Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum.

A clone encoding farnesyl diphosphate synthase (FPPS) was obtained by PCR from a cDNA library made from young leaves of Artemisia annua. A cDNA clone encoding the tobacco epi-aristolochene synthase (eAS) was kindly supplied by J. Chappell (University of Kentucky, Lexington, KY, USA). Two fusions were constructed, i.e. FPPS/eAS and eAS/FPPS. The stop codon of the N-terminal enzyme was removed and replaced by a short peptide (Gly-Ser-Gly) to introduce a linker between the two ORFs. These two fusions and the two single cDNA clones were separately introduced into a bacterial expression vector (pET32). Escherichia coli was transformed with the expression vectors and enzymatically active soluble proteins were obtained after induction with isopropyl thio-beta-d-thiogalactoside. The recombinant enzymes were purified using immobilized metal affinity chromatography on Co2+ columns. The fusion enzymes produced epi-aristolochene from isopentenyl diphosphate through a coupled reaction. The Km values of FPPS and eAS for isopentenyl diphosphate and farnesyl diphosphate, respectively, were essentially the same for the single and fused enzymes. The bifunctional enzymes showed a more efficient conversion of isopentenyl diphosphate to epi-aristolochene than the corresponding amount of single enzymes

Effect of Salt Stress on Purslane and Potential Health Benefits: Oxalic Acid and Fatty Acids Profile

Polyunsaturated fatty acids (PUFAs) are crucial for human health and nutrition since they cannot be synthesized in the body and hence must be provided by the diet. Portulaca oleracea L. (purslane) is the eighth most commonly distributed plant in the world. It is a heat- and drought-tolerant plant and our previous study demonstrates that their leaf provide high amounts of antioxidants, minerals, vitamins and proteins. In the present study, we set out to characterize the oxalic acid and the fatty acid composition (particularly the ratio 3/6) in saline stress studies on two purslane cultivars cultured in a hydroponics system, with the main goal of determine the effects of the exposure of the plant to saline environment on those constituents in leaves (edible part of the plant). Five saline treatments were applied: 20, 40, 60, 90, and 120 mmol NaCl; the control had only the base nutritive solution. In both purslanes, a sharp decrease on total oxalic acid concentration was observed when the salt concentration increase in the hydroponic solution after 30 days of saline stress. Palmitic acid (C16:0) linoleic acid (C18:2) and linolenic acid (C18:3) here the major fatty acids detected and they amounts slightly increases with the increase of salinity until approximately 40 mmol of NaCl. The ratio of ω6 to ω3 was similar in both cultivars and did not change with saline stress. Consumption of purslane will contribute to dietary ω3 PUFA intake, with benefits to human health

Fatty acids profile of selected Artemisia spp. plants: health promotion

In the present study we report the fatty acids profile of thirteen species of Artemisia, a hardy herb or shrub, analyzed by gas chromatography connected to a mass detector (GC-MS) for their nutritional value and their potential exploitation as a new source of essential fatty acids. Total lipids content ranged from 3.31 ± 0.19 to 17.78 ± 0.27 mg/g (fresh weight). The three most abundant fatty acids were C16:0, C18:2ω6 and C18:3ω3. Unsaturated fatty acids predominated in all the Artemisia species are studied with the α-linolenic acid (ALA) and linoleic acid (LA), which are essential for normal human growth, health promotion, and disease prevention. The predominant ω3 PUFA acid in all Artemisia species analyzed, was linolenic acid, with Artemisia gmellini, Artemisia ludoviciana and Artemisia vulgaris, showing higher amounts of this fatty acid, all thirteen species, analyzed in this study, were also rich in oleic acid (ω9) and linoleic acid (ω6), accounted for 50–70% of total PUFA. The ratio of ω3 PUFA to ω6 PUFA was similar in all species, varying from 1.0 to 3.0. Identifying Artemisia species as newer sources of PUFAs and enriching or optimizing the ω3FAs in known plant sources offer us ways of increasing the availability of ω3FAs in the food supply

Omega-3 fatty acid desaturase genes isolated from Purslane (Portulaca oleracea L.): expression in different tissues and response to cold and wound stress

Two full-length cDNA clones PoleFAD7 and PoleFAD8, encoding plastidial omega-3 fatty acid desaturases were isolated from purslane (Portulaca oleracea). The encoded enzymes convert linoleic to alpha-linolenic acid (C18:3n-3). Three histidine clusters characteristic of fatty acid desaturases, a putative chloroplast transit peptide in the N-terminal, and three putative transmembrane domains were identified in the sequence. Both genes were expressed in all analyzed tissues showing different levels of expression. PoleFAD7 was up-regulated by wounding but not by low temperature. PoleFAD8 was up-regulated by cold stress but not by wounding. Total fatty acid and linolenic acid content were higher both, in wounded and intact leaves of plants exposed to low temperature