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.     

Multiscale Metabolic Modeling: Dynamic Flux Balance Analysis on a Whole-Plant Scale

Plant metabolism is characterized by a unique complexity on the cellular, tissue, and organ levels. On a whole-plant scale, changing source and sink relations accompanying plant development add another level of complexity to metabolism. With the aim of achieving a spatiotemporal resolution of source-sink interactions in crop plant metabolism, a multiscale metabolic modeling (MMM) approach was applied that integrates static organ-specific models with a whole-plant dynamic model. Allowing for a dynamic flux balance analysis on a whole-plant scale, the MMM approach was used to decipher the metabolic behavior of source and sink organs during the generative phase of the barley (Hordeum vulgare) plant. It reveals a sink-to-source shift of the barley stem caused by the senescence-related decrease in leaf source capacity, which is not sufficient to meet the nutrient requirements of sink organs such as the growing seed. The MMM platform represents a novel approach for the in silico analysis of metabolism on a whole-plant level, allowing for a systemic, spatiotemporally resolved understanding of metabolic processes involved in carbon partitioning, thus providing a novel tool for studying yield stability and crop improvement.Plants are of vital significance as a source of food (Grusak and DellaPenna, 1999; Rogalski and Carrer, 2011), feed (Lu et al., 2011), energy (Tilman et al., 2006; Parmar et al., 2011), and feedstocks for the chemical industry (Metzger and Bornscheuer, 2006; Kinghorn et al., 2011). Given the close connection between plant metabolism and the usability of plant products, there is a growing interest in understanding and predicting the behavior and regulation of plant metabolic processes. In order to increase crop quality and yield, there is a need for methods guiding the rational redesign of the plant metabolic network (Schwender, 2009).Mathematical modeling of plant metabolism offers new approaches to understand, predict, and modify complex plant metabolic processes. In plant research, the issue of metabolic modeling is constantly gaining attention, and different modeling approaches applied to plant metabolism exist, ranging from highly detailed quantitative to less complex qualitative approaches (for review, see Giersch, 2000; Morgan and Rhodes, 2002; Poolman et al., 2004; Rios-Estepa and Lange, 2007).A widely used modeling approach is flux balance analysis (FBA), which allows the prediction of metabolic capabilities and steady-state fluxes under different environmental and genetic backgrounds using (non)linear optimization (Orth et al., 2010). Assuming steady-state conditions, FBA has the advantage of not requiring the knowledge of kinetic parameters and, therefore, can be applied to model detailed, large-scale systems. In recent years, the FBA approach has been applied to several different plant species, such as maize (Zea mays; Dal’Molin et al., 2010; Saha et al., 2011), barley (Hordeum vulgare; Grafahrend-Belau et al., 2009b; Melkus et al., 2011; Rolletschek et al., 2011), rice (Oryza sativa; Lakshmanan et al., 2013), Arabidopsis (Arabidopsis thaliana; Poolman et al., 2009; de Oliveira Dal’Molin et al., 2010; Radrich et al., 2010; Williams et al., 2010; Mintz-Oron et al., 2012; Cheung et al., 2013), and rapeseed (Brassica napus; Hay and Schwender, 2011a, 2011b; Pilalis et al., 2011), as well as algae (Boyle and Morgan, 2009; Cogne et al., 2011; Dal’Molin et al., 2011) and photoautotrophic bacteria (Knoop et al., 2010; Montagud et al., 2010; Boyle and Morgan, 2011). These models have been used to study different aspects of metabolism, including the prediction of optimal metabolic yields and energy efficiencies (Dal’Molin et al., 2010; Boyle and Morgan, 2011), changes in flux under different environmental and genetic backgrounds (Grafahrend-Belau et al., 2009b; Dal’Molin et al., 2010; Melkus et al., 2011), and nonintuitive metabolic pathways that merit subsequent experimental investigations (Poolman et al., 2009; Knoop et al., 2010; Rolletschek et al., 2011). Although FBA of plant metabolic models was shown to be capable of reproducing experimentally determined flux distributions (Williams et al., 2010; Hay and Schwender, 2011b) and generating new insights into metabolic behavior, capacities, and efficiencies (Sweetlove and Ratcliffe, 2011), challenges remain to advance the utility and predictive power of the models.Given that many plant metabolic functions are based on interactions between different subcellular compartments, cell types, tissues, and organs, the reconstruction of organ-specific models and the integration of these models into interacting multiorgan and/or whole-plant models is a prerequisite to get insight into complex plant metabolic processes organized on a whole-plant scale (e.g. source-sink interactions). Almost all FBA models of plant metabolism are restricted to one cell type (Boyle and Morgan, 2009; Knoop et al., 2010; Montagud et al., 2010; Cogne et al., 2011; Dal’Molin et al., 2011), one tissue or one organ (Grafahrend-Belau et al., 2009b; Hay and Schwender, 2011a, 2011b; Pilalis et al., 2011; Mintz-Oron et al., 2012), and only one model exists taking into account the interaction between two cell types by specifying the interaction between mesophyll and bundle sheath cells in C4 photosynthesis (Dal’Molin et al., 2010). So far, no model representing metabolism at the whole-plant scale exists.Considering whole-plant metabolism raises the problem of taking into account temporal and environmental changes in metabolism during plant development and growth. Although classical static FBA is unable to predict the dynamics of metabolic processes, as the network analysis is based on steady-state solutions, time-dependent processes can be taken into account by extending the classical static FBA to a dynamic flux balance analysis (dFBA), as proposed by Mahadevan et al. (2002). The static (SOA) and dynamic optimization approaches introduced in this work provide a framework for analyzing the transience of metabolism by integrating kinetic expressions to dynamically constrain exchange fluxes. Due to the requirement of knowing or estimating a large number of kinetic parameters, so far dFBA has only been applied to a plant metabolic model once, to study the photosynthetic metabolism in the chloroplasts of C3 plants by a simplified model of five biochemical reactions (Luo et al., 2009). Integrating a dynamic model into a static FBA model is an alternative approach to perform dFBA.In this study, a multiscale metabolic modeling (MMM) approach was applied with the aim of achieving a spatiotemporal resolution of cereal crop plant metabolism. To provide a framework for the in silico analysis of the metabolic dynamics of barley on a whole-plant scale, the MMM approach integrates a static multiorgan FBA model and a dynamic whole-plant multiscale functional plant model (FPM) to perform dFBA. The performance of the novel whole-plant MMM approach was tested by studying source-sink interactions during the seed developmental phase of barley plants.     

Evolution of Conifer Diterpene Synthases: Diterpene Resin Acid Biosynthesis in Lodgepole Pine and Jack Pine Involves Monofunctional and Bifunctional Diterpene Synthases

Abscisic acid (ABA) induces stomatal closure and inhibits light-induced stomatal opening. The mechanisms in these two processes are not necessarily the same. It has been postulated that the ABA receptors involved in opening inhibition are different from those involved in closure induction. Here, we provide evidence that four recently identified ABA receptors (PYRABACTIN RESISTANCE1 [PYR1], PYRABACTIN RESISTANCE-LIKE1 [PYL1], PYL2, and PYL4) are not sufficient for opening inhibition in Arabidopsis (Arabidopsis thaliana). ABA-induced stomatal closure was impaired in the pyr1/pyl1/pyl2/pyl4 quadruple ABA receptor mutant. ABA inhibition of the opening of the mutant’s stomata remained intact. ABA did not induce either the production of reactive oxygen species and nitric oxide or the alkalization of the cytosol in the quadruple mutant, in accordance with the closure phenotype. Whole cell patch-clamp analysis of inward-rectifying K⁺ current in guard cells showed a partial inhibition by ABA, indicating that the ABA sensitivity of the mutant was not fully impaired. ABA substantially inhibited blue light-induced phosphorylation of H⁺-ATPase in guard cells in both the mutant and the wild type. On the other hand, in a knockout mutant of the SNF1-related protein kinase, srk2e, stomatal opening and closure, reactive oxygen species and nitric oxide production, cytosolic alkalization, inward-rectifying K⁺ current inactivation, and H⁺-ATPase phosphorylation were not sensitive to ABA.The phytohormone abscisic acid (ABA), which is synthesized in response to abiotic stresses, plays a key role in the drought hardiness of plants. Reducing transpirational water loss through stomatal pores is a major ABA response (Schroeder et al., 2001). ABA promotes the closure of open stomata and inhibits the opening of closed stomata. These effects are not simply the reverse of one another (Allen et al., 1999; Wang et al., 2001; Mishra et al., 2006).A class of receptors of ABA was identified (Ma et al., 2009; Park et al., 2009; Santiago et al., 2009; Nishimura et al., 2010). The sensitivity of stomata to ABA was strongly decreased in quadruple and sextuple mutants of the ABA receptor genes PYRABACTIN RESISTANCE/PYRABACTIN RESISTANCE-LIKE/REGULATORY COMPONENT OF ABSCISIC ACID RECEPTOR (PYR/PYL/RCAR; Nishimura et al., 2010; Gonzalez-Guzman et al., 2012). The PYR/PYL/RCAR receptors are involved in the early ABA signaling events, in which a sequence of interactions of the receptors with PROTEIN PHOSPHATASE 2Cs (PP2Cs) and subfamily 2 SNF1-RELATED PROTEIN KINASES (SnRK2s) leads to the activation of downstream ABA signaling targets in guard cells (Cutler et al., 2010; Kim et al., 2010; Weiner et al., 2010). Studies of Commelina communis and Vicia faba suggested that the ABA receptors involved in stomatal opening are not the same as the ABA receptors involved in stomatal closure (Allan et al., 1994; Anderson et al., 1994; Assmann, 1994; Schwartz et al., 1994). The roles of PYR/PYL/RCAR in either stomatal opening or closure remained to be elucidated.Blue light induces stomatal opening through the activation of plasma membrane H⁺-ATPase in guard cells that generates an inside-negative electrochemical gradient across the plasma membrane and drives K⁺ uptake through voltage-dependent inward-rectifying K⁺ channels (Assmann et al., 1985; Shimazaki et al., 1986; Blatt, 1987; Schroeder et al., 1987; Thiel et al., 1992). Phosphorylation of the penultimate Thr of the plasma membrane H⁺-ATPase is a prerequisite for blue light-induced activation of the H⁺-ATPase (Kinoshita and Shimazaki, 1999, 2002). ABA inhibits H⁺-ATPase activity through dephosphorylation of the penultimate Thr in the C terminus of the H⁺-ATPase in guard cells, resulting in prevention of the opening (Goh et al., 1996; Zhang et al., 2004; Hayashi et al., 2011). Inward-rectifying K⁺ currents (I_Kin) of guard cells are negatively regulated by ABA in addition to through the decline of the H⁺ pump-driven membrane potential difference (Schroeder and Hagiwara, 1989; Blatt, 1990; McAinsh et al., 1990; Schwartz et al., 1994; Grabov and Blatt, 1999; Saito et al., 2008). This down-regulation of ion transporters by ABA is essential for the inhibition of stomatal opening.A series of second messengers has been shown to mediate ABA-induced stomatal closure. Reactive oxygen species (ROS) produced by NADPH oxidases play a crucial role in ABA signaling in guard cells (Pei et al., 2000; Zhang et al., 2001; Kwak et al., 2003; Sirichandra et al., 2009; Jannat et al., 2011). Nitric oxide (NO) is an essential signaling component in ABA-induced stomatal closure (Desikan et al., 2002; Guo et al., 2003; Garcia-Mata and Lamattina, 2007; Neill et al., 2008). Alkalization of cytosolic pH in guard cells is postulated to mediate ABA-induced stomatal closure in Arabidopsis (Arabidopsis thaliana) and Pisum sativum and Paphiopedilum species (Irving et al., 1992; Gehring et al., 1997; Grabov and Blatt, 1997; Suhita et al., 2004; Gonugunta et al., 2008). These second messengers transduce environmental signals to ion channels and ion transporters that create the driving force for stomatal movements (Ward et al., 1995; MacRobbie, 1998; Garcia-Mata et al., 2003).In this study, we examined the mobilization of second messengers, the inactivation of I_Kin, and the suppression of H⁺-ATPase phosphorylation evoked by ABA in Arabidopsis mutants to clarify the downstream signaling events of ABA signaling in guard cells. The mutants included a quadruple mutant of PYR/PYL/RCARs, pyr1/pyl1/pyl2/pyl4, and a mutant of a SnRK2 kinase, srk2e.     

The Rubisco Small Subunit Is Involved in Tobamovirus Movement and Tm-22-Mediated Extreme Resistance

The multifunctional movement protein (MP) of Tomato mosaic tobamovirus (ToMV) is involved in viral cell-to-cell movement, symptom development, and resistance gene recognition. However, it remains to be elucidated how ToMV MP plays such diverse roles in plants. Here, we show that ToMV MP interacts with the Rubisco small subunit (RbCS) of Nicotiana benthamiana in vitro and in vivo. In susceptible N. benthamiana plants, silencing of NbRbCS enabled ToMV to induce necrosis in inoculated leaves, thus enhancing virus local infectivity. However, the development of systemic viral symptoms was delayed. In transgenic N. benthamiana plants harboring Tobacco mosaic virus resistance-2² (Tm-2²), which mediates extreme resistance to ToMV, silencing of NbRbCS compromised Tm-2²-dependent resistance. ToMV was able to establish efficient local infection but was not able to move systemically. These findings suggest that NbRbCS plays a vital role in tobamovirus movement and plant antiviral defenses.Plant viruses use at least one movement protein (MP) to facilitate viral spread between plant cells via plasmodesmata (PD; Lucas and Gilbertson, 1994; Ghoshroy et al., 1997). Among viral MPs, the MP of tobamoviruses, such as Tobacco mosaic virus (TMV) and its close relative Tomato mosaic virus (ToMV), is the best characterized. TMV MP specifically accumulates in PD and modifies the plasmodesmatal size exclusion limit in mature source leaves or tissues (Wolf et al., 1989; Deom et al., 1990; Ding et al., 1992). TMV MP and viral genomic RNA form a mobile ribonucleoprotein complex that is essential for cell-to-cell movement of viral infection (Watanabe et al., 1984; Deom et al., 1987; Citovsky et al., 1990, 1992; Kiselyova et al., 2001; Kawakami et al., 2004; Waigmann et al., 2007). TMV MP also enhances intercellular RNA silencing (Vogler et al., 2008) and affects viral symptom development, host range, and host susceptibility to virus (Dardick et al., 2000; Bazzini et al., 2007). Furthermore, ToMV MP is identified as an avirulence factor that is recognized by tomato (Solanum lycopersicum) resistance proteins Tobacco mosaic virus resistance-2 (Tm-2) and Tm-2² (Meshi et al., 1989; Lanfermeijer et al., 2004). Indeed, tomato Tm-2² confers extreme resistance against TMV and ToMV in tomato plants and even in heterologous tobacco (Nicotiana tabacum) plants (Lanfermeijer et al., 2003, 2004).To date, several host factors that interact with TMV MP have been identified. These TMV MP-binding host factors include cell wall-associated proteins such as pectin methylesterase (Chen et al., 2000), calreticulin (Meshi et al., 1989), ANK1 (Ueki et al., 2010), and the cellular DnaJ-like protein MPIP1 (Shimizu et al., 2009). Many cytoskeletal components such as actin filaments (McLean et al., 1995), microtubules (Heinlein et al., 1995), and the microtubule-associated proteins MPB2C (Kragler et al., 2003) and EB1a (Brandner et al., 2008) also interact with TMV MP. Most of these factors are involved in TMV cell-to-cell movement.Rubisco catalyzes the first step of CO₂ assimilation in photosynthesis and photorespiration. The Rubisco holoenzyme is a heteropolymer consisting of eight large subunits (RbCLs) and eight small subunits (RbCSs). RbCL was reported to interact with the coat protein of Potato virus Y (Feki et al., 2005). Both RbCS and RbCL were reported to interact with the P3 proteins encoded by several potyviruses, including Shallot yellow stripe virus, Onion yellow dwarf virus, Soybean mosaic virus, and Turnip mosaic virus (Lin et al., 2011). Proteomic analysis of the plant-virus interactome revealed that RbCS participates in the formation of virus complexes of Rice yellow mottle virus (Brizard et al., 2006). However, the biological function of Rubisco in viral infection remains unknown.In this study, we show that RbCS plays an essential role in virus movement, host susceptibility, and Tm-2²-mediated extreme resistance in the ToMV-host plant interaction.     

The Endocytosis of Cellulose Synthase in Arabidopsis Is Dependent on μ2, a Clathrin-Mediated Endocytosis Adaptin

Clathrin-mediated endocytosis (CME) is the best-characterized type of endocytosis in eukaryotic cells. Plants appear to possess all of the molecular components necessary to carry out CME; however, functional characterization of the components is still in its infancy. A yeast two-hybrid screen identified μ2 as a putative interaction partner of CELLULOSE SYNTHASE6 (CESA6). Arabidopsis (Arabidopsis thaliana) μ2 is homologous to the medium subunit 2 of the mammalian ADAPTOR PROTEIN COMPLEX2 (AP2). In mammals, the AP2 complex acts as the central hub of CME by docking to the plasma membrane while concomitantly recruiting cargo proteins, clathrin triskelia, and accessory proteins to the sites of endocytosis. We confirmed that μ2 interacts with multiple CESA proteins through the μ-homology domain of μ2, which is involved in specific interactions with endocytic cargo proteins in mammals. Consistent with its role in mediating the endocytosis of cargos at the plasma membrane, μ2-YELLOW FLUORESCENT PROTEIN localized to transient foci at the plasma membrane, and loss of μ2 resulted in defects in bulk endocytosis. Furthermore, loss of μ2 led to increased accumulation of YELLOW FLUORESCENT PROTEIN-CESA6 particles at the plasma membrane. Our results suggest that CESA represents a new class of CME cargo proteins and that plant cells might regulate cellulose synthesis by controlling the abundance of active CESA complexes at the plasma membrane through CME.Cellulose microfibrils, as the major load-bearing polymers in plant cell walls, are the predominant component that enforces asymmetric cell expansion (Green, 1962). In higher plants, cellulose is synthesized by multimeric rosettes, which are also referred to as cellulose synthase complexes (CSCs; Kimura et al., 1999). Genetic and coimmunoprecipitation studies have indicated that CELLULOSE SYNTHASE1 (CESA1), CESA3, and CESA6-like (CESA6, CESA2, CESA5, and CESA9) isoforms are constituents of CSCs during primary cell wall synthesis (Persson et al., 2005; Desprez et al., 2007; Persson et al., 2007; Wang et al., 2008), whereas CESA4, CESA7, and CESA8 are implicated in the cellulose synthesis of secondary cell walls (Taylor et al., 1999, 2003; Brown et al., 2005). Knowledge about cellulose synthesis has recently been enhanced by the development of a system whereby the dynamics of CESA can be imaged in living cells (Paredez et al., 2006; Desprez et al., 2007). In agreement with earlier transmission electron microscopy studies in which rosettes were visualized in Golgi cisternae, vesicles, and at the plasma membrane (Haigler and Brown, 1986), fluorescent protein tagging of CESA has identified CESA localization at the plasma membrane, in Golgi bodies, and in small intracellular compartments (Paredez et al., 2006; Desprez et al., 2007; Crowell et al., 2009; Gutierrez et al., 2009; Gu et al., 2010; Lei et al., 2012; Li et al., 2012b).Assuming that cellulose synthesis occurs solely at the plasma membrane, the trafficking of CSCs to and from the plasma membrane may act as a significant regulatory mechanism. Although the mechanistic details of CESA trafficking are lacking, live cell imaging has shown that CESA localizes to various subcellular compartments. A subset of CESAs colocalize with markers of the trans-Golgi network (TGN)/early endosome (EE), an organelle that is part of both the secretory and endocytic pathways in Arabidopsis (Arabidopsis thaliana; Dettmer et al., 2006; Lam et al., 2007; Crowell et al., 2009, 2010; Viotti et al., 2010). CESAs also localize to microtubule-associated cellulose synthase compartments (MASCs) and small CESA-containing compartments (SmaCCs). The exact function of SmaCCs/MASCs is unknown, but it has been proposed that SmaCCs/MASCs might result from the internalization of CSCs or might act in the delivery of CSCs to the plasma membrane (Crowell et al., 2009, 2010; Gutierrez et al., 2009).Clathrin-mediated endocytosis (CME) has been shown to be a major endocytic pathway in Arabidopsis (Holstein, 2002; Samaj et al., 2005; Dhonukshe et al., 2007; Kleine-Vehn and Friml, 2008; Chen et al., 2011; Beck et al., 2012; Wang et al., 2013), although there is also evidence of clathrin-independent endocytosis mechanisms (Bandmann and Homann, 2012). The function of many CME proteins has been extensively characterized in mammals (McMahon and Boucrot, 2011), and homologs of many CME components are encoded by the Arabidopsis genome, including multiple copies of clathrin H chain and clathrin light chain (CLC), all four subunits of the heterotetrameric ADAPTOR PROTEIN COMPLEX2 (AP2) complex, dynamin-related proteins, and accessory proteins such as AP180 (Holstein, 2002; Chen et al., 2011); however, many CME components have yet to be characterized in plants.It has been suggested that CME might also function in controlling cell wall metabolism. For example, dividing and growing cells internalize cross-linked cell wall pectins, which might allow for cell wall remodeling (Baluska et al., 2002, 2005; Samaj et al., 2004). Moreover, the importance of endocytosis for cell wall morphogenesis is apparent from the functional characterization of proteins involved in CME. A dynamin-related protein, DRP1A, plays a significant role in endocytosis and colocalizes with CLC (Collings et al., 2008; Konopka and Bednarek, 2008). Defective endocytosis in RADIAL SWELLING9 (rsw9) plants, which contain a mutation in DRP1A, results in cellulose deficiency and defects in cell elongation (Collings et al., 2008). A mutation in rice, brittle culm3 (bc3), was mapped to the dynamin-related gene OsDRP2A, which has been proposed to function in CME. The brittle-culm phenotype in this mutant was attributed to cellulose deficiency (Xiong et al., 2010). Although the abundance of OsCESA4 was also altered in bc3, it remains unclear whether the cellulose deficiency of either bc3 or rsw9 results directly from perturbations in CESA trafficking.To identify proteins involved in the regulation of cellulose biosynthesis, a yeast two-hybrid (Y2H) screen was performed in which the central domain of CESA6 (CESA6CD) was used as bait to screen an Arabidopsis complementary DNA library for potential interaction partners of CESA6 (Gu et al., 2010; Gu and Somerville, 2010). The Y2H screen identified μ2 as a putative interaction partner of CESA6CD. The mammalian homolog of μ2 is the medium subunit of the tetrameric AP2, which acts as the core of the CME machinery by docking to the plasma membrane while concomitantly recruiting cargo proteins, clathrin triskelia, and accessory proteins to the sites of endocytosis (Jackson et al., 2010; McMahon and Boucrot, 2011; Cocucci et al., 2012). In this study, we provide evidence that μ2 plays a role in CME in Arabidopsis, that CESAs are a new set of CME cargo proteins, and that plant cells might regulate cellulose synthesis by controlling the abundance of CSCs at the plasma membrane through CME. To our knowledge, this study is the first to show the affect of an AP2 complex component on endocytosis in plants and the first to visualize an AP2 complex component in living plant cells. Furthermore, our data suggest that the role of AP2 in plants may differ from what has been shown in animals.     

Rhamnolipids Elicit Defense Responses and Induce Disease Resistance against Biotrophic, Hemibiotrophic, and Necrotrophic Pathogens That Require Different Signaling Pathways in Arabidopsis and Highlight a Central Role for Salicylic Acid

Plant resistance to phytopathogenic microorganisms mainly relies on the activation of an innate immune response usually launched after recognition by the plant cells of microbe-associated molecular patterns. The plant hormones, salicylic acid (SA), jasmonic acid, and ethylene have emerged as key players in the signaling networks involved in plant immunity. Rhamnolipids (RLs) are glycolipids produced by bacteria and are involved in surface motility and biofilm development. Here we report that RLs trigger an immune response in Arabidopsis (Arabidopsis thaliana) characterized by signaling molecules accumulation and defense gene activation. This immune response participates to resistance against the hemibiotrophic bacterium Pseudomonas syringae pv tomato, the biotrophic oomycete Hyaloperonospora arabidopsidis, and the necrotrophic fungus Botrytis cinerea. We show that RL-mediated resistance involves different signaling pathways that depend on the type of pathogen. Ethylene is involved in RL-induced resistance to H. arabidopsidis and to P. syringae pv tomato whereas jasmonic acid is essential for the resistance to B. cinerea. SA participates to the restriction of all pathogens. We also show evidence that SA-dependent plant defenses are potentiated by RLs following challenge by B. cinerea or P. syringae pv tomato. These results highlight a central role for SA in RL-mediated resistance. In addition to the activation of plant defense responses, antimicrobial properties of RLs are thought to participate in the protection against the fungus and the oomycete. Our data highlight the intricate mechanisms involved in plant protection triggered by a new type of molecule that can be perceived by plant cells and that can also act directly onto pathogens.In their environment, plants are challenged by potentially pathogenic microorganisms. In response, they express a set of defense mechanisms including preformed structural and chemical barriers, as well as an innate immune response quickly activated after microorganism perception (Boller and Felix, 2009). Plant innate immunity is triggered after recognition by pattern recognition receptors of conserved pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs, respectively) or by plant endogenous molecules released by pathogen invasion and called danger-associated molecular patterns (Boller and Felix, 2009; Dodds and Rathjen, 2010). This first step of recognition leads to the activation of MAMP-triggered immunity (MTI). Successful pathogens can secrete effectors that interfere or suppress MTI, resulting in effector-triggered susceptibility. A second level of perception involves the direct or indirect recognition by specific receptors of pathogen effectors leading to effector-triggered immunity (ETI; Boller and Felix, 2009; Dodds and Rathjen, 2010). Whereas MTI and ETI are thought to involve common signaling network, ETI is usually quantitatively stronger than MTI and associated with more sustained and robust immune responses (Katagiri and Tsuda, 2010; Tsuda and Katagiri, 2010).The plant hormones, salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) have emerged as key players in the signaling networks involved in MTI and ETI (Robert-Seilaniantz et al., 2007; Tsuda et al., 2009; Katagiri and Tsuda, 2010; Mersmann et al., 2010; Tsuda and Katagiri, 2010; Robert-Seilaniantz et al., 2011). Interactions between these signal molecules allow the plant to activate and/or modulate an appropriate spectrum of responses, depending on the pathogen lifestyle, necrotroph or biotroph (Glazebrook, 2005; Koornneef and Pieterse, 2008). It is assumed that JA and ET signaling pathways are important for resistance to necrotrophic fungi including Botrytis cinerea and Alternaria brassicicola (Thomma et al., 2001; Ferrari et al., 2003; Glazebrook, 2005). Infection of Arabidopsis (Arabidopsis thaliana) with B. cinerea causes the induction of the JA/ET responsive gene PLANT DEFENSIN1.2 (PDF1.2; Penninckx et al., 1996; Zimmerli et al., 2001). Induction of PDF1.2 by B. cinerea is blocked in ethylene-insensitive2 (ein2) and coronatine-insensitive1 (coi1) mutants that are respectively defective in ET and JA signal transduction pathways. Moreover, ein2 and coi1 plants are highly susceptible to B. cinerea infection (Thomma et al., 1998; Thomma et al., 1999). JA/ET-dependent responses do not seem to be usually induced during resistance to biotrophs, but they can be effective if they are stimulated prior to pathogen challenge (Glazebrook, 2005). Plants impaired in SA signaling are highly susceptible to biotrophic and hemibiotrophic pathogens. Following pathogen infection, SA hydroxylase (NahG), enhanced disease susceptibility5 (eds5), or SA induction-deficient2 (sid2) plants are unable to accumulate high SA levels and they display heightened susceptibility to Pseudomonas syringae pv tomato (Pst), Hyaloperonospora arabidopsidis, or Erysiphe orontii (Delaney et al., 1994; Lawton et al., 1995; Wildermuth et al., 2001; Nawrath et al., 2002; Vlot et al., 2009). Mutants that are insensitive to SA, such as nonexpressor of PATHOGENESIS-RELATED (PR) genes1 (npr1), have enhanced susceptibility to these pathogens (Cao et al., 1994; Glazebrook et al., 1996; Shah et al., 1997; Dong, 2004). According to some reports, plant defense against necrotrophs also involves SA. Arabidopsis plants expressing the nahG gene and infected with B. cinerea show larger lesions compared with wild-type plants (Govrin and Levine, 2002). In tobacco (Nicotiana tabacum), acidic isoforms of PR3 and PR5 gene that are specifically induced by SA (Ménard et al., 2004) are up-regulated after challenge by B. cinerea (El Oirdi et al., 2010). Resistance to some necrotrophs like Fusarium graminearum involves both SA and JA signaling pathways (Makandar et al., 2010). It is assumed that SA and JA signaling can be antagonistic (Bostock, 2005; Koornneef and Pieterse, 2008; Pieterse et al., 2009; Thaler et al., 2012). In Arabidopsis, SA inhibits JA-dependent resistance against A. brassicicola or B. cinerea (Spoel et al., 2007; Koornneef et al., 2008). Recent studies demonstrated that ET modulates the NPR1-mediated antagonism between SA and JA (Leon-Reyes et al., 2009; Leon-Reyes et al., 2010a) and suppression by SA of JA-responsive gene expression is targeted at a position downstream of the JA biosynthesis pathway (Leon-Reyes et al., 2010b). Synergistic effects of SA- and JA-dependent signaling are also well documented (Schenk et al., 2000; van Wees et al., 2000; Mur et al., 2006) and induction of some defense responses after pathogen challenge requires intact JA, ET, and SA signaling pathways (Campbell et al., 2003).Isolated MAMPs trigger defense responses that also require the activation of SA, JA, and ET signaling pathways (Tsuda et al., 2009; Katagiri and Tsuda, 2010). For instance, treatment with the flagellin peptide flg22 induces many SA-related genes including SID2, EDS5, NPR1, and PR1 (Ferrari et al., 2007; Denoux et al., 2008), causes SA accumulation (Tsuda et al., 2008; Wang et al., 2009), and activates ET signaling (Bethke et al., 2009; Mersmann et al., 2010). Local application of lipopolysaccharides elevates the level of SA (Mishina and Zeier, 2007). The oomycete Pep13 peptide induces defense responses in potato (Solanum tuberosum) that require both SA and JA (Halim et al., 2009). Although signaling networks induced by isolated MAMPs are well documented, the contribution of SA, JA, and ET in MAMP- or PAMP-induced resistance to biotrophs and necrotrophs is poorly understood.Rhamnolipids (RLs) are glycolipids produced by various bacteria species including some Pseudomonas and Burkholderia species. They are essential for bacterial surface motility and biofilm development (Vatsa et al., 2010; Chrzanowski et al., 2012). RLs are potent stimulators of animal immunity (Vatsa et al., 2010). They have recently been shown to elicit plant defense responses and to induce resistance against B. cinerea in grapevine (Vitis vinifera; Varnier et al., 2009). They also participate to biocontrol activity of the plant beneficial bacteria Pseudomonas aeruginosa PNA1 against oomycetes (Perneel et al., 2008). However, the signaling pathways used by RLs to stimulate plant innate immunity are not known. To gain more insights into RL-induced MTI, we investigated RL-triggered defense responses and resistance to the necrotrophic fungus B. cinerea, the biotroph oomycete H. arabidopsidis, and the hemibiotroph bacterium Pst in Arabidopsis. Our results show that RLs trigger an innate immune response in Arabidopsis that protects the plant against these different lifestyle pathogens. We demonstrate that RL-mediated resistance involves separated signaling sectors that depend on the type of pathogen. In plants challenged by RLs, SA has a central role and participates to the restriction of the three pathogens. ET is fully involved in RL-induced resistance to the biotrophic oomycete and to the hemibiotrophic bacterium whereas JA is essential for the resistance to the necrotrophic fungus.     

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.