ARGONAUTE1-binding Tudor domain proteins function in small interfering RNA production for RNA-directed DNA methylation.

Transposable elements (TEs) contribute to plant evolution, development, and adaptation to environmental changes, but the regulatory mechanisms are largely unknown. RNA-directed DNA methylation (RdDM) is 1 TE regulatory mechanism in plants. Here, we identified that novel ARGONAUTE 1 (AGO1)-binding Tudor domain proteins Precocious dissociation of sisters C/E (PDS5C/E) are involved in 24-nt siRNA production to establish RdDM on TEs in Arabidopsis thaliana. PDS5 family proteins are subunits of the eukaryote-conserved cohesin complex. However, the double mutant lacking angiosperm-specific subfamily PDS5C and PDS5E (pds5c/e) exhibited different developmental phenotypes and transcriptome compared with those of the double mutant lacking eukaryote-conserved subfamily PDS5A and PDS5B (pds5a/b), suggesting that the angiosperm-specific PDS5C/E subfamily has a unique function in angiosperm plants. Proteome and imaging analyses revealed that PDS5C/E interact with AGO1. The pds5c/e double mutant had defects in 24-nt siRNA accumulation and CHH DNA methylation on TEs. In addition, some lncRNAs that accumulated in the pds5c/e mutant were targeted by AGO1-loading 21-nt miRNAs and 21-nt siRNAs. These results indicate that PDS5C/E and AGO1 participate in 24-nt siRNA production for RdDM in the cytoplasm. These findings indicate that angiosperm plants evolved a new regulator, the PDS5C/E subfamily, to control the increase in TEs during angiosperm evolution.

A rich and bountiful harvest: Key discoveries in plant cell biology.

The field of plant cell biology has a rich history of discovery, going back to Robert Hooke's discovery of cells themselves. The development of microscopes and preparation techniques has allowed for the visualization of subcellular structures, and the use of protein biochemistry, genetics, and molecular biology has enabled the identification of proteins and mechanisms that regulate key cellular processes. In this review, seven senior plant cell biologists reflect on the development of this research field in the past decades, including the foundational contributions that their teams have made to our rich, current insights into cell biology. Topics covered include signaling and cell morphogenesis, membrane trafficking, cytokinesis, cytoskeletal regulation, and cell wall biology. In addition, these scientists illustrate the pathways to discovery in this exciting research field.

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.

Aerial (+)-borneol modulates root morphology, auxin signalling and meristematic activity in Arabidopsis roots.

One of the characteristic aspects of odour sensing in humans is the activation of olfactory receptors in a slightly different manner in response to different enantiomers. Here, we focused on whether plants showed enantiomer-specific response similar to that in humans. We exposed Arabidopsis seedlings to methanol (control) and (+)- or (-)-borneol, and found that only (+)-borneol reduced the root length. Furthermore, the root-tip width was more increased upon (+)-borneol exposure than upon (-)-borneol exposure. In addition, root-hair formation was observed near the root tip in response to (+)-borneol. Auxin signalling was strongly reduced in the root tip following exposure to (+)-borneol, but was detected following exposure to (-)-borneol and methanol. Similarly, in the root tip, the activity of cyclin B1:1 was detected on exposure to (-)-borneol and methanol, but not on exposure to (+)-borneol, indicating that (+)-borneol inhibits the meristematic activity in the root. These results partially explain the (+)-borneol-specific reduction in the root length of Arabidopsis. Our results indicate the presence of a sensing system specific for (+)-borneol in Arabidopsis.

Galactoglucomannan structure of Arabidopsis seed-coat mucilage in GDP-mannose synthesis impaired mutants.

Cell-wall polysaccharides are synthesized from nucleotide sugars by glycosyltransferases. However, in what way the level of nucleotide sugars affects the structure of the polysaccharides is not entirely clear. guanosine diphosphate (GDP)-mannose (GDP-Man) is one of the major nucleotide sugars in plants and serves as a substrate in the synthesis of mannan polysaccharides. GDP-Man is synthesized from mannose 1-phosphate and GTP by a GDP-Man pyrophosphorylase, VITAMIN C DEFECTIVE1 (VTC1), which is positively regulated by the interacting protein KONJAC1 (KJC1) in Arabidopsis. Since seed-coat mucilage can serve as a model of the plant cell wall, we examined the influence of vtc1 and kjc1 mutations on the synthesis of mucilage galactoglucomannan. Sugar composition analysis showed that mannose content in adherent mucilage of kjc1 and vtc1 mutants was only 42% and 11% of the wild-type, respectively, indicating a drastic decrease of galactoglucomannan. On the other hand, structural analysis based on specific oligosaccharides released by endo-ß-1,4-mannanase indicated that galactoglucomannan had a patterned glucomannan backbone consisting of alternating residues of glucose and mannose and the frequency of α-galactosyl branches was also similar to the wild type structure. These results suggest that the structure of mucilage galactoglucomannan is mainly determined by properties of glycosyltransferases rather than the availability of nucleotide sugars.

Arabidopsis FLYING SAUCER 2 Functions Redundantly with FLY1 to Establish Normal Seed Coat Mucilage.

Following exposure to water, mature Arabidopsis seeds are surrounded by a gelatinous capsule, termed mucilage. The mucilage consists of pectin-rich polysaccharides, which are produced in epidermal cells of the seed coat. Although pectin is a major component of plant cell walls, its biosynthesis and biological functions are not fully understood. Previously, we reported that a transmembrane RING E3 ubiquitin ligase, FLYING SAUCER 1 (FLY1) regulates the degree of pectin methyl esterification for mucilage capsule formation. The Arabidopsis thaliana genome has a single FLY1 homolog, FLY2. In this study, we show that the FLY2 protein functions in mucilage modification together with FLY1. FLY2 was expressed in seed coat epidermal cells during mucilage synthesis, but its expression level was much lower than that of FLY1. While fly2 showed no obvious difference in mucilage capsule formation from wild type, the fly1 fly2 double mutants showed more severe defects in mucilage than fly1 alone. FLY2-EYFP that was expressed under the control of the FLY1 promoter rescued fly1 mucilage, showing that FLY2 has the same molecular function as FLY1. FLY2-EYFP colocalized with marker proteins of Golgi apparatus (sialyltransferase-mRFP) and late endosome (mRFP-ARA7), indicating that as FLY1, FLY2 controls pectin modification by functioning in these endomembrane organelles. Furthermore, phylogenetic analysis suggests that FLY1 and FLY2 originated from a common ancestral gene by gene duplication prior to the emergence of Brassicaceae. Taken together, our findings suggest that FLY2 functions in the Golgi apparatus and/or the late endosome of seed coat epidermal cells in a manner similar to FLY1.

NAI2 and TSA1 Drive Differentiation of Constitutive and Inducible ER Body Formation in Brassicaceae.

Brassicaceae and closely related species develop unique endoplasmic reticulum (ER)-derived structures called ER bodies, which accumulate ß-glucosidases/myrosinases that are involved in chemical defense. There are two different types of ER bodies: ER bodies constitutively present in seedlings (cER bodies) and ER bodies in rosette leaves induced by treatment with the wounding hormone jasmonate (JA) (iER bodies). Here, we show that At-α whole-genome duplication (WGD) generated the paralogous genes NAI2 and TSA1, which consequently drive differentiation of cER bodies and iER bodies in Brassicaceae plants. In Arabidopsis, NAI2 is expressed in seedlings where cER bodies are formed, whereas TSA1 is expressed in JA-treated leaves where iER bodies are formed. We found that the expression of NAI2 in seedlings and the JA inducibility of TSA1 are conserved across other Brassicaceae plants. The accumulation of NAI2 transcripts in Arabidopsis seedlings is dependent on the transcription factor NAI1, whereas the JA induction of TSA1 in rosette leaves is dependent on MYC2, MYC3 and MYC4. We discovered regions of microsynteny, including the NAI2/TSA1 genes, but the promoter regions are differentiated between TSA1 and NAI2 genes in Brassicaceae. This suggests that the divergence of function between NAI2 and TSA1 occurred immediately after WGD in ancestral Brassicaceae plants to differentiate the formation of iER and cER bodies. Our findings indicate that At-α WGD enabled diversification of defense strategies, which may have contributed to the massive diversification of Brassicaceae plants.

Stress granule formation is induced by a threshold temperature rather than a temperature difference in Arabidopsis.

Stress granules, a type of cytoplasmic RNA granule in eukaryotic cells, are induced in response to various environmental stresses, including high temperature. However, how high temperatures induce the formation of these stress granules in plant cells is largely unknown. Here, we characterized the process of stress granule formation in Arabidopsis thaliana by combining live imaging and electron microscopy analysis. In seedlings grown at 22°C, stress granule formation was induced at temperatures above a critical threshold level of 34°C in the absence of transpiration. The threshold temperature was the same, regardless of whether the seedlings were grown at 22°C or 4°C. High-resolution live imaging microscopy revealed that stress granule formation is not correlated with the sizes of pre-existing RNA processing bodies (P-bodies) but that the two structures often associated rapidly. Immunoelectron microscopy revealed a previously unidentified characteristic of the fine structures of Arabidopsis stress granules and P-bodies: the lack of ribosomes and the presence of characteristic electron-dense globular and filamentous structures. These results provide new insights into the universal nature of stress granules in eukaryotic cells.

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.

Vacuolar processing enzymes in the plant life cycle.

Vacuolar processing enzyme (VPE) is a cysteine-type endopeptidase that has a substrate-specificity for asparagine or aspartic acid residues and cleaves peptide bonds at their carboxyl-terminal side. Various vacuolar proteins are synthesized as larger proprotein precursors, and VPE is an important initiator of maturation and activation of these proteins. It mediates programmed cell death (PCD) by provoking vacuolar rupture and initiating the proteolytic cascade leading to PCD. Vacuolar processing enzyme also possesses a peptide ligation activity, which is responsible for producing cyclic peptides in several plant species. These unique functions of VPE support developmental and environmental responses in plants. The number of VPE homologues is higher in angiosperm species, indicating that there has been differentiation and specialization of VPE function over the course of evolution. Angiosperm VPEs are separated into two major types: the γ-type VPEs, which are expressed mainly in vegetative organs, and the ß-type VPEs, whose expression occurs mainly in storage organs; in eudicots, the δ-type VPEs are further separated within γ-type VPEs. This review also considers the importance of processing and peptide ligation by VPE in vacuolar protein maturation.

ANGUSTIFOLIA Regulates Actin Filament Alignment for Nuclear Positioning in Leaves.

During dark adaptation, plant nuclei move centripetally toward the midplane of the leaf blade; thus, the nuclei on both the adaxial and abaxial sides become positioned at the inner periclinal walls of cells. This centripetal nuclear positioning implies that a characteristic cell polarity exists within a leaf, but little is known about the mechanism underlying this process. Here, we show that ANGUSTIFOLIA (AN) and ACTIN7 regulate centripetal nuclear positioning in Arabidopsis (Arabidopsis thaliana) leaves. Two mutants defective in the positioning of nuclei in the dark were isolated and designated as unusual nuclear positioning1 (unp1) and unp2 In the dark, nuclei of unp1 were positioned at the anticlinal walls of adaxial and abaxial mesophyll cells and abaxial pavement cells, whereas the nuclei of unp2 were positioned at the anticlinal walls of mesophyll and pavement cells on both the adaxial and abaxial sides. unp1 was caused by a dominant-negative mutation in ACTIN7, and unp2 resulted from a recessive mutation in AN Actin filaments in unp1 were fragmented and reduced in number, which led to pleiotropic defects in nuclear morphology, cytoplasmic streaming, and plant growth. The mutation in AN caused aberrant positioning of nuclei-associated actin filaments at the anticlinal walls. AN was detected in the cytosol, where it interacted physically with plant-specific dual-specificity tyrosine phosphorylation-regulated kinases (DYRKPs) and itself. The DYRK inhibitor (1Z)-1-(3-ethyl-5-hydroxy-2(3H)-benzothiazolylidene)-2-propanone significantly inhibited dark-induced nuclear positioning. Collectively, these results suggest that the AN-DYRKP complex regulates the alignment of actin filaments during centripetal nuclear positioning in leaf cells.

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.

The nuclear envelope protein KAKU4 determines the migration order of the vegetative nucleus and sperm cells in pollen tubes.

A putative component protein of the nuclear lamina, KAKU4, modulates nuclear morphology in Arabidopsis thaliana seedlings, but its physiological significance is unknown. KAKU4 was highly expressed in mature pollen grains, each of which has a vegetative cell and two sperm cells. KAKU4 protein was highly abundant on the envelopes of vegetative nuclei and less abundant on the envelopes of sperm cell nuclei in pollen grains and elongating pollen tubes. Vegetative nuclei are irregularly shaped in wild-type pollen. However, KAKU4 deficiency caused them to become more spherical. After a pollen grain germinates, the vegetative nuclei and sperm cells enter and move along the pollen tube. In the wild type, the vegetative nucleus preceded the sperm cell nuclei in >90% of the pollen tubes, whereas, in kaku4 mutants, the vegetative nucleus preceded the sperm cell nuclei in only about half of the pollen tubes. kaku4 pollen was less competitive for fertilization than wild-type pollen after pollination. These results led us to hypothesize that the nuclear shape in vegetative cells of pollen grains affects the orderly migration of the vegetative nucleus and sperm cells in pollen tubes.

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.

Excess sterols disrupt plant cellular activity by inducing stress-responsive gene expression.

Sterols are important lipid constituents of cellular membranes in plants and other organisms. Sterol homeostasis is under strict regulation in plants because excess sterols negatively impact plant growth. HIGH STEROL ESTER 1 (HISE1) functions as a negative regulator of sterol accumulation. If sterol production exceeds a certain threshold, excess sterols are detoxified via conversion to sterol esters by PHOSPHOLIPID STEROL ACYL TRANSFERASE 1 (PSAT1). We previously reported that the Arabidopsis thaliana double mutant hise1-3 psat1-2 shows 1.5-fold higher sterol content than the wild type and consequently a severe growth defect. However, the specific defects caused by excess sterol accumulation in plants remain unknown. In this study, we investigated the effects of excess sterols on plants by analyzing the phenotypes and transcriptomes of the hise1-3 psat1-2 double mutant. Transcriptomic analysis revealed that 435 genes were up-regulated in hise1-3 psat1-2 leaves compared with wild-type leaves. Gene ontology (GO) enrichment analysis revealed that abiotic and biotic stress-responsive genes including RESPONSIVE TO DESICCATION 29B/LOW-TEMPERATURE-INDUCED 65 (RD29B/LTI65) and COLD-REGULATED 15A (COR15A) were up-regulated in hise1-3 psat1-2 leaves compared with wild-type leaves. Expression levels of senescence-related genes were also much higher in hise1-3 psat1-2 leaves than in wild-type leaves. hise1-3 psat1-2 leaves showed early senescence, suggesting that excess sterols induce senescence of leaves. In the absence of sucrose, hise1-3 psat1-2 exhibited defects in seedling growth and root elongation. Together, our data suggest that excess sterol accumulation disrupts cellular activities of vegetative organs including leaves and roots, resulting in multiple damages to plants.

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.

tRNA Wobble Modification Affects Leaf Cell Development in Arabidopsis thaliana.

The tRNA modification at the wobble position of Lys, Glu and Gln (wobbleU* modification) is responsible for the fine-tuning of protein translation efficiency and translation rate. This modification influences organism function in accordance with growth and environmental changes. However, the effects of wobbleU* modification at the cellular, tissue, or individual level have not yet been elucidated. In this study, we show that sulfur modification of wobbleU* of the tRNAs affects leaf development in Arabidopsis thaliana. The sulfur modification was impaired in the two wobbleU*-modification mutants: the URM1-like protein-defective mutant and the Elongator complex-defective mutants. Analyses of the mutant phenotypes revealed that the deficiency in the wobbleU* modification increased the airspaces in the leaves and the leaf size without affecting the number and the area of palisade mesophyll cells. On the other hand, both mutants exhibited increased number of leaf epidermal pavement cells but with reduced cell size. The deficiency in the wobbleU* modification also delayed the initiation of the endoreduplication processes of mesophyll cells. The phenotype of ASYMMETRIC LEAVES2-defective mutant was enhanced in the Elongator-defective mutants, while it was unchanged in the URM1-like protein-defective mutant. Collectively, the findings of this study suggest that the tRNA wobbleU* modification plays an important role in leaf morphogenesis by balancing the development between epidermal and mesophyll tissues.

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.