Disruption of Fumarylacetoacetate Hydrolase Causes Spontaneous Cell Death under Short-Day Conditions in Arabidopsis

Fumarylacetoacetate hydrolase (FAH) hydrolyzes fumarylacetoacetate to fumarate and acetoacetate, the final step in the tyrosine (Tyr) degradation pathway that is essential to animals. Deficiency of FAH in animals results in an inborn lethal disorder. However, the role for the Tyr degradation pathway in plants remains to be elucidated. In this study, we isolated an Arabidopsis (Arabidopsis thaliana) short-day sensitive cell death1 (sscd1) mutant that displays a spontaneous cell death phenotype under short-day conditions. The SSCD1 gene was cloned via a map-based cloning approach and found to encode an Arabidopsis putative FAH. The spontaneous cell death phenotype of the sscd1 mutant was completely eliminated by further knockout of the gene encoding the putative homogentisate dioxygenase, which catalyzes homogentisate into maleylacetoacetate (the antepenultimate step) in the Tyr degradation pathway. Furthermore, treatment of Arabidopsis wild-type seedlings with succinylacetone, an abnormal metabolite caused by loss of FAH in the Tyr degradation pathway, mimicked the sscd1 cell death phenotype. These results demonstrate that disruption of FAH leads to cell death in Arabidopsis and suggest that the Tyr degradation pathway is essential for plant survival under short-day conditions.Programmed cell death (PCD) has been defined as a sequence of genetically regulated events that lead to the elimination of specific cells, tissues, or whole organs (Lockshin and Zakeri, 2004). In plants, PCD is essential for developmental processes and defense responses (Dangl et al., 1996; Greenberg, 1996; Durrant et al., 2007). One well-characterized example of plant PCD is the hypersensitive response occurring during incompatible plant-pathogen interactions (Lam, 2004), which results in cell death to form visible lesions at the site of infection by an avirulent pathogen and consequently limits the pathogen spread (Morel and Dangl, 1997).To date, a large number of mutants that display spontaneous cell death lesions have been identified in barley (Hordeum vulgare), maize (Zea mays), rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana; Marchetti et al., 1983; Wolter et al., 1993; Dietrich et al., 1994; Gray et al., 1997). Because lesions form in the absence of pathogen infection, these mutants have been collectively termed as lesion-mimic mutants. Many genes with regulatory roles in PCD and defense responses, including LESION SIMULATING DISEASE1, ACCELERATED CELL DEATH11, and VASCULAR ASSOCIATED DEATH1, have been cloned and characterized (Dietrich et al., 1997; Brodersen et al., 2002; Lorrain et al., 2004).The appearance of spontaneous cell death lesions in some lesion-mimic mutants is dependent on photoperiod. For example, the Arabidopsis mutant lesion simulating disease1 and myoinositol-1-phosphate synthase1 show lesions under long days (LD; Dietrich et al., 1994; Meng et al., 2009), whereas the lesion simulating disease2, lesion initiation1, enhancing RPW8-mediated HR-like cell death1, and lag one homolog1 display lesions under short days (SD; Dietrich et al., 1994; Ishikawa et al., 2003; Wang et al., 2008; Ternes et al., 2011).Blockage of some metabolic pathways in plants may cause cell death and result in lesion formation. For example, the lesion-mimic phenotypes in the Arabidopsis mutants lesion initiation2 and accelerated cell death2 and the maize mutant lesion mimic22 result from an impairment of porphyrin metabolism (Hu et al., 1998; Ishikawa et al., 2001; Mach et al., 2001). Deficiency in fatty acid, sphingolipid, and myoinositol metabolism also causes cell death in Arabidopsis (Mou et al., 2000; Liang et al., 2003; Wang et al., 2008; Meng et al., 2009; Donahue et al., 2010; Berkey et al., 2012).Tyr degradation is an essential five-step pathway in animals (Lindblad et al., 1977). First, Tyr aminotransferase catalyzes the conversion of Tyr into 4-hydroxyphenylpyruvate, which is further transformed into homogentisate by 4-hydroxyphenylpyruvate dioxygenase. Through the sequential action of homogentisate dioxygenase (HGO), maleylacetoacetate isomerase (MAAI), and fumarylacetoacetate hydrolase (FAH), homogentisate is catalyzed to generate fumarate and acetoacetate (Lindblad et al., 1977). Blockage of this pathway in animals results in metabolic disorder diseases (Lindblad et al., 1977; Ruppert et al., 1992; Grompe et al., 1993). For example, human FAH deficiency causes hereditary tyrosinemia type I (HT1), an inborn lethal disease (St-Louis and Tanguay, 1997). Although the homologous genes putatively encoding these enzymes exist in plants (Dixon et al., 2000; Lopukhina et al., 2001; Dixon and Edwards, 2006), it is unclear whether this pathway is essential for plant growth and development.In this study, we report the isolation and characterization of a recessive short-day sensitive cell death1 (sscd1) mutant in Arabidopsis. Map-based cloning of the corresponding gene revealed that SSCD1 encodes the Arabidopsis putative FAH. Further knockout of the gene encoding the Arabidopsis putative HGO completely eliminated the spontaneous cell death phenotype in the sscd1 mutant. Furthermore, we found that treatment of Arabidopsis wild-type seedlings with succinylacetone, an abnormal metabolite caused by loss of FAH in the Tyr degradation pathway (Lindblad et al., 1977), is able to mimic the sscd1 cell death phenotype. These results demonstrate that disruption of FAH leads to cell death in Arabidopsis and suggest that the Tyr degradation pathway is essential for plant survival under SD.     

Site-Specific Nitrosoproteomic Identification of Endogenously S-Nitrosylated Proteins in Arabidopsis

Nitric oxide (NO) regulates multiple developmental events and stress responses in plants. A major biologically active species of NO is S-nitrosoglutathione (GSNO), which is irreversibly degraded by GSNO reductase (GSNOR). The major physiological effect of NO is protein S-nitrosylation, a redox-based posttranslational modification mechanism by covalently linking an NO molecule to a cysteine thiol. However, little is known about the mechanisms of S-nitrosylation-regulated signaling, partly due to limited S-nitrosylated proteins being identified. In this study, we identified 1,195 endogenously S-nitrosylated peptides in 926 proteins from the Arabidopsis (Arabidopsis thaliana) by a site-specific nitrosoproteomic approach, which, to date, is the largest data set of S-nitrosylated proteins among all organisms. Consensus sequence analysis of these peptides identified several motifs that contain acidic, but not basic, amino acid residues flanking the S-nitrosylated cysteine residues. These S-nitrosylated proteins are involved in a wide range of biological processes and are significantly enriched in chlorophyll metabolism, photosynthesis, carbohydrate metabolism, and stress responses. Consistently, the gsnor1-3 mutant shows the decreased chlorophyll content and altered photosynthetic properties, suggesting that S-nitrosylation is an important regulatory mechanism in these processes. These results have provided valuable resources and new clues to the studies on S-nitrosylation-regulated signaling in plants.Nitric oxide (NO), a gaseous signaling molecule, plays important regulatory roles in higher plants, including seed dormancy and germination, root development and hypocotyl elongation, floral transition, senescence and cell death, phytohormone signaling, and responses to abiotic and biotic stresses (He et al., 2004; Besson-Bard et al., 2008; Hong et al., 2008; Neill et al., 2008; Leitner et al., 2009; Feng et al., 2013). S-Nitrosoglutathione (GSNO) is a major biologically active form of reactive nitrogen species (RNS) and functions as a primary NO donor. The endogenous GSNO homeostasis is highly dynamic, and the GSNO level is negatively regulated by GSNO reductase (GSNOR), an evolutionally conserved enzyme catalyzing irreversibly degrading GSNO (Liu et al., 2001). Mutations in the GSNOR gene cause the elevated GSNO level and consequently severe abnormalities under physiological and pathological conditions in various species (Liu et al., 2004; Feechan et al., 2005; Que et al., 2005; Lee et al., 2008; Chen et al., 2009; Moore et al., 2009; Kwon et al., 2012).In Arabidopsis (Arabidopsis thaliana), GSNOR1 is a single-copy gene, and the enzymatic activity of the encoded protein has been biochemically characterized (Sakamoto et al., 2002). Genetic studies revealed that the gsnor1-1 and gsnor1-2 mutants are gain-of-function mutations with increased GSNOR activity and a decreased cellular S-nitrosothiol level. Conversely, gsnor1-3 is a loss-of-function mutant with a significantly increased S-nitrosothiol level (Feechan et al., 2005). The defense responses mediated by distinct resistance (R) genes are significantly impaired in the gsnor1-3 mutant, and GSNOR1 functions as a positive regulator of the salicylic acid-regulated signaling network in the defense response (Feechan et al., 2005). In a genetic screen for thermotolerance-defective mutants, the sensitive to hot temperatures5 (hot5) mutant was characterized as having decreased heat acclimation and was shown to be allelic to gsnor1, indicating the importance of GSNOR1-regulated NO homeostasis in the regulation of the abiotic stress response (Lee et al., 2008). In an independent genetic screen for the oxidative stress-related mutants, the paraquat resistant2 (par2) mutant was also identified to be allelic to gsnor1, which showed an anti-cell death phenotype and multiple developmental defects, revealing the critical role of GSNOR1/HOT5/PAR2 in the regulation of oxidative stress-induced cell death (Chen et al., 2009). Similar to gsnor1-3, the hot5 and par2 allelic mutants also accumulate the significantly increased level of NO. As a result of this defect, these gsnor1/hot5/par2 mutants show a pleiotropic phenotype, with severe developmental abnormalities in both reproductive and vegetative stages (Lee et al., 2008; Chen et al., 2009; Kwon et al., 2012). These studies highlight the critical role of GSNOR1/HOT5/PAR2-modulated NO homeostasis in diverse physiological processes, including plant growth and development as well as in responses to both biotic and abiotic stresses. However, little is known about the underpinning molecular mechanisms of the NO-modulated signaling in various physiological processes.A major physiological effect of NO is executed by protein S-nitrosylation, a reversible posttranslational modification by covalent addition of an NO molecule onto a Cys thiol to form S-nitrosothiol (Jaffrey et al., 2001; Stamler et al., 2001). S-Nitrosothiols are dynamically labile in response to the intracellular redox status, allowing protein S-nitrosylation as a highly sensitive mechanism in the regulation of cellular signaling (Stamler et al., 2001; Hess et al., 2005). Emerging evidence indicates that S-nitrosylation regulates the function of the modified proteins by various mechanisms, including enzymatic activity, stability, subcellular localization, three-dimensional conformation changes, protein-protein interaction, and ligand binding (Hess et al., 2005; Wang et al., 2006; Astier et al., 2011; Gupta, 2011; Hess and Stamler, 2012). In Arabidopsis, S-nitrosylation has been shown as an important mechanism in regulating the stress responses. The activity of Met adenosyltransferase1 (MAT1), which catalyzes S-adenosyl-Met synthesis, was shown to be inhibited by S-nitrosylation (Lindermayr et al., 2006). S-nitrosylation negatively regulates the activity of a peroxynitrite detoxification enzyme, peroxiredoxin II E (PrxII E), and an NADPH oxidase, thereby modulating the oxidative stress in the defense response (Romero-Puertas et al., 2007; Yun et al., 2011). Moreover, S-nitrosylation has also been shown to regulate the conformational changes of NONEXPRESSOR OF PATHOGEN-RELATED1 (NPR1), a master regulator of the defense response, and the activity of SALICYLIC ACID-BINDING PROTEIN3 (SABP3), a key enzyme for salicylic acid biosynthesis (Tada et al., 2008; Wang et al., 2009). In addition, S-nitrosylation of TRANSPORT INHIBITOR RESPONSE1 (TIR1) and Arabidopsis Histidine Phosphotransfer Protein1 (AHP1), two key signaling components of the auxin and cytokinin pathways, respectively, plays an important role in regulating respective phytohormone signaling (Terrile et al., 2012; Feng et al., 2013). These studies illustrate the importance of S-nitrosylation in the regulation of diverse physiological processes in plants.S-Nitrosylation has been considered as one of the most important posttranslational modification mechanisms (Lane et al., 2001; Stamler et al., 2001; Hess et al., 2005). A growing number of S-nitrosylated proteins have been identified using the proteomic approach. To date, the S-nitrosoproteomic studies have identified more than 2,200 S-nitrosylated proteins, covering more than 4,100 S-nitrosylated Cys residues. Of those S-nitrosylated proteins, more than 95% were identified from mammals (Lee et al., 2012). Several proteomic studies in Arabidopsis identified a number of S-nitrosylated proteins (Lindermayr et al., 2005; Romero-Puertas et al., 2008; Palmieri et al., 2010; Fares et al., 2011; Puyaubert et al., 2014). In GSNO-treated cell suspension cultures and NO-treated leaves derived from Arabidopsis, 63 and 52 S-nitrosylated proteins were identified, which are involved in stress response, redox homeostasis, cytoskeleton organization, metabolic processes, and cellular signaling (Lindermayr et al., 2005). In an independent study, 16 S-nitrosylated proteins were identified from Arabidopsis seedlings undergoing the hypersensitive response (Romero-Puertas et al., 2008). In another independent analysis, 46 S-nitrosylated proteins were identified from cultured Arabidopsis suspension cells (Fares et al., 2011). In a more specific analysis, 11 mitochondria proteins were identified to be S-nitrosylated and/or glutathionylated (Palmieri et al., 2010). More recently, 62 endogenously S-nitrosylated proteins were identified from Arabidopsis seedlings (Puyaubert et al., 2014). Notably, a large number of the S-nitrosylated proteins are repeatedly identified in these analyses, thus confirming the validation of each study. Because of the labile nature of S-nitrosylation, most of the S-nitrosoproteomic studies used the protein samples treated with NO donors or the protein extracts prepared from NO donor-treated cells or tissues. The Arabidopsis gsnor1-3 mutants accumulate an excessive amount of NO (Feechan et al., 2005; Lee et al., 2008; Chen et al., 2009), and the identification of S-nitrosylated proteins in gsnor1-3 should depict a more comprehensive map of S-nitrosoproteome in Arabidopsis, and provide important clues on the molecular basis of the pleiotropic phenotype of the mutant.Because of the labile and dynamic nature of protein S-nitrosylation, large-scale identification of endogenously S-nitrosylated proteins remains technically challenging. At present, two major methods for identification of S-nitrosoproteome are shotgun and site-specific nitrosoproteomic analysis, both of which are based on the biotin-switch method and mass spectrometry (Jaffrey et al., 2001; Hao et al., 2006; Torta et al., 2008). In the shotgun analysis, S-nitrosylated proteins were first biotinylated, enriched by affinity-chromatography, and then identified by mass spectrometry. Although the method is relatively simple, the number of S-nitrosylated proteins identified by shotgun proteomics is often few due to various technical limitations (Torta et al., 2008). The identification capacity of nitrosoproteomics was greatly improved by the site-specific strategy, in which biotinylated proteins were first digested by trypsin and the enriched peptides were then characterized by mass spectrometry (Hao et al., 2006; Chen et al., 2010). Moreover, S-nitrosylated Cys residues can also be identified from site-specific nitrosoproteomic analysis.In this study, we performed a large-scale, site-specific proteomic analysis of endogenously S-nitrosylated proteins in Arabidopsis wild-type and gsnor1-3 seedlings, and identified 1,195 endogenously S-nitrosylated peptides in 926 proteins from the model plant species, representing the largest data set thus far reported in any organisms and providing important resources for future studies on S-nitrosylation-regulated signaling in plants.     

Polyamine Oxidase5 Regulates Arabidopsis Growth through Thermospermine Oxidase Activity

The major plant polyamines (PAs) are the tetraamines spermine (Spm) and thermospermine (T-Spm), the triamine spermidine, and the diamine putrescine. PA homeostasis is governed by the balance between biosynthesis and catabolism; the latter is catalyzed by polyamine oxidase (PAO). Arabidopsis (Arabidopsis thaliana) has five PAO genes, AtPAO1 to AtPAO5, and all encoded proteins have been biochemically characterized. All AtPAO enzymes function in the back-conversion of tetraamine to triamine and/or triamine to diamine, albeit with different PA specificities. Here, we demonstrate that AtPAO5 loss-of-function mutants (pao5) contain 2-fold higher T-Spm levels and exhibit delayed transition from vegetative to reproductive growth compared with that of wild-type plants. Although the wild type and pao5 are indistinguishable at the early seedling stage, externally supplied low-dose T-Spm, but not other PAs, inhibits aerial growth of pao5 mutants in a dose-dependent manner. Introduction of wild-type AtPAO5 into pao5 mutants rescues growth and reduces the T-Spm content, demonstrating that AtPAO5 is a T-Spm oxidase. Recombinant AtPAO5 catalyzes the conversion of T-Spm and Spm to triamine spermidine in vitro. AtPAO5 specificity for T-Spm in planta may be explained by coexpression with T-Spm synthase but not with Spm synthase. The pao5 mutant lacking T-Spm oxidation and the acl5 mutant lacking T-Spm synthesis both exhibit growth defects. This study indicates a crucial role for T-Spm in plant growth and development.Polyamines (PAs) are low-molecular mass aliphatic amines that are present in almost all living organisms. Cellular PA concentrations are governed primarily by the balance between biosynthesis and catabolism. In plants, the major PAs are the diamine putrescine (Put), the triamine spermidine (Spd), and the tetraamines spermine (Spm) and thermospermine (T-Spm; Kusano et al., 2008; Alcázar et al., 2010; Mattoo et al., 2010; Takahashi and Kakehi, 2010; Tiburcio et al., 2014). Put is synthesized from Orn by Orn decarboxylase and/or from Arg by three sequential reactions catalyzed by Arg decarboxylase (ADC), agmatine iminohydrolase, and N-carbamoylputrescine amidohydrolase. Arabidopsis (Arabidopsis thaliana) does not contain an ORNITHINE DECARBOXYLASE gene (Hanfrey et al., 2001) and synthesizes Put from Arg via the ADC pathway. Put is further converted to Spd via an aminopropyltransferase reaction catalyzed by spermidine synthase (SPDS). In this reaction, an aminopropyl residue is transferred to Put from decarboxylated S-adenosyl-Met, which is synthesized by S-adenosyl-Met decarboxylase (SAMDC; Kusano et al., 2008). Spd is then converted to Spm or T-Spm, reactions catalyzed in Arabidopsis by spermine synthase (SPMS; encoded by SPMS) or thermospermine synthase (encoded by Acaulis5 [ACL5]), respectively (Hanzawa et al., 2000; Knott et al., 2007; Kakehi et al., 2008; Naka et al., 2010). A recent review reports that T-Spm is ubiquitously present in the plant kingdom (Takano et al., 2012).The PA catabolic pathway has been extensively studied in mammals. Spm and Spd acetylation by Spd/Spm-N¹-acetyltransferase (Enzyme Commission no. 2.3.1.57) precedes the catabolism of PAs and is a rate-limiting step in the catabolic pathway (Wallace et al., 2003). A mammalian polyamine oxidase (PAO), which requires FAD as a cofactor, oxidizes N¹-acetyl Spm and N¹-acetyl Spd at the carbon on the exo-side of the N⁴-nitrogen to produce Spd and Put, respectively (Wang et al., 2001; Vujcic et al., 2003; Wu et al., 2003; Cona et al., 2006). Mammalian spermine oxidases (SMOs) perform oxidation of the carbon on the exo-side of the N⁴-nitrogen to produce Spd, 3-aminopropanal, and hydrogen peroxide (Vujcic et al., 2002; Cervelli et al., 2003; Wang et al., 2003). Thus, mammalian PAOs and SMOs are classified as back-conversion (BC)-type PAOs.In plants, Spm, T-Spm, and Spd are catabolized by PAO. Plant PAOs derived from maize (Zea mays) and barley (Hordeum vulgare) catalyze terminal catabolism (TC)-type reactions (Tavladoraki et al., 1998). TC-type PAOs oxidize the carbon at the endo-side of the N⁴-nitrogen of Spm and Spd to produce N-(3-aminopropyl)-4-aminobutanal and 4-aminobutanal, respectively, plus 1,3-diaminopropane and hydrogen peroxide (Cona et al., 2006; Angelini et al., 2008, 2010). The Arabidopsis genome contains five PAO genes, designated as AtPAO1 to AtPAO5. Four recombinant AtPAOs, AtPAO1 to AtPAO4, have been homogenously purified and characterized (Tavladoraki et al., 2006; Kamada-Nobusada et al., 2008; Moschou et al., 2008; Takahashi et al., 2010; Fincato et al., 2011, 2012). AtPAO1 to AtPAO4 possess activities that convert Spm (or T-Spm) to Spd, called partial BC, or they convert Spm (or T-Spm) first to Spd and subsequently to Put, called full BC. Ahou et al. (2014) report that recombinant AtPAO5 also catalyzes a BC-type reaction. Therefore, all Arabidopsis PAOs are BC-type enzymes (Kamada-Nobusada et al., 2008; Moschou et al., 2008; Takahashi et al., 2010; Fincato et al., 2011, 2012; Ahou et al., 2014). Four of the seven PAOs in rice (Oryza sativa; OsPAO1, OsPAO3, OsPAO4, and OsPAO5) catalyze BC-type reactions (Ono et al., 2012; Liu et al., 2014a), whereas OsPAO7 catalyzes a TC-type reaction (Liu et al., 2014b). OsPAO2 and OsPAO6 remain to be characterized, but may catalyze TC-type reactions based on their structural similarity with OsPAO7. Therefore, plants possess both TC-type and BC-type PAOs.PAs are involved in plant growth and development. Recent molecular genetic analyses in Arabidopsis indicate that metabolic blocks at the ADC, SPDS, or SAMDC steps lead to embryo lethality (Imai et al., 2004; Urano et al., 2005; Ge et al., 2006). Potato (Solanum tuberosum) plants with suppressed SAMDC expression display abnormal phenotypes (Kumar et al., 1996). It was also reported that hydrogen peroxide derived from PA catabolism affects root development and xylem differentiation (Tisi et al., 2011). These studies indicate that flux through metabolic and catabolic PA pathways is required for growth and development. The Arabidopsis acl5 mutant, which lacks T-Spm synthase activity, displays excessive differentiation of xylem tissues and a dwarf phenotype, especially in stems (Hanzawa et al., 2000; Kakehi et al., 2008, 2010). An allelic ACL5 mutant (thickvein [tkv]) exhibits a similar phenotype as that of acl5 (Clay and Nelson, 2005). These results indicate that T-Spm plays an important role in Arabidopsis xylem differentiation (Vera-Sirera et al., 2010; Takano et al., 2012).Here, we demonstrate that Arabidopsis pao5 mutants contain 2-fold higher T-Spm levels and exhibit aerial tissue growth retardation approximately 50 d after sowing compared with that of wild-type plants. Growth inhibition of pao5 stems and leaves at an early stage of development is induced by growth on media containing low T-Spm concentrations. Complementation of pao5 with AtPAO5 rescues T-Spm-induced growth inhibition. We confirm that recombinant AtPAO5 catalyzes BC of T-Spm (or Spm) to Spd. Our data strongly suggest that endogenous T-Spm levels in Arabidopsis are fine tuned, and that AtPAO5 regulates T-Spm homeostasis through a T-Spm oxidation pathway.     

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.The seed is a complex, specific structure that allows a quiescent plant embryo to cope with unfavorable germinating conditions and also permits dissemination of the species. To achieve these functions, seeds accumulate reserve compounds that will ensure the survival of the embryo and fuel the growth of the plantlet upon germination. Accumulation of lipids occurs in many eukaryotic cells and is a rather common means of storing carbon and energy. Lipid droplets (LDs) can be found in all eukaryotes, such as yeast (Saccharomyces cerevisiae; Leber et al., 1994), mammals (Murphy, 2001; Hodges and Wu, 2010), Caenorhabditis elegans (Zhang et al., 2010; Mak, 2012), Drosophila melanogaster (Beller et al., 2006, 2010), and plants (Hsieh and Huang, 2004), but also in prokaryotes (Wältermann et al., 2005). The basic structure of an LD is a core of neutral lipids covered by a phospholipid monolayer. LDs differ between species by the set of proteins covering their surface, the nature of the lipids stored, and their turnover. Nevertheless, they apparently always ensure the same function in the cell (i.e. energy storage; Murphy, 2012). In Brassicacea species such as Arabidopsis (Arabidopsis thaliana), seed reserves are mainly composed of carbohydrates, proteins, and lipids (Baud et al., 2002). The lipids are primarily stored as triacylglycerols (TAGs) in LDs, more commonly called oil bodies (OBs; Hsieh and Huang, 2004; Chapman et al., 2012; Chapman and Ohlrogge, 2012) of diameter 0.5 to 2 µm (Tzen et al., 1993).The protein composition of seed OBs has been determined for several plant species, including Brassica napus (Katavic et al., 2006; Jolivet et al., 2009) and Arabidopsis (Jolivet et al., 2004; D’Andréa et al., 2007; Vermachova et al., 2011). In Arabidopsis, 10 proteins have been identified, and seed-specific oleosins represent up to 79% of the OB proteins (Jolivet et al., 2004; D’Andréa et al., 2007; Vermachova et al., 2011). Oleosins are rather small proteins of 18.5 to 21.2 kD with a specific and highly conserved central hydrophobic domain of 72 amino acid residues flanked by hydrophilic domains of variable size and amino acid composition (Qu and Huang, 1990; Tzen et al., 1990, 1992; Huang, 1996; Hsieh and Huang, 2004). It is generally agreed that oleosins cover the OB surface, with their central hydrophobic domain inserted in the TAG through the phospholipid layer (Tzen and Huang, 1992). Besides their structural function in OBs, oleosins may serve as docking stations for other proteins at its surface (Wilfling et al., 2013) and may participate in the biosynthesis and mobilization of plant oils (Parthibane et al., 2012a, 2012b). Oleosins are probably involved in OB stability (Leprince et al., 1998; Shimada et al., 2008) and in the regulation of OB repulsion (Heneen et al., 2008), preventing the coalescence of OBs into a single organelle (Schmidt and Herman, 2008). Nevertheless, the precise functions of oleosins in OB biogenesis and dynamics have not yet been established.Global analysis of seed lipids can be performed using gas chromatography (Li et al., 2006), which allows the precise determination of both lipid content and fatty acid composition. Recently, direct organelle mass spectrometry has been used to visualize the lipid composition of cotton (Gossypium hirsutum) seed OBs (Horn et al., 2011). Nevertheless, in both cases, the methods are destructive. To observe lipid accumulation at the subcellular level, well-known nondestructive techniques for lipid visualization have been adapted to seeds. Third harmonic generation microscopy (Débarre et al., 2006) and label-free coherent anti-Stokes Raman scattering microscopy (Paar et al., 2012) allow dyeless observation of LDs but require very specific equipment. Magnetic resonance imaging enables topographic analysis of lipid distribution in cereal grains (Neuberger et al., 2008) and in submillimeter-sized seeds like those of tobacco (Nicotiana tabacum; Fuchs et al., 2013). Nevertheless, the use of fluorescent dyes such as Nile Red (Greenspan and Fowler, 1985), BODIPY (Pagano et al., 1991), or LipidTOX (Invitrogen) associated with confocal microscopy is also a powerful way to monitor LDs in living organisms.Despite knowledge accumulated on this topic (Brasaemle and Wolins, 2012; Chapman et al., 2012), little is known about OB dynamics during seed maturation. In this article, we investigate this question by monitoring the evolution of OBs in living Arabidopsis embryos over time. This analysis showed a marked change in OB size at 9 to 10 d after flowering (DAF). We then examined single, double, and triple mutants of the major oleosins found in developing seeds (OLE1 [At4g25140], OLE2 [At5g40420], and OLE4 [At3g01570]; Jolivet et al., 2004). We analyzed the OB dynamics in these mutant backgrounds as if they would contain only these three proteins. We show that the lack of specific oleosins influences the dynamics and distribution of OBs during seed maturation, which in turn affects lipid accumulation. These results pave the way for analyzing specific functions of oleosins in the synthesis, growth, and evolution of OBs.