Euphorbia Lagascae Research Articles

Characterization of SCP‐2 from Euphorbia lagascae reveals that a single Leu/Met exchange enhances sterol transfer activity

Sterol carrier protein-2 (SCP-2) is a small intracellular basic protein domain implicated in peroxisomal beta-oxidation. We extend our knowledge of plant SCP-2 by characterizing SCP-2 from Euphorbia lagascae. This protein consists of 122 amino acids including a PTS1 peroxisomal targeting signal. It has a molecular mass of 13.6 kDa and a pI of 9.5. It shares 67% identity and 84% similarity with SCP-2 from Arabidopsis thaliana. Proteomic analysis revealed that E. lagascae SCP-2 accumulates in the endosperm during seed germination. It showed in vitro transfer activity of BODIPY-phosphatidylcholine (BODIPY-PC). The transfer of BODIPY-PC was almost completely inhibited after addition of phosphatidylinositol, palmitic acid, stearoyl-CoA and vernolic acid, whereas sterols only had a very marginal inhibitory effect. We used protein modelling and site-directed mutagenesis to investigate why the BODIPY-PC transfer mediated by E. lagascae SCP-2 is not sensitive to sterols, whereas the transfer mediated by A. thaliana SCP-2 shows sterol sensitivity. Protein modelling suggested that the ligand-binding cavity of A. thaliana SCP-2 has four methionines (Met12, 14, 15 and 100), which are replaced by leucines (Leu11, 13, 14 and 99) in E. lagascae SCP-2. Changing Leu99 to Met99 was sufficient to convert E. lagascae SCP-2 into a sterol-sensitive BODIPY-PC-transfer protein, and correspondingly, changing Met100 to Leu100 abolished the sterol sensitivity of A. thaliana SCP-2.

Apoptosis induction and modulation of P-glycoprotein mediated multidrug resistance by new macrocyclic lathyrane-type diterpenoids

The macrocyclic lathyrane diterpenes, latilagascenes D–F ( 1– 3) and jolkinol B ( 4), were isolated from the methanol extract of Euphorbia lagascae, and evaluated for multidrug resistance reversing activity on mouse lymphoma cells. All compounds displayed very strong activity compared with that of the positive control, verapamil. The structure–activity relationship is discussed. The evaluation of compounds 1 and 4, and of latigascenes A-C ( 5– 7), isolated from the same species, as apoptosis-inducers was also carried out. Compound 1 was the most active. Furthermore, in the model of combination chemotherapy, the interaction between the doxorubicine and latilagascene B ( 6) was studied in vitro, on human MDR1 gene transfected mouse lymphoma cells, showing that the type of interaction was synergistic. Latilagascenes D–F ( 1– 3) are new compounds whose structures were established on the basis of spectroscopic methods, including 2D NMR experiments (COSY, HMQC, HMBC and NOESY).

New Macrocyclic Lathyrane Diterpenes, fromEuphorbia lagascae,as Inhibitors of Multidrug Resistance of Tumour Cells

The new macrocyclic lathyrane diterpenes latilagascenes A and B ( 1 and 2), the diacetylated derivative of 2, latilagascene C ( 3), and the known diterpenes ent-16alpha,17-dihydroxyatisan-3-one ( 4) and ent-16alpha,17-dihydroxykauran-3-one ( 5), isolated from the methanol extract of Euphorbia lagascae, were examined for their effects on the reversal of multidrug resistance (MDR) on mouse lymphoma cells. Among the active lathyrane derivatives 1 - 3, compound 2 displayed the highest inhibition of rhodamine 123 efflux of human MDR1 gene transfected mouse lymphoma cells when compared to the untreated cells or the positive control verapamil. The new compounds are the first macrocyclic lathyrane diterpenes showing oxidation at C-16, whose structures were characterized by extensive spectroscopic methods, including 2D NMR experiments ( (1)H- (1)H COSY, HMQC, HMBC and NOESY). The known phenolic compounds vanillic acid ( 6), p-salicylic acid ( 7), isofraxidin ( 8) and cleomiscosin A ( 9) were also isolated from this species.

Quantitative Deuterium Isotopic Profiling at Natural Abundance Indicates Mechanistic Differences for Δ12-Epoxidase and Δ12-Desaturase in Vernonia galamensis

Quantitative (2)H NMR spectroscopy can determine the natural abundance ((2)H/(1)H) ratio at each site of a molecule. In natural products, variation in these values is related to the reaction mechanisms in the pertinent biosynthetic pathway. For the first time, this novel approach has been exploited to probe for mechanistic differences in the introduction of different functionalities into a long-chain fatty acid. Vernolic acid, a major component of the seed oil of Vernonia galamensis, contains both an epoxide and a desaturation. The site-specific isotopic distribution ((2)H/(1)H)(i) has been determined for both vernolic acid and linoleic acid isolated from the same V. galamensis oil. It is found that the ((2)H/(1)H) ratio of vernolic acid shows a pattern along the entire length of the chain, consistent with linoleic acid being its immediate precursor. Notably, the C13 relates to the C13 of linoleic acid but not to the C13 of oleic acid. Furthermore, the C12 and C13 positions in vernolic acid are less depleted, consistent with a change in hybridization state from sp(2) to sp(3). However, the C11 position shows a marked relative enrichment in the vernolic acid, implying that it plays a role in the epoxidase but not the desaturase mechanism. Thus, although it can be concluded that the catalytic mechanisms for the epoxidase and desaturase activities are similar, marked differences in the residual ((2)H/(1)H) patterns indicate that the reaction mechanisms are not identical.

Low‐Valent Titanium Mediated Synthesis of Hydroxystilbenoids: Some New Observations.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Indehiscence Expression and Capsule Anatomy in Vernola

Euphorbia lagascae Spreng. is a potential new industrial crop because of the synthesis of vernolic acid in about 65% of the total fatty acids in its seed oil. Breeding research in the 1990s gave rise to the indehiscent Vernola cultivar as opposed to a complete dehiscence of the wild types. With the aim to evaluate the environmental influence in the expression of the nonshattering trait, field trials were sown in spring and autumn in Murcia, Spain, and Ancona, Italy, during 2001 and 2002. To further examine the basis of partial indehiscence, a capsule wall histological study was also performed. There was a significant decrease (P < 0.01) from 86.9 to 67.9% in the number of capsules retained per plant in the spring sowing, when mister sprinklers were used to increase relative humidity (RH) during pod formation. Overall, 37.9% indehiscent capsules per plant were retained in the autumn cycle vs. 77.4% in the spring cycle, indicating a strong influence of the growing season, which was associated to a combined effect of higher moisture and lower temperatures during capsule development. Transversal sections of carpel walls from Vernola capsules showed that the physical reason of the partial dehiscence observed in this cultivar is a small portion of the mesocarp remaining near the valve sutures, which makes the fruits susceptible to a slower opening under some environmental conditions.

Anti-nutritive constituents in oilseed crops from Italy

Eight different oilseed crops ( Brassica carinata, Camelina sativa, Coriandrum sativum, Euphorbia lagascae, Lepidium sativum, Lesquerella fendleri, Madia sativa, Vernonia galamensis) grown in Italy were investigated regarding anti-nutritive compounds, such as glucosinolates, sinapine, inositol phosphates and condensed tannins, which can adversely affect the nutritional value of residues from the oilseed processing. In all seeds at least one anti-nutritive compound was found, which possibly could lower the nutritive value, but in most cases a real negative effect is not to be expected. The existence and the concentration of the different anti-nutritive components varied in the different seeds. Glucosinolates and sinapine were found only in seeds of B. carinata, L. sativum, C. sativa and L. fendleri, whereas condensed tannins and inositol phosphates appeared in all seeds. In the different seeds the amount ranged from 0.2 mg/g ( L. fendleri) to 13.1 mg/g ( L. sativum) for sinapine, from 0.4 mg/g ( E. lagascae) to 19.6 mg/g ( L. fendleri) for condensed tannins, from 6.6 mg/g ( E. lagascae) to 23.1 mg/g ( B. carinata) for inositol hexa-phosphate as well as from 18.7 μmol/g ( C. sativa) to 164.6 μmol/g ( L. sativum) for glucosinolates.

Localization of nonspecific lipid transfer proteins correlate with programmed cell death responses during endosperm degradation in Euphorbia lagascae seedlings.

When the storage materials have been depleted, the endosperm cells undergo programmed cell death. Very little is known about how the components of the dying cells are recycled and used by the growing seedling. To learn more about endosperm degradation and nutrient recycling, we isolated soluble proteins from the endosperm of Euphorbia lagascae seedlings collected 2, 4, and 6 d after sowing. The protein extracts were subjected to two-dimensional gel electrophoresis. Proteins that increased in amount in the endosperm with time were selected for further analysis with mass spectrometry. We successfully identified 17 proteins, which became more abundant by time during germination. Among these proteins were three E. lagascae lipid transfer proteins (ElLTPs), ElLTP1, ElLTP2, and ElLTP3. Detailed expressional studies were performed on ElLTP1 and ElLTP2. ElLTP1 transcripts were detected in endosperm and cotyledons, whereas ElLTP2 transcripts were only detected in endosperm. Western blots confirmed that ElLTP1 and ElLTP2 accumulate during germination. Immunolocalization experiments showed that ElLTP1 was present in the vessels of the developing cotyledons, and also in the alloplastic space in the endosperm. ElLTP2 formed a concentration gradient in the endosperm, with higher amounts in the inner regions close to the cotyledons, and lesser amounts in the outer regions of the endosperm. On the basis of these data, we propose that ElLTP1 and ElLTP2 are involved in recycling of endosperm lipids, or that they act as protease inhibitors protecting the growing cotyledons from proteases released during programmed cell death.

A germination-specific epoxide hydrolase from Euphorbia lagascae.

The endosperm of Euphorbia lagascae Spreng. seeds contains high levels of the epoxidated fatty acid vernolic acid [12(S),13(R)-epoxy-12-octadecenoic acid]. To learn more about the function and metabolism of vernolic acid, we have initiated an investigation into the gene activities involved in the processes of vernolic acid mobilization and oxidation during germination. Here we report the cloning and characterization of a soluble epoxide hydrolase from E. lagascae. Epoxide hydrolases are a group of functionally related enzymes that catalyze the co-factor-independent hydrolysis of epoxides to their corresponding vicinal diols by the addition of a water molecule. A detailed investigation of the transcription pattern of the E. lagascae epoxide hydrolase gene shows that it is induced during germination. Results from in situ hybridization show that the gene is expressed in the cotyledons and in the hypocotyl. We conclude that soluble epoxide hydrolase plays an important role during germination of E. lagascae seeds.

Characterization of germination-specific lipid transfer proteins from Euphorbia lagascae.

The endosperm of Euphorbia lagascae Spreng. seeds contains high levels of the epoxidated fatty acid vernolic acid ( cis-12-epoxyoctadeca-cis-9-enoic acid). To obtain transgenic oilcrops producing high levels of vernolic acid, better knowledge of its endogenous metabolism is needed. In this paper we study the gene activities involved in the mobilization and oxidation of vernolic acid during germination. A cDNA library was constructed from mRNA isolated from germinating E. lagascae seeds. Over 300 cDNA clones were partially characterized by DNA sequencing. Of the sequenced cDNAs, 18% encoded proteins with a putative function related to the metabolism of lipids or fatty acids. Among these cDNAs were genes coding for lipase, thiolase, acyl-CoA reductase and epoxide hydrolase. Of the sequenced clones, 4.5% encoded lipid-transfer proteins (LTPs), indicating the high abundance of such proteins during germination. We isolated the full-length sequences of the E. lagascae cDNAs encoding the LTPs ElLTP1 and ElLTP2. These proteins share only 38% identity, but both show high similarity to LTPs from other plant species. Both sequences contain eight cysteine residues, which are conserved in most plant LTPs. Expression analysis revealed that both genes were specifically expressed during germination.

Transgenic Production of Epoxy Fatty Acids by Expression of a Cytochrome P450 Enzyme from Euphorbia lagascae Seed

Seed oils of a number of Asteraceae and Euphorbiaceae species are enriched in 12-epoxyoctadeca-cis-9-enoic acid (vernolic acid), an unusual 18-carbon Δ12-epoxy fatty acid with potential industrial value. It has been previously demonstrated that the epoxy group of vernolic acid is synthesized by the activity of a Δ12-oleic acid desaturase-like enzyme in seeds of the Asteraceae Crepis palaestina and Vernonia galamensis. In contrast, results from metabolic studies have suggested the involvement of a cytochrome P450 enzyme in vernolic acid synthesis in seeds of the Euphorbiaceae species Euphorbia lagascae. To clarify the biosynthetic origin of vernolic acid in E. lagascae seed, an expressed sequence tag analysis was conducted. Among 1,006 randomly sequenced cDNAs from developingE. lagascae seeds, two identical expressed sequence tags were identified that encode a cytochrome P450 enzyme classified as CYP726A1. Consistent with the seed-specific occurrence of vernolic acid in E. lagascae, mRNA corresponding to theCYP726A1 gene was abundant in developing seeds, but was not detected in leaves. In addition, expression of the E. lagascae CYP726A1 cDNA in Saccharomyces cerevisiae was accompanied by production of vernolic acid in cultures supplied with linoleic acid and an epoxy fatty acid tentatively identified as 12-epoxyoctadeca-9,15-dienoic acid (12-epoxy-18:2Δ9,15) in cultures supplied with α-linolenic acid. Consistent with this, expression of CYP726A1 in transgenic tobacco (Nicotiana tabacum) callus or somatic soybean (Glycine max) embryos resulted in the accumulation of vernolic acid and 12-epoxy-18:2Δ9,15. Overall, these results conclusively demonstrate that Asteraceae species and the Euphorbiaceae E. lagascae have evolved structurally unrelated enzymes to generate the Δ12-epoxy group of vernolic acid.

Transgenic production of epoxy fatty acids by expression of a cytochrome P450 enzyme from Euphorbia lagascae seed.

Seed oils of a number of Asteraceae and Euphorbiaceae species are enriched in 12-epoxyoctadeca-cis-9-enoic acid (vernolic acid), an unusual 18-carbon Delta(12)-epoxy fatty acid with potential industrial value. It has been previously demonstrated that the epoxy group of vernolic acid is synthesized by the activity of a Delta(12)-oleic acid desaturase-like enzyme in seeds of the Asteraceae Crepis palaestina and Vernonia galamensis. In contrast, results from metabolic studies have suggested the involvement of a cytochrome P450 enzyme in vernolic acid synthesis in seeds of the Euphorbiaceae species Euphorbia lagascae. To clarify the biosynthetic origin of vernolic acid in E. lagascae seed, an expressed sequence tag analysis was conducted. Among 1,006 randomly sequenced cDNAs from developing E. lagascae seeds, two identical expressed sequence tags were identified that encode a cytochrome P450 enzyme classified as CYP726A1. Consistent with the seed-specific occurrence of vernolic acid in E. lagascae, mRNA corresponding to the CYP726A1 gene was abundant in developing seeds, but was not detected in leaves. In addition, expression of the E. lagascae CYP726A1 cDNA in Saccharomyces cerevisiae was accompanied by production of vernolic acid in cultures supplied with linoleic acid and an epoxy fatty acid tentatively identified as 12-epoxyoctadeca-9,15-dienoic acid (12-epoxy-18:2Delta(9,15)) in cultures supplied with alpha-linolenic acid. Consistent with this, expression of CYP726A1 in transgenic tobacco (Nicotiana tabacum) callus or somatic soybean (Glycine max) embryos resulted in the accumulation of vernolic acid and 12-epoxy-18:2Delta(9,15). Overall, these results conclusively demonstrate that Asteraceae species and the Euphorbiaceae E. lagascae have evolved structurally unrelated enzymes to generate the Delta(12)-epoxy group of vernolic acid.

Characterization of a Euphorbia lagascae epoxide hydrolase gene that is induced early during germination

In Euphorbia lagascae the major fatty acid in triacylglycerol is the epoxidated fatty acid vernolic acid (cis-12-epoxyoctadeca-cis-9-enoic acid). The enzymic reactions occurring during the catabolism of epoxidated fatty acids during germination are not known, but it seems likely that the degradation requires the activity of an epoxide hydrolase. Epoxide hydrolases are a group of functionally related enzymes that catalyse the cofactor-independent hydrolysis of epoxides to their corresponding vicinal diols by the addition of a water molecule. Here we report the cloning and characterization of an epoxide hydrolase gene from E. lagascae. The structure of the gene is unusual since it lacks introns. A detailed investigation of the transcription pattern of the epoxide hydrolase gene shows that the gene is induced during germination. We have used in situ hybridization to identify in which tissues the gene is expressed during germination. We speculate that this epoxide hydrolase enzyme is involved in the catabolism of epoxidated fatty acids during germination of E. lagascae seeds.

Characterization of a Euphorbia lagascae epoxide hydrolase gene that is induced early during germination

In Euphorbia lagascae the major fatty acid in triacylglycerol is the epoxidated fatty acid vernolic acid (cis- 12-epoxyoctadeca-cis-9-enoic acid). The enzymic reactions occurring during the catabolism of epoxidated fatty acids during germination are not known, but it seems likely that the degradation requires the activity of an epoxide hydrolase. Epoxide hydrolases are a group of functionally related enzymes that catalyse the cofactor-independent hydrolysis of epoxides to their corresponding vicinal diols by the addition of a water molecule. Here we report the cloning and characterization of an epoxide hydrolase gene from E. lagascae. The structure of the gene is unusual since it lacks introns. A detailed investigation of the transcription pattern of the epoxide hydrolase gene shows that the gene is induced during germination. We have used in situ hybridization to identify in which tissues the gene is expressed during germination. We speculate that this epoxide hydrolase enzyme is involved in the catabolism of epoxidated fatty acids during germination of E. lagascae seeds.

The involvement of phospholipid:diacylglycerol acyltransferases in triacylglycerol production

We have characterized three CoA-independent types of enzyme, phospholipases, phospholipid:diacylglycerol acyltransferases (PDATs) and cholinephosphotransferases, responsible for the removal of unusual fatty acids from phosphatidylcholine (PC) in microsomal preparations from developing oil seeds. The metabolism of sn-2-[(14)C]acyl-PC was monitored in microsomal preparations from various oilseeds having either medium-chain, acetylenic, epoxy or hydroxy fatty acids as their major fatty acids in the oil. The results indicate that PDAT plays a major role in removing ricinoleic acid and vernolic acid from phospholipids in Ricinus communis and Crepis palaestina seeds, respectively. However, vernolic, crepenynic and capric acids are primarily removed from phospholipids by phospholipases in Euphorbia lagascae, Crepis rubra and elm seeds, respectively. Further, we show that significant PDAT activity is also present in vegetative tissues of Arabidopsis thaliana.

Investigation into the Prospects of Five Novel Oilseed Crops within Europe

The economic potential in Europe of five novel oilseed crops for applications within the chemical industry, particularly in paints and lubricants, was investigated within a multidisciplinary integrated research and development project. The entire production chain was subjected to a study. The following five oilseed crops were evaluated: Lunaria annua, Calendula officinalis, Dimorphotheca pluvialis, Lesquerella grandiflora and Euphorbia lagascae. It was concluded that Calendula was nearest to commercialization, and that Calendula oil showed excellent behaviour in several paint formulations. Lunaria and Euphorbia oils showed good lubricating performance, but feasible commercial production of these oils does not seem realistic in the near future.

Piceatannol, a Stilbene Phytochemical, Inhibits Mitochondrial F0F1-ATPase Activity by Targeting the F1 Complex

Piceatannol is a stilbene phytochemical from the seeds of Euphorbia lagascae, previously identified as an antileukemic principle. Piceatannol is considered an inhibitor of several tyrosine kinases. We recently reported that resveratrol, another stilbene phytoalexin from grape seeds, was an inhibitor of ATP synthase. Here, we demonstrated that piceatannol potently inhibited the rat brain mitochondrial F0F1-ATPase activity in both solubilized and submitochondrial preparations (IC50 of 8–9 μM), while having relatively small effect on the Na+, K+-ATPase activity of porcine cerebral cortex (no effect up to 7 μM). Piceatannol inhibited the ATPase activity of the purified rat liver F1 with IC50 of about 4 μM, while resveratrol was slightly less active (IC50 of about 14 μM). Our results indicate that piceatannol and resveratrol inhibit the F-type ATPase by targeting the F1 sector, which is located to the inner membrane of mitochondria and plasma membrane of normal endothelial cells and several cancer cell lines. This mechanism could potentially contribute to the multiple effects of these chemopreventive phytochemicals.

Variation in agronomic characteristics and seed oil composition of new oilseed crops in central Italy

The following new potential oilseed crops for industrial uses have been considered for agronomic traits and seed oil composition during a 3 year trial in Central Italy: Calendula officinalis (calendic acid); Camelina sativa and Lepidium sativum (linolenic acid); Coriandrum sativum (petroselinic acid); Euphorbia lagascae and Vernonia galamensis (epoxy fatty acids); Madia sativa (linoleic acid); Lesquerella fendleri (hydroxy fatty acids). Agronomic characteristics were examined in replicated field trials and phenological and morphological assessments were made. Seed oil content was determined by the Soxhlet method. Fatty acids were determined by gas liquid chromatography after transesterification to the methyl esters. A range of variation within most species was established for genetic differences in such traits as date of flowering, plant height, seed yield and oil percentage. Mean oil content in Camelina sativa, Lepidium sativum, Madia sativa and Coriandrum sativum was also influenced by the climatic conditions occurring in the different years, decreasing in the driest seasons. Some species ( Coriandrum sativum, Madia sativa, Vernonia galamensis, Euphorbia lagascae and Lesquerella fendleri) produced high levels (more than 50%) of a single fatty acid in their seeds. This finding is favourable for industrial uses by minimising dowstream processing costs. On the other hand, other species ( Lepidium sativum, Camelina sativa, Calendula officinalis) contained a mixture of fatty acids in their seeds. Some of these species showed good agronomic traits, including tolerance to drought and to high temperature, making them potentially suitable for cultivation in the Mediterranean area. For most of them, further breeding work should be directed towards increasing seed oil content and eliminating certain wild characteristics such as fruit dehiscence.

Antinutritive Compounds in Different Oilseeds

AbstractDifferent oilseeds, usable as renewable resources, are analyzed for their content of condensed tannins, glucosinolates, phytic acid and its degradation products as well as for sinapine, and the results are presented. Seeds of sunflower, flax, coriander, Euphorbia lagascae, iberic. dragon head, safflor, Camelina sativa, soy, mustard, hemp and rapeseeds are used. Apart from hemp, coriander and Euphorbia lagascae different cultivars or genotypes of each species are investigated. The variation of the results of each cultivar or genotype is shown. Tannins and phytic acid are found in different amounts in all oilseeds, whereas sinapine and glucosinolates are limited to seeds from species belonging to the plant family Brassicaceae.

Supercritical fluid chromatographic analysis of new crop seed oils and their reactions

AbstractSupercritical fluid chromatography (SFC) with an open tubular column of nonpolar stationary phase separated triglycerides from crambe, meadowfoam,Euphorbia lagascae, and vernonia oils based on their molecular weight. The triglyceride compositions were consistent with the literature. SFC proved also to be a valuable tool in analyzing lipase‐catalyzed transesterification reactions where lesquerella oil and estolides were among the substrates employed. Analyte molecular weights could be estimated from a retention time‐ (or elution density‐) molecular eeight calibration curve. An increase in isothermal column temperature during SFC pressure or density programming improved the resolution of high‐molecular‐weight (>600 Da) analytes but yielded poorer resolution for analytes of molecular weight <200. A simultaneous pressure and temperature ramping program proved superior in enhancing resolution in several instances.