Lipid turnover through lipophagy in the newly identified extremophilic green microalga Chlamydomonas urium.

Autophagy is a central degradative pathway highly conserved among eukaryotes, including microalgae, which remains unexplored in extremophilic organisms. In this study, we described and characterized autophagy in the newly identified extremophilic green microalga Chlamydomonas urium, which was isolated from an acidic environment. The nuclear genome of C. urium was sequenced, assembled and annotated in order to identify autophagy-related genes. Transmission electron microscopy, immunoblotting, metabolomic and photosynthetic analyses were performed to investigate autophagy in this extremophilic microalga. The analysis of the C. urium genome revealed the conservation of core autophagy-related genes. We investigated the role of autophagy in C. urium by blocking autophagic flux with the vacuolar ATPase inhibitor concanamycin A. Our results indicated that inhibition of autophagic flux in this microalga resulted in a pronounced accumulation of triacylglycerols and lipid droplets (LDs). Metabolomic and photosynthetic analyses indicated that C. urium cells with impaired vacuolar function maintained an active metabolism. Such effects were not observed in the neutrophilic microalga Chlamydomonas reinhardtii. Inhibition of autophagic flux in C. urium uncovered an active recycling of LDs through lipophagy, a selective autophagy pathway for lipid turnover. This study provided the metabolic basis by which extremophilic algae are able to catabolize lipids in the vacuole.

Phosphorus Availability Regulates TORC1 Signaling via LST8 in Chlamydomonas.

Target of rapamycin complex 1 (TORC1) is a central regulator of cell growth. It balances anabolic and catabolic processes in response to nutrients, growth factors, and energy availability. Nitrogen- and carbon-containing metabolites have been shown to activate TORC1 in yeast, animals, and plants. Here, we show that phosphorus (P) regulates TORC1 signaling in the model green alga Chlamydomonas (Chlamydomonas reinhardtii) via LST8, a conserved TORC1 subunit that interacts with the kinase domain of TOR. P starvation results in a sharp decrease in LST8 abundance and downregulation of TORC1 activity. A hypomorphic lst8 mutation resulted in decreased LST8 abundance, and it both reduced TORC1 signaling and altered the cellular response to P starvation. Additionally, we found that LST8 levels and TORC1 activity were not properly regulated in a mutant defective in the transcription factor PSR1, which is the major mediator of P deprivation responses in Chlamydomonas. Unlike wild-type cells, the psr1 mutant failed to downregulate LST8 abundance and TORC1 activity when under P limitation. These results identify PSR1 as an upstream regulator of TORC1 and demonstrate that TORC1 is a key component in P signaling in Chlamydomonas.

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.

Chloroplast Damage Induced by the Inhibition of Fatty Acid Synthesis Triggers Autophagy in Chlamydomonas.

Fatty acids are synthesized in the stroma of plant and algal chloroplasts by the fatty acid synthase complex. Newly synthesized fatty acids are then used to generate plastidial lipids that are essential for chloroplast structure and function. Here, we show that inhibition of fatty acid synthesis in the model alga Chlamydomonas reinhardtii activates autophagy, a highly conserved catabolic process by which cells degrade intracellular material under adverse conditions to maintain cell homeostasis. Treatment of Chlamydomonas cells with cerulenin, a specific fatty acid synthase inhibitor, stimulated lipidation of the autophagosome protein ATG8 and enhanced autophagic flux. We found that inhibition of fatty acid synthesis decreased monogalactosyldiacylglycerol abundance, increased lutein content, down-regulated photosynthesis, and increased the production of reactive oxygen species. Electron microscopy revealed a high degree of thylakoid membrane stacking in cerulenin-treated cells. Moreover, global transcriptomic analysis of these cells showed an up-regulation of genes encoding chloroplast proteins involved in protein folding and oxidative stress and the induction of major catabolic processes, including autophagy and proteasome pathways. Thus, our results uncovered a link between lipid metabolism, chloroplast integrity, and autophagy through a mechanism that involves the activation of a chloroplast quality control system.

Autophagic flux is required for the synthesis of triacylglycerols and ribosomal protein turnover in Chlamydomonas.

Autophagy is an intracellular catabolic process that allows cells to recycle unneeded or damaged material to maintain cellular homeostasis. This highly dynamic process is characterized by the formation of double-membrane vesicles called autophagosomes, which engulf and deliver the cargo to the vacuole. Flow of material through the autophagy pathway and its degradation in the vacuole is known as autophagic flux, and reflects the autophagic degradation activity. A number of assays have been developed to determine autophagic flux in yeasts, mammals, and plants, but it has not been examined yet in algae. Here we analyzed autophagic flux in the model green alga Chlamydomonas reinhardtii. By monitoring specific autophagy markers such as ATG8 lipidation and using immunofluorescence and electron microscopy techniques, we show that concanamycin A, a vacuolar ATPase inhibitor, blocks autophagic flux in Chlamydomonas. Our results revealed that vacuolar lytic function is needed for the synthesis of triacylglycerols and the formation of lipid bodies in nitrogen- or phosphate-starved cells. Moreover, we found that concanamycin A treatment prevented the degradation of ribosomal proteins RPS6 and RPL37 under nitrogen or phosphate deprivation. These results indicate that autophagy might play an important role in the regulation of lipid metabolism and the recycling of ribosomal proteins under nutrient limitation in Chlamydomonas.

Molecular and biochemical characterization of the OLE-1 high-oleic castor seed (Ricinus communis L.) mutant.

MAIN CONCLUSION: The natural OLE-1 high-oleic castor mutant has been characterized, demonstrating that point mutations in the FAH12 gene are responsible for the high-oleic phenotype. The contribution of each mutation was evaluated by heterologous expression in yeast, and lipid studies in developing OLE-1 seeds provided new evidence of unusual fatty acids channeling into TAGs. Ricinus communis L. is a plant of the Euphorbiaceae family well known for producing seeds whose oil has a very high ricinoleic (12-hydroxyoctadecenoic) acid content. Castor oil is considered the only commercially renewable source of hydroxylated fatty acids, which have many applications as chemical reactants. Accordingly, there has been great interest in the field of plant lipid biotechnology to define how ricinoleic acid is synthesized, which could also provide information that might serve to increase the content of other unusual fatty acids in oil crops. Accordingly, we set out to study the biochemistry of castor oil synthesis by characterizing a natural castor bean mutant deficient in ricinoleic acid synthesis (OLE-1). This mutant accumulates high levels of oleic acid and displays remarkable alterations in its seed lipid composition. To identify enzymes that are critical for this phenotype in castor oil, we cloned and sequenced the oleate desaturase (FAD2) and hydroxylase (FAH12) genes from wild-type and OLE-1 castor bean plants and analyzed their expression in different tissues. Heterologous expression in yeast confirmed that three modifications to the OLE-1 FAH12 protein were responsible for its weaker hydroxylase activity. In addition, we studied the expression of the genes involved in this biosynthetic pathway at different developmental stages, as well as that of other genes involved in lipid biosynthesis, both in wild-type and mutant seeds.

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

MAIN CONCLUSION: Two sunflower hydroxyacyl-[acyl carrier protein] dehydratases evolved into two different isoenzymes showing distinctive expression levels and kinetics' efficiencies. ß-Hydroxyacyl-[acyl carrier protein (ACP)]-dehydratase (HAD) is a component of the type II fatty acid synthase complex involved in 'de novo' fatty acid biosynthesis in plants. This complex, formed by four intraplastidial proteins, is responsible for the sequential condensation of two-carbon units, leading to 16- and 18-C acyl-ACP. HAD dehydrates 3-hydroxyacyl-ACP generating trans-2-enoyl-ACP. With the aim of a further understanding of fatty acid biosynthesis in sunflower (Helianthus annuus) seeds, two ß-hydroxyacyl-[ACP] dehydratase genes have been cloned from developing seeds, HaHAD1 (GenBank HM044767) and HaHAD2 (GenBank GU595454). Genomic DNA gel blot analyses suggest that both are single copy genes. Differences in their expression patterns across plant tissues were detected. Higher levels of HaHAD2 in the initial stages of seed development inferred its key role in seed storage fatty acid synthesis. That HaHAD1 expression levels remained constant across most tissues suggest a housekeeping function. Heterologous expression of these genes in E. coli confirmed both proteins were functional and able to interact with the bacterial complex 'in vivo'. The large increase of saturated fatty acids in cells expressing HaHAD1 and HaHAD2 supports the idea that these HAD genes are closely related to the E. coli FabZ gene. The proposed three-dimensional models of HaHAD1 and HaHAD2 revealed differences at the entrance to the catalytic tunnel attributable to Phe166/Val1159, respectively. HaHAD1 F166V was generated to study the function of this residue. The 'in vitro' enzymatic characterization of the three HAD proteins demonstrated all were active, with the mutant having intermediate K m and V max values to the wild-type proteins.

Acyl carrier proteins from sunflower (Helianthus annuus L.) seeds and their influence on FatA and FatB acyl-ACP thioesterase activities.

MAIN CONCLUSION: The kinetics of acyl-ACP thioesterases from sunflower importantly changed when endogenous ACPs were used. Sunflower FatB was much more specific towards saturated acyl-ACPs when assayed with them. Acyl carrier proteins (ACPs) are small (~9 kDa), soluble, acidic proteins involved in fatty acid synthesis in plants and bacteria. ACPs bind to fatty acids through a thioester bond, generating the acyl-ACP lipoproteins that are substrates for fatty acid synthase (FAS) complexes, and that are required for fatty acid chain elongation, acting as important intermediates in de novo fatty acid synthesis in plants. Plants, usually express several ACP isoforms with distinct functionalities. We report here the cloning of three ACPs from developing sunflower seeds: HaACP1, HaACP2, and HaACP3. These proteins were plastidial ACPs expressed strongly in seeds, and as such they are probably involved in the synthesis of sunflower oil. The recombinant sunflower ACPs were expressed in bacteria but they were lethal to the prokaryote host. Thus, they were finally produced using the GST gene fusion system, which allowed the apo-enzyme to be produced and later activated to the holo form. Radiolabelled acyl-ACPs from the newly cloned holo-ACP forms were also synthesized and used to characterize the activity of recombinant sunflower FatA and FatB thioesterases, important enzymes in plant fatty acids synthesis. The activity of these enzymes changed significantly when the endogenous ACPs were used. Thus, FatA importantly increased its activity levels, whereas FatB displayed a different specificity profile, with much high activity levels towards saturated acyl-CoA derivatives. All these data pointed to an important influence of the ACP moieties on the activity of enzymes involved in lipid synthesis.

Temperature effect on triacylglycerol species in seed oil from high stearic sunflower lines with different genetic backgrounds.

BACKGROUND: This study characterized the influence of temperature during grain filling on the saturated fatty acid distribution in triacylglycerol molecules from high stearic sunflower lines with different genetic backgrounds. Two growth chamber experiments were conducted with day/night temperatures of 16/16, 26/16, 26/26 and 32/26 °C. RESULTS: In all genotypes, independently of the genetic background, higher temperatures increased palmitic and oleic acid and reduced linoleic acid concentrations. Increasing night temperature produced an increase in saturated-unsaturated-saturated species, indicating a more symmetrical distribution of saturated fatty acids. The solid fat index was more affected by temperature during grain filling in lines with high linoleic than high oleic background. Higher variations in symmetry among night temperatures were observed in lines with high oleic background, which are more stable in fatty acid composition. CONCLUSION: The effect of temperature on triacylglycerol composition is not completely explained by its effect on fatty acid composition. Thus night temperature affects oil properties via its effects on fatty acid synthesis and on the distribution of fatty acids in the triacylglycerol molecules. © 2016 Society of Chemical Industry.

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

MAIN CONCLUSION: Enoyl-[acyl carrier protein]-reductases from sunflower. A major factor contributing to the amount of fatty acids in plant oils are the first steps of their synthesis. The intraplastidic fatty acid biosynthetic pathway in plants is catalysed by type II fatty acid synthase (FAS). The last step in each elongation cycle is carried out by the enoyl-[ACP]-reductase, which reduces the dehydrated product of ß-hydroxyacyl-[ACP] dehydrase using NADPH or NADH. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus) seeds, two enoyl-[ACP]-reductase genes have been identified and cloned from developing seeds with 75 % identity: HaENR1 (GenBank HM021137) and HaENR2 (HM021138). The two genes belong to the ENRA and ENRB families in dicotyledons, respectively. The genetic duplication most likely originated after the separation of di- and monocotyledons. RT-qPCR revealed distinct tissue-specific expression patterns. Highest expression of HaENR1 was in roots, stems and developing cotyledons whereas that of H a ENR2 was in leaves and early stages of seed development. Genomic DNA gel blot analyses suggest that both are single-copy genes. In vivo activity of the ENR enzymes was tested by complementation experiments with the JP1111 fabI(ts) E. coli strain. Both enzymes were functional demonstrating that they interacted with the bacterial FAS components. That different fatty acid profiles resulted infers that the two Helianthus proteins have different structures, substrate specificities and/or reaction rates. The latter possibility was confirmed by in vitro analysis with affinity-purified heterologous-expressed enzymes that reduced the crotonyl-CoA substrate using NADH with different V max.

Effect of a mutagenized acyl-ACP thioesterase FATA allele from sunflower with improved activity in tobacco leaves and Arabidopsis seeds.

The substrate specificity of the acyl-acyl carrier protein (ACP) thioesterases significantly determines the type of fatty acids that are exported from plastids. Thus, designing acyl-ACP thioesterases with different substrate specificities or kinetic properties would be of interest for plant lipid biotechnology to produce oils enriched in specialty fatty acids. In the present work, the FatA thioesterase from Helianthus annuus was used to test the impact of changes in the amino acids present in the binding pocket on substrate specificity and catalytic efficiency. Amongst all the mutated enzymes studied, Q215W was especially interesting as it had higher specificity towards saturated acyl-ACP substrates and higher catalytic efficiency compared to wild-type H. annuus FatA. Null, wild type and high-efficiency alleles were transiently expressed in tobacco leaves to check their effect on lipid biosynthesis. Expression of active FatA thioesterases altered the composition of leaf triacylglycerols but did not alter total lipid content. However, the expression of the wild type and the high-efficiency alleles in Arabidopsis thaliana transgenic seeds resulted in a strong reduction in oil content and an increase in total saturated fatty acid content. The role and influence of acyl-ACP thioesterases in plant metabolism and their possible applications in lipid biotechnology are discussed.

Sunflower (Helianthus annuus) long-chain acyl-coenzyme A synthetases expressed at high levels in developing seeds.

Long chain fatty acid synthetases (LACSs) activate the fatty acid chains produced by plastidial de novo biosynthesis to generate acyl-CoA derivatives, important intermediates in lipid metabolism. Oilseeds, like sunflower, accumulate high levels of triacylglycerols (TAGs) in their seeds to nourish the embryo during germination. This requires that sunflower seed endosperm supports very active glycerolipid synthesis during development. Sunflower seed plastids produce large amounts of fatty acids, which must be activated through the action of LACSs, in order to be incorporated into TAGs. We cloned two different LACS genes from developing sunflower endosperm, HaLACS1 and HaLACS2, which displayed sequence homology with Arabidopsis LACS9 and LACS8 genes, respectively. These genes were expressed at high levels in developing seeds and exhibited distinct subcellular distributions. We generated constructs in which these proteins were fused to green fluorescent protein and performed transient expression experiments in tobacco cells. The HaLACS1 protein associated with the external envelope of tobacco chloroplasts, whereas HaLACS2 was strongly bound to the endoplasmic reticulum. Finally, both proteins were overexpressed in Escherichia coli and recovered as active enzymes in the bacterial membranes. Both enzymes displayed similar substrate specificities, with a very high preference for oleic acid and weaker activity toward stearic acid. On the basis of our findings, we discuss the role of these enzymes in sunflower oil synthesis.

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.

Hydroxycitrate delays early mortality in mice and promotes muscle regeneration while inducing a rich hepatic energetic status.

ATP citrate lyase (ACLY) inhibitors have the potential of modulating central processes in protein, carbohydrate, and lipid metabolism, which can have relevant physiological consequences in aging and age-related diseases. Here, we show that hepatic phospho-active ACLY correlates with overweight and Model for End-stage Liver Disease score in humans. Wild-type mice treated chronically with the ACLY inhibitor potassium hydroxycitrate exhibited delayed early mortality. In AML12 hepatocyte cultures, the ACLY inhibitors potassium hydroxycitrate, SB-204990, and bempedoic acid fostered lipid accumulation, which was also observed in the liver of healthy-fed mice treated with potassium hydroxycitrate. Analysis of soleus tissue indicated that potassium hydroxycitrate produced the modulation of wound healing processes. In vivo, potassium hydroxycitrate modulated locomotor function toward increased wire hang performance and reduced rotarod performance in healthy-fed mice, and improved locomotion in mice exposed to cardiotoxin-induced muscle atrophy. Our findings implicate ACLY and ACLY inhibitors in different aspects of aging and muscle regeneration.

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.

Metabolic reprogramming by Acly inhibition using SB-204990 alters glucoregulation and modulates molecular mechanisms associated with aging.

ATP-citrate lyase is a central integrator of cellular metabolism in the interface of protein, carbohydrate, and lipid metabolism. The physiological consequences as well as the molecular mechanisms orchestrating the response to long-term pharmacologically induced Acly inhibition are unknown. We report here that the Acly inhibitor SB-204990 improves metabolic health and physical strength in wild-type mice when fed with a high-fat diet, while in mice fed with healthy diet results in metabolic imbalance and moderated insulin resistance. By applying a multiomic approach using untargeted metabolomics, transcriptomics, and proteomics, we determined that, in vivo, SB-204990 plays a role in the regulation of molecular mechanisms associated with aging, such as energy metabolism, mitochondrial function, mTOR signaling, and folate cycle, while global alterations on histone acetylation are absent. Our findings indicate a mechanism for regulating molecular pathways of aging that prevents the development of metabolic abnormalities associated with unhealthy dieting. This strategy might be explored for devising therapeutic approaches to prevent metabolic diseases.

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.

Evaluation of high oleic-high stearic sunflower hard stearins for cocoa butter equivalent formulation.

Cocoa butter equivalents (CBEs) are produced from vegetable fats by blending palm mid fraction (PMF) and tropical butters coming from shea, mango kernel or kokum fat. In this regard, high oleic-high stearic (HOHS) sunflower hard stearins from solvent fractionation can be used in CBE production since their compositions and physical properties are similar to those found in the above-mentioned tropical butters. In this work, three sunflower hard stearins (SHS) ranging from 65% to 95% of disaturated triacylglycerols and a shea stearin (used as reference) were blended with PMF to evaluate their potential use in CBEs formulation. Isosolid phase diagrams of mixtures of PMF/SHS showed eutectic formation for SHS 65 and SHS 80, but monotectic behaviour with softening effect for SHS 95. Three CBEs from SHS and shea stearin were formulated according to phase behaviour diagrams and solid fat content data at 25 °C. Isosolid phase diagrams of mixtures of these CBEs with cocoa butter showed no eutectic behaviour. Therefore, CBEs elaborated from SHS exhibited full compatibility with cocoa butter.

The Sunflower WRINKLED1 Transcription Factor Regulates Fatty Acid Biosynthesis Genes through an AW Box Binding Sequence with a Particular Base Bias.

Sunflower is an important oilseed crop in which the biochemical pathways leading to seed oil synthesis and accumulation have been widely studied. However, how these pathways are regulated is less well understood. The WRINKLED1 (WRI1) transcription factor is considered a key regulator in the control of triacylglycerol biosynthesis, acting through the AW box binding element (CNTNG(N)7CG). Here, we identified the sunflower WRI1 gene and characterized its activity in electrophoretic mobility shift assays. We studied its role as a co-regulator of sunflower genes involved in plastidial fatty acid synthesis. Sunflower WRI1-targets included genes encoding the pyruvate dehydrogenase complex, the α-CT and BCCP genes, genes encoding ACPs and the fatty acid synthase complex, together with the FATA1 gene. As such, sunflower WRI1 regulates genes involved in seed plastidial fatty acid biosynthesis in a coordinated manner, establishing a WRI1 push and pull strategy that drives oleic acid synthesis for its export into the cytosol. We also determined the base bias at the N positions in the active sunflower AW box motif. The sunflower AW box is sequence-sensitive at the non-conserved positions, enabling WRI1-binding. Moreover, sunflower WRI1 could bind to a non-canonical AW-box motif, opening the possibility of searching for new target genes.