Possible crosstalk between the Arabidopsis TSPO-related protein and the transcription factor WRINKLED1.

This study uncovers a regulatory interplay between WRINKLED1 (WRI1), a master transcription factor for glycolysis and lipid biosynthesis, and Translocator Protein (TSPO) expression in Arabidopsis thaliana seeds. We identified potential WRI1-responsive elements upstream of AtTSPO through bioinformatics, suggesting WRI1's involvement in regulating TSPO expression. Our analyses showed a significant reduction in AtTSPO levels in wri1 mutant seeds compared to wild type, establishing a functional link between WRI1 and TSPO. This connection extends to the coordination of seed development and lipid metabolism, with both WRI1 and AtTSPO levels decreasing post-imbibition, indicating their roles in seed physiology. Further investigations into TSPO's impact on fatty acid synthesis revealed that TSPO misexpression alters WRI1's post-translational modifications and significantly enhances seed oil content. Additionally, we noted a decrease in key reserve proteins, including 12 S globulin and oleosin 1, in seeds with TSPO misexpression, suggesting a novel energy storage strategy in these lines. Our findings reveal a sophisticated network involving WRI1 and AtTSPO, highlighting their crucial contributions to seed development, lipid metabolism, and the modulation of energy storage mechanisms in Arabidopsis.

Lipid droplets are versatile organelles involved in plant development and plant response to environmental changes.

Since decades plant lipid droplets (LDs) are described as storage organelles accumulated in seeds to provide energy for seedling growth after germination. Indeed, LDs are the site of accumulation for neutral lipids, predominantly triacylglycerols (TAGs), one of the most energy-dense molecules, and sterol esters. Such organelles are present in the whole plant kingdom, from microalgae to perennial trees, and can probably be found in all plant tissues. Several studies over the past decade have revealed that LDs are not merely simple energy storage compartments, but also dynamic structures involved in diverse cellular processes like membrane remodeling, regulation of energy homeostasis and stress responses. In this review, we aim to highlight the functions of LDs in plant development and response to environmental changes. In particular, we tackle the fate and roles of LDs during the plant post-stress recovery phase.

Reserve lipids and plant autophagy.

Autophagy is a universal mechanism that facilitates the degradation of unwanted cytoplasmic components in eukaryotic cells. In this review, we highlight recent developments in the investigation of the role of autophagy in lipid homeostasis in plants by comparison with algae, yeast, and animals. We consider the storage compartments that form the sources of lipids in plants, and the roles that autophagy plays in the synthesis of triacylglycerols and in the formation and maintenance of lipid droplets. We also consider the relationship between lipids and the biogenesis of autophagosomes, and the role of autophagy in the degradation of lipids in plants.

Arabidopsis seedlings display a remarkable resilience under severe mineral starvation using their metabolic plasticity to remain self-sufficient for weeks.

During the life cycle of plants, seedlings are considered vulnerable because they are at the interface between the highly stress tolerant seed embryos and the established plant, and must develop rapidly, often in a challenging environment, with limited access to nutrients and light. Using a simple experimental system, whereby the seedling stage of Arabidopsis is considerably prolonged by nutrient starvation, we analysed the physiology and metabolism of seedlings maintained in such conditions up to 4 weeks. Although development was arrested at the cotyledon stage, there was no sign of senescence and seedlings remained viable for weeks, yielding normal plants after transplantation. Photosynthetic activity compensated for respiratory carbon losses, and energy dissipation by photorespiration and alternative oxidase appeared important. Photosynthates were essentially stored as organic acids, while the pool of free amino acids remained stable. Seedlings lost the capacity to store lipids in cytosolic lipid droplets, but developed large plastoglobuli. Arabidopsis seedlings arrested in their development because of mineral starvation displayed therefore a remarkable resilience, using their metabolic and physiological plasticity to maintain a steady state for weeks, allowing resumption of development when favourable conditions ensue.

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.

Lipid droplet mobilization: The different ways to loosen the purse strings.

Cytosolic lipid droplets are dynamic lipid-storage organelles that play a crucial role as reservoirs of metabolic energy and membrane precursors. These organelles are present in virtually all cell types, from unicellular to pluricellular organisms. Despite similar structural organization, lipid droplets are heterogeneous in morphology, distribution and composition. The protein repertoire associated to lipid droplet controls the organelle dynamics. Distinct structural lipid droplet proteins are associated to specific lipolytic pathways. The role of these structural lipid droplet-associated proteins in the control of lipid droplet degradation and lipid store mobilization is discussed. The control of the strictly-regulated lipolysis in lipid-storing tissues is compared between mammals and plants. Differences in the cellular regulation of lipolysis between lipid-storing tissues and other cell types are also discussed.

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.

Specialization of oleosins in oil body dynamics during seed development in Arabidopsis seeds.

Oil bodies (OBs) are seed-specific lipid storage organelles that allow the accumulation of neutral lipids that sustain plantlet development after the onset of germination. OBs are covered with specific proteins embedded in a single layer of phospholipids. Using fluorescent dyes and confocal microscopy, we monitored the dynamics of OBs in living Arabidopsis (Arabidopsis thaliana) embryos at different stages of development. Analyses were carried out with different genotypes: the wild type and three mutants affected in the accumulation of various oleosins (OLE1, OLE2, and OLE4), three major OB proteins. Image acquisition was followed by a detailed statistical analysis of OB size and distribution during seed development in the four dimensions (x, y, z, and t). Our results indicate that OB size increases sharply during seed maturation, in part by OB fusion, and then decreases until the end of the maturation process. In single, double, and triple mutant backgrounds, the size and spatial distribution of OBs are modified, affecting in turn the total lipid content, which suggests that the oleosins studied have specific functions in the dynamics of lipid accumulation.

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.

Oil body proteins sequentially accumulate throughout seed development in Brassica napus.

Despite the importance of seed oil bodies (OBs) as enclosed compartments for oil storage, little is known about lipid and protein accumulation in OBs during seed formation. OBs from rapeseed (Brassica napus) consist of a triacylglycerol (TAG) core surrounded by a phospholipid monolayer embedded with integral proteins which confer high stability to OBs in the mature dry seed. In the present study, we investigated lipid and protein accumulation patterns throughout seed development (from 5 to 65 days after pollination [DAP]) both in the whole seed and in purified OBs. Deposition of the major proteins (oleosins, caleosins and steroleosins) into OBs was assessed through (i) gene expression pattern, (ii) proteomics analysis, and (iii) protein immunodetection. For the first time, a sequential deposition of integral OB proteins was established. Accumulation of oleosins and caleosins was observed starting from early stages of seed development (12-17 DAP), while steroleosins accumulated later (~25 DAP) onwards.

The fatty acid desaturase 3 gene encodes for different FADS3 protein isoforms in mammalian tissues.

In 2000, Marquardt et al. (A. Marquardt, H. Stöhr, K. White, and B. H. F. Weber. 2000. cDNA cloning, genomic structure, and chromosomal localization of three members of the human fatty acid desaturase family. Genomics. 66: 176-183.) described the genomic structure of the fatty acid desaturase (FADS) cluster in humans. This cluster includes the FADS1 and FADS2 genes encoding, respectively, for the Delta 5- and Delta 6-desaturases involved in polyunsaturated fatty acid biosynthesis. A third gene, named FADS3, has recently been identified but no functional role has yet been attributed to the putative FADS3 protein. In this study, we investigated the FADS3 occurrence in rat tissues by using two specific polyclonal antibodies directed against the N-terminal and C-terminal ends of rat FADS3. Our results showed three potential protein isoforms of FADS3 (75 kDa, 51 kDa, and 37 kDa) present in a tissue-dependent manner. The occurrence of these FADS3 isoforms did not depend on the mRNA level determined by real-time PCR. In parallel, mouse tissues were also tested and showed the same three FADS3 isoforms but with a different tissue distribution. Finally, we reported the existence of FADS3 in human cells and tissues but different new isoforms were identified. To conclude, we showed in this study that FADS3 does exist under multiple protein isoforms depending on the mammalian tissues. These results will help further investigations to determine the physiological function of FADS3.

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.

Selective one-step extraction of Arabidopsis thaliana seed oleosins using organic solvents.

Oleosins are hydrophobic proteins from oleaginous seeds, surrounding and stabilizing oil bodies. They are known to display interesting interfacial properties. Specific sera were raised against four different A. thaliana oleosins and used in dot-blot assays for oleosin quantification. These assays were used to set up extraction of oleosins from A. thaliana seeds. One mixture of chloroform/methanol gave optimal oleosin extraction. Extracted proteins represented 9% of seed proteins and were identified by immunoblot and proteomic analyses. Oleosins accounted for 79% of the extracted proteins. This simple one-step procedure allows selective extraction and concentration of oleosins from seeds without tedious oil body purification. Oleosin extract was indeed used to demonstrate the presence of the rare oleosin S5 in mature seeds. Moreover, this method will be useful to investigate the potential use of oleosins as emulsifier and to question their possible allergenicity.

Structural properties of caleosin: a MS and CD study.

We have investigated the covalent and secondary solution structure of caleosin, a 27-kDa protein also called ATS1 or AtClo1 (At4g26740) found within Arabidopsis thaliana seed lipid bodies. The native protein was partly phosphorylated at S225. Purified bacterially expressed caleosin (recClo) was not phosphorylated; cysteine residues C221 and C230 were connected by a disulfide bridge. In solution it exists as a mixture of predominant monomers and covalent dimers. We have used recClo as a model for the study of AtClo1 secondary structure. recClo is folded in aqueous solution (16% alpha-helix, 29% beta-sheet), its secondary structure being dramatically influenced by the polarity of media, as deduced from CD spectra measured in the presence of increasing concentrations of various aliphatic alcohols.

Myristic acid increases delta6-desaturase activity in cultured rat hepatocytes.

In order to study the effects of saturated fatty acids on delta6-desaturase activity, rat hepatocytes in primary culture were incubated with lauric (C12:0), myristic (C14:0) or palmitic (C16:0) acids. After optimization, the standard in vitro conditions for the measurement of delta6-desaturase activity were as follows: 60 micromol x L(-1) alpha-linolenic acid (C18:3n-3), reaction time of 20 min and protein content of 0.4 mg. Data showed that cell treatment with 0.5 mmol x L(-1) myristic acid during 43 h specifically increased delta6-desaturase activity. This improvement, reproducible for three substrates of delta6-desaturase, i.e. oleic acid (C18:1n-9), linoleic acid (C18:2n-6) and alpha-linoleic acid (C18:3n-3) was dose-dependent in the range 0.1-0.5 mmol x L(-1) myristic acid concentration.

Protein composition of oil bodies in Arabidopsis thaliana ecotype WS.

Till now, only scattered data are available in the literature, which describes the protein content of plant oil bodies. Especially, the proteins closely associated with the model plant Arabidopsis thaliana oil bodies have never been previously purified and characterized. Oil bodies have been purified using flotation techniques, combined with incubations under high salt concentration, in the presence of detergents and urea in order to remove non-specifically trapped proteins. The identity and integrity of the oil bodies have been characterized. Oil bodies exhibited hydrodynamic diameters close to 2.6 microm, and a ratio fatty acid-protein content near 20. The proteins composing these organelles were extracted, separated by SDS-PAGE, digested by trypsin, and their peptides were subsequently analyzed by nano-chromatography-mass spectrometry (nano-LC-MS/MS). This led to the identification of a limited number of proteins: four different oleosins, ATS1, a protein homologous to calcium binding protein, a 11-beta-hydroxysteroid dehydrogenase-like protein, a probable aquaporin and a glycosylphosphatidylinositol-anchored protein with no known function. The two last proteins were till now never identified in plant oil bodies. Structural proteins (oleosins) represented up to 79% of oil body proteins and the 18.5 kDa oleosin was the most abundant among them.

Lipid accumulation, lipid body formation, and acyl coenzyme A oxidases of the yeast Yarrowia lipolytica.

Yarrowia lipolytica contains five acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX5 genes, that catalyze the limiting step of peroxisomal beta-oxidation. In this study, we analyzed morphological changes of Y. lipolytica growing in an oleic acid medium and the effect of POX deletions on lipid accumulation. Protrusions involved in the uptake of lipid droplets (LDs) from the medium were seen in electron micrographs of the surfaces of wild-type cells grown on oleic acid. The number of protrusions and surface-bound LDs increased during growth, but the sizes of the LDs decreased. The sizes of intracellular lipid bodies (LBs) and their composition depended on the POX genotype. Only a few, small, intracellular LBs were observed in the mutant expressing only Aox4p (Deltapox2 Deltapox3 Deltapox5), but strains expressing either Aox3p or both Aox3p and Aox4p had the same number of LBs as did the wild type. In contrast, strains expressing either Aox2p or both Aox2p and Aox4p formed fewer, but larger, LBs than did the wild type. The size of the LBs increased proportionately with the amount of triacylglycerols in the LBs of the mutants. In summary, Aox2p expression regulates the size of cellular triacylglycerol pools and the size and number of LBs in which these fatty acids accumulate.

Distinct roles of endoplasmic reticulum cytochrome b5 and fused cytochrome b5-like domain for rat Delta6-desaturase activity.

The Delta6-desaturase catalyzes key steps in long-chain polyunsaturated fatty acid biosynthesis. Although the gene coding for this enzyme has been isolated in diverse animal species, the protein structure remains poorly characterized. In this work, rat Delta6-desaturase expressed in COS-7 cells was shown to localize in the endoplasmic reticulum. As the enzyme contains an N-terminal cytochrome b5-like domain, we investigated by site-directed mutagenesis the role of this domain in the enzyme activity. The typical HPGG motif of the cytochrome b5-like domain, and particularly histidine in this motif, is required for the activity of the enzyme, whatever the substrate. Neither endogenous COS-7 cytochrome b5 nor coexpressed rat endoplasmic reticulum cytochrome b5 could rescue the activity of mutated forms of Delta6-desaturase. Moreover, when rat endoplasmic reticulum cytochrome b5 was coexpressed with wild-type desaturase, both proteins interacted and Delta6-desaturase activity was significantly increased. The identified interaction between these proteins is not dependent on the desaturase HPGG motif. These data suggest distinct and essential roles for both the desaturase cytochrome b5-like domain and free endoplasmic reticulum cytochrome b5 for Delta6-desaturase activity.