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By being at the interface of the exocytic and endocytic pathways, the plant trans-Golgi network (TGN) is a multitasking and highly diversified organelle. Despite governing vital cellular processes, the TGN remains one of the most uncharacterized organelle of plant cells. In this review, we highlight recent studies that have contributed new insights and to the generation of markers needed to answer several important questions on the plant TGN. Several drugs specifically affecting proteins critical for the TGN functions have been extremely useful for the identification of mutants of the TGN in the pursuit to understand how the morphology and the function of this organelle are controlled. In addition to these chemical tools, we review emerging microscopy techniques that help visualize the TGN at an unpreceded resolution and appreciate the heterogeneity and dynamics of this organelle in plant cells.

Defining convergent and divergent mechanisms underlying the biogenesis and function of endomembrane organelles is fundamentally important in cell biology. In all eukaryotes, the Trans-Golgi Network (TGN) is the hub where the exocytic and endocytic pathways converge. To gain knowledge in the mechanisms underlying TGN biogenesis and function, we characterized TGNap1, a protein encoded by a plant gene of unknown function conserved with metazoans. We demonstrate that TGNap1 is a TGN protein required for the homeostasis of biosynthetic and endocytic traffic pathways. We also show that TGNap1 binds Rab6, YIP4 and microtubules. Finally, we establish that TGNap1 contributes to microtubule-dependent biogenesis, tracking and function of a TGN subset, likely through interaction with Rab6 and YIP4. Our results identify an important trafficking determinant at the plant TGN and reveal an unexpected reliance of post-Golgi traffic homeostasis and organelle biogenesis on microtubules in plants., The plant trans-Golgi network comprises functionally distinct subcompartments. Here Renna et al. show that the TGNap1 protein acts in microtubule-dependent biogenesis, tracking and function of a TGN subset, highlighting diversification within the plant TGN.

Summary: Through yet-undefined mechanisms, the plant endoplasmic reticulum (ER) has a critical role in endocytosis. The plant ER establishes a close association with endosomes and contacts the plasma membrane (PM) at ER-PM contact sites (EPCSs) demarcated by the ER membrane-associated VAMP-associated-proteins (VAP). Here, we investigated two plant VAPs, VAP27-1 and VAP27-3, and found an interaction with clathrin and a requirement for the homeostasis of clathrin dynamics at endocytic membranes and endocytosis. We also demonstrated direct interaction of VAP27-proteins with phosphatidylinositol-phosphate lipids (PIPs) that populate endocytic membranes. These results support that, through interaction with PIPs, VAP27-proteins bridge the ER with endocytic membranes and maintain endocytic traffic, likely through their interaction with clathrin. : Stefano et al. demonstrate that plant VAPs (VAP27-1 and VAP27-3) interact with clathrin and with phosphatidylinositol-phosphate lipids (PIPs) that populate endocytic membranes. The findings support a model showing that, through interaction with PIPs and clathrin, VAP27-proteins bridge the ER with endocytic membranes and maintain endocytic traffic. Keywords: endosplasmic reticulum, endocytosis, clathrin, trans-golgi network, ER-PM contact sites

Arabidopsis thaliana calmodulin binding transcription activator (CAMTA) factors repress the expression of genes involved in salicylic acid (SA) biosynthesis and SA-mediated immunity in healthy plants grown at warm temperature (22°C). This repression is overcome in plants exposed to low temperature (4°C) for more than a week and in plants infected by biotrophic and hemibiotrophic pathogens. Here, we present evidence that CAMTA3-mediated repression of SA pathway genes in nonstressed plants involves the action of an N-terminal repression module (NRM) that acts independently of calmodulin (CaM) binding to the IQ and CaM binding (CaMB) domains, a finding that is contrary to current thinking that CAMTA3 repression activity requires binding of CaM to the CaMB domain. Induction of SA pathway genes in response to low temperature did not occur in plants expressing only the CAMTA3-NRM region of the protein. Mutational analysis provided evidence that the repression activity of the NRM was suppressed by action of the IQ and CaMB domains responding to signals generated in response to low temperature. Plants expressing the CAMTA3-NRM region were also impaired in defense against the bacterial hemibiotrophic pathogen Pseudomonas syringae pv tomato DC3000. Our results indicate that the regulation of CAMTA3 repression activity by low temperature and pathogen infection involves related mechanisms, but with distinct differences.

Summary The endoplasmic reticulum (ER) is an essential organelle that spreads throughout the cytoplasm as one interconnected network of narrow tubules and dilated cisternae that enclose a single lumen. The ER network undergoes extensive remodeling, which critically depends on membrane-cytoskeleton interactions [1]. In plants, the ER is also highly mobile, and its streaming contributes significantly to the movement of other organelles [2, 3]. The remodeling and motility of the plant ER rely mainly on actin [4] and to a minor extent on microtubules [5]. Although a three-way interaction between the ER, cytosolic myosin-XI, and F-actin mediates the plant ER streaming [6], the mechanisms underlying stable interaction of the ER membrane with actin are unknown. Early electron microscopy studies suggested a direct attachment of the plant ER with actin filaments [7, 8], but it is plausible that yet-unknown proteins facilitate anchoring of the ER membrane with the cytoskeleton. We demonstrate here that SYP73, a member of the plant Syp7 subgroup of SNARE proteins [9] containing actin-binding domains, is a novel ER membrane-associated actin-binding protein. We show that overexpression of SYP73 causes a striking rearrangement of the ER over actin and that, similar to mutations of myosin-XI [4, 10, 11], loss of SYP73 reduces ER streaming and affects overall ER network morphology and plant growth. We propose a model for plant ER remodeling whereby the dynamic rearrangement and streaming of the ER network depend on the propelling action of myosin-XI over actin coupled with a SYP73-mediated bridging, which dynamically anchors the ER membrane with actin filaments.

The availability of more specific dyes for a subset of endomembrane compartments, combined with the development of genetically encoded probes and advanced microscopy technologies, makes live cell imaging an approach that goes beyond the microscopically observation of cell structure. Here we describe the latest improved techniques to investigate protein-protein interaction, protein topology, and protein dynamics.Furthermore, we depict new technical approaches to identify mutants for chloroplast morphology and distribution through the tracking of chlorophyll fluorescence, as well as mutants for chloroplast movement.

In plant cells, vacuoles are extremely important for growth and development, and influence important cellular functions as photosynthesis, respiration, and transpiration. Plant cells contain lytic and storage vacuoles, whose size can be different depending on cell type and tissue developmental stage. One of the main roles of vacuoles is to regulate the cell turgor in response to different stimuli. Thus, studying the morphology, dynamics, and physiology of vacuole is fundamentally important to advance knowledge in plant cell biology at large. The availability of fluorescent probes allows marking vacuoles in multiple ways. These may be fast, when using commercially available chemical dyes, or relatively slow, in the case of specific genetically encoded markers based on proteins directed either to the membrane of the vacuole (tonoplast) or to the vacuole lumen. Any of these approaches provides useful information about the morphology and physiology of the vacuole.

The unfolded protein response (UPR) is an ancient signaling pathway designed to protect cells from the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER). Because misregulation of the UPR is potentially lethal, a stringent surveillance signaling system must be in place to modulate the UPR. The major signaling arms of the plant UPR have been discovered and rely on the transcriptional activity of the transcription factors bZIP60 and bZIP28 and on the kinase and ribonuclease activity of IRE1, which splices mRNA to activate bZIP60. Both bZIP28 and bZIP60 modulate UPR gene expression to overcome ER stress. In this study, we demonstrate at a genetic level that the transcriptional role of bZIP28 and bZIP60 in ER-stress responses is antagonized by nonexpressor of PR1 genes 1 (NPR1), a critical redox-regulated master regulator of salicylic acid (SA)-dependent responses to pathogens, independently of its role in SA defense. We also establish that the function of NPR1 in the UPR is concomitant with ER stress-induced reduction of the cytosol and translocation of NPR1 to the nucleus where it interacts with bZIP28 and bZIP60. Our results support a cellular role for NPR1 as well as a model for plant UPR regulation whereby SA-independent ER stress-induced redox activation of NPR1 suppresses the transcriptional role of bZIP28 and bZIP60 in the UPR.

Xyloglucan is a polysaccharide that has important roles in the formation and function of the walls that surround growing land plant cells. Many of these plants synthesize xyloglucan that contains galactose in two different side chains (L and F), which exist in distinct molecular environments. However, little is known about the contribution of these side chains to xyloglucan function. Here, we show that Arabidopsis (Arabidopsis thaliana) mutants devoid of the F side chain galactosyltransferase MURUS3 (MUR3) form xyloglucan that lacks F side chains and contains much less galactosylated xylose than its wild-type counterpart. The galactose-depleted xyloglucan is dysfunctional, as it leads to mutants that are dwarfed with curled rosette leaves, short petioles, and short inflorescence stems. Moreover, cell wall matrix polysaccharides, including xyloglucan and pectin, are not properly secreted and instead accumulate within intracellular aggregates. Near-normal growth is restored by generating mur3 mutants that produce no detectable amounts of xyloglucan. Thus, cellular processes are affected more by the presence of the dysfunctional xyloglucan than by eliminating xyloglucan altogether. To identify structural features responsible for xyloglucan dysfunction, xyloglucan structure was modified in situ by generating mur3 mutants that lack specific xyloglucan xylosyltransferases (XXTs) or that overexpress the XYLOGLUCAN L-SIDE CHAIN GALACTOSYLTRANSFERASE2 (XLT2) gene. Normal growth was restored in the mur3-3 mutant overexpressing XLT2 and in mur3-3 xxt double mutants when the dysfunctional xyloglucan was modified by doubling the amounts of galactosylated side chains. Our study assigns a role for galactosylation in normal xyloglucan function and demonstrates that altering xyloglucan side chain structure disturbs diverse cellular and physiological processes.

The availability of more specific dyes for a subset of endomembrane compartments, combined with the development of genetically encoded probes and advanced microscopy technologies, makes live cell imaging an approach that goes beyond the microscopically observation of cell structure. Here we describe the latest improved techniques to investigate protein-protein interaction, protein topology, and protein dynamics.Furthermore, we depict new technical approaches to identify mutants for chloroplast morphology and distribution through the tracking of chlorophyll fluorescence, as well as mutants for chloroplast movement.

In plant cells, vacuoles are extremely important for growth and development, and influence important cellular functions as photosynthesis, respiration, and transpiration. Plant cells contain lytic and storage vacuoles, whose size can be different depending on cell type and tissue developmental stage. One of the main roles of vacuoles is to regulate the cell turgor in response to different stimuli. Thus, studying the morphology, dynamics, and physiology of vacuole is fundamentally important to advance knowledge in plant cell biology at large. The availability of fluorescent probes allows marking vacuoles in multiple ways. These may be fast, when using commercially available chemical dyes, or relatively slow, in the case of specific genetically encoded markers based on proteins directed either to the membrane of the vacuole (tonoplast) or to the vacuole lumen. Any of these approaches provides useful information about the morphology and physiology of the vacuole.

Leaf architecture is determined by cell shape, size, and density. As plant cells are enclosed by a rigid cell wall, changes to leaf architecture have to occur through downstream genetic systems that induce alterations in (1) cell wall composition, (2) synthesis, assembly, and orientation of cytoskeletal elements and/or (3) the degree of cross-linkage between wall components in response to upstream developmental and environmental cues. This chapter reviews how leaf architecture is influenced by molecular mechanisms that modulate the above wall modification processes. Upstream signaling systems such as salicylic (SA), jasmonic (JA), and gibberellic (GA) acid have significant effects on leaf architecture. GA promotes and JA and SA suppress growth. Leaf architectural changes are brought about by these upstream systems in concert or in an interactive manner, and the associated downstream molecular systems that are involved in executing changes to cell wall properties will be discussed. Evidence will be provided to show that xyloglucan endotransglucosylase/hydrolase and pectin methyltransferase/pectin methylesterase/pectin methylesterase inhibitor systems are key downstream execution points of leaf architectural changes common to different upstream molecular systems. Optimization of leaf architecture maximizes light interception, gas exchange properties, and photosynthesis. In addition, plant growth has been shown to be more sensitive to leaf area than to area-based photosynthesis rate. Therefore, understanding genes and molecular mechanisms that affect cell wall properties and leaf architecture has broader implications in terms of crop improvement, and candidate genes that can be manipulated to optimize leaf architecture in order to maximize net carbon assimilation and plant growth will be proposed.

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.

N-myristoylation is a crucial irreversible eukaryotic lipid modification allowing a key subset of proteins to be targeted at the periphery of specific membrane compartments. Eukaryotes have conserved N-myristoylation enzymes, involving one or two N-myristoyltransferases (NMT1 and NMT2), among which NMT1 is the major enzyme. In the postembryonic developmental stages, defects in NMT1 lead to aberrant cell polarity, flower differentiation, fruit maturation, and innate immunity; however, no specific NMT1 target responsible for such deficiencies has hitherto been identified. Using a confocal microscopy forward genetics screen for the identification of Arabidopsis thaliana secretory mutants, we isolated STINGY, a recessive mutant with defective Golgi traffic and integrity. We mapped STINGY to a substitution at position 160 of Arabidopsis NMT1 (NMT1A160T). In vitro kinetic studies with purified NMT1A160T enzyme revealed a significant reduction in its activity due to a remarkable decrease in affinity for both myristoyl-CoA and peptide substrates. We show here that this recessive mutation is responsible for the alteration of Golgi traffic and integrity by predominantly affecting the Golgi membrane/cytosol partitioning of ADP-ribosylation factor proteins. Our results provide important functional insight into N-myristoylation in plants by ascribing postembryonic functions of Arabidopsis NMT1 that involve regulation of the functional and morphological integrity of the plant endomembranes.

The mechanisms underlying the organization and dynamics of plant endomembranes are largely unknown. Arabidopsis RHD3, a distant member of the dynamin superfamily, has recently been implicated in plant ER morphology and Golgi movement through analyses of dominant-negative mutants of the putative GTPase domain in a heterologous system. Whether RHD3 is indispensable for ER architecture and what role regions other than the putative GTPase domain play in RHD3 function are unanswered questions. Here we characterized an EMS mutant, gom8, with disrupted Golgi movement and positioning and compromised ER shape and dynamics. gom8 mapped to a missense mutation in the RHD3 hairpin loop domain, causing accumulation of the mutant protein into large structures, a markedly different distribution compared with wild-type RHD3 over the ER network. Despite the GOM8 distribution, tubules fused in the peripheral ER of the gom8 mutant. These data imply that integrity of the hairpin region is important for the subcellular distribution of RHD3, and that reduced availability of RHD3 over the ER can cause ER morphology defects, but does not prevent peripheral fusion between tubules. This was confirmed by evidence that gom8 was phenocopied in an RHD3 null background. Furthermore, we established that the region encompassing the RHD3 hairpin domain and the C-terminal cytosolic domain is necessary for RHD3 function. We conclude that RHD3 is important in ER morphology, but is dispensable for peripheral ER tubulation in an endogenous context, and that its activity relies on the C-terminal region in addition to the GTPase domain.

Eukaryotic cells use COPII-coated carriers for endoplasmic reticulum (ER)-to-Golgi protein transport. Selective cargo capture into ER-derived carriers is largely driven by the SEC24 component of the COPII coat. The Arabidopsis genome encodes three AtSEC24 genes with overlapping expression profiles but it is yet to be established whether the AtSEC24 proteins have overlapping roles in plant growth and development. Taking advantage of Arabidopsis thaliana as a model plant system for studying gene function in vivo, through reciprocal crosses, pollen characterization, and complementation tests, evidence is provided for a role for AtSEC24A in the male gametophyte. It is established that an AtSEC24A loss-of-function mutation is tolerated in the female gametophyte but that it causes defects in pollen leading to failure of male transmission of the AtSEC24A mutation. These data provide a characterization of plant SEC24 family in planta showing incompletely overlapping functions of the AtSEC24 isoforms. The results also attribute a novel role to SEC24 proteins in a multicellular model system, specifically in male fertility.

ARF-GTPases are important proteins that control membrane trafficking events. Their activity is largely influenced by the interplay between guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), which facilitate the activation or inactivation of ARF-GTPases, respectively. There are 15 predicted proteins that contain an ARF-GAP domain within the Arabidopsis thaliana genome, and these are classified as ARF-GAP domain (AGD) proteins. The function and subcellular distribution of AGDs, including the ability to activate ARF-GTPases in vivo, that remain largely uncharacterized to date. Here we show that AGD5 is localised to the trans-Golgi network (TGN), where it co-localises with ARF1, a crucial GTPase that is involved in membrane trafficking and which was previously shown to be distributed on Golgi and post-Golgi structures of unknown nature. Taking advantage of the in vivo AGD5-ARF1 interaction at the TGN, we show that mutation of an arginine residue that is critical for ARF-GAP activity of AGD5 leads to longer residence of ARF1 on the membranes, as expected if GTP hydrolysis on ARF1 was impaired due to a defective GAP. Our results establish the nature of the post-Golgi compartments in which ARF1 localises, as well as identifying the role of AGD5 in vivo as a TGN-localised GAP. Furthermore, in vitro experiments established the promiscuous interaction between AGD5 and the plasma membrane-localised ADP ribosylation factor B (ARFB), confirming that ARF-GAP specificity for ARF-GTPases within the cell environment may be spatially regulated.

The secretory pathway encloses functionally interlinked organelles for the synthesis and deposition of most of the building blocks of eukaryotic cells, such as lipids, proteins and sugars. The coat protein complex II (COPII) is a specialized protein complex for the transport between secretory organelles, specifically from the endoplasmic reticulum (ER) to the Golgi apparatus. This review focuses on the developments on COPII research in the plant system. Here, we address the most recent advances in the distribution and regulation of ER-to-Golgi protein transport intermediates and functional analyses of COPII isoforms. New studies support that such isoforms might not be functionally redundant and that they might have unanticipated roles in maintaining the integrity of the ER.

A central question in cell biology is how the identity of organelles is established and maintained. Here, we report on GOLD36, an EMS mutant identified through a screen for partial displacement of the Golgi marker, ST-GFP, to other organelles. GOLD36 showed partial distribution of ST-GFP into a modified endoplasmic reticulum (ER) network, which formed bulges and large skein-like structures entangling Golgi stacks. GOLD36 showed defects in ER protein export as evidenced by our observations that, besides the partial retention of Golgi markers in the ER, the trafficking of a soluble bulk-flow marker to the cell surface was also compromised. Using a combination of classical mapping and next-generation DNA sequencing approaches, we linked the mutant phenotype to a missense mutation of a proline residue in position 80 to a leucine residue in a small endomembrane protein encoded by the gold36 locus (At1g54030). Subcellular localization analyses indicated that GOLD36 is a vacuolar protein and that its mutated form is retained in the ER. Interestingly also, a gold36 knock-out mutant mirrored the GOLD36 subcellular phenotype. These data indicate that GOLD36 is a protein destined to post-ER compartments and suggest that its export from the ER is a requirement to ensure steady-state maintenance of the organelle's organization and functional activity in relation to other secretory compartments. We speculate that GOLD36 may be a factor that is necessary for ER integrity because of its ability to limit deleterious effects of other secretory proteins on the ER.

The plant endoplasmic reticulum (ER) contains functionally distinct subdomains at which cargo molecules are packed into transport carriers. To study these ER export sites (ERES), we used tobacco (Nicotiana tabacum) leaf epidermis as a model system and tested whether increased cargo dosage leads to their de novo formation. We have followed the subcellular distribution of the known ERES marker based on a yellow fluorescent protein (YFP) fusion of the Sec24 COPII coat component (YFP-Sec24), which, differently from the previously described ERES marker, tobacco Sar1-YFP, is visibly recruited at ERES in both the presence and absence of overexpressed membrane cargo. This allowed us to quantify variation in the ERES number and in the recruitment of Sec24 to ERES upon expression of cargo. We show that increased synthesis of membrane cargo leads to an increase in the number of ERES and induces the recruitment of Sec24 to these ER subdomains. Soluble proteins that are passively secreted were found to leave the ER with no apparent up-regulation of either the ERES number or the COPII marker, showing that bulk flow transport has spare capacity in vivo. However, de novo ERES formation, as well as increased recruitment of Sec24 to ERES, was found to be dependent on the presence of the diacidic ER export motif in the cytosolic domain of the membrane cargo. Our data suggest that the plant ER can adapt to a sudden increase in membrane cargo-stimulated secretory activity by signal-mediated recruitment of COPII machinery onto existing ERES, accompanied by de novo generation of new ERES.

Eukaryotic cells internalize cargo at the plasma membrane via endocytosis, a vital process that is accomplished through a complex network of endosomal organelles. In mammalian cells, the ER is in close association with endosomes and regulates their fission. Nonetheless, the physiological role of such interaction on endocytosis is yet unexplored. Here, we probed the existence of ER–endosome association in plant cells and assayed its physiological role in endocytosis. Through live-cell imaging and electron microscopy studies, we established that endosomes are extensively associated with the plant ER, supporting conservation of interaction between heterotypic organelles in evolutionarily distant kingdoms. Furthermore, by analyzing ER–endosome dynamics in genetic backgrounds with defects in ER structure and movement, we also established that the ER network integrity is necessary for homeostasis of the distribution and streaming of various endosome populations as well as for efficient endocytosis. These results support a novel model that endocytosis homeostasis depends on a spatiotemporal control of the endosome dynamics dictated by the ER membrane network.

The endomembrane system plays essential roles in plant development, but the proteome responsible for its function and organization remains largely uncharacterized in plants. Here, we identified and characterized the HYPERSENSITIVE TO LATRUNCULIN B1 (HLB1) protein isolated through a forward-genetic screen in Arabidopsis thaliana for mutants with heightened sensitivity to actin-disrupting drugs. HLB1 is a plant-specific tetratricopeptide repeat domain-containing protein of unknown function encoded by a single Arabidopsis gene. HLB1 associated with the trans-Golgi network (TGN)/early endosome (EE) and tracked along filamentous actin, indicating that it could link post-Golgi traffic with the actin cytoskeleton in plants. HLB1 was found to interact with the ADP-ribosylation-factor guanine nucleotide exchange factor, MIN7/BEN1 (HOPM INTERACTOR7/BREFELDIN A-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1) by coimmunoprecipitation. The min7/ben1 mutant phenocopied the mild root developmental defects and latrunculin B hypersensitivity of hlb1, and analyses of a hlb1/ min7/ben1 double mutant showed that hlb1 and min7/ben1 operate in common genetic pathways. Based on these data, we propose that HLB1 together with MIN7/BEN1 form a complex with actin to modulate the function of the TGN/EE at the intersection of the exocytic and endocytic pathways in plants.

ARF GTPases play a central role in regulating membrane dynamics and protein transport in eukaryotic cells. ARF-like (ARL) proteins are close relatives of the ARF regulators of vesicular transport, but their function in plant cells is poorly characterized. Here, by means of live cell imaging and site-directed mutagenesis, we have investigated the cellular function of the plant GTPase ARL1. We provide direct evidence for a role of this ARL family member in the association of a plant golgin with the plant Golgi apparatus. Our data reveal the existence of key residues within the conserved GRIP-domain of the golgin and within the GTPase ARL1 that are central to ARL1-GRIP interaction. Mutations of these residues abolish the interaction of GRIP with the GTP-bound ARL1 and induce a redistribution of GRIP into the cytosol. This indicates that the localization of GRIP to the Golgi apparatus is strongly influenced by the interaction of GRIP with Golgi-localized ARL1. Our results assign a cellular role to a member of the Arabidopsis ARL family in the plant secretory pathway and propose mechanisms for localization of peripheral golgins to the plant Golgi apparatus.

Golgins are a family of coiled-coil proteins that are associated with the Golgi apparatus. They are necessary for tethering events in membrane fusion and may act as structural support for Golgi cisternae. Here we report on the identification of an Arabidopsis golgin which is a homologue of CASP, a known transmembrane mammalian and yeast golgin. Similar to its homologues, the plant CASP contains a long N-terminal coiled-coil region protruding into the cytosol and a C-terminal transmembrane domain with amino acid residues which are highly conserved across species. Through fluorescent protein tagging experiments, we show that plant CASP localizes at the plant Golgi apparatus and that the C-terminus of this protein is sufficient for its localization, as has been shown for its mammalian counterpart. In addition, we demonstrate that the plant CASP is able to localize at the mammalian Golgi apparatus. However, mutagenesis of a conserved tyrosine in the transmembrane domain revealed that it is necessary for ER export and Golgi localization of the Arabidopsis CASP in mammalian cells, but is not required for its correct localization in plant cells. These data suggest that mammalian and plant cells have different mechanisms for concentrating CASP in the Golgi apparatus.

The transport of proteins between the endoplasmic reticulum (ER) and the Golgi apparatus in plants is an exciting and constantly expanding topic, which has attracted much attention in recent years. The study of protein transport within the secretory pathway is a relatively new field, dating back to the 1970s for mammalian cells and considerably later for plants. This may explain why COPI- and COPII-mediated transport between the ER and the Golgi in plants is only now becoming clear, while the existence of these pathways in other organisms is relatively well documented. We summarize current knowledge of these protein transport routes, as well as highlighting key differences between those of plant systems and those of mammals and yeast. These differences have necessitated the study of plant-specific aspects of protein transport in the early secretory pathway, and this review discusses recent developments in this area. Advances in live-cell-imaging technology have allowed the observation of protein movement in vivo, giving a new insight into many of the processes involved in vesicle formation and protein trafficking. The use of these new technologies has been combined with more traditional methods, such as protein biochemistry and electron microscopy, to increase our understanding of the transport routes in the cell.

Green fluorescent protein (GFP) allows the direct visualization of gene expression and sub cellular localization of fusion proteins in living cells. Many GFP variants have been developed to solve stability and emission problems. In this report the localization of different GFP fusion proteins, targeted to vacuoles, was studied in Nicotiana tabacum cv SR1. Even if a strong emission variant of the plant adapted GFP was used, no fluorescence was detected in differentiated tissues of N. tabacum with few exceptions. This model plant does not appear a good experimental system for the use of GFPs as vacuolar markers compared to Arabidopsis thaliana. In spite of this, our observations have evidenced a peculiar pattern of separated vacuoles in guard cells, providing new elements in the understanding of the vacuolar system organization.

BiFC (Bimolecular Fluorescence Complementation) is one of the most widely used techniques to study protein-protein interactions as well as protein topology in living cells. This method allows the visualization of protein interactions or the analysis of their topology in the cell compartments where the proteins belong, without changing their chemical properties, as often occurs after mixing the content of different cellular compartments in cell extracts. Several laboratories use this method because it is relatively easy to perform; however, sometimes a positive protein-protein interaction BiFC signal (i.e., reconstitution of fluorescence of interacting protein pairs) does not necessarily mean that the tested proteins are actually interacting in vivo in a specific way. Here we describe the BiFC approach for assessing protein-protein interactions and for establishing protein topology and we discuss how to best perform this method to avoid false positive results when studying protein interactions in plant cells.

BiFC (Bimolecular Fluorescence Complementation) is one of the most widely used techniques to study protein-protein interactions as well as protein topology in living cells. This method allows the visualization of protein interactions or the analysis of their topology in the cell compartments where the proteins belong, without changing their chemical properties, as often occurs after mixing the content of different cellular compartments in cell extracts. Several laboratories use this method because it is relatively easy to perform; however, sometimes a positive protein-protein interaction BiFC signal (i.e., reconstitution of fluorescence of interacting protein pairs) does not necessarily mean that the tested proteins are actually interacting in vivo in a specific way. Here we describe the BiFC approach for assessing protein-protein interactions and for establishing protein topology and we discuss how to best perform this method to avoid false positive results when studying protein interactions in plant cells.

Cytoplasmic streaming is crucial for cell homeostasis and expansion but the precise driving forces are largely unknown. In plants, partial loss of cytoplasmic streaming due to chemical and genetic ablation of myosins supports the existence of yet-unknown motors for organelle movement. Here we tested a role of the endoplasmic reticulum (ER) as propelling force for cytoplasmic streaming during cell expansion. Through quantitative live-cell analyses in wild-type Arabidopsis thaliana cells and mutants with compromised ER structure and streaming, we demonstrate that cytoplasmic streaming undergoes profound changes during cell expansion and that it depends on motor forces co-exerted by the ER and the cytoskeleton.

Photosynthesis occurs in mesophyll cells of specialized organs such as leaves. The rigid cell wall encapsulating photosynthetic cells controls the expansion and distribution of cells within photosynthetic tissues. The relationship between photosynthesis and plant growth is affected by leaf area. However, the underlying genetic mechanisms affecting carbon partitioning to different aspects of leaf growth are not known. To fill this gap, we analyzed Arabidopsis plants with altered levels of pectin methylesterification, which is known to modulate cell wall plasticity and plant growth. Pectin methylesterification levels were varied through manipulation of cotton Golgi-related (CGR) 2 or 3 genes encoding two functionally redundant pectin methyltransferases. Increased levels of methylesterification in a line over-expressing CGR2 (CGR2OX) resulted in highly expanded leaves with enhanced intercellular air spaces; reduced methylesterification in a mutant lacking both CGR-genes 2 and 3 (cgr2/3) resulted in thin but dense leaf mesophyll that limited CO2 diffusion to chloroplasts. Leaf, root, and plant dry weight were enhanced in CGR2OX but decreased in cgr2/3. Differences in growth between wild type and the CGR-mutants can be explained by carbon partitioning but not by variations in area-based photosynthesis. Therefore, photosynthesis drives growth through alterations in carbon partitioning to new leaf area growth and leaf mass per unit leaf area; however, CGR-mediated pectin methylesterification acts as a primary factor in this relationship through modulation of the expansion and positioning of the cells in leaves, which in turn drive carbon partitioning by generating dynamic carbon demands in leaf area growth and leaf mass per unit leaf area.

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see(1,2)), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

Two envelope membranes delimit plastids, the defining organelles of plant cells. The inner and outer envelope membranes are unique in their protein and lipid composition. Several studies have attempted to establish the proteome of these two membranes; however, differentiating between them is difficult due to their close proximity. Here, we describe a novel approach to distinguish the localization of proteins between the two membranes using a straightforward approach based on live cell imaging coupled with transient expression. We base our approach on analyses of the distribution of GFP-fusions, which were aimed to verify outer envelope membrane proteomics data. To distinguish between outer envelope and inner envelope protein localization, we used AtTOC64-GFP and AtTIC40-GFP, as respective controls. During our analyses, we observed membrane proliferations and loss of chloroplast shape in conditions of protein over-expression. The morphology of the proliferations varied in correlation with the suborganellar distribution of the over-expressed proteins. In particular, while layers of membranes built up in the inner envelope membrane, the outer envelope formed long extensions into the cytosol. Using electron microscopy, we showed that these extensions were stromules, a dynamic feature of plastids. Since the behavior of the membranes is different and is related to the protein localization, we propose that in vivo studies based on the analysis of morphological differences of the membranes can be used to distinguish between inner and outer envelope localizations of proteins. To demonstrate the applicability of this approach, we demonstrated the localization of AtLACS9 to the outer envelope membrane. We also discuss protein impact on membrane behavior and regulation of protein insertion into membranes, and provide new hypotheses on the formation of stromules.

The mechanisms underlying the organization and dynamics of plant endomembranes are largely unknown. Arabidopsis RHD3, a distant member of the dynamin superfamily, has recently been implicated in plant ER morphology and Golgi movement through analyses of dominant-negative mutants of the putative GTPase domain in a heterologous system. Whether RHD3 is indispensable for ER architecture and what role regions other than the putative GTPase domain play in RHD3 function are unanswered questions. Here we characterized an EMS mutant, gom8, with disrupted Golgi movement and positioning and compromised ER shape and dynamics. gom8 mapped to a missense mutation in the RHD3 hairpin loop domain, causing accumulation of the mutant protein into large structures, a markedly different distribution compared with wild-type RHD3 over the ER network. Despite the GOM8 distribution, tubules fused in the peripheral ER of the gom8 mutant. These data imply that integrity of the hairpin region is important for the subcellular distribution of RHD3, and that reduced availability of RHD3 over the ER can cause ER morphology defects, but does not prevent peripheral fusion between tubules. This was confirmed by evidence that gom8 was phenocopied in an RHD3 null background. Furthermore, we established that the region encompassing the RHD3 hairpin domain and the C-terminal cytosolic domain is necessary for RHD3 function. We conclude that RHD3 is important in ER morphology, but is dispensable for peripheral ER tubulation in an endogenous context, and that its activity relies on the C-terminal region in addition to the GTPase domain.

ARF-GTPases are important proteins that control membrane trafficking events. Their activity is largely influenced by the interplay between guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), which facilitate the activation or inactivation of ARF-GTPases, respectively. There are 15 predicted proteins that contain an ARF-GAP domain within the Arabidopsis thaliana genome, and these are classified as ARF-GAP domain (AGD) proteins. The function and subcellular distribution of AGDs, including the ability to activate ARF-GTPases in vivo, that remain largely uncharacterized to date. Here we show that AGD5 is localised to the trans-Golgi network (TGN), where it co-localises with ARF1, a crucial GTPase that is involved in membrane trafficking and which was previously shown to be distributed on Golgi and post-Golgi structures of unknown nature. Taking advantage of the in vivo AGD5-ARF1 interaction at the TGN, we show that mutation of an arginine residue that is critical for ARF-GAP activity of AGD5 leads to longer residence of ARF1 on the membranes, as expected if GTP hydrolysis on ARF1 was impaired due to a defective GAP. Our results establish the nature of the post-Golgi compartments in which ARF1 localises, as well as identifying the role of AGD5 in vivo as a TGN-localised GAP. Furthermore, in vitro experiments established the promiscuous interaction between AGD5 and the plasma membrane-localised ADP ribosylation factor B (ARFB), confirming that ARF-GAP specificity for ARF-GTPases within the cell environment may be spatially regulated.

A central question in cell biology is how the identity of organelles is established and maintained. Here, we report on GOLD36, an EMS mutant identified through a screen for partial displacement of the Golgi marker, ST-GFP, to other organelles. GOLD36 showed partial distribution of ST-GFP into a modified endoplasmic reticulum (ER) network, which formed bulges and large skein-like structures entangling Golgi stacks. GOLD36 showed defects in ER protein export as evidenced by our observations that, besides the partial retention of Golgi markers in the ER, the trafficking of a soluble bulk-flow marker to the cell surface was also compromised. Using a combination of classical mapping and next-generation DNA sequencing approaches, we linked the mutant phenotype to a missense mutation of a proline residue in position 80 to a leucine residue in a small endomembrane protein encoded by the gold36 locus (At1g54030). Subcellular localization analyses indicated that GOLD36 is a vacuolar protein and that its mutated form is retained in the ER. Interestingly also, a gold36 knock-out mutant mirrored the GOLD36 subcellular phenotype. These data indicate that GOLD36 is a protein destined to post-ER compartments and suggest that its export from the ER is a requirement to ensure steady-state maintenance of the organelle's organization and functional activity in relation to other secretory compartments. We speculate that GOLD36 may be a factor that is necessary for ER integrity because of its ability to limit deleterious effects of other secretory proteins on the ER.

The study of electrical network systems, integrated with chemical signaling networks, is becoming a common trend in contemporary biology. Classical techniques are limited to the assessment of signals from doublets or triplets of cells at a fixed temporal bin width. At present, full characteristics of the electrical network distribution and dynamics in plant cells and tissues has not been established. Here, a 60-channels multielectrode array (MEA) is applied to study spatiotemporal characteristics of the electrical network activity of the root apex. Both intense spontaneous electrical activities and stimulation-elicited bursts of locally propagating electrical signals have been observed. Propagation of the spikes indicates the existence of excitable traveling waves in plants, similar to those observed in non-nerve electrogenic tissues of animals. Obtained data reveal synchronous electric activities of root cells emerging in a specific root apex region. The dynamic electrochemical activity of root apex cells is proposed to continuously integrate internal and external signaling for developmental adaptations in a changing environment.

*† ‡ Summary Trafficking of secretory proteins between the endoplasmic reticulum (ER) and the Golgi apparatus depends on coat protein complexes I (COPI) and II (COPII) machineries. To date, full characterization of the distribution and dynamics of these machineries in plant cells remains elusive. Furthermore, except for a presumed linkage between COPI and COPII for the maintenance of ER protein export, the mechanisms by which COPI influences COPII-mediated protein transport from the ER in plant cells are largely uncharacterized. Here we dissect the dynamics of COPI in intact cells using live-cell imaging and fluorescence recovery after photobleaching analyses to provide insights into the distribution of COPI and COPII machineries and the mechanisms by which COPI influences COPII-mediated protein export from the ER. We found that Arf1 and coatomer are dynamically associated with the Golgi apparatus and that the COPII coat proteins Sec24 and Sec23 localize at ER export sites that track with the Golgi apparatus in tobacco leaf epidermal cells. Arf1 is also localized at additional structures that originate from the Golgi apparatus but that lack coatomer, supporting the model that Arf1 also has a coatomer-independent role for post-Golgi protein transport in plants. When ER to Golgi protein transport is inhibited by mutations that hamper Arf1-GTPase activity without directly disrupting the COPII machinery for ER protein export, Golgi markers are localized in the ER and the punctate distribution of Sec24 and Sec23 at the ER export sites is lost. These findings suggest that Golgi membrane protein distribution is maintained by the balanced action of COPI and COPII systems, and that Arf1-coatomer is most likely indirectly required for forward trafficking out of the ER due to its role in recycling components that are essential for differentiation of the ER export domains formed by the Sar1-COPII system.

In yeast and mammals, amino acid motifs in the cytosolic tails of transmembrane domains play a role in protein trafficking by facilitating export from the endoplasmic reticulum (ER). However, little is known about ER export signals of membrane proteins in plants. Therefore, we investigated the role of diacidic motifs in the ER export of Golgi-localized membrane proteins. We show that diacidic motifs perform a significant function in the export of transmembrane proteins to the Golgi apparatus, as mutations of these signals impede the efficient anterograde transport of multispanning, type II, and type I proteins. Furthermore, we demonstrate that diacidic motifs instigate the export of proteins that reside in the ER due to the lengths of their transmembrane domains. However, not all of the diacidic motifs in the cytosolic tails of the proteins studied were equally important in ER export. Transport of Golgi proteins was disrupted only by mutagenesis of specific diacidic signals, suggesting that the protein environment of these signals affects their function. Our findings indicate that diacidic ER export motifs are present and functional in plant membrane proteins and that they are dominant over transmembrane domain length in determining the export of proteins from the ER in plant cells.

The transport of proteins between the endoplasmic reticulum (ER) and the Golgi apparatus in plants is an exciting and constantly expanding topic, which has attracted much attention in recent years. The study of protein transport within the secretory pathway is a relatively new field, dating back to the 1970s for mammalian cells and considerably later for plants. This may explain why COPI- and COPII-mediated transport between the ER and the Golgi in plants is only now becoming clear, while the existence of these pathways in other organisms is relatively well documented. We summarize current knowledge of these protein transport routes, as well as highlighting key differences between those of plant systems and those of mammals and yeast. These differences have necessitated the study of plant-specific aspects of protein transport in the early secretory pathway, and this review discusses recent developments in this area. Advances in live-cell-imaging technology have allowed the observation of protein movement in vivo, giving a new insight into many of the processes involved in vesicle formation and protein trafficking. The use of these new technologies has been combined with more traditional methods, such as protein biochemistry and electron microscopy, to increase our understanding of the transport routes in the cell.