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.

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.

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.

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.

Characterization and impact of sunflower plastidial octanoyltransferases (Helianthus annuus L.) on oil composition.

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.

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.

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.

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.

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.

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.

Characterization and function of a sunflower (Helianthus annuus L.) Class II acyl-CoA-binding protein.

Acyl-CoA-binding proteins (ACBP) bind to long-chain acyl-CoA esters and phospholipids, enhancing the activity of different acyltransferases in animals and plants. Nevertheless, the role of these proteins in the synthesis of triacylglycerols (TAGs) remains unclear. Here, we cloned a cDNA encoding HaACBP1, a Class II ACBP from sunflower (Helianthus annuus), one of the world's most important oilseed crop plants. Transcriptome analysis of this gene revealed strong expression in developing seeds from 16 to 30 days after flowering. The recombinant protein (rHaACBP1) was expressed in Escherichia coli and purified to be studied by in vitro isothermal titration calorimetry and for phospholipid binding. Its high affinity for saturated palmitoyl-CoA (16:0-CoA; KD 0.11 µM) and stearoyl-CoA (18:0-CoA; KD 0.13 µM) esters suggests that rHaACBP1 could act in acyl-CoA transfer pathways that involve saturated acyl derivatives. Furthermore, rHaACBP1 also binds to both oleoyl-CoA (18:1-CoA; KD 6.4 µM) and linoleoyl-CoA (18:2-CoA; KD 21.4 µM) esters, the main acyl-CoA substrates used to synthesise the TAGs that accumulate in sunflower seeds. Interestingly, rHaACBP1 also appears to bind to different species of phosphatidylcholines (dioleoyl-PC and dilinoleoyl-PC), glycerolipids that are also involved in TAG synthesis, and while it interacts with dioleoyl-PA, this is less prominent than its binding to the PC derivative. Expression of rHaACBP in yeast alters its fatty acid composition, as well as the composition and size of the host acyl-CoA pool. These results suggest that HaACBP1 may potentially fulfil a role in the transport and trafficking of acyl-CoAs during sunflower seed development.

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).

Impact of sunflower (Helianthus annuus L.) plastidial lipoyl synthases genes expression in glycerolipids composition of transgenic Arabidopsis plants.

Lipoyl synthases are key enzymes in lipoic acid biosynthesis, a co-factor of several enzyme complexes involved in central metabolism. Plant pyruvate dehydrogenase complex (PDH), located in mitochondria and plastids, catalyses the first step of fatty acid biosynthesis in these organelles. Among their different components, the E2 subunit requires the lipoic acid prosthetic group to be active. De novo lipoic acid biosynthesis is achieved by the successive action of two enzymes on octanoyl-ACP: octanoyltransferase (LIP2) and lipoyl synthase (LIP1). In this study, two plastidial lipoyl synthase genes from sunflower (Helianthus annuus L.) were identified (HaLIP1p1 and HaLIP1p2), sequenced and cloned in a heterologous production system (Escherichia coli). Gene expression studies revealed similar expression patterns for both isoforms, with a slight predominance of HaLIP1p1 in vegetative tissues and mature seeds. Tertiary structural models for these enzymes indicate they both have the same theoretical catalytic sites, using lipoyl-lys and 5-deoxyadenosine as docking substrates. The fatty acid profile of E. coli cells overexpressing HaLIP1p1 and HaLIP1p2 did not present major differences, and the in vivo activity of both proteins was confirmed by complementation of an E. coli JW0623 mutant in which lipoyl synthase is defective. Although no significant differences were detected in the total fatty acid composition of transgenic Arabidopsis thaliana seeds overexpressing any of both proteins, a lipidomic analysis revealed a redistribution of the glycerolipid species, accompanied with increased phosphatidylethanolamine (PE) content and a decrease in diacyglycerols (DAG) and phosphatidylcholine (PC). Depletion of the SAM co-factor caused by HaLIP1p1 and HaLIP1p2 overexpression in transgenic plants could explain this remodelling through its effects on PC synthesis.

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.

Agrobacterium-Mediated Transient Gene Expression in Developing Ricinus communis Seeds: A First Step in Making the Castor Oil Plant a Chemical Biofactory.

The castor oil plant represents a promising platform to produce oils with industrial applications. However, its use in biotechnology is limited by the absence of a well-established procedure to transform it, and a poor understanding of gene regulation and promoter use in this species. As such, a method has been developed to express proteins or hairpin-RNA in this plant, a method based on the direct injection of Agrobacterium into the developing endosperm of castor oil fruit, enabling different constructs and promoters to be tested. This method produces a high rate of transformation and a good proportion of viable seeds that express reporter genes for up to 20 days after infiltration (DAI). Gene expression under the control of different promoters was tested by quantitative real-time polymerase chain reaction and by directly assaying the activity of the galactouronidase reporter gene, which proved to be strongest when driven by the glycinin promoter. Constructs expressing a fatty acid elongase from Lesquerella fendleri were tested, the expression of which provoked an important increase in the lesquerolic acid in the castor oil endosperm at 5 and 10 DAI, although this fatty acid did not accumulate significantly in the final mature seeds. The nature of this response could reflect the poor availability of substrates for this enzyme. In the light of this data, the potential of this technique to test promoters and different constructs in castor oil plants and other oilseeds is discussed.

Lipidomic Analysis of Plastidial Octanoyltransferase Mutants of Arabidopsis thaliana.

Plant de novo fatty acid synthesis takes place in the plastid using acetyl-coenzyme A (acetyl-CoA) as the main precursor. This first intermediate is produced from pyruvate through the action of the plastidial pyruvate dehydrogenase complex (PDH), which catalyses the oxidative decarboxylation of pyruvate to produce acetyl-CoA, CO2, and NADH. For the proper functioning of this complex, lipoic acid is required to be bound to the dihydrolipoamide S-acetyltransferase E2 subunit of PDH. Octanoyltransferase (LIP2; EC 2.3.1.181) and lipoyl synthase (LIP1; EC 2.8.1.8) are the enzymes involved in the biosynthesis of this essential cofactor. In Arabidopsis plastids, an essential lipoyl synthase (AtLIP1p) and two redundant octanoyltransferases (AtLIP2p1 and AtLIP2p2) have been described. In the present study, the lipidomic characterization of Arabidopsis octanoyltransferase mutants reveals new insight into the lipoylation functions within plastid metabolism. Lipids and fatty acids from mature seeds and seedlings from Atlip2p1 and Atlip2p2 mutants were analysed by gas chromatography (GC) and liquid chromatography-electrospray ionization high-resolution mass spectrometry (LC-ESI-HRMS2), the analysis revealed changes in fatty acid profiles that showed similar patterns in both mutant seeds and seedlings and in the lipid species containing those fatty acids. Although both mutants showed similar tendencies, the lack of the AtLIP2p2 isoform produced a more acute variation in its lipids profile. These changes in fatty acid composition and the increase in their content per seed point to the interference of octanoyltransferases in the fatty acid synthesis flux in Arabidopsis thaliana seeds.

Inadequate control of thyroid hormones sensitizes to hepatocarcinogenesis and unhealthy aging.

An inverse correlation between thyroid hormone levels and longevity has been reported in several species and reduced thyroid hormone levels have been proposed as a biomarker for healthy aging and metabolic fitness. However, hypothyroidism is a medical condition associated with compromised health and reduced life expectancy. Herein, we show, using wild-type and the Pax8 ablated model of hypothyroidism in mice, that hyperthyroidism and severe hypothyroidism are associated with an overall unhealthy status and shorter lifespan. Mild hypothyroid Pax8 +/- mice were heavier and displayed insulin resistance, hepatic steatosis and increased prevalence of liver cancer yet had normal lifespan. These pathophysiological conditions were precipitated by hepatic mitochondrial dysfunction and oxidative damage accumulation. These findings indicate that individuals carrying mutations on PAX8 may be susceptible to develop liver cancer and/or diabetes and raise concerns regarding the development of interventions aiming to modulate thyroid hormones to promote healthy aging or lifespan in mammals.