Trans-Golgi Network-Located AP1 Gamma Adaptins Mediate Dileucine Motif-Directed Vacuolar Targeting in Arabidopsis

Membrane proteins on the tonoplast are indispensible for vacuolar functions in plants. However, how these proteins are transported to the vacuole and how they become separated from plasma membrane proteins remain largely unknown. In this study, we used Arabidopsis thaliana vacuolar ion transporter1 (VIT1) as a reporter to study the mechanisms of tonoplast targeting. We showed that VIT1 reached the tonoplast through a pathway involving the endoplasmic reticulum (ER), Golgi, trans-Golgi network (TGN), prevacuolar compartment, and tonoplast. VIT1 contains a putative N-terminal dihydrophobic type ER export signal, and its N terminus has a conserved dileucine motif (EKQTLL), which is responsible for tonoplast targeting. In vitro peptide binding assays with synthetic VIT1 N terminus identified adaptor protein complex-1 (AP1) subunits that interacted with the dileucine motif. A deficiency of AP1 gamma adaptins in Arabidopsis cells caused relocation of tonoplast proteins containing the dileucine motif, such as VIT1 and inositol transporter1, to the plasma membrane. The dileucine motif also effectively rerouted the plasma membrane protein SCAMP1 to the tonoplast. Together with subcellular localization studies showing that AP1 gamma adaptins localize to the TGN, we propose that the AP1 complex on the TGN mediates tonoplast targeting of membrane proteins with the dileucine motif.     

Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase

The presence of a large central vacuole is one of the hallmarks of a prototypical plant cell, and the multiple functions of this compartment require massive fluxes of molecules across its limiting membrane, the tonoplast. Transport is assumed to be energized by the membrane potential and the proton gradient established by the combined activity of two proton pumps, the vacuolar H⁺-pyrophosphatase (V-PPase) and the vacuolar H⁺-ATPase (V-ATPase). Exactly how labor is divided between these two enzymes has remained elusive. Here, we provide evidence using gain- and loss-of-function approaches that lack of the V-ATPase cannot be compensated for by increased V-PPase activity. Moreover, we show that increased V-ATPase activity during cold acclimation requires the presence of the V-PPase. Most importantly, we demonstrate that a mutant lacking both of these proton pumps is conditionally viable and retains significant vacuolar acidification, pointing to a so far undetected contribution of the trans-Golgi network/early endosome-localized V-ATPase to vacuolar pH.     

Traffic of Human α-Mannosidase in Plant Cells Suggests the Presence of a New Endoplasmic Reticulum-to-Vacuole Pathway without Involving the Golgi Complex

The transport of secretory proteins from the endoplasmic reticulum to the vacuole requires sorting signals as well as specific transport mechanisms. This work is focused on the transport in transgenic tobacco (Nicotiana tabacum) plants of a human α-mannosidase, MAN2B1, which is a lysosomal enzyme involved in the turnover of N-linked glycoproteins and can be used in enzyme replacement therapy. Although ubiquitously expressed, α-mannosidases are targeted to lysosomes or vacuoles through different mechanisms according to the organisms in which these proteins are produced. In tobacco cells, MAN2B1 reaches the vacuole even in the absence of mannose-6-phosphate receptors, which are responsible for its transport in animal cells. We report that MAN2B1 is targeted to the vacuole without passing through the Golgi complex. In addition, a vacuolar targeting signal that is recognized in plant cells is located in the MAN2B1 amino-terminal region. Indeed, when this amino-terminal domain is removed, the protein is retained in the endoplasmic reticulum. Moreover, when this domain is added to a plant-secreted protein, the resulting fusion protein is partially redirected to the vacuole. These results strongly suggest the existence in plants of a new type of vacuolar traffic that can be used by leaf cells to transport vacuolar proteins.Acidic α-mannosidases (EC 3.2.1.24) are exoglycosidases responsible for the removal of α-linked Man residues in the catabolism of glycoproteins (Daniel et al., 1994). These enzymes are secretory proteins that perform their function within the lysosomes in mammalian cells and into the vacuoles of yeast (Saccharomyces cerevisiae) and plant cells. Moreover, acidic α-mannosidases have also been described in microorganisms (Santacruz-Tinoco et al., 2010). The secretory proteins normally move from the endoplasmic reticulum (ER) to the target compartment using either vesicles or direct connections between compartments (Vitale and Hinz, 2005). These types of proteins need an N-terminal signal peptide to be inserted into the ER, which is removed in the ER lumen by signal peptidases. Once in the ER, secretory proteins, in the absence of other types of sorting signals, are secreted out of the cell (Jurgens, 2004). With regard to acidic α-mannosidases, while the primary structure of these proteins is highly conserved among various kingdoms, the way in which they are targeted to their final compartment inside the cell differs in eukaryotic cells. In animal cells, these hydrolases are transported to lysosomes thanks to trans-Golgi mannose 6-phosphate receptors (MPRs) that recognize the phosphorylation of a specific residue of Man (Man-6-P) in the glucidic structure of the protein. Hence, the phosphorylated oligosaccharide side chains act as targeting signals for the lysosomal compartment (Thomas, 2001; Hansen et al., 2004). Two types of MPRs have been identified with molecular masses of 46 kD (cation-dependent MPR) and 300 kD (cation-independent MPR). MPRs are also present on the cell surface, and at least the cation-independent MPR is capable of endocytosing extracellular lysosomal hydrolases (Díaz and Pfeffer, 1998). In yeast, these enzymes reach the vacuolar localization by both cytoplasm-to-vacuole targeting and autophagy pathways (Hutchins and Klionsky, 2001). In plants, vacuolar α-mannosidase follows the classic secretory pathway involving the ER-Golgi system to reach their final destination (Faye et al., 1998).Recently, a functional human α-mannosidase (MAN2B1) has been expressed in stably transformed tobacco (Nicotiana tabacum) plants to develop an enzyme-replacement therapy for α-mannosidosis, which is a rare lysosomal storage disease caused by mutations in the MAN2B1 gene (De Marchis et al., 2011). In the human cells, MAN2B1 is synthesized as a high-M_r precursor that is posttranslationally modified by N-glycosylation, disulfide bridge formation, proteolysis, zinc binding, and homodimer formation (Tollersrud et al., 1997). Similarly, in transgenic plants, recombinant MAN2B1, provided with a plant signal peptide, is synthesized as a 110-kD precursor that undergoes specific posttranslational modifications including N-glycosylation and proteolytic maturation in the vacuole, producing four processed forms with apparent molecular masses of 70, 40, 32, and 18 kD. Unexpectedly, recombinant MAN2B1 in tobacco, instead of being secreted due to the absence in plants of MPRs (Gaudreault and Beevers, 1984), is targeted to the vacuole (De Marchis et al., 2011). Conversely, another human lysosomal enzyme, glucocerebrosidase, when produced in Arabidopsis (Arabidopsis thaliana) seeds, is mainly secreted in the apoplast, and only a minor fraction of the protein is detected in protein storage vacuoles (PSVs; He et al., 2012). Indeed, to facilitate glucocerebrosidase targeting to the vacuoles of carrot (Daucus carota) cells, Shaaltiel and colleagues (2007) added a seven-amino acid vacuole-targeting signal to the C terminus of the protein. Therefore, in this study, we tried to understand which route is used by the soluble lysosomal MAN2B1 in tobacco to reach the vacuoles.Mammalian lysosomes are considered equivalent to plant lytic vacuoles (LVs), but plants also contain PSVs for reserve accumulation, even if the distinction between different vacuoles is debated (Frigerio et al., 2008). In plants, regardless of the type of vacuole (LV or PSV), soluble vacuolar proteins reach the vacuole through the Golgi apparatus (Hwang, 2008). The transport of most secretory proteins from the ER to the Golgi complex is coat protein II mediated before reaching their final destinations. From the Golgi apparatus, vacuolar proteins reach the vacuole either through electron-opaque vesicles or via clathrin-coated vesicles (Vitale and Hinz, 2005). Plant vacuolar sorting signals and vacuolar sorting receptors that enable this traffic have recently been described (Hwang, 2008; De Marcos Lousa et al., 2012). There are certainly exceptions to this main vacuolar sorting mechanism, characterized by proteins that travel directly from the ER to the vacuole, bypassing the Golgi system, but these polypeptides are either membrane proteins or proteins that form insoluble aggregates. For example, the vacuolar storage proteins of pumpkin (Cucurbita maxima) reach PSVs via precursor-accumulating vesicles, bypassing the Golgi complex (Hara-Nishimura et al., 1998). In addition, the route that bypasses the Golgi system seems to be linked to the specific transport of proteins that form large aggregates (Herman and Schmidt, 2004; Herman, 2008). Cereal prolamins, when aggregated in the ER in large polymers, can also be transported directly from the ER to PSVs, apparently by autophagy (Levanony et al., 1992; Reyes et al., 2011). Moreover, many vacuolar enzymes are stored in ER-derived vesicles, which, under certain circumstances such as programmed cell death or seed germination, are directly fused with the vacuolar compartment (Hayashi et al., 2001; Rojo et al., 2003).We show that MAN2B1, when expressed in tobacco, reaches the vacuole of leaf cells while bypassing the Golgi and that the N-terminal domain of MAN2B1 has a cryptic vacuolar targeting signal. Indeed, the removal of 200 amino acids from the N terminus prevents MAN2B1 vacuolar delivery, and, when fused with a secreted protein, this N-terminal domain is able to redirect this protein to the vacuole by a transport mechanism without involving the Golgi apparatus. Therefore, this study describes an alternative route followed by plant soluble vacuolar proteins to reach the vacuole directly from the ER, without passing through the Golgi complex.     

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.     

MAIGO5 Functions in Protein Export from Golgi-Associated Endoplasmic Reticulum Exit Sites in Arabidopsis

Plant cells face unique challenges to efficiently export cargo from the endoplasmic reticulum (ER) to mobile Golgi stacks. Coat protein complex II (COPII) components, which include two heterodimers of Secretory23/24 (Sec23/24) and Sec13/31, facilitate selective cargo export from the ER; however, little is known about the mechanisms that regulate their recruitment to the ER membrane, especially in plants. Here, we report a protein transport mutant of Arabidopsis thaliana, named maigo5 (mag5), which abnormally accumulates precursor forms of storage proteins in seeds. mag5-1 has a deletion in the putative ortholog of the Saccharomyces cerevisiae and Homo sapiens Sec16, which encodes a critical component of ER exit sites (ERESs). mag mutants developed abnormal structures (MAG bodies) within the ER and exhibited compromised ER export. A functional MAG5/SEC16A–green fluorescent protein fusion localized at Golgi-associated cup-shaped ERESs and cycled on and off these sites at a slower rate than the COPII coat. MAG5/SEC16A interacted with SEC13 and SEC31; however, in the absence of MAG5/SEC16A, recruitment of the COPII coat to ERESs was accelerated. Our results identify a key component of ER export in plants by demonstrating that MAG5/SEC16A is required for protein export at ERESs that are associated with mobile Golgi stacks, where it regulates COPII coat turnover.     

Degradation of the Endoplasmic Reticulum by Autophagy during Endoplasmic Reticulum Stress in Arabidopsis

In this article, we show that the endoplasmic reticulum (ER) in Arabidopsis thaliana undergoes morphological changes in structure during ER stress that can be attributed to autophagy. ER stress agents trigger autophagy as demonstrated by increased production of autophagosomes. In response to ER stress, a soluble ER marker localizes to autophagosomes and accumulates in the vacuole upon inhibition of vacuolar proteases. Membrane lamellae decorated with ribosomes were observed inside autophagic bodies, demonstrating that portions of the ER are delivered to the vacuole by autophagy during ER stress. In addition, an ER stress sensor, INOSITOL-REQUIRING ENZYME-1b (IRE1b), was found to be required for ER stress–induced autophagy. However, the IRE1b splicing target, bZIP60, did not seem to be involved, suggesting the existence of an undiscovered signaling pathway to regulate ER stress–induced autophagy in plants. Together, these results suggest that autophagy serves as a pathway for the turnover of ER membrane and its contents in response to ER stress in plants.     

Functional Identification of Sorting Receptors Involved in Trafficking of Soluble Lytic Vacuolar Proteins in Vegetative Cells of Arabidopsis

In eukaryotic cells, protein trafficking plays an essential role in biogenesis of proteins that belong to the endomembrane compartments. In this process, an important step is the sorting of organellar proteins depending on their final destinations. For vacuolar proteins, vacuolar sorting receptors (VSRs) and receptor homology-transmembrane-RING H2 domain proteins (RMRs) are thought to be responsible. Arabidopsis (Arabidopsis thaliana) contains seven VSRs. Among them, VSR1, VSR3, and VSR4 are involved in sorting storage proteins targeted to the protein storage vacuole (PSV) in seeds. However, the identity of VSRs for soluble proteins of the lytic vacuole in vegetative cells remains controversial. Here, we provide evidence that VSR1, VSR3, and VSR4 are involved in sorting soluble lytic vacuolar and PSV proteins in vegetative cells. In protoplasts from leaf tissues of vsr1vsr3 and vsr1vsr4 but not vsr5vsr6, and rmr1rmr2 and rmr3rmr4 double mutants, soluble lytic vacuolar (Arabidopsis aleurain-like protein:green fluorescent protein [GFP] and carboxypeptidase Y:GFP and PSV (phaseolin) proteins, but not the vacuolar membrane protein Arabidopsis βFructosidase4:GFP, exhibited defects in their trafficking; they accumulated to the endoplasmic reticulum with an increased secretion into medium. The trafficking defects in vsr1vsr4 protoplasts were rescued by VSR1 or VSR4 but not VSR5 or AtRMR1. Furthermore, of the luminal domain swapping mutants between VSR1 and VSR5, the mutant with the luminal domain of VSR1, but not that of VSR5, rescued the trafficking defects of Arabidopsis aleurain-like protein:GFP and phaseolin in vsr1vsr4 protoplasts. Based on these results, we propose that VSR1, VSR3, and VSR4, but not other VSRs, are involved in sorting soluble lytic vacuolar and PSV proteins for their trafficking to the vacuoles in vegetative cells.Two different types of vacuoles have been identified in plant cells. One of them is the lytic vacuole (LV) that is present in vegetative cells, and the other is the protein storage vacuole (PSV) that is present in seed cells (Frigerio et al., 2008; Zouhar and Rojo, 2009; De Marcos Lousa et al., 2012). These two types of vacuoles have different functions. The LV carries out various functions such as osmotic pressure regulation, various hydrolytic activities, detoxification, and homeostasis of calcium and sodium ions. For some of these aspects LV is analogous to the vacuole in yeast (Saccharomyces cerevisiae) or lysosomes in animal cells. In contrast, the PSV is unique in plants and stores a large amount of proteins and minerals that are necessary for seed germination. To perform these functions, vacuoles need a large number of proteins.The organellar proteins destined for vacuoles have to be transported from the endoplasmic reticulum (ER) via a process called protein trafficking. This has been extensively studied in many different eukaryotic cell types, including plant cells. In general, proteins that belong to various endomembrane compartments are cotranslationally translocated into the ER and then transported through the Golgi apparatus and other intermediate compartments depending on their final destinations (Jurgens, 2004; Jolliffe et al., 2005; Sato and Nakano, 2007; Hwang and Robinson, 2009; Reyes et al., 2011). Vesicles are used to transport proteins from one compartment to another. Another important aspect is the specific targeting of organellar proteins. For this, organellar proteins carry a specific sorting or targeting signal that can be a sequence motif generated intrinsically or added posttranslationally (Hadlington and Denecke, 2000; Robinson et al., 2005; Hwang, 2008). The sequence motifs are recognized specifically by sorting receptors localized at the organelles that serve as donor compartments in trafficking pathways (Bassham and Raikhel, 2000; De Marcos Lousa et al., 2012).Two different types of sorting receptors, receptor homology-transmembrane-RING H2 domain proteins (RMRs) and vacuolar sorting receptors (VSRs), have been shown to be involved in the trafficking of vacuolar proteins. It has been proposed that RMRs function as a sorting receptor for storage proteins (Park et al., 2005; Hinz et al., 2007; Wang et al., 2011a). RMRs are type I membrane proteins and those in the luminal domain specifically interact with the C-terminal vacuolar sorting sequence (ctVSS) of storage proteins (Park et al., 2005; Shen et al., 2011). In addition, overexpression of an AtRMR1 deletion mutant inhibits the trafficking of phaseolin to the PSV, but not the protein trafficking to the LV, in protoplasts from leaf cells (Park et al., 2005). VSRs have been identified from various plant species and shown to specifically interact with the sorting motif of vacuolar proteins, which is known as the sequence-specific vacuolar sorting signal (ssVSS) or N-terminal propeptide (Ahmed et al., 1997; Hadlington and Denecke, 2000; Masclaux et al., 2005; Robinson et al., 2005; Hwang, 2008). In plant cells, the majority of VSRs localize to the prevacuolar compartment (PVC), which is the intermediate organelle between the trans-Golgi network (TGN) and vacuole (Tse et al., 2004; daSilva et al., 2005; Miao et al., 2006). In addition, a minor portion of VSR1 localizes to the TGN in plant cells, which supports the notion that VSRs recycle to the TGN from the PVC for sorting of their cargo proteins (Kim et al., 2010). Recent studies in plant cells questioned this concept and proposed other mechanisms for sorting vacuolar proteins. In the alternative proposal, sorting of vacuolar proteins may occur at the ER, and the VSRs may recycle from the TGN to the ER (Castelli and Vitale, 2005; Niemes et al., 2010). VSRs that were once thought to function as sorting receptors at the TGN for the LV proteins (daSilva et al., 2005; Foresti et al., 2010; Kim et al., 2010) have an additional function in the protein trafficking to the PSV in seed cells (Shimada et al., 2003; Zouhar et al., 2010). By using a genetic approach, it has been shown that among seven Arabidopsis (Arabidopsis thaliana) VSRs, VSR1, VSR3, and VSR4 play a role in trafficking of 12S globulins and 2S albumins in seed cells.The VSR isoforms involved in the protein trafficking to the PSV also exist in vegetative tissues (Laval et al., 1999; Kim et al., 2010; Zouhar et al., 2010). Mutations in both VSR1 and VSR4 cause secretion of AtAleurain, but not other LV proteins, into the apoplasts. Thus, it is not clearly understood what is the physiological role of AtVSRs in vegetative tissues (except for their role in vacuolar trafficking of AtAleurain), and what are the VSRs of other vacuolar proteins. In previous studies, it was demonstrated that overexpression of mutant forms of VSR1, VSR2, or BP80 of pea (Pisum sativum), a close homolog of VSR3 and VSR4, in protoplasts from wild-type plants affects trafficking of proteins to the LV (daSilva et al., 2005; Foresti et al., 2010; Kim et al., 2010). In this study, we utilized various VSR and RMR mutant plants and examined the effect of these mutations on the trafficking of LV and PSV proteins in protoplasts. These studies demonstrated that VSR1, VSR3, and VSR4, but not other VSRs and RMRs, are involved in trafficking of soluble LV and PSV proteins in vegetative cells. Further, the luminal domain but not the cytosolic tail of VSRs contains the determinant for the sorting specificity.     

The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis

Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1.Plant vacuoles are vital organelles for maintaining cell volume and cell turgor, regulating ion homeostasis and pH, disposing toxic materials, and storing and degrading unwanted proteins (Marty, 1999). To perform these diverse functions, vacuoles require an array of different and complex proteins. These proteins are synthesized at the endoplasmic reticulum (ER) and are transported to the vacuole through the vacuolar trafficking pathway. Perturbation of the vacuolar trafficking machinery affects many cellular processes, including tropisms, responses to pathogens, cytokinesis, hormone transport, and signal transduction (Surpin and Raikhel, 2004). The vacuolar trafficking system is comprised of several compartments: the ER, the Golgi apparatus, the trans-Golgi network (TGN), the prevacuolar compartment (PVC), and the vacuole. Vacuolar proteins synthesized at the ER are transported to the cis-Golgi via coat protein complex II (COPII) vesicles and are then transported to the TGN through the Golgi apparatus. In the TGN, proteins are sorted for delivery to their respective locations according to their targeting signal. Vacuolar proteins carrying a vacuolar sorting signal are thought to be recognized by vacuolar sorting receptors (VSRs), which are mainly located in the PVC, although sorting of vacuolar proteins may also occur at the ER and VSRs can be recycled from the TGN to the ER (Castelli and Vitale, 2005; Niemes et al., 2010). Multiple studies suggest that plant VSRs serve as sorting receptors both for lytic vacuole proteins (daSilva et al., 2005; Foresti et al., 2006; Kim et al., 2010) and for storage vacuole proteins (Shimada et al., 2003; Fuji et al., 2007; Zouhar et al., 2010).Osmotic stress is commonly associated with many environmental stresses, including drought, cold, and high soil salinity, that have a severe impact on the productivity of agricultural plants worldwide. Therefore, understanding how plants perceive and respond to osmotic stress is critical for improving plant resistance to abiotic stresses (Zhu, 2002; Fujita et al., 2013). It has long been recognized that osmotic stress can activate several signaling pathways that lead to changes in gene expression and metabolism. One important regulator of these signaling pathways is the phytohormone abscisic acid (ABA), which accumulates in response to osmotic stress. ABA regulates many critical processes, such as seed dormancy, stomatal movement, and adaptation to environmental stress (Finkelstein and Gibson, 2002; Xiong and Zhu, 2003; Cutler et al., 2010). De novo synthesis of ABA is of primary importance for increasing ABA levels in response to abiotic stress. ABA is synthesized through the cleavage of a C40 carotenoid originating from the 2-C-methyl-d-erythritol-4-phosphate pathway, followed by a conversion from zeaxanthin to violaxanthin catalyzed by the zeaxanthin epoxidase ABA1 and then to neoxanthin catalyzed by the neoxanthin synthase ABA4. Subsequently, a 9-cis-epoxycarotenoid dioxygenase (NCED) cleaves the violaxanthin and neoxanthin to xanthoxin. Xanthoxin, in turn, is oxidized by a short-chain alcohol dehydrogenase (ABA2) to abscisic aldehyde, which is converted to ABA by abscisic acid aldehyde oxidase3 (AAO3) using a molybdenum cofactor activated by the molybdenum cofactor sulfurase (ABA3; Nambara and Marion-Poll, 2005). In this pathway, it is generally thought that the cleavage step catalyzed by NCED is the rate-limiting step (Iuchi et al., 2000, 2001; Qin and Zeevaart, 2002; Xiong and Zhu, 2003). In Arabidopsis (Arabidopsis thaliana), five members of the NCED family (NCED2, NCED3, NCED5, NCED6, and NCED9) have been characterized (Tan et al., 2003). Of those, NCED3 has been suggested to play a crucial role in ABA biosynthesis, and its expression is induced by dehydration and osmotic stress (Iuchi et al., 2000, 2001; Qin and Zeevaart, 2002; Xiong and Zhu, 2003). Thus, understanding how the NCED3 gene is activated in response to osmotic stress is important for the elucidation of the mechanisms that govern plant acclimation to abiotic stress.We have used the firefly luciferase reporter gene driven by the stress-responsive NCED3 promoter to enable the genetic dissection of plant responses to osmotic stress (Wang et al., 2011). Here, we report the characterization of a unique regulator of ABA biosynthesis, 9-cis Epoxycarotenoid Dioxygenase Defective2 (CED2). The ced2 mutants are impaired in osmotic stress tolerance and are defective in the expression of genes required for ABA synthesis and consequently osmotic stress-induced ABA accumulation. The CED2 gene encodes VSR1, previously known to be involved in vacuolar trafficking but not known to be critical for osmotic stress induction of ABA biosynthesis and osmotic stress tolerance. Our study further suggests that intracellular pH changes might act as an early stress response signal triggering osmotic stress-activated ABA biosynthesis.     

Reduced Tonoplast Fast-Activating and Slow-Activating Channel Activity Is Essential for Conferring Salinity Tolerance in a Facultative Halophyte,Quinoa

Halophyte species implement a “salt-including” strategy, sequestering significant amounts of Na⁺ to cell vacuoles. This requires a reduction of passive Na⁺ leak from the vacuole. In this work, we used quinoa (Chenopodium quinoa) to investigate the ability of halophytes to regulate Na⁺-permeable slow-activating (SV) and fast-activating (FV) tonoplast channels, linking it with Na⁺ accumulation in mesophyll cells and salt bladders as well as leaf photosynthetic efficiency under salt stress. Our data indicate that young leaves rely on Na⁺ exclusion to salt bladders, whereas old ones, possessing far fewer salt bladders, depend almost exclusively on Na⁺ sequestration to mesophyll vacuoles. Moreover, although old leaves accumulate more Na⁺, this does not compromise their leaf photochemistry. FV and SV channels are slightly more permeable for K⁺ than for Na⁺, and vacuoles in young leaves express less FV current and with a density unchanged in plants subjected to high (400 mm NaCl) salinity. In old leaves, with an intrinsically lower density of the FV current, FV channel density decreases about 2-fold in plants grown under high salinity. In contrast, intrinsic activity of SV channels in vacuoles from young leaves is unchanged under salt stress. In vacuoles of old leaves, however, it is 2- and 7-fold lower in older compared with young leaves in control- and salt-grown plants, respectively. We conclude that the negative control of SV and FV tonoplast channel activity in old leaves reduces Na⁺ leak, thus enabling efficient sequestration of Na⁺ to their vacuoles. This enables optimal photosynthetic performance, conferring salinity tolerance in quinoa species.The increasing problem of global land salinization (Flowers, 2004; Rengasamy, 2006) and its associated multibillion dollar losses in agricultural production require a better understanding of the key physiological mechanisms that confer salinity tolerance in crops. One effective way of gaining such knowledge comes from studying halophytes (Glenn et al., 1999; Flowers and Colmer, 2008; Shabala and Mackay, 2011).One of the prominent features of halophytes is their ability to efficiently sequester cytosolically toxic Na⁺ to the cell vacuole. The classic view is that this sequestration is achieved by tonoplast Na⁺/H⁺ antiporters (Barkla et al., 1995; Flowers and Colmer, 2008), a process energized by both vacuolar H⁺ pumps: ATPase (Ayala et al., 1996; Vera-Estrella et al., 1999; Wang et al., 2001) and pyrophosphatase (Parks et al., 2002; Vera-Estrella et al., 2005; Guo et al., 2006; Krebs et al., 2010). However, recent studies have added more complexity to the relationship between Na⁺/H⁺ antiporters and vacuolar Na⁺ sequestration, assigning a role to the transporter in the regulation of K⁺ and H⁺ homeostasis (for review, see Rodríguez-Rosales et al., 2009; Jiang et al., 2010; Bassil et al., 2011). Vacuolar Na⁺/H⁺ antiporters encoded by NHX genes have been shown to also act as K⁺/H⁺ antiporters, with a relatively weak selectivity between Na⁺ and K⁺. The Na⁺/K⁺ selectivity ratio, in turn, is regulated by vacuolar calmodulin in a pH- and Ca²⁺-dependent manner (Yamaguchi et al., 2005). Consequently, other transporters, in addition to and different from NHX, are likely to be involved in vacuolar Na⁺ sequestration. In addition, salt-induced up-regulation of Na⁺/H⁺ antiporter expression levels has been observed in leaves but not in roots (Cosentino et al., 2010), suggesting the importance of Na⁺ exclusion and intracellular sequestration, primarily in photosynthesizing cells. Thus, tissue- and species-specific differences in the respective mechanisms should be considered as well.Whatever the actual mechanisms are for intracellular Na⁺ sequestration, efficient Na⁺ pumping into vacuole is only one side of the coin. To confer salinity tolerance, toxic Na⁺ ions must be prevented from leaking back into the cytosol. Indeed, given the at least 4- to 5-fold concentration gradient between the vacuole and the cytosol (Shabala and Mackay, 2011) and a zero or slightly negative cytosol-to-vacuole voltage difference across the tonoplast, Na⁺ leakage from the vacuole is thermodynamically favorable. Thus, to avoid energy-consuming futile Na⁺ cycling between the cytosol and the vacuole, and to achieve efficient vacuolar sequestration of toxic Na⁺, passive tonoplast Na⁺ conductance has to be kept to an absolute minimum. This implies strict and efficient control over Na⁺-permeable tonoplast channels.Two major types of Na⁺-permeable channels are present in the tonoplast, the slow-activating (SV) and fast-activating (FV) vacuolar channels. The SV channel is permeable to both monovalent and divalent cations and is activated by cytosolic Ca²⁺ and positive vacuolar voltage (Hedrich and Neher, 1987; Ward and Schroeder, 1994; Pottosin et al., 1997, 2001). The FV channel is permeable for monovalent cations only, is activated by large voltages of either sign, and is inhibited by divalent cations from either side of the membrane (Tikhonova et al., 1997; Brüggemann et al., 1999a, 1999b). In Arabidopsis (Arabidopsis thaliana), SV channels are shown to be encoded by a TPC1 (for two-pore channel1) protein (Peiter et al., 2005; Pottosin and Schönknecht, 2007; Hedrich and Marten, 2011). Importantly, recent studies on mammalian two-pore channels have suggested that endolysosomal TPCs are, in fact, Na⁺-selective channels (Wang et al., 2012). In contrast, the molecular identity of FV channels remains elusive. Both SV and FV channels are ubiquitous and abundant (up to several copies per μm²) in plant tissues, including mesophyll cell vacuoles (Pottosin and Muñiz, 2002; Pottosin and Schönknecht, 2007). SV and FV channel activity is strongly controlled at physiologically attainable conditions (physiological tonoplast voltages and vacuolar and cytosolic divalent and polyvalent cation concentrations). Importantly, even with 0.1% to 1% of the total population of channels open at any one time, impressive monovalent cation currents in the range of tens of pA per vacuole can be conducted. This is equivalent to a current mediated by the whole vacuole population of H⁺ pumps (Hedrich et al., 1988). Thus, under saline conditions, SV and FV channel activity probably needs to be further reduced.Early attempts to unravel any dramatic differences between the properties of tonoplast cation channels in salt-tolerant and salt-sensitive plants did not yield a clear outcome. Ivashikina and Hedrich (2005) studied the voltage dependence of the SV channels in vacuoles from Arabidopsis cell culture and found that an increase in luminal Na⁺/K⁺ ratio, mimicking the accumulation of Na⁺ in vacuoles during salt stress, shifted the threshold for SV activation to positive potentials, reducing SV channel open probability under saline conditions. Maathuis and coworkers (1992) found significant SV channel activity in leaf vacuoles isolated from the extreme halophyte Suaeda maritima, even when plants were grown under high (200 mm) NaCl conditions. The estimated activity of the transporter at physiologically relevant cytosolic Ca²⁺ levels and relatively small transmembrane voltage differences was low. Thus, the authors suggested that, rather than possessing some specific salt-induced control over the SV channel, the transporter’s low activity would mean that even under highly saline conditions, it would consume only about 30% of the H⁺-ATPase-generated power. Further studies from this laboratory demonstrated that voltage gating, unitary conductance, and Na⁺/K⁺ selectivity (P_K = P_Na) of SV channels from roots of Plantago media (salt sensitive) and Plantago maritima (salt tolerant) were essentially the same (Maathuis and Prins, 1990). However, when both species were grown under saline conditions, the SV channel activity greatly diminished. Yet, based on the original data of this study, it is not possible to decipher whether the SV channel activity in the two species was the same or different under control conditions and whether it was a statistically significant difference between the salt-induced decrease in the open probability of SV channels between P. media and P. maritima. As for FV channels, we are not aware of a single study on their properties/expression in relation to the salt tolerance.While the total number of halophytic species is relatively small compared with glycophytes, it still amounts to at least several thousand species (Glenn et al., 1999; Flowers et al., 2010). Moreover, halophytes are present in about one-half of higher plant families (Flowers and Colmer, 2008). These species possess a wide range of anatomical and morphological features that may potentially enable their superior performance under saline conditions (Shabala and Mackay, 2011). Nonetheless, the extent to which the above considerations could be extrapolated to all halophytes remains to be assessed. In this work, we used quinoa (Chenopodium quinoa) mesophyll leaf vacuoles to address some of these issues. Quinoa is a facultative halophyte species that originates from the Andean region of South America and was domesticated for human consumption some 3,000 to 4,000 years ago. It can grow under extreme saline conditions with a soil electrical conductivity exceeding 40 dS m⁻¹, approximately 500 mm NaCl (Jacobsen et al., 2003; Razzaghi et al., 2011). Optimal plant growth is usually observed at NaCl concentrations of around 100 mm (Hariadi et al., 2011), but this may be genotype specific (Adolf et al., 2012). Quinoa possesses some degree of leaf succulence as well as epidermal bladder cells (EBC), so it has the potential to employ two different sequestration strategies for cytosolic Na⁺ exclusion: internal (e.g. vacuolar sequestration) and external (sequestration in EBC). This makes quinoa an excellent model species to investigate the role of vacuolar Na⁺ sequestration in the overall salinity tolerance in this crop plant as well as to determine the contribution of SV and FV channels in this process. Here, we report a highly significant difference in SV and FV channel activity between old and young leaves of quinoa plants, a difference that is further enhanced under saline conditions. We conclude that the ability of quinoa plants to control ion leak via SV and FV tonoplast channels is essential for conferring salinity tolerance in this species. The possible implications of these findings for crop breeding for salinity tolerance are discussed.     

Rapid Structural Changes and Acidification of Guard Cell Vacuoles during Stomatal Closure Require Phosphatidylinositol 3,5-Bisphosphate

Rapid stomatal closure is essential for water conservation in plants and is thus critical for survival under water deficiency. To close stomata rapidly, guard cells reduce their volume by converting a large central vacuole into a highly convoluted structure. However, the molecular mechanisms underlying this change are poorly understood. In this study, we used pH-indicator dyes to demonstrate that vacuolar convolution is accompanied by acidification of the vacuole in fava bean (Vicia faba) guard cells during abscisic acid (ABA)–induced stomatal closure. Vacuolar acidification is necessary for the rapid stomatal closure induced by ABA, since a double mutant of the vacuolar H⁺-ATPase vha-a2 vha-a3 and vacuolar H⁺-PPase mutant vhp1 showed delayed stomatal closure. Furthermore, we provide evidence for the critical role of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂] in changes in pH and morphology of the vacuole. Single and double Arabidopsis thaliana null mutants of phosphatidylinositol 3-phosphate 5-kinases (PI3P5Ks) exhibited slow stomatal closure upon ABA treatment compared with the wild type. Moreover, an inhibitor of PI3P5K reduced vacuolar acidification and convolution and delayed stomatal closure in response to ABA. Taken together, these results suggest that rapid ABA-induced stomatal closure requires PtdIns(3,5)P₂, which is essential for vacuolar acidification and convolution.     

Composition,Assembly, and Trafficking of a Wheat Xylan Synthase Complex

Xylans play an important role in plant cell wall integrity and have many industrial applications. Characterization of xylan synthase (XS) complexes responsible for the synthesis of these polymers is currently lacking. We recently purified XS activity from etiolated wheat (Triticum aestivum) seedlings. To further characterize this purified activity, we analyzed its protein composition and assembly. Proteomic analysis identified six main proteins: two glycosyltransferases (GTs) TaGT43-4 and TaGT47-13; two putative mutases (TaGT75-3 and TaGT75-4) and two non-GTs; a germin-like protein (TaGLP); and a vernalization related protein (TaVER2). Coexpression of TaGT43-4, TaGT47-13, TaGT75-3, and TaGT75-4 in Pichia pastoris confirmed that these proteins form a complex. Confocal microscopy showed that all these proteins interact in the endoplasmic reticulum (ER) but the complexes accumulate in Golgi, and TaGT43-4 acts as a scaffold protein that holds the other proteins. Furthermore, ER export of the complexes is dependent of the interaction between TaGT43-4 and TaGT47-13. Immunogold electron microscopy data support the conclusion that complex assembly occurs at specific areas of the ER before export to the Golgi. A di-Arg motif and a long sequence motif within the transmembrane domains were found conserved at the NH₂-terminal ends of TaGT43-4 and homologous proteins from diverse taxa. These conserved motifs may control the forward trafficking of the complexes and their accumulation in the Golgi. Our findings indicate that xylan synthesis in grasses may involve a new regulatory mechanism linking complex assembly with forward trafficking and provide new insights that advance our understanding of xylan biosynthesis and regulation in plants.It is believed that Golgi-localized, multiprotein complexes synthesize plant hemicellulosic polysaccharides, including xylans. Such complexes are not well characterized in plants (Zeng et al., 2010; Atmodjo et al., 2011; Chou et al., 2012), which is in sharp contrast with mammalian and yeast cells (Jungmann and Munro, 1998; McCormick et al., 2000; Giraudo et al., 2001). Xylans are the most abundant plant hemicellulosic polysaccharides on Earth and play an important role in the integrity of cell walls, which is a key factor in plant growth. Any mutations affecting xylan backbone biosynthesis seem to result in abnormal growth of plants due mostly to thinning and weakening of secondary xylem walls, described as the irregular xylem (irx) phenotype. Thus, characterizing the xylan synthase complex (XSC) would have an impact on plant improvement, as well as many industrial applications related to food, feed, and biofuel production (Yang and Wyman, 2004; Faik, 2010). Although the Arabidopsis (Arabidopsis thaliana) irx mutants have revealed the involvement of several glycosyltransferase (GT) gene families in xylan biosynthesis (Brown et al., 2007, 2009; Lee et al., 2007, 2010; Wu et al., 2009, 2010), no XSCs have been purified/isolated from Arabidopsis tissues, and we still do not know whether some of the identified Arabidopsis GTs can assemble into functional XSCs. Furthermore, if GTs do assemble into XSCs, we don’t know the mechanisms by which plant cells control their assembly and cellular trafficking. In contrast to dicots, xylan synthase activity was recently immunopurified from etiolated wheat (Triticum aestivum) microsomes (Zeng et al., 2010). This purified wheat XS activity was shown to catalyze three activities, xylan-glucuronosyltransferase (XGlcAT), xylan-xylosyltransferase (XXylT), and xylan-arabinofuranosyltranferase (XAT), which work synergistically to synthesize xylan-type polymers in vitro (Zeng et al., 2008, 2010). This work focuses on describing protein composition, assembly, and trafficking of this purified wheat XS activity.In all eukaryotes, proteins of the secretory pathway (including GTs) are synthesized in the endoplasmic reticulum (ER) and modified as they go through the Golgi cisternae. Most proteins exit the ER from ER export sites (ERESs; Hanton et al., 2009) and use a signal-based sorting mechanism that allows them to be selectively recruited into vesicles coated by coat protein II complexes (Barlowe, 2003; Beck et al., 2008). For many Golgi-resident type II membrane proteins, di-Arg motifs, such as RR, RXR, and RRR located in their cytosolic NH₂-terminal ends, have been shown to be required for their ER export (Giraudo et al., 2003; Czlapinski and Bertozzi, 2006; Schoberer et al., 2009; Tu and Banfield, 2010). Interestingly, di-Arg motifs located ∼40 amino acids from the membrane on the cytosolic side can also be used to retrieve some type II ER-resident proteins from cis-Golgi (Schutze et al., 1994; Hardt et al., 2003; Boulaflous et al., 2009). In contrast to the signal-based sorting mechanism involved in trafficking between the ER and Golgi, the steady-state localization/retention of proteins (including GTs) in the Golgi is thought to occur through vesicular cycling. Cycling is influenced by various mechanisms, including the length and composition of the transmembrane domain (TMD) of type II GTs (Bretscher and Munro, 1993; Colley, 1997; van Vliet et al., 2003; Sousa et al., 2003; Sharpe et al., 2010), and the oligomerization/aggregation of GTs (kin hypothesis), which suggests that formation of homo- or heterooligomers of GTs in the Golgi may prevent their recruitment into clathrin-coated vesicles (Machamer, 1991; Nilsson et al., 1993; Weisz et al., 1993; Cole et al., 1996). Some Golgi-resident GTs are predicted to have a cleavable NH₂-terminal secretion signal peptide (SP) and would therefore exist as soluble proteins in the Golgi lumen. To maintain their proper Golgi localization, these processed GTs are likely part of multiprotein complexes anchored to integral membrane proteins. The fact that homologs of many of the trafficking proteins from mammalian and yeast cells are found in plants indicates that trafficking machineries of the plant secretory pathway are likely conserved (d’Enfert et al., 1992; Bar-Peled and Raikhel, 1997; Batoko et al., 2000; Pimpl et al., 2000; Phillipson et al., 2001; Hawes et al., 2008).It is becoming increasingly evident that understanding the mechanisms controlling protein-protein interaction, sorting, and trafficking of polysaccharide synthases (including XSCs) will help elucidate how plants regulate cell wall synthesis and deposition during their development. To this end, we believe that the purified wheat XS activity (Zeng et al., 2010) is an excellent model for this type of study. In this work, proteomics was used to determine the protein composition of the purified XS activity. Confocal microscopy and immunogold transmission electron microscopy (TEM) were used to investigate the assembly and trafficking of the complex. Our proteomics data showed that the purified activity contains two GTs, TaGT43-4 and TaGT47-13, two putative mutases, TaGT75-3 and TaGT75-4, and two non-GT proteins: a germin-like protein (TaGLP) belonging to cupin superfamily and a protein specific to monocots annotated as wheat vernalization-related protein 2 (TaVER2). Microscopy analyses revealed that all these proteins interact in the ER, but the assembled complexes accumulate in the Golgi. Export of these complexes from the ER is controlled by the interaction between TaGT43-4 and TaGT47-13. Characterization of the wheat XSC and its trafficking furthers our understanding of xylan biosynthesis in grasses and helps elucidate how polysaccharide synthase complexes are assembled, sorted, and maintained in different compartments of the secretory pathway.     

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.     

Microtubules Contribute to Tubule Elongation and Anchoring of Endoplasmic Reticulum,Resulting in High Network Complexity in Arabidopsis

The endoplasmic reticulum (ER) is a network of tubules and sheet-like structures in eukaryotic cells. Some ER tubules dynamically change their morphology, and others form stable structures. In plants, it has been thought that the ER tubule extension is driven by the actin-myosin machinery. Here, we show that microtubules also contribute to the ER tubule extension with an almost 20-fold slower rate than the actin filament-based ER extension. Treatment with the actin-depolymerizing drug Latrunculin B made it possible to visualize the slow extension of the ER tubules in transgenic Arabidopsis (Arabidopsis thaliana) plants expressing ER-targeted green fluorescent protein. The ER tubules elongated along microtubules in both directions of microtubules, which have a distinct polarity. This feature is similar to the kinesin- or dynein-driven ER tubule extension in animal cells. In contrast to the animal case, ER tubules elongating with the growing microtubule ends were not observed in Arabidopsis. We also found the spots where microtubules are stably colocalized with the ER subdomains during long observations of 1,040 s, suggesting that cortical microtubules contribute to provide ER anchoring points. The anchoring points acted as the branching points of the ER tubules, resulting in the formation of multiway junctions. The density of the ER tubule junction positively correlated with the microtubule density in both elongating cells and mature cells of leaf epidermis, showing the requirement of microtubules for formation of the complex ER network. Taken together, our findings show that plants use microtubules for ER anchoring and ER tubule extension, which establish fine network structures of the ER within the cell.The endoplasmic reticulum (ER) is a complex network composed of tubules and sheet structures. The ER network’s morphology changes dynamically by elongation and shrinkage of tubules, sheet expansion, and sliding junctions. For example, an ER tubule elongates straight forward from a cisterna and subsequently, fuses to another cisterna, producing a linkage between two cisternae. If an elongating tubule fails to fuse to another cisterna, the tubule contracts into the original cisterna. However, the ER has stable anchoring points that associate with other cellular structures, such as the plasma membrane or cytoskeleton. When an elongating ER tubule reaches an association point, it forms a stable ER anchor (i.e. establishment of the ER anchoring points forms stable ER tubules). Hence, increasing the number of ER anchoring points produces fine ER meshwork.ER dynamics in eukaryotes depend on the cytoskeleton. In plants, major contributors for ER organization are actin filaments (Quader et al., 1989; Knebel et al., 1990; Lichtscheidl and Hepler, 1996; Sparkes et al., 2009a) and the actin-associated motor proteins (myosins; Prokhnevsky et al., 2008; Peremyslov et al., 2010; Ueda et al., 2010). However, it had generally been thought that microtubules are not involved in ER organization in plants, because microtubule-depolymerizing drugs do not induce obvious changes in the ER network (Quader et al., 1989; Knebel et al., 1990; Lichtscheidl and Hepler, 1996; Sparkes et al., 2009a). Nevertheless, involvement of microtubules in plant ER organization has been suspected from several electron microscopy observations that showed microtubules located close to the ER membrane in Vicia faba guard cells, Nicotiana alata pollen tubes, and Funaria hygrometrica caulonemata (Lancelle et al., 1987; Hepler et al., 1990; McCauley and Hepler, 1992).Foissner et al. (2009) have suggested that microtubules are involved in motility and orientation of cortical ER in Characean algae (Nitella translucens, Nitella flexilis, Nitella hyalina, and Nitella pseudoflabellata) internodal cells. Characean cortical ER is spatially separated from inner cytoplasmic streaming by the middle layer of fixed chloroplasts. The cortical ER forms a tight meshwork of predominantly transverse ER tubules that frequently coalign with microtubules, and microtubule depolymerization reduces the transverse ER tubules and increases mesh size (Foissner et al., 2009). Consistently, Hamada et al. (2012) have shown in Arabidopsis (Arabidopsis thaliana) that microtubule depolymerization increases mesh size in young elongating cells. In addition, stable ER tubule junctions are often colocalized with cortical microtubules (Hamada et al., 2012), suggesting that microtubules stabilize ER tubule junctions to form fine ER meshes. Oryzalin-induced ER nodulation (Langhans et al., 2009) was not observed in our experimental conditions.Here, we showed that ER tubules elongate along microtubules in plant cells. In addition, we revealed that the ER is stably anchored to defined points on cortical microtubules. The stable anchoring points are the basis of various ER shapes, such as three-way, two-way, or dead-end ER tubules. These microtubule-ER interactions, together with the actin-myosin system, contribute to ER network organization.     

Putative Glycosyltransferases and Other Plant Golgi Apparatus Proteins Are Revealed by LOPIT Proteomics

The Golgi apparatus is the central organelle in the secretory pathway and plays key roles in glycosylation, protein sorting, and secretion in plants. Enzymes involved in the biosynthesis of complex polysaccharides, glycoproteins, and glycolipids are located in this organelle, but the majority of them remain uncharacterized. Here, we studied the Arabidopsis (Arabidopsis thaliana) membrane proteome with a focus on the Golgi apparatus using localization of organelle proteins by isotope tagging. By applying multivariate data analysis to a combined data set of two new and two previously published localization of organelle proteins by isotope tagging experiments, we identified the subcellular localization of 1,110 proteins with high confidence. These include 197 Golgi apparatus proteins, 79 of which have not been localized previously by a high-confidence method, as well as the localization of 304 endoplasmic reticulum and 208 plasma membrane proteins. Comparison of the hydrophobic domains of the localized proteins showed that the single-span transmembrane domains have unique properties in each organelle. Many of the novel Golgi-localized proteins belong to uncharacterized protein families. Structure-based homology analysis identified 12 putative Golgi glycosyltransferase (GT) families that have no functionally characterized members and, therefore, are not yet assigned to a Carbohydrate-Active Enzymes database GT family. The substantial numbers of these putative GTs lead us to estimate that the true number of plant Golgi GTs might be one-third above those currently annotated. Other newly identified proteins are likely to be involved in the transport and interconversion of nucleotide sugar substrates as well as polysaccharide and protein modification.The Golgi apparatus is the central organelle in the secretory pathway, and in higher plants it is involved in the biosynthesis and transport of cell wall matrix polysaccharides, glycoproteins, proteoglycans, and glycolipids as well as in protein trafficking to different subcellular compartments. The last decade has produced substantial findings on the function of the Golgi apparatus: insights into the protein trafficking at the endoplasmic reticulum (ER)/Golgi interface, Golgi structural maintenance, its involvement in endocytosis, and its behavior during cell division (for review, see Faso et al., 2009). However, despite its importance, only a small proportion of the Golgi proteome has been studied: relatively few Golgi proteins have been localized, and even fewer have been functionally characterized.The Golgi apparatus is thought to contain a large and diverse group of membrane-bound glycosyltransferases (GTs). The current view is that different GT activities are required for synthesis of the linkage between different donor and acceptor sugars. Having in mind the diversity of linkage types found in cell wall polysaccharides, the number of different GTs involved is likely to be very large. For instance, it has been estimated that for the biosynthesis of pectin alone, the action of 65 different enzymatic activities is needed (Caffall and Mohnen, 2009). By the end of the year 2011, 468 Arabidopsis (Arabidopsis thaliana) sequences had been annotated in the Carbohydrate-Active EnZymes (CAZy) GT database (Cantarel et al., 2009; http://www.cazy.org). We estimate that two-thirds of these CAZy-classified GTs may be targeted to the Golgi. The remaining one-third are cytosolic or plastidic enzymes involved in processes including, secondary metabolism or starch synthesis. The reported sequences are classified into 43 CAZy families based on amino acid sequence similarities within which at least one member has been biochemically characterized. Each family is likely to have a common structural fold, and three-dimensional (3-D) structures have been resolved for 20 of these 43 families. These are divided mostly into two structural classes, having either a GT-A fold or a GT-B fold (Unligil and Rini, 2000; Bourne and Henrissat, 2001). Moreover, most of the structurally uncharacterized GT families are predicted to adopt either the GT-A or GT-B fold based on 3-D structural homology modeling (Coutinho et al., 2003; Lairson et al., 2008). Despite this conserved 3-D structure, different GT families have very low or undetectable sequence similarities. Consequently, predicting novel GTs based solely on their amino acid sequence similarities is not always achievable, and structural homology searches have also proven useful (Hansen et al., 2009).The length and properties of the transmembrane domain (TMD) of endomembrane proteins appear to play a role in protein sorting and location within the secretory pathway and can be used to predict protein localization (Hanton et al., 2005; Sharpe et al., 2010). In order to perform such predictions, a high number of experimentally localized proteins is required, but only limited data sets have been available for plants to date.In order to identify the most abundant CAZy-classified GTs as well as novel putative GTs, in this work we rigorously extended our proteomic studies of the Golgi apparatus. We have previously developed a high-throughput mass spectrometry (MS)-based quantitative proteomics technique for localization of organelle proteins by isotope tagging (LOPIT; Dunkley et al., 2004, 2006). Here, we report new LOPIT data sets and apply a new method of combining them with published LOPIT data sets, localizing an unprecedented number of plant organelle proteins. We have analyzed the TMD properties of the proteins assigned to the ER, Golgi, and plasma membrane (PM) and determined the organelle-specific features. Structural prediction analysis of the Golgi-localized proteins with unknown functions assessed the protein sequences for the potential to fold similarly to known GT structures. We found that the Golgi contains a substantial number of candidate GT families that have no characterized functions. These results yield a broader understanding of the Golgi function and its biochemical properties.     

The KEEP ON GOING Protein of Arabidopsis Regulates Intracellular Protein Trafficking and Is Degraded during Fungal Infection

In plants, the trans-Golgi network and early endosomes (TGN/EE) function as the central junction for major endomembrane trafficking events, including endocytosis and secretion. Here, we demonstrate that the KEEP ON GOING (KEG) protein of Arabidopsis thaliana localizes to the TGN/EE and plays an essential role in multiple intracellular trafficking processes. Loss-of-function keg mutants exhibited severe defects in cell expansion, which correlated with defects in vacuole morphology. Confocal microscopy revealed that KEG is required for targeting of plasma membrane proteins to the vacuole. This targeting process appeared to be blocked at the step of multivesicular body (MVB) fusion with the vacuolar membrane as the MVB-associated small GTPase ARA6 was also blocked in vacuolar delivery. In addition, loss of KEG function blocked secretion of apoplastic defense proteins, indicating that KEG plays a role in plant immunity. Significantly, KEG was degraded specifically in cells infected by the fungus Golovinomyces cichoracearum, suggesting that this pathogen may target KEG to manipulate the host secretory system as a virulence strategy. Taking these results together, we conclude that KEG is a key component of TGN/EE that regulates multiple post-Golgi trafficking events in plants, including vacuole biogenesis, targeting of membrane-associated proteins to the vacuole, and secretion of apoplastic proteins.     

The Cytosolic Nucleoprotein of the Plant-Infecting Bunyavirus Tomato Spotted Wilt Recruits Endoplasmic Reticulum–Resident Proteins to Endoplasmic Reticulum Export Sites

In contrast with animal-infecting viruses, few known plant viruses contain a lipid envelope, and the processes leading to their membrane envelopment remain largely unknown. Plant viruses with lipid envelopes include viruses of the Bunyaviridae, which obtain their envelope from the Golgi complex. The envelopment process is predominantly dictated by two viral glycoproteins (Gn and Gc) and the viral nucleoprotein (N). During maturation of the plant-infecting bunyavirus Tomato spotted wilt, Gc localizes at endoplasmic reticulum (ER) membranes and becomes ER export competent only upon coexpression with Gn. In the presence of cytosolic N, Gc remains arrested in the ER but changes its distribution from reticular into punctate spots. Here, we show that these areas correspond to ER export sites (ERESs), distinct ER domains where glycoprotein cargo concentrates prior to coat protein II vesicle–mediated transport to the Golgi. Gc concentration at ERES is mediated by an interaction between its cytoplasmic tail (CT) and N. Interestingly, an ER-resident calnexin provided with Gc-CT was similarly recruited to ERES when coexpressed with N. Furthermore, disruption of actin filaments caused the appearance of a larger amount of smaller ERES loaded with N-Gc complexes, suggesting that glycoprotein cargo concentration acts as a trigger for de novo synthesis of ERES.     

Reticulomics: Protein-Protein Interaction Studies with Two Plasmodesmata-Localized Reticulon Family Proteins Identify Binding Partners Enriched at Plasmodesmata,Endoplasmic Reticulum,and the Plasma Membrane

The endoplasmic reticulum (ER) is a ubiquitous organelle that plays roles in secretory protein production, folding, quality control, and lipid biosynthesis. The cortical ER in plants is pleomorphic and structured as a tubular network capable of morphing into flat cisternae, mainly at three-way junctions, and back to tubules. Plant reticulon family proteins (RTNLB) tubulate the ER by dimerization and oligomerization, creating localized ER membrane tensions that result in membrane curvature. Some RTNLB ER-shaping proteins are present in the plasmodesmata (PD) proteome and may contribute to the formation of the desmotubule, the axial ER-derived structure that traverses primary PD. Here, we investigate the binding partners of two PD-resident reticulon proteins, RTNLB3 and RTNLB6, that are located in primary PD at cytokinesis in tobacco (Nicotiana tabacum). Coimmunoprecipitation of green fluorescent protein-tagged RTNLB3 and RTNLB6 followed by mass spectrometry detected a high percentage of known PD-localized proteins as well as plasma membrane proteins with putative membrane-anchoring roles. Förster resonance energy transfer by fluorescence lifetime imaging microscopy assays revealed a highly significant interaction of the detected PD proteins with the bait RTNLB proteins. Our data suggest that RTNLB proteins, in addition to a role in ER modeling, may play important roles in linking the cortical ER to the plasma membrane.The endoplasmic reticulum (ER) is a multifunctional organelle (Hawes et al., 2015) and is the site of secretory protein production, folding, and quality control (Brandizzi et al., 2003) and lipid biosynthesis (Wallis and Browse, 2010), but it is also involved in many other aspects of day-to-day plant life, including auxin regulation (Friml and Jones, 2010) and oil and protein body formation (Huang, 1996; Herman, 2008). The cortical ER network displays a remarkable polygonal arrangement of motile tubules that are capable of morphing into small cisternae, mainly at the three-way junctions of the ER network (Sparkes et al., 2009). The cortical ER network of plants has been shown to play multiple roles in protein trafficking (Palade, 1975; Vitale and Denecke, 1999) and pathogen responses (for review, see Pattison and Amtmann, 2009; Beck et al., 2012).In plants, the protein family of reticulons (RTNLBs) contributes significantly to tubulation of the ER (Tolley et al., 2008, 2010; Chen et al., 2012). RTNLBs are integral ER membrane proteins that feature a C-terminal reticulon homology domain (RHD) that contains two major hydrophobic regions. These regions form two V-shaped transmembrane wedges joined together via a cytosolic loop, with the C and N termini of the protein facing the cytosol. RTNLBs can dimerize or oligomerize, creating localized tensions in the ER membrane, inducing varying degrees of membrane curvature (Sparkes et al., 2010). Hence, RTNLBs are considered to be essential in maintaining the tubular ER network.The ability of RTNLBs to constrict membranes is of interest in the context of cell plate development and the formation of primary plasmodesmata (PD; Knox et al., 2015). PD formation involves extensive remodeling of the cortical ER into tightly furled tubules to form the desmotubules, axial structures that run through the PD pore (Overall and Blackman, 1996; Ehlers and Kollmann, 2001). At only 15 nm in diameter, the desmotubule is one of the most constricted membrane structures found in nature, with no animal counterparts (Tilsner et al., 2011). PD are membrane-rich structures characterized by a close association of the plasma membrane (PM) with the ER. The forces that model the ER into desmotubules, however, are poorly understood. RTNLBs are excellent candidates for this process and can constrict fluorescent protein-labeled ER membranes into extremely fine tubules (Sparkes et al., 2010). We showed recently that two of the RTNLBs present in the PD proteome, RTNLB3 and RTNLB6 (Fernandez-Calvino et al., 2011), are present in primary PD at cytokinesis (Knox et al., 2015). However, nothing is known of the proteins that interact with RTNLBs identified in the PD proteome or that may link RTNLBs to the PM. To date, the only protein shown to bind to plant RTNLBs is RHD3-LIKE2, the plant homolog of the ER tubule fusion protein ATLASTIN (Lee et al., 2013).Here, we used a dual approach to identify interacting partners of RTNLB3 and RTNLB6 (Fernandez-Calvino et al., 2011; Knox et al.., 2015). First, we used GFP immunoprecipitation assays coupled to mass spectrometry (MS) to identify proteins potentially binding to RTNLB3 and RTNLB6. Second, from the proteins we identified, we conducted a detailed Förster resonance energy transfer by fluorescence lifetime imaging microscopy (FRET-FLIM) analysis to confirm prey-bait interactions in vivo.The application of time-resolved fluorescence spectroscopy to imaging biological systems has allowed the design and implementation of fluorescence lifetime imaging microscopy (FLIM). The technique allows measuring and determining the space map of picosecond fluorescence decay at each pixel of the image through confocal single and multiphoton excitation. The general fluorescence or Förster resonance energy transfer (FRET) to determine the colocalization of two color chromophores can now be improved to determine physical interactions using FRET-FLIM and protein pairs tagged with appropriate GFP fluorophores and monomeric red fluorescent protein (mRFP). FRET-FLIM measures the reduction in the excited-state lifetime of GFP (donor) fluorescence in the presence of an acceptor fluorophore (e.g. mRFP) that is independent of the problems associated with steady-state intensity measurements. The observation of such a reduction is an indication that the two proteins are within a distance of 1 to 10 nm, thus indicating a direct physical interaction between the two protein fusions (Osterrieder et al., 2009; Sparkes et al., 2010; Schoberer and Botchway, 2014). It was shown previously that a reduction of as little as approximately 200 ps in the excited-state lifetime of the GFP-labeled protein represents quenching through a protein-protein interaction (Stubbs et al., 2005).Our interaction data identified a large percentage (40%) of ER proteins, including other RTNLB family members. However, we also found a relatively large number (25%) of proteins present in the published PD proteome (Fernandez-Calvino et al., 2011) and a surprisingly high proportion (35%) of PM proteins. Of the PD-resident proteins we identified, a significant number were shown previously to be targets of viral movement proteins (MPs) or proteins present within lipid rafts, consistent with the view that PD are lipid-rich microdomains (Bayer et al., 2014). Additional proteins identified suggested roles for RTNLBs in transport and pathogen defense. We suggest that RTNLBs may play key roles in anchoring and/or signaling between the cortical ER and PM.     

SAUR Inhibition of PP2C-D Phosphatases Activates Plasma Membrane H+-ATPases to Promote Cell Expansion in Arabidopsis

The plant hormone auxin promotes cell expansion. Forty years ago, the acid growth theory was proposed, whereby auxin promotes proton efflux to acidify the apoplast and facilitate the uptake of solutes and water to drive plant cell expansion. However, the underlying molecular and genetic bases of this process remain unclear. We have previously shown that the SAUR19-24 subfamily of auxin-induced SMALL AUXIN UP-RNA (SAUR) genes promotes cell expansion. Here, we demonstrate that SAUR proteins provide a mechanistic link between auxin and plasma membrane H⁺-ATPases (PM H⁺-ATPases) in Arabidopsis thaliana. Plants overexpressing stabilized SAUR19 fusion proteins exhibit increased PM H⁺-ATPase activity, and the increased growth phenotypes conferred by SAUR19 overexpression are dependent upon normal PM H⁺-ATPase function. We find that SAUR19 stimulates PM H⁺-ATPase activity by promoting phosphorylation of the C-terminal autoinhibitory domain. Additionally, we identify a regulatory mechanism by which SAUR19 modulates PM H⁺-ATPase phosphorylation status. SAUR19 as well as additional SAUR proteins interact with the PP2C-D subfamily of type 2C protein phosphatases. We demonstrate that these phosphatases are inhibited upon SAUR binding, act antagonistically to SAURs in vivo, can physically interact with PM H⁺-ATPases, and negatively regulate PM H⁺-ATPase activity. Our findings provide a molecular framework for elucidating auxin-mediated control of plant cell expansion.     

Trafficking of Vacuolar Sorting Receptors: New Data and New Problems

Vacuolar sorting receptors bind cargo ligands early in the secretory pathway and show that multivesicular body-vacuole fusion requires a Rab5/Rab7 GTPase conversion with consequences for retromer binding.To serve the purposes of controlled protein turnover, eukaryotic cells compartmentalize the required acid hydrolases in specialized digestive organelles: lysosomes in animals and vacuoles in yeasts and plants. Therefore, a reliable system must be in operation to prevent such proteolytic enzymes being released at the cell surface. Such a mechanism requires that acid hydrolases be identified and diverted away from the secretory flow to the plasma membrane (PM). This process is facilitated by receptors that recognize specific motifs in the hydrolases that are absent in secretory proteins. The most well-known example of this is the mannosyl 6-phosphate receptor (MPR), which is responsible for the sorting of lysosomal enzymes; indeed, it has become a paradigm for protein sorting in most cell biology textbooks. It entails the recognition of phosphomannan cargo ligands by MPRs in the trans-Golgi network (TGN) followed by the sequestration of the MPR-ligand complexes into specific transport vectors (clathrin-coated vesicles [CCVs]). These are then transported to an endosomal compartment (the early endosome [EE]) having a more acidic pH than the TGN, thereby causing the ligands to separate from the MPRs. The MPRs are subsequently recycled back to the TGN via retromer-coated carriers for another round of trafficking (for review, see Braulke and Bonifacino, 2009; Seaman, 2012).Many plant scientists support a scenario for the sorting of soluble vacuolar proteins and the trafficking of their receptors (vacuolar sorting receptors [VSRs]) that closely resembles that of the MPR system of mammalian cells (Hwang, 2008; De Marcos Lousa et al., 2012; Kang et al., 2012; Sauer et al., 2013; Xiang et al., 2013). This working model is based on three key observations: (1) VSRs were first identified in detergent-solubilized CCV fractions isolated from developing pea (Pisum sativum) cotyledons; (2) CCVs are regularly seen budding off the TGN in thin-sectioned plant cells; and (3) depending on the organism, VSRs and VSR-reporter constructs are found concentrated either in the TGN or in multivesicular prevacuolar compartments (PVCs) under steady-state conditions (Robinson and Pimpl, 2014a, 2014b, and refs. therein). Unfortunately, information on VSRs has not been obtained from a single experimental system. Although much work on Arabidopsis (Arabidopsis thaliana) VSR mutants has been published (for review, see De Marcos Lousa et al., 2012) and the majority of immunogold electron microscopic localization experiments have been performed in Arabidopsis, the majority of the fluorescence localizations, particularly with regard to VSR trafficking, have been carried out by transient expression in tobacco (Nicotiana tabacum; agroinfiltration for leaves and electroporation for protoplasts). Nevertheless, it should be stressed that sorting motifs for acid hydrolases and their corresponding receptors in the three major eukaryotic organismal groups differ considerably (Robinson et al., 2012). In addition, the secretory and endocytic pathways of plant cells contrast significantly with mammalian cells, the most important distinctions being (1) the lack of an intermediate compartment between the endoplasmic reticulum (ER) and the Golgi apparatus in plants, (2) that plants have motile Golgi stacks rather than a perinuclear Golgi complex, and (3) the absence of an independent EE in plants, the function of which is assumed by the TGN (Contento and Bassham, 2012). While these differences do not automatically negate the validity of the above working model for VSR trafficking, they at least legitimize a more thorough analysis of the supporting data than has previously been the case (Robinson and Pimpl, 2014a, 2014b).The principal issues at stake are as follows. Where do VSRs bind and release their cargo ligands? What is the actual mechanism resulting in the separation of secretory from vacuolar cargo molecules? What is/are the precise role(s) of TGN-derived CCVs? And where does retromer pick up VSRs and where are they delivered to? The impact of several new publications on these points of dispute is the subject of this article.