Carine Deruyffelaere

Zita Purkrtova

Isabelle Bouchez

Boris Collet

Jean-Luc Cacas

Thierry Chardot

Jean-Luc Gallois

Sabine D'andrea
email: sabine.dandrea@inra.fr

PUX10 is a CDC48A Adaptor Protein that Regulates the Extraction of Ubiquitinated Oleosins from Seed Lipid Droplets in Arabidopsis

Post-germinative 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 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 (LDAD) system. Importantly, PUX10 is also the first determinant of a LD subpopulation described in plants, suggesting functional differentiation of LDs in Arabidopsis seedlings.

Introduction

Essentially all cell types are able to synthesize and store neutral lipids. Storage lipids are packaged into specialized organelles called lipid droplets (LDs; also referred to as oil bodies, lipid bodies or oleosomes). Originally recognized for their importance in energy storage, LDs also play important roles in many cellular processes, including lipid transport and membrane synthesis, protein storage and degradation, lipid signaling, and stress response [START_REF] Chapman | Acquisition of membrane lipids by differentiating glyoxysomes: role of lipid bodies[END_REF][START_REF] Chapman | Biogenesis and functions of lipid droplets in plants: Thematic Review Series: Lipid Droplet Synthesis and Metabolism: from Yeast to Man[END_REF][START_REF] Murphy | The dynamic roles of intracellular lipid droplets: from archaea to mammals[END_REF][START_REF] Arrese | Lipid Droplets as Signaling Platforms Linking Metabolic and Cellular Functions[END_REF][START_REF] Welte | Lipid droplet functions beyond energy storage[END_REF][START_REF] Huang | Plant Lipid Droplets and Their Associated Proteins: Potential for Rapid Advances[END_REF].

The structure of LDs is unique among organelles and conserved across kingdoms, with a core of neutral lipids (mainly triacylglycerols and sterol esters) encircled by a phospholipid monolayer that contains specific proteins. Among the array of proteins identified in LDs isolated from different cell types and organisms, a specific set of structural proteins predominates. They belong to the oleosin (OLE) family in plant seeds, to the perilipin (PLIN) family in mammals, and to the major lipid droplet protein (MLDP) family in microalgae [START_REF] D'andrea | Lipid droplet mobilization: The different ways to loosen the purse strings[END_REF][START_REF] Goold | Microalgal lipid droplets: composition, diversity, biogenesis and functions[END_REF][START_REF] Bersuker | Establishing the lipid droplet proteome: Mechanisms of lipid droplet protein targeting and degradation[END_REF].

In plants, besides the seed-and pollen-specific oleosins [START_REF] Huang | Bioinformatics Reveal Five Lineages of Oleosins and the Mechanism of Lineage Evolution Related to Structure/Function from Green Algae to Seed Plants[END_REF], a class of structural LD-associated proteins called lipid-droplet associated proteins (LDAPs; also abbreviated as SRPs) has recently been identified in non-seed tissues such as leaves and oleaginous fruit tissues [START_REF] Gidda | Lipid dropletassociated proteins (LDAPs) are involved in the compartmentalization of lipophilic compounds in plant cells[END_REF][START_REF] Horn | Identification of a new class of lipid droplet-associated proteins in plants[END_REF][START_REF] Kim | Arabidopsis Small Rubber Particle Protein Homolog SRPs Play Dual Roles as Positive Factors for Tissue Growth and Development and in Drought Stress Responses[END_REF]. Despite the absence of primary sequence similarity, these different families of structural LD proteins share a common role in controlling lipid mobilization.

Overexpression and deficiency experiments support this notion. Indeed, overexpression or ectopic expression of oleosins, perilipins or LDAPs consistently induces the accumulation of neutral lipids [START_REF] Brasaemle | Perilipin A increases triacylglycerol storage by decreasing the rate of triacylglycerol hydrolysis[END_REF][START_REF] Jamme | Single cell synchrotron FT-IR microspectroscopy reveals a link between neutral lipid and storage carbohydrate fluxes in S. cerevisiae[END_REF][START_REF] Gidda | Lipid Droplet-Associated Proteins (LDAPs) Are Required for the Dynamic Regulation of Neutral Lipid Compartmentation in Plant Cells[END_REF]. This accumulation could result from either reduced lipolysis or increased LD formation, but the latter hypothesis is unlikely in plants because oleosin or LDAP deficiency induces limited to no decrease in triacylglycerol accumulation [START_REF] Miquel | Specialization of Oleosins in Oil Body Dynamics during Seed Development in Arabidopsis Seeds[END_REF][START_REF] Gidda | Lipid Droplet-Associated Proteins (LDAPs) Are Required for the Dynamic Regulation of Neutral Lipid Compartmentation in Plant Cells[END_REF]. Oleosins, perilipins and LDAPs may prevent lipolysis by covering the entire LD surface and shielding triacylglycerols from lipases. They can also regulate lipolysis by interacting with cytoplasmic lipases and regulatory proteins, a welldocumented role for perilipins but probably not fulfilled by oleosins [START_REF] D'andrea | Lipid droplet mobilization: The different ways to loosen the purse strings[END_REF][START_REF] Sztalryd | The perilipin family of lipid droplet proteins: Gatekeepers of intracellular lipolysis[END_REF]. How these major LD proteins are removed from the LD surface to allow the turnover of storage lipids remains largely an unanswered question. Part of the answer depends on how these proteins interact with the LD surface. According to [START_REF] Kory | Targeting Fat: Mechanisms of Protein Localization to Lipid Droplets[END_REF], LDassociated proteins can be classified into two groups based on how they target LDs.

Class II LD proteins access the LD surface from the cytosol and bind through amphipathic helices or domains. PLINs are recognized as class II LD proteins [START_REF] Kory | Targeting Fat: Mechanisms of Protein Localization to Lipid Droplets[END_REF][START_REF] Rowe | Conserved Amphipathic Helices Mediate Lipid Droplet Targeting of Perilipins 1-3[END_REF]. LDAPs may belong to this class as they localize predominantly to the cytosol when overexpressed in cells lacking abundant LDs, but the mechanism for their LD association is currently unknown [START_REF] Gidda | Lipid Droplet-Associated Proteins (LDAPs) Are Required for the Dynamic Regulation of Neutral Lipid Compartmentation in Plant Cells[END_REF]. By contrast, class I LD proteins are co-translationally inserted into the endoplasmic reticulum (ER) and translocated from the ER to nascent LDs (which originated by the accumulation of neutral lipids between the two leaflets of the ER membrane bilayer) or to expanding LDs through ER-LD membrane bridges [START_REF] Wilfling | Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets[END_REF]. Class I proteins are embedded into the LD monolayer by hydrophobic sequences that frequently adopt a hairpin topology. Contrary to class II proteins, they cannot translocate from LDs to the cytosol and must be removed from LDs by a specific, yet unknown mechanism.

Oleosins are the first identified and best structurally described class I LD proteins. Cotranslationally inserted in ER-budding LDs, they exclusively localize to LDs through a hydrophobic hairpin domain that deeply penetrates into the LD core [START_REF] Beaudoin | Targeting and membrane-insertion of a sunflower oleosin in vitro and in Saccharomyces cerevisiae: the central hydrophobic domain contains more than one signal sequence, and directs oleosin insertion into the endoplasmic reticulum membrane using a signal anchor sequence mechanism[END_REF][START_REF] Huang | Unique Motifs and Length of Hairpin in Oleosin Target the Cytosolic Side of Endoplasmic Reticulum and Budding Lipid Droplet[END_REF]. This hairpin, consisting of a conserved 72residue hydrophobic region with a central proline knot, is indeed too long to be stable in the phospholipid bilayer of a membrane. Seed oleosins have short hydrophilic N-and Cterminal peptides flanking the central hydrophobic hairpin and exposed to the LD surface [START_REF] Jolivet | Structural proteomics: Topology and relative accessibility of plant lipid droplet associated proteins[END_REF]. This amphipathic tri-block structure is probably responsible for the ability of oleosins to reduce interfacial tension at the oil/water interface in vitro [START_REF] Roux | Oleosins of Arabidopsis thaliana: expression in Escherichia coli, purification, and functional properties[END_REF] and to stabilize LDs in vivo. In mature seeds, oleosin deficiency is indeed associated with giant LDs instead of normal submicrometer-sized LDs, showing that oleosins control LD size and stability by preventing LD coalescence during desiccation [START_REF] Siloto | The accumulation of oleosins determines the size of seed oilbodies in Arabidopsis[END_REF][START_REF] Schmidt | Suppression of soybean oleosin produces micro-oil bodies that aggregate into oil body/ER complexes[END_REF][START_REF] Miquel | Specialization of Oleosins in Oil Body Dynamics during Seed Development in Arabidopsis Seeds[END_REF].

In Arabidopsis thaliana seeds, the degradation of the oleosin coat surrounding LDs begins during seed germination [START_REF] Deruyffelaere | Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis[END_REF]. Oleosin proteolysis thus starts before lipid mobilization, which occurs post-germinatively to sustain seedling growth before the onset of photosynthesis. This observation suggests that proteolysis of oleosins is a prerequisite for lipolysis and brings up the question of how these class I LD proteins are degraded. Previous studies have shown that oleosins are marked for degradation by ubiquitination [START_REF] Hsiao | Ubiquitination of oleosin-H and caleosin in sesame oil bodies after seed germination[END_REF][START_REF] Deruyffelaere | Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis[END_REF]. The ubiquitination profile of oleosins in germinating seeds is surprisingly complex. Three distinct and exclusive motifs can be attached to the most abundant Arabidopsis oleosins OLE1 and OLE2: monoubiquitin, K48-linked diubiquitin chain, and K63-linked diubiquitin chain [START_REF] Deruyffelaere | Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis[END_REF]. These different ubiquitinations may channel modified oleosins towards specific degradation pathways according to ubiquitination type. One of these pathways was identified as the ubiquitin-proteasome pathway, which specifically degrades oleosins marked by the addition of a K48-linked diubiquitin. Once ubiquitinated, these oleosins are extracted from the LD coat before proteasomal degradation [START_REF] Deruyffelaere | Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis[END_REF]. However, the molecular mechanism underlying oleosin extraction still remains unknown.

Proteomic analyses of seed LDs revealed two candidate proteins that could be involved in this mechanism. One candidate is a putative ubiquitin-binding protein belonging to the plant UBX-domain containing (PUX) protein family, named PUX10. The other candidate is a putative PUX10 interactant called cell division cycle 48 homolog A (CDC48A).

CDC48A is a chaperone-like ATPase known for its role in the ER-associated degradation (ERAD) of misfolded proteins [START_REF] Liu | Endoplasmic reticulum-mediated protein quality control in Arabidopsis[END_REF]. We showed that PUX10 mutations impair the degradation of all three types of ubiquitinated oleosins, and prevent the dislocation of ubiquitinated oleosins from LDs, which precedes LD degradation. Our results also revealed that PUX10 is an integral LD protein that acts as 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 ERAD-like degradation machinery, which we named LD-associated degradation (LDAD).

Results

PUX10 and CDC48A, two candidates for oleosin extraction from LDs

To gain insight into the proteins involved in LD dynamics in seeds, we previously performed a proteomics analysis of LDs isolated from Arabidopsis thaliana seeds at various post-germinative stages [START_REF] Deruyffelaere | Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis[END_REF]. A search for candidates involved in the degradation of ubiquitinated oleosins only resulted in the identification of a putative ubiquitin-binding protein called PUX10 and the CDC48A ATPase (Supplemental Data Set 1 and Supplemental Figure 1). The latter protein belongs to a family of highly conserved ATPases named AAA-ATPases (ATPases associated with various cellular activities) involved in many processes ensuring cellular homeostasis, including protein degradation by the ubiquitin-proteasome system (UPS) and by autophagy [START_REF] Barthelme | Origin and Functional Evolution of the Cdc48/p97/VCP AAA+ Protein Unfolding and Remodeling Machine[END_REF]. Members of the CDC48 family (called Cdc48 in yeast and p97 or VCP in metazoans) are considered to mediate these functions through their ubiquitin-selective segregase activity that separates ubiquitinated proteins from binding partners or cellular structures [START_REF] Jentsch | Cdc48 (p97): a "molecular gearbox" in the ubiquitin pathway?[END_REF]. CDC48 proteins indirectly associate with ubiquitinated substrates through cofactors. These substraterecruiting cofactors bind CDC48s via specific interaction domains like the ubiquitin regulatory X (UBX) domain.

PUX10 contains a putative UBX domain at its C terminus (Figure 1A and Supplemental Figure 2), indicating that it may act as a CDC48A cofactor. The Arabidopsis genome encodes 16 UBX-containing proteins (called PUX1-16) and four of them (PUX1, PUX4, PUX5 and PUX7) have so far been shown to interact with CDC48A [START_REF] Rancour | Plant UBX Domain-containing Protein 1, PUX1, Regulates the Oligomeric Structure and Activity of Arabidopsis CDC48[END_REF][START_REF] Park | Protein domain-domain interactions and requirements for the negative regulation of Arabidopsis CDC48/p97 by the plant ubiquitin regulatory X (UBX) domain-containing protein, PUX1[END_REF][START_REF] Gallois | Functional characterization of the plant ubiquitin regulatory X (UBX) domain-containing protein AtPUX7 in Arabidopsis thaliana[END_REF]. PUX10 also contains a UBA-like domain, which is known to bind ubiquitin [START_REF] Hurley | Ubiquitin-binding domains[END_REF]. As such, it appears that PUX10 may have a role in the degradation of oleosins as an adaptor bridging ubiquitinated oleosins to CDC48A. The hypothesis of a role of PUX10 in LD degradation is strengthened by the observation that PUX10 is the closest putative Arabidopsis ortholog of mammalian FAF2/UBXD8 and yeast Ubx2p, two LD-associated proteins known to control LD dynamics [START_REF] Suzuki | Derlin-1 and UBXD8 are engaged in dislocation and degradation of lipidated ApoB-100 at lipid droplets[END_REF][START_REF] Wang | The ubiquitin-like (UBX)-domain-containing protein Ubx2/Ubxd8 regulates lipid droplet homeostasis[END_REF][START_REF] Olzmann | Spatial regulation of UBXD8 and p97/VCP controls ATGL-mediated lipid droplet turnover[END_REF]. Indeed, PUX10 clusters in the same phylogenetic group as FAF2/UBXD8 and Ubx2p, unlike the other Arabidopsis PUXs (Supplemental Figure 3; Supplemental Dataset 2). PUX10 is also the only Arabidopsis PUX containing a putative membrane-anchoring domain, as predicted by CCTOP algorithms (http://cctop.enzim.ttk.mta.hu). The two membrane-anchoring domains detected in PUX10 (amino acid residues 102-117 and 120-141, highlighted in yellow in Supplemental Figure 2) may form a hydrophobic hairpin, which is a characteristic structure of class I LD proteins [START_REF] Thiam | The biophysics and cell biology of lipid droplets[END_REF][START_REF] Kory | Targeting Fat: Mechanisms of Protein Localization to Lipid Droplets[END_REF]. Such a hairpin domain, sometimes referred to as the HP domain, is present in UBXD8 and Ubx2p and required for membrane and LD targeting [START_REF] Lee | Identification of Ubxd8 protein as a sensor for unsaturated fatty acids and regulator of triglyceride synthesis[END_REF][START_REF] Wang | The ubiquitin-like (UBX)-domain-containing protein Ubx2/Ubxd8 regulates lipid droplet homeostasis[END_REF]. Moreover, PUX10 displays a domain architecture similar to that of UBXD8 and Ubx2p, with the hydrophobic region located between the N-terminal UBA domain and the central UAS domain (Figure 1A and Supplemental Figure 2). Taken together, these data highlight PUX10 and CDC48A as good candidates for controlling oleosin degradation during LD mobilization. To explore their role in LD dynamics, we have undertaken functional analyses using T-DNA mutants. Because CDC48A-deficient mutants were reported to be lethal [START_REF] Park | In Planta Analysis of the Cell Cycle-Dependent Localization of AtCDC48A and Its Critical Roles in Cell Division, Expansion, and Differentiation[END_REF], we focused our study on PUX10 mutants.

Isolation and characterization of pux10 mutants

Two T-DNA insertion mutants were isolated from the SAIL and FLAG collections and called pux10-1 (SAIL_1187) and pux10-2 (FLAG_211E02). The T-DNA inserts of pux10-1 and pux10-2 mutants mapped to the first and third exons, respectively (Figure 1A (left)). We analyzed PUX10 expression by RT-PCR in mutant backgrounds. As expected, RT-PCR amplification using primers on either side of the T-DNA insertion site occurred in Columbia (Col-0) and Wassilewskija (Ws) accessions but not in pux10-1 or pux10-2 mutants (Figure 1A (right); F4-R3 and F5-R2 PCRs for pux10-1; F4-R3 and F3-R3 PCRs for pux10-2). However, DNA fragments were amplified from PUX10 transcripts in mutant backgrounds when primers located upstream (F5-R2 for pux10-2) or downstream (F3-R3 for pux10-1, and F9-R3 for pux10-1 and pux10-2) of the T-DNA insertion site were used (Figure 1A). This result indicates that pux10-1 and pux10-2 may not be knockout mutants, as modified PUX10 transcripts are synthesized and may possibly be translated into aberrant proteins in these insertional lines.

The effect of PUX10 mutation on lipid accumulation during seed maturation was assessed by measuring the average weight and the fatty acid (FA) content of pux10-1 and pux10-2 mature seeds. No significant difference between mutant genotypes and their respective wild types was observed for these parameters (Supplemental Figure 4A-B). By contrast, FA composition was altered in both pux10 mutants (Supplemental Figure 4C). The relative abundance of linoleic acid (C18:2) slightly but significantly decreased with a concurrent increase in linolenic acid (C18:3) levels. Consequently, the ratio of C18:3 to C18:2 was consistently increased in pux10 mutants (Supplemental Figure 4D), suggesting a role for PUX10 in polyunsaturated FA metabolism during seed maturation. We also analyzed phenotypic effects of the mutations affecting PUX10 on seed germination and plant growth. Germination rates were similar between pux10 mutants and their wild-type counterparts (Supplemental Figure 4E). The absence of a developmental phenotype thus allowed rigorous comparison between mutant and wildtype plants at different stages of seed germination and seedling growth.

PUX10 mutation impairs oleosin degradation in young seedlings

During germination and early seedling growth, oleosins are targeted for degradation by ubiquitination [START_REF] Deruyffelaere | Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis[END_REF]. To explore the potential role of PUX10 in the degradation of ubiquitinated oleosins, we evaluated the content of ubiquitinated oleosins in pux10 and wild-type seedlings at various post-germinative stages. We analyzed by immunoblotting the three types of oleosin ubiquitinations previously described in germinating seeds [START_REF] Deruyffelaere | Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis[END_REF]. Oleosins marked for proteasomal degradation by the addition of a K48-linked diubiquitin (hereafter referred to as OLEs-K48Ub2) over-accumulated in pux10-1 and pux10-2 germinating seeds compared to their respective wild types (Apu2 immunoblots; Figure 1B). This accumulation was not only more intense but also lasted longer, suggesting that PUX10 is required for OLEs-K48Ub2 to enter the UPS degradative pathway. The mutations in PUX10 also reduced the degradation of the two other types of ubiquitin-oleosin conjugates, i.e. monoubiquitinated oleosins (OLEs-Ub) and oleosins marked with a K63-linked diubiquitin (OLEs-K63Ub2). Indeed, immunodetection of OLEs-Ub and OLEs-K63Ub2 using FK2 and Apu3 antibodies, respectively, revealed a strong accumulation of these two types of ubiquitinated oleosins in 24-h-to 50-h-old seedlings, associated with PUX10 mutation (Apu3 and FK2 immunoblots; Figure 1B). Overall, these results demonstrate that PUX10 is involved in the degradation of all types of ubiquitinated oleosins, without discriminating between the three ubiquitination motifs borne by oleosins. It is noteworthy that both insertional mutations in PUX10 substantially reduced, but did not block, the degradation of ubiquitinated oleosins. This partial phenotype suggests that ubiquitinated oleosins could be proteolyzed by a yet unidentified PUX10-independent degradation pathway (this hypothesis is detailed in the discussion). An alternative explanation is that pux10-1 and pux10-2 are not null mutants but hypomorphic ones, as suggested by the RT-PCR analysis previously described (Figure 1A).

To confirm the role of PUX10 in the ubiquitin-dependent proteolysis of oleosins, we analyzed the degradation of native oleosins during post-germinative seedling growth.

Between 32 h and 50 h of germination, the gradual hydrolysis of oleosins was slower in pux10-1 and pux10-2 mutants than in their corresponding wild-type seedlings, as shown by Coomassie-stained SDS-PAGE of seed proteins (Figure 1B; Coom. panels) and by specific immunodetection of the two major oleosins, OLE1 and OLE2 (Figure 1B; OLE1 and OLE2 immunoblots). Quantification of OLE1 in 41-h-old seedlings confirmed the significant reduction of OLE1 hydrolysis in the pux10-1 mutant compared to Col0 (Figure 1C; left panel). We conclude that PUX10 mutation impairs the degradation of ubiquitinated oleosins, which results in a significant reduction of oleosin hydrolysis in young seedlings.

PUX10 mutation alters LD size but not oil mobilization in young seedlings

To further characterize the effects of PUX10 on post-germinative LD degradation, we analyzed the oil content of mutant and wild-type seedlings by measuring eicosanoic acid (C20:1), a FA specific to storage oil. Eicosanoic acid content was not significantly different between pux10-1 and Col0 seedlings at 41 h of germination (Figure 1C; right panel), contrary to OLE1 content (Figure 1C; left panel). The degradation of the oleosin coat surrounding LDs and shielding oil from lipases is probably not impaired enough to prevent lipolysis in pux10-1. Although PUX10 mutation did not significantly alter the amount of lipids packed in LDs, it had a major effect on LD size. To compare LD morphology between mutants and wild types, we stained neutral lipids of 32-h-old seedlings using LipidTOX Deep Red and then imaged LDs in epidermal cells of the lower hypocotyl by confocal laser-scanning microscopy (CLSM). Bright-field microscopy was also helpful to visualize LD rim. The left panel in Figure 1D displays representative images showing that LD size was smaller in mutants than in wild types. To quantify this difference, LD diameters were measured using ImageJ on several merged bright-field and confocal images. The boxplot representation of diameter distribution in Figure 1D (right panel) shows that LD size was significantly different between pux10 mutants (0.69±0.27 and 0.70±0.23 µm for pux10-1 and pux10-2, respectively) and their corresponding wild types (1.85±0.61 and 1.62±0.53 µm for Col0 and Ws, respectively) as estimated by t-test (p<0.0001). This result suggests that PUX10 mutation prevents the increase in LD size occurring during seed germination. LD morphology is indeed dramatically modified during germination [START_REF] Miquel | Specialization of Oleosins in Oil Body Dynamics during Seed Development in Arabidopsis Seeds[END_REF]. Whereas LDs are very small in dry mature seeds (they cannot be individually distinguished by CLSM), they increase in size and reduce in number after seed imbibition and germination. Such a process most probably involves LD fusions that could be promoted by an increase in interfacial tension due to oleosin removal from the LD surface. We therefore hypothesize that the effect of PUX10 mutation on LD size is indirect and results from the partial blockade of oleosin degradation.

Phenotypical restoration by PUX10-GFP and PUX10-myc

A transgenic complementation strategy was employed to confirm that the loss of function of PUX10 is responsible for the mutant phenotype. The pux10-1 mutant was transformed with constructs containing ProPUX10:PUX10 (a 2907-bp genomic fragment encompassing 628 bp upstream of the coding sequence) and allowing the native promoter-driven expression of PUX10 C-terminally fused to GFP or myc. One stable homozygous transgenic line was studied for each construct. Regardless of the tag, the restored expression of PUX10 was able to abolish the accumulation of ubiquitinated oleosins observed in 41-h-old pux10-1 seedlings (Apu2, Apu3, and FK2 immunoblots in Figure 2A). Moreover, oleosin level in transgenic seedlings was similar to that of wildtype seedlings and lower than that of pux10-1 seedlings (OLE1 and OLE2 immunoblots in Figure 2A), showing that PUX10 expression was able to restore normal oleosin degradation in the pux10-1 mutant. Finally, we analyzed the capacity of PUX10-GFP to complement the LD size phenotype of pux10-1 seedlings (Figure 2B). LDs of 32-h-old seedlings were significantly larger in the ProPUX10:PUX10-GFP/pux10-1 transgenic line (1.38±0.56 µm in diameter) than in the pux10-1 mutant (0.69±0.27 µm) as determined by t-test (p<0.0001). Expression of PUX10-GFP in the pux10-1 mutant was therefore able to restore the increase in LD size occurring during seed germination in the wild-type background. The restoration was partial since LD size was smaller in transgenic (1.38±0.56 µm) than in wild-type (1.85±0.61 µm) seedlings, suggesting that PUX10 activity might be slightly decreased by the tag fused at its C-terminus, close to the UBA domain. Collectively, these results unambiguously demonstrate that the phenotype displayed by pux10-1 seedlings is caused by the mutation affecting PUX10.

PUX10 associates with LDs during germination

Since the PUX10-GFP fusion protein complements the pux10-1 loss-of-function, it is highly likely that the translational fusion is targeted similarly as the endogenous PUX10.

Accordingly, we characterized the temporal and tissue-specific regulation of PUX10 and its subcellular localization by CLSM imaging of the fluorescent PUX10-GFP expressed in ProPUX10:PUX10-GFP/pux10-1 transgenic lines. Three distinct homozygous transgenic lines of Arabidopsis were analyzed (lines #5, #7, and #12). As these three lines displayed similar patterns of localization (Supplemental Figure 5), a more detailed analysis was conducted on only one of them (line #12), which was the transgenic line characterized in the complementation experiment.

Our previous analysis of the LD proteome revealed an association of PUX10 with LDs in germinated seeds (Supplemental Figure 1). To confirm this subcellular localization, the fluorescent signal of PUX10-GFP relative to that of LipidTOX-stained LDs was imaged in transgenic seeds by CLSM. At the beginning of germination, PUX10-GFP labelled punctate structures, less numerous in cotyledons than in the hypocotyl (Figure 3A; 4 h of germination). As these submicrometer-sized structures were scattered throughout LD clusters, they were assumed to be LDs that could not be individually distinguished by CLSM. This hypothesis was confirmed by imaging seeds at later stages of germination, when LDs get larger. In the hypocotyl and cotyledon epidermises of young seedlings at 24 h (Figure 3A) and 32 h of germination (Figure 4), the fluorescent PUX10-GFP uniformly labeled the entire circumference of LDs, as expected for a LD-associated protein. Notably, PUX10-free LDs (empty arrowheads) were present together with PUX10-coated LDs (white arrowheads) within a given cell (Figure 3A; 24 h of germination). We thus conclude that PUX10 exclusively localizes to LDs and only associates with a subset of LDs in the cotyledons and hypocotyl of germinating seeds.

Moreover, PUX10 labelling was more intense at 24 h than at 4 h of germination, indicating an increase in PUX10 level during germination. This up-regulation was consistent with transcriptomic data retrieved from the eFP browser (Figure 3B) and was confirmed by immunoblot quantification of PUX10-GFP in transgenic ProPUX10:PUX10-GFP/pux10-1 seeds/seedlings (Figure 3C). Overall, these results indicate that PUX10 is progressively expressed and unevenly targeted to cotyledon and hypocotyl LDs as germination progresses.

To characterize the tissue-specific regulation of PUX10, PUX10-GFP labeling was examined in different tissues of 32-h-old seedlings, revealing a brighter fluorescence in the radicle, collet (also called hypocotyl-radicle transition zone), and root hairs than in the cotyledons and hypocotyl (Figure 4; left panel). In the radicle and collet (including root hairs), PUX10 encircled the LipidTOX-stained lipid core, indicating that PUX10 was targeted to the surface of LDs in these tissues (Figure 4; right panels). PUX10 was therefore associated with LDs in newly germinated seedlings, whatever the tissue. However, we noticed major differences in PUX10 intracellular distribution between seedling tissues. Whereas PUX10 was associated with most, if not all, the LDs in the radicle, collet and root hairs, it was targeted to only a subset of LDs in the cotyledons and hypocotyl. Moreover, the intracellular positioning of LDs varied substantially between cell types. LDs localized to the cell periphery in cotyledon and hypocotyl epidermises, as they did in dry mature seeds. By contrast, cells from the hypocotylradicle transition zone (including root hairs) and from the radicle exhibited one or a few central clustered pools of LDs (Figure 4; right panels).

PUX10 localization changes from LDs to chloroplasts during seed development

Publicly available expression data indicate that PUX10 transcripts are not only present in dry and imbibed seeds but also in developing seeds, albeit to a lower extent (Figure 3B). This observation prompted us to decipher PUX10 expression and localization during seed development. Figure 3A shows that PUX10 was indeed expressed during embryogenesis, at 7 and 9 days after flowering (DAF), and during seed maturation, at 12 DAF. Surprisingly, PUX10 localization dramatically changed during the transition between seed embryogenesis and maturation. In the cotyledons and hypocotyl of embryos at the torpedo and upturned-U stages (7 and 9 DAF, respectively), PUX10 was targeted to the periphery of LDs. Three days later, in the mid-phase of oil accumulation [START_REF] Baud | An integrated overview of seed development in Arabidopsis thaliana ecotype WS[END_REF], PUX10-GFP was no longer localized to LDs but labeled the chloroplast periphery, suggesting its association with the chloroplast membranes (Figure 3A; 12 DAF). We also observed a faint reticulated PUX10 fluorescence, distinct from the ring-shaped pattern typical of LD-associated proteins, suggesting an endoplasmic reticulum (ER) localization of PUX10 at 12 DAF. Along with the modification of PUX10 targeting, major changes occurred on LD structure during the embryogenesis-maturation transition. Whereas LDs were organized in central clusters during seed embryogenesis (7 and 9 DAF), they increased in size and were pushed towards the cell periphery at the maturation phase (12 DAF). These observations revealed that LDs are highly dynamic organelles during seed development and that PUX10 specifically associates with LDs during embryogenesis but no longer during seed maturation.

The hydrophobic HP domain of PUX10 is a membrane-anchoring sequence required for LD localization

To confirm the in planta association of PUX10 to LDs, mCherry fluorescent PUX10 was transiently expressed in Nicotiana benthamiana leaves together with GFP-tagged Arabidopsis OLE1 (GFP-OLE1), a LD marker. Consistent with its localization to LDs in Arabidopsis early developing embryos and young seedlings, PUX10 was localized to OLE1-labeled LDs in N. benthamiana leaves (Figure 5A). Time-lapse imaging of GFP-PUX10 in N. benthamiana leaves revealed that PUX10-labeled LDs were dynamic organelles, permanently connected to and continuously moving along the ER (Supplemental Movie 1). Besides this association with LDs, PUX10 was localized to the chloroplast periphery (Figure 5A), as already observed in Arabidopsis developing seeds at the maturation phase (12 DAF; Figure 3A). Using transgenic N. benthamiana expressing an ER fluorescent marker, we observed that GFP-tagged PUX10 was also marginally localized to the ER (Figure 5B). Overall, these results show that GFP-PUX10 and mCherry-PUX10 fusions transiently expressed in N. benthamiana under the 35S promoter localize to the same subcellular compartments as the PUX10-GFP fusion expressed in Arabidopsis under the PUX10 native promoter. These data reinforce the conclusion that PUX10 can be targeted to the membrane of LDs, chloroplasts, or ER. PUX10 possesses two consecutive hydrophobic regions (I 102 -V 117 and I 121 -F 141 ) that may form a membrane-anchoring hairpin-structured domain. To decipher the role of this hydrophobic domain, hereafter called HP domain because of its putative hairpin structure, we examined the effect of deleting this region on PUX10 localization in N. benthamiana cells. Whereas full-length PUX10 was targeted to LD, ER and chloroplast membranes (Figure 5B), a mutated version of PUX10 devoid of the HP domain (DI 102 -F 141 ) was diffusely distributed in the cytoplasm, as expected for a cytosolic protein (Figure 5C). These results indicate that the HP domain is required for the association of PUX10 with monolayer and bilayer membranes.

The UBX domain of PUX10 promotes CDC48A association with LDs

The analysis of the LD proteome described above identified the AAA-ATPase CDC48A as a putative PUX10 interactant (Supplemental Data Set 1 and Supplemental Figure 1).

To demonstrate this hypothesis, we first investigated whether CDC48A is associated with LDs in germinating seeds and young seedlings, especially as such a LD localization of CDC48A was not observed by Park and co-workers [START_REF] Park | In Planta Analysis of the Cell Cycle-Dependent Localization of AtCDC48A and Its Critical Roles in Cell Division, Expansion, and Differentiation[END_REF].

These authors analyzed the localization of CDC48A in complemented Atcdc48A mutant seedlings expressing YFP-AtCDC48A under the native CDC48A promoter (ProCDC48A:YFP-CDC48A/Atcdc48A line). They only reported a nuclear and cytoplasmic localization of CDC48A and mentioned its recruitment to the division midzone during cytokinesis. To specifically assess the issue of CDC48A association with LDs, we reexamined CDC48A localization in seedlings from this ProCDC48A:YFP-CDC48A/Atcdc48A transgenic line, after fluorescent staining of LDs. Figure 6A shows that YFP-CDC48A fusion protein uniformly labeled the surface of some, but not all, LDs in cotyledon and hypocotyl epidermises of 32-h-old seedlings. To rule out a mislocalization artifact due to the presence of the GFP tag, we analyzed the association of the native CDC48A protein with purified LDs. Using specific antibodies, CDC48A was immunodetected in LDs isolated from dry mature and germinated seeds (Figure 7A), confirming the association of CDC48A to LDs. To characterize the biophysical interaction between CDC48A and LDs, purified LDs were washed in 8 M urea/carbonate buffer to release non-integral proteins. Under these conditions, CDC48A was released from LDs (Figure 7A), which is consistent with previous data showing that it is a peripheral membrane protein [START_REF] Rancour | Characterization of AtCDC48. Evidence for Multiple Membrane Fusion Mechanisms at the Plane of Cell Division in Plants[END_REF]. By contrast, PUX10 remained in the LD fraction after the washing step (Figure 7A), demonstrating that PUX10 is an integral LD membrane protein.

Both CDC48A and PUX10 are associated with a subset of LDs in Arabidopsis seedlings. This observation suggests that CDC48A and PUX10 may co-localize to some LDs during early seedling growth. To investigate this hypothesis, PUX10-GFP and mCherry-CDC48A protein fusions were transiently co-expressed in N. benthamiana leaves. As shown in Figure 6B (upper panel), fluorescent CDC48A co-localized with PUX10 to dots previously identified as LDs (in Figure 5A) and to chloroplast membranes. CDC48A also localized in the cytosol, albeit to a weaker extent. Disruption of the UBX domain of PUX10 abrogated the co-localization of CDC48A with PUX10 on LDs and chloroplast membranes (Figure 6B; middle panel), showing that the UBX domain is required for PUX10-mediated targeting of cytosolic CDC48A to membranes.

To confirm that PUX10 mediates the membrane targeting of CDC48A, CDC48A was expressed without PUX10. In the absence of PUX10, CDC48A was indeed exclusively cytosolic and did not localize to OLE1-labeled LDs or chloroplast membranes (Figure 6B; lower panel). However, the role of PUX10 in mediating CDC48A targeting to LDs could not be confirmed in Arabidopsis seeds. The association of CDC48A with LDs, measured by CDC48A immunodetection in purified seed LDs, was not impaired in pux10 mutants (Figure 7B). To explain this apparent discrepancy, we hypothesize that PUX10 may not be the only LD-associated protein able to bind CDC48A. CDC48A could be targeted to LDs by interaction with an unknown LD protein distinct from PUX10 and expressed in Arabidopsis seedlings but not in N. benthamiana leaves. Consistent with this hypothesis, we observed a lack of association between CDC48A and PUX10 levels in purified LDs at different stages of seed germination, as measured by LD proteomics (Supplemental Figure 1) and immunoblots (Figure 7A). Overall, we conclude that PUX10 is an integral LD protein promoting CDC48A association with LDs, although this targeting mechanism of CDC48A to LDs may not be the only one involved in seedlings.

PUX10 can bridge ubiquitin to CDC48A through its UBA and UBX domains

The presence of both UBX and UBA domains in PUX10 strongly suggests a role as an adaptor bridging CDC48A to the ubiquitin moiety of ubiquitinated proteins. We used a yeast three-hybrid assay to reveal the formation of a ternary complex between ubiquitin, PUX10 and CDC48A. The expression of PUX10 in a yeast strain expressing ubiquitin and CDC48A allowed yeast growth on selective medium (Figure 8A), showing that PUX10 is necessary and sufficient to bridge CDC48A to ubiquitin. The deletion of either the UBA or the UBX domain suppressed this interaction (Figure 8A), indicating that these domains are involved in PUX10 interaction with ubiquitin or CDC48A. To ensure that the UBA and UBX domains are sufficient for PUX10 interaction with its partners, we investigated the ability of these domains to bind either ubiquitin or CDC48A, using the yeast two-hybrid method. The PUX10 N-terminus region (M 1 -S 44 ) encompassing the UBA domain (F 11 -T 42 , as determined on NCBI CD Search) was sufficient to bind ubiquitin but not CDC48A (Figure 8B). Conversely, the PUX10 C-terminus region (E 398 -N 480 ) encompassing the UBX domain (E 398 -E 478 , as determined on NCBI CD Search) was sufficient for the specific interaction with CDC48A but did not bind ubiquitin. These two pairs of interaction, i.e., ubiquitin-UBA domain and CDC48A-UBX domain, were confirmed in both orientations of bait and target (Figure 8B).

The interaction between PUX10 and CDC48A was also demonstrated in planta using bimolecular fluorescence complementation. The in vivo formation of a stable PUX10-CDC48A complex, visualized by the emission of YFP fluorescence, occurred in LDs and chloroplast membranes of agrobacterium-inoculated N. benthamiana epidermal cells (Figure 8C; magnified views in the right panel). This interaction is consistent with the subcellular localizations of both PUX10 and CDC48A previously observed in Figures 5 and6. Moreover, we confirmed in planta that the interaction of PUX10 with CDC48A is mediated by its UBX domain, as co-expression of YFP N -PUX10DUBX and YFP C -CDC48A resulted in significantly fewer fluorescent punctae compared to co-expression of full-length proteins (Figure 8D).

PUX10 is required for segregation of the ubiquitinated oleosins

In germinated seeds, OLEs-K48Ub2 is extracted from the phospholipid monolayer surrounding LDs before reaching the cytosolic proteasome for degradation [START_REF] Deruyffelaere | Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis[END_REF]. The extraction of ubiquitinated oleosins from LDs may be catalyzed by the CDC48A ATPase. Numerous studies in animals and yeast have indeed reported that CDC48 ATPases have the ability to physically disassemble ubiquitinated proteins from binding partners or cellular structures with the help of associated cofactors [START_REF] Meyer | Emerging functions of the VCP/p97 AAA-ATPase in the ubiquitin system[END_REF]. We thus hypothesized that PUX10 could be the cofactor promoting CDC48A activity to disassemble OLEs-K48Ub2 from LDs in germinated seeds. To investigate this possibility, we analyzed the intracellular distribution of the ubiquitinated oleosins over-accumulated in pux10-1 seedlings. Most of the OLEs-K48Ub2 accumulated in pux10-1 seedlings were present in the LD fraction, showing that PUX10 mutation interferes with the segregation of OLEs-K48Ub2 from LDs (Figure 9; Apu2 immunoblot). By contrast, accumulation of OLEs-K48Ub2 due to MG132-induced proteasome blockade was recovered in the insoluble protein fraction (P2 pellet) of wild-type seedling extracts (Figure 9; Apu2 immunoblot). This result confirmed that proteasome inhibition blocks the degradation of OLEs-K48Ub2 after their extraction from LDs. Extracted OLEs-K48Ub2 were no longer degraded by the proteasome and consequently aggregated within the cytosol due to their hydrophobicity.

We also analyzed the intracellular distribution of the two other types of ubiquitin-oleosin conjugates, i.e., OLEs-Ub and OLEs-K63Ub2. In the pux10-1 mutant, over-accumulated OLEs-Ub and OLEs-K63Ub2 were essentially associated with LDs (Figure 9; FK2 and Apu3 immunoblots), indicating that PUX10 is required for the dislocation of OLEs-Ub and OLEs-K63Ub2 from LDs. MG132 did not impair the degradation of these two types of ubiquitin-oleosin conjugates, suggesting that oleosins modified by the covalent binding of a monoubiquitin or a K63-linked diubiquitin are not targeted to the UPS. In conclusion, this experiment demonstrated that PUX10 is required for the segregation of all three types of ubiquitinated oleosins from LDs, regardless of the topology of ubiquitination. It also showed that, once dislocated from the LDs to the cytosol, ubiquitinated oleosins are targeted to distinct degradation pathways according to the topology of their ubiquitin moiety. Whereas the UPS hydrolyses OLEs-K48Ub2, the degradation pathways of OLEs-Ub and OLEs-K63Ub2 are currently unknown.

Discussion

A significant remodeling of the LD proteome occurs during the transition from seed dormancy to postgerminative growth. In the quiescent cells of mature seeds, LDs are coated and stabilized by oleosins. When seeds germinate, oleosins are progressively degraded [START_REF] Deruyffelaere | Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis[END_REF] and replaced by LDAPs, which are the structural LD proteins recently identified in non-seed cells [START_REF] Gidda | Lipid Droplet-Associated Proteins (LDAPs) Are Required for the Dynamic Regulation of Neutral Lipid Compartmentation in Plant Cells[END_REF]. Why are oleosins replaced by LDAPs at the LD surface? When overexpressed in leaves, both families of proteins are able to increase the accumulation of neutral lipids and to compartmentalize them into normal-sized LDs, showing that they function similarly and as expected for structural LD proteins [START_REF] Winichayakul | In Vivo Packaging of Triacylglycerols Enhances Arabidopsis Leaf Biomass and Energy Density[END_REF][START_REF] Vanhercke | Metabolic engineering of biomass for high energy density: oilseed-like triacylglycerol yields from plant leaves[END_REF][START_REF] Gidda | Lipid Droplet-Associated Proteins (LDAPs) Are Required for the Dynamic Regulation of Neutral Lipid Compartmentation in Plant Cells[END_REF]. However, oleosins prevent other proteins, such as LDAPs in leaves or native LD proteins in yeast, from targeting to LDs [START_REF] Jamme | Single cell synchrotron FT-IR microspectroscopy reveals a link between neutral lipid and storage carbohydrate fluxes in S. cerevisiae[END_REF][START_REF] Gidda | Lipid Droplet-Associated Proteins (LDAPs) Are Required for the Dynamic Regulation of Neutral Lipid Compartmentation in Plant Cells[END_REF]. These results suggest that oleosins impair LD remodeling and maintain the organelle in a quiescent state. Therefore, the degradation of oleosins is likely required for LDs to enter the dynamic state associated with seedling growth. The present study focused on deciphering the molecular machinery controlling oleosin degradation, and thus represents an important early step in understanding the subcellular processes involved in the transition from quiescent to dynamic LDs during germination.

Oleosins are degraded by a novel protein degradation pathway called LDassociated degradation (LDAD)

Here, we demonstrated that PUX10 is an integral LD protein that acts as an adaptor directly linking CDC48A to ubiquitin conjugates. The UBX and UBA domains of PUX10 were shown to mediate PUX10 interaction with CDC48A and ubiquitin conjugates, respectively. PUX10 is therefore the fifth Arabidopsis PUX protein shown to be a functional cofactor of CDC48A, out of 16. More importantly, PUX10 is the first PUX protein whose ubiquitinated substrates have been identified. The accumulation of ubiquitinated oleosins and the reduction of oleosin degradation observed in pux10-1 and pux10-2 seedlings demonstrate that ubiquitin-conjugated oleosins are specific substrates of PUX10.

We also provided evidence that CDC48A and PUX10 are part of a protein machinery segregating ubiquitinated oleosins from LDs. Our findings indicate that PUX10 is required for the extraction of all types of ubiquitinated oleosins from the LD surface into the cytosol. Given that CDC48A co-localizes and interacts with PUX10 on LDs (Figures 6 and8), and that CDC48A belongs to a family of ubiquitous proteins known for their ability to physically disassemble ubiquitinated proteins from membranes or complexes [START_REF] Bodnar | Toward an understanding of the Cdc48/p97 ATPase[END_REF], the role of PUX10 in this dislocation process most likely occurs by promoting the interaction of CDC48A with ubiquitinated oleosins. Intriguingly, our data revealed that PUX10 is not able to discriminate the different ubiquitin motifs conjugated to oleosins. Mutations in PUX10 indeed blocked not only the proteasomal degradation of OLEs-K48Ub2, but also the proteasomal-independent degradation of OLEs-K63Ub2 and OLEs-Ub. Some yet unknown CDC48A cofactors, which selectively bind either K48Ub2-, K63Ub2-or Ub-conjugated oleosins, are probably required to mediate specificity for degradation pathways.

Cellular functions of CDC48A homologs in yeast and mammals are well documented, starting with their role in the selective extraction of misfolded proteins from the ER, a key step in the degradation process known as ERAD. Besides ERAD, yeast Cdc48 and mammalian p97/VCP are also involved in dislocating proteins from the mitochondrial outer membrane, in a process termed mitochondria-associated degradation (MAD) [START_REF] Xu | The AAA-ATPase p97 is essential for outer mitochondrial membrane protein turnover[END_REF][START_REF] Braun | With the Help of MOM: Mitochondrial Contributions to Cellular Quality Control[END_REF]. In plants, there is no evidence of a MAD pathway to date, but several components of the ERAD process have been identified, including Arabidopsis CDC48A [START_REF] Muller | Conserved ERAD-like quality control of a plant polytopic membrane protein[END_REF][START_REF] Begue | Structure and functions of the chaperone-like p97/CDC48 in plants[END_REF], uncovering similar mechanisms of ER-associated protein degradation in plants and in yeast or mammals [START_REF] Muller | Conserved ERAD-like quality control of a plant polytopic membrane protein[END_REF][START_REF] Liu | Endoplasmic reticulum-mediated protein quality control in Arabidopsis[END_REF]). An ERAD-like machinery is also associated with plastids derived by secondary endosymbiosis (called complex plastids), in secondary red algal endosymbionts such as diatoms. Contrary to ERAD and MAD, this Cdc48-containing machinery, called symbiont-specific ERAD-like machinery (SELMA), is not involved in protein degradation but is required for the transport of nuclear-encoded proteins across the periplastidial membrane of complex plastids [START_REF] Agrawal | Genetic Evidence that an Endosymbiont-derived Endoplasmic Reticulum-associated Protein Degradation (ERAD) System Functions in Import of Apicoplast Proteins[END_REF][START_REF] Lau | N-terminal lysines are essential for protein translocation via a modified ERAD system in complex plastids[END_REF]. Besides the ER, mitochondria and plastids, the present study thus identifies LDs as another organelle targeted by an ERAD-like CDC48-containing machinery. Together with CDC48A, PUX10 would be a core component of this putative machinery, which we name LD-associated degradation (LDAD).

This ubiquitin-and PUX10-dependent system is the first molecular mechanism of LD protein degradation identified in plants. This pathway is distinct from the ones previously identified in mammals. The first mechanism described to control protein turnover in mammalian LDs involves ARF1 and the coat protein complex I (COPI), two components of the vesicular trafficking pathway [START_REF] Soni | Coatomer-dependent protein delivery to lipid droplets[END_REF][START_REF] Takashima | GBF1-Arf-COPI-ArfGAP-mediated Golgi-to-ER transport involved in regulation of lipid homeostasis[END_REF]. The ARF1-COPI machinery associates with LDs and releases portions of the LD surface by budding nano-LDs. This budding process triggers LD-ER connections, which are used for protein relocalization [START_REF] Wilfling | Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets[END_REF]. More recently, PLIN2 and PLIN3, the major LD proteins in non-adipose tissues, were shown to be degraded by chaperonemediated autophagy (CMA), a highly selective form of autophagy [START_REF] Kaushik | Degradation of lipid droplet-associated proteins by chaperonemediated autophagy facilitates lipolysis[END_REF]. Intriguingly, although neither of these pathways is dependent on ubiquination, several mammalian LD proteins are ubiquitinated and degraded by the UPS, such as PLIN1 and PLIN2, the mammalian LD gatekeepers [START_REF] Xu | Posttranslational regulation of adipose differentiation-related protein by the ubiquitin/proteasome pathway[END_REF][START_REF] Xu | Degradation of perilipin is mediated through ubiquitinationproteasome pathway[END_REF].

Moreover, several proteins of the ubiquitination machinery are associated with LDs in yeast and mammals [START_REF] Bersuker | Establishing the lipid droplet proteome: Mechanisms of lipid droplet protein targeting and degradation[END_REF], including p97/VCP/Cdc48 and the counterparts of PUX10. These latter, namely human FAF2/UBXD8 and Saccharomyces cerevisiae Ubx2p, can relocalize from the ER to LDs and regulate LD dynamics [START_REF] Zehmer | Targeting sequences of UBXD8 and AAM-B reveal that the ER has a direct role in the emergence and regression of lipid droplets[END_REF][START_REF] Wang | The ubiquitin-like (UBX)-domain-containing protein Ubx2/Ubxd8 regulates lipid droplet homeostasis[END_REF][START_REF] Olzmann | Spatial regulation of UBXD8 and p97/VCP controls ATGL-mediated lipid droplet turnover[END_REF], besides their role in ERAD. The prominent presence of proteins related to the ubiquitin system in mammalian LDs was recently confirmed by mapping human LD proteomes using a proximity labelling strategy [START_REF] Bersuker | A Proximity Labeling Strategy Provides Insights into the Composition and Dynamics of Lipid Droplet Proteomes[END_REF]. Overall, these data raise the possibility that the LDAD pathway, uncovered herein in plants, might also be present in mammals and yeast. Demonstrating this hypothesis will require identifying LDassociated proteins regulated by ubiquitination and accumulated in some LDADdeficient mutants. One candidate substrate of the putative mammalian LDAD is Apo-B100, which translocates to LDs before its proteasomal degradation by a UBXD8dependent mechanism [START_REF] Suzuki | Derlin-1 and UBXD8 are engaged in dislocation and degradation of lipidated ApoB-100 at lipid droplets[END_REF].

PUX10 and CDC48A discriminate subpopulations of LDs in seedlings

The comprehensive analysis of PUX10 localization revealed distinct targeting pathways during seed development and germination. While PUX10 is exclusively associated with LDs in germinating seeds and young seedlings, it shows a more complex localization pattern in developing seeds. During embryogenesis, PUX10 is mainly associated with LDs but also localizes to the ER. This dual steady-state localization to LDs and the ER was also observed by transient expression in N. benthamiana leaves, and previously reported for the yeast and mammalian homologs of PUX10 [START_REF] Wang | The ubiquitin-like (UBX)-domain-containing protein Ubx2/Ubxd8 regulates lipid droplet homeostasis[END_REF][START_REF] Olzmann | Spatial regulation of UBXD8 and p97/VCP controls ATGL-mediated lipid droplet turnover[END_REF]. The hydrophobic HP domain of PUX10 serves as a membrane anchor and is required for this dual localization. This domain may form a hairpin with arms of 15-20 residues, which are short enough for PUX10 to partition between the cytoplasmic leaflet of the ER and the LD monolayer, according to [START_REF] Huang | Unique Motifs and Length of Hairpin in Oleosin Target the Cytosolic Side of Endoplasmic Reticulum and Budding Lipid Droplet[END_REF]. The targeting patterns of PUX10 in developing and germinating seeds are consistent with the prevailing model of LD targeting for class I LD proteins [START_REF] Kory | Targeting Fat: Mechanisms of Protein Localization to Lipid Droplets[END_REF]. This model posits that class I LD proteins are co-translationally inserted into the ER membrane and transferred either directly to the nascent LDs emerging from ER subdomains, or to some mature LDs through specific ER-LD membrane bridges. In developing seeds, PUX10 is associated with the pool of LDs, suggesting its insertion into the LD monolayer during organelle biogenesis. In the cotyledons and hypocotyl of germinated seeds, PUX10 is progressively expressed and sequentially translocated to a specific subset of LDs, possibly through ER-LD connections. The molecular machinery by which PUX10 is translocated to LDs is currently unknown. However, the translocation mechanism is probably distinct from the ARF1/COPI-mediated mechanism described in mammals [START_REF] Wilfling | Arf1/COPI machinery acts directly on lipid droplets and enables their connection to the ER for protein targeting[END_REF], since proteins of the ARF family were not identified in the LD proteome of Arabidopsis seedlings (Pyc et al., 2017b).

Unexpectedly, we observed a dramatic change of PUX10 localization during the transition between embryogenesis and seed maturation. At 12 DAF, PUX10 exclusively localizes to the chloroplast periphery, which is a localization also observed in N. benthamiana leaves. However, while PUX10 targeting pathways to LD monolayer and to chloroplast membranes are both functional in N. benthamiana epidermal cells, they are mutually exclusive and sequentially activated during seed development and germination. Thus, PUX10 partitioning between LDs and chloroplasts is subject to a developmental control, which represents a potentially important mechanism for regulating LD dynamics in seeds.

In considering the functional significance of the interaction between PUX10 and CDC48A, the prevailing hypothesis is that PUX10 serves as an anchor for targeting the cytoplasmic CDC48A to LDs. Although tantalizingly simple, this hypothesis might not represent the whole story, since the association of CDC48A with LDs is not disrupted in pux10-1 and pux10-2 seedlings. A similar observation was reported for Ubx2 and Cdc48 in yeast ERAD, suggesting an evolutionary-conserved functional redundancy of membrane-recruitment factors for CDC48s [START_REF] Neuber | Ubx2 links the Cdc48 complex to ER-associated protein degradation[END_REF]. Thus, another LDrecruitment factor for CDC48A might exist in young seedlings, in addition to PUX10. This hypothesis can also explain the partial loss-of-function associated with PUX10 deficiency in pux10-1 and pux10-2 mutants. However, the putative LD-recruitment factor would only partially compensate for PUX10 deficiency as the pux10-1 and pux10-2 seedlings exhibit a major reduction of oleosin degradation. These results suggest that PUX10 is required to facilitate correct orientation of ubiquitinated oleosin and CDC48A for efficient substrate binding and subsequent processing, while it is not mandatory for the recruitment of CDC48A to LDs in seedlings.

One important finding of the present study is the coexistence of distinct LD subpopulations within a single cell, suggesting a functional differentiation of LDs in plants. Morphological and compositional heterogeneity within the LD pool of a single cell have been described for several years in animals and yeast, but not in plants [START_REF] Wolins | S3-12, Adipophilin, and TIP47 Package Lipid in Adipocytes[END_REF][START_REF] Beller | Characterization of the Drosophila lipid droplet subproteome[END_REF][START_REF] Wilfling | Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets[END_REF][START_REF] Zhang | Morphologically and Functionally Distinct Lipid Droplet Subpopulations[END_REF][START_REF] Thiam | The why, when and how of lipid droplet diversity[END_REF][START_REF] Eisenberg-Bord | Identification of seipin-linked factors that act as determinants of a lipid droplet subpopulation[END_REF]. In cotton (Gossypium hirsutum) and Arabidopsis plants, [START_REF] Horn | Visualization of Lipid Droplet Composition by Direct Organelle Mass Spectrometry[END_REF] reported the uneven distribution of triacylglycerol species among individual LDs isolated from seed or leaf tissues, suggesting that LD heterogeneity may also exist in plants, at least at the tissue level [START_REF] Horn | Visualization of Lipid Droplet Composition by Direct Organelle Mass Spectrometry[END_REF]. Such a heterogeneity was proved at the cell level in the present study. PUX10 and CDC48A, which exclusively localize on a subset of LDs in seedling cotyledons and hypocotyls, are indeed determinants of a LD subpopulation in Arabidopsis.

Undoubtedly, it will be interesting to characterize the difference in protein and lipid compositions between the PUX10-free and PUX10-coated LD subpopulations, and to examine if proteins and organelles involved in seed LD dynamics, such as LDAPs, LDIP, SEIPINs, SDP1 and peroxisomes, are preferentially or exclusively associated with a specific subset of LDs [START_REF] Kelly | Seed storage oil mobilization is important but not essential for germination or seedling establishment in Arabidopsis[END_REF][START_REF] Cai | Arabidopsis SEIPIN Proteins Modulate Triacylglycerol Accumulation and Influence Lipid Droplet Proliferation[END_REF][START_REF] Thazar-Poulot | Peroxisome extensions deliver the Arabidopsis SDP1 lipase to oil bodies[END_REF][START_REF] Gidda | Lipid Droplet-Associated Proteins (LDAPs) Are Required for the Dynamic Regulation of Neutral Lipid Compartmentation in Plant Cells[END_REF]Pyc et al., 2017a).

A working model of the LDAD pathway

The complete proteolysis of type I LD proteins, such as oleosins, requires unmasking of hydrophobic domains embedded in the phospholipid monolayer. Our results shed light on a mechanism able to dislocate these proteins from the LD surface. PUX10 and CDC48A are central to this mechanism. PUX10 is a LD-anchored scaffolding protein that bridges the CDC48A segregase to the substrate, i.e., ubiquitinated oleosins, to promote the dislocation of ubiquitinated oleosins from the phospholipid monolayer (Figure 10). This mechanism is reminiscent of the ERAD translocation process but localizes to LDs; we therefore propose to name it LDAD. Further characterization of the LDAD pathway will require the identification of additional proteins of the LDAD machinery, besides PUX10 and CDC48A. This includes proteins involved in predislocation events, such as ubiquitin ligases, and in post-dislocation events, such as substrate-processing cofactors controlling ubiquitin chain editing or recruiting specific proteolytic systems according to the ubiquitin mark borne by oleosins.

Methods

Plant material, germination and growth conditions

Arabidopsis thaliana seeds of the Ws and Col0 ecotypes and of the T-DNA mutant line pux10-2 (FLAG_211E02) were obtained from the Arabidopsis thaliana resource center for genomics at the Institut Jean-Pierre Bourgin (http://publiclines.versailles.inra.fr/). The T-DNA mutant line pux10-1 (SAIL_1187) was ordered from the NASC (http://arabidopsis.info). The pux10-1 and pux10-2 mutants are in Col0 and Ws genetic backgrounds, respectively. The Arabidopsis ProCDC48A:YFP-CDC48A/Atcdc48A line (expressing YFP-CDC48A) was kindly provided by S.Y. Bednarek [START_REF] Park | In Planta Analysis of the Cell Cycle-Dependent Localization of AtCDC48A and Its Critical Roles in Cell Division, Expansion, and Differentiation[END_REF].

The pux10-1 and pux10-2 T-DNA insertion lines were genotyped using the primers pux10_SAIL_for/ pux10_SAIL_rev and pux10_FLAG_for/ pux10_FLAG_rev, respectively (see Supplemental Table 1 for primer sequences). DNA insertion positions within PUX10 were determined by DNA sequencing of the PCR-amplified products obtained with T-DNA-specific primers and primers on either side of the PUX10 insertion site. The location of insertion was mapped between positions 6642476 and 6642398 of Chr4 for pux10-1 and at position 6641494 of Chr4 for pux10-2. Homozygous pux10-1 and pux10-2 plants were backcrossed twice to wild-type Col-0 and Ws ecotype plants, respectively. RT-PCR analyses were ultimately carried out to analyze gene expression in mutant backgrounds. The sequences of primers used for PCR amplifications are listed in Supplemental Table 1.

For the selection of Arabidopsis transgenic plants, seeds were surface-sterilized, stratified for 7 2h at 4°C in the dark and germinated on full-strength Gamborg B5 medium, pH 5.6 (Duchefa Biochemie), containing 0.8% (w/v) agar, 1% (w/v) sucrose, and the appropriate selection agent. After 14 days of selection in a growth cabinet (16h-light/8-h-dark cycle), plantlets were transferred to compost. Arabidopsis thaliana wildtype and transgenic plants were grown on soil in long-day conditions (16-h-light/8-hdark cycle) in greenhouse or growth chamber. Control seeds were grown side by side with experimental seeds for each experiment. For biochemical analyses and imaging, seeds were cold stratified and germinated on water-imbibed paper at 25°C under continuous light, as described in [START_REF] Deruyffelaere | Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis[END_REF]. Seeds were collected just after stratification (imbibed seeds; 0 h of germination) or after 24-72 h of incubation at 25°C (24-72 h of germination). When indicated, seeds were germinated in water containing 100 µM MG132 (Sigma-Aldrich) or 0.1% DMSO as a solvent control.

For germination assays, seeds were incubated at 25°C, with 8 h light daily, on three sheets of absorbent paper (paper circles, Schleicher and Schuell) wetted with distilled water. Germination was scored daily based on radicle emergence.

Agrobacterium-mediated transient expression in Nicotiana benthamiana leaves

Agrobacterium tumefaciens strain C58C1pMP90 was transformed by electroporation with pMDC32-mCherry-PUX10, pGWB6-GFP-PUX10, pGWB6-GFP-PUX10-DUBX, pGWB6-GFP-PUX10c-DHP, pMDC32-mCherry-CDC48A or pB7WGF2-GFP-OLE1 binary plasmid. After overnight culture at 28°C in LB medium containing antibiotics (rifampicin 50 µg/mL, gentamycin 50 µg/mL, kanamycin 50 µg/mL), agrobacteria were centrifuged and resuspended in infiltration buffer (10 mM MgCl2, 10 mM 2-(Nmorpholino)ethanesulfonic acid (MES) pH 5.7) at 0.1 OD600 for each construct.

Agrobacteria infiltration was performed at the abaxial face of leaves from 4-week-old plants using a syringe, 2 to 3 d before imaging. When indicated, N. benthamiana plants expressing an ER resident red fluorescent protein (RFP-ER) were used instead of wildtype plants [START_REF] Martin | Transient expression in Nicotiana benthamiana fluorescent marker lines provides enhanced definition of protein localization, movement and interactions in planta[END_REF].

Agrobacterium-mediated stable transformation and selection of transgenic lines

Arabidopsis mutant pux10-1 plants were transformed by the floral dip method with the Agrobacterium tumefaciens strain C58C1pMP90 containing the pGWB4-ProPUX10:PUX10-GFP or pGWB16-ProPUX10:PUX10-myc binary vector [START_REF] Clough | Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[END_REF]. Hygromycin-resistant transgenic plants were obtained and were selfpollinated to obtain F2 progenies. Seeds from homozygous F2 plants were used for visualization of the fluorescent protein and for immunoblot analysis.

Plasmid constructions

discontinuous iodixanol density gradient, and recovered in the floating fraction, as described in [START_REF] Deruyffelaere | Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis[END_REF]. To analyze the protein association to LDs, freshly isolated LDs were diluted into 100 mM Na carbonate containing 8 M urea (pH 10), or alternatively, into SH buffer. Samples were incubated at 4°C for 30 min on a rotating agitator. Washed LDs were re-isolated by ultracentrifugation on a discontinuous iodixanol density gradient, according to the same protocol as used for the first isolation.

Protein analysis by SDS-PAGE and immunoblotting

Total seed/seedling protein extracts were prepared by direct homogenization in 2X NuPAGE LDS Sample Buffer (Thermo Fisher Scientific) containing 0.1 M dithiothreitol, as described in [START_REF] Deruyffelaere | Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis[END_REF]. LD proteins were precipitated with cold acetone containing 10% (v/v) trichloroacetic acid and solubilized in 2X LDS buffer containing 200 mM Tris base and 0.1 M dithiothreitol. Protein extracts were incubated at 37°C for 10 min, separated by electrophoresis in NuPAGE gels with MOPS SDS running buffer, and blotted in NuPAGE transfer buffer (Thermo Fisher Scientific). For anti-PUX10 or -OLEs immunoblot, proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P 0.45 mm, Millipore). For anti-CDC48A immunoblot, proteins were blotted onto nitrocellulose membrane (BAS-85, Schleicher and Schuell). Saturation and incubation with antibodies were carried out according to D' Andrea et al. (2007). Rabbit anti-oleosin sera were used as previously described [START_REF] D'andrea | Selective one-step extraction of Arabidopsis thaliana seed oleosins using organic solvents[END_REF]. Rabbit antibodies recognizing polyubiquitin chains linked at Lys48 (clone Apu2, cat. 05-1307, lot 2840426) or Lys63 (clone Apu3, cat. 05-1308, lot 2923061) and mouse antibody against ubiquitinated proteins (clone FK2, cat. 04-263, lot 2475581) were obtained from Millipore and used at 1:1,000 dilution for Apu2 and FK2 and at 1:500 dilution for Apu3.

Chicken antiserum specific to Arabidopsis CDC48A was kindly given by S.Y. Bednarek and used according to [START_REF] Park | In Planta Analysis of the Cell Cycle-Dependent Localization of AtCDC48A and Its Critical Roles in Cell Division, Expansion, and Differentiation[END_REF]. Luminescence from peroxidase activity, revealed using Super Signal West Dura Extended Duration Substrate (Pierce), was recorded using a LAS-3000 imaging system and quantified with MultiGauge software, both from Fujifilm. After immunodetection, proteins were stained on membranes with amido black 10B [START_REF] Gershoni | Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a positively charged membrane filter[END_REF] to control protein loading.

Fatty acid analysis by gas chromatography

Total fatty acid content and composition of mature or germinated seeds were determined by direct transmethylation followed by gas chromatography with flame ionization detection (GC-FID) as previously described [START_REF] Browse | Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue[END_REF], with the minor modifications described in [START_REF] Deruyffelaere | Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis[END_REF]. For each batch of seeds, FA analysis was performed in triplicate or quadruplicate on 30 100 seeds/seedlings.

Confocal Laser Scanning Microscopy (CSLM)

For visualizing LDs, Arabidopsis seedlings were incubated in HCS LipidTOX™ Deep Red Neutral Lipid Stain (ThermoFisher Scientific, 500-fold dilution in water) for 30 min before imaging. Arabidopsis seeds/seedlings and agro-infiltrated N. benthamiana leaves were imaged with a Leica SP8 confocal laser scanning microscope. GFP and chloroplast autofluorescence were excited with the 488-nm line of an argon laser, mCherry and RFP with a 561-nm diode laser, YFP was excited with the 514-nm line of an argon laser, and LipidTOX Deep Red with a 633-nm helium/neon laser.

Fluorescence emission was detected between 495-510 nm for GFP, 522-550 nm for YFP, 600-625 nm for mCherry and RFP, and 637-650 for LipidTOX Deep Red.

Chloroplast autofluorescence was imaged between 670-700 nm. For multi-labelling studies, detection was performed in a sequential line-scanning mode. The apparent diameter of LDs observed by CLSM and bright-field microscopy was estimated using ImageJ software (http://rsbweb.nih.gov/ij/), by manually drawing the diameter using the "line" tool and measuring it with the "measure" function of the software.

Protein-protein interaction studies in yeast and in planta

Yeast interaction assays were carried out using the Matchmaker GAL4 two-hybrid system 3 from Clontech, according to protocols described in the Yeast Protocols Handbook (Clontech). For yeast two-hybrid (Y2H) analyses, pGADT7-and pGBKT7derived plasmids were introduced by lithium acetate transformation into PJ69-4A and PJ69-4a strains, respectively. For yeast three-hybrid (Y3H) analyses, PJ69-4A clone transformed with pGADT7-CDC48A was re-transformed with the pARC351-derived vectors. Transformed yeasts were selected on SD medium lacking specific amino-acids, according to the auxotrophy genes carried by the cloning vectors. After appropriate mating between PJ69-4A and PJ69-4 a clones, diploid cells were selected on SD medium lacking leucine and tryptophane (SD-LW) for Y2H assays, and on SD medium lacking leucine, tryptophane and uracyl (SD-LWU) for Y3H assays. Protein interactions were assayed by growing diploid cells on selective medium lacking leucine, tryptophane and histidine (SD-LWH) for Y2H assays, and on medium lacking leucine, tryptophane, uracyl and histidine (SD-LWUH) for Y3H assays.

Protein interactions were tested in planta using a bimolecular fluorescence complementation (BiFC) assay. Leaf epidermal cells of N. benthamiana plants expressing ER-RFP were transiently co-transformed with agrobacteria harboring pBiFP3-YFP C -CDC48A [START_REF] Gallois | Functional characterization of the plant ubiquitin regulatory X (UBX) domain-containing protein AtPUX7 in Arabidopsis thaliana[END_REF] and pBiFP2-YFP N -PUX10 or pBiFP2-YFP N -PUX10-DUBX (Supplemental Table 2). Two days after infiltration, YFP fluorescence was imaged by CLSM. The interaction between the MADS box transcription factors DEFICIENS and GLOBOSA was used as a positive control and carried out by co-infiltration with the pBIFP2-DEF and pBIFP3-GLO constructs [START_REF] Ferrario | The MADS box gene FBP2 is required for SEPALLATA function in petunia[END_REF]. For BiFC quantification, fluorescent punctae were numbered in several areas of transformed epidermal cells, identical in size and imaged using the same CLSM acquisition settings, as previously described [START_REF] Stefano | BiFC for protein-protein interactions and protein topology: discussing an integrative approach for an old technique[END_REF].

Accession numbers

The Arabidopsis Genome Initiative locus identifiers for the genes mentioned in this article are PUX10 (At4g10790), PUX7 (At1g14570), CDC48A (At3g09840), EF1a (At5g60390), OLE1 (At4g25140), OLE2 (At5g40420).

Protein sequence data for the alignment of PUX10 can be found in the UniProt data library under accession numbers Q9M0N1(PUX10), Q94JZ8 (PUX7), F4JPR7 (PUX8), Q4V3D3 (PUX9), Q9ZW74 (PUX11), Q9LUG7 (PUX12), Q9C5G7 (PUX13), P0DKI4 (PUX14), Q94HV8 (PUX15), P0DKI5 (PUX16), Q9UNN5 (human FAF1), Q96CS3 (human FAF2/UBXD8), Q04228 (S. cerevisiae Ubx2p), and Q12229 (S. cerevisiae Ubx3p).

Bertrand Dubreucq for plasmid gifts. We are grateful to Philippe Guerche, Olivier Lamotte, Loïc Rajjou, Elodie Akary and Marine Froissard for helpful discussion. Aline Ballot, Laura Doucerain, and Antoine Moniot are acknowledged for participating in the experiments during their internship.

The IJPB benefits from the support of the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS). Table 1). EF1aA4 (EF1) gene expression was used as a constitutive control. (B) PUX10 mutation induces the accumulation of all three types of ubiquitinated oleosins and slows down the degradation of oleosins during post-germinative growth. Proteins were extracted from mutant and wild-type seeds/seedlings at various stages of germination (0 to 72 h). The three types of ubiquitin-oleosin conjugates were detected by immunoblotting with antiubiquitination antibodies (Apu2, specific to K48-linked polyubiquitinated proteins; Apu3, specific to K63-linked polyubiquitinated proteins; FK2, detecting monoubiquitinated proteins). Oleosin content was analyzed by SDS-PAGE followed by Coomassie blue staining (Coom.) or immunoblotting with anti-OLE1 or anti-OLE2 serum (OLE1 or OLE2). Total protein extracts from an equivalent amount of seedlings were loaded on each lane. Red asterisks mark non specific labeling of seed storage proteins in FK2 immunoblot.

(C) Oleosin degradation associated to post-germinative growth is reduced in pux10-1 mutant. Col0 and pux10-1 seeds were germinated in water for 41 h. OLE1 content per seedling was measured using quantitative immunoblot (mean ± SD from four independent seed batches). The asterisk indicates a significant genotype difference (Kruskal-Wallis test; p < 0.02). Eicosanoic acid (C20:1), a marker for storage oil, was measured by gas chromatography on the same batches of 41-h-old seedlings (G41) and compared to mature dry seed content (DS). C20:1 content was not significantly different in pux10-1 compared to wild-type Col0 (n=16; four samples of 30 seeds / seed batch x four independent seed batches) as determined by Kruskal-Wallis test (p > 0.05).

(D) PUX10 mutation prevents the increase in lipid droplet (LD) size occurring during early seedling growth. LDs of 32-h-old seedlings were stained with LipidTOX Deep Red and visualized in the hypocotyl epidermis next to the radicle by fluorescent CLSM (green) and bright-field (BF) microscopy (grey). Representative images of pux10-1 and pux10-2 seedlings are compared with those of Col0 and Ws seedlings. Scale bars represent 10 µm. The boxplot with Tukey-style whiskers shows the LD diameter distribution of pux10-1, pux10-2 and respective wild-type seedlings. Black lines represent medians, top and bottom ends of the boxes represent the first and third quartiles, respectively. Letters above the boxes indicate groups with significant differences as determined by t-test (p<0,0001; n>450 from 3-4 individuals). Oleosins (OLEs) are marked for degradation by the addition of either a monoubiquitin (Ub), a K48linked diubiquitin (K48Ub 2 ) or a K63-linked diubiquitin (K63Ub 2 ). The E2 ubiquitin-conjugating and E3 ligase enzymes involved in oleosin ubiquitinations are yet unknown. In germinating seeds, PUX10 associates with LDs through its hydrophobic HP domain. PUX10 bridges the CDC48A segregase to ubiquitinated oleosins through its UBX and UBA domains, respectively, to promote the dislocation of all three types of ubiquitinated oleosins from the LD semi-membrane. Once dislocated from the LD surface, K48Ub 2 -conjugated oleosins are degraded by the proteasome. The fate of Uband K63Ub 2 -conjugated oleosins is currently undetermined.

Figure 1 .

 1 Figure 1. PUX10 mutation alters LD dynamics in germinated seeds. (A) Molecular characterization of two pux10 mutants. Top left panel shows a schematic representation of PUX10 gene (At4g10790) with exons represented by blue boxes. Bottom left panel displays the domain architecture of PUX10 proteinshowing the UBA-like domain in green (M 1 -S 44 ), the hydrophobic sequences of the HP domain in yellow (I 102 -V 117 and A 120 -F 141 ), the UAS domain in blue (L 170 -R 295 ) and the UBX domain in pink (E 398 -I 477 ). The position of the pux10-1 and pux10-2 T-DNA insertion mutations is indicated by red triangle on the gene schematics. Sequenced T-DNA borders are indicated by green arrow (RB, right border; LB, left border). Note that the pux10-1 T-DNA insert is flanked by two LB borders. Accumulation of PUX10 mRNA in mutant and corresponding wild-type backgrounds was analyzed by RT-PCR on 24-h-germinated seedlings (right panel) using primers indicated as purple arrows on the PUX10 gene structure (primer sequences are listed in Supplemental Table1). EF1aA4 (EF1) gene expression was used as a constitutive control. (B) PUX10 mutation induces the accumulation of all three types of ubiquitinated oleosins and slows down the degradation of oleosins during post-germinative growth. Proteins were extracted from mutant and wild-type seeds/seedlings at various stages of germination (0 to 72 h). The three types of ubiquitin-oleosin conjugates were detected by immunoblotting with antiubiquitination antibodies (Apu2, specific to K48-linked polyubiquitinated proteins; Apu3, specific to K63-linked polyubiquitinated proteins; FK2, detecting monoubiquitinated proteins). Oleosin content was analyzed by SDS-PAGE followed by Coomassie blue staining (Coom.) or immunoblotting with anti-OLE1 or anti-OLE2 serum (OLE1 or OLE2). Total protein extracts from an equivalent amount of seedlings were loaded on each lane. Red asterisks mark non specific labeling of seed storage proteins in FK2 immunoblot. (C) Oleosin degradation associated to post-germinative growth is reduced in pux10-1 mutant. Col0 and pux10-1 seeds were germinated in water for 41 h. OLE1 content per seedling was measured using quantitative immunoblot (mean ± SD from four independent seed batches). The asterisk indicates a significant genotype difference (Kruskal-Wallis test; p < 0.02). Eicosanoic acid (C20:1), a marker for storage oil, was measured by gas chromatography on the same batches of 41-h-old seedlings (G41) and compared to mature dry seed content (DS). C20:1 content was not significantly different in pux10-1 compared to wild-type Col0 (n=16; four samples of 30 seeds / seed batch x four independent seed batches) as determined by Kruskal-Wallis test (p > 0.05). (D) PUX10 mutation prevents the increase in lipid droplet (LD) size occurring during early seedling growth. LDs of 32-h-old seedlings were stained with LipidTOX Deep Red and visualized in the hypocotyl epidermis next to the radicle by fluorescent CLSM (green) and bright-field (BF) microscopy (grey). Representative images of pux10-1 and pux10-2 seedlings are compared with those of Col0 and Ws seedlings. Scale bars represent 10 µm. The boxplot with Tukey-style whiskers shows the LD diameter distribution of pux10-1, pux10-2 and respective wild-type seedlings. Black lines represent medians, top and bottom ends of the boxes represent the first and third quartiles, respectively. Letters above the boxes indicate groups with significant differences as determined by t-test (p<0,0001; n>450 from 3-4 individuals).

Figure 2 .Figure 3 .

 23 Figure 2. Stable expression of PUX10-GFP or PUX10-myc rescues the phenotype of pux10-1.(A) Expression of PUX10-GFP or PUX10-myc restores the ability to degrade ubiquitinated oleosins in pux10-1 seedlings. Seeds from Col0, pux10-1, and stable Arabidopsis ProPUX10:PUX10-GFP/pux10-1 and ProPUX10:PUX10-myc/pux10-1 transgenic lines were germinated for 41 h. Proteins were extracted from seedlings and analyzed by immunoblot with anti-GFP, anti-myc, Apu2, Apu3, FK2, anti-OLE1, and anti-OLE2 antibodies to detect PUX10-GFP, PUX10-myc, OLEs-K48Ub 2 , OLEs-K63Ub 2 , OLEs-Ub, OLE1, and OLE2, respectively. Total protein extracts from an equivalent amount of seedlings were loaded on each lane. (B) PUX10-GFP expression partially restores LD morphology in pux10-1 mutant. The boxplot shows the distribution of LD diameters measured in 32-h-old seedlings of the ProPUX10:PUX10-GFP/pux10-1 transgenic line, compared to those of pux10-1 and Col0 (reported from Figure1D). Letters above the boxes indicate groups with significant differences as determined by t-test (p<0,0001; n>450 from 3 individuals).

Figure 4 .Figure 5 .Figure 6 .Figure 7 .Figure 8 .

 45678 Figure 4. PUX10 localizes to LDs in the different tissues of young seedlings. Stable Arabidopsis ProPUX10:PUX10-GFP/pux10-1 transgenic seeds were germinated for 32 h, labeled with LipidTOX Deep Red to stain LDs (magenta) and imaged by confocal microscopy. (A) Bright-field/GFP overlay image of the whole seedling was reconstituted by joining three images recorded using the same settings. (B) Magnified images of PUX10-GFP localization in different seedling tissues. Upper central panels show chloroplast (Cp) autofluorescence (blue) merged to LipidTOX fluorescence (magenta). Lower central panel shows bright-field (BF) image merged to LipidTOX fluorescence. Scale bar = 10 µm.

Figure 9 .Figure 10 .

 910 Figure9. The dislocation of ubiquitinated oleosins from LDs is prevented in pux10-1 mutant. Arabidopsis pux10-1 or Col0 seeds were germinated for 41 h in water containing 0.2% DMSO as a solvent control or 100 µM MG132. Seedling homogenates (Total) were centrifuged at 1,000 x g to remove cell debris (pellet P1) and then fractionated by centrifugation into three fractions (25,000 x g pellet, P2; floating fat layer containing LDs, LD; the soluble fraction was not analyzed because oleosins are water insoluble). Protein samples fractionated from an equivalent number of seedlings were loaded on each lane and analyzed by immunoblotting with the anti-ubiquitination antibodies Apu2, Apu3, or FK2.

Acknowledgments

We thank Olivier Grandjean from the Imaging and Cytology platform of IJPB for assistance with confocal microscopy and technical support for the BiFC experiments.

We are grateful to Patrick Grillot for taking care of the plants. We also thank Sebastian Bednarek for providing the Arabidopsis line expressing YFP-CDC48A and the antiserum specific to CDC48A, and Valérie Gaudin, Nathalie Simoncello, Hakim Mireau and

Plant and yeast expression vectors were constructed using Gateway cloning technology as described in the Gateway technology instruction manual (Invitrogen, http://www.lifetechnologies.com). To generate entry vectors with full-length or truncated versions of PUX10 and full-length OLE1, PUX10 cDNA or gene and OLE1 cDNA were amplified by PCR using attB-flanked primers and the Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) before BP clonase recombination into the pDONR207 vector (Invitrogen). The pENTR-PUX10c-DHP plasmid was generated by mutagenesis of pENTR-PUX10-stop using a Q5 site-directed Mutagenesis Kit (Clontech) to remove the transmembrane-coding region. Various expression vectors were obtained by LR reaction between these entry vectors and a set of Gateway-based destination vectors. The destination vectors used in this study are as follows: pGWB6 for 35S-driven N-terminal GFP fusions and pGWB4 for C-terminal GFP fusions w/o promoter [START_REF] Nakagawa | Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation[END_REF]; pB7WGF2 for 35S-driven N-terminal GFP fusions [START_REF] Karimi | GATEWAY vectors for Agrobacterium-mediated plant transformation[END_REF]; pDEST-GADT7 and pDEST-GBKT7 for yeast two-hybrid interaction studies [START_REF] Rossignol | Arabidopsis POT1A interacts with TERT-V(I8), an N-terminal splicing variant of telomerase[END_REF]; pARC351 for yeast three-hybrid interaction studies [START_REF] Ferrario | The MADS box gene FBP2 is required for SEPALLATA function in petunia[END_REF]; pBIFP2 and pBIFP3 for bimolecular fluorescent complementation experiments [START_REF] Azimzadeh | Arabidopsis TONNEAU1 proteins are essential for preprophase band formation and interact with centrin[END_REF]; and pMDC32-mCherry for 35Sdriven N-terminal mCherry fusions. The latter binary vector was constructed by inserting the mCherry gene, amplified using mCherry_Nter and mCherry_Cter primers, between the KpnI and AscI sites of pMDC32 [START_REF] Curtis | A gateway cloning vector set for high-throughput functional analysis of genes in planta[END_REF], and was kindly provided by Valérie Gaudin and Nathalie Simoncello (INRA de Versailles, France).

Primer sequences are listed in Supplemental Table 1. More detailed information about plasmid construction is available in Supplemental Table 2. All constructs were confirmed by DNA sequencing.

LD isolation

Using a glass-glass conical tissue grinder, dry or germinated seeds were ground in seed homogenization buffer (SH buffer; 100 mM HEPES pH 7.5, 500 mM sorbitol, 150 mM KCl, 1 mM MgCl2, 1 mM EDTA) containing protease inhibitors (complete mini EDTA-free, Roche) and 10 mM N-ethylmaleimide to prevent deubiquitinase activity. LDs Supplemental Table 1. Primers used in this study. 2. Constructs used in this study.

Supplemental Table

Supplemental