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.

Vitamin E prevents lipid peroxidation and iron accumulation in PLA2G6-Associated Neurodegeneration.

BACKGROUND: PLA2G6-Associated Neurodegeneration (PLAN) is a rare neurodegenerative disease with autosomal recessive inheritance, which belongs to the NBIA (Neurodegeneration with Brain Iron Accumulation) group. Although the pathogenesis of the disease remains largely unclear, lipid peroxidation seems to play a central role in the pathogenesis. Currently, there is no cure for the disease. OBJECTIVE: In this work, we examined the presence of lipid peroxidation, iron accumulation and mitochondrial dysfunction in two cellular models of PLAN, patients-derived fibroblasts and induced neurons, and assessed the effects of α-tocopherol (vitamin E) in correcting the pathophysiological alterations in PLAN cell cultures. METHODS: Pathophysiological alterations were examined in fibroblasts and induced neurons generated by direct reprograming. Iron and lipofuscin accumulation were assessed using light and electron microscopy, as well as biochemical analysis techniques. Reactive Oxygen species production, lipid peroxidation and mitochondrial dysfunction were measured using specific fluorescent probes analysed by fluorescence microscopy and flow cytometry. RESULTS: PLAN fibroblasts and induced neurons clearly showed increased lipid peroxidation, iron accumulation and altered mitochondrial membrane potential. All these pathological features were reverted with vitamin E treatment. CONCLUSIONS: PLAN fibroblasts and induced neurons reproduce the main pathological alterations of the disease and provide useful tools for disease modelling. The main pathological alterations were corrected by Vitamin E supplementation in both models, suggesting that blocking lipid peroxidation progression is a critical therapeutic target.

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.

Tailoring the composition of novel wax esters in the seeds of transgenic Camelina sativa through systematic metabolic engineering.

The functional characterization of wax biosynthetic enzymes in transgenic plants has opened the possibility of producing tailored wax esters (WEs) in the seeds of a suitable host crop. In this study, in addition to systematically evaluating a panel of WE biosynthetic activities, we have also modulated the acyl-CoA substrate pool, through the co-expression of acyl-ACP thioesterases, to direct the accumulation of medium-chain fatty acids. Using this combinatorial approach, we determined the additive contribution of both the varied acyl-CoA pool and biosynthetic enzyme substrate specificity to the accumulation of non-native WEs in the seeds of transgenic Camelina plants. A total of fourteen constructs were prepared containing selected FAR and WS genes in combination with an acyl-ACP thioesterase. All enzyme combinations led to the successful production of wax esters, of differing compositions. The impact of acyl-CoA thioesterase expression on wax ester accumulation varied depending on the substrate specificity of the WS. Hence, co-expression of acyl-ACP thioesterases with Marinobacter hydrocarbonoclasticus WS and Marinobacter aquaeolei FAR resulted in the production of WEs with reduced chain lengths, whereas the co-expression of the same acyl-ACP thioesterases in combination with Mus musculus WS and M. aquaeolei FAR had little impact on the overall final wax composition. This was despite substantial remodelling of the acyl-CoA pool, suggesting that these substrates were not efficiently incorporated into WEs. These results indicate that modification of the substrate pool requires careful selection of the WS and FAR activities for the successful high accumulation of these novel wax ester species in Camelina seeds.

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.

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.

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.

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.

An integrative "omics" approach identifies new candidate genes to impact aroma volatiles in peach fruit.

BACKGROUND: Ever since the recent completion of the peach genome, the focus of genetic research in this area has turned to the identification of genes related to important traits, such as fruit aroma volatiles. Of the over 100 volatile compounds described in peach, lactones most likely have the strongest effect on fruit aroma, while esters, terpenoids, and aldehydes have minor, yet significant effects. The identification of key genes underlying the production of aroma compounds is of interest for any fruit-quality improvement strategy. RESULTS: Volatile (52 compounds) and gene expression (4348 genes) levels were profiled in peach fruit from a maturity time-course series belonging to two peach genotypes that showed considerable differences in maturation characteristics and postharvest ripening. This data set was analyzed by complementary correlation-based approaches to discover the genes related to the main aroma-contributing compounds: lactones, esters, and phenolic volatiles, among others. As a case study, one of the candidate genes was cloned and expressed in yeast to show specificity as an ω-6 Oleate desaturase, which may be involved in the production of a precursor of lactones/esters. CONCLUSIONS: Our approach revealed a set of genes (an alcohol acyl transferase, fatty acid desaturases, transcription factors, protein kinases, cytochromes, etc.) that are highly associated with peach fruit volatiles, and which could prove useful in breeding or for biotechnological purposes.

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.

A large decrease of cytosolic triosephosphate isomerase in transgenic potato roots affects the distribution of carbon in primary metabolism.

Triosephosphate isomerase (TPI, EC 5.3.1.1) catalyzes the interconversion of dihydroxyacetone-P and glyceraldehyde 3-P in the glycolytic pathway. A constitutively expressed antisense construct for cytosolic TPI was introduced into potato (Solanum tuberosum) using Agrobacterium rhizogenes to examine the metabolic effects of a reduction in cytosolic TPI in roots. We obtained a population of transgenic root clones displaying ~36 to 100 % of the TPI activity found in control clones carrying an empty binary vector. Ion exchange chromatography and immunoblot analysis showed that the antisense strategy significantly decreased the cytosolic TPI isoform, while levels of plastidial TPI activity remained apparently unaffected. Transgenic roots were characterized with respect to the activity of glycolytic enzymes, their metabolite contents and carbon fluxes. Metabolite profiling of sugars, organic acids, amino acids and lipids showed elevated levels of sucrose, glucose, fructose, fumarate, isocitrate, 4-aminobutyrate, alanine, glycine, aromatic amino acids and saturated long chain fatty acids in roots containing the lowest TPI activity. Labelings with (14)C-glucose, (14)C-sucrose and (14)C-acetate indicated that a reduction of cytosolic TPI activity in roots increased carbon metabolism through the pentose phosphate pathway, O(2) uptake and catabolism of sucrose to CO(2), and capacity for lipid synthesis. These results demonstrate that a large reduction of cytosolic TPI alters the distribution of carbon in plant primary metabolism.

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.

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 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.

Proteome analysis of cold acclimation in sunflower.

Cold acclimation is the phenomenon in which plants are exposed to low, but nonfreezing, temperatures before exposure to drastic temperatures. To investigate how sunflower plants adjust their metabolism during cold treatment, a comparative proteomic approach, based on spectral counting data, was adopted to identify differentially expressed proteins in leaves of freezing susceptible (Hopi) and tolerant (PI 543006 and BSD-2-691) lines after cold acclimation. In total 718, 675, and 769 proteins were confidently identified by tandem mass spectrometry in Hopi, PI 543006, and BSD-2-691 sunflower lines. Tolerant lines PI 543006 and BSD-2-691 showed the highest number of differentially expressed proteins, as 43, 72, and 168 proteins changed their expression in Hopi, PI 543006, and BSD-2-691 sunflower lines, respectively, at 95% confidence. Cold-responsive proteins were mostly involved in metabolism, protein synthesis, energy, and defense processes in all sunflower lines studied. Hierarchical clustering of all differentially expressed proteins resulted in the characterization of 14 different patterns of expression across Hopi, PI 543006, and BSD-2-691 and indicated that tolerant lines showed different proteome responses to cold acclimation.