Plant ESCRT protein ALIX coordinates with retromer complex in regulating receptor-mediated sorting of soluble vacuolar proteins.

Vacuolar proteins play essential roles in plant physiology and development, but the factors and the machinery regulating their vesicle trafficking through the endomembrane compartments remain largely unknown. We and others have recently identified an evolutionarily conserved plant endosomal sorting complex required for transport (ESCRT)-associated protein apoptosis-linked gene-2 interacting protein X (ALIX), which plays canonical functions in the biogenesis of the multivesicular body/prevacuolar compartment (MVB/PVC) and in the sorting of ubiquitinated membrane proteins. In this study, we elucidate the roles and underlying mechanism of ALIX in regulating vacuolar transport of soluble proteins, beyond its conventional ESCRT function in eukaryotic cells. We show that ALIX colocalizes and physically interacts with the retromer core subunits Vps26 and Vps29 in planta. Moreover, double-mutant analysis reveals the genetic interaction of ALIX with Vps26 and Vps29 for regulating trafficking of soluble vacuolar proteins. Interestingly, depletion of ALIX perturbs membrane recruitment of Vps26 and Vps29 and alters the endosomal localization of vacuolar sorting receptors (VSRs). Taken together, ALIX functions as a unique retromer core subcomplex regulator by orchestrating receptor-mediated vacuolar sorting of soluble proteins.

The Arabidopsis katamari2 Mutant Exhibits a Hypersensitive Seedling Arrest Response at the Phase Transition from Heterotrophic to Autotrophic Growth.

Young seedlings use nutrients stored in the seeds to grow and acquire photosynthetic potential. This process, called seedling establishment, involves a developmental phase transition from heterotrophic to autotrophic growth. Some membrane-trafficking mutants of Arabidopsis (Arabidopsis thaliana), such as the katamari2 (kam2) mutant, exhibit growth arrest during seedling development, with a portion of individuals failing to develop true leaves on sucrose-free solid medium. However, the reason for this seedling arrest is unclear. In this study, we show that seedling arrest is a temporal growth arrest response that occurs not only in kam2 but also in wild-type (WT) Arabidopsis; however, the threshold for this response is lower in kam2 than in the WT. A subset of the arrested kam2 seedlings resumed growth after transfer to fresh sucrose-free medium. Growth arrest in kam2 on sucrose-free medium was restored by increasing the gel concentration of the medium or covering the surface of the medium with a perforated plastic sheet. WT Arabidopsis seedlings were also arrested when the gel concentration of sucrose-free medium was reduced. RNA sequencing revealed that transcriptomic changes associated with the rate of seedling establishment were observed as early as 4 d after sowing. Our results suggest that the growth arrest of both kam2 and WT seedlings is an adaptive stress response and is not simply caused by the lack of a carbon source in the medium. This study provides a new perspective on an environmental stress response under unfavorable conditions during the phase transition from heterotrophic to autotrophic growth in Arabidopsis.

Fluorescent protein-based imaging and tissue-specific RNA-seq analysis of Arabidopsis hydathodes.

Hydathodes are typically found at leaf teeth in vascular plants and are involved in water release to the outside. Although morphological and physiological analysis of hydathodes has been performed in various plants, little is known about the genes involved in hydathode function. In this study, we performed fluorescent protein-based imaging and tissue-specific RNA-seq analysis in Arabidopsis hydathodes. We used the enhancer trap line E325, which has been reported to express green fluorescent protein (GFP) at its hydathodes. We found that E325-GFP was expressed in small cells found inside the hydathodes (named E cells) that were distributed between the water pores and xylem ends. No fluorescence of the phloem markers pSUC2:GFP and pSEOR1:SEOR1-YFP was observed in the hydathodes. These observations indicate that Arabidopsis hydathodes are composed of three major components: water pores, xylem ends, and E cells. In addition, we performed transcriptome analysis of the hydathode using the E325-GFP line. Microsamples were collected from GFP-positive or -negative regions of E325 leaf margins with a needle-based device (~130 µm in diameter). RNA-seq was performed with each single microsample using a high-throughput library preparation method called Lasy-Seq. We identified 72 differentially expressed genes. Among them, 68 genes showed significantly higher and four genes showed significantly lower expression in the hydathode. Our results provide new insights into the molecular basis for hydathode physiology and development.

The chemical compound bubblin induces stomatal mispatterning in Arabidopsis by disrupting the intrinsic polarity of stomatal lineage cells.

Stem cell polarization is a crucial step in asymmetric cell division, which is a universal system for generating cellular diversity in multicellular organisms. Several conventional genetics studies have attempted to elucidate the mechanisms underlying cell polarization in plants, but it remains largely unknown. In plants, stomata, which are valves for gas exchange, are generated through several rounds of asymmetric divisions. In this study, we identified and characterized a chemical compound that affects stomatal stem cell polarity. High-throughput screening for bioactive molecules identified a pyridine-thiazole derivative, named bubblin, which induced stomatal clustering in Arabidopsis epidermis. Bubblin perturbed stomatal asymmetric division, resulting in the generation of two identical daughter cells. Both cells continued to express the stomatal fate determinant SPEECHLESS, and then differentiated into mispatterned stomata. Bubblin-treated cells had a defect in the polarized localization of BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), which is required for asymmetric cell fate determination. Our results suggest that bubblin induces stomatal lineage cells to divide without BASL-dependent pre-mitotic establishment of polarity. Bubblin is a potentially valuable tool for investigating cell polarity establishment in stomatal asymmetric division.

Structural and functional relationships between plasmodesmata and plant endoplasmic reticulum-plasma membrane contact sites consisting of three synaptotagmins.

Synaptotagmin 1 (SYT1) has been recognised as a tethering factor of plant endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (EPCSs) and partially localises to around plasmodesmata (PD). However, other components of EPCSs associated with SYT1 and functional links between the EPCSs and PD have not been identified. We explored interactors of SYT1 by immunoprecipitation and mass analysis. The dynamics, morphology and spatial arrangement of the ER in Arabidopsis mutants lacking the EPCS components were investigated using confocal microscopy and electron microscopy. PD permeability of EPCS mutants was assessed using a virus movement protein and free green fluorescent protein (GFP) as indicators. We identified two additional components of the EPCSs, SYT5 and SYT7, that interact with SYT1. The mutants of the three SYTs were defective in the anchoring of the ER to the PM. The ER near the PD entrance appeared to be weakly squeezed in the triple mutant compared with the wild-type. The triple mutant suppressed cell-to-cell movement of the virus movement protein, but not GFP diffusion. We revealed major additional components of EPCS associated with SYT1 and suggested that the EPCSs arranged around the PD squeeze the ER to regulate active transport via PD.

Leaf Endoplasmic Reticulum Bodies Identified in Arabidopsis Rosette Leaves Are Involved in Defense against Herbivory.

ER bodies are endoplasmic reticulum (ER)-derived organelles specific to the order Brassicales and are thought to function in plant defense against insects and pathogens. ER bodies are generally classified into two types: constitutive ER bodies in the epidermal cells of seedlings, and wound-inducible ER bodies in rosette leaves. Herein, we reveal a third type of ER body found in Arabidopsis (Arabidopsis thaliana) rosette leaves and designate them "leaf ERbodies" (L-ER bodies). L-ER bodies constitutively occurred in specific cells of the rosette leaves: marginal cells, epidermal cells covering the midrib, and giant pavement cells. The distribution of L-ER bodies was closely associated with the expression profile of the basic helix-loop-helix transcription factor NAI1, which is responsible for constitutive ER-body formation. L-ER bodies were seldom observed in nai1 mutant leaves, indicating that NAI1 is involved in L-ER body formation. Confocal imaging analysis revealed that L-ER bodies accumulated two types of ß-glucosidases: PYK10, the constitutive ER-body ß-glucosidase; and BETA-GLUCOSIDASE18 (BGLU18), the wound-inducible ER-body ß-glucosidase. Combined with the absence of L-ER bodies in the bglu18 pyk10 mutant, these results indicate that BGLU18 and PYK10 are the major components of L-ER bodies. A subsequent feeding assay with the terrestrial isopod Armadillidium vulgare revealed that bglu18 pyk10 leaves were severely damaged as a result of herbivory. In addition, the bglu18 pyk10 mutant was defective in the hydrolysis of 4-methoxyindol-3-ylmethyl glucosinolate These results suggest that L-ER bodies are involved in the production of defensive compound(s) from 4-methoxyindol-3-ylmethyl glucosinolate that protect Arabidopsis leaves against herbivory attack.

Polar Localization of the Borate Exporter BOR1 Requires AP2-Dependent Endocytosis.

Boron (B) is an essential element in plants but is toxic when it accumulates to high levels. In root cells of Arabidopsis (Arabidopsis thaliana), the borate exporter BOR1 is polarly localized in the plasma membrane toward the stele side for directional transport of B. Upon high-B supply, BOR1 is rapidly internalized and degraded in the vacuole. The polar localization and B-induced vacuolar sorting of BOR1 are mediated by endocytosis from the plasma membrane. To dissect the endocytic pathways mediating the polar localization and vacuolar sorting, we investigated the contribution of the clathrin adaptor protein, ADAPTOR PROTEIN2 (AP2) complex, to BOR1 trafficking. In the mutants lacking µ- or σ-subunits of the AP2 complex, the polar localization and constitutive endocytosis of BOR1 under low-B conditions were dramatically disturbed. A coimmunoprecipitation assay showed association of the AP2 complex with BOR1, while it was independent of YxxΦ sorting motifs, which are in a cytosolic loop of BOR1. A yeast two-hybrid assay supported the interaction of the AP2 complex µ-subunit with the C-terminal tail but not with the YxxΦ motifs in the cytosolic loop of BOR1. Intriguingly, lack of the AP2 subunit did not affect the B-induced rapid internalization/vacuolar sorting of BOR1. Consistent with defects in the polar localization, the AP2 complex mutants showed hypersensitivity to B deficiency. Our results indicate that AP2-dependent endocytosis maintains the polar localization of BOR1 to support plant growth under low-B conditions, whereas the B-induced vacuolar sorting of BOR1 is mediated through an AP2-independent endocytic pathway.

Arabidopsis ECHIDNA protein is involved in seed coloration, protein trafficking to vacuoles, and vacuolar biogenesis.

Flavonoids are a major group of plant-specific metabolites that determine flower and seed coloration. In plant cells, flavonoids are synthesized at the cytosolic surface of the endoplasmic reticulum and are sequestered in the vacuole. It is possible that membrane trafficking, including vesicle trafficking and organelle dynamics, contributes to flavonoid transport and accumulation. However, the underlying mechanism has yet to be fully elucidated. Here we show that the Arabidopsis ECHIDNA protein plays a role in flavonoid accumulation in the vacuole and protein trafficking to the vacuole. We found defective pigmentation patterns in echidna seed, possibly caused by reduced levels of proanthocyanidins, which determine seed coloration. The echidna mutant has defects in protein sorting to the protein storage vacuole as well as vacuole morphology. These findings indicate that ECHIDNA is involved in the vacuolar trafficking pathway as well as the previously described secretory pathway. In addition, we found a genetic interaction between echidna and green fluorescent seed 9 (gfs9), a membrane trafficking factor involved in flavonoid accumulation. Our findings suggest that vacuolar trafficking and/or vacuolar development, both of which are collectively regulated by ECHIDNA and GFS9, are required for flavonoid accumulation, resulting in seed coat pigmentation.

Polar Localization of the NIP5;1 Boric Acid Channel Is Maintained by Endocytosis and Facilitates Boron Transport in Arabidopsis Roots.

Boron uptake in Arabidopsis thaliana is mediated by nodulin 26-like intrinsic protein 5;1 (NIP5;1), a boric acid channel that is located preferentially on the soil side of the plasma membrane in root cells. However, the mechanism underlying this polar localization is poorly understood. Here, we show that the polar localization of NIP5;1 in epidermal and endodermal root cells is mediated by the phosphorylation of Thr residues in the conserved TPG (ThrProGly) repeat in the N-terminal region of NIP5;1. Although substitutions of Ala for three Thr residues in the TPG repeat did not affect lateral diffusion in the plasma membrane, these substitutions inhibited endocytosis and strongly compromised the polar localization of GFP-NIP5;1. Consistent with this, the polar localization was compromised in µ subunit mutants of the clathrin adaptor AP2. The Thr-to-Ala substitutions did not affect the boron transport activity of GFP-NIP5;1 in Xenopus laevis oocytes but did inhibit the ability to complement boron translocation to shoots and rescue growth defects in nip5;1-1 mutant plants under boron-limited conditions. These results demonstrate that the polar localization of NIP5;1 is maintained by clathrin-mediated endocytosis, is dependent on phosphorylation in the TPG repeat, and is necessary for the efficient transport of boron in roots.

Identification of Periplasmic Root-Cap Mucilage in Developing Columella Cells of Arabidopsis thaliana.

Plant roots secrete various substances with diverse functions against both plants and microbes in the rhizosphere. A major secretory substance is root-cap mucilage, whose functions have been well characterized, albeit mainly in crops. However, little is currently known about the developmental mechanisms of root-cap mucilage. Here, we show the accumulation and extrusion of root-cap mucilage in Arabidopsis. We found propidium iodide (PI) stainable structures between the plasma membrane and cell wall in the sixth layer of columella cells (c6) from the quiescent center. Ruthenium red staining and PI staining with calcium ions suggested that the structure comprises in part pectin polysaccharides. Electron microscopy revealed that the structure had a meshwork of electron-dense filaments that resembled periplasmic mucilage in other plants. In the c6 cells, we also observed many large vesicles with denser meshwork filaments to periplasmic mucilage, which likely mediate the transport of mucilage components. Extruded mucilage was observed outside a partially degraded cell wall in the c7 cells. Moreover, we found that the Class IIB NAC transcription factors BEARSKIN1 (BRN1) and BRN2, which are known to regulate the terminal differentiation of columella cells, were required for the efficient accumulation of root-cap mucilage in Arabidopsis. Taken together, our findings reveal the accumulation of and dynamic changes in periplasmic mucilage during columella cell development in Arabidopsis.

Synaptotagmin-Associated Endoplasmic Reticulum-Plasma Membrane Contact Sites Are Localized to Immobile ER Tubules.

The plant endoplasmic reticulum (ER), which is morphologically divided into tubules and sheets, seems to flow continuously as a whole, but locally, mobile and immobile regions exist. In eukaryotes, the ER physically and functionally interacts with the plasma membrane (PM) at domains called ER-PM contact sites (EPCSs). Extended synaptotagmin family proteins are concentrated in the cortical ER to form one type of EPCS; however, it is unclear whether the localization of extended synaptotagmin corresponds to the EPCS and where in the cortical ER the EPCSs are formed. Here, we analyzed the spatiotemporal localization of SYNAPTOTAGMIN1 (SYT1), a synaptotagmin in Arabidopsis (Arabidopsis thaliana), to investigate the precise distribution of SYT1-associated EPCSs in the cortical ER. Three-dimensional imaging using superresolution confocal live imaging microscopy demonstrated that SYT1 was specifically localized to the ER-PM boundary. Time-lapse imaging revealed that SYT1 was distributed to immobile ER tubules, but not to mobile tubules. Moreover, SYT1 was frequently localized to the edges of ER sheets that were transformed into immobile ER tubules over time. A lower intracellular calcium ion concentration resulted in an increased EPCS area and disrupted the ER network. Finally, SYT1 deficiency caused a reduction of the immobile tubules and enlargement of the ER meshes. Taken together, our findings show that SYT1-associated EPCS are distributed to immobile tubules and play an important role in the formation of the tubular ER network. This provides important insight into the relationship between the function and dynamics/morphology of the cortical ER.

Involvement of Adapter Protein Complex 4 in Hypersensitive Cell Death Induced by Avirulent Bacteria.

Plant immunity to avirulent bacterial pathogens is associated with subcellular membrane dynamics including fusion between the vacuolar and plasma membranes, resulting in hypersensitive cell death. Here, we report that ADAPTOR PROTEIN COMPLEX-4 (AP-4) subunits are involved in plant immunity associated with hypersensitive cell death. We isolated a mutant with a defect in resistance to an avirulent strain of Pseudomonas syringae pv. tomato (Pto) DC3000 avrRpm1 from a vacuolar protein sorting mutant library of Arabidopsis (Arabidopsis thaliana). The mutant was identical to gfs4-1, which has a mutation in the gene encoding the AP-4 subunit AP4B. Thus, we focused on AP4B and another subunit, AP4E. All of the mutants (ap4b-3, ap4b-4, ap4e-1, and ap4e-2) were defective in hypersensitive cell death and resistance to Pto DC3000 with the type III effector AvrRpm1 or AvrRpt2, both of which are recognized on the plasma membrane, while they showed slightly enhanced susceptibility to the type-III-secretion-deficient P. syringae strain hrcC On the other hand, both ap4b-3 and ap4b-4 showed no defect in resistance to Pto DC3000 with the type III effector AvrRps4, which is recognized in the cytosol and does not induce hypersensitive cell death. Upon infection with Pto DC3000 avrRpt2, the ap4b-3 and ap4b-4 leaf cells did not show fusion between vacuolar and plasma membranes, whereas the wild-type leaf cells did. These results suggest that AP-4 contributes to cell death-associated immunity, possibly via membrane fusion, after type III effector-recognition on the plasma membrane.

Endoplasmic Reticulum (ER) Membrane Proteins (LUNAPARKs) are Required for Proper Configuration of the Cortical ER Network in Plant Cells.

The endoplasmic reticulum (ER) is a large network made of membranous cisternae and tubules, which accounts for a large proportion of the total lipid bilayer endomembrane of the cell. In mammals and yeast, LUNAPARK proteins are preferentially localized at the three-way junctions of the ER network, stabilizing the junctions and establishing the ER architecture. We identified two Arabidopsis homologs and designated them LNPA and LNPB. Subcellular localization analysis with a non-dimerizable type of green fluorescent protein (GFP) revealed that both LNPA and LNPB are predominantly distributed throughout the ER, but not preferentially localized at the three-way junctions. Quantitative analysis of the network in the double mutant lnpa lnpb revealed that deficiency of LNPA and LNPB caused the cortical ER to develop poor ER cisternae and a less dense tubular network. These phenotypes are opposite to those of LNP-deficient mutants of yeast and mammals. Despite the importance of cysteine residues in the zinc finger motif of the yeast LNP homolog (Lnp1p), the corresponding cysteine residues of LNPA were not necessary for the stabilization of ER morphology because replacing the four cysteine residues in the zinc finger motif of the LNPA protein with alanine residues did not affect its function. A significant phenotype of lnpa lnpb is generation of large spherical structures from the ER. Formation of the structures might reduce the amounts of the ER membrane to be used for generating the network, resulting in poor development of the ER network. Taken together, our results suggest that plant LNPs function differently from those in yeast and mammals: they function to distribute ER membranes appropriately throughout the cells.

The AP-1 Complex is Required for Proper Mucilage Formation in Arabidopsis Seeds.

The adaptor protein (AP) complexes play crucial roles in vesicle formation in post-Golgi trafficking. Land plants have five types of AP complexes (AP-1 to AP-5), each of which consists of two large subunits, one medium subunit and one small subunit. Here, we show that the Arabidopsis AP-1 complex mediates the polarized secretion and accumulation of a pectic polysaccharide called mucilage in seed coat cells. Previously, a loss-of-function mutant of AP1M2, the medium subunit of AP-1, has been shown to display deleterious growth defects because of defective cytokinesis. To investigate the function of AP-1 in interphase, we generated transgenic Arabidopsis plants expressing AP1M2-GFP (green fluorescent protein) under the control of the cytokinesis-specific KNOLLE (KN) promoter in the ap1m2 background. These transgenic plants, designated pKN lines, successfully rescued the cytokinesis defect and dwarf phenotype of ap1m2. pKN lines showed reduced mucilage extrusion from the seed coat. Furthermore, abnormal accumulation of mucilage was found in the vacuoles of the outermost integument cells of pKN lines. During seed development, the accumulation of AP1M2-GFP was greatly reduced in the integument cells of pKN lines. These results suggest that trans-Golgi network (TGN)-localized AP-1 is involved in the trafficking of mucilage components to the outer surface of seed coat cells. Our study highlights an evolutionarily conserved function of AP-1 in polarized sorting in eukaryotic cells.

The Adaptor Complex AP-4 Regulates Vacuolar Protein Sorting at the trans-Golgi Network by Interacting with VACUOLAR SORTING RECEPTOR1.

Adaptor protein (AP) complexes play critical roles in protein sorting among different post-Golgi pathways by recognizing specific cargo protein motifs. Among the five AP complexes (AP-1-AP-5) in plants, AP-4 is one of the most poorly understood; the AP-4 components, AP-4 cargo motifs, and AP-4 functional mechanism are not known. Here, we identify the AP-4 components and show that the AP-4 complex regulates receptor-mediated vacuolar protein sorting by recognizing VACUOLAR SORTING RECEPTOR1 (VSR1), which was originally identified as a sorting receptor for seed storage proteins to target protein storage vacuoles in Arabidopsis (Arabidopsis thaliana). From the vacuolar sorting mutant library GREEN FLUORESCENT SEED (GFS), we isolated three gfs mutants that accumulate abnormally high levels of VSR1 in seeds and designated them as gfs4, gfs5, and gfs6. Their responsible genes encode three (AP4B, AP4M, and AP4S) of the four subunits of the AP-4 complex, respectively, and an Arabidopsis mutant (ap4e) lacking the fourth subunit, AP4E, also had the same phenotype. Mass spectrometry demonstrated that these four proteins form a complex in vivo. The four mutants showed defects in the vacuolar sorting of the major storage protein 12S globulins, indicating a role for the AP-4 complex in vacuolar protein transport. AP4M bound to the tyrosine-based motif of VSR1. AP4M localized at the trans-Golgi network (TGN) subdomain that is distinct from the AP-1-localized TGN subdomain. This study provides a novel function for the AP-4 complex in VSR1-mediated vacuolar protein sorting at the specialized domain of the TGN.

Phosphorylation of the C Terminus of RHD3 Has a Critical Role in Homotypic ER Membrane Fusion in Arabidopsis.

The endoplasmic reticulum (ER) consists of dynamically changing tubules and cisternae. In animals and yeast, homotypic ER membrane fusion is mediated by fusogens (atlastin and Sey1p, respectively) that are membrane-associated dynamin-like GTPases. In Arabidopsis (Arabidopsis thaliana), another dynamin-like GTPase, ROOT HAIR DEFECTIVE3 (RHD3), has been proposed as an ER membrane fusogen, but direct evidence is lacking. Here, we show that RHD3 has an ER membrane fusion activity that is enhanced by phosphorylation of its C terminus. The ER network was RHD3-dependently reconstituted from the cytosol and microsome fraction of tobacco (Nicotiana tabacum) cultured cells by exogenously adding GTP, ATP, and F-actin. We next established an in vitro assay system of ER tubule formation with Arabidopsis ER vesicles, in which addition of GTP caused ER sac formation from the ER vesicles. Subsequent application of a shearing force to this system triggered the formation of tubules from the ER sacs in an RHD-dependent manner. Unexpectedly, in the absence of a shearing force, Ser/Thr kinase treatment triggered RHD3-dependent tubule formation. Mass spectrometry showed that RHD3 was phosphorylated at multiple Ser and Thr residues in the C terminus. An antibody against the RHD3 C-terminal peptide abolished kinase-triggered tubule formation. When the Ser cluster was deleted or when the Ser residues were replaced with Ala residues, kinase treatment had no effect on tubule formation. Kinase treatment induced the oligomerization of RHD3. Neither phosphorylation-dependent modulation of membrane fusion nor oligomerization has been reported for atlastin or Sey1p. Taken together, we propose that phosphorylation-stimulated oligomerization of RHD3 enhances ER membrane fusion to form the ER network.

Myrosin cell development is regulated by endocytosis machinery and PIN1 polarity in leaf primordia of Arabidopsis thaliana.

Myrosin cells, which accumulate myrosinase to produce toxic compounds when they are ruptured by herbivores, form specifically along leaf veins in Arabidopsis thaliana. However, the mechanism underlying this pattern formation is unknown. Here, we show that myrosin cell development requires the endocytosis-mediated polar localization of the auxin-efflux carrier PIN1 in leaf primordia. Defects in the endocytic/vacuolar SNAREs (syp22 and syp22 vti11) enhanced myrosin cell development. The syp22 phenotype was rescued by expressing SYP22 under the control of the PIN1 promoter. Additionally, myrosin cell development was enhanced either by lacking the activator of endocytic/vacuolar RAB5 GTPase (VPS9A) or by PIN1 promoter-driven expression of a dominant-negative form of RAB5 GTPase (ARA7). By contrast, myrosin cell development was not affected by deficiencies of vacuolar trafficking factors, including the vacuolar sorting receptor VSR1 and the retromer components VPS29 and VPS35, suggesting that endocytic pathway rather than vacuolar trafficking pathway is important for myrosin cell development. The phosphomimic PIN1 variant (PIN1-Asp), which is unable to be polarized, caused myrosin cells to form not only along leaf vein but also in the intervein leaf area. We propose that Brassicales plants might arrange myrosin cells near vascular cells in order to protect the flux of nutrients and water via polar PIN1 localization.

The Novel Nuclear Envelope Protein KAKU4 Modulates Nuclear Morphology in Arabidopsis.

In animals, the nuclear lamina is a fibrillar meshwork on the inner surface of the nuclear envelope, composed of coiled-coil lamin proteins and lamin binding membrane proteins. Plants also have a meshwork on the inner surface of the nuclear envelope, but little is known about its composition other than the presence of members of the CROWDED NUCLEI (CRWN) protein family, possible plant lamin analogs. Here, we describe a candidate lamina component, based on two Arabidopsis thaliana mutants (kaku2 and kaku4) with aberrant nuclear morphology. The responsible gene in kaku2 encodes CRWN1, and the responsible gene in kaku4 encodes a plant-specific protein of unknown function (KAKU4) that physically interacts with CRWN1 and its homolog CRWN4. Immunogold labeling revealed that KAKU4 localizes at the inner nuclear membrane. KAKU4 deforms the nuclear envelope in a dose-dependent manner, in association with nuclear membrane invagination and stack formation. The KAKU4-dependent nuclear envelope deformation was enhanced by overaccumulation of CRWN1, although KAKU4 can deform the nuclear envelope even in the absence of CRWN1 and/or CRWN4. Together, these results suggest that plants have evolved a unique lamina-like structure to modulate nuclear shape and size.

FAMA is an essential component for the differentiation of two distinct cell types, myrosin cells and guard cells, in Arabidopsis.

Brassicales plants, including Arabidopsis thaliana, have an ingenious two-compartment defense system, which sequesters myrosinase from the substrate glucosinolate and produces a toxic compound when cells are damaged by herbivores. Myrosinase is stored in vacuoles of idioblast myrosin cells. The molecular mechanism that regulates myrosin cell development remains elusive. Here, we identify the basic helix-loop-helix transcription factor FAMA as an essential component for myrosin cell development along Arabidopsis leaf veins. FAMA is known as a regulator of stomatal development. We detected FAMA expression in myrosin cell precursors in leaf primordia in addition to stomatal lineage cells. FAMA deficiency caused defects in myrosin cell development and in the biosynthesis of myrosinases THIOGLUCOSIDE GLUCOHYDROLASE1 (TGG1) and TGG2. Conversely, ectopic FAMA expression conferred myrosin cell characteristics to hypocotyl and root cells, both of which normally lack myrosin cells. The FAMA interactors ICE1/SCREAM and its closest paralog SCREAM2/ICE2 were essential for myrosin cell development. DNA microarray analysis identified 32 candidate genes involved in myrosin cell development under the control of FAMA. This study provides a common regulatory pathway that determines two distinct cell types in leaves: epidermal guard cells and inner-tissue myrosin cells.

A direction-selective local-thresholding method, DSLT, in combination with a dye-based method for automated three-dimensional segmentation of cells and airspaces in developing leaves.

Quantifying the anatomical data acquired from three-dimensional (3D) images has become increasingly important in recent years. Visualization and image segmentation are essential for acquiring accurate and detailed anatomical data from images; however, plant tissues such as leaves are difficult to image by confocal or multi-photon laser scanning microscopy because their airspaces generate optical aberrations. To overcome this problem, we established a staining method based on Nile Red in silicone-oil solution. Our staining method enables color differentiation between lipid bilayer membranes and airspaces, while minimizing any damage to leaf development. By repeated applications of our staining method we performed time-lapse imaging of a leaf over 5 days. To counteract the drastic decline in signal-to-noise ratio at greater tissue depths, we also developed a local thresholding method (direction-selective local thresholding, DSLT) and an automated iterative segmentation algorithm. The segmentation algorithm uses the DSLT to extract the anatomical structures. Using the proposed methods, we accurately segmented 3D images of intact leaves to single-cell resolution, and measured the airspace volumes in intact leaves.