Functional characterization and structural modelling of Helianthus annuus (sunflower) ketoacyl-CoA synthases and their role in seed oil composition.

MAIN CONCLUSION: The enzymes HaKCS1 and HaKCS2 are expressed in sunflower seeds and contribute to elongation of C18 fatty acids, resulting in the C20-C24 fatty acids in sunflower oil. Most plant fatty acids are produced by plastidial soluble fatty acid synthases that produce fatty acids of up to 18 carbon atoms. However, further acyl chain elongations can take place in the endoplasmic reticulum, catalysed by membrane-bound synthases that act on acyl-CoAs. The condensing enzymes of these complexes are the ketoacyl-CoA synthase (KCSs), responsible for the synthesis of very long chain fatty acids (VLCFAs) and their derivatives in plants, these including waxes and cuticle hydrocarbons, as well as fatty aldehydes. Sunflower seeds accumulate oil that contains around 2-3% of VLCFAs and studies of the fatty acid elongase activity in developing sunflower embryos indicate that two different KCS isoforms drive the synthesis of these fatty acids. Here, two cDNAs encoding distinct KCSs were amplified from RNAs extracted from developing sunflower embryos and named HaKCS1 and HaKCS2. These genes are expressed in developing seeds during the period of oil accumulation and they are clear candidates to condition sunflower oil synthesis. These two KCS cDNAs complement a yeast elongase null mutant and when expressed in yeast, they alter the host's fatty acid profile, proving the encoded KCSs are functional. The structure of these enzymes was modelled and their contribution to the presence of VLCFAs in sunflower oil is discussed based on the results obtained.

Coupling the pretreatment and hydrolysis of lignocellulosic biomass by the expression of beta-xylosidases.

Thermochemical pretreatment and enzymatic hydrolysis are the areas contributing most to the operational costs of second generation ethanol in lignocellulosic biorefineries. The improvement of lignocellulosic enzyme cocktails has been significant in the recent years. Although the needs for the reduction of the energy intensity and chemical consumption in the pretreatment step are well known, the reduction of the severity of the process strongly affects the enzymatic hydrolysis yield. To explore the formulation requirements of the well known cellulolytic cocktail from Myceliophthora thermophila on mild pretreated raw materials, this cocktail was tested on steam exploded corn stover without acid impregnation. The low hemicellulose yield and significant accumulation of xylobiose compared with the standard pretreated material obtained with dilute acid impregnation evidenced a clear limitation in the conversion of xylan to xylose. In order to complement the beta-xylosidase limitation, a selection of enzymes was expressed and tested in this fungus. A controlled expression of xylosidases from Aspergillus nidulans, Aspergillus fumigatus, and Fusarium oxysporum allowed recovering hemicellulose yields reached with standard acid treated material. The results underline the need of parallel development of the pretreatment process with the optimization of the formulation of the enzymatic cocktails.

Arabidopsis seeds altered in the circadian clock protein TOC1 are characterized by higher level of linolenic acid.

The circadian clock plays a critical role in regulating plant physiology and metabolism. However, the way in which the clock impacts the regulation of lipid biosynthesis in seeds is partially understood. In the present study, we characterized the seed fatty acid (FA) and glycerolipid (GL) compositions of pseudo-response regulator mutants. Among these mutants, toc1 (timing of cab expression 1) exhibited the most significant differences compared to control plants. These included an increase in total FA content, characterized by elevated levels of linolenic acid (18:3) along with a reduction in linoleic acid (18:2). Furthermore, our findings revealed that toc1 developing seeds showed increased expression of genes related to FA metabolism. Our results show a connection between TOC1 and lipid metabolism in Arabidopsis seeds.

Characterisation of fatty acyl reductases of sunflower (Helianthus annuus L.) seed.

Long and very long chain fatty alcohols are produced from their corresponding acyl-CoAs through the activity of fatty acyl reductases (FARs). Fatty alcohols are important components of the cuticle that protects aerial plant organs, and they are metabolic intermediates in the synthesis of the wax esters in the hull of sunflower (Helianthus annuus) seeds. Genes encoding 4 different FARs (named HaFAR2, HaFAR3, HaFAR4 and HaFAR5) were identified using BLAST, and studies showed that four of the genes were expressed in seed hulls. In this study, the structure and location of sunflower FAR proteins were determined. They were also expressed exogenously in Saccharomyces cerevisiae to evaluate their substrate specificity based on the fatty alcohols synthesized by the transformed yeasts. Three of the four enzymes tested showed activity in yeast. HaFAR3 produced C18, C20 and C22 saturated alcohols, whereas HaFAR4 and HaFAR5 produced C24 and C26 saturated alcohols. The involvement of these genes in the synthesis of sunflower seed wax esters was addressed by considering the results obtained.

A GC/MS method for the rapid determination of disaturated triacylglycerol positional isomers.

The properties of Triacylglycerols (TAGs) depend on their fatty acid composition and distribution. The presence of saturated fatty acids at the different positions of TAGs is important in determining the melting and tempering profile of many solid and plastic fats. The distribution of fatty acids of a fat can vary depending on its origin and processing. Here we developed a method to determine the composition of positional isomers of disaturated TAGs involved in food formulations using a GC/MS based method that requires no prior purification of the TAG species. The method is based on the different breakages that disaturated TAGs undergo in the MS detector and that permit a rapid determination of the regioisomer distribution of all major TAG species in a crude fat. This approach could facilitate the characterization of a large variety of fats, oils and butter of interest in many food formulations.

Soil physicochemical properties associated with the yield and phytochemical composition of the edible halophyte Crithmum maritimum.

There is growing interest in the consumption of halophytes due to their excellent nutritional profile and antioxidant properties, and their cultivation offers viable alternatives in the face of irreversible global salinization of soils. Nevertheless, abiotic factors strongly influence their phytochemical composition, and little is known about how growing conditions can produce plants with the best nutritional and functional properties. Crithmum maritimum is an edible halophyte with antioxidant properties and considerable potential for sustainable agriculture in marginal environments. However, it is found naturally in contrasting habitats with variable soil physicochemical properties and the extent to which edaphic factors can influence plant performance, accumulation of phytochemicals and their quality remains unknown. We investigated the influence of soil physicochemical properties (texture, pH, electrical conductivity, organic matter content and mineral element concentrations) on growth and reproductive performance, nutritional traits, and the accumulation of specific metabolites in C. maritimum. Soil, leaf and seed samples were taken from eight C. maritimum populations located on the southern coasts of Spain and Portugal. We found greater vegetative growth and seed production in coarser, sandier soils with lower microelement concentrations. The nutritional traits of leaves varied, with soil organic matter and macronutrient content associated with reduced leaf Na, protein and phenolic (mainly flavonoid) concentrations, whereas soils with lower pH and Fe concentrations, and higher clay content yielded plants with lower leaf Zn concentration and greater accumulation of hydroxycinnamic acids. The nutritional value of the seed oil composition appeared to be enhanced in soils with coarser texture and lower microelement concentrations. The accumulation of specific phenolic compounds in the seed was influenced by a wide range of soil properties including texture, pH and some microelements. These findings will inform the commercial cultivation of C. maritimum, particularly in the economic exploitation of poorly utilized, saline soils.

Reduced expression of FatA thioesterases in Arabidopsis affects the oil content and fatty acid composition of the seeds.

Acyl-acyl carrier protein (ACP) thioesterases are enzymes that control the termination of intraplastidial fatty acid synthesis by hydrolyzing the acyl-ACP complexes. Among the different thioesterase gene families found in plants, the FatA-type fulfills a fundamental role in the export of the C18 fatty acid moieties that will be used to synthesize most plant glycerolipids. A reverse genomic approach has been used to study the FatA thioesterase in seed oil accumulation by screening different mutant collections of Arabidopsis thaliana for FatA knockouts. Two mutants were identified with T-DNA insertions in the promoter region of each of the two copies of FatA present in the Arabidopsis genome, from which a double FatA Arabidopsis mutant was made. The expression of both forms of FatA thioesterases was reduced in this double mutant (fata1 fata2), as was FatA activity. This decrease did not cause any evident morphological changes in the mutant plants, although the partial reduction of this activity affected the oil content and fatty acid composition of the Arabidopsis seeds. Thus, dry mutant seeds had less triacylglycerol content, while other neutral lipids like diacylglycerols were not affected. Furthermore, the metabolic flow of the different glycerolipid species into seed oil in the developing seeds was reduced at different stages of seed formation in the fata1 fata2 line. This diminished metabolic flow induced increases in the proportion of linolenic and erucic fatty acids in the seed oil, in a similar way as previously reported for the wri1 Arabidopsis mutant that accumulates oil poorly. The similarities between these two mutants and the origin of their phenotype are discussed in function of the results.

Metabolism and accumulation of hydroxylated fatty acids by castor (Ricinus comunis) seed microsomes.

Castor beans accumulate large amounts of triacylglycerols (TAGs) in the seed endosperm. This oil contains hydroxylated ricinoleic levels close to 90%, which is unique among oil seeds. The capacity to accumulate such high levels of such an unusual fatty acids is due to its specific accumulation and channeling. Here, the ability of the castor biosynthetic machinery to accumulate unusual fatty acids in the form of TAGs was investigated, focusing on ricinoleic acid and the structurally analogous lesquerolic and coriolic fatty acids. The metabolism of different radioactive precursors in active membrane fractions from castor bean's were studied, and the rates and accumulation of these fatty acids provided evidence of the different mechanisms involved in the accumulation of hydroxylated fatty acids in this species. In particular, these studies highlighted the potential of castor to accumulate unusual fatty acids other than ricinoleic acid, showing that castor endosperm can efficiently accumulate lesquerolic acid.

Sunflower (Helianthus annuus) fatty acid synthase complex: ß-Ketoacyl-[acyl carrier protein] reductase genes.

Fatty acids play many roles in plants, but the function of some key genes involved in fatty acid biosynthesis in plant development are not yet properly understood. Here, we clone two ß-ketoacyl-[ACP] reductase (KAR) genes from sunflower, HaKAR1 and HaKAR2, and characterize their functional roles. The enzymes cloned were the only two copies present in the sunflower genome. Both displayed a high degree of similarity, but their promoters infer different regulation. The two sunflower KAR genes were constitutively expressed in all tissues examined, being maximum in developing cotyledons at the start of oil synthesis. Over-expression of HaKAR1 in E. coli changed the fatty acid composition by promoting the elongation of C16:0 to C18:0 fatty acids. The enzymatic characterization of HaKAR1 revealed similar kinetic parameters to homologues from other oil accumulating species. The results point to a partially functional redundancy between HaKAR1 and HaKAR2. This study clearly revealed that these genes play a prominent role in de novo fatty acids synthesis in sunflower seeds.

Characterization of Helianthus annuus Lipoic Acid Biosynthesis: The Mitochondrial Octanoyltransferase and Lipoyl Synthase Enzyme System.

Lipoic acid (LA, 6,8-dithiooctanoic acid) is a sulfur containing coenzyme essential for the activity of several key enzymes involved in oxidative and single carbon metabolism in most bacteria and eukaryotes. LA is synthetized by the concerted activity of the octanoyltransferase (LIP2, EC 2.3.1.181) and lipoyl synthase (LIP1, EC 2.8.1.8) enzymes. In plants, pyruvate dehydrogenase (PDH), 2-oxoglutarate dehydrogenase or glycine decarboxylase are essential complexes that need to be lipoylated. These lipoylated enzymes and complexes are located in the mitochondria, while PDH is also present in plastids where it provides acetyl-CoA for de novo fatty acid biosynthesis. As such, lipoylation of PDH could regulate fatty acid synthesis in both these organelles. In the present work, the sunflower LIP1 and LIP2 genes (HaLIP1m and HaLIP2m) were isolated sequenced, cloned, and characterized, evaluating their putative mitochondrial location. The expression of these genes was studied in different tissues and protein docking was modeled. The genes were also expressed in Escherichia coli and Arabidopsis thaliana, where their impact on fatty acid and glycerolipid composition was assessed. Lipidomic studies in Arabidopsis revealed lipid remodeling in lines overexpressing these enzymes and the involvement of both sunflower proteins in the phenotypes observed is discussed in the light of the results obtained.

Genome-Wide Mapping of Histone H3 Lysine 4 Trimethylation (H3K4me3) and Its Involvement in Fatty Acid Biosynthesis in Sunflower Developing Seeds.

Histone modifications are of paramount importance during plant development. Investigating chromatin remodeling in developing oilseeds sheds light on the molecular mechanisms controlling fatty acid metabolism and facilitates the identification of new functional regions in oil crop genomes. The present study characterizes the epigenetic modifications H3K4me3 in relationship with the expression of fatty acid-related genes and transcription factors in developing sunflower seeds. Two master transcriptional regulators identified in this analysis, VIV1 (homologous to Arabidopsis ABI3) and FUS3, cooperate in the regulation of WRINKLED 1, a transcriptional factor regulating glycolysis, and fatty acid synthesis in developing oilseeds.

Interoperability with Moby 1.0--it's better than sharing your toothbrush!

The BioMoby project was initiated in 2001 from within the model organism database community. It aimed to standardize methodologies to facilitate information exchange and access to analytical resources, using a consensus driven approach. Six years later, the BioMoby development community is pleased to announce the release of the 1.0 version of the interoperability framework, registry Application Programming Interface and supporting Perl and Java code-bases. Together, these provide interoperable access to over 1400 bioinformatics resources worldwide through the BioMoby platform, and this number continues to grow. Here we highlight and discuss the features of BioMoby that make it distinct from other Semantic Web Service and interoperability initiatives, and that have been instrumental to its deployment and use by a wide community of bioinformatics service providers. The standard, client software, and supporting code libraries are all freely available at http://www.biomoby.org/.

Functional Characterization of Lysophosphatidylcholine: Acyl-CoA Acyltransferase Genes From Sunflower (Helianthus annuus L.).

Lysophosphatidylcholine acyltransferase (LPCAT, EC 2.3.1.23) is an evolutionarily conserved key enzyme in the Lands cycle that catalyzes acylation of lysophosphatidylcholine (LPC) to produce phosphatidylcholine (PC), the main phospholipid in cellular membranes. In this study, three LPCAT genes from sunflower were identified and the corresponding proteins characterized. These HaLPCAT genes encoded functionally active enzymes that were able to complement a deficient yeast mutant. Moreover, enzymatic assays were carried out using microsomal preparations of the yeast cells. When acyl specificities were measured in the forward reaction, these enzymes exhibited a substrate preference for unsaturated acyl-CoAs, especially for linolenoyl-CoA, while in the reverse reaction, linoleoyl or linolenoyl acyl groups were transferred from PC to acyl-CoA to a similar extent. Expression levels of LPCAT genes were studied revealing distinct tissue-specific expression patterns. In summary, this study suggests that the combined forward and reverse reactions catalyzed by sunflower LPCATs facilitate acyl-exchange between the sn-2 position of PC and the acyl-CoA pool. Sunflower LPCATs displayed different characteristics, which could point to different functionalities, favoring the enrichment of seed triacylglycerols (TAGs) with polyunsaturated fatty acid (PUFA).

New Insights Into Sunflower (Helianthus annuus L.) FatA and FatB Thioesterases, Their Regulation, Structure and Distribution.

Sunflower seeds (Helianthus annuus L.) accumulate large quantities of triacylglycerols (TAG) between 12 and 28 days after flowering (DAF). This is the period of maximal acyl-acyl carrier protein (acyl-ACP) thioesterase activity in vitro, the enzymes that terminate the process of de novo fatty acid synthesis by catalyzing the hydrolysis of the acyl-ACPs synthesized by fatty acid synthase. Fatty acid thioesterases can be classified into two families with distinct substrate specificities, namely FatA and FatB. Here, some new aspects of these enzymes have been studied, assessing how both enzymes contribute to the acyl composition of sunflower oil, not least through the changes in their expression during the process of seed filling. Moreover, the binding pockets of these enzymes were modeled based on new data from plant thioesterases, revealing important differences in their volume and geometry. Finally, the subcellular location of the two enzymes was evaluated and while both possess an N-terminal plastid transit peptide, only in FatB contains a hydrophobic sequence that could potentially serve as a transmembrane domain. Indeed, using in vivo imaging and organelle fractionation, H. annuus thioesterases, HaFatA and HaFatB, appear to be differentially localized in the plastid stroma and membrane envelope, respectively. The divergent roles fulfilled by HaFatA and HaFatB in oil biosynthesis are discussed in the light of our data.

Species-environment interactions changed by introduced herbivores in an oceanic high-mountain ecosystem.

Summit areas of oceanic islands constitute some of the most isolated ecosystems on earth, highly vulnerable to climate change and introduced species. Within the unique high-elevation communities of Tenerife (Canary Islands), reproductive success and thus long-term survival of species may depend on environmental suitability as well as threat by introduced herbivores. By experimentally modifying the endemic and vulnerable species Viola cheiranthifolia along its entire altitudinal occurrence range, we studied plant performance, autofertility, pollen limitation and visitation rate and the interactive effect of grazing by non-native rabbits on them. We assessed the grazing effects by recording (1) the proportion of consumed plants and flowers along the gradient, (2) comparing fitness traits of herbivore-excluded plants along the gradient, and (3) comparing fitness traits, autofertility and pollen limitation between plants excluded from herbivores with unexcluded plants at the same locality. Our results showed that V. cheiranthifolia performance is mainly affected by inter-annual and microhabitat variability along the gradient, especially in the lowest edge. Despite the increasingly adverse environmental conditions, the plant showed no pollen limitation with elevation, which is attributed to the increase in autofertility levels (≥ 50% of reproductive output) and decrease in competition for pollinators at higher elevations. Plant fitness is, however, extremely reduced owing to the presence of non-native rabbits in the area (consuming more than 75% of the individuals in some localities), which in turn change plant trait-environment interactions along the gradient. Taken together, these findings indicate that the elevational variation found on plant performance results from the combined action of non-native rabbits with the microhabitat variability, exerting intricate ecological influences that threaten the survival of this violet species.

Update on XplorMed: A web server for exploring scientific literature.

As scientific literature databases like MEDLINE increase in size, so does the time required to search them. Scientists must frequently inspect long lists of references manually, often just reading the titles. XplorMed is a web tool that aids MEDLINE searching by summarizing the subjects contained in the results, thus allowing users to focus on subjects of interest. Here we describe new features added to XplorMed during the last 2 years (http://www.bork.embl-heidelberg.de/xplormed/).

Multiple positional nystagmus suggests multiple canal involvement in benign paroxysmal vertigo.

CONCLUSION: Video-oculography demonstrates a higher occurrence of atypical positional nystagmus in patients with benign paroxysmal positional vertigo (BPPV). This includes anterior and horizontal canal variants and multiple positional nystagmus, suggesting combined lesions affecting several canals. OBJECTIVE: To analyse the video-oculographic findings of positional tests in patients with BPPV. MATERIAL AND METHODS: Seventy individuals with symptoms of BPPV and positional nystagmus were included in this study. The diagnosis was based on a history of brief episodes of vertigo and the presence of positional nystagmus as confirmed by video-oculographic examination during the Dix-Hallpike test, the McClure test or the head-hanging manoeuvre. Patients were treated by means of different particle repositioning manoeuvres according to the affected canal (Epley's manoeuvre for the posterior or anterior canals and Lempert's manoeuvre for the lateral canal) and the effectiveness was evaluated at 7 and 30 days. RESULTS: Twenty-nine individuals (41.43%) presented an affected unilateral posterior canal. Fifteen patients (21.43%) presented a pure horizontal direction-changing positional nystagmus consistent with a diagnosis of horizontal canal BPPV. Twelve individuals (17.14%) presented a unilateral down-beating nystagmus, suggesting possible anterior canal BPPV. In addition, 14 patients (20%) showed multiple positional nystagmus during the examination corresponding to simultaneous multi-canal BPPV, 5 had bilateral posterior canal BPPV and 2 presented a positional down-beating nystagmus in both left and right Dix-Hallpike manoeuvres and the head-hanging manoeuvre, which is highly suggestive of anterior canal BPPV. However, seven individuals showed positional horizontal and vertical side-changing nystagmus that could not be explained by single-canal BPPV. These patients with multiple positional nystagmus showed changing patterns of positional nystagmus at follow-up.