Variation in Expression of the HECT E3 Ligase UPL3 Modulates LEC2 Levels, Seed Size, and Crop Yields in Brassica napus.

Identifying genetic variation that increases crop yields is a primary objective in plant breeding. We used association analyses of oilseed rape/canola (Brassica napus) accessions to identify genetic variation that influences seed size, lipid content, and final crop yield. Variation in the promoter region of the HECT E3 ligase gene BnaUPL3 C03 made a major contribution to variation in seed weight per pod, with accessions exhibiting high seed weight per pod having lower levels of BnaUPL3 C03 expression. We defined a mechanism in which UPL3 mediated the proteasomal degradation of LEC2, a master transcriptional regulator of seed maturation. Accessions with reduced UPL3 expression had increased LEC2 protein levels, larger seeds, and prolonged expression of lipid biosynthetic genes during seed maturation. Natural variation in BnaUPL3 C03 expression appears not to have been exploited in current B napus breeding lines and could therefore be used as a new approach to maximize future yields in this important oil crop.

PUX10 Is a CDC48A Adaptor Protein That Regulates the Extraction of Ubiquitinated Oleosins from Seed Lipid Droplets in Arabidopsis.

Postgerminative mobilization of neutral lipids stored in seed lipid droplets (LDs) is preceded by the degradation of oleosins, the major structural LD proteins that stabilize LDs in dry seeds. We previously showed that Arabidopsis thaliana oleosins are marked for degradation by ubiquitination and are extracted from LDs before proteolysis. However, the mechanisms underlying the dislocation of these LD-anchored proteins from the LD monolayer are yet unknown. Here, we report that PUX10, a member of the plant UBX-domain containing (PUX) protein family, is an integral LD protein that associates with a subpopulation of LDs during seed germination. In pux10 mutant seedlings, PUX10 deficiency impaired the degradation of ubiquitinated oleosins and prevented the extraction of ubiquitinated oleosins from LDs. We also showed that PUX10 interacts with ubiquitin and CDC48A, the AAA ATPase Cell Division Cycle 48, through its UBA and UBX domains, respectively. Collectively, these results strongly suggest that PUX10 is an adaptor recruiting CDC48A to ubiquitinated oleosins, thus facilitating the dislocation of oleosins from LDs by the segregase activity of CDC48A. We propose that PUX10 and CDC48A are core components of a LD-associated degradation machinery, which we named the LD-associated degradation system. Importantly, PUX10 is also the first determinant of a LD subpopulation described in plants, suggesting functional differentiation of LDs in Arabidopsis seedlings.

Increasing medium chain fatty acids production in Yarrowia lipolytica by metabolic engineering.

BACKGROUND: Oleaginous yeast Yarrowia lipolytica is an organism of choice for the development of biofuel and oleochemicals. It has become a chassis for metabolic engineering in order to produce targeted lipids. Understanding the function of key-enzymes involved in lipid metabolism is essential to design better routes for enhanced lipid production and for strains producing lipids of interest. Because medium chain fatty acids (MCFA) are valuable compounds for biokerosene production, we previously generated strains capable of producing MCFA up to 12% of total lipid content (Rigouin et al. in ACS Synth Biol 6:1870-1879, 2017). In order to improve accumulation and content of C14 fatty acid (FA), the elongation, degradation and accumulation of these MCFA in Yarrowia lipolytica were studied. RESULTS: We brought evidence of the role of YALI0F0654 (YlELO1) protein in the elongation of exogenous or de novo synthesized C14 FA into C16 FA and C18 FA. YlELO1 deletion into a αFAS_I1220W expressing strain leads to the sole production of C14 FA. However, because this strain does not provide the FA essential for its growth, it requires being cultivated with essential fatty acids and C14 FA yield is limited. To promote MCFA accumulation in Y. lipolytica without compromising the growth, we overexpressed a plant diglyceride acyltransferase specific for MCFA and reached an accumulation of MCFA up to 45% of total lipid content. CONCLUSION: We characterized the role of YlELO1 in Y. lipolytica by proving its involvement in Medium chain fatty acids elongation. We showed that MCFA content can be increased in Yarrowia lipolytica by promoting their accumulation into a stable storage form (triacylglycerides) to limit their elongation and their degradation.

Lipids containing medium-chain fatty acids are specific to post-whole genome duplication Saccharomycotina yeasts.

BACKGROUND: Yeasts belonging to the subphylum Saccharomycotina have been used for centuries in food processing and, more recently, biotechnology. Over the past few decades, these yeasts have also been studied in the interest of their potential to produce oil to replace fossil resources. Developing yeasts for massive oil production requires increasing yield and modifying the profiles of the fatty acids contained in the oil to satisfy specific technical requirements. For example, derivatives of medium-chain fatty acids (MCFAs, containing 6-14 carbons) are used for the production of biodiesels, cleaning products, lubricants and cosmetics. Few studies are available in the literature on the production of MCFAs in yeasts. RESULTS: We analyzed the MCFA content in Saccharomyces cerevisiae grown in various conditions. The results revealed that MCFAs preferentially accumulated when cells were grown on synthetic media with a high C/N ratio at low temperature (23 °C). Upon screening deletion mutant strains for genes encoding lipid droplet-associated proteins, we found two genes, LOA1 and TGL3, involved in MCFA homeostasis. A phylogenetic analysis on 16 Saccharomycotina species showed that fatty acid profiles differed drastically among yeasts. Interestingly, MCFAs are only present in post-whole genome duplication yeast species. CONCLUSIONS: In this study, we produced original data on fatty acid diversity in yeasts. We demonstrated that yeasts are amenable to genetic and metabolic engineering to increase their MCFA production. Furthermore, we revealed that yeast lipid biodiversity has not been fully explored, but that yeasts likely harbor as-yet-undiscovered strains or enzymes that can contribute to the production of high-value fatty acids for green chemistry.

Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis.

In oleaginous seeds, lipids--stored in organelles called oil bodies (OBs)--are degraded post-germinatively to provide carbon and energy for seedling growth. To date, little is known about how OB coat proteins, known as oleosins, control OB dynamics during seed germination. Here, we demonstrated that the sequential proteolysis of the five Arabidopsis thaliana oleosins OLE1-OLE5 begins just prior to lipid degradation. Several post-translational modifications (e.g. phosphorylation and ubiquination) of oleosins were concomitant with oleosin degradation. Phosphorylation occurred only on the minor OLE5 and on an 8 kDa proteolytic fragment of OLE2. A combination of immunochemical and proteomic approaches revealed ubiquitination of the four oleosins OLE1-OLE4 at the onset of OB mobilization. Ubiquitination topology was surprisingly complex. OLE1 and OLE2 were modified by three distinct and predominantly exclusive motifs: monoubiquitin, K48-linked diubiquitin (K48Ub(2)) and K63-linked diubiquitin. Ubiquitinated oleosins may be channeled towards specific degradation pathways according to ubiquitination type. One of these pathways was identified as the ubiquitin-proteasome pathway. A proteasome inhibitor (MG132) reduced oleosin degradation and induced cytosolic accumulation of K48Ub(2)-oleosin aggregates. These results indicate that K48Ub(2)-modified oleosins are selectively extracted from OB coat and degraded by the proteasome. Proteasome inhibition also reduced lipid hydrolysis, providing in vivo evidence that oleosin degradation is required for lipid mobilization.

The structural organization of seed oil bodies could explain the contrasted oil extractability observed in two rapeseed genotypes.

MAIN CONCLUSION: The protein, phospholipid and sterol composition of the oil body surface from the seeds of two rapeseed genotypes was compared in order to explain their contrasted oil extractability. In the mature seeds of oleaginous plants, storage lipids accumulate in specialized structures called oil bodies (OBs). These organelles consist of a core of neutral lipids surrounded by a phospholipid monolayer in which structural proteins are embedded. The physical stability of OBs is a consequence of the interactions between proteins and phospholipids. A detailed study of OB characteristics in mature seeds as well as throughout seed development was carried out on two contrasting rapeseed genotypes Amber and Warzanwski. These two accessions were chosen because they differ dramatically in (1) crushing ability, (2) oil extraction yield and, (3) the stability of purified OBs. Warzanwski has higher crushing ability, better oil extraction yield and less stable purified OBs than Amber. OB morphology was investigated in situ using fluorescence microscopy, transmission electron microscopy and pulsed field gradient NMR. During seed development, OB diameter first increased and then decreased 30 days after pollination in both Amber and Warzanwski embryos. In mature seeds, Amber OBs were significantly smaller. The protein, phospholipid and sterol composition of the hemi-membrane was compared between the two accessions. Amber OBs were enriched with H-oleosins and steroleosins, suggesting increased coverage of the OB surface consistent with their higher stability. The nature and composition of phospholipids and sterols in Amber OBs suggest that the hemi-membrane would have a more rigid structure than that of Warzanwski OBs.

N-terminus of seed caleosins is essential for lipid droplet sorting but not for lipid accumulation.

Caleosin, a calcium-binding protein associated with plant lipid droplets, stimulates lipid accumulation when heterologously expressed in Saccharomyces cerevisiae. Accumulated lipids are stored in cytoplasmic lipid droplets that are stabilised by incorporated caleosin. We designed a set of mutants affecting putative crucial sites for caleosin function and association with lipid droplets, i.e. the N-terminus, the EF-hand motif and the proline-knot motif. We investigated the effect of introduced mutations on caleosin capacity to initiate lipid accumulation and on caleosin sorting within cell as well as on its association with lipid droplets. Our results strongly suggest that the N-terminal domain is essential for proper protein sorting and targeting to lipid droplets but not for enhancing lipid accumulation.

Fold of an oleosin targeted to cellular oil bodies.

In cells, from bacteria to plants or mammals, lipids are stored in natural emulsions called oil bodies (OBs). This organelle is surrounded by a phospholipid monolayer which is thought to contain integral proteins involved in its stabilization. The insertion and fold of these proteins into the phospholipid monolayer are poorly understood. In seed OBs, the most abundant integral proteins are oleosins, which contain a 70-residue central hydrophobic domain. The secondary structure of solubilized oleosins varies greatly from mainly alpha helices to a predominantly beta sheets depending on the detergent used. To study the fold of integral membrane proteins inserted in a cellular OB environment, S3 protein, the major Arabidopsis thaliana seed oleosin, was targeted to Saccharomyces cerevisiae OBs. The diameter of purified yeast OBs harboring S3 or S3 fused with the Green Fluorescent Protein (GFP) was smaller and more homogeneous than plant OBs. Comparison of the secondary structure of S3 and S3-GFP was used to validate the structure of folded S3. Circular dichroism using synchrotron radiation indicated that S3 and S3-GFP in yeast OBs contain mainly beta secondary structures. While yeast OBs are chemically different to A. thaliana seed OBs, this approach allowed the secondary structure of S3 in OB particles to be determined for the first time.

Crop seed oil bodies: from challenges in protein identification to an emerging picture of the oil body proteome.

Oleaginous seeds store lipids in specialized structures called oil bodies (OBs). These organelles consist of a core of neutral lipids bound by proteins embedded in a phospholipid monolayer. OB proteins are well conserved in plants and have long been grouped into only two categories: structural proteins or enzymes. Recent work, however, which identified other classes of proteins associated with OBs, clearly shows that this classification is obsolete. Proteomics-mediated OB protein identification is facilitated in plants for which the genome is sequenced and annotated. However, it is not clear whether this knowledge can be dependably transposed to less well-characterized plants, including the well-established commercial sources of seed oil as well as the many others being proposed as novel sources for biodiesel, especially in Africa and Asia. Toward an update of the current data available on OB proteins this review discusses (i) the specific difficulties for proteomic studies of organelles; (ii) a 2012 census of the proteins found in seed OBs from various crops; (iii) the oleosin composition of OBs and their role in organelle stability; (iv) PTM of OB proteins as an emerging field of investigation; and finally we describe the emerging model of the OB proteome from oilseed crops.

High water solubility and fold in amphipols of proteins with large hydrophobic regions: oleosins and caleosin from seed lipid bodies.

Seed lipid bodies constitute natural emulsions stabilized by specialized integral membrane proteins, among which the most abundant are oleosins, followed by the calcium binding caleosin. These proteins exhibit a triblock structure, with a highly hydrophobic central region comprising up to 71 residues. Little is known on their three-dimensional structure. Here we report the solubilization of caleosin and of two oleosins in aqueous solution, using various detergents or original amphiphilic polymers, amphipols. All three proteins, insoluble in water buffers, were maintained soluble either by anionic detergents or amphipols. Neutral detergents were ineffective. In complex with amphipols the oleosins and caleosin contain more beta and less alpha secondary structures than in the SDS detergent, as evaluated by synchrotron radiation circular dichroism. These are the first reported structural results on lipid bodies proteins maintained in solution with amphipols, a promising alternative to notoriously denaturing detergents.

Plant organelle proteomics: collaborating for optimal cell function.

Organelle proteomics describes the study of proteins present in organelle at a particular instance during the whole period of their life cycle in a cell. Organelles are specialized membrane bound structures within a cell that function by interacting with cytosolic and luminal soluble proteins making the protein composition of each organelle dynamic. Depending on organism, the total number of organelles within a cell varies, indicating their evolution with respect to protein number and function. For example, one of the striking differences between plant and animal cells is the plastids in plants. Organelles have their own proteins, and few organelles like mitochondria and chloroplast have their own genome to synthesize proteins for specific function and also require nuclear-encoded proteins. Enormous work has been performed on animal organelle proteomics. However, plant organelle proteomics has seen limited work mainly due to: (i) inter-plant and inter-tissue complexity, (ii) difficulties in isolation of subcellular compartments, and (iii) their enrichment and purity. Despite these concerns, the field of organelle proteomics is growing in plants, such as Arabidopsis, rice and maize. The available data are beginning to help better understand organelles and their distinct and/or overlapping functions in different plant tissues, organs or cell types, and more importantly, how protein components of organelles behave during development and with surrounding environments. Studies on organelles have provided a few good reviews, but none of them are comprehensive. Here, we present a comprehensive review on plant organelle proteomics starting from the significance of organelle in cells, to organelle isolation, to protein identification and to biology and beyond. To put together such a systematic, in-depth review and to translate acquired knowledge in a proper and adequate form, we join minds to provide discussion and viewpoints on the collaborative nature of organelles in cell, their proper function and evolution.

Identification and characterization of DGA2, an acyltransferase of the DGAT1 acyl-CoA:diacylglycerol acyltransferase family in the oleaginous yeast Yarrowia lipolytica. New insights into the storage lipid metabolism of oleaginous yeasts.

Triacylglycerols (TAG) and steryl esters (SE) are the principal storage lipids in all eukaryotic cells. In yeasts, these storage lipids accumulate within special organelles known as lipid bodies (LB). In the lipid accumulation-oriented metabolism of the oleaginous yeast Yarrowia lipolytica, storage lipids are mostly found in the form of TAG, and only small amounts of SE accumulate. We report here the identification of a new DAG acyltransferase gene, DGA2, homologous to the ARE genes of Saccharomyces cerevisiae. This gene encodes a member of the type 1 acyl-CoA:diacylglycerol acyltransferase family (DGAT1), which has not previously been identified in yeasts, but is commonly found in mammals and plants. Unlike the Are proteins in S. cerevisiae, Dga2p makes a major contribution to TAG synthesis via an acyl-CoA-dependent mechanism and is not involved in SE synthesis. This enzyme appears to affect the size and morphology of LB, suggesting a direct role of storage lipid proteins in LB formation. We report that the Are1p of Y. lipolytica was essential for sterol esterification, as deletion of the encoding gene (ARE1) completely abolished SE synthesis. Unlike its homologs in yeasts, YlARE1 has no DAG acyltransferase activity. We also reconsider the role and function of all four acyltransferase enzymes involved in the final step of neutral lipid synthesis in this oleaginous yeast.

New protein isoforms identified within Arabidopsis thaliana seed oil bodies combining chymotrypsin/trypsin digestion and peptide fragmentation analysis.

Plant seed oil bodies, subcellular lipoprotein inclusions providing storage reserves, are composed of a neutral lipid core surrounded by a phospholipid monolayer with several integrated proteins that play a significant role in stabilization of the particles and probably also in lipid mobilization. Oil bodies' proteins are generally very hydrophobic, due to the long uncharged sequences anchoring them into the lipid core, which makes them extremely difficult to handle and to digest successfully. Although oil bodies have been intensively studied during last decades, not all their proteins have been identified yet. To overcome the problems connected with their identification, a method based on SDS-PAGE, in-gel digestion and LC-MS/MS analysis was used. Digestion was carried out with trypsin and chymotrypsin, single or in combination, which increased significantly the number of identified peptides, namely the hydrophobic ones. Thanks to this methodology it was possible to achieve an extensive coverage of proteins studied, to analyze their N-terminal modifications and moreover, to detect four new oil bodies' protein isoforms, which demonstrates the complexity of oil bodies' protein composition.

Deciphering the properties of hemp seed oil bodies for food applications: Lipid composition, microstructure, surface properties and physical stability.

Hemp seed oil bodies (HSOBs) are of growing interest in response to the demand of consumers for healthy and natural plant-based food formulations. In this study, we used minimal processing including aqueous extraction by grinding and centrifugation to obtain HSOBs. We determined the lipid composition of HSBOs, their microstructure, and the impact of the homogenization pressure, pH and minerals on their surface properties and the physical stability of the emulsions. HSOBs contain high levels of well-balanced PUFA with LA/ALA = 2.9, γ-tocopherol, lutein and phytosterols. The mean diameter of HSOBs was 2.3 ± 0.1 µm with an isoelectric point in the range of pH 4.4 to 4.6. Homogenization of hemp seed extracts induced a decrease in the size of HSOBs but did not eliminate the sedimentation of the protein bodies composed of the globulin edestin. By changing the surface properties of HSOBs, pH values below 6 and NaCl induced the aggregation of HSOBs, while CaCl2 induced both aggregation and membrane-fusion mediated coalescence of HSOBs by involving probably the anionic phospholipids together with membrane proteins. This study will contribute to extend the range of novel food products and designed emulsions containing hemp seed proteins and oil bodies.

Protein composition of oil bodies from mature Brassica napus seeds.

Seed oil bodies (OBs) are intracellular particles storing lipids as food or biofuel reserves in oleaginous plants. Since Brassica napus OBs could be easily contaminated with protein bodies and/or myrosin cells, they must be purified step by step using floatation technique in order to remove non-specifically trapped proteins. An exhaustive description of the protein composition of rapeseed OBs from two double-zero varieties was achieved by a combination of proteomic and genomic tools. Genomic analysis led to the identification of sequences coding for major seed oil body proteins, including 19 oleosins, 5 steroleosins and 9 caleosins. Most of these proteins were also identified through proteomic analysis and displayed a high level of sequence conservation with their Arabidopsis thaliana counterparts. Two rapeseed oleosin orthologs appeared acetylated on their N-terminal alanine residue and both caleosins and steroleosins displayed a low level of phosphorylation.

Regulation of HSD1 in seeds of Arabidopsis thaliana.

The hydroxysteroid dehydrogenase HSD1, identified in the proteome of oil bodies from mature Arabidopsis seeds, is encoded by At5g50600 and At5g50700, two gene copies anchored on a duplicated region of chromosome 5. Using a real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) approach, the accumulation of HSD1 mRNA was shown to be specifically and highly induced in oil-accumulating tissues of maturing seeds. HSD1 mRNA disappeared during germination. The activity of HSD1 promoter and the localization of HSD1 transcripts by in situ hybridization were consistent with this pattern. A complementary set of molecular and genetic analyses showed that HSD1 is a target of LEAFY COTYLEDON2, a transcriptional regulator able to bind the promoter of HSD1. Immunoblot analyses and immunolocalization experiments using anti-AtHSD1 antibodies established that the pattern of HSD1 deposition faithfully reflected mRNA accumulation. At the subcellular level, the study of HSD1:GFP fusion proteins showed the targeting of HSD1 to the surface of oil bodies. Transgenic lines overexpressing HSD1 were then obtained to test the importance of proper transcriptional regulation of HSD1 in seeds. Whereas no impact on oil accumulation could be detected, transgenic seeds exhibited lower cold and light requirements to break dormancy, germinate and mobilize storage lipids. Interestingly, overexpressors of HSD1 over-accumulated HSD1 protein in seeds but not in vegetative organs, suggesting that post-transcriptional regulations exist that prevent HSD1 accumulation in tissues deprived of oil bodies.

Heterologous expression of AtClo1, a plant oil body protein, induces lipid accumulation in yeast.

Proteomic approaches on lipid bodies have led to the identification of proteins associated with this compartment, showing that, rather than the inert fat depot, lipid droplets appear as complex dynamic organelles with roles in metabolism control and cell signaling. We focused our investigations on caleosin [Arabidopsis thaliana caleosin 1 (AtClo1)], a minor protein of the Arabidopsis thaliana seed lipid body. AtClo1 shares an original triblock structure, which confers to the protein the capacity to insert at the lipid body surface. In addition, AtClo1 possesses a calcium-binding domain. The study of plants deficient in caleosin revealed its involvement in storage lipid degradation during seed germination. Using Saccharomyces cerevisiae as a heterologous expression system, we investigated the potential role of AtClo1 in lipid body biogenesis and filling. The green fluorescent protein-tagged protein was correctly targeted to lipid bodies. We observed an increase in the number and size of lipid bodies. Moreover, transformed yeasts accumulated more fatty acids (+46.6%). We confirmed that this excess of fatty acids was due to overaccumulation of lipid body neutral lipids, triacylglycerols and steryl esters. We showed that the original intrinsic properties of AtClo1 protein were sufficient to generate a functional lipid body membrane and to promote overaccumulation of storage lipids in yeast oil bodies.

Control of lipid accumulation in the yeast Yarrowia lipolytica.

A genomic comparison of Yarrowia lipolytica and Saccharomyces cerevisiae indicates that the metabolism of Y. lipolytica is oriented toward the glycerol pathway. To redirect carbon flux toward lipid synthesis, the GUT2 gene, which codes for the glycerol-3-phosphate dehydrogenase isomer, was deleted in Y. lipolytica in this study. This Delta gut2 mutant strain demonstrated a threefold increase in lipid accumulation compared to the wild-type strain. However, mobilization of lipid reserves occurred after the exit from the exponential phase due to beta-oxidation. Y. lipolytica contains six acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX6 genes, that catalyze the limiting step of peroxisomal beta-oxidation. Additional deletion of the POX1 to POX6 genes in the Delta gut2 strain led to a fourfold increase in lipid content. The lipid composition of all of the strains tested demonstrated high proportions of FFA. The size and number of the lipid bodies in these strains were shown to be dependent on the lipid composition and accumulation ratio.

Structure and function of seed lipid-body-associated proteins.

Many organisms among the different kingdoms store reserve lipids in discrete subcellular organelles called lipid bodies. In plants, lipid bodies can be found in seeds but also in fruits (olives, ...), and in leaves (plastoglobules). These organelles protect plant lipid reserves against oxidation and hydrolysis until seed germination and seedling establishment. They can be stabilized by specific structural proteins, namely the oleosins and caleosins, which act as natural emulsifiers. Considering the putative role of some of them in controlling the size of lipid bodies, these proteins may constitute important targets for seed improvement both in term of oil seed yield and optimization of technological processes for extraction of oil and storage proteins. We present here an overview of the data on the structure of these proteins, which are scarce, and sometimes contradictory and on their functional roles.

Arabidopsis thaliana DGAT3 is a [2Fe-2S] protein involved in TAG biosynthesis.

Acyl-CoA:diacylglycerol acyltransferases 3 (DGAT3) are described as plant cytosolic enzymes synthesizing triacylglycerol. Their protein sequences exhibit a thioredoxin-like ferredoxin domain typical of a class of ferredoxins harboring a [2Fe-2S] cluster. The Arabidopsis thaliana DGAT3 (AtDGAT3; At1g48300) protein is detected in germinating seeds. The recombinant purified protein produced from Escherichia coli, although very unstable, exhibits DGAT activity in vitro. A shorter protein version devoid of its N-terminal putative chloroplast transit peptide, Δ46AtDGAT3, was more stable in vitro, allowing biochemical and spectroscopic characterization. The results obtained demonstrate the presence of a [2Fe-2S] cluster in the protein. To date, AtDGAT3 is the first metalloprotein described as a DGAT.