Clathrin Light Chains Regulate Clathrin-Mediated Trafficking,Auxin Signaling,and Development in Arabidopsis

Plant clathrin-mediated membrane trafficking is involved in many developmental processes as well as in responses to environmental cues. Previous studies have shown that clathrin-mediated endocytosis of the plasma membrane (PM) auxin transporter PIN-FORMED1 is regulated by the extracellular auxin receptor AUXIN BINDING PROTEIN1 (ABP1). However, the mechanisms by which ABP1 and other factors regulate clathrin-mediated trafficking are poorly understood. Here, we applied a genetic strategy and time-resolved imaging to dissect the role of clathrin light chains (CLCs) and ABP1 in auxin regulation of clathrin-mediated trafficking in Arabidopsis thaliana. Auxin was found to differentially regulate the PM and trans-Golgi network/early endosome (TGN/EE) association of CLCs and heavy chains (CHCs) in an ABP1-dependent but TRANSPORT INHIBITOR RESPONSE1/AUXIN-BINDING F-BOX PROTEIN (TIR1/AFB)-independent manner. Loss of CLC2 and CLC3 affected CHC membrane association, decreased both internalization and intracellular trafficking of PM proteins, and impaired auxin-regulated endocytosis. Consistent with these results, basipetal auxin transport, auxin sensitivity and distribution, and root gravitropism were also found to be dramatically altered in clc2 clc3 double mutants, resulting in pleiotropic defects in plant development. These results suggest that CLCs are key regulators in clathrin-mediated trafficking downstream of ABP1-mediated signaling and thus play a critical role in membrane trafficking from the TGN/EE and PM during plant development.     

The Small GTPase ROP10 of Medicago truncatula Is Required for Both Tip Growth of Root Hairs and Nod Factor-Induced Root Hair Deformation

Rhizobia preferentially enter legume root hairs via infection threads, after which root hairs undergo tip swelling, branching, and curling. However, the mechanisms underlying such root hair deformation are poorly understood. Here, we showed that a type II small GTPase, ROP10, of Medicago truncatula is localized at the plasma membrane (PM) of root hair tips to regulate root hair tip growth. Overexpression of ROP10 and a constitutively active mutant (ROP10CA) generated depolarized growth of root hairs, whereas a dominant negative mutant (ROP10DN) inhibited root hair elongation. Inoculated with Sinorhizobium meliloti, the depolarized swollen and ballooning root hairs exhibited extensive root hair deformation and aberrant infection symptoms. Upon treatment with rhizobia-secreted nodulation factors (NFs), ROP10 was transiently upregulated in root hairs, and ROP10 fused to green fluorescent protein was ectopically localized at the PM of NF-induced outgrowths and curls around rhizobia. ROP10 interacted with the kinase domain of the NF receptor NFP in a GTP-dependent manner. Moreover, NF-induced expression of the early nodulin gene ENOD11 was enhanced by the overexpression of ROP10 and ROP10CA. These data suggest that NFs spatiotemporally regulate ROP10 localization and activity at the PM of root hair tips and that interactions between ROP10 and NF receptors are required for root hair deformation and continuous curling during rhizobial infection.     

Insights into the Localization and Function of the Membrane Trafficking Regulator GNOM ARF-GEF at the Golgi Apparatus in Arabidopsis

GNOM is one of the most characterized membrane trafficking regulators in plants, with crucial roles in development. GNOM encodes an ARF-guanine nucleotide exchange factor (ARF-GEF) that activates small GTPases of the ARF (ADP ribosylation factor) class to mediate vesicle budding at endomembranes. The crucial role of GNOM in recycling of PIN auxin transporters and other proteins to the plasma membrane was identified in studies using the ARF-GEF inhibitor brefeldin A (BFA). GNOM, the most prominent regulator of recycling in plants, has been proposed to act and localize at so far elusive recycling endosomes. Here, we report the GNOM localization in context of its cellular function in Arabidopsis thaliana. State-of-the-art imaging, pharmacological interference, and ultrastructure analysis show that GNOM predominantly localizes to Golgi apparatus. Super-resolution confocal live imaging microscopy identified GNOM and its closest homolog GNOM-like 1 at distinct subdomains on Golgi cisternae. Short-term BFA treatment stabilizes GNOM at the Golgi apparatus, whereas prolonged exposures results in GNOM translocation to trans-Golgi network (TGN)/early endosomes (EEs). Malformed TGN/EE in gnom mutants suggests a role for GNOM in maintaining TGN/EE function. Our results redefine the subcellular action of GNOM and reevaluate the identity and function of recycling endosomes in plants.     

On the Inside

Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls.Plant cell expansion and anisotropic cell growth are driven by vacuolar turgor pressure and cell wall extensibility, which in a dynamic and restrictive manner direct cell morphogenesis (Baskin, 2005). Cellulose is the major load-bearing component of the cell wall and is thus a major determinant for anisotropic growth (Baskin, 2001). Cellulose is made up of β-1,4-linked glucan chains that may aggregate to form microfibrils holding 18 to 36 chains (Somerville, 2006; Fernandes et al., 2011; Jarvis, 2013; Newman et al., 2013; Thomas et al., 2013). In contrast to cell wall structural polysaccharides, including pectin and hemicellulose, which are synthesized by Golgi-localized enzymes, cellulose is synthesized at the plasma membrane (PM) by cellulose synthase complexes (CSCs; Somerville, 2006; Scheller and Ulvskov, 2010; Atmodjo et al., 2013). The cellulose synthases (CESAs) are the principal catalytic units of cellulose biosynthesis and in higher plants are organized into globular rosettes (Haigler and Brown, 1986). For their biosynthetic function, each primary cell wall CSC requires a minimum of three catalytic CESA proteins (Desprez et al., 2007; Persson et al., 2007).On the basis of observations that cellulose microfibrils align with cortical microtubules (MTs) and that MT disruption leads to a loss of cell expansion, it was hypothesized that cortical MTs guide the deposition and, therefore, the orientation of cellulose (Green, 1962; Ledbetter and Porter, 1963; Baskin, 2001; Bichet et al., 2001; Sugimoto et al., 2003; Baskin et al., 2004; Wasteneys and Fujita, 2006). Confocal microscopy of CESA fluorescent fusions has advanced our understanding of CESA trafficking and dynamics. CSCs are visualized as small particles moving within the plane of the PM, with an average velocity of approximately 200 to 400 nm min⁻¹. Their movement in linear tracks along cortical MTs (Paredez et al., 2006) supports the MT-cellulose alignment hypothesis.Our current understanding of cellulose synthesis suggests that CESAs are assembled into CSCs in either the endoplasmic reticulum (ER) or the Golgi apparatus and trafficked by vesicles to the PM (Bashline et al., 2014; McFarlane et al., 2014). The presence of CESAs in isolated Golgi and vesicles from the trans-Golgi network (TGN) has been established by proteomic studies (Dunkley et al., 2006; Drakakaki et al., 2012; Nikolovski et al., 2012; Parsons et al., 2012; Groen et al., 2014). Their localization at the TGN has been corroborated by electron microscopy and colocalization with TGN markers, such as vacuolar H⁺-ATP synthase subunit a1 (VHA-a1), and the Soluble NSF Attachment Protein Receptor (SNARE) protein SYNTAXIN OF PLANTS41 (SYP41), SYP42, and SYP61 (Crowell et al., 2009; Gutierrez et al., 2009; Drakakaki et al., 2012). A population of post-Golgi compartments carrying CSCs, referred to as microtubule-associated cellulose synthase compartments (MASCs) or small cellulose synthase compartments (SmaCCs), may be associated with MTs or actin filaments and are thought to be directly involved in either CSC delivery to, or internalization from, the PM (Crowell et al., 2009; Gutierrez et al., 2009).In addition to the CESAs, auxiliary proteins have been identified that play a vital role in the cellulose-synthesizing machinery. These include COBRA (Roudier et al., 2005), the endoglucanase KORRIGAN1 (KOR1; Lane et al., 2001; Lei et al., 2014b; Vain et al., 2014), and the recently identified POM-POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (POM2/CSI1; Gu et al., 2010; Bringmann et al., 2012). The latter protein functions as a linker between the cortical MTs and CSCs, as genetic lesions in POM2/CSI1 result in a lower incidence of coalignment between CSCs and cortical MTs (Bringmann et al., 2012). Given the highly regulated process of cellulose biosynthesis and deposition, it can be expected that many more accessory proteins participate in the delivery of CSCs and their interaction with MTs. Identification of these unique CSC-associated proteins can ultimately provide clues for the mechanisms behind cell growth and cell shape formation.Arabidopsis (Arabidopsis thaliana) mutants with defects in the cellulose biosynthetic machinery exhibit a loss of anisotropic growth, which results in organ swelling. This phenotype may be used as a diagnostic tool in genetic screens to identify cellulose biosynthetic and CSC auxiliary proteins (Mutwil et al., 2008). Chemical inhibitors complement genetic lesions to perturb, study, and control the cellular and physiological function of proteins (Drakakaki et al., 2009). A plethora of bioactive small molecules have been identified, and their analytical use contributes to our understanding of cellulose biosynthesis and CESA subcellular behavior (for review, see Brabham and Debolt, 2012). Small molecule treatment can induce distinct characteristic subcellular CESA patterns that can be broadly grouped into three categories (Brabham and Debolt, 2012). The first is characterized by the depletion of CESAs from the PM and their accumulation in cytosolic compartments, as observed for the herbicide isoxaben {N-[3-(1-ethyl-1-methylpropyl)-5-isoxazolyl]-2,6-dimethyoxybenzamide}, CGA 325615 [1-cyclohexyl-5-(2,3,4,5,6-pentafluorophe-noxyl)-1λ4,2,4,6-thiatriazin-3-amine], thaxtomin A (4-nitroindol-3-yl containing 2,5-dioxopiperazine), AE F150944 [N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-di-amine], and quinoxyphen [4-(2-bromo-4,5-dimethoxyphenyl)-3,4-dihydro-¹H-benzo-quinolin-2-one]; (Paredez et al., 2006; Bischoff et al., 2009; Crowell et al., 2009; Gutierrez et al., 2009; Harris et al., 2012). The second displays hyperaccumulation of CESAs at the PM, as seen for the herbicides dichlobenil (2,6-dichlorobenzonitrile) and indaziflam {N-[(1R,2S)-2,3-dihydro-2,6-dimethyl-¹H-inden-1-yl)-6-(1-fluoroethyl]-1,3,5-triazine-2,4-diamine} (Herth, 1987; DeBolt et al., 2007b; Brabham et al., 2014). The third exhibits disturbance of both CESAs and MTs and alters CESA trajectories at the PM, as exemplified by morlin (7-ethoxy-4-methylchromen-2-one; DeBolt et al., 2007a). Unique compounds inducing a phenotype combining CESA accumulation in intermediate compartments and disruption of CSC-MT interactions can contribute to both the identification of the accessory proteins linking CSCs with MTs and the vesicular delivery mechanisms of CESAs.In this study, we identified and characterized a unique cellulose deposition inhibitor, the small molecule CESA TRAFFICKING INHIBITOR (CESTRIN), which affects the localization pattern of CSCs and their interacting proteins in a unique way. The induction of cytoplasmic CESTRIN bodies might provide further clues for trafficking routes that carry CESAs to the PM.     

Determining the Subcellular Location of Synthesis and Assembly of the Cell Wall Polysaccharide (1,3; 1,4)-β-d-Glucan in Grasses

The current dogma for cell wall polysaccharide biosynthesis is that cellulose (and callose) is synthesized at the plasma membrane (PM), whereas matrix phase polysaccharides are assembled in the Golgi apparatus. We provide evidence that (1,3;1,4)-β-d-glucan (mixed-linkage glucan [MLG]) does not conform to this paradigm. We show in various grass (Poaceae) species that MLG-specific antibody labeling is present in the wall but absent over Golgi, suggesting it is assembled at the PM. Antibodies to the MLG synthases, cellulose synthase-like F6 (CSLF6) and CSLH1, located CSLF6 to the endoplasmic reticulum, Golgi, secretory vesicles, and the PM and CSLH1 to the same locations apart from the PM. This pattern was recreated upon expression of VENUS-tagged barley (Hordeum vulgare) CSLF6 and CSLH1 in Nicotiana benthamiana leaves and, consistent with our biochemical analyses of native grass tissues, shown to be catalytically active with CSLF6 and CSLH1 in PM-enriched and PM-depleted membrane fractions, respectively. These data support a PM location for the synthesis of MLG by CSLF6, the predominant enzymatically active isoform. A model is proposed to guide future experimental approaches to dissect the molecular mechanism(s) of MLG assembly.     

Specific Membrane Lipid Composition Is Important for Plasmodesmata Function in Arabidopsis

Plasmodesmata (PD) are nano-sized membrane-lined channels controlling intercellular communication in plants. Although progress has been made in identifying PD proteins, the role played by major membrane constituents, such as the lipids, in defining specialized membrane domains in PD remains unknown. Through a rigorous isolation of “native” PD membrane fractions and comparative mass spectrometry-based analysis, we demonstrate that lipids are laterally segregated along the plasma membrane (PM) at the PD cell-to-cell junction in Arabidopsis thaliana. Remarkably, our results show that PD membranes display enrichment in sterols and sphingolipids with very long chain saturated fatty acids when compared with the bulk of the PM. Intriguingly, this lipid profile is reminiscent of detergent-insoluble membrane microdomains, although our approach is valuably detergent-free. Modulation of the overall sterol composition of young dividing cells reversibly impaired the PD localization of the glycosylphosphatidylinositol-anchored proteins Plasmodesmata Callose Binding 1 and the β-1,3-glucanase PdBG2 and altered callose-mediated PD permeability. Altogether, this study not only provides a comprehensive analysis of the lipid constituents of PD but also identifies a role for sterols in modulating cell-to-cell connectivity, possibly by establishing and maintaining the positional specificity of callose-modifying glycosylphosphatidylinositol proteins at PD. Our work emphasizes the importance of lipids in defining PD membranes.     

Hitching a Ride on Vesicles: Cauliflower Mosaic Virus Movement Protein Trafficking in the Endomembrane System

The transport of a viral genome from cell to cell is enabled by movement proteins (MPs) targeting the cell periphery to mediate the gating of plasmodesmata. Given their essential role in the development of viral infection, understanding the regulation of MPs is of great importance. Here, we show that cauliflower mosaic virus (CaMV) MP contains three tyrosine-based sorting signals that interact with an Arabidopsis (Arabidopsis thaliana) μA-adaptin subunit. Fluorophore-tagged MP is incorporated into vesicles labeled with the endocytic tracer N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide. The presence of at least one of the three endocytosis motifs is essential for internalization of the protein from the plasma membrane to early endosomes, for tubule formation, and for CaMV infection. In addition, we show that MP colocalizes in vesicles with the Rab GTPase AtRAB-F2b, which is resident in prevacuolar late endosomal compartments that deliver proteins to the vacuole for degradation. Altogether, these results demonstrate that CaMV MP traffics in the endocytic pathway and that virus viability depends on functional host endomembranes.Membrane trafficking is essential in eukaryotic cells. Cellular membranes serve as a delivery system for newly synthesized proteins such as transporters and receptors exiting the endoplasmic reticulum after proper folding. They then transit through the Golgi complex, reaching the plasma membrane (PM) or the tonoplast via intermediate endomembrane compartments. Receptors and transporters returning from the PM are either recycled or targeted to the vacuole for degradation. Delivery and recycling sorting pathways overlap in the trans-Golgi network (TGN)/early endosome (EE), an intermediate compartment for both exocytosis and endocytosis (Reyes et al., 2011). In plant systems, the endoplasmic reticulum and PM provide membrane continuity between cells through the connections made by plasmodesmata (PD), cytoplasmic channels that regulate traffic in the symplasm (Maule et al., 2011).The selective transport of macromolecules between different compartments of the endomembrane system is mediated by coat proteins promoting the generation of small cargo-trafficking coated vesicles (Spang, 2008). The recognition and recruitment of cargo proteins are mediated by so-called adaptor complexes (AP complexes [AP-1–AP-4]; Robinson, 2004) one of which, AP-1, is localized on the TGN/EE and endosomes, whereas AP-2 is in the PM. The μ-subunit of AP complexes is devoted to cargo protein selection via a specific and well-characterized interaction with a Tyr-sorting signal, YXXΦ, where Φ is a bulky hydrophobic residue and X is any amino acid (Bonifacino and Dell’Angelica, 1999). YXXΦ motifs are present in the cytoplasmic tail of many proteins integral to the PM and TGN/EE and have been found in the movement proteins (MPs) of some viruses (Laporte et al., 2003; Haupt et al., 2005). Plant viruses are obligate parasites that exploit host components to move within the cell and from cell to cell into the vascular system for systemic invasion of the host. Virus movement, which requires the passage of macromolecules through PD connections, is mediated by one or more virus-encoded MPs with the help of the host cytoskeleton and/or endomembranes (Harries et al., 2010). While most MPs act to increase the size exclusion limit of PD to facilitate the passage of the viral nucleoprotein complex, other MPs are assembled in tubules that pass inside highly modified PD and transport encapsidated particles through their lumen.Here, we focus on this second group of tubule-forming MPs and examine the intracellular trafficking of cauliflower mosaic virus (CaMV) MP. The MP encoded by CaMV forms tubules guiding encapsidated virus particle cell-to-cell transport via an indirect MP-virion interaction (Stavolone et al., 2005; Sánchez-Navarro et al., 2010). However, how CaMV MP (and the other tubule-forming MPs) targets the PM and forms tubules remains to be elucidated. Tubule-forming MPs do not require an intact cytoskeleton for PM targeting (Huang et al., 2000; Pouwels et al., 2002) and/or tubule formation (Laporte et al., 2003). However, suppression of tubule formation upon treatment with brefeldin A (BFA), a specific inhibitor of secretion or endocytosis, suggests the involvement of the endomembrane system in correct functioning of some tubule-forming MPs (Huang et al., 2000; Laporte et al., 2003). In this study, we examined the three Tyr-sorting motifs in CaMV MP and show that each of the three domains interacts directly with subunit μ of an Arabidopsis (Arabidopsis thaliana) AP complex. Mutations in these domains revert in the viral context to maintain CaMV viability. MP is found in endosomal compartments labeled by AtRAB-F2b (ARA7) and N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64). The presence of at least one functional YXXΦ domain is essential for the localization of MP to endosomes and for tubule assembly but is not required for MP targeting to the PM. We provide several lines of evidence to show CaMV MP trafficking in the endocytic pathway. Our findings are discussed in the light of the recent demonstration that the TGN/EE functions as a major hub controlling secretory and endocytic pathways in plants.     

Phosphatidylinositol 4,5-Bisphosphate Influences PIN Polarization by Controlling Clathrin-Mediated Membrane Trafficking in Arabidopsis

The functions of the minor phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] during vegetative plant growth remain obscure. Here, we targeted two related phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) PIP5K1 and PIP5K2, which are expressed ubiquitously in Arabidopsis thaliana. A pip5k1 pip5k2 double mutant with reduced PtdIns(4,5)P₂ levels showed dwarf stature and phenotypes suggesting defects in auxin distribution. The roots of the pip5k1 pip5k2 double mutant had normal auxin levels but reduced auxin transport and altered distribution. Fluorescence-tagged auxin efflux carriers PIN-FORMED (PIN1)–green fluorescent protein (GFP) and PIN2-GFP displayed abnormal, partially apolar distribution. Furthermore, fewer brefeldin A–induced endosomal bodies decorated by PIN1-GFP or PIN2-GFP formed in pip5k1 pip5k2 mutants. Inducible overexpressor lines for PIP5K1 or PIP5K2 also exhibited phenotypes indicating misregulation of auxin-dependent processes, and immunolocalization showed reduced membrane association of PIN1 and PIN2. PIN cycling and polarization require clathrin-mediated endocytosis and labeled clathrin light chain also displayed altered localization patterns in the pip5k1 pip5k2 double mutant, consistent with a role for PtdIns(4,5)P₂ in the regulation of clathrin-mediated endocytosis. Further biochemical tests on subcellular fractions enriched for clathrin-coated vesicles (CCVs) indicated that pip5k1 and pip5k2 mutants have reduced CCV-associated PI4P 5-kinase activity. Together, the data indicate an important role for PtdIns(4,5)P₂ in the control of clathrin dynamics and in auxin distribution in Arabidopsis.     

Revisiting Plant Plasma Membrane Lipids in Tobacco: A Focus on Sphingolipids

The lipid composition of plasma membrane (PM) and the corresponding detergent-insoluble membrane (DIM) fraction were analyzed with a specific focus on highly polar sphingolipids, so-called glycosyl inositol phosphorylceramides (GIPCs). Using tobacco (Nicotiana tabacum) ‘Bright Yellow 2’ cell suspension and leaves, evidence is provided that GIPCs represent up to 40 mol % of the PM lipids. Comparative analysis of DIMs with the PM showed an enrichment of 2-hydroxylated very-long-chain fatty acid-containing GIPCs and polyglycosylated GIPCs in the DIMs. Purified antibodies raised against these GIPCs were further used for immunogold-electron microscopy strategy, revealing the distribution of polyglycosylated GIPCs in domains of 35 ± 7 nm in the plane of the PM. Biophysical studies also showed strong interactions between GIPCs and sterols and suggested a role for very-long-chain fatty acids in the interdigitation between the two PM-composing monolayers. The ins and outs of lipid asymmetry, raft formation, and interdigitation in plant membrane biology are finally discussed.Eukaryotic plasma membranes (PMs) are composed of three main classes of lipids, glycerolipids, sphingolipids, and sterols, which may account for up to 100,000 different molecular species (Yetukuri et al., 2008; Shevchenko and Simons, 2010). Overall, all glycerolipids share the same molecular moieties in plants, animals, and fungi. By contrast, sterols and sphingolipids are different and specific to each kingdom. For instance, the plant PM contains an important number of sterols, among which β-sitosterol, stigmasterol, and campesterol predominate (Furt et al., 2011). In addition to free sterols, phytosterols can be conjugated to form steryl glycosides (SG) and acyl steryl glycosides (ASG) that represent up to approximately 15% of the tobacco (Nicotiana tabacum) PM (Furt et al., 2010). As for sphingolipids, sphingomyelin, the major phosphosphingolipid in animals, which harbors a phosphocholine as a polar head, is not detected in plants. Glycosyl inositol phosphorylceramides (GIPCs) are the major class of sphingolipids in plants, but they are absent in animals (Sperling and Heinz, 2003; Pata et al., 2010). Sphingolipidomic approaches identified up to 200 plant sphingolipids (for review, see Pata et al., 2010; Cacas et al., 2013).Although GIPCs belong to one of the earliest classes of plant sphingolipids that were identified in the late 1950s (Carter et al., 1958), only a few GIPCs have been structurally characterized to date because of their high polarity and a limited solubility in typical lipid extraction solvents. For these reasons, they were systematically omitted from published plant PM lipid composition. GIPCs are formed by the addition of an inositol phosphate to the ceramide moiety, the inositol headgroup of which can then undergo several glycosylation steps. The dominant glycan structure, composed of a hexose-GlcA linked to the inositol, is called series A. Polar heads containing three to seven sugars, so-called series B to F, have been identified and appeared to be species specific (Buré et al., 2011; Cacas et al., 2013; Mortimer et al., 2013). The ceramide moiety of GIPCs consists of a long-chain base (LCB), mainly t18:0 (called phytosphingosine) or t18:1 compounds (for review, see Pata et al., 2010), to which is amidified a very-long-chain fatty acid (VLCFA), the latter of which is mostly 2-hydroxylated (hVLCFA) with an odd or even number of carbon atoms. In plants, little is known about the subcellular localization of GIPCs. It is assumed, however, that they would be highly represented in the PM (Worrall et al., 2003; Sperling et al., 2005), even if this remains to be experimentally proven. The main argument supporting such an assumption is the strong enrichment of trihydroxylated LCB (t18:n) in detergent-insoluble membrane (DIM) fractions (Borner et al., 2005; Lefebvre et al., 2007), LCB being known to be predominant in GIPC’s core structure as aforementioned.In addition to this chemical complexity, lipids are not evenly distributed within the PM. Sphingolipids and sterols can preferentially interact with each other and segregate to form microdomains dubbed the membrane raft (Simons and Toomre, 2000). The membrane raft hypothesis suggests that lipids play a regulatory role in mediating protein clustering within the bilayer by undergoing phase separation into liquid-disordered and liquid-ordered phases. The liquid-ordered phase, termed the membrane raft, was described as enriched in sterol and saturated sphingolipids and is characterized by tight lipid packing. Proteins, which have differential affinities for each phase, may become enriched in, or excluded from, the liquid-ordered phase domains to optimize the rate of protein-protein interactions and maximize signaling processes. In animals, rafts have been implicated in a huge range of cellular processes, such as hormone signaling, membrane trafficking in polarized epithelial cells, T cell activation, cell migration, and the life cycle of influenza and human immunodeficiency viruses (Simons and Ikonen, 1997; Simons and Gerl, 2010). In plants, evidence is increasing that rafts are also involved in signal transduction processes and membrane trafficking (for review, see Mongrand et al., 2010; Simon-Plas et al., 2011; Cacas et al., 2012a).Moreover, lipids are not evenly distributed between the two leaflets of the PM. Within the PM of eukaryotic cells, sphingolipids are primarily located in the outer monolayer, whereas unsaturated phospholipids are predominantly exposed on the cytosolic leaflet. This asymmetrical distribution has been well established in human red blood cells, in which the outer leaflet contains sphingomyelin, phosphatidylcholine, and a variety of glycolipids like gangliosides. By contrast, the cytoplasmic leaflet is composed mostly of phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and their phosphorylated derivatives (Devaux and Morris, 2004). With regard to sphingolipids and glycerolipids, the asymmetry of the former is established during their biosynthesis and that of the latter requires ATPases such as the aminophospholipid translocase that transports lipids from the outer to the inner leaflet as well as multiple drug resistance proteins that transport phosphatidylcholine in the opposite direction (Devaux and Morris, 2004). This ubiquitous scheme encountered in animal cells could apply in plant cells as proposed (Tjellstrom et al., 2010). Indeed, the authors showed that there is a pronounced transverse lipid asymmetry in root at the PM. Phospholipids and galactolipids dominate the cytosolic leaflet, whereas the apoplastic leaflet is enriched in sphingolipids and sterols.From such a high diversity of the plant PM thus arises the question of the respective contribution of lipids to membrane suborganization. Our group recently tackled this aspect by characterizing the order level of liposomes prepared from various plant lipids and labeled with the environment-sensitive probe di-4-ANEPPDHQ (Grosjean et al., 2015). Fluorescence spectroscopy experiments showed that, among phytosterols, campesterol exhibits the strongest ability to order model membranes. In agreement with these data, spatial analysis of the membrane organization through multispectral confocal microscopy pointed to the strong ability of campesterol to promote liquid-ordered domain formation and organize their spatial distribution at the membrane surface. Conjugated sterols also exhibit a striking ability to order membranes. In addition, GIPCs enhance the sterol-induced ordering effect by emphasizing the formation and increasing the size of sterol-dependent ordered domains.The aim of this study was to reinvestigate the lipid composition and organization of the PM with a particular focus on GIPCs using tobacco leaves and cv Bright Yellow 2 (BY-2) cell cultures as models. Analyzing all membrane lipid classes at once, including sphingolipids, is challenging because they all display dramatically different chemical polarity, from very apolar (like free sterols) to highly polar (like polyglycosylated GIPCs) molecules. Most lipid extraction techniques published thus far use a chloroform/methanol mixture and phase partition to remove contaminants, resulting in the loss GIPCs, which remain in the aqueous phase, unextracted in the insoluble pellet, or at the interphase (Markham et al., 2006). In order to gain access to both glycerolipid and sphingolipid species at a glance, we developed a protocol whereby the esterifed or amidified fatty acids were hydrolyzed from the glycerol backbone (glycerolipids) or the LCB (sphingolipids) of membrane lipids, respectively. Fatty acids were then analyzed by gas chromatography-mass spectrometry (GC-MS) with appropriate internal standards for quantification. We further proposed that the use of methyl tert-butyl ether (MTBE) ensures the extraction of all classes of plant polar lipids. Our results indicate that GIPCs represent up to 40 mol % of total tobacco PM lipids. Interestingly, polyglycolyslated GIPCs are 5-fold enriched in DIMs of BY-2 cells when compared with the PM. Further investigation led us to develop a preparative purification procedure that allowed us to obtain enough material to raise antibodies against GIPCs. Using immunogold labeling on PM vesicles, it was found that polyglycosylated GIPCs cluster in membrane nanodomains, strengthening the idea that lateral nanosegregation of sphingolipids takes place at the PM in plants. Multispectral confocal microscopy was performed on vesicles prepared using GIPCs, phospholipids, and sterols and labeled with the environment-sensitive probe di-4-ANEPPDHQ. Our results show that, despite different fatty acid and polar head compositions, GIPCs extracted from tobacco leaves and BY-2 cells have a similar intrinsic propensity of enhancing vesicle global order together with sterols. Assuming that GIPCs are mostly present in the outer leaflet of the PM, interactions between sterols and sphingolipids were finally studied by the Langmuir monolayer technique, and the area of a single molecule of GIPC, or in interaction with phytosterols, was calculated. Using the calculation docking method, the energy of interaction between GIPCs and phytosterols was determined. A model was proposed in which GIPCs and phytosterols interact together to form liquid-ordered domains and in which the VLCFAs of GIPCs promote the interdigitation of the two membrane leaflets. The implications of domain formation and the asymmetrical distribution of lipids at the PM in plants are also discussed. Finally, we propose a model that reconsiders the intricate organization of the plant PM bilayer.     

Trafficking of Vacuolar Proteins: The Crucial Role of Arabidopsis Vacuolar Protein Sorting 29 in Recycling Vacuolar Sorting Receptor

The retromer is involved in recycling lysosomal sorting receptors in mammals. A component of the retromer complex in Arabidopsis thaliana, vacuolar protein sorting 29 (VPS29), plays a crucial role in trafficking storage proteins to protein storage vacuoles. However, it is not known whether or how vacuolar sorting receptors (VSRs) are recycled from the prevacuolar compartment (PVC) to the trans-Golgi network (TGN) during trafficking to the lytic vacuole (LV). Here, we report that VPS29 plays an essential role in the trafficking of soluble proteins to the LV from the TGN to the PVC. maigo1-1 (mag1-1) mutants, which harbor a knockdown mutation in VPS29, were defective in trafficking of two soluble proteins, Arabidopsis aleurain-like protein (AALP):green fluorescent protein (GFP) and sporamin:GFP, to the LV but not in trafficking membrane proteins to the LV or plasma membrane or via the secretory pathway. AALP:GFP and sporamin:GFP in mag1-1 protoplasts accumulated in the TGN but were also secreted into the medium. In mag1-1 mutants, VSR1 failed to recycle from the PVC to the TGN; rather, a significant proportion was transported to the LV; VSR1 overexpression rescued this defect. Moreover, endogenous VSRs were expressed at higher levels in mag1-1 plants. Based on these results, we propose that VPS29 plays a crucial role in recycling VSRs from the PVC to the TGN during the trafficking of soluble proteins to the LV.     

Glycosylphosphatidylinositol (GPI) Modification Serves as a Primary Plasmodesmal Sorting Signal

Plasmodesmata (Pd) are membranous channels that serve as a major conduit for cell-to-cell communication in plants. The Pd-associated β-1,3-glucanase (BG_pap) and CALLOSE BINDING PROTEIN1 (PDCB1) were identified as key regulators of Pd conductivity. Both are predicted glycosylphosphatidylinositol-anchored proteins (GPI-APs) carrying a conserved GPI modification signal. However, the subcellular targeting mechanism of these proteins is unknown, particularly in the context of other GPI-APs not associated with Pd. Here, we conducted a comparative analysis of the subcellular targeting of the two Pd-resident and two unrelated non-Pd GPI-APs in Arabidopsis (Arabidopsis thaliana). We show that GPI modification is necessary and sufficient for delivering both BG_pap and PDCB1 to Pd. Moreover, the GPI modification signal from both Pd- and non-Pd GPI-APs is able to target a reporter protein to Pd, likely to plasma membrane microdomains enriched at Pd. As such, the GPI modification serves as a primary Pd sorting signal in plant cells. Interestingly, the ectodomain, a region that carries the functional domain in GPI-APs, in Pd-resident proteins further enhances Pd accumulation. However, in non-Pd GPI-APs, the ectodomain overrides the Pd targeting function of the GPI signal and determines a specific GPI-dependent non-Pd localization of these proteins at the plasma membrane and cell wall. Domain-swap analysis showed that the non-Pd localization is also dominant over the Pd-enhancing function mediated by a Pd ectodomain. In conclusion, our results indicate that segregation between Pd- and non-Pd GPI-APs occurs prior to Pd targeting, providing, to our knowledge, the first evidence of the mechanism of GPI-AP sorting in plants.Plant cells are interconnected with cross-wall membranous channels called plasmodesmata (Pd). Recent studies have shown that the region of the plasma membrane (PM) lining the Pd channel is a specialized membrane microdomain whose lipid and protein composition differs from the rest of the PM (Tilsner et al., 2011, 2016; Bayer et al., 2014; González-Solís et al., 2014; Grison et al., 2015). In a similar manner, the cell wall domain surrounding the Pd channel is specialized and, unlike the rest of the cell wall, is devoid of cellulose, rich in pectin, and contains callose (an insoluble β-1,3-glucan; Zavaliev et al., 2011; Knox and Benitez-Alfonso, 2014). In response to physiological signals, callose can be transiently deposited and degraded at Pd, which provides a mechanism for controlling the Pd aperture in diverse developmental and stress-related processes (Zavaliev et al., 2011). Control of Pd functioning is mediated by proteins that are specifically targeted to Pd. Plasmodesmal proteins localized to the PM domain of Pd can be integral transmembrane proteins, such as Pd-localized proteins (Thomas et al., 2008), the receptor kinase ARABIDOPSIS CRINKLY4 (Stahl et al., 2013), and callose synthases (Vatén et al., 2011). Alternatively, Pd proteins can associate with the membrane through a lipid modification like myristoylation (e.g. remorins; Raffaele et al., 2009) or be attached by a glycosylphosphatidylinositol (GPI) anchor (e.g. Pd-associated β-1,3-glucanases [BG_pap]; Levy et al., 2007; Rinne et al., 2011; Benitez-Alfonso et al., 2013), Pd-associated callose-binding proteins (PDCBs; Simpson et al., 2009), and LYSIN MOTIF DOMAIN-CONTAINING PROTEIN2 (LYM2; Faulkner et al., 2013).Among the known Pd proteins involved in Pd-specific callose degradation is BG_pap, a cell wall enzyme carrying a glycosyl hydrolase family 17 (GH17) module as its functional domain (Levy et al., 2007). Another group of proteins controlling callose dynamics at Pd are PDCBs that harbor a callose-binding domain termed carbohydrate-binding module 43 (CBM43), implicated in stabilizing callose at Pd (Simpson et al., 2009). Some β-1,3-glucanases may combine the two callose-modifying activities by harboring both GH17 and CBM43 functional domains, and several such proteins were shown to localize to Pd (Rinne et al., 2011; Benitez-Alfonso et al., 2013; Gaudioso-Pedraza and Benitez-Alfonso, 2014). A distinct feature of BG_pap and PDCBs is that both are predicted glycosylphosphatidylinositol-anchored proteins (GPI-APs). The GPI anchor is a form of posttranslational modification common to many cell surface proteins in all eukaryotes. GPI-APs are covalently attached to the outer leaflet of the PM through the GPI anchor. The basic structure of the anchor consists of ethanolamine phosphate, followed by a glycan chain of three Man residues and glucosamine, followed by phosphatidylinositol lipid moiety (EtNP-6Manα1-2Manα1-6Manα1-4GlcNα1-6myoinositol-1-P-lipid; Ferguson et al., 2009). All predicted GPI-APs carry an N-terminal secretion signal peptide (SP) similar to other secreted proteins. Distinctly, GPI-APs also carry a structurally conserved 25- to 30-residue C-terminal GPI attachment signal, which typically begins with a small amino acid (e.g. Ala, Asn, Asp, Cys, Gly, or Ser) termed omega, followed by a spacer region of five to 10 polar residues, and ending with a transmembrane segment of 15 to 20 hydrophobic residues (Ferguson et al., 2009). The entire region between the N-terminal and the C-terminal signals of a GPI-AP is termed the ectodomain and carries the protein’s functional domain(s). The GPI modification process takes place in the lumenal face of the endoplasmic reticulum (ER) in a cotranslational manner. Upon translocation into the ER, a GPI-AP is stabilized in the ER membrane by its C-terminal signal, which is concurrently cleaved after the omega amino acid, and a preassembled GPI anchor is covalently attached to the C terminus of the omega amino acid. After attachment to a protein, the GPI anchor undergoes a series of modifications (remodeling), both at the glucan chain and at the lipid moiety. Such remodeling is crucial for the sorting of GPI-APs in the secretory pathway and the subsequent lateral heterogeneity at the PM (Kinoshita, 2015). In particular, the addition of saturated fatty acid chains to the lipid moiety of the anchor leads to the enriched accumulation of GPI-APs in the PM microdomains, also termed lipid rafts (Muñiz and Zurzolo, 2014). In Arabidopsis (Arabidopsis thaliana), GPI modification has been predicted for 210 proteins of diverse functions at the PM or the cell wall or both (Borner et al., 2002). Despite extensive research on the GPI modification pathway and the function of GPI-APs in mammalian and yeast cells, such knowledge in plant systems is scarce. In particular, despite an emerging role of GPI-APs in the regulation of the cell wall domain of Pd, their subcellular targeting and compartmentalization mechanism have not been studied. In addition, it is not known how the targeting mechanism of Pd-resident GPI-APs is different from that of other classes of GPI-APs, which are not localized to Pd.In this study, we investigated the subcellular targeting mechanism of Pd-associated callose-modifying GPI-APs, BG_pap and PDCB1, and compared it with that of two unrelated non-Pd GPI-APs, ARABINOGALACTAN PROTEIN4 (AGP4) and LIPID TRANSFER PROTEIN1 (LTPG1). Using sequential fluorescent labeling of protein domains, we found that the C-terminal GPI modification signal present in both Pd- and non-Pd GPI-APs can function as a primary signal in targeting proteins to the Pd-enriched PM domain. Moreover, we show that while the GPI signal is sufficient for Pd targeting, the ectodomains in BG_pap and PDCB1 further enhance their accumulation at Pd. In contrast, the ectodomains in non-Pd GPI-APs mediate exclusion of the proteins from the Pd-enriched targeting pathway. The Pd exclusion effect was found to be dominant over the Pd-targeting function of the GPI signal and the Pd-enhancing function of the Pd ectodomain, and it possibly occurs prior to PM localization. Our findings thus uncover a novel Pd-targeting signal and provide, to our knowledge, the first evidence of the cellular mechanism that regulates the sorting of GPI-APs in plants.     

A New LxxxA Motif in the Transmembrane Helix3 of Maize Aquaporins Belonging to the Plasma Membrane Intrinsic Protein PIP2 Group Is Required for Their Trafficking to the Plasma Membrane

Aquaporins play important roles in maintaining plant water status under challenging environments. The regulation of aquaporin density in cell membranes is essential to control transcellular water flows. This work focuses on the maize (Zea mays) plasma membrane intrinsic protein (ZmPIP) aquaporin subfamily, which is divided into two sequence-related groups (ZmPIP1s and ZmPIP2s). When expressed alone in mesophyll protoplasts, ZmPIP2s are efficiently targeted to the plasma membrane, whereas ZmPIP1s are retained in the endoplasmic reticulum (ER). A protein domain-swapping approach was utilized to demonstrate that the transmembrane domain3 (TM3), together with the previously identified N-terminal ER export diacidic motif, account for the differential localization of these proteins. In addition to protoplasts, leaf epidermal cells transiently transformed by biolistic particle delivery were used to confirm and refine these results. By generating artificial proteins consisting of a single transmembrane domain, we demonstrated that the TM3 of ZmPIP1;2 or ZmPIP2;5 discriminates between ER and plasma membrane localization, respectively. More specifically, a new LxxxA motif in the TM3 of ZmPIP2;5, which is highly conserved in plant PIP2s, was shown to regulate its anterograde routing along the secretory pathway, particularly its export from the ER.Aquaporins are of major importance to plant physiology, being essential for the regulation of transcellular water movement during growth and development (Maurel et al., 2008; Gomes et al., 2009; Heinen et al., 2009; Prado and Maurel, 2013; Chaumont and Tyerman, 2014). Aquaporins are small membrane proteins consisting of six transmembrane (TM) domains connected by five loops (A–E), and N and C termini facing the cytosol (Fig. 1A). They assemble as homotetramers and/or heterotetramers in the membrane, with each monomer acting as an independent water channel (Murata et al., 2000; Fetter et al., 2004; Gomes et al., 2009). Aquaporins form a highly divergent protein family in plants (Chaumont et al., 2001; Johanson et al., 2001), and this work focuses on the maize (Zea mays) plasma membrane intrinsic protein (ZmPIP) family (Chaumont et al., 2001). The regulation of the subcellular localization of these proteins is a key process controlling their density in the plasma membrane (PM) and, hence, their physiological roles (Hachez et al., 2013).Open in a separate windowFigure 1.Swapping TM3 of ZmPIP2;5 with that of ZmPIP1;2 retains the protein in intracellular structures. A, Cartoons representing the chimeric proteins composed of ZmPIP2;5, in which each TM has been replaced by the corresponding TM from ZmPIP1;2. All proteins are drawn with the cytosolic domains facing down. ZmPIP2;5 and ZmPIP1;2 portions are shown in black and white, respectively. All chimeras were fused to the C terminus of mYFP, which is not displayed for clarity purposes. B, Confocal microscopy images of maize mesophyll protoplasts transiently coexpressing mYFP-tagged ZmPIP2;5-PIP1;2 TM chimeric proteins (green) and the ER marker mCFP:HDEL (cyan). FM4-64 was added as a PM marker (red). Arrowheads in image 13 indicate accumulation of the protein in punctate structures that are not labeled by mCFP:HDEL. The localization patterns of the proteins of interest are representative of a total of at least 22 cells from three independent experiments. C, Confocal microscopy images of a maize mesophyll protoplast transiently expressing mYFP:ZmPIP2;5-TM3_PIP1;2 (green) and ST:mCFP (magenta). Arrowheads indicate colocalization in Golgi stacks. The images are representative of a total of 17 cells from two independent experiments. Bar = 5 µm.PIP aquaporins cluster in two groups (PIP1s and PIP2s), which are highly conserved across species (Kammerloher et al., 1994; Chaumont et al., 2000, 2001; Johanson et al., 2001; Anderberg et al., 2012). We previously showed that the maize PIP1 and PIP2 isoforms exhibit different water channel activities when expressed in Xenopus laevis oocytes, with only PIP2s increasing the membrane water permeability coefficient (P_f; Chaumont et al., 2000). However, when ZmPIP1 and ZmPIP2 are coexpressed, the isoforms physically interact to modify their stability and trafficking to the oocyte membrane, and synergistically increase the oocyte P_f (Fetter et al., 2004). Similar synergistic interactions between PIP1s and PIP2s have been reported in numerous plant species (Temmei et al., 2005; Mahdieh et al., 2008; Vandeleur et al., 2009; Bellati et al., 2010; Ayadi et al., 2011; Horie et al., 2011; Yaneff et al., 2014).PIPs were originally thought to be exclusively localized in the PM and were named accordingly (Kammerloher et al., 1994). However, recent experiments have shown that not all PIPs are located to the PM under all conditions, and that regulation of PIP subcellular localization is a highly dynamic process involving protein interactions (Boursiac et al., 2005, 2008; Zelazny et al., 2007, 2009; Uehlein et al., 2008; Besserer et al., 2012; Luu et al., 2012). When expressed singly in maize leaf mesophyll protoplasts, fluorescently tagged ZmPIP1s and ZmPIP2s differ in their subcellular localization. ZmPIP1s are retained in the endoplasmic reticulum (ER), whereas ZmPIP2s are targeted to the PM (Zelazny et al., 2007). However, upon coexpression, ZmPIP1s are relocalized from the ER to the PM, where they perfectly colocalize with ZmPIP2s. This relocalization results from their physical interaction as demonstrated by Förster resonance energy transfer/fluorescence lifetime imaging microscopy and immunoprecipitation experiments (Zelazny et al., 2007). These results indicate that ZmPIP2s, but not ZmPIP1s, possess signals that allow them to be delivered to the PM, and that hetero-oligomerization is required for ZmPIP1 trafficking to the PM. Interestingly, a diacidic motif (DxE, Asp-any amino acid-Glu) located in the N terminus of ZmPIP2;4, ZmPIP2;5, and Arabidopsis (Arabidopsis thaliana) AtPIP2;1 was shown to be required to exit the ER (Zelazny et al., 2009; Sorieul et al., 2011). Diacidic motifs interact with Secretory protein24, which is thought to be the main cargo-selection protein of the Coat proteinII complex that mediates vesicle formation at ER export sites (Miller et al., 2003). However, not all PM-localized PIP2s contain a diacidic ER export signal (Zelazny et al., 2009). In addition, swapping the N-terminal region of ER-retained ZmPIP1;2 with that of PM-localized ZmPIP2;5, which contains the functional diacidic motif, is not sufficient to trigger ER export of the protein (Zelazny et al., 2009). This result suggests that other export signals might be present in PIP2s and/or ER retention signals might be present in PIP1s elsewhere than in the N terminus.To identify new signals regulating ZmPIP1 and ZmPIP2 protein trafficking along the secretory pathway, we used a protein domain swapping-based approach and identified the TM3 as an important region that discriminates between ER-retained ZmPIP1;2 and PM-localized ZmPIP2;5. Specific mutations in the TM3 region of ZmPIP2;5 allowed the identification of a new ZmPIP2-conserved LxxxA motif, which regulates its export from the ER.     

MTV1 and MTV4 Encode Plant-Specific ENTH and ARF GAP Proteins That Mediate Clathrin-Dependent Trafficking of Vacuolar Cargo from the Trans-Golgi Network

Many soluble proteins transit through the trans-Golgi network (TGN) and the prevacuolar compartment (PVC) en route to the vacuole, but our mechanistic understanding of this vectorial trafficking step in plants is limited. In particular, it is unknown whether clathrin-coated vesicles (CCVs) participate in this transport step. Through a screen for modified transport to the vacuole (mtv) mutants that secrete the vacuolar protein VAC2, we identified MTV1, which encodes an EPSIN N-TERMINAL HOMOLOGY protein, and MTV4, which encodes the ADP ribosylation factor GTPase-activating protein NEVERSHED/AGD5. MTV1 and NEV/AGD5 have overlapping expression patterns and interact genetically to transport vacuolar cargo and promote plant growth, but they have no apparent roles in protein secretion or endocytosis. MTV1 and NEV/AGD5 colocalize with clathrin at the TGN and are incorporated into CCVs. Importantly, mtv1 nev/agd5 double mutants show altered subcellular distribution of CCV cargo exported from the TGN. Moreover, MTV1 binds clathrin in vitro, and NEV/AGD5 associates in vivo with clathrin, directly linking these proteins to CCV formation. These results indicate that MTV1 and NEV/AGD5 are key effectors for CCV-mediated trafficking of vacuolar proteins from the TGN to the PVC in plants.     

HAPLESS13, the Arabidopsis μ1 Adaptin,Is Essential for Protein Sorting at the trans-Golgi Network/Early Endosome

In plant cells, secretory and endocytic routes intersect at the trans-Golgi network (TGN)/early endosome (EE), where cargos are further sorted correctly and in a timely manner. Cargo sorting is essential for plant survival and therefore necessitates complex molecular machinery. Adaptor proteins (APs) play key roles in this process by recruiting coat proteins and selecting cargos for different vesicle carriers. The µ1 subunit of AP-1 in Arabidopsis (Arabidopsis thaliana) was recently identified at the TGN/EE and shown to be essential for cytokinesis. However, little was known about other cellular activities affected by mutations in AP-1 or the developmental consequences of such mutations. We report here that HAPLESS13 (HAP13), the Arabidopsis µ1 adaptin, is essential for protein sorting at the TGN/EE. Functional loss of HAP13 displayed pleiotropic developmental defects, some of which were suggestive of disrupted auxin signaling. Consistent with this, the asymmetric localization of PIN-FORMED2 (PIN2), an auxin transporter, was compromised in the mutant. In addition, cell morphogenesis was disrupted. We further demonstrate that HAP13 is critical for brefeldin A-sensitive but wortmannin-insensitive post-Golgi trafficking. Our results show that HAP13 is a key link in the sophisticated trafficking network in plant cells.Plant cells contain sophisticated endomembrane compartments, including the endoplasmic reticulum, the Golgi, the trans-Golgi network (TGN)/early endosome (EE), the prevacuolar compartments/multivesicular bodies (PVC/MVB), various types of vesicles, and the plasma membrane (PM; Ebine and Ueda, 2009; Richter et al., 2009). Intracellular protein sorting between the various locations in the endomembrane system occurs in both secretory and endocytic routes (Richter et al., 2009; De Marcos Lousa et al., 2012). Vesicles in the secretory route start at the endoplasmic reticulum, passing through the Golgi before reaching the TGN/EE, while vesicles in the endocytic route start from the PM before reaching the TGN/EE (Dhonukshe et al., 2007; Viotti et al., 2010). The TGN/EE in Arabidopsis (Arabidopsis thaliana) is an independent and highly dynamic organelle transiently associated with the Golgi (Dettmer et al., 2006; Lam et al., 2007; Viotti et al., 2010), distinct from the animal TGN. Once reaching the TGN/EE, proteins delivered by their vesicle carriers are subject to further sorting, being incorporated either into vesicles that pass through the PVC/MVB before reaching the vacuole for degradation or into vesicles that enter the secretory pathway for delivery to the PM (Ebine and Ueda, 2009; Richter et al., 2009). Therefore, the TGN/EE is a critical sorting compartment that lies at the intersection of the secretory and endocytic routes.Fine-tuned control of intracellular protein sorting at the TGN/EE is essential for plant development (Geldner et al., 2003; Dhonukshe et al., 2007, 2008; Richter et al., 2007; Kitakura et al., 2011; Wang et al., 2013). An auxin gradient is crucial for pattern formation in plants, whose dynamic maintenance requires the polar localization of auxin efflux carrier PINs through endocytic recycling (Geldner et al., 2003; Blilou et al., 2005; Paciorek et al., 2005; Abas et al., 2006; Jaillais et al., 2006; Dhonukshe et al., 2007; Kleine-Vehn et al., 2008). Receptor-like kinases (RLKs) have also been recognized as major cargos undergoing endocytic trafficking, which are either recycled back to the PM or sent for vacuolar degradation (Geldner and Robatzek, 2008; Irani and Russinova, 2009). RLKs are involved in most if not all developmental processes of plants (De Smet et al., 2009).Intracellular protein sorting relies on sorting signals within cargo proteins and on the molecular machinery that recognizes sorting signals (Boehm and Bonifacino, 2001; Robinson, 2004; Dhonukshe et al., 2007). Adaptor proteins (AP) play a key role (Boehm and Bonifacino, 2001; Robinson, 2004) in the recognition of sorting signals. APs are heterotetrameric protein complexes composed of two large subunits (β and γ/α/δ/ε), a small subunit (σ), and a medium subunit (µ) that is crucial for cargo selection (Boehm and Bonifacino, 2001). APs associate with the cytoplasmic side of secretory and endocytic vesicles, recruiting coat proteins and recognizing sorting signals within cargo proteins for their incorporation into vesicle carriers (Boehm and Bonifacino, 2001). Five APs have been identified so far, classified by their components, subcellular localization, and function (Boehm and Bonifacino, 2001; Robinson, 2004; Hirst et al., 2011). Of the five APs, AP-1 associates with the TGN or recycling endosomes (RE) in yeast and mammals (Huang et al., 2001; Robinson, 2004), mediating the sorting of cargo proteins to compartments of the endosomal-lysosomal system or to the basolateral PM of polarized epithelial cells (Gonzalez and Rodriguez-Boulan, 2009). Knockouts of AP-1 components in multicellular organisms resulted in embryonic lethality (Boehm and Bonifacino, 2001; Robinson, 2004).We show here that the recently identified Arabidopsis µ1 adaptin AP1M2 (Park et al., 2013; Teh et al., 2013) is a key component in the cellular machinery mediating intracellular protein sorting at the TGN/EE. AP1M2 was previously named HAPLESS13 (HAP13), whose mutant allele hap13 showed male gametophytic lethality (Johnson et al., 2004). In recent quests for AP-1 in plants, HAP13/AP1M2 was confirmed as the Arabidopsis µ1 adaptin based on its interaction with other components of the AP-1 complex as well as its localization at the TGN (Park et al., 2013; Teh et al., 2013). A novel mutant allele of HAP13/AP1M2, ap1m2-1, was found to be defective in the intracellular distribution of KNOLLE, leading to defective cytokinesis (Park et al., 2013; Teh et al., 2013). However, it was not clear whether HAP13/AP1M2 mediated other cellular activities and their developmental consequences. Using the same mutant allele, we found that functional loss of HAP13 (hap13-1/ap1m2-1) resulted in a full spectrum of growth defects, suggestive of compromised auxin signaling and of defective RLK signaling. Cell morphogenesis was also disturbed in hap13-1. Importantly, hap13-1 was insensitive to brefeldin A (BFA) washout, indicative of defects in guanine nucleotide exchange factors for ADP-ribosylation factor (ArfGEF)-mediated post-Golgi trafficking. Furthermore, HAP13/AP1M2 showed evolutionarily conserved function during vacuolar fusion, providing additional support to its identity as a µ1 adaptin. These results demonstrate the importance of the Arabidopsis µ1 adaptin for intracellular protein sorting centered on the TGN/EE.     

Patterning and Lifetime of Plasma Membrane-Localized Cellulose Synthase Is Dependent on Actin Organization in Arabidopsis Interphase Cells

The actin and microtubule cytoskeletons regulate cell shape across phyla, from bacteria to metazoans. In organisms with cell walls, the wall acts as a primary constraint of shape, and generation of specific cell shape depends on cytoskeletal organization for wall deposition and/or cell expansion. In higher plants, cortical microtubules help to organize cell wall construction by positioning the delivery of cellulose synthase (CesA) complexes and guiding their trajectories to orient newly synthesized cellulose microfibrils. The actin cytoskeleton is required for normal distribution of CesAs to the plasma membrane, but more specific roles for actin in cell wall assembly and organization remain largely elusive. We show that the actin cytoskeleton functions to regulate the CesA delivery rate to, and lifetime of CesAs at, the plasma membrane, which affects cellulose production. Furthermore, quantitative image analyses revealed that actin organization affects CesA tracking behavior at the plasma membrane and that small CesA compartments were associated with the actin cytoskeleton. By contrast, localized insertion of CesAs adjacent to cortical microtubules was not affected by the actin organization. Hence, both actin and microtubule cytoskeletons play important roles in regulating CesA trafficking, cellulose deposition, and organization of cell wall biogenesis.Plant cells are surrounded by a flexible yet durable extracellular matrix that makes up the cell wall. This structure offers mechanical strength that counters osmotically driven turgor pressure, is an important factor for water movement in plants, acts as a physical barrier against pathogens (Somerville et al., 2004), and is a determining factor for plant cell morphogenesis. Hence, the cell wall plays a central role in plant biology.Two main types of cell walls can typically be distinguished: the primary and the secondary cell wall. The major load-bearing component in both of these cell walls is the β-1,4-linked glucan polymer cellulose (Somerville et al., 2004). Cellulose polymers are synthesized by plasma membrane (PM)-localized cellulose synthase (CesA) complexes (Mueller and Brown, 1980), which contain several CesA subunits with similar amino acid sequences (Mutwil et al., 2008a). The primary wall CesA complexes are believed to be assembled in the Golgi and are subsequently delivered to the PM via vesicular trafficking (Gutierrez et al., 2009), sometimes associated with Golgi pausing (Crowell et al., 2009). Furthermore, the primary wall CesA complexes are preferentially inserted into the PM at sites that coincide with cortical microtubules (MTs), which subsequently guide cellulose microfibril deposition (Gutierrez et al., 2009). Hence, the cortical MT array is a determinant for multiple aspects of primary wall cellulose production.The actin cytoskeleton plays a crucial role in organized deposition of cell wall polymers in many cell types, including cellulose-related polymers and pectins in tip-growing cells, such as pollen tubes and root hairs (Hu et al., 2003; Chen et al., 2007). Thus, actin-depolymerizing drugs and genetic manipulation of ACTIN genes impair directed expansion of tip-growing cells and long-distance transport of Golgi bodies with vesicles to growing regions (Ketelaar et al., 2003; Szymanski, 2005). In diffusely growing cells in roots and hypocotyls, loss of anisotropic growth has also been observed in response to mutations to vegetative ACTIN genes and to actin-depolymerizing and -stabilizing drugs (Baluska et al., 2001; Kandasamy et al., 2009). While actin is clearly important for cell wall assembly, it is less clear what precise roles it plays.One well-known function of actin in higher plants is to support intracellular movement of cytoplasmic organelles via actomyosin-based motility (Geisler et al., 2008; Szymanski, 2009). During primary wall synthesis in interphase cells, treatment with the actin assembly inhibitor latrunculin B (LatB) led to inhibition of Golgi motility and pronounced inhomogenities in CesA density at the PM (Crowell et al., 2009; Gutierrez et al., 2009) that coincided with the density of underlying and immobile Golgi bodies (Gutierrez et al., 2009). These results suggested that Golgi motility is important for CesA distribution (Gutierrez et al., 2009). The actin cytoskeleton also appears to be important for secondary wall cellulose microfibril deposition. For example, longitudinal actin filaments (AFs) define the movement of secondary wall CesA-containing Golgi bodies in developing xylem vessels (Wightman and Turner, 2008). In addition, it has been proposed that the AFs also can regulate the delivery of the secondary wall CesA complex to the PM via pausing of the Golgi (Wightman and Turner, 2008). It is therefore clear that actin organization is important for CesA distribution and for the pattern of cellulose microfibril deposition.Despite the above findings, very few reports have undertaken detailed studies to elucidate the role of the actin cytoskeleton in the distribution and trafficking of specific proteins in plant cells. Here, we have investigated the intracellular trafficking of CesA-containing vesicles and delivery of CesAs to the PM, in the context of the actin cytoskeleton. We quantitatively demonstrate that the organization of the actin cytoskeleton regulates CesA-containing Golgi distribution and the exocytic and endocytic rate of the CesAs. However, actin organization has no effect on the localized insertion of CesAs at sites of MTs at the PM.     

ROP3 GTPase Contributes to Polar Auxin Transport and Auxin Responses and Is Important for Embryogenesis and Seedling Growth in Arabidopsis

ROP GTPases are crucial for the establishment of cell polarity and for controlling responses to hormones and environmental signals in plants. In this work, we show that ROP3 plays important roles in embryo development and auxin-dependent plant growth. Loss-of-function and dominant-negative (DN) mutations in ROP3 induced a spectrum of similar defects starting with altered cell division patterning during early embryogenesis to postembryonic auxin-regulated growth and developmental responses. These resulted in distorted embryo development, defective organ formation, retarded root gravitropism, and reduced auxin-dependent hypocotyl elongation. Our results showed that the expression of AUXIN RESPONSE FACTOR5/MONOPTEROS and root master regulators PLETHORA1 (PLT1) and PLT2 was reduced in DN-rop3 mutant embryos, accounting for some of the observed patterning defects. ROP3 mutations also altered polar localization of auxin efflux proteins (PINs) at the plasma membrane (PM), thus disrupting auxin maxima in the root. Notably, ROP3 is induced by auxin and prominently detected in root stele cells, an expression pattern similar to those of several stele-enriched PINs. Our results demonstrate that ROP3 is important for maintaining the polarity of PIN proteins at the PM, which in turn ensures polar auxin transport and distribution, thereby controlling plant patterning and auxin-regulated responses.     

The Arabidopsis Synaptotagmin1 Is Enriched in Endoplasmic Reticulum-Plasma Membrane Contact Sites and Confers Cellular Resistance to Mechanical Stresses

Eukaryotic endoplasmic reticulum (ER)-plasma membrane (PM) contact sites are evolutionarily conserved microdomains that have important roles in specialized metabolic functions such as ER-PM communication, lipid homeostasis, and Ca²⁺ influx. Despite recent advances in knowledge about ER-PM contact site components and functions in yeast (Saccharomyces cerevisiae) and mammals, relatively little is known about the functional significance of these structures in plants. In this report, we characterize the Arabidopsis (Arabidopsis thaliana) phospholipid binding Synaptotagmin1 (SYT1) as a plant ortholog of the mammal extended synaptotagmins and yeast tricalbins families of ER-PM anchors. We propose that SYT1 functions at ER-PM contact sites because it displays a dual ER-PM localization, it is enriched in microtubule-depleted regions at the cell cortex, and it colocalizes with Vesicle-Associated Protein27-1, a known ER-PM marker. Furthermore, biochemical and physiological analyses indicate that SYT1 might function as an electrostatic phospholipid anchor conferring mechanical stability in plant cells. Together, the subcellular localization and functional characterization of SYT1 highlights a putative role of plant ER-PM contact site components in the cellular adaptation to environmental stresses.Land plants are sessile organisms that are persistently challenged by physicomechanical forces that arise from the external environment. Some of the most common mechanical stimuli perceived by plants, collectively termed thigmostimuli (the Greek prefix thigmo means touch), are those induced by gradients in pressure (e.g. wind or tidal flows), by the gravity vector (e.g. ice and snow accumulation), or by direct impact with inanimate objects and/or living organisms (e.g. raindrops, hailstones, insects, canopy rubbing; Telewski, 2006). Mechanical stresses are not only exerted by the environment but are also intrinsic to the biophysics of plant growth (Landrein and Hamant, 2013). For instance, plants experience progressive mechanical self-loading as they increase in size or bear fruit (Almeras et al., 2004), intricate stress patterns are generated by different expansion rates between particular plant tissues (Mirabet et al., 2011; Sampathkumar et al., 2014), and plant cells, which are physically restrained by a rigid cell wall, generate turgor pressure characterized by circumferential tensile forces and radial compressive forces toward the plasma membrane (Telewski, 2006).Under mechanical stresses, different cell types, including meristematic, expanding, and fully differentiated cells, undergo physiological changes based on the sensing and integration of various mechanical signals (Monshausen and Haswell, 2013). Although it is well established that plants sense and respond to mechanical cues, our understanding of the various molecular mechanisms by which this is accomplished is limited, and most of our knowledge relies on comparisons with mechanosensors and transduction pathways identified in Escherichia coli and mammalian cells (Arnadóttir and Chalfie, 2010). Currently, two nonmutually exclusive mechanosensing models, namely, the ion channel and the tensegrity models, coexist in the plant literature. In the ion channel model, plant homologs of the bacterial mechanosensitive channel of small conductance and putative stretch-activated Ca²⁺-permeable channels are gated in response to mechanical forces and trigger a signaling cascade through the rapid influx of extracellular Ca²⁺ toward the cytosol (Arnadóttir and Chalfie, 2010; Jensen and Haswell, 2012; Sukharev and Sachs, 2012; Kurusu et al., 2013). In the tensegrity model, plant cells operate as self-supporting structures stabilized by a dynamic prestress state in which all elements are in isometric tension (Fuller, 1961). In such a structure, the mechanical disturbance of any individual element allows stress signals to propagate and be transduced at relatively distant locations (Ingber, 2008). Thus, the mechanically stable cell walls provide structural support, and the constant remodeling of the underlying cytoskeleton in response to mechanical disturbances acts as a tensegrity sensor (Komis et al., 2002; Berghöfer et al., 2009; Nick, 2013).In this article, we characterize the Arabidopsis (Arabidopsis thaliana) type I anchor Synaptotagmin1 (SYT1/SYTA/NTMC2T1.1 [hereafter SYT1]; Craxton, 2010; Yamazaki et al., 2010; Lewis and Lazarowitz, 2010) as an important component required to withstand mechanical stress in plant cells. SYT1 belongs to a five-member family in Arabidopsis and shares a common modular structure with different members of the mammalian extended synaptotagmins (E-Syts) and yeast (Saccharomyces cerevisiae) tricalbins families of organelle tethers (Manford et al., 2012; Fig. 1A; Supplemental Fig. S1). These proteins act as molecular bridges between the ER and the PM at sites where both cellular membranes are in close proximity, called ER-PM contact sites. These specialized microdomains carry out important roles in organelle communication, lipid and Ca²⁺ homeostasis, and intracellular signaling in animal and yeast cells (Toulmay and Prinz, 2011; Helle et al., 2013; Prinz, 2014). In plants, these ER-PM contact sites have been morphologically described for decades (Staehelin, 1997), but their physiological roles have not been thoroughly characterized. Furthermore, the identity of molecular components at these sites has remained elusive until the recent characterization of the ER-PM localized Vesicle-Associated Protein27-1 (VAP27) and NETWORKED 3C (NET3C) markers in Arabidopsis (Wang et al., 2014).Open in a separate windowFigure 1.SYT1 displays dual endoplasmic reticulum (ER)-plasma membrane (PM) localization. A, Schematic representation of the SYT1 structure and functional domains. TM, Transmembrane domain; SMP, synaptotagmin-like-mitochondrial-lipid binding domain; C2A and C2B, Ca²⁺ and phospholipid binding domains. B and C, Coimmunolocalization of the endogenous SYT1 and the ER marker HDEL-GFP (B) or the PM marker PIN2-GFP (C) in 5-d-old root epidermal cells. Scale bars = 10 μm. D and E, Subcellular localization of the SYT1proSYT1-GFP marker (SYT1-GFP) in leaves. Images were acquired at the cortical (D) or equatorial regions (E) of 8-d-old leaf epidermal cells. Costaining with propidium iodide (PI) was used to facilitate the visualization of the cortical regions. Scale bars = 20 μm.In previous reports, SYT1 has been described as an essential component for PM integrity maintenance, especially under conditions of high potential for membrane disruption such as freezing or salt stresses (Schapire et al., 2008, 2009; Yamazaki et al., 2008). In these reports, SYT1 was proposed to act as a Ca²⁺-dependent regulator of membrane fusion, in analogy to the classical animal SYTs that mediate Ca²⁺-triggered vesicle fusion during neurotransmission (Carr and Munson, 2007). Another report highlighted a role for SYT1 in viral spreading from cell to cell (Lewis and Lazarowitz, 2010). The recent characterization of mammalian E-Syts in stress tolerance (Herdman et al., 2014) and the phylogenetic relationships of tricalbins and E-Syts with SYT1 (Craxton, 2010; Yamazaki et al., 2010) led us to hypothesize that SYT1 could be a functional ortholog of E-Syts and tricalbins in plants. Consistently, we found that it is localized in specific ER-PM subdomains in cortical cytoskeleton-depleted regions, it colocalizes with the VAP27 marker, and it anchors negatively charged phospholipids through its PM-targeted C2 domains. Additionally, we show that SYT1 loss of function causes mechanical instability at the tissue and cellular level without altering the gross ER morphology. Based on these findings, we conclude that SYT1 acts at ER-PM contact sites as part of structural platforms adjacent to the cortical cytoskeleton that are required for mechanical stress tolerance in Arabidopsis.     

Manipulation of Mitogen-Activated Protein Kinase Kinase Signaling in the Arabidopsis Stomatal Lineage Reveals Motifs That Contribute to Protein Localization and Signaling Specificity

When multiple mitogen-activated protein kinase (MAPK) components are recruited recurrently to transduce signals of different origins, and often opposing outcomes, mechanisms to enforce signaling specificity are of utmost importance. These mechanisms are largely uncharacterized in plant MAPK signaling networks. The Arabidopsis thaliana stomatal lineage was previously used to show that when rendered constitutively active, four MAPK kinases (MKKs), MKK4/5/7/9, are capable of perturbing stomatal development and that these kinases comprise two pairs, MKK4/5 and MKK7/9, with both overlapping and divergent functions. We characterized the contributions of specific structural domains of these four “stomatal” MKKs to MAPK signaling output and specificity both in vitro and in vivo within the three discrete cell types of the stomatal lineage. These results verify the influence of functional docking (D) domains of MKKs on MAPK signal output and identify novel regulatory functions for previously uncharacterized structures within the N termini of MKK4/5. Beyond this, we present a novel function of the D-domains of MKK7/9 in regulating the subcellular localization of these kinases. These results provide tools to broadly assess the extent to which these and additional motifs within MKKs function to regulate MAPK signal output throughout the plant.