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1.  Cell Polarity and Patterning by PIN Trafficking through Early Endosomal Compartments in Arabidopsis thaliana 
PLoS Genetics  2013;9(5):e1003540.
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.
Author Summary
Auxin is a unique plant hormone, which is actively and directionally transported in plant tissues. Transported auxin locally accumulates in the plant body and triggers a multitude of responses, including organ formation and patterning. Therefore, regulation of the directional auxin transport is very important in multiple aspects of plant development. The PIN-FORMED (PIN) family of auxin transporters is known to localize at specific sides of cells and export auxin from the cells, enabling the directional transport of auxin in the tissues. PIN proteins are rapidly shuttling between the plasma membrane and intracellular compartments, potentially allowing dynamic changes of the asymmetric localization according to developmental and environmental cues. Here, we discovered that a mutation in the Sec1/Munc18 family protein VPS45 abolishes its own early endosomal localization and compromises intracellular trafficking of PIN proteins. By genetic and pharmacological inhibition of early endosomal trafficking, we also revealed that another early endosomal protein, ARF GEF BEN1, is involved in early endosomal trafficking at a distinct step. Furthermore, we showed that these components play crucial roles in polar localization and dynamic repolarization of PIN proteins, which underpin various developmental processes. These findings highlight the indispensable roles of early endosomal components in regulating PIN polarity and plant architecture.
doi:10.1371/journal.pgen.1003540
PMCID: PMC3667747  PMID: 23737757
2.  The physiological role of SYP4 in the salinity and osmotic stress tolerances 
Plant Signaling & Behavior  2012;7(9):1118-1120.
The trans-Golgi network (TGN) contains multiple sorting domains and acts as the compartment for cargo sorting. Recent evidence indicates that the TGN also functions as an early endosome, the first compartment in the endocytic pathway in plants. The SYP4 group, plant Qa-SNAREs localized on the TGN, regulates both secretory and vacuolar transport pathways. Consistent with a secretory role, SYP4 proteins are required for extracellular resistance to fungal pathogens. However, the physiological role of SYP4 in abiotic stress remains unknown. Here, we report the phenotypes of a syp4-mutant in regard to salinity and osmotic response, and describe the physiological roles of the SYP4 group in the abiotic stress response.
doi:10.4161/psb.21307
PMCID: PMC3489641  PMID: 22899062
SNARE; SYP4; TGN; salinity stress
3.  cis-Golgi proteins accumulate near the ER exit sites and act as the scaffold for Golgi regeneration after brefeldin A treatment in tobacco BY-2 cells 
Molecular Biology of the Cell  2012;23(16):3203-3214.
Particular cis-Golgi proteins accumulate in novel punctate structures close to ERES by BFA treatment in tobacco BY-2 cells. These structures reassemble first to form cis-Golgi after BFA removal, and the Golgi stacks regenerate in the cis-to-trans order. This indicates that the punctate structures act as the scaffold for Golgi regeneration.
The Golgi apparatus forms stacks of cisternae in many eukaryotic cells. However, little is known about how such a stacked structure is formed and maintained. To address this question, plant cells provide a system suitable for live-imaging approaches because individual Golgi stacks are well separated in the cytoplasm. We established tobacco BY-2 cell lines expressing multiple Golgi markers tagged by different fluorescent proteins and observed their responses to brefeldin A (BFA) treatment and BFA removal. BFA treatment disrupted cis, medial, and trans cisternae but caused distinct relocalization patterns depending on the proteins examined. Medial- and trans-Golgi proteins, as well as one cis-Golgi protein, were absorbed into the endoplasmic reticulum (ER), but two other cis-Golgi proteins formed small punctate structures. After BFA removal, these puncta coalesced first, and then the Golgi stacks regenerated from them in the cis-to-trans order. We suggest that these structures have a property similar to the ER-Golgi intermediate compartment and function as the scaffold of Golgi regeneration.
doi:10.1091/mbc.E12-01-0034
PMCID: PMC3418314  PMID: 22740633
4.  Flowering Time Modulation by a Vacuolar SNARE via FLOWERING LOCUS C in Arabidopsis thaliana 
PLoS ONE  2012;7(7):e42239.
The transition of plant growth from vegetative to reproductive phases is one of the most important and dramatic events during the plant life cycle. In Arabidopsis thaliana, flowering promotion involves at least four genetically defined regulatory pathways, including the photoperiod-dependent, vernalization-dependent, gibberellin-dependent, and autonomous promotion pathways. Among these regulatory pathways, the vernalization-dependent and autonomous pathways are integrated by the expression of FLOWERING LOCUS C (FLC), a negative regulator of flowering; however, the upstream regulation of this locus has not been fully understood. The SYP22 gene encodes a vacuolar SNARE protein that acts in vacuolar and endocytic trafficking pathways. Loss of SYP22 function was reported to lead to late flowering in A. thaliana plants, but the mechanism has remained completely unknown. In this study, we demonstrated that the late flowering phenotype of syp22 was due to elevated expression of FLC caused by impairment of the autonomous pathway. In addition, we investigated the DOC1/BIG pathway, which is also suggested to regulate vacuolar/endosomal trafficking. We found that elevated levels of FLC transcripts accumulated in the doc1-1 mutant, and that syp22 phenotypes were exaggerated with a double syp22 doc1-1 mutation. We further demonstrated that the elevated expression of FLC was suppressed by ara6-1, a mutation in the gene encoding plant-unique Rab GTPase involved in endosomal trafficking. Our results indicated that vacuolar and/or endocytic trafficking is involved in the FLC regulation of flowering time in A. thaliana.
doi:10.1371/journal.pone.0042239
PMCID: PMC3407077  PMID: 22848750
5.  Qualitative difference between “bulb” membranes and other vacuolar membranes 
Plant Signaling & Behavior  2011;6(12):1914-1917.
“Bulb” is a mobile and complex structure appearing in vacuolar membrane of plant cell. We recently reported new fluorescent marker lines for bulbs and bulb-less mutants. We tried multicolor visualization of vacuolar membrane to show distinct segregation of bulb-positive protein (γTIP or AtVAM3) and bulb-negative protein (AtRab75). Unexpectedly, GFP-AtRab75 resulted to localize in bulb under the condition of co-expression with TagRFP-AtVAM3. The signal intensities of GFP-AtRab75 and TagRFP-AtVAM3 were quantified and compared. The result indicates that TagRFP-AtVAM3 is concentrated in bulb than GFP-AtRab75.
doi:10.4161/psb.6.12.18061
PMCID: PMC3337177  PMID: 22105033
AtRab75; AtVam3; plant growth; Rab-GTPase; SNARE; vacuolar membranes; “bulb”
6.  Endosomal trafficking pathway regulated by ARA6, a RAB5 GTPase unique to plants 
Small GTPases  2012;3(1):23-27.
Lineage-specific expansion, followed by functional diversification of key components that act in membrane trafficking, is thought to contribute to lineage-specific diversification of organelles and membrane trafficking pathways. Indeed, recent comparative genomic studies have indicated that specific expansion of RAB and SNARE molecules occurred independently in various eukaryotic lineages over evolutionary history. However, experimental verification of this notion is difficult, because detailed functional analyses of RAB and SNARE proteins uniquely acquired by specific lineages are essential to understanding how new membrane trafficking pathways may have evolved. Recently, we found that a plant-specific RAB GTPase, ARA6, and a plant-unique R-SNARE, VAMP727, mediate a trafficking pathway from endosomes to the plasma membrane in Arabidopsis thaliana. Although a similar endosomal trafficking pathway was also reported in animals, the molecular machineries acting in these trafficking systems differ between animals and plants. Thus, trafficking pathways from endosomes to the plasma membrane appear to have been acquired independently in animal and plant systems. We further demonstrated that the ARA6-mediated trafficking pathway is required for the proper salt-stress response of A. thaliana. These results indicate that acquisition of a new membrane trafficking pathway may be associated with maximization of the fitness of each organism in a lineage-specific manner.
doi:10.4161/sgtp.18299
PMCID: PMC3398913  PMID: 22710734
ARA6; Arabidopsis thaliana; Rab5; SNARE; endosome; stress response
7.  Generation of cell polarity in plants links endocytosis, auxin distribution and cell fate decisions 
Nature  2008;456(7224):962-966.
Dynamically polarized membrane proteins define different cell boundaries and have an important role in intercellular communication—a vital feature of multicellular development. Efflux carriers for the signalling molecule auxin from the PIN family1 are landmarks of cell polarity in plants and have a crucial involvement in auxin distribution-dependent development including embryo patterning, organogenesis and tropisms2–7. Polar PIN localization determines the direction of intercellular auxin flow8, yet the mechanisms generating PIN polarity remain unclear. Here we identify an endocytosis-dependent mechanism of PIN polarity generation and analyse its developmental implications. Real-time PIN tracking showed that after synthesis, PINs are initially delivered to the plasma membrane in a non-polar manner and their polarity is established by subsequent endocytic recycling. Interference with PIN endocytosis either by auxin or by manipulation of the Arabidopsis Rab5 GTPase pathway prevents PIN polarization. Failure of PIN polarization transiently alters asymmetric auxin distribution during embryogenesis and increases the local auxin response in apical embryo regions. This results in ectopic expression of auxin pathway-associated root-forming master regulators in embryonic leaves and promotes homeotic transformation of leaves to roots. Our results indicate a two-step mechanism for the generation of PIN polar localization and the essential role of endocytosis in this process. It also highlights the link between endocytosis-dependent polarity of individual cells and auxin distribution-dependent cell fate establishment for multicellular patterning.
doi:10.1038/nature07409
PMCID: PMC2692841  PMID: 18953331

Results 1-7 (7)