Authorship：Last author,　Corresponding author   Publisher：Cold Spring Harbor Laboratory  

Abstract

Tip growth is essential for plant growth and reproduction. However, it remains elusive how highly polarized tip-growing cells coordinate the organization of intracellular components. Here, we show that plant-specific armadillo-repeat containing kinesin, MpARK, mediates organelle transport and microtubule convergence in tip growing rhizoids in the early diverging land plantMarchantia polymorpha. MpARK is required for anterograde transport of organelles to maintain their position. Furthermore, MpARK participates in the formation of microtubule foci at the rhizoid apex to stabilize growth direction. MpARK-dependent rhizoid growth is essential for plant anchorage and soil holding capacity. Thus, ARK might be a principal organelle transporter and intracellular organizer in the primitive rooting cells for the adaptation to terrestrial environments. Our findings suggest that ARK is functionally comparable to canonical anterograde kinesins in animals and fungi.

DOI： 10.1101/2022.07.08.499237

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1080/19420889.2022.2095137

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Agrobacterium-mediated transient gene expression is a rapid and useful approach for characterizing functions of gene products in planta. However, the practicability of the method in the model liverwort Marchantia polymorpha has not yet been thoroughly described. Here we report a simple and robust method for Agrobacterium-mediated transient transformation of Marchantia thalli and its applicability. When thalli of M. polymorpha were co-cultured with Agrobacterium tumefaciens carrying GUS genes, GUS staining was observed primarily in assimilatory filaments and rhizoids. GUS activity was detected 2 days after infection and saturated 3 days after infection. We were able to transiently co-express fluorescently tagged proteins with proper localizations. Furthermore, we demonstrate that our method can be used as a novel pathosystem to study liverwort-bacteria interactions. We also provide evidence that air chambers support bacterial colonization.

DOI： 10.1093/pcp/pcab126

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Ethylene is a gaseous phytohormone involved in various physiological processes including fruit ripening, senescence, root hair development, and stress responses. Recent genomics have suggested that most homologous genes of ethylene biosynthesis and signaling are conserved from algae to angiosperms, whereas function and biosynthesis of ethylene remain unknown in basal plants. Here, we examined physiological effects of ethylene, an ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), and an inhibitor of ethylene perception, silver thiosulfate (STS), in a basal land plant Marchantia polymorpha. M. polymorpha plants biosynthesized ethylene and treatment with high concentrations of ACC slightly promoted ethylene production. ACC remarkably suppressed growth of thalli (vegetative organs) and rhizoids (root-hair-like cells), whereas exogenous ethylene slightly promoted thallus growth. STS suppressed thallus growth and induced ectopic rhizoid formation on the dorsal surface of thalli. Thus, ACC and ethylene have different effects on vegetative growth of M. polymorpha. We generated single and double mutants of ACC synthase-like (ACSL) genes, MpACSL1 and MpACSL2. They did not show obvious defects in thallus growth, ACC content, and ethylene production, indicating that MpACSL genes are not essential for the vegetative growth and the biosynthesis of ACC and ethylene. The gene expression analysis suggested involvement of MpACSL1 and MpACSL2 in stress responses. Collectively, our results imply ethylene-independent function of ACC and the absence of ACC-mediated ethylene biosynthesis in M. polymorpha.

DOI： 10.1093/pcp/pcab042

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Key message We found that mutations in a Ca2+-permeable mechanosensitive channel MCA1, an ethylene-regulated microtubule-associated protein WDL5, and a versatile co-receptor BAK1 affect root growth response to mechanical stress. Plant root tips exposed to mechanical impedance show a temporal reduction in the elongation growth. The process involves a transient Ca2+ increase in the cytoplasm followed by ethylene signaling. To dissect the molecular mechanisms underlying this response, we examined the root growth of a series of Arabidopsis mutants with potentially altered response to mechanical stress after transfer from vertical to horizontal plates that were covered by dialysis membrane as an impedance. Among the plant hormone-response mutants tested, the ethylene-insensitive mutant ein3 was confirmed to show no growth reduction after the transfer. The root growth reduction was attenuated in a mutant of MCA1 encoding a Ca2+-permeable mechanosensitive channel and that of WDL5 encoding an ethylene-regulated microtubule-associated protein. We also found that the growth reduction was enhanced in a mutant of BAK1 encoding a co-receptor that pairs with numerous leucine-rich repeat receptor kinases to modulate growth and immunity. These results suggest the root growth reduction in response to mechanical stress involves ethylene-mediated microtubule reorganization and also transmembrane receptor-mediated signal transduction.

DOI： 10.1007/s00299-020-02653-6

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

Xylemin (6) and its designed structural analogues 18-23, N-(4-aminobutyl)alkylamines, were synthesized by 2-nitrobenzenesulfonamide (Ns) strategy. Investigation of the improved synthesis of 20-23 resulted in the development of one-step synthesis of these analogues from the commercially available corresponding ketones. Biological assessment of the synthetic molecules elucidated that xylemin (6) and the analogue N-(4-aminobutyl)cyclopentylamine (21) promoted the expression level of thermospermine synthase ACAULIS5 (ACL5) and enhanced xylem formation. In addition, xylemin (6) was found to significantly promote lateral root formation, whereas xylemin analogues 18-23 including 21 did not. These results indicate that the analogue 21 has the potential as a novel inhibitor of thermospermine synthesis to work specifically in xylem differentiation.

DOI： 10.1002/ejoc.202000322

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Growth variability generates mechanical conflicts in tissues. In plants, cortical microtubules usually align with maximal tensile stress direction, thereby mechanically reinforcing cell walls, and channeling growth rate and direction. How this is achieved remains largely unknown and likely involves microtubule regulators. The NIMA-related microtubule-associated kinase NEK6 phosphorylates tubulin, leading to the depolymerization of a subset of microtubules. We found that cortical microtubules exhibit a hyper-response to mechanical stress in the nek6 mutant. This response contributes to local cell protrusions in slow-growing regions of the nek6 mutant hypocotyl. When growth amplitude is higher, the hyper-alignment of microtubules leads to variable, stop-and-go, phenotypes, resulting in wavy hypocotyl shapes. After gravistimulation or touch, the nek6 mutant also exhibits a hyperbent hypocotyl phenotype, consistent with an enhanced perception of its own deformation. Strikingly, we find that NEK6 exhibits a novel form of polarity, being recruited at the ends of a subset of microtubules at cell edges. This pattern can be modified after local ablation, matching the new maximal tensile stress directions. We propose that NEK6 depolymerizes cortical microtubules that best align with maximal tensile stress to generate a noisier network of microtubules. This prevents an overreaction of microtubules to growth fluctuations and, instead, promotes the buffering of growth variations.

DOI： 10.1016/j.cub.2020.02.024

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© 2019 The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. Plants generally possess a strong ability to regenerate organs; for example, in tissue culture, shoots can regenerate from callus, a clump of actively proliferating, undifferentiated cells. Processing of pre-mRNA and ribosomal RNAs is important for callus formation and shoot regeneration. However, our knowledge of the roles of RNA quality control via the nonsense-mediated mRNA decay (NMD) pathway in shoot regeneration is limited. Here, we examined the shoot regeneration phenotypes of the low-beta-amylase1 (lba1)/upstream frame shift1-1 (upf1-1) and upf3-1 mutants, in which the core NMD components UPF1 and UPF3 are defective. These mutants formed callus from hypocotyl explants normally, but this callus behaved abnormally during shoot regeneration: the mutant callus generated numerous adventitious root structures instead of adventitious shoots in an auxin-dependent manner. Quantitative RT-PCR and microarray analyses showed that the upf mutations had widespread effects during culture on shoot-induction medium. In particular, the expression patterns of early auxin response genes, including those encoding AUXIN/INDOLE ACETIC ACID (AUX/IAA) family members, were significantly affected in the upf mutants. Also, the upregulation of shoot apical meristem-related transcription factor genes, such as CUP-SHAPED COTYLEDON1 (CUC1) and CUC2, was inhibited in the mutants. Taken together, these results indicate that NMD-mediated transcriptomic regulation modulates the auxin response in plants and thus plays crucial roles in the early stages of shoot regeneration.

DOI： 10.1093/pcp/pcz154

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DOI： 10.1111/tpj.14448

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DOI： 10.3390/plants8080269

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DOI： 10.3389/fpls.2019.00564

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Mechanical sensing is one of the most fundamental processes for sessile plants to survive and grow. The response is known to involve calcium elevation in the cell. Arabidopsis seedlings grown horizontally on agar plates covered with a dialysis membrane show a 2-fold reduction in root growth compared with those grown vertically, a response to mechanical stress generated due to gravitropism of the root. To understand the molecular mechanism of how plant roots sense and respond to mechanical stimuli, we screened chemical libraries for compounds that affect the horizontal root growth in this experimental system and found that, while having no effect on root gravitropism, omeprazole known as a proton pump inhibitor significantly enhanced the mechanical stress-induced root growth reduction especially in lower pH media. In contrast, omeprazole reversed neither the alleviation of the mechanical stress-induced growth reduction caused by calcium depletion nor the insensitivity to the mechanical stress in the ethylene signaling mutant ein2. Together with the finding that omeprazole increased expression of touch-induced genes and ETHYLENE RESPONSE FACTOR1, our results suggest that the target of omeprazole mediates ethylene signaling in the root growth response to mechanical stress.

DOI： 10.1093/pcp/pcy131

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Tip growth is driven by turgor pressure and mediated by the polarized accumulation of cellular materials. How a single polarized growth site is established and maintained is unclear. Here, we analyzed the function of NIMA-related protein kinase 1 (MpNEK1) in the liverwort Marchantia polymorpha In the wild type, rhizoid cells differentiate from the ventral epidermis and elongate through tip growth to form hair-like protrusions. In Mpnek1 knockout mutants, rhizoids underwent frequent changes in growth direction, resulting in a twisted and/or spiral morphology. The functional MpNEK1-Citrine protein fusion localized to microtubule foci in the apical growing region of rhizoids. Mpnek1 knockouts exhibited increases in both microtubule density and bundling in the apical dome of rhizoids. Treatment with the microtubule-stabilizing drug taxol phenocopied the Mpnek1 knockout. These results suggest that MpNEK1 directs tip growth in rhizoids through microtubule organization. Furthermore, MpNEK1 expression rescued ectopic outgrowth of epidermal cells in the Arabidopsis thaliana nek6 mutant, strongly supporting an evolutionarily conserved NEK-dependent mechanism of directional growth. It is possible that such a mechanism contributed to the evolution of the early rooting system in land plants.

DOI： 10.1242/dev.154617

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Plant cortical microtubules align perpendicular to the growth axis to determine the direction of cell growth. However, it remains unclear how plant cells form well-organized cortical microtubule arrays in the absence of a centrosome. In this study, we investigated the functions of Arabidopsis NIMA-related kinase 6 (NEK6), which regulates microtubule organization during anisotropic cell expansion. Quantitative analysis of hypocotyl cell growth in the nek6-1 mutant demonstrated that NEK6 suppresses ectopic outgrowth and promotes cell elongation in different regions of the hypocotyl. Loss of NEK6 function led to excessive microtubule waving and distortion, implying that NEK6 suppresses the aberrant cortical microtubules. Live cell imaging showed that NEK6 localizes to the microtubule lattice and to the shrinking plus and minus ends of microtubules. In agreement with this observation, the induced overexpression of NEK6 reduced and disorganized cortical microtubules and suppressed cell elongation. Furthermore, we identified five phosphorylation sites in beta-tubulin that serve as substrates for NEK6 in vitro. Alanine substitution of the phosphorylation site Thr166 promoted incorporation of mutant beta-tubulin into microtubules. Taken together, these results suggest that NEK6 promotes directional cell growth through phosphorylation of beta-tubulin and the resulting destabilization of cortical microtubules.

DOI： 10.1038/s41598-017-08453-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Microtubules are dynamic filaments, the assembly and disassembly of which are under precise control of various associated proteins, including motor proteins and regulatory enzymes. In Arabidopsis thaliana, two such proteins are the ARMADILLO-REPEAT KINESIN 1 (ARK1), which promotes microtubule disassembly, and the NIMA-RELATED KINASE 6 (NEK6), which has a role in organizing microtubule arrays. Previous yeast two-hybrid and in vitro pull-down assays determined that NEK6 can interact with ARK1 through the latter protein's Armadillo-repeat (ARM) cargo domain. To explore the function of the ARM domain, we generated fluorescent reporter fusion proteins to ARK1 lacking the ARM domain (ARK1 Delta ARM-GFP) and to the ARM domain alone (ARM-GFP). Both of these constructs strongly associated with the growing plus ends of microtubules, but only ARK1 Delta ARM-GFP was capable of inducing microtubule catastrophe and rescuing the ark1-1 root hair phenotype. These results indicate that neither the ARM domain nor NEK6's putative interaction with it is required for ARK1 to induce microtubule catastrophe. In further exploration of the ARK1-NEK6 relationship, we demonstrated that, despite evidence that NEK6 can phosphorylate ARK1 in vitro, the in vivo distribution and function of ARK1 were not affected by the loss of NEK6, and vice versa. Moreover, NEK6 and ARK1 were found to have overlapping but non-identical distribution on microtubules, and hormone treatments known to affect NEK6 activity did not stimulate interaction. These findings suggest that ARK1 and NEK6 function independently in microtubule dynamics and cell morphogenesis. Despite the results of this functional analysis, we found that overexpression of the ARM domain led to complete loss of NEK6 transcription, suggesting that the ARM domain might have a regulatory role in NEK6 expression.

DOI： 10.1093/pcp/pcx070

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The acaulis5 (acl5) mutant of Arabidopsis thaliana is defective in the biosynthesis of thermospermine and shows a dwarf phenotype associated with excess xylem differentiation. SAC51 was identified from a dominant suppressor of acl5, sac51-d, and encodes a basic helix-loop-helix protein. The sac51-d mutant has a premature termination codon in an upstream open reading frame (uORF) that is conserved among all four members of the SAC51 family, SAC51 and SACL1-SACL3. This suggests that thermospermine cancels the inhibitory effect of the uORF in main ORF translation. Another suppressor, sac57-d, has a mutation in the conserved uORF of SACL3. To define further the function of the SAC51 family in the thermospermine response, we analyzed T-DNA insertion mutants of each gene. Although sacl1-1 may not be a null allele, the quadruple mutant showed a semi-dwarf phenotype but with an increased level of thermospermine and decreased sensitivity to exogenous thermospermine that normally represses xylem differentiation. The sac51-1 sacl3-1 double mutant was also insensitive to thermospermine. These results suggest that SAC51 and SACL3 play a key role in thermospermine-dependent negative control of thermospermine biosynthesis and xylem differentiation. Using 5' leader-GUS (beta-glucuronidase) fusion constructs, however, we detected a significant enhancement of the GUS activity by thermospermine only in SAC51 and SACL1 constructs. Furthermore, while acl5-1 sac51-1 showed the acl5 dwarf phenotype, acl5-1 sacl3-1 exhibited an extremely tiny-plant phenotype. These results suggest a complex regulatory network for the thermospermine response in which SAC51 and SACL3 function in parallel pathways.

DOI： 10.1093/pcp/pcw113

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS MEDIA SA  

Polyamines are small basic compounds present in all living organisms and act in a variety of biological processes. However, the mechanism of polyamine sensing, signaling and response in relation to other metabolic pathways remains to be fully addressed in plant cells. As one approach, we isolated Arabidopsis mutants that show increased resistance to spermine in terms of chlorosis. We show here that two of the mutants have a point mutation in a nitrate transporter gene of the NRT1/PTR family (NPF), NRT1.3 (AtNPF6.4). These mutants also exhibit increased resistance to putrescine and spermidine while loss-of-function mutants of the two closest homologs of NRT1.3, root-specific NRT1.1 (AtNPF6.3) and petiole-specific NRT1.4 (AtNPF6.2), were shown to have a normal sensitivity to polyamines. When the GUS reporter gene was expressed under the control of the NRT1.3 promoter, GUS staining was observed in leaf mesophyll cells and stem cortex cells but not in the epidermis, suggesting that NRT1.3 specifically functions in parenchymal tissues. We further found that the aerial part of the mutant seedling has normal levels of polyamines but shows reduced uptake of norspermidine compared with the wild type. These results suggest that polyamine transport or metabolism is associated with nitrate transport in the parenchymal tissue of the shoot.

DOI： 10.3389/fpls.2016.00834

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

The xylem conducts water and minerals from the root to the shoot and provides mechanical strength to the plant body. The vascular precursor cells of the procambium differentiate to form continuous vascular strands, from which xylem and phloem cells are generated in the proper spatiotemporal pattern. Procambium formation and xylem differentiation are directed by auxin. In angiosperms, thermospermine, a structural isomer of spermine, suppresses xylem differentiation by limiting auxin signalling. However, the process of auxin-inducible xylem differentiation has not been fully elucidated and remains difficult to manipulate. Here, we found that an antagonist of spermidine can act as an inhibitor of thermospermine biosynthesis and results in excessive xylem differentiation, which is a phenocopy of a thermospermine-deficient mutant acaulis5 in Arabidopsis thaliana. We named this compound xylemin owing to its xylem-inducing effect. Application of a combination of xylemin and thermospermine to wild-type seedlings negates the effect of xylemin, whereas co-treatment with xylemin and a synthetic proauxin, which undergoes hydrolysis to release active auxin, has a synergistic inductive effect on xylem differentiation. Thus, xylemin may serve as a useful transformative chemical tool not only for the study of thermospermine function in various plant species but also for the control of xylem induction and woody biomass production.

DOI： 10.1038/srep21487

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Language：English   Publisher：SPRINGER JAPAN KK  

Microtubules are highly dynamic structures that control the spatiotemporal pattern of cell growth and division. Microtubule dynamics are regulated by reversible protein phosphorylation involving both protein kinases and phosphatases. Never in mitosis A (NIMA)-related kinases (NEKs) are a family of serine/threonine kinases that regulate microtubule-related mitotic events in fungi and animal cells (e.g. centrosome separation and spindle formation). Although plants contain multiple members of the NEK family, their functions remain elusive. Recent studies revealed that NEK6 of Arabidopsis thaliana regulates cell expansion and morphogenesis through beta-tubulin phosphorylation and microtubule destabilization. In addition, plant NEK members participate in organ development and stress responses. The present phylogenetic analysis indicates that plant NEK genes are diverged from a single NEK6-like gene, which may share a common ancestor with other kinases involved in the control of microtubule organization. On the contrary, another mitotic kinase, polo-like kinase, might have been lost during the evolution of land plants. We propose that plant NEK members have acquired novel functions to regulate cell growth, microtubule organization, and stress responses.

DOI： 10.1007/s10265-015-0751-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Abscisic acid (ABA) regulates seed maturation, germination and various stress responses in plants. The roles of ABA in cellular growth and morphogenesis, however, remain to be explored. Here, we report that ABA induces the ectopic outgrowth of epidermal cells in Arabidopsis thaliana. Seedlings of A. thaliana germinated and grown in the presence of ABA developed ectopic protrusions in the epidermal cells of hypocotyls, petioles and cotyledons. One protrusion was formed in the middle of each epidermal cell. In the hypocotyl epidermis, two types of cell files are arranged alternately into non-stoma cell files and stoma cell files, ectopic protrusions being restricted to the non-stoma cell files. This suggests the presence of a difference in the degree of sensitivity to ABA or in the capacity of cells to form protrusions between the two cell files. The ectopic outgrowth was suppressed in ABA insensitive mutants, whereas it was enhanced in ABA hypersensitive mutants. Interestingly, ABA-induced ectopic outgrowth was also suppressed in mutants in which microtubule organization was compromised. Furthermore, cortical microtubules were disorganized and depolymerized by the ABA treatment. These results suggest that ABA signaling induces ectopic outgrowth in epidermal cells through microtubule reorganization.

DOI： 10.1038/srep11364

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

The plant hormone auxin is a key morphogenetic signal that controls many aspects of plant growth and development. Cellular auxin levels are coordinately regulated by multiple processes, including auxin biosynthesis and the polar transport and metabolic pathways. The auxin concentration gradient determines plant organ positioning and growth responses to environmental cues. Auxin transport systems play crucial roles in the spatiotemporal regulation of the auxin gradient. This auxin gradient has been analyzed using SCF-type E3 ubiquitin-ligase complex-based auxin biosensors in synthetic auxin-responsive reporter lines. However, the contributions of auxin biosynthesis and metabolism to the auxin gradient have been largely elusive. Additionally, the available information on subcellular auxin localization is still limited. Here we designed fluorescently labeled auxin analogs that remain active for auxin transport but are inactive for auxin signaling and metabolism. Fluorescent auxin analogs enable the selective visualization of the distribution of auxin by the auxin transport system. Together with auxin biosynthesis inhibitors and an auxin biosensor, these analogs indicated a substantial contribution of local auxin biosynthesis to the formation of auxin maxima at the root apex. Moreover, fluorescent auxin analogs mainly localized to the endoplasmic reticulum in cultured cells and roots, implying the presence of a subcellular auxin gradient in the cells. Our work not only provides a useful tool for the plant chemical biology field but also demonstrates a new strategy for imaging the distribution of small-molecule hormones.

DOI： 10.1073/pnas.1408960111

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS RESEARCH FOUNDATION  

Thermospermine, a structural isomer of spermine, is widely distributed in the plant kingdom and has been shown to play a role in repressing xylem differentiation by studies of its deficient mutant, acaulis5 (acl5), in Arabidopsis. Our results of microarray and real-time PCR analyses revealed that, in addition to a number of genes involved in xylem differentiation, genes related to auxin signaling were up-regulated in acl5 seedlings. These genes include MONOPTEROS, an auxin response factor gene, which acts as a master switch for auxin-dependent procambium formation, and its target genes. Their expression was reduced by exogenous treatment with thermospermine or by transgenic induction of the ACL5 gene. We examined the effect of synthetic polyamines on the expression of these auxin-related genes and on the vascular phenotype of acl5, and found that tetramines containing the NC3NC3N chain could mimic the effect of thermospermine but longer polyamines containing the same chain had little or no such effect. We also found that thermospermine had an inhibitory effect on lateral root formation in wild-type seedlings and it was mimicked by synthetic tetramines with the NC3NC3N chain. These results suggest the importance of the NC3NC3N chain of thermospermine in its action in-modulating auxin signaling.

DOI： 10.3389/fpls.2014.00094

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NIMA-related kinase 6 (NEK6) regulates cellular expansion and morphogenesis through microtubule organizaiton in Arabidopsis thaliana. Loss-of-function mutations in NEK6 (nek6/ibo1) cause ectopic outgrowth and microtubule disorganization in epidermal cells. We recently found that NEK6 forms homodimers and heterodimers with NEK4 and NEK5 to destabilize cortical microtubules possibly by direct binding to microtubules and the β-tubulin phosphorylation. Here, we identified a new allele of NEK6 and further analyzed the morphological phenotypes of nek6/ibo1 mutants, along with alleles of nek4 and nek5 mutants. Phenotypic analysis demonstrated that NEK6 is required for the directional growth of roots and hypocotyls, petiole elongation, cell file formation, and trichome morphogenesis. In addition, nek4, nek5, and nek6/ibo1 mutants were hypersensitive to microtubule inhibitors such as propyzamide and taxol. These results suggest that plant NEKs function in directional cell growth and organ development through the regulation of microtubule organization.

DOI： 10.4161/psb.22412

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Thermospermine, a structural isomer of spermine, is synthesized by a thermospermine synthase designated ACAULIS5 (ACL5). Thermospermine-deficient acl5 mutant of Arabidopsis thaliana shows severe dwarfism and excessive xylem differentiation. By screening for compounds that affect xylem differentiation in the acl5 mutant, we identified auxin analogs that remarkably enhanced xylem vessel differentiation in the acl5 mutant but not in the wild type. The xyleminducing effect of auxin analogs was clearly suppressed by thermospermine, indicating that auxin-inducible xylem differentiation is normally limited by thermospermine. Here, we further characterized xylem-inducing effect of auxin analogs in various organs. Auxin analogs promoted protoxylem differentiation in roots and cotyledons in the acl5 mutant. Our results indicate that the opposite action between thermospermine and auxin in xylem differentiation is common in different organs and also suggest that thermospermine might be required for the suppression of protoxylem differentiation. © 2012 Landes Bioscience.

DOI： 10.4161/psb.20784

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Thermospermine, a structural isomer of spermine, is produced through the action of ACAULIS5 (ACL5) and suppresses xylem differentiation in Arabidopsis thaliana. To elucidate the molecular basis of the function of thermospermine, we screened chemical libraries for compounds that can modulate xylem differentiation in the acl5 mutant, which is deficient in thermospermine and shows a severe dwarf phenotype associated with excessive proliferation of xylem vessels. We found that the isooctyl ester of a synthetic auxin, 2,4-D, remarkably enhanced xylem vessel differentiation in acl5 seedlings. 2,4-D, 2,4-D analogs and IAA analogs, including 4-chloro IAA (4-Cl-IAA) and IAA ethyl ester, also enhanced xylem vessel formation, while IAA alone had little or no obvious effect on xylem differentiation. These effects of auxin analogs were observed only in the acl5 mutant but not in the wild type, and were suppressed by the anti-auxin, p-chlorophenoxyisobutyric acid (PCIB) and alpha-(phenyl ethyl-2-one)-IAA (PEO-IAA), and also by thermospermine. Furthermore, the suppressor of acaulis51-d (sac51-d) mutation, which causes SAC51 overexpression in the absence of thermospermine and suppresses the dwarf phenotype of acl5, also suppressed the effect of auxin analogs in acl5. These results suggest that the auxin signaling that promotes xylem differentiation is normally limited by SAC51-mediated thermospermine signaling but can be continually stimulated by exogenous auxin analogs in the absence of thermospermine. The opposite action between thermospermine and auxin may fine-tune the timing and spatial pattern of xylem differentiation.

DOI： 10.1093/pcp/pcs017

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NimA-related kinase 6 (NEK6) has been implicated in microtubule regulation to suppress the ectopic outgrowth of epidermal cells; however, its molecular functions remain to be elucidated. Here, we analyze the function of NEK6 and other members of the NEK family with regard to epidermal cell expansion and cortical microtubule organization. The functional NEK6-green fluorescent protein fusion localizes to cortical microtubules, predominantly in particles that exhibit dynamic movement along microtubules. The kinase-dead mutant of NEK6 (ibo1-1) exhibits a disturbance of the cortical microtubule array at the site of ectopic protrusions in epidermal cells. Pharmacological studies with microtubule inhibitors and quantitative analysis of microtubule dynamics indicate excessive stabilization of cortical microtubules in ibo1/nek6 mutants. In addition, NEK6 directly binds to microtubules in vitro and phosphorylates beta-tubulin. NEK6 interacts and co-localizes with NEK4 and NEK5 in a transient expression assay. The ibo1-3 mutation markedly reduces the interaction between NEK6 and NEK4 and increases the interaction between NEK6 and NEK5. NEK4 and NEK5 are required for the ibo1/nek6 ectopic outgrowth phenotype in epidermal cells. These results demonstrate that NEK6 homodimerizes and forms heterodimers with NEK4 and NEK5 to regulate cortical microtubule organization possibly through the phosphorylation of beta-tubulins.

DOI： 10.1111/j.1365-313X.2011.04652.x

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In higher plants, many extracellular proteins are involved in developmental processes, including cell-cell signaling and cell wall construction. Xylogen is an extracellular arabinogalactan protein (AGP) isolated from Zinnia elegans xylogenic culture medium, which promotes xylem cell differentiation. Xylogen has a unique structure, containing a non-specific lipid transfer protein (nsLTP) domain and AGP domains. We searched for xylogen-type genes in the genomes of land plants, including Arabidopsis thaliana, to further our knowledge of xylogen-type genes as functional extracellular proteins in plants. We found that many xylogen-type genes, including 13 Arabidopsis genes, comprise a gene family in land plants, including Populus trichocarpa, Vitis vinifera, Lotus japonicus, Oryza sativa, Selaginella moellendorffii and Physcomitrella patens. The genes shared an N-terminal signal peptide sequence, a distinct nsLTP domain, one or more AGP domains and a glycosylphosphatidylinositol (GPI)-anchored sequence. We analyzed transgenic plants harboring promoter::GUS (beta-glucuronidase) constructs to test expression of the 13 Arabidopsis xylogen-type genes, and detected a diversity of gene family members with related expression patterns. AtXYP2 was the best candidate as the Arabidopsis counterpart of the Zinnia xylogen gene. We observed two distinct expression patterns for several genes, with some anther specific and others preferentially expressed in the endodermis/pericycle. We conclude that xylogen-type genes, which may have diverse functions, form a novel chimeric AGP gene family with a distinct nsLTP domain.

DOI： 10.1093/pcp/pcr060

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Thermospermine is a structural isomer of spermine and is required for stem elongation in Arabidopsis thaliana. We noted the C3C3 arrangement of carbon chains in thermospermine (C3C3C4), which is not present in spermine (C3C4C3), and examined if it is functionally replaced with norspermine (C3C3C3) or not. Exogenous application of norspermine to acl5, a mutant defective in the synthesis of thermospermine, partially suppressed its dwarf phenotype, and down-regulated the level of the acl5 transcript which is much higher than that of the ACL5 transcript in the wild type. Furthermore, in the Zinnia culture, differentiation of mesophyll cells into tracheary elements was blocked by thermospermine and norspermine but not by spermine. Our results indicate that norspermine can functionally substitute for thermospermine. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2010.05.035

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

Phytosulfokine (PSK) is a sulfated peptide hormone required for the proliferation and differentiation of plant cells. Here, we characterize the physiological roles of PSK in transdifferentiation of isolated mesophyll cells of zinnia (Zinnia elegans 'Canary Bird') into tracheary elements (TEs). Transcripts for a zinnia PSK precursor gene, ZePSK1, show two peaks of expression during TE differentiation; the first accumulation is transiently induced in response to wounding at 24 h of culture, and the second accumulation is induced in the final stage of TE differentiation and is dependent on endogenous brassinosteroids. Chlorate, a potent inhibitor of peptide sulfation, is successfully applied as an inhibitor of PSK action. Chlorate significantly suppresses TE differentiation. The chlorate-induced suppression of TE differentiation is overcome by exogenously applied PSK. In the presence of chlorate, expression of stress-related genes for proteinase inhibitors and a pathogenesis-related protein is enhanced and changed from a transient to a continuous pattern. On the contrary, administration of PSK significantly reduces the accumulation of transcripts for the stress-related genes. Even in the absence of auxin and cytokinin, addition of PSK suppresses stress-related gene expression. Microarray analysis reveals 66 genes down-regulated and 42 genes up-regulated in the presence of PSK. The large majority of down-regulated genes show significant similarity to various families of stress-related proteins, including chitinases, phenylpropanoid biosynthesis enzymes, 1-aminocyclopropane-1-carboxylic acid synthase, and receptor-like protein kinases. These results suggest the involvement of PSK in the attenuation of stress response and healing of wound-activated cells during the early stage of TE differentiation.

DOI： 10.1104/pp.109.135954

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Suppressor of gene silencing 3 (SGS3) is involved in RNA-dependent RNA polymerase 6 (RDR6)-dependent small-interfering RNA (siRNA) pathways in Arabidopsis. However, the roles of SGS3 in those pathways are unclear. Here, we show that SGS3 interacts and colocalizes with RDR6 in cytoplasmic granules. Interestingly, the granules containing SGS3 and RDR6 (named SGS3/RDR6-bodies) were distinct from the processing bodies where mRNAs are decayed and/or stored. Microscopic analyses and complementation experiments using SGS3-deletion mutants suggested that proper localization of SGS3 is important for its function. These results provide novel insights into RDR6-dependent siRNA formation in plants.

DOI： 10.1016/j.febslet.2009.03.055

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT  

MicroRNAs (miRNAs) are sequence-specific negative regulators of gene expression generated by DICER-LIKE1 (DCL1) with the assistance of a double-stranded RNA-binding protein, HYPONASTIC LEAVES1 (HYL1), in Arabidopsis. To achieve a better understanding of miRNA biogenesis, we isolated hyl1 suppressors. Our genetic screening identified a novel semidominant mutation in DCL1 (dcl1-13), which causes an amino acid substitution of Glu-395 with Lys in the ATPase/DExH-box RNA helicase domain. This mutation confers significant restoration from the developmental abnormality and a reduction in the level of miRNA in the loss-of-function mutant of HYL1. However, the dcl1-13 single mutant, exhibiting a decreased number of leaves, showed a slight decrease in miRNA accumulation. Thus, the effect of the dcl1-13 mutation is HYL1 dependent: it promotes miRNA processing in the absence of HYL1, but conversely, impairs it in the presence of HYL1. Our results suggest significant roles of the helicase domain of DCL1, which remain unclear to date, possibly in relation with HYL1.

DOI： 10.1261/rna.1297109

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

To study cellular morphogenesis genetically, we isolated loss-of-function mutants of Arabidopsis thaliana, designated ibo1. The ibo1 mutations cause local outgrowth in the middle of epidermal cells of the hypocotyls and petioles, resulting in the formation of a protuberance. In Arabidopsis, the hypocotyl epidermis differentiates into two alternate cell files, the stoma cell file and the non-stoma cell file, by a mechanism involving TRANSPARENT TESTA GLABRA1 (TTG1) and GLABRA2 (GL2). The ectopic protuberances of the ibo1 mutants were preferentially induced in the non-stoma cell files, which express GL2. TTG1-dependent epidermal patterning is required for protuberance formation in ibo1, suggesting that IBO1 functions downstream from epidermal cell specification. Pharmacological and genetic analyses demonstrated that ethylene promotes protuberance formation in ibo1, implying that IBO1 acts antagonistically to ethylene to suppress radial outgrowth. IBO1 is identical to NEK6, which encodes a Never In Mitosis A (NIMA)-related protein kinase (Nek) with sequence similarity to Neks involved in microtubule organization in fungi, algae, and animals. The ibo1-1 mutation, in which a conserved Glu residue in the activation loop is substituted by Arg, completely abolishes its kinase activity. The intracellular localization of GFP-tagged NEK6 showed that NEK6 mainly accumulates in cytoplasmic spots associated with cortical microtubules and with a putative component of the gamma-tubulin complex. The localization of NEK6 is regulated by the C-terminal domain, which is truncated in the ibo1-2 allele. These results suggest that the role of NEK6 in the control of cellular morphogenesis is dependent on its kinase action and association with the cortical microtubules.

DOI： 10.1111/j.1365-313X.2008.03445.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Although decapping is an important process in eukaryotic mRNA turnover, little is known about this process in plants. Here, we identified Arabidopsis thaliana decapping proteins AtDCP1 and AtDCP2 and showed that (I) AtDCP2 is an active decapping enzyme, (II) AtDCP1 interacts with itself, (III) AtDCP1 and AtDCP2 are localized to cytoplasmic foci (putative Arabidopsis processing body), and (IV) AtDCP1 and AtDCP2 are essential for post-embryonic development. Our findings provide new insights into the role of decapping-dependent mRNA turnover. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2007.04.051

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC ADVANCEMENT SCIENCE  

In plants and animals, small peptide ligands that signal in cell-cell communication have been suggested to be a crucial component of development. A bioassay of single-cell transdifferentation demonstrates that a dodecapeptide with two hydroxyproline residues is the functional product of genes from the CLE family, which includes CLAVATA3 in Arabidopsis. The dodecapeptide suppresses xylem cell development at a concentration of 10(-11) M and promotes cell division. An application, corresponding to all 26 Arabidopsis CLE protein family members, of synthetic dodecapeptides reveals two counteracting signaling pathways involved in stem cell fate.

DOI： 10.1126/science.1128436

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Inductive cell-cell interactions are essential for controlling cell fate determination in both plants and animals(1); however, the chemical basis of inductive signals in plants remains little understood. A proteoglycan-like factor named xylogen mediates local and inductive cell-cell interactions required for xylem differentiation in Zinnia cells cultured in vitro(2,)3. Here we describe the purification of xylogen and cloning of its complementary DNA, and present evidence for its role in planta. The polypeptide backbone of xylogen is a hybrid-type molecule with properties of both arabinogalactan proteins and nonspecific lipid-transfer proteins. Xylogen predominantly accumulates in the meristem, procambium and xylem. In the xylem, xylogen has a polar localization in the cell walls of differentiating tracheary elements. Double knockouts of Arabidopsis lacking both genes that encode xylogen proteins show defects in vascular development: discontinuous veins, improperly interconnected vessel elements and simplified venation. Our results suggest that the polar secretion of xylogen draws neighbouring cells into the pathway of vascular differentiation to direct continuous vascular development, thereby identifying a molecule that mediates an inductive cell cell interaction involved in plant tissue differentiation.

DOI： 10.1038/nature02619

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER-VERLAG TOKYO  

Precise control of vascular development is essential for the life and evolution of terrestrial plants. Molecular mechanisms underlying this control is still unclear but should involve cell-cell interactions. Here, we will summarize recent findings on the role of cell-cell interactions in vascular cell differentiation, with particular emphasis on the studies with the zinnia xylogenic culture.

DOI： 10.1007/pl00014014

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：ELSEVIER SCIENCE BV  

Vascular plants have developed a complex network of vascular systems through the plant body, allowing efficient transport of water, nutrients and signals. To understand molecular mechanisms of vascular pattern formation, we have made two approaches. First we have isolated Arabidopsis mutants with defects in vascular pattern formation. Microscopic and genetic examination of the cotyledonary venation of 3400 M3 lines led to the identification of 8 mutant lines whose abnormality was caused by mutations in 7 genetic loci designated VAN1-VAN7. Morphological analysis of van mutants indicated that van1-van6 mutations caused fragmentation of lateral veins in cotyledons and of tertiary veins in rosette leaves, but did less injurious effects on the formation of their main veins or of vasculatures in hypocotyls and roots. van mutants were further characterized using pAthb8::GUS and pTED3::GUS as molecular markers of provascular cells and tracheary element precursor cells, respectively. As a result, it was revealed that most of van mutants lacked provascular cells at the disconnection points of the vascular network even at walking stick stage of embryogenesis. These results suggest that VAN genes are involved in the spatial control of provascular tissue differentiation, which realizes a continuous network of the vascular system. Second, we have analyzed regulation of cell-cell communication that may be involved in continuous formation of the vascular system using Zinnia cell culture. For this purpose, we developed two culture methods, thin-sheet culture and microbead culture. These culture methods indicated the presence of a high-molecular weight proteinaceous substance that promotes tracheary element differentiation. An improved microbead culture method brought about the partial purification of the substance, revealing that it is an arabinogalactan protein. Based on these results, we will discuss molecular mechanism of vascular pattern formation.

DOI： 10.1016/s0921-0423(01)80055-1

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Local intercellular communication is involved in tracheary element (TE) differentiation of zinnia (Zinnia elegans L.) mesophyll cells and mediated by a proteinous macro-molecule, which was designated xylogen. To characterize and isolate xylogen, a bioassay system to monitor the activity of xylogen was developed, in which mesophyll cells were embedded in microbeads of agarose gel at a low (2.0-4.3×104 cells ml-1) or high density (8.0-9.0×104 cells ml-1) and microbeads of different cell densities were cultured together in a liquid medium to give a total density of 2.1- 2.5×104 cells ml-1. Without any additives, the frequency of TE differentiation was much smaller in the low-density microbeads than in the high-density microbeads. This low level of TE differentiation in the low-density microbeads was attributable to the shortage of xylogen. When cultures were supplemented with conditioned medium (CM) prepared from zinnia cell suspensions undergoing TE differentiation, the frequency of TE differentiation in the low-density microbeads increased remarkably, indicating the activity of xylogen in the CM. The xylogen activity in CM was sensitive to proteinase treatments. Xylogen was bound to galactose-specific lectins such as Ricinus communis agglutinin and peanut agglutinin, and precipitated by β-glucosyl Yariv reagent. These results indicate that xylogen is a kind of arabinogalactan protein.

DOI： 10.1093/pcp/pce014

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The transdifferentiation of isolated mesophyll cells of zinnia (Zinnia elegans L.) into tracheary elements (TEs) has been well studied as a model of plant cell differentiation. In order to investigate intercellular communication in this phenomenon, two types of culture method were developed, in which mesophyll cells were embedded in a thin sheet of agarose gel and cultured on solid medium, or embedded in microbeads of agarose gel and cultured in liquid medium. A statistical analysis of the two-dimensional distribution of TEs in the thin-sheet cultures demonstrated their aggregation. In the microbead cultures, the frequency of TE differentiation was shown to depend on the local cell density (the cell density in each microbead): TE differentiation required local cell densities of more than 105 cells ml-1. These results suggest that TE differentiation involves cell-cell communication mediated by a locally acting diffusible factor. This presumptive factor was characterized by applying a modified version of the sheet culture, which used two sheets of different cell densities, a low-density sheet and a high-density sheet. Differentiation of TEs in the former could be induced only by bringing it into contact with the latter. Insertion of a 25-kDa-cutoff membrane between the high-density and low-density sheets severely suppressed such induction of TEs in the low-density sheet while a 300-kDa-cutoff membrane suppressed induction only slightly. Insertion of agarose sheets containing immobilized pronase E or trypsin also interfered with the induction of TEs in the low-density sheets. Thus, a proteinaceous macromolecule of 25-300 kDa in molecular weight was assumed to mediate the local intercellular communication required for TE differentiation. This substance was designated "xylogen" with reference to its xylogenic activity. The time of requirement for xylogen during TE differentiation was assessed by experiments in which cells in the low-density sheet were separated from xylogen produced in the high-density sheet at various times by insertion of a 25-kDa-cutoff membrane between the two sheets, and was estimated to be from the 36th hour to the 60th hour of culture (12-36 h before visible thickening of secondary cell walls of TEs).

DOI： 10.1007/s004250000482

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

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Herein, we report a novel method for the determination of polyamines in a sample extracted from Arabidopsis thaliana by capillary electrophoresis (CE) using salicylaldehyde-5-sulfonate (SAS) as a derivatizing reagent. An aldehyde group of SAS forms a Schiff base with amino groups of aliphatic polyamines, resulting in an anionic species with an absorption band in the ultraviolet region. The derivatization method was straightforward since the derivatives were formed by mixing a sample with the derivatizing reagent at a neutral pH. In addition, the negative charges induced by SAS led to a high resolution with a short analysis time. This method permitted the separation of five polyamines, which play important roles in plants. However, further improvement in sensitivity was needed for the determination of the polyamines in plant samples. Therefore, the CE method was coupled with solid-phase extraction (SPE) using an ion-pairing formation with sodium dodecyl benzene sulfonate. The SPE method improved the concentration limits of detection to sub-mu M levels, which corresponded with a 10-fold enhancement. The calibration curves for cadaverine, putrescine, and spermidine were linear with concentrations that ranged from 1 to 20 mu M and correlation coefficients (R-2) were greater than 0.998. The proposed method was applied to the determination of spermidine in a plant sample, Arabidopsis thaliana.

DOI： 10.1039/c3ay26360f

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Language：English   Publisher：IOP PUBLISHING LTD  

We studied the tolerance of living organisms, such as a small animal (Milnesium tardigradum), a small crustacean (Artemia), non-vascular plants or moss (Ptichomitrium and Venturiella), and a vascular plant (Trifolium) to the extremely high hydrostatic pressure of 7.5 GPa. It turned out that most of the high pressure exposed seeds of white clover were alive. Those exposed to 7.5 GPa for up to 1 day and seeded on agar germinated roots. Those exposed for up to 1 hour and seeded on soil germinated stems and leaves. Considering the fact that proteins begins to unfold around 0.3 GPa, it seems difficult to understand that all the living samples which have been investigated can survive after exposure to 7.5 GPa.

DOI： 10.1088/1742-6596/377/1/012053

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DOI： 10.18978/jscrp.39.1_50

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Language：English   Publisher：SPRINGER-VERLAG TOKYO  

Precise control of vascular development is essential for the life and evolution of terrestrial plants. Molecular mechanisms underlying this control is still unclear but should involve cell-cell interactions. Here, we will summarize recent findings on the role of cell-cell interactions in vascular cell differentiation, with particular emphasis on the studies with the zinnia xylogenic culture.

DOI： 10.1007/PL00014014

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Language：English   Publisher：Japanese Society of Plant Physiologists  

Local intercellular communication is involved in tracheary element (TE) differentiation of zinnia (Zinnia elegans L.) mesophyll cells and mediated by a proteinous macro-molecule, which was designated xylogen. To characterize and isolate xylogen, a bioassay system to monitor the activity of xylogen was developed, in which mesophyll cells were embedded in microbeads of agarose gel at a low (2.0-4.3×104 cells ml-1) or high density (8.0-9.0×104 cells ml-1) and microbeads of different cell densities were cultured together in a liquid medium to give a total density of 2.1- 2.5×104 cells ml-1. Without any additives, the frequency of TE differentiation was much smaller in the low-density microbeads than in the high-density microbeads. This low level of TE differentiation in the low-density microbeads was attributable to the shortage of xylogen. When cultures were supplemented with conditioned medium (CM) prepared from zinnia cell suspensions undergoing TE differentiation, the frequency of TE differentiation in the low-density microbeads increased remarkably, indicating the activity of xylogen in the CM. The xylogen activity in CM was sensitive to proteinase treatments. Xylogen was bound to galactose-specific lectins such as Ricinus communis agglutinin and peanut agglutinin, and precipitated by β-glucosyl Yariv reagent. These results indicate that xylogen is a kind of arabinogalactan protein.

DOI： 10.1093/pcp/pce014

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The transdifferentiation of isolated mesophyll cells of zinnia (Zinnia elegans L.) into tracheary elements (TEs) has been well studied as a model of plant cell differentiation. In order to investigate intercellular communication in this phenomenon, two types of culture method were developed, in which mesophyll cells were embedded in a thin sheet of agarose gel and cultured on solid medium, or embedded in microbeads of agarose gel and cultured in liquid medium. A statistical analysis of the two-dimensional distribution of TEs in the thin-sheet cultures demonstrated their aggregation. In the microbead cultures, the frequency of TE differentiation was shown to depend on the local cell density (the cell density in each microbead): TE differentiation required local cell densities of more than 105 cells ml-1. These results suggest that TE differentiation involves cell-cell communication mediated by a locally acting diffusible factor. This presumptive factor was characterized by applying a modified version of the sheet culture, which used two sheets of different cell densities, a low-density sheet and a high-density sheet. Differentiation of TEs in the former could be induced only by bringing it into contact with the latter. Insertion of a 25-kDa-cutoff membrane between the high-density and low-density sheets severely suppressed such induction of TEs in the low-density sheet while a 300-kDa-cutoff membrane suppressed induction only slightly. Insertion of agarose sheets containing immobilized pronase E or trypsin also interfered with the induction of TEs in the low-density sheets. Thus, a proteinaceous macromolecule of 25-300 kDa in molecular weight was assumed to mediate the local intercellular communication required for TE differentiation. This substance was designated "xylogen" with reference to its xylogenic activity. The time of requirement for xylogen during TE differentiation was assessed by experiments in which cells in the low-density sheet were separated from xylogen produced in the high-density sheet at various times by insertion of a 25-kDa-cutoff membrane between the two sheets, and was estimated to be from the 36th hour to the 60th hour of culture (12-36 h before visible thickening of secondary cell walls of TEs).

DOI： 10.1007/s004250000482

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Language：English   Publisher：ELSEVIER SCIENCE BV  

Vascular plants have developed a complex network of vascular systems through the plant body, allowing efficient transport of water, nutrients and signals. To understand molecular mechanisms of vascular pattern formation, we have made two approaches. First we have isolated Arabidopsis mutants with defects in vascular pattern formation. Microscopic and genetic examination of the cotyledonary venation of 3400 M3 lines led to the identification of 8 mutant lines whose abnormality was caused by mutations in 7 genetic loci designated VAN1-VAN7. Morphological analysis of van mutants indicated that van1-van6 mutations caused fragmentation of lateral veins in cotyledons and of tertiary veins in rosette leaves, but did less injurious effects on the formation of their main veins or of vasculatures in hypocotyls and roots. van mutants were further characterized using pAthb8::GUS and pTED3::GUS as molecular markers of provascular cells and tracheary element precursor cells, respectively. As a result, it was revealed that most of van mutants lacked provascular cells at the disconnection points of the vascular network even at walking stick stage of embryogenesis. These results suggest that VAN genes are involved in the spatial control of provascular tissue differentiation, which realizes a continuous network of the vascular system. Second, we have analyzed regulation of cell-cell communication that may be involved in continuous formation of the vascular system using Zinnia cell culture. For this purpose, we developed two culture methods, thin-sheet culture and microbead culture. These culture methods indicated the presence of a high-molecular weight proteinaceous substance that promotes tracheary element differentiation. An improved microbead culture method brought about the partial purification of the substance, revealing that it is an arabinogalactan protein. Based on these results, we will discuss molecular mechanism of vascular pattern formation.

DOI： 10.1016/S0921-0423(01)80055-1

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Event date： 2020.10.27

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Venue：オンライン  

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Venue：名古屋  

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Venue：大阪  

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Venue：奈良先端大, 生駒, 奈良  

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Event date： 2018.9.14 - 2018.9.16

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Venue：岡山大学・資源植物科学研究所  

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Event date： 2017.12.16 - 2017.12.18

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Venue：Okazaki, Japan  

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Venue：岡山大学 津島キャンパス  

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Venue：岡山大学 津島キャンパス  

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Event date： 2017.11.17 - 2017.11.18

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Venue：岡山大学 津島キャンパス  

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Event date： 2017.10.23 - 2017.10.25

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Venue：Lyon, France  

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Venue：野田  

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Event date： 2017.3.16 - 2017.3.18

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2017.3.16 - 2017.3.18

Language：Japanese   Presentation type：Poster presentation  

Venue：鹿児島大学  

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Event date： 2017.3.16 - 2017.3.18

Language：Japanese   Presentation type：Poster presentation  

Venue：鹿児島大学  

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Event date： 2016.9.16 - 2016.9.18

Presentation type：Oral presentation (general)  

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Event date： 2016.5.29 - 2016.6.1

Language：English   Presentation type：Poster presentation  

Venue：Heidelberg, Germany  

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Event date： 2016.5.29 - 2016.6.1

Language：English   Presentation type：Poster presentation  

Venue：Heidelberg, Germany  

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Event date： 2016.3.18 - 2016.3.20

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：岩手大学  

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Event date： 2016.3.18 - 2016.3.20

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：岩手大学  

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Event date： 2015.11.27 - 2015.11.28

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：静岡・三島・遺伝研  

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Event date： 2015.9.6 - 2015.9.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：新潟  

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Event date： 2015.8.27 - 2015.8.28

Language：English   Presentation type：Poster presentation  

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Event date： 2015.6.26

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：横浜・慶應義塾?学?上キャンパス・厚?棟?会議室  

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Event date： 2015.3.17

Language：English   Presentation type：Symposium, workshop panel (nominated)  

Venue：東京農業大学  

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Event date： 2015.3.16 - 2015.3.18

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京農業大学  

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Event date： 2015.3.16 - 2015.3.18

Language：Japanese   Presentation type：Poster presentation  

Venue：東京農業大学  

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Event date： 2015.3.16 - 2015.3.18

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京農業大学  

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Event date： 2014.12.8 - 2014.12.10

Presentation type：Poster presentation  

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Event date： 2014.12.8 - 2014.12.10

Language：English   Presentation type：Poster presentation  

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Event date： 2014.12.8 - 2014.12.10

Language：English   Presentation type：Poster presentation  

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Event date： 2014.7.28 - 2014.8.1

Language：English   Presentation type：Oral presentation (general)  

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Event date： 2014.7.28 - 2014.8.1

Language：English   Presentation type：Poster presentation  

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Event date： 2014.3.18

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：富山大学  

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Event date： 2014.3.18

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：富山大学  

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Event date： 2013.3.21 - 2013.3.23

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：岡山大学 津島キャンパス  

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Event date： 2012.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京都産業大学  

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Event date： 2011.6.22 - 2011.6.25

Language：English   Presentation type：Oral presentation (general)  

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Event date： 2011.3.20 - 2011.3.22

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2011.3

Language：English   Presentation type：Oral presentation (invited, special)  

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Event date： 2010.9.9

Language：Japanese  

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Event date： 2010.9.9

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2010.7

Language：English   Presentation type：Poster presentation  

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Event date： 2010.7

Language：English   Presentation type：Poster presentation  

Venue：Shizuoka, Japan  

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Event date： 2010.3

Language：Japanese   Presentation type：Poster presentation  

Venue：熊本大学 熊本市  

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Event date： 2009.6.30 - 2009.7.4

Language：English   Presentation type：Poster presentation  

Venue：Edinburgh, Scotland, UK  

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Event date： 2009.3.21 - 2009.3.24

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：名古屋  

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Event date： 2008.12.9 - 2008.12.12

Language：Japanese   Presentation type：Poster presentation  

Venue：神戸  

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Event date： 2008.7.23 - 2008.7.27

Language：English   Presentation type：Poster presentation  

Venue：Montreal, Canada.  

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Event date： 2008.3.20 - 2008.3.22

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌  

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Event date： 2007.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：横浜  

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Event date： 2007.11.29

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：奈良先端科学技術大学院大学（NAIST）  

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Event date： 2007.9.7

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：野田 千葉  

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Event date： 2007.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：野田 千葉  

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Event date： 2007.8.31

Presentation type：Oral presentation (invited, special)  

Venue：八王子セミナーハウス  

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Event date： 2007.8.24

Language：English   Presentation type：Oral presentation (invited, special)  

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Event date： 2007.3.29

Language：Japanese   Presentation type：Poster presentation  

Venue：松山 愛媛  

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Event date： 2007.3.23 - 2007.3.28

Language：English   Presentation type：Poster presentation  

Venue：Idaho, USA  

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Event date： 2006.9.14 - 2006.9.16

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：熊本  

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Event date： 2006.3.20 - 2006.3.22

Language：Japanese   Presentation type：Poster presentation  

Venue：筑波  

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