The TGN/EE SNARE protein SYP61 and the ubiquitin ligase ATL31 cooperatively regulate plant responses to carbon/nitrogen conditions in Arabidopsis.

Ubiquitination is a post-translational modification involving the reversible attachment of the small protein ubiquitin to a target protein. Ubiquitination is involved in numerous cellular processes, including the membrane trafficking of cargo proteins. However, the ubiquitination of the trafficking machinery components and their involvement in environmental responses are not well understood. Here, we report that the Arabidopsis thaliana trans-Golgi network/early endosome localized SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein SYP61 interacts with the transmembrane ubiquitin ligase ATL31, a key regulator of resistance to disrupted carbon (C)/nitrogen/(N)-nutrient conditions. SYP61 is a key component of membrane trafficking in Arabidopsis. The subcellular localization of ATL31 was disrupted in knockdown mutants of SYP61, and the insensitivity of ATL31-overexpressing plants to high C/low N-stress was repressed in these mutants, suggesting that SYP61 and ATL31 cooperatively function in plant responses to nutrient stress. SYP61 is ubiquitinated in plants, and its ubiquitination level is upregulated under low C/high N-nutrient conditions. These findings provide important insights into the ubiquitin signaling and membrane trafficking machinery in plants.

Trapa bispinosa Roxb. Pericarp Extract Exerts 5α-Reductase Inhibitory Activity in Castrated Benign Prostatic Hyperplasia Model Mice.

Trapa bispinosa Roxb. pericarp extract (TBE) has a polyphenol-rich composition and exhibits potent antioxidant and anti-glycation activities in vitro. In the present study, we investigated the inhibitory effects of TBE on 5α-reductase in vitro using LNCaP cells and in vivo using a mouse model of castrated benign prostatic hyperplasia. TBE showed concentration-dependent inhibitory effects in the 5α-reductase (5αR) activity assay. In a reporter assay using AR-Luc/LNCaP cells, TBE inhibited the activity induced by testosterone, but not that induced by dihydrotestosterone. TBE also suppressed prostate cell proliferation, prostate-specific antigens, and transmembrane protease serine 2 expression in a castrated benign prostatic hyperplasia mouse model. In addition, ellagic acid, but not gallic acid, decreased 5αR and AR-Luc activities. Together, these results suggest a potential role for TBE in benign prostatic hyperplasia through inhibition of 5αR.

Transient activity of the florigen complex during the floral transition in Arabidopsis thaliana.

FLOWERING LOCUS T (FT) is an essential component of florigen in Arabidopsis thaliana Transcription of FT is induced in leaves, and the resulting FT protein is transported to the shoot apex, in which it initiates floral development. Previous analyses suggest that, together with the b-ZIP transcription factor FD, FT regulates the transcription of downstream targets such as APETALA1 (AP1) in floral anlagen. However, conclusive in vivo evidence that FT is transported to the shoot apex to form an FT-FD complex is lacking. Here, using an innovative in vivo imaging technique, we show that the FT-FD complex and AP1 colocalise in floral anlagen. In addition, the FT-FD complex disappears soon after the floral transition owing to a reduction in FD transcripts in the shoot apex. We further show that misinduction of FD activity after the transition leads to defective reproductive development. Taken together, our results indicate that the FT-FD complex functions as a transient stimulus and imply that a regulatory mechanism exists during the floral transition that reduces FT-FD complex levels via modulation of FD expression.

RAB GTPases and SNAREs at the trans-Golgi network in plants.

Membrane traffic is a fundamental cellular system to exchange proteins and membrane lipids among single membrane-bound organelles or between an organelle and the plasma membrane in order to keep integrity of the endomembrane system. RAB GTPases and SNARE proteins, the key regulators of membrane traffic, are conserved broadly among eukaryotic species. However, genome-wide analyses showed that organization of RABs and SNAREs that regulate the post-Golgi transport pathways is greatly diversified in plants compared to other model eukaryotes. Furthermore, some organelles acquired unique properties in plant lineages. Like in other eukaryotic systems, the trans-Golgi network of plants coordinates secretion and vacuolar transport; however, uniquely in plants, it also acts as a platform for endocytic transport and recycling. In this review, we focus on RAB GTPases and SNAREs that function at the TGN, and summarize how these regulators perform to control different transport pathways at the plant TGN. We also highlight the current knowledge of RABs and SNAREs' role in regulation of plant development and plant responses to environmental stimuli.

Characterization and Identification of Bioactive Polyphenols in the Trapabispinosa Roxb. Pericarp Extract.

In this study, we present the isolation and characterization of the structure of six gallotannins (1-6), three ellagitannins (7-9), a neolignan glucoside (10), and three related polyphenolic compounds (gallic acid, 11 and 12) from Trapa bispinosa Roxb. pericarp extract (TBE). Among the isolates, the structure of compound 10 possessing a previously unclear absolute configuration was unambiguously determined through nuclear magnetic resonance and circular dichroism analyses. The α-glucosidase activity and glycation inhibitory effects of the isolates were evaluated. Decarboxylated rugosin A (8) showed an α-glucosidase inhibitory activity, while hydrolyzable tannins revealed stronger antiglycation activity than that of the positive control. Furthermore, the identification and quantification of the TBE polyphenols were investigated by high-performance liquid chromatography coupled to ultraviolet detection and electrospray ionization mass spectrometry analysis, indicating the predominance of gallic acid, ellagic acid, and galloyl glucoses showing marked antiglycation properties. These findings suggest that there is a potential food industry application of polyphenols in TBE as a functional food with antidiabetic and antiglycation activities.

The Golgi entry core compartment functions as a COPII-independent scaffold for ER-to-Golgi transport in plant cells.

Many questions remain about how the stacked structure of the Golgi is formed and maintained. In our previous study, we challenged this question using tobacco BY-2 cells and revealed that, upon Brefeldin A (BFA) treatment, previously undescribed small punctate structures containing a particular subset of cis-Golgi proteins are formed adjacent to the ER-exit sites and act as scaffolds for Golgi regeneration after BFA removal. In this study, we analyzed these structures further. The proteins that localize to these punctate structures originate from the cis-most cisternae. 3D time-lapse observations show that the trans-Golgi marker is transported through these structures during Golgi regeneration. These data indicate that the cis-most cisternae have a specialized region that receives cargo from the ER, which becomes obvious upon BFA treatment. Expression of a dominant mutant form of SAR1 does not affect the formation of the punctate structures. We propose to call these punctate structures the 'Golgi entry core compartment' (GECCO). They act as receivers for the rest of the Golgi materials and are formed independently of the COPII machinery.This article has an associated First Person interview with the first author of the paper.

A Golgi-Released Subpopulation of the Trans-Golgi Network Mediates Protein Secretion in Arabidopsis.

Spatiotemporal coordination of protein trafficking among organelles is essential for eukaryotic cells. The post-Golgi interface, including the trans-Golgi network (TGN), is a pivotal hub for multiple trafficking pathways. The Golgi-released independent TGN (GI-TGN) is a compartment described only in plant cells, and its cellular and physiological roles remain elusive. In Arabidopsis (Arabidopsis thaliana), the SYNTAXIN OF PLANTS (SYP) 4 group Qa-SNARE (soluble N-ethylmaleimide) membrane fusion proteins are shared components of TGN and GI-TGN and regulate secretory and vacuolar transport. Here we reveal that GI-TGNs mediate the transport of the R-SNARE VESICLE-ASSOCIATED MEMBRANE PROTEIN (VAMP) 721 to the plasma membrane. In interactions with a nonadapted powdery mildew pathogen, the SYP4 group of SNAREs is required for the dynamic relocation of VAMP721 to plant-fungus contact sites via GI-TGNs, thereby facilitating complex formation with its cognate SNARE partner PENETRATION1 to restrict pathogen entry. Furthermore, quantitative proteomic analysis of leaf apoplastic fluid revealed constitutive and pathogen-inducible secretion of cell wall-modification enzymes in a SYP4- and VAMP721-dependent manner. Hence, the GI-TGN acts as a transit compartment between the Golgi apparatus and the plasma membrane. We propose a model in which the GA-TGN matures into the GI-TGN and then into secretory vesicles by increasing the abundance of VAMP721-dependent secretory pathway components.

Enrichment of Phosphatidylinositol 4,5-Bisphosphate in the Extra-Invasive Hyphal Membrane Promotes Colletotrichum Infection of Arabidopsis thaliana.

Pathogenic fungi from the genus Colletotrichum form invasive hyphae; the hyphae are surrounded by an extra-invasive hyphal membrane (EIHM), which is continuous with the plant plasma membrane. Although the EIHM plays a crucial role as the interface between plant and fungal cells, its precise function during Colletotrichum infection remains elusive. Here, we show that enrichment of phosphoinositides (PIs) has a crucial role in Colletotrichum infection. We observed the localization of PIs in Arabidopsis thaliana cells infected by A. thaliana-adapted Colletotrichum higginsianum (Ch), and found that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] was extremely enriched in the EIHM during Ch infection. We also found that phosphatidylinositol 4-phosphate-5 kinase (PIP5K), which catalyzes production of PI(4,5)P2, also accumulated at the EIHM. The overexpression of PIP5K3 in A. thaliana increased hyphal invasion by Ch. An exocytic factor, EXO84b, was targeted to the EIHM during Ch infection, although endocytic factors such as CLATHRIN LIGHT CHAIN 2 and FLOTILLIN 1 did not. Intriguingly, the interfacial membranes between A. thaliana and powdery mildew- or downy mildew-causing pathogens did not accumulate PI(4,5)P2. These results suggest that Ch could modify the PI(4,5)P2 levels in the EIHM to increase the exocytic membrane/protein supply of the EIHM for successful infection. Our results also suggest that PI(4,5)P2 biosynthesis is a promising target for improved defense against Colletotrichum infection.

A Missense Mutation in the NSF Gene Causes Abnormal Golgi Morphology in Arabidopsis thaliana.

The Golgi apparatus is a key station of glycosylation and membrane traffic. It consists of stacked cisternae in most eukaryotes. However, the mechanisms how the Golgi stacks are formed and maintained are still obscure. The model plant Arabidopsis thaliana provides a nice system to observe Golgi structures by light microscopy, because the Golgi in A. thaliana is in the form of mini-stacks that are distributed throughout the cytoplasm. To obtain a clue to understand the molecular basis of Golgi morphology, we took a forward-genetic approach to isolate A. thaliana mutants that show abnormal structures of the Golgi under a confocal microscope. In the present report, we describe characterization of one of such mutants, named #46-3. The #46-3 mutant showed pleiotropic Golgi phenotypes. The Golgi size was in majority smaller than the wild type, but varied from very small ones, sometimes without clear association of cis and trans cisternae, to abnormally large ones under a confocal microscope. At the ultrastructual level by electron microscopy, queer-shaped large Golgi stacks were occasionally observed. By positional mapping, genome sequencing, and complementation and allelism tests, we linked the mutant phenotype to the missense mutation D374N in the NSF gene, encoding the N-ethylmaleimide-sensitive factor (NSF), a key component of membrane fusion. This residue is near the ATP-binding site of NSF, which is very well conserved in eukaryotes, suggesting that the biochemical function of NSF is important for maintaining the normal morphology of the Golgi.Key words: Golgi morphology, N-ethylmaleimide-sensitive factor (NSF), Arabidopsis thaliana.

The trans-Golgi Network and the Golgi Stacks Behave Independently During Regeneration After Brefeldin A Treatment in Tobacco BY-2 Cells.

The trans-Golgi network (TGN) plays an essential role in intracellular membrane trafficking. In plant cells, recent live-cell imaging studies have revealed the dynamic behavior of the TGN independent from the Golgi apparatus. In order to better understand the relationships between the two organelles, we examined their dynamic responses to the reagent brefeldin A (BFA) and their recovery after BFA removal. Golgi markers responded to BFA similarly over a range of concentrations, whereas the behavior of the TGN was BFA concentration dependent. The TGN formed aggregates at high concentrations of BFA; however, TGN proteins relocalized to numerous small vesicular structures dispersed throughout the cytoplasm at lower BFA concentrations. During recovery from weak BFA treatment, the TGN started to regenerate earlier than the completion of the Golgi. The regeneration of the two organelles proceeded independently of each other for a while, and eventually was completed by their association. Our data suggest that there is some degree of autonomy for the regeneration of the TGN and the Golgi in tobacco BY-2 cells.

Physiological Roles of Plant Post-Golgi Transport Pathways in Membrane Trafficking.

Membrane trafficking is the fundamental system through which proteins are sorted to their correct destinations in eukaryotic cells. Key regulators of this system include RAB GTPases and soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs). Interestingly, the numbers of RAB GTPases and SNAREs involved in post-Golgi transport pathways in plant cells are larger than those in animal and yeast cells, suggesting that plants have evolved unique and complex post-Golgi transport pathways. The trans-Golgi network (TGN) is an important organelle that acts as a sorting station in the post-Golgi transport pathways of plant cells. The TGN also functions as the early endosome, which is the first compartment to receive endocytosed proteins. Several endocytosed proteins on the plasma membrane (PM) are initially targeted to the TGN/EE, then recycled back to the PM or transported to the vacuole for degradation. The recycling and degradation of the PM localized proteins is essential for the development and environmental responses in plant. The present review describes the post-Golgi transport pathways that show unique physiological functions in plants.

SNARE Molecules in Marchantia polymorpha: Unique and Conserved Features of the Membrane Fusion Machinery.

The membrane trafficking pathway has been diversified in a specific way for each eukaryotic lineage, probably to fulfill specific functions in the organisms. In green plants, comparative genomics has supported the possibility that terrestrialization and/or multicellularization could be associated with the elaboration and diversification of membrane trafficking pathways, which have been accomplished by an expansion of the numbers of genes required for machinery components of membrane trafficking, including soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. However, information regarding membrane trafficking pathways in basal land plant lineages remains limited. In the present study, we conducted extensive analyses of SNARE molecules, which mediate membrane fusion between target membranes and transport vesicles or donor organelles, in the liverwort, Marchantia polymorpha. The M. polymorpha genome contained at least 34 genes for 36 SNARE proteins, comprising fundamental sets of SNARE proteins that are shared among land plant lineages with low degrees of redundancy. We examined the subcellular distribution of a major portion of these SNARE proteins by expressing Citrine-tagged SNARE proteins in M. polymorpha, and the results showed that some of the SNARE proteins were targeted to different compartments from their orthologous products in Arabidopsis thaliana. For example, MpSYP12B was localized to the surface of the oil body, which is a unique organelle in liverworts. Furthermore, we identified three VAMP72 members with distinctive structural characteristics, whose N-terminal extensions contain consensus sequences for N-myristoylation. These results suggest that M. polymorpha has acquired unique membrane trafficking pathways associated with newly acquired machinery components during evolution.

RABA members act in distinct steps of subcellular trafficking of the FLAGELLIN SENSING2 receptor.

Cell surface proteins play critical roles in the perception of environmental stimuli at the plasma membrane (PM) and ensuing signal transduction. Intracellular localization of such proteins must be strictly regulated, which requires elaborate integration of exocytic and endocytic trafficking pathways. Subcellular localization of Arabidopsis thaliana FLAGELLIN SENSING2 (FLS2), a receptor that recognizes bacterial flagellin, also depends on membrane trafficking. However, our understanding about the mechanisms involved is still limited. In this study, we visualized ligand-induced endocytosis of FLS2 using green fluorescent protein (GFP)-tagged FLS2 expressed in Nicotiana benthamiana. Upon treatment with the flg22 peptide, internalized FLS2-GFP from the PM was transported to a compartment with properties intermediate between the trans-Golgi network (TGN) and the multivesicular endosome. This compartment gradually discarded the TGN characteristics as it continued along the trafficking pathway. We further found that FLS2 endocytosis involves distinct RABA/RAB11 subgroups at different steps. Moreover, we demonstrated that transport of de novo-synthesized FLS2 to the PM also involves a distinct RABA/RAB11 subgroup. Our results demonstrate the complex regulatory system for properly localizing FLS2 and functional differentiation in RABA members in endo- and exocytosis.

Cell polarity and patterning by PIN trafficking through early endosomal compartments in Arabidopsis thaliana.

PIN-FORMED (PIN) proteins localize asymmetrically at the plasma membrane and mediate intercellular polar transport of the plant hormone auxin that is crucial for a multitude of developmental processes in plants. PIN localization is under extensive control by environmental or developmental cues, but mechanisms regulating PIN localization are not fully understood. Here we show that early endosomal components ARF GEF BEN1 and newly identified Sec1/Munc18 family protein BEN2 are involved in distinct steps of early endosomal trafficking. BEN1 and BEN2 are collectively required for polar PIN localization, for their dynamic repolarization, and consequently for auxin activity gradient formation and auxin-related developmental processes including embryonic patterning, organogenesis, and vasculature venation patterning. These results show that early endosomal trafficking is crucial for cell polarity and auxin-dependent regulation of plant architecture.

The powdery mildew resistance protein RPW8.2 is carried on VAMP721/722 vesicles to the extrahaustorial membrane of haustorial complexes.

Plants employ multiple cell-autonomous defense mechanisms to impede pathogenesis of microbial intruders. Previously we identified an exocytosis defense mechanism in Arabidopsis against pathogenic powdery mildew fungi. This pre-invasive defense mechanism depends on the formation of ternary protein complexes consisting of the plasma membrane-localized PEN1 syntaxin, the adaptor protein SNAP33 and closely sequence-related vesicle-resident VAMP721 or VAMP722 proteins. The Arabidopsis thaliana resistance to powdery mildew 8.2 protein (RPW8.2) confers disease resistance against powdery mildews upon fungal entry into host cells and is specifically targeted to the extrahaustorial membrane (EHM), which envelops the haustorial complex of the fungus. However, the secretory machinery involved in trafficking RPW8.2 to the EHM is unknown. Here we report that RPW8.2 is transiently located on VAMP721/722 vesicles, and later incorporated into the EHM of mature haustoria. Resistance activity of RPW8.2 against the powdery mildew Golovinomyces orontii is greatly diminished in the absence of VAMP721 but only slightly so in the absence of VAMP722. Consistent with this result, trafficking of RPW8.2 to the EHM is delayed in the absence of VAMP721. These findings implicate VAMP721/722 vesicles as key components of the secretory machinery for carrying RPW8.2 to the plant-fungal interface. Quantitative fluorescence recovery after photobleaching suggests that vesicle-mediated trafficking of RPW8.2-yellow fluorescent protein (YFP) to the EHM occurs transiently during early haustorial development and that lateral diffusion of RPW8.2-YFP within the EHM exceeds vesicle-mediated replenishment of RPW8.2-YFP in mature haustoria. Our findings imply the engagement of VAMP721/722 in a bifurcated trafficking pathway for pre-invasive defense at the cell periphery and post-invasive defense at the EHM.

Sex-specific posttranslational regulation of the gamete fusogen GCS1 in the isogamous volvocine alga Gonium pectorale.

Male and female, generally defined based on differences in gamete size and motility, likely have multiple independent origins, appearing to have evolved from isogamous organisms in various eukaryotic lineages. Recent studies of the gamete fusogen GCS1/HAP2 indicate that this protein is deeply conserved across eukaryotes, and its exclusive and/or functional expression generally resides in males or in male homologues. However, little is known regarding the conserved or primitive molecular traits of males and females within eukaryotes. Here, using morphologically indistinguishable isogametes of the colonial volvocine Gonium pectorale, we demonstrated that GCS1 is differently regulated between the sexes. G. pectorale GCS1 molecules in one sex (homologous to male) are transported from the gamete cytoplasm to the protruded fusion site, whereas those of the other sex (females) are quickly degraded within the cytoplasm upon gamete activation. This molecular trait difference might be conserved across various eukaryotic lineages and may represent male and female prototypes originating from a common eukaryotic ancestor.

Qa-SNAREs localized to the trans-Golgi network regulate multiple transport pathways and extracellular disease resistance in plants.

In all eukaryotic cells, a membrane-trafficking system connects the post-Golgi organelles, such as the trans-Golgi network (TGN), endosomes, vacuoles, and the plasma membrane. This complex network plays critical roles in several higher-order functions in multicellular organisms. The TGN, one of the important organelles for protein transport in the post-Golgi network, functions as a sorting station, where cargo proteins are directed to the appropriate post-Golgi compartments. Unlike its roles in animal and yeast cells, the TGN has also been reported to function like early endosomal compartments in plant cells. However, the physiological roles of the TGN functions in plants are not understood. Here, we report a study of the SYP4 group (SYP41, SYP42, and SYP43), which represents the plant orthologs of the Tlg2/syntaxin16 Qa-SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) that localizes on the TGN in yeast and animal cells. The SYP4 group regulates the secretory and vacuolar transport pathways in the post-Golgi network and maintains the morphology of the Golgi apparatus and TGN. Consistent with a secretory role, SYP4 proteins are required for extracellular resistance responses to a fungal pathogen. We also reveal a plant cell-specific higher-order role of the SYP4 group in the protection of chloroplasts from salicylic acid-dependent biotic stress.

Arabidopsis RABA1 GTPases are involved in transport between the trans-Golgi network and the plasma membrane, and are required for salinity stress tolerance.

RAB GTPases are key regulators of membrane traffic. Among them, RAB11, a widely conserved sub-group, has evolved in a unique way in plants; plant RAB11 members show notable diversity, whereas yeast and animals have only a few RAB11 members. Fifty-seven RAB GTPases are encoded in the Arabidopsis thaliana genome, 26 of which are classified in the RAB11 group (further divided into RABA1-RABA6 sub-groups). Although several plant RAB11 members have been shown to play pivotal roles in plant-unique developmental processes, including cytokinesis and tip growth, molecular and physiological functions of the majority of RAB11 members remain unknown. To reveal precise functions of plant RAB11, we investigated the subcellular localization and dynamics of the largest sub-group of Arabidopsis RAB11, RABA1, which has nine members. RABA1 members reside on mobile punctate structures adjacent to the trans-Golgi network and co-localized with VAMP721/722, R-SNARE proteins that operate in the secretory pathway. In addition, the constitutive-active mutant of RABA1b, RABA1b(Q72L) , was present on the plasma membrane. The RABA1b -containing membrane structures showed actin-dependent dynamic motion . Vesicles labeled by GFP-RABA1b moved dynamically, forming queues along actin filaments. Interestingly, Arabidopsis plants whose four major RABA1 members were knocked out, and those expressing the dominant-negative mutant of RABA1B, exhibited hypersensitivity to salinity stress. Altogether, these results indicate that RABA1 members mediate transport between the trans-Golgi network and the plasma membrane, and are required for salinity stress tolerance.

Dynamic behavior of the trans-golgi network in root tissues of Arabidopsis revealed by super-resolution live imaging.

The trans-Golgi network (TGN) is an important organelle for protein transport at the post-Golgi network, which functions as a sorting station that directs cargo proteins to a variety of destinations including post-Golgi compartments and the extracellular space. However, the functions and dynamics of the TGN in plant cells have not been well understood yet. To elucidate the dynamics of the plant TGN, we established transgenic plants expressing green fluorescent protein (GFP)-SYP43, the ortholog of Tlg2/syntaxin16, which is localized to the TGN in yeast and mammalian cells, under the control of the native promoter as a TGN marker. Observation by confocal laser scanning microscopy and super-resolution confocal live imaging microscopy revealed two types of TGN in Arabidopsis root: the GA-TGNs (Golgi-associated TGNs), located on the trans-side of the Golgi apparatus, and the GI-TGNs (Golgi-released independent TGNs), located away from the Golgi apparatus and behaving independently. The GI-TGNs is derived from a population of GA-TGNs by segregation, although the core of the GA-TGN remains even after the generation of the GI-TGN. We further found that the abundance of the GI-TGNs differs between observed tissues. Our results indicate that the dynamic features of the TGN in plant cells differ from those of animal and yeast cells.