Stepwise strategy based on 1H-NMR fingerprinting in combination with chemometrics to determine the content of vegetable oils in olive oil mixtures.

1H NMR fingerprinting of edible oils and a set of multivariate classification and regression models organised in a decision tree is proposed as a stepwise strategy to assure the authenticity and traceability of olive oils and their declared blends with other vegetable oils (VOs). Oils of the 'virgin olive oil' and 'olive oil' categories and their mixtures with the most common VOs, i.e. sunflower, high oleic sunflower, hazelnut, avocado, soybean, corn, refined palm olein and desterolized high oleic sunflower oils, were studied. Partial least squares (PLS) discriminant analysis provided stable and robust binary classification models to identify the olive oil type and the VO in the blend. PLS regression afforded models with excellent precisions and acceptable accuracies to determine the percentage of VO in the mixture. The satisfactory performance of this approach, tested with blind samples, confirm its potential to support regulations and control bodies.

Characterization of the S. cerevisiae inp51 mutant links phosphatidylinositol 4,5-bisphosphate levels with lipid content, membrane fluidity and cold growth.

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and its derivatives diphosphoinositol phosphates (DPIPs) play key signaling and regulatory roles. However, a direct function of these molecules in lipid and membrane homeostasis remains obscure. Here, we have studied the cold tolerance phenotype of yeast cells lacking the Inp51-mediated phosphoinositide-5-phosphatase. Genetic and biochemical approaches showed that increased metabolism of PI(4,5)P2 reduces the activity of the Pho85 kinase by increasing the levels of the DPIP isomer 1-IP7. This effect was key in the cold tolerance phenotype. Indeed, pho85 mutant cells grew better than the wild-type at 15 °C, and lack of this kinase abolished the inp51-mediated cold phenotype. Remarkably, reduced Pho85 function by loss of Inp51 affected the activity of the Pho85-regulated target Pah1, the yeast phosphatidate phosphatase. Cells lacking Inp51 showed reduced Pah1 abundance, derepression of an INO1-lacZ reporter, decreased content of triacylglycerides and elevated levels of phosphatidate, hallmarks of the pah1 mutant. However, the inp51 phenotype was not associated to low Pah1 activity since deletion of PAH1 caused cold sensitivity. In addition, the inp51 mutant exhibited features not shared by pah1, including a 40%-reduction in total lipid content and decreased membrane fluidity. These changes may influence the activity of membrane-anchored and/or associated proteins since deletion of INP51 slows down the transit to the vacuole of the fluorescent dye FM4-64. In conclusion, our work supports a model in which changes in the PI(4,5)P2 pool affect the 1-IP7 levels modulating the activity of Pho85, Pah1 and likely additional Pho85-controlled targets, and regulate lipid composition and membrane properties.

Non-redundant Contribution of the Plastidial FAD8 ω-3 Desaturase to Glycerolipid Unsaturation at Different Temperatures in Arabidopsis.

Plastidial ω-3 desaturase FAD7 is a major contributor to trienoic fatty acid biosynthesis in the leaves of Arabidopsis plants. However, the precise contribution of the other plastidial ω-3 desaturase, FAD8, is poorly understood. Fatty acid and lipid analysis of several ω-3 desaturase mutants, including two insertion lines of AtFAD7 and AtFAD8, showed that FAD8 partially compensated the disruption of the AtFAD7 gene at 22 °C, indicating that FAD8 was active at this growth temperature, contrasting to previous observations that circumscribed the FAD8 activity at low temperatures. Our data revealed that FAD8 had a higher selectivity for 18:2 acyl-lipid substrates and a higher preference for lipids other than galactolipids, particularly phosphatidylglycerol, at any of the temperatures studied. Differences in the mechanism controlling AtFAD7 and AtFAD8 gene expression at different temperatures were also detected. Confocal microscopy and biochemical analysis of FAD8-YFP over-expressing lines confirmed the chloroplast envelope localization of FAD8. Co-localization experiments suggested that FAD8 and FAD7 might be located in close vicinity in the envelope membrane. FAD8-YFP over-expressing lines showed a specific increase in 18:3 fatty acids at 22 °C. Together, these results indicate that the function of both plastidial ω-3 desaturases is coordinated in a non-redundant manner.

The evolutionary conserved oil body associated protein OBAP1 participates in the regulation of oil body size.

A transcriptomic approach has been used to identify genes predominantly expressed in maize (Zea mays) scutellum during maturation. One of the identified genes is oil body associated protein1 (obap1), which is transcribed during seed maturation predominantly in the scutellum, and its expression decreases rapidly after germination. Proteins similar to OBAP1 are present in all plants, including primitive plants and mosses, and in some fungi and bacteria. In plants, obap genes are divided in two subfamilies. Arabidopsis (Arabidopsis thaliana) genome contains five genes coding for OBAP proteins. Arabidopsis OBAP1a protein is accumulated during seed maturation and disappears after germination. Agroinfiltration of tobacco (Nicotiana benthamiana) epidermal leaf cells with fusions of OBAP1 to yellow fluorescent protein and immunogold labeling of embryo transmission electron microscopy sections showed that OBAP1 protein is mainly localized in the surface of the oil bodies. OBAP1 protein was detected in the oil body cellular fraction of Arabidopsis embryos. Deletion analyses demonstrate that the most hydrophilic part of the protein is responsible for the oil body localization, which suggests an indirect interaction of OBAP1 with other proteins in the oil body surface. An Arabidopsis mutant with a transfer DNA inserted in the second exon of the obap1a gene and an RNA interference line against the same gene showed a decrease in the germination rate, a decrease in seed oil content, and changes in fatty acid composition, and their embryos have few, big, and irregular oil bodies compared with the wild type. Taken together, our findings suggest that OBAP1 protein is involved in the stability of oil bodies.

Pathogen and Circadian Controlled 1 (PCC1) regulates polar lipid content, ABA-related responses, and pathogen defence in Arabidopsis thaliana.

Pathogen and Circadian Controlled 1 (PCC1) was previously characterized as a regulator of defence against pathogens and stress-activated transition to flowering. Plants expressing an RNA interference construct for the PCC1 gene (iPCC1 plants) showed a pleiotropic phenotype. They were hypersensitive to abscisic acid (ABA) as shown by reduced germination potential and seedling establishment, as well as reduced stomatal aperture and main root length in ABA-supplemented media. In addition, iPCC1 plants displayed alterations in polar lipid contents and their corresponding fatty acids. Importantly, a significant reduction in the content of phosphatidylinositol (PI) was observed in iPCC1 leaves when compared with wild-type plants. A trend in reduced levels of 18:0 and increased levels of 18:2 and particularly 18:3 was also detected in several classes of polar lipids. The enhanced ABA-mediated responses and the reduced content of PI might be responsible for iPCC1 plants displaying a complex pattern of defence against pathogens of different lifestyles. iPCC1 plants were more susceptible to the hemi-biotrophic oomycete pathogen Phytophthora brassicae and more resistant to the necrotrophic fungal pathogen Botrytis cinerea compared with wild-type plants.

De novo assembly and functional annotation of the olive (Olea europaea) transcriptome.

Olive breeding programmes are focused on selecting for traits as short juvenile period, plant architecture suited for mechanical harvest, or oil characteristics, including fatty acid composition, phenolic, and volatile compounds to suit new markets. Understanding the molecular basis of these characteristics and improving the efficiency of such breeding programmes require the development of genomic information and tools. However, despite its economic relevance, genomic information on olive or closely related species is still scarce. We have applied Sanger and 454 pyrosequencing technologies to generate close to 2 million reads from 12 cDNA libraries obtained from the Picual, Arbequina, and Lechin de Sevilla cultivars and seedlings from a segregating progeny of a Picual × Arbequina cross. The libraries include fruit mesocarp and seeds at three relevant developmental stages, young stems and leaves, active juvenile and adult buds as well as dormant buds, and juvenile and adult roots. The reads were assembled by library or tissue and then assembled together into 81 020 unigenes with an average size of 496 bases. Here, we report their assembly and their functional annotation.

Contribution of the different omega-3 fatty acid desaturase genes to the cold response in soybean.

This study analysed the contribution of each omega-3 desaturase to the cold response in soybean. Exposure to cold temperatures (5 °C) did not result in great modifications of the linolenic acid content in leaf membrane lipids. However, an increase in the GmFAD3A transcripts was observed both in plant leaves and soybean cells whereas no changes in GmFAD3B or GmFAD3C expression levels were detected. This increase was reversible and accompanied by the accumulation of an mRNA encoding a truncated form of GmFAD3A (GmFAD3A-T), which originated from alternative splicing of GmFAD3A in response to cold. When the expression of plastidial omega-3 desaturases was analysed, a transient accumulation of GmFAD7-2 mRNA was detected upon cold exposure in mature soybean trifoliate leaves while GmFAD7-1 transcripts remained unchanged. No modification of the GmFAD8-1 and GmFAD8-2 transcripts was observed. The functionality of GmFAD3A, GmFAD3B, GmFAD3C and GmFAD3A-T was examined by heterologous expression in yeast. No activity was detected with GmFAD3A-T, consistent with the absence of one of the His boxes necessary for desaturase activity. The linolenic acid content of Sacharomyces cerevisiae cells overexpressing GmFAD3A or GmFAD3B was higher when the cultures were incubated at cooler temperatures, suggesting that reticular desaturases of the GmFAD3 family, and more specifically GmFAD3A, may play a role in the cold response, even in leaves. The data point to a regulatory mechanism of omega-3 fatty acid desaturases in soybean affecting specific isoforms in both the plastid and the endoplasmic reticulum to maintain appropriate levels of linolenic acid under low temperature conditions.

Increasing omega-3 desaturase expression in tomato results in altered aroma profile and enhanced resistance to cold stress.

One of the drawbacks in improving the aroma properties of tomato (Solanum lycopersicum) fruit is the complexity of this organoleptic trait, with a great variety of volatiles contributing to determine specific quality features. It is well established that the oxylipins hexanal and (Z)-hex-3-enal, synthesized through the lipoxygenase pathway, are among the most important aroma compounds and impart in a correct proportion some of the unique fresh notes in tomato. Here, we confirm that all enzymes responsible for the synthesis of these C6 compounds are present and active in tomato fruit. Moreover, due to the low odor threshold of (Z)-hex-3-enal, small changes in the concentration of this compound could modify the properties of the tomato fruit aroma. To address this possibility, we have overexpressed the omega-3 fatty acid desaturases FAD3 and FAD7 that catalyze the conversion of linoleic acid (18:2) to linolenic acid (18:3), the precursor of hexenals and its derived alcohols. Transgenic OE-FAD tomato plants exhibit altered fatty acid composition, with an increase in the 18:3/18:2 ratio in leaves and fruits. These changes provoke a clear variation in the C6 content that results in a significant alteration of the (Z)-hex-3-enal/hexanal ratio that is particularly important in ripe OE-FAD3FAD7 fruits. In addition to this effect on tomato volatile profile, OE-FAD tomato plants are more tolerant to chilling. However, the different behaviors of OE-FAD plants underscore the existence of separate fatty acid fluxes to ensure plant survival under adverse conditions.

Fluidization of membrane lipids enhances the tolerance of Saccharomyces cerevisiae to freezing and salt stress.

Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Delta(9) position. We expressed two sunflower (Helianthus annuus) oleate Delta(12) desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Delta(9,12), the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15 degrees C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp(+) or Trp(-) strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30 degrees C or 15 degrees C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains.