Integument-Specific Transcriptional Regulation in the Mid-Stage of Flax Seed Development Influences the Release of Mucilage and the Seed Oil Content.

Flax (Linum usitatissimum L.) seed oil, which accumulates in the embryo, and mucilage, which is synthesized in the seed coat, are of great economic importance for food, pharmaceutical as well as chemical industries. Theories on the link between oil and mucilage production in seeds consist in the spatio-temporal competition of both compounds for photosynthates during the very early stages of seed development. In this study, we demonstrate a positive relationship between seed oil production and seed coat mucilage extrusion in the agronomic model, flax. Three recombinant inbred lines were selected for low, medium and high mucilage and seed oil contents. Metabolite and transcript profiling (1H NMR and DNA oligo-microarrays) was performed on the seeds during seed development. These analyses showed main changes in the seed coat transcriptome during the mid-phase of seed development (25 Days Post-Anthesis), once the mucilage biosynthesis and modification processes are thought to be finished. These transcriptome changes comprised genes that are putatively involved in mucilage chemical modification and oil synthesis, as well as gibberellic acid (GA) metabolism. The results of this integrative biology approach suggest that transcriptional regulations of seed oil and fatty acid (FA) metabolism could occur in the seed coat during the mid-stage of seed development, once the seed coat carbon supplies have been used for mucilage biosynthesis and mechanochemical properties of the mucilage secretory cells.

A water-soluble polysaccharide from Anethum graveolens seeds: Structural characterization, antioxidant activity and potential use as meat preservative.

A novel water-soluble polysaccharide named AGP1 was successfully isolated from seeds of Anethum graveolens by hot water extraction and further purified by DEAE-Sepharose chromatography. AGP1 has a relative molecular weight of 2.1 104 Da determined by Ultra-high-performance liquid chromatography (UHPLC). The AGP1 characterization was investigated by chemical and instrumental analysis including gas chromatography mass spectrometry (GC-MS), Fourier transform infrared (FT-IR) spectroscopy and X-ray diffraction. Results showed that AGP1 was mainly composed of glucose, galactose, mannose and arabinose in a molar percent of 54.3, 23.8, 14.7 and 7.2, respectively. The thermogravimetry analysis (TGA) and the differential scanning calorimetry (DSC) were used and showed that AGP1 has good thermal stability until 275 °C. Moreover, the purified polysaccharide demonstrated an appreciable in vitro antioxidant potential. The addition of the AGP1, particularly at 0.3% (w/w), in turkey sausages instead of ascorbic acid, as preservative, reduced the lipid peroxidation, preserved the pH and color and improved the bacterial stability during cold storage at 4 °C for 12 days. Overall, the results showed that the AGP1 deserves to be developed as functional and bioactive components for the food and nutraceutical industries.

A metabolic engineering strategy for producing conjugated linoleic acids using the oleaginous yeast Yarrowia lipolytica.

Conjugated linoleic acids (CLAs) have been found to have beneficial effects on human health when used as dietary supplements. However, their availability is limited because pure, chemistry-based production is expensive, and biology-based fermentation methods can only create small quantities. In an effort to enhance microbial production of CLAs, four genetically modified strains of the oleaginous yeast Yarrowia lipolytica were generated. These mutants presented various genetic modifications, including the elimination of ß-oxidation (pox1-6∆), the inability to store lipids as triglycerides (dga1∆ dga2∆ are1∆ lro1∆), and the overexpression of the Y. lipolytica ∆12-desaturase gene (YlFAD2) under the control of the constitutive pTEF promoter. All strains received two copies of the pTEF-oPAI or pPOX-oPAI expression cassettes; PAI encodes linoleic acid isomerase in Propionibacterium acnes. The strains were cultured in neosynthesis or bioconversion medium in flasks or a bioreactor. The strain combining the three modifications mentioned above showed the best results: when it was grown in neosynthesis medium in a flask, CLAs represented 6.5% of total fatty acids and in bioconversion medium in a bioreactor, and CLA content reached 302 mg/L. In a previous study, a CLA degradation rate of 117 mg/L/h was observed in bioconversion medium. Here, by eliminating ß-oxidation, we achieved a much lower rate of 1.8 mg/L/h.

Quantifying ¹³C-labeling in free sugars and starch by GC-MS.

We describe an approach to extract (13)C-labeled sugars (glucose, fructose, maltose, sucrose, myo-inositol as well as glucose from starch) from plant tissues and to analyze their isotopomer distribution by gas chromatography-mass spectrometry (GC-MS). Sugars are derivatized with N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) into their Si(CH3)3 derivatives. Electronic and chemical ionizations are used to obtain suitable fragments for metabolic flux analysis (MFA). Unique fragments are identified by computer simulation and experimental verification with labeled standards. Linear equations for separating information from glucosyl and fructosyl moieties of sucrose are presented. Finally, mass distributions are corrected for natural isotope abundance using a home-written program. The method is illustrated by sugar isotopomer analysis of (13)C-labeled rapeseed embryos.

Gas chromatography-mass spectrometry analysis of 13C labeling in sugars for metabolic flux analysis.

Metabolic flux analysis, using 13C labeled substrates, has become a powerful methodology for quantifying intracellular fluxes. Most often, analysis is restricted to nuclear magnetic resonance or mass spectrometry measurement of 13C label incorporation into protein amino acids. However, amino acid isotopomer distribution insufficiently covers the entire network of central metabolism, especially in plant cells with highly compartmented metabolism, and analysis of other metabolites is required. Analysis of label in saccharides provides complementary data to better define fluxes around hexose, pentose, and triose phosphate pools. Here, we propose a gas chromatography-mass spectrometry (GC-MS) method to analyze 13C labeling in glucose and fructose moieties of sucrose, free glucose, fructose, maltose, inositol, and starch. Our results show that saccharide labeling for isotopomer quantification is better analyzed by chemical ionization than by electron ionization. The structure of the generated fragments was simulated and validated using labeled standards. The method is illustrated by analysis of saccharides extracted from developing rapeseed (Brassica napus L.) embryos. It is shown that glucose 6-phosphate isomerase and plastidial glucose 6-phosphate transport reactions are not at equilibrium, and light is shed on the pathways leading to fructose, maltose, and inositol synthesis.

Agrobacterium-mediated transformation of Ruta graveolens L.

Agrobacterium tumefaciens is used to develop a genetic transformation method for a medicinal plant Ruta graveolens. The direct plant regeneration strategy is preferred to callus line establishment. In vitro seedlings, 2- -to 3-wk-old, are used to excise hypocotyls and co-cultivated for 3 d with A. tumefaciens strain C58C1Rif containing plasmid pTDE4 harbouring neomycin phosphotransferase (npt II, kanamycin resistance) and beta-glucuronidase encoding genes. The Southern blot analysis has shown that 78% kanamycin resistant plants contain gene encoding beta-glucuronidase. The GUS histochemical assay shows that 67% transgenic plants exhibit the corresponding enzymatic activity. Routine transformation efficiency of R. graveolens L. is 11% and could reach up to 22%. Transgenic plants are grown in the greenhouse within 4 months after the initial seedlings.