cancer Model: Important Role of Oxylipins.

Lung cancer is currently the leading cause of cancer death worldwide; it is often diagnosed at an advanced stage and bears poor prognosis. It has been shown that diet is an important environmental factor that contributes to the risk and mortality of several types of cancers. Intake of ω-3 and ω-6 PUFAs plays an important role in cancer risk and progression. Current Western populations have high consumption of ω-6 PUFAs with a ratio of ω-6/ω-3 PUFAs at 15:1 to 16.7:1 This high consumption of ω-6 PUFAs is related to increased cancer risk and progression. However, whether a diet rich in ω-6 PUFAs can contribute to tumor aggressiveness has not been well investigated. We used a murine model of pulmonary squamous cell carcinoma to study the aggressiveness of tumors in mice fed with a diet rich in ω-6 PUFAs and its relationship with oxylipins. Our results shown that the mice fed a diet rich in ω-6 showed a marked increase in proliferation, angiogenesis and pro-inflammatory markers and decreased expression of pro-apoptotic proteins in their tumors. Oxylipin profiling revealed an upregulation of various pro-tumoral oxylipins including PGs, HETEs, DiHETrEs and HODEs. These results demonstrate for the first time that high intake of ω-6 PUFAs in the diet enhances the malignancy of tumor cells by histological changes on tumor dedifferentiation and increases cell proliferation, angiogenesis, pro-inflammatory oxylipins and molecular aggressiveness targets such as NF-κB p65, YY1, COX-2 and TGF-ß.

Developing a platform for production of the oxylipin KODA in plants.

KODA (9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid) is a plant oxylipin involved in recovery from stress. As an agrichemical, KODA helps maintain crop production under various environmental stresses. In plants, KODA is synthesized from α-linolenic acids via 9-lipoxygenase (9-LOX) and allene oxide synthase (AOS), although the amount is usually low, except in the free-floating aquatic plant Lemna paucicostata. To improve KODA biosynthetic yield in other plants such as Nicotiana benthamiana and Arabidopsis thaliana, we developed a system to overproduce KODA in vivo via ectopic expression of L. paucicostata 9-LOX and AOS. The transient expression in N. benthamiana showed that the expression of these two genes is sufficient to produce KODA in leaves. However, stable expression of 9-LOX and AOS (with consequent KODA production) in Arabidopsis plants succeeded only when the two proteins were targeted to plastids or the endoplasmic reticulum/lipid droplets. Although only small amounts of KODA could be detected in crude leaf extracts of transgenic Nicotiana or Arabidopsis plants, subsequent incubation of the extracts increased KODA abundance over time. Therefore, KODA production in transgenic plants stably expressing 9-LOX and AOS requires specific sub-cellular localization of these two enzymes and incubation of crude leaf extracts, which liberates α-linolenic acid via breakdown of endogenous lipids.

AUXIN RESPONSE FACTOR 16 (StARF16) regulates defense gene StNPR1 upon infection with necrotrophic pathogen in potato.

KEY MESSAGE: We demonstrate a new regulatory mechanism in the jasmonic acid (JA) and salicylic acid (SA) mediated crosstalk in potato defense response, wherein, miR160 target StARF16 (a gene involved in growth and development) binds to the promoter of StNPR1 (a defense gene) and negatively regulates its expression to suppress the SA pathway. Overall, our study establishes the importance of StARF16 in regulation of StNPR1 during JA mediated defense response upon necrotrophic pathogen interaction. Plants employ antagonistic crosstalk between salicylic acid (SA) and jasmonic acid (JA) to effectively defend them from pathogens. During biotrophic pathogen attack, SA pathway activates and suppresses the JA pathway via NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1). However, upon necrotrophic pathogen attack, how JA-mediated defense response suppresses the SA pathway, is still not well-understood. Recently StARF10 (AUXIN RESPONSE FACTOR), a miR160 target, has been shown to regulate SA and binds to the promoter of StGH3.6 (GRETCHEN HAGEN3), a gene proposed to maintain the balance between the free SA and auxin in plants. In the current study, we investigated the role of StARF16 (a miR160 target) in the regulation of the defense gene StNPR1 in potato upon activation of the JA pathway. We observed that a negative correlation exists between StNPR1 and StARF16 upon infection with the pathogen. The results were further confirmed through the exogenous application of SA and JA. Using yeast one-hybrid assay, we demonstrated that StARF16 binds to the StNPR1 promoter through putative ARF binding sites. Additionally, through protoplast transfection and chromatin immunoprecipitation experiments, we showed that StARF16 could bind to the StNPR1 promoter and regulate its expression. Co-transfection assays using promoter deletion constructs established that ARF binding sites are present in the 2.6 kb sequence upstream to the StNPR1 gene and play a key role in its regulation during infection. In summary, we demonstrate the importance of StARF16 in the regulation of StNPR1, and thus SA pathway, during JA-mediated defense response upon necrotrophic pathogen interaction.

Effect of Drought and Methyl Jasmonate Treatment on Primary and Secondary Isoprenoid Metabolites Derived from the MEP Pathway in the White Spruce Picea glauca.

White spruce (Picea glauca) emits monoterpenes that function as defensive signals and weapons after herbivore attack. We assessed the effects of drought and methyl jasmonate (MeJA) treatment, used as a proxy for herbivory, on monoterpenes and other isoprenoids in P. glauca. The emission of monoterpenes was significantly increased after MeJA treatment compared to the control, but drought suppressed the MeJA-induced increase. The composition of the emitted blend was altered strongly by stress, with drought increasing the proportion of oxygenated compounds and MeJA increasing the proportion of induced compounds such as linalool and (E)-ß-ocimene. In contrast, no treatment had any significant effect on the levels of stored monoterpenes and diterpenes. Among other MEP pathway-derived isoprenoids, MeJA treatment decreased chlorophyll levels by 40%, but had no effect on carotenoids, while drought stress had no impact on either of these pigment classes. Of the three described spruce genes encoding 1-deoxy-D-xylulose-5-phosphate synthase (DXS) catalyzing the first step of the MEP pathway, the expression of only one, DXS2B, was affected by our treatments, being increased by MeJA and decreased by drought. These findings show the sensitivity of monoterpene emission to biotic and abiotic stress regimes, and the mediation of the response by DXS genes.