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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.

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.

Climate change is threatening ecosystem functioning and sustainability worldwide. In silvopastoral systems, ongoing warmer and drier conditions could impact productivity and quality of pastures (in terms of protein content and digestibility), with important economic consequences. However, the extent to which climate change could alter other nutritional traits with a potential role in livestock nutrition and production, such as essential fatty acids and amino acids, is a question that remains poorly known. We designed a field manipulative experiment of increased temperature (+2–3 °C) and rainfall exclusion (−30%) aimed to evaluate the influence of forecasted climate variations on the yield and nutritional composition of pastures (i.e. proteins, lipids, fibers, amino acids, fatty acids and digestibility) in a Mediterranean silvopastoral system. To test whether scattered trees typical of these ecosystems could buffer the effects of higher aridity, experimental plots were installed under trees and in open grasslands. First, we found that plant communities under tree canopies were less productive but exhibited higher quality than those located in open grasslands, likely due to the shade and higher soil fertility provided by trees. Both climatic stressors had a significant influence on pasture productivity and nutritional composition. Thus, pastures subjected to rainfall reduction produced less biomass with higher content in nitrogen, proteins, essential amino acids and lipids, likely as a mechanism of plant tolerance to water stress. In contrast, warming increased plant productivity and enhanced the proportion of unsaturated fatty acids, likely mediated by alterations in plant community composition. Finally, our results suggest that trees might slow the impact of climate change on productivity and specific amino acids in pasture. These results could be also applied for the design of management strategies to ensure the ecologic and economic value of silvopastoral ecosyste

The objective of the present study was to analyse the fruit yield and the lipid and phenolic composition of the seeds from the edible halophyte Crithmum maritimum growing in different types of soil with variable physicochemical properties. Seed oil content ranged between 8.39–11.66 % and showed high nutritional value, and the phenolic composition (423−603 mg TPC 100 g−1) presented high levels of quercetin-type compounds with high pharmaceutical interest. The strong conservation of these traits, together with a high and stable fruit production, suggests that C. maritimum could be cultivated in soils with no agronomical relevance to produce seeds as an accessible and environmentally friendly source of good quality oil and phenolics for industrial purposes. © 2022 Elsevier Inc.

Crithmum maritimum L. is an edible halophyte with large potential in human nutrition field. However, it is unclear whether its nutritional value is maintained throughout the contrasting habitats where it commonly grows (cliffs, sandy and rocky beaches) and the nutritional profile of cultivated plants still remains uncertain. In this work, we provided for the first time a comparison of the nutritional profile of C. maritimum across its different type of habitats in the south of Spain and between wild plants and plant material under optimal growing conditions. The protein, amino acids, lipids, fatty acids, minerals composition and phenolic content of plants were analysed. Plants under field conditions exhibited a nutritionally balanced composition (3.8–6.2 g protein/100 g DW, 4.9–7.5 mg lipids/g WW, 3.9–5.0 g Na/100 g DW), with high phenolic content (30.2–48.0 mg/g DW) regardless of the variability of the contrasting habitats. In contrast, under optimal conditions, C. maritimum showed a greater protein and lipid content (10.2 g/100 g DW and 9.6 mg/g WW, respectively), and lower sodium accumulation (1.2 g/100 g DW), allowing a greater consumption of this halophyte without exceeding the daily intake recommendations. Conversely, phenolics were strongly decreased in these plants (6.1 mg/g DW) likely due to the absence of stress factors. © 2021 Elsevier Inc.

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

The hull of the sunflower is a ligneous tissue that protects the embryo and cotyledons, and it is rich in wax esters (WEs). WEs are extracted from whole sunflower seeds together with the oil, conferring an undesirable turbidity on this product which must be removed through cooling and filtration for commercialisation. The analysis of sunflower oil WEs is hampered by the presence of significant amounts of sterol esters that co-elute with them in chromatography. Thus, we established here different methods based on GC/MS to analyse the WE content and composition of sunflower hulls, kernels and oil, and that of industrially crystallised sunflower WEs. Hence, using a combination of MS scanning and specific ion monitoring it was possible to quantify and characterise sunflower WEs, overcoming interference by other lipids in the sample. The results revealed that sunflower WEs were formed by saturated species from C40 to C60. The use of the new method for the analysis of WEs from species other than sunflower was also discussed.

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.

Plant bioactive metabolites play a pivotal role in protection against environmental stresses and offer great potential for nutraceutical exploitation. In this sense, halophytes present an exceptional opportunity for sustainable saline agriculture since they can massively accumulate bioactive metabolites under stressful conditions. We investigated the responses of the halophyte Crithmum maritimum to increasing salinity, nutrient deficient and their interaction. We hypothesized that the concentration of bioactive compounds would increase together with salinity and nutrient deficient, and that the interaction between these two stresses would produce synergic effects on bioactive compounds accumulation. We studied the tolerance and foliar accumulation of nutrients and bioactive compounds (amino acids, phenols, terpenes and fatty acids) for C. maritimum in response to salinity and nutrient deprivation in greenhouse controlled conditions. The high salinity tolerance of C. maritimum was not diminished by the interaction of salinity with nutrient limitation. Mineral nutrition deficit and saturated fatty acid accumulation were induced at 200–500 mM NaCl, and accumulation of polyphenol 5-caffeoylquinic acid and some amino acids were identified as nutrient deficiency markers. C. maritimum responded mainly to increasing salinity by reducing foliar accumulation of five terpenes and total lipids, and to nutrient limitation by increasing the concentration of vaccenic acid and 5-caffeoylquinic acid. The interaction between salinity and nutrient availability provoked antagonistic and synergistic effects on the accumulation of terpenes and fatty acids. C. maritimum showed high physiological phenotypic plasticity in the response to salt stress, nutrient deficiency and their combination.

Prosthetic lipoyl groups are essential for the metabolic activity of several multienzyme complexes in most organisms. In plants, octanoyltransferase (LIP2) and lipoyl synthase (LIP1) enzymes in the mitochondria and plastids participate in the de novo synthesis of lipoic acid, and in the attachment of the lipoyl cofactors to their specific targets. In plastids, the lipoylated pyruvate dehydrogenase complex catalyzes the synthesis of the acetyl-CoA that is required for de novo fatty acid synthesis. Since lipoic acid transport across plastid membranes has not been demonstrated, these organelles require specific plastidial LIP1 and LIP2 activities for the in situ synthesis of this cofactor. Previously, one essential LIP1 enzyme and two redundant LIP2 enzymes have been identified within Arabidopsis chloroplasts. In this study, two plastidial sunflower (Helianthus annuus L.) LIP2 genes (HaLIP2p1 and HaLIP2p2) were identified, cloned and characterized. The expression of these genes in different tissues was studied and the tertiary structure of the peptides they encode was modeled by protein docking. These genes were overexpressed in Escherichia coli and their impact on bacterial fatty acid synthesis was studied. Finally, transgenic Arabidopsis plants overexpressing HaLIP2p1 were generated and their seed lipid profiles analyzed. The lipid composition of the transgenic seeds, particularly their TAG species, differed from that of wild-type plants, revealing a relationship between lipoic acid synthesis and the accumulation of storage lipids in Arabidopsis seeds.

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.

Koelreuteria paniculata is a deciduous tree, popular in temperate regions for its ornamental value, which accumulates unusual cyanolipids in its seeds. The seed oil of this plant is rich in the unusual cis-11-eicosenoic fatty acid (20:1, or gondoic acid), a monounsaturated oil of interest to the oleochemical industry. In higher plants, de novo fatty acid biosynthesis takes place in the plastids, a process that is terminated by hydrolysis of the thioester bond between the acyl moiety and the ACP by acyl-ACP thioesterases. The specificity of acyl-ACP thioesterases is fundamental in controlling the fatty acid composition of seed oil. To determine the mechanisms involved in fatty acid biosynthesis in K. paniculata seeds, we isolated, cloned and sequenced two cDNAs encoding acyl-ACP thioesterases in this plant, KpFatA and KpFatB. Both of them were expressed heterologously in Escherichia coli and characterized with different acyl-ACP substrates. The K. paniculata FatB2 displayed unusual substrate specificity, so that unlike most FatB2 type enzymes, it displayed preference for oleoyl-ACP instead of palmitoyl-ACP. This specificity was consistent with the changes in E. coli and N. benthamiana fatty acid composition following heterologous expression of this enzyme. KpFatB also showed certain genetic divergence relative to other FatB-type thioesterases and when modelled, its structure revealed differences at the active site. Together, these results suggest that this thioesterase could be a new class of FatB not described previously.

In the present study, we describe the molecular and biochemical characterization of sunflower (Helianthus annuus L.) enolase (ENO, EC 4.2.1.11) proteins, which catalyze the formation of phosphoenolpyruvate, the penultimate intermediate in the glycolytic pathway. We cloned and characterized three cDNAs encoding different ENO isoforms from developing sunflower seeds. Studies using fluorescently tagged ENOs confirmed the predicted subcellular localization of ENO isoforms: HaENO1 in the plastid while HaENO2 and HaENO3 were found in the cytosol. The cDNAs were used to express the corresponding 6(His)-tagged proteins in Escherichia coli. The proteins were purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Recombinant HaENO1 and HaENO2, but not HaENO3 were shown to have enolase activity, in agreement with data obtained with the Arabidopsis homolog proteins. Site directed mutagenesis of several critical amino acids was used to attempt to recover enolase activity in recombinant HaENO3, resulting in very small increases that were not additive. A kinetic characterization of the two active isoforms showed that pH had similar effect on their velocity, that they had similar affinity for 2-phosphoglycerate, but that the kcat/Km of the plastidial enzyme was higher than that of the cytosolic isoform. Even though HaENO2 was always the most highly expressed transcript, the levels of expression of the three ENO genes were remarkably distinct in all the vegetative and reproductive tissues studied. This indicates that in seeds the conversion of 2-phosphoglycerate to phosphoenolpyruvate takes place through the cytosolic and the plastidial pathways therefore both routes could contribute to the supply of carbon for lipid synthesis. The identity of the main source of carbon during the period of stored products synthesis is discussed.

Fats based on stearic acid could be a healthier alternative to existing oils especially hydrogenated fractions of oils or palm, but only a few non-tropical species produce oils with these characteristics. In this regard, newly developed high stearic oil seed crops could be a future source of fats and hard stocks rich in stearic and oleic fatty acids. These oil crops have been obtained either by breeding and mutagenesis or by suppression of desaturases using RNA interference. The present review depicts the molecular and biochemical bases for the accumulation of stearic acid in sunflower. Moreover, aspects limiting the accumulation of stearate in the seeds of this species are reviewed. This included data obtained from the characterization of genes and enzymes related to fatty acid biosynthesis and triacylglycerol assembly. Future improvements and uses of these oils are also discussed. © 2014 Elsevier Ltd. All rights reserved.

The mechanisms by which macadamia nuts accumulate the unusual palmitoleic and asclepic acyl moieties, which constitute up to 20% of the fatty acids in some varieties, are still unknown. Acyl-acyl carrier protein (ACP) thioesterases (EC 3.1.2.14) are intraplastidial enzymes that terminate the synthesis of fatty acids in plants and that facilitate the export of the acyl moieties to the endoplasmic reticulum where they can be used in the production of glycerolipids. Here, we have investigated the possible role of acyl-ACP thioesterase activity in the composition of macadamia kernel oil. Accordingly, two acyl-ACP thioesterases were cloned from developing macadamia kernels, one of the FatA type and the other of the FatB type. These enzymes were heterologously expressed in Escherichia coli, and the recombinant thioesterases were purified, characterized kinetically and assayed with a variety of substrates, demonstrating the high specificity of macadamia FatA towards 16:1-ACP. Acyl-ACP thioesterase activity was also characterized in crude extracts from two different varieties of macadamia, Cate and Beaumont, which accumulate different amounts of n-7 fatty acids. The impact of acyl-ACP thioesterase activities on the oil composition of these kernels is discussed in the light of these results. © 2010 Elsevier Masson SAS.

Acyl-acyl carrier protein (ACP) thioesterases are enzymes that terminate the intraplastidial fatty acid synthesis in plants by hydrolyzing the acyl-ACP intermediates and releasing free fatty acids to be incorporated into glycerolipids. These enzymes are classified in two families, FatA and FatB, which differ in amino acid sequence and substrate specificity. In the present work, both FatA and FatB thioesterases were cloned, sequenced and characterized from castor (Ricinus communis) seeds, a crop of high interest in oleochemistry. Single copies of FatA and FatB were found in castor resulting to be closely related with those of Jatropha curcas. The corresponding mature proteins were heterologously expressed in Escherichia coli for biochemical characterization after purification, resulting in high catalytic efficiency of RcFatA on oleoyl-ACP and palmitoleoyl-ACP and high efficiencies of RcFatB for oleoyl-ACP and palmitoyl-ACP. The expression profile of these genes displayed the highest levels in expanding tissues that typically are very active in lipid biosynthesis such as developing seed endosperm and young expanding leaves. The contribution of these two enzymes to the synthesis of castor oil is discussed. © 2010 Elsevier Ltd.