The SYP123-VAMP727 SNARE complex delivers secondary cell wall components for root hair shank hardening in Arabidopsis.

The extended tubular shape of root hairs is established by tip growth and concomitant hardening. Here, we demonstrate that a syntaxin of plants (SYP)123-vesicle-associated membrane protein (VAMP)727-dependent secretion system delivers secondary cell wall components for hardening the subapical zone and shank of Arabidopsis (Arabidopsis thaliana) root hairs. We found increased SYP123 localization at the plasma membrane (PM) of the subapical and shank zones compared with the tip region in elongating root hairs. Inhibition of phosphatidylinositol (PtdIns)(3,5)P2 production impaired SYP123 localization at the PM and SYP123-mediated root hair shank hardening. Moreover, root hair elongation in the syp123 mutant was insensitive to a PtdIns(3,5)P2 synthesis inhibitor. SYP123 interacts with both VAMP721 and VAMP727. syp123 and vamp727 mutants exhibited reduced shank cell wall stiffness due to impaired secondary cell wall component deposition. Based on these results, we conclude that SYP123 is involved in VAMP721-mediated conventional secretion for root hair elongation as well as in VAMP727-mediated secretory functions for the delivery of secondary cell wall components to maintain root hair tubular morphology.

Remodeling of organelles and microtubules during spermiogenesis in the liverwort Marchantia polymorpha.

Gametogenesis is an essential event for sexual reproduction in various organisms. Bryophytes employ motile sperm (spermatozoids) as male gametes, which locomote to the egg cells to accomplish fertilization. The spermatozoids of bryophytes harbor distinctive morphological characteristics, including a cell body with a helical shape and two flagella. During spermiogenesis, the shape and cellular contents of the spermatids are dynamically reorganized. However, the reorganization patterns of each organelle remain obscure. In this study, we classified the developmental processes during spermiogenesis in the liverwort Marchantia polymorpha according to changes in cellular and nuclear shapes and flagellar development. We then examined the remodeling of microtubules and the reorganization of endomembrane organelles. The results indicated that the state of glutamylation of tubulin changes during formation of the flagella and spline. We also found that the plasma membrane and endomembrane organelles are drastically reorganized in a precisely regulated manner, which involves the functions of endosomal sorting complexes required for transport (ESCRT) machineries in endocytic and vacuolar transport. These findings are expected to provide useful indices to classify developmental and subcellular processes of spermiogenesis in bryophytes.

Longin R-SNARE is retrieved from the plasma membrane by ANTH domain-containing proteins in Arabidopsis.

The plasma membrane (PM) acts as the interface between intra- and extracellular environments and exhibits a tightly regulated molecular composition. The composition and amount of PM proteins are regulated by balancing endocytic and exocytic trafficking in a cargo-specific manner, according to the demands of specific cellular states and developmental processes. In plant cells, retrieval of membrane proteins from the PM depends largely on clathrin-mediated endocytosis (CME). However, the mechanisms for sorting PM proteins during CME remain ambiguous. In this study, we identified a homologous pair of ANTH domain-containing proteins, PICALM1a and PICALM1b, as adaptor proteins for CME of the secretory vesicle-associated longin-type R-SNARE VAMP72 group. PICALM1 interacted with the SNARE domain of VAMP72 and clathrin at the PM. The loss of function of PICALM1 resulted in faulty retrieval of VAMP72, whereas general endocytosis was not considerably affected by this mutation. The double mutant of PICALM1 exhibited impaired vegetative development, indicating the requirement of VAMP72 recycling for normal plant growth. In the mammalian system, VAMP7, which is homologous to plant VAMP72, is retrieved from the PM via the interaction with a clathrin adaptor HIV Rev-binding protein in the longin domain during CME, which is not functional in the plant system, whereas retrieval of brevin-type R-SNARE members is dependent on a PICALM1 homolog. These results indicate that ANTH domain-containing proteins have evolved to be recruited distinctly for recycling R-SNARE proteins and are critical to eukaryote physiology.

Phylogenetic distribution and expression pattern analyses identified a divergent basal body assembly protein involved in land plant spermatogenesis.

Land plant spermatozoids commonly possess characteristic structures such as the spline, which consists of a microtubule array, the multilayered structure (MLS) in which the uppermost layer is a continuum of the spline, and multiple flagella. However, the molecular mechanisms underpinning spermatogenesis remain to be elucidated. We successfully identified candidate genes involved in spermatogenesis, deeply divergent BLD10s, by computational analyses combining multiple methods and omics data. We then examined the functions of BLD10s in the liverwort Marchantia polymorpha and the moss Physcomitrium patens. MpBLD10 and PpBLD10 are required for normal basal body (BB) and flagella formation. Mpbld10 mutants exhibited defects in remodeling of the cytoplasm and nucleus during spermatozoid formation, and thus MpBLD10 should be involved in chromatin reorganization and elimination of the cytoplasm during spermiogenesis. We identified orthologs of MpBLD10 and PpBLD10 in diverse Streptophyta and found that MpBLD10 and PpBLD10 are orthologous to BLD10/CEP135 family proteins, which function in BB assembly. However, BLD10s evolved especially quickly in land plants and MpBLD10 might have acquired additional functions in spermatozoid formation through rapid molecular evolution.

Distinct sets of tethering complexes, SNARE complexes, and Rab GTPases mediate membrane fusion at the vacuole in Arabidopsis.

Membrane trafficking plays pivotal roles in various cellular activities and higher-order functions of eukaryotes and requires tethering factors to mediate contact between transport intermediates and target membranes. Two evolutionarily conserved tethering complexes, homotypic fusion and protein sorting (HOPS) and class C core vacuole/endosome tethering (CORVET), are known to act in endosomal/vacuolar transport in yeast and animals. Both complexes share a core subcomplex consisting of Vps11, Vps18, Vps16, and Vps33, and in addition to this core, HOPS contains Vps39 and Vps41, whereas CORVET contains Vps3 and Vps8. HOPS and CORVET subunits are also conserved in the model plant Arabidopsis. However, vacuolar trafficking in plants occurs through multiple unique transport pathways, and how these conserved tethering complexes mediate endosomal/vacuolar transport in plants has remained elusive. In this study, we investigated the functions of VPS18, VPS3, and VPS39, which are core complex, CORVET-specific, and HOPS-specific subunits, respectively. Impairment of these tethering proteins resulted in embryonic lethality, distinctly altering vacuolar morphology and perturbing transport of a vacuolar membrane protein. CORVET interacted with canonical RAB5 and a plant-specific R-soluble NSF attachment protein receptor (SNARE), VAMP727, which mediates fusion between endosomes and the vacuole, whereas HOPS interacted with RAB7 and another R-SNARE, VAMP713, which likely mediates homotypic vacuolar fusion. These results indicate that CORVET and HOPS act in distinct vacuolar trafficking pathways in plant cells, unlike those of nonplant systems that involve sequential action of these tethering complexes during vacuolar/lysosomal trafficking. These results highlight a unique diversification of vacuolar/lysosomal transport that arose during plant evolution, using evolutionarily conserved tethering components.

Transcriptional switch for programmed cell death in pith parenchyma of sorghum stems.

Pith parenchyma cells store water in various plant organs. These cells are especially important for producing sugar and ethanol from the sugar juice of grass stems. In many plants, the death of pith parenchyma cells reduces their stem water content. Previous studies proposed that a hypothetical D gene might be responsible for the death of stem pith parenchyma cells in Sorghum bicolor, a promising energy grass, although its identity and molecular function are unknown. Here, we identify the D gene and note that it is located on chromosome 6 in agreement with previous predictions. Sorghum varieties with a functional D allele had stems enriched with dry, dead pith parenchyma cells, whereas those with each of six independent nonfunctional D alleles had stems enriched with juicy, living pith parenchyma cells. D expression was spatiotemporally coupled with the appearance of dead, air-filled pith parenchyma cells in sorghum stems. Among D homologs that are present in flowering plants, Arabidopsis ANAC074 also is required for the death of stem pith parenchyma cells. D and ANAC074 encode previously uncharacterized NAC transcription factors and are sufficient to ectopically induce programmed death of Arabidopsis culture cells via the activation of autolytic enzymes. Taken together, these results indicate that D and its Arabidopsis ortholog, ANAC074, are master transcriptional switches that induce programmed death of stem pith parenchyma cells. Thus, targeting the D gene will provide an approach to breeding crops for sugar and ethanol production.

Deep Imaging Analysis in VISUAL Reveals the Role of YABBY Genes in Vascular Stem Cell Fate Determination.

Stem cells undergo cell division and differentiation to ensure organized tissue development. Because plant cells are immobile, plant stem cells ought to decide their cell fate prior to differentiation, to locate specialized cells in the correct position. In this study, based on a chemical screen, we isolated a novel secondary cell wall indicator BF-170, which binds to lignin and can be used to image in vitro and in situ xylem development. Use of BF-170 to observe the vascular differentiation pattern in the in vitro vascular cell induction system, VISUAL, revealed that adaxial mesophyll cells of cotyledons predominantly generate ectopic xylem cells. Moreover, phloem cells are abundantly produced on the abaxial layer, suggesting the involvement of leaf adaxial-abaxial polarity in determining vascular cell fate. Analysis of abaxial polarity mutants highlighted the role of YAB3, an abaxial cell fate regulator, in suppressing xylem and promoting phloem differentiation on the abaxial domains in VISUAL. Furthermore, YABBY family genes affected in vivo vascular development during the secondary growth. Our results denoted the possibility that such mediators of spatial information contribute to correctly determine the cell fate of vascular stem cells, to conserve the vascular pattern of land plants.

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.

RAB GTPases in the Basal Land Plant Marchantia polymorpha.

The RAB GTPase is an evolutionarily conserved machinery component of membrane trafficking, which is the fundamental system for cell viability and higher order biological functions. The composition of RAB GTPases in each organism is closely related to the complexity and organization of the membrane trafficking pathway, which has been developed uniquely to realize the organism-specific membrane trafficking system. Comparative genomics has suggested that terrestrialization and/or multicellularization were associated with the expansion of membrane trafficking pathways in green plants, which has yet to be validated in basal land plant lineages. To obtain insight into the diversification of membrane trafficking systems in green plants, we analyzed RAB GTPases encoded in the genome of the liverwort Marchantia polymorpha in a comprehensive manner. We isolated all genes for RAB GTPases in Marchantia and analyzed their expression patterns and subcellular localizations in thallus cells. While a majority of MpRAB GTPases exhibited a ubiquitous expression pattern, specific exceptions were also observed; MpRAB2b, which contains a sequence similar to an intraflagellar transport protein at the C-terminal region; and MpRAB23, which has been secondarily lost in angiosperms, were specifically expressed in the male reproductive organ. MpRAB21, which is another RAB GTPase whose homolog is absent in Arabidopsis, exhibited endosomal localization with RAB5 members in Marchantia. These results suggest that Marchantia possesses unique membrane trafficking pathways involving a unique repertoire of RAB GTPases.

Triphosphate Tunnel Metalloenzyme Function in Senescence Highlights a Biological Diversification of This Protein Superfamily.

The triphosphate tunnel metalloenzyme (TTM) superfamily comprises a group of enzymes that hydrolyze organophosphate substrates. They exist in all domains of life, yet the biological role of most family members is unclear. Arabidopsis (Arabidopsis thaliana) encodes three TTM genes. We have previously reported that AtTTM2 displays pyrophosphatase activity and is involved in pathogen resistance. Here, we report the biochemical activity and biological function of AtTTM1 and diversification of the biological roles between AtTTM1 and 2 Biochemical analyses revealed that AtTTM1 displays pyrophosphatase activity similar to AtTTM2, making them the only TTMs characterized so far to act on a diphosphate substrate. However, knockout mutant analysis showed that AtTTM1 is not involved in pathogen resistance but rather in leaf senescence. AtTTM1 is transcriptionally up-regulated during leaf senescence, and knockout mutants of AtTTM1 exhibit delayed dark-induced and natural senescence. The double mutant of AtTTM1 and AtTTM2 did not show synergistic effects, further indicating the diversification of their biological function. However, promoter swap analyses revealed that they functionally can complement each other, and confocal microscopy revealed that both proteins are tail-anchored proteins that localize to the mitochondrial outer membrane. Additionally, transient overexpression of either gene in Nicotiana benthamiana induced senescence-like cell death upon dark treatment. Taken together, we show that two TTMs display the same biochemical properties but distinct biological functions that are governed by their transcriptional regulation. Moreover, this work reveals a possible connection of immunity-related programmed cell death and senescence through novel mitochondrial tail-anchored proteins.

Modulation of Plant RAB GTPase-Mediated Membrane Trafficking Pathway at the Interface Between Plants and Obligate Biotrophic Pathogens.

RAB5 is a small GTPase that acts in endosomal trafficking. In addition to canonical RAB5 members that are homologous to animal RAB5, land plants harbor a plant-specific RAB5, the ARA6 group, which regulates trafficking events distinct from canonical RAB5 GTPases. Here, we report that plant RAB5, both canonical and plant-specific members, accumulate at the interface between host plants and biotrophic fungal and oomycete pathogens. Biotrophic fungi and oomycetes colonize living plant tissues by establishing specialized infection hyphae, the haustorium, within host plant cells. We found that Arabidopsis thaliana ARA6/RABF1, a plant-specific RAB5, is localized to the specialized membrane that surrounds the haustorium, the extrahaustorial membrane (EHM), formed by the A. thaliana-adapted powdery mildew fungus Golovinomyces orontii Whereas the conventional RAB5 ARA7/RABF2b was also localized to the EHM, endosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and RAB5-activating proteins were not, which suggests that the EHM has modified endosomal characteristic. The recruitment of host RAB5 to the EHM was a property shared by the barley-adapted powdery mildew fungus Blumeria graminis f.sp. hordei and the oomycete Hyaloperonospora arabidopsidis, but the extrahyphal membrane surrounding the hypha of the hemibiotrophic fungus Colletotrichum higginsianum at the biotrophic stage was devoid of RAB5. The localization of RAB5 to the EHM appears to correlate with the functionality of the haustorium. Our discovery sheds light on a novel relationship between plant RAB5 and obligate biotrophic pathogens.

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.

Plasmodium Rab5b is secreted to the cytoplasmic face of the tubovesicular network in infected red blood cells together with N-acylated adenylate kinase 2.

BACKGROUND: Rab5 GTPase regulates membrane trafficking between the plasma membrane and endosomes and harbours a conserved C-terminal isoprenyl modification that is necessary for membrane recruitment. Plasmodium falciparum encodes three Rab5 isotypes, and one of these, Rab5b (PfRab5b), lacks the C-terminal modification but possesses the N-terminal myristoylation motif. PfRab5b was reported to localize to the parasite periphery. However, the trafficking pathway regulated by PfRab5b is unknown. METHODS: A complementation analysis of Rab5 isotypes was performed in Plasmodium berghei. A constitutively active PfRab5b mutant was expressed under the regulation of a ligand-dependent destabilization domain (DD)-tag system in P. falciparum. The localization of PfRab5b was evaluated after removing the ligand followed by selective permeabilization of the membrane with different detergents. Furthermore, P. falciparum N-terminally myristoylated adenylate kinase 2 (PfAK2) was co-expressed with PfRab5b, and trafficking of PfAK2 to the parasitophorous vacuole membrane was examined by confocal microscopy. RESULTS: PfRab5b complemented the function of PbRab5b, however, the conventional C-terminally isoprenylated Rab5, PbRab5a or PbRab5c, did not. The constitutively active PfRab5b mutant localized to the cytosol of the parasite and the tubovesicular network (TVN), a region that extends from the parasitophorous vacuole membrane (PVM) in infected red blood cells (iRBCs). By removing the DD-ligand, parasite cytosolic PfRab5b signal disappeared and a punctate structure adjacent to the endoplasmic reticulum (ER) and parasite periphery accumulated. The peripheral PfRab5b was sensitive to extracellular proteolysis after treatment with streptolysin O, which selectively permeabilizes the red blood cell plasma membrane, indicating that PfRab5b localized on the iRBC cytoplasmic face of the TVN. Transport of PfAK2 to the PVM was abrogated by overexpression of PfRab5b, and PfAK2 accumulated in the punctate structure together with PfRab5b. CONCLUSION: N-myristoylated Plasmodium Rab5b plays a role that is distinct from that of conventional mammalian Rab5 isotypes. PfRab5b localizes to a compartment close to the ER, translocated to the lumen of the organelle, and co-localizes with PfAK2. PfRab5b and PfAK2 are then transported to the TVN, and PfRab5b localizes on the iRBC cytoplasmic face of TVN. These data demonstrate that PfRab5b is transported from the parasite cytosol to TVN together with N-myristoylated PfAK2 via an uncharacterized membrane-trafficking pathway.

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.

Interactomics of Qa-SNARE in Arabidopsis thaliana.

Membrane trafficking in plants is involved in cellular development and the adaptation to various environmental changes. SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) proteins mediate the fusion between vesicles and organelles to facilitate transport cargo proteins in cells. To characterize further the SNARE protein networks in cells, we carried out interactome analysis of SNARE proteins using 12 transgenic Arabidopsis thaliana plants expressing green fluorescent protein (GFP)-tagged Qa-SNAREs (SYP111, SYP121, SYP122, SYP123, SYP132, SYP21, SYP22, SYP31, SYP32, SYP41, SYP42 and SYP43). Microsomal fractions were prepared from each transgenic root, and subjected to immunoprecipitation (IP) using micromagnetic beads coupled to anti-GFP antibodies. To identify Qa-SNARE-interacting proteins, all immunoprecipitated products were then subjected to mass spectrometric (IP-MS) analysis. The IP-MS data revealed not only known interactions of SNARE proteins, but also unknown interactions. The IP-MS results were next categorized by gene ontology analysis. The data revealed that categories of cellular component organization, the cytoskeleton and endosome were enriched in the SYP2, SYP3 and SYP4 groups. In contrast, transporter activity was classified specifically in the SYP132 group. We also identified a novel interaction between SYP22 and VAMP711, which was validated using co-localization analysis with confocal microscopy and IP. Additional novel SNARE-interacting proteins play roles in vesicle transport and lignin biosynthesis, and were identified as membrane microdomain-related proteins. We propose that Qa-SNARE interactomics is useful for understanding SNARE interactions across the whole cell.

Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis.

Cytokinesis represents the final stage of eukaryotic cell division during which the cytoplasm becomes partitioned between daughter cells. The process differs to some extent between animal and plant cells, but proteins of the syntaxin family mediate membrane fusion in the plane of cell division in diverse organisms. How syntaxin localization is kept in check remains elusive. Here, we report that localization of the Arabidopsis KNOLLE syntaxin in the plane of cell division is maintained by sterol-dependent endocytosis involving a clathrin- and DYNAMIN-RELATED PROTEIN1A-dependent mechanism. On genetic or pharmacological interference with endocytosis, KNOLLE mis-localizes to lateral plasma membranes after cell-plate fusion. Fluorescence-loss-in-photo-bleaching and fluorescence-recovery-after-photo-bleaching experiments reveal lateral diffusion of GFP-KNOLLE from the plane of division to lateral membranes. In an endocytosis-defective sterol biosynthesis mutant displaying lateral KNOLLE diffusion, KNOLLE secretory trafficking remains unaffected. Thus, restriction of lateral diffusion by endocytosis may serve to maintain specificity of syntaxin localization during late cytokinesis.

Generation of cell polarity in plants links endocytosis, auxin distribution and cell fate decisions.

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 family are landmarks of cell polarity in plants and have a crucial involvement in auxin distribution-dependent development including embryo patterning, organogenesis and tropisms. Polar PIN localization determines the direction of intercellular auxin flow, 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.

Dynamic behavior of clathrin in Arabidopsis thaliana unveiled by live imaging.

Clathrin-coated vesicles (CCV) are necessary for selective transport events, including receptor-mediated endocytosis on the plasma membrane and cargo molecule sorting in the trans-Golgi network (TGN). Components involved in CCV formation include clathrin heavy and light chains and several adaptor proteins that are conserved among plants. Clathrin-dependent endocytosis has been shown to play an integral part in plant endocytosis. However, little information is known about clathrin dynamics in living plant cells. In this study, we have visualized clathrin in Arabidopsis thaliana by tagging clathrin light chain with green fluorescent protein (CLC-GFP). Quantitative evaluations of colocalization demonstrate that the majority of CLC-GFP is localized to the TGN, and a minor population is associated with multivesicular endosomes and the Golgi trans-cisternae. Live imaging further demonstrated the presence of highly dynamic clathrin-positive tubules and vesicles, which appeared to mediate interactions between the TGNs. CLC-GFP is also targeted to cell plates and the plasma membrane. Although CLC-GFP colocalizes with a dynamin isoform at the plasma membrane, these proteins exhibit distinct distributions at newly forming cell plates. This finding indicates independent functions of CLC (clathrin light chains) and dynamin during the formation of cell plates. We have also found that brefeldin A and wortmannin treatment causes distinctly different alterations in the dynamics and distribution of clathrin-coated domains at the plasma membrane. This could account for the different effects of these drugs on plant endocytosis.

A bHLH heterodimer regulates germ cell differentiation in land plant gametophytes.

Land plants are a monophyletic group of photosynthetic eukaryotes that diverged from streptophyte algae about 470 million years ago. During both the alternating haploid and diploid stages of the life cycle, land plants form multicellular bodies.1,2,3,4 The haploid multicellular body (gametophyte) produces progenitor cells that give rise to gametes and the reproductive organs.5,6,7,8 In the liverwort Marchantia polymorpha, differentiation of the initial cells of gamete-producing organs (gametangia) from the gametophyte is regulated by MpBONOBO (MpBNB), a member of the basic helix-loop-helix (bHLH) transcription factor subfamily VIIIa. In Arabidopsis thaliana, specification of generative cells in developing male gametophytes (pollen) requires redundant action of BNB1 and BNB2.9 Subfamily XI bHLHs, such as LOTUS JAPONICUS ROOTHAIRLESS LIKE1 (LRL1)/DEFECTIVE REGION OF POLLEN1 (DROP1) and LRL2/DROP2 in A. thaliana and the single LRL/DROP protein MpLRL in M. polymorpha, are the evolutionarily conserved regulators of rooting system development.10 Although the role of LRL1/DROP1 and LRL2/DROP2 in gametogenesis remains unclear, their loss leads to the formation of abnormal pollen devoid of sperm cells.11 Here, we show that BNBs and LRL/DROPs co-localize to gametophytic cell nuclei and form heterodimers. LRL1/DROP1 and LRL2/DROP2 act redundantly to regulate BNB expression for generative cell specification in A. thaliana after asymmetric division of the haploid microspore. MpLRL is required for differentiation of MpBNB-expressing gametangium initial cells in M. polymorpha gametophytes. Our findings suggest that broadly expressed LRL/DROP stabilizes BNB expression, leading to the formation of an evolutionarily conserved bHLH heterodimer, which regulates germ cell differentiation in the haploid gametophyte of land plants.

Genetic identification of Arabidopsis RID2 as an essential factor involved in pre-rRNA processing.

A temperature-sensitive mutant of Arabidopsis, root initiation defective 2-1 (rid2-1), is characterized by peculiar defects in callus formation. To gain insights into the requirements for the reactivation of cell division, we analyzed this mutant and isolated the gene responsible, RID2. The phenotypes of rid2-1 in tissue culture and in seedlings indicated that the rid2 mutation has various (acute and non-acute) inhibitory effects on different aspects of cell proliferation. This suggests that the RID2 function is not directly involved in every cycle of cell division, but is related to 'vitality', supporting cell proliferation. The rid2-1 mutation was shown to cause nucleolar vacuolation and excessive accumulation of various intermediates of pre-rRNA processing. Positional cloning of the RID2 gene revealed that it encodes an evolutionarily conserved methyltransferase-like protein, which was found to localize in the nucleus, with accumulation being most evident in the nucleolus. It can be inferred from these findings that RID2 contributes to the nucleolar activity for pre-rRNA processing, probably through some methylation reaction.