Authorship：Corresponding author   Publisher：Cold Spring Harbor Laboratory  

Abstract

Thermospermine acts in negative regulation of xylem differentiation through enhancing mRNA translation of the members of the SAC51 gene family in Arabidopsis. These mRNAs contain conserved upstream open-reading-frames (uORFs) that are inhibitory to the main ORF translation. To address the mode of action of thermospermine in this process, we have isolated mutants that are insensitive to thermospermine, named its. We show here that four genes responsible for the mutants, its1 to its4, encode a homologue of SPOUT RNA methyl transferase, an rRNA pseudouridine synthase CBF5/NAP57, a putative spliceosome disassembly factor STIPL1/NTR1, and a plant-specific RNA-binding protein PHIP1, respectively. While these mutants except its1 are almost normal in appearance, they enhance the dwarf phenotype of a mutant of ACL5 defective in thermospermine biosynthesis, resulting in tiny-sized plants reminiscent of a double knockout of ACL5 and SACL3, a member of the SAC51 family. We confirmed that the GUS reporter activity from the SAC51 5’-GUS fusion transcript was severely reduced in all of these mutants. These results unveil the importance of RNA processing and modification for the translation of transcripts containing regulatory uORFs.

DOI： 10.1101/2022.09.19.508594

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DOI： 10.1080/19420889.2022.2095137

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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   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

In plants, many of the enzymes in polyamine metabolism are encoded by multiple genes, whose expressions are differentially regulated under different physiological conditions. For comprehensive understanding of their regulation during the seedling growth stage, we examined the expression of polyamine metabolic genes in response to polyamines and stress-related plant hormones in Arabidopsis thaliana. While confirming previous findings such as induction of many of the genes by abscisic acid, induction of arginase genes and a copper amine oxidase gene, CuAOα3, by methyl jasmonate, that of an arginine decarboxylase gene, ADC2, and a spermine synthase gene, SPMS, by salicylic acid, and negative feedback regulation of thermospermine biosynthetic genes by thermospermine, our results showed that expressions of most of the genes are not responsive to exogenous polyamines. We thus examined expression of OsPAO6, which encodes an apoplastic polyamine oxidase and is strongly induced by polyamines in rice, by using the promoter-GUS fusion in transgenic Arabidopsis seedlings. The GUS activity was increased by treatment with methyl jasmonate but neither by polyamines nor by other plant hormones, suggesting a difference in the response to polyamines between Arabidopsis and rice. Our results provide a framework to study regulatory modules directing expression of each polyamine metabolic gene.

DOI： 10.3390/cells10123283

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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

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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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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The differentiation of distinct cell types in appropriate patterns is a fundamental process in the development of multicellular organisms. In Arabidopsis thaliana, protoderm/epidermis differentiates as a single cell layer at the outermost position. However, little is known about the molecular nature of the positional signals that achieve correct epidermal cell differentiation. Here, we propose that very-long-chain fatty acid-containing ceramides (VLCFA-Cers) mediate positional signals by stimulating the function of ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1), a master regulator of protoderm/epidermis differentiation, during lateral root development. We show that VLCFA-Cers, which are synthesized predominantly in the outermost cells, bind to the lipid-binding domain of ATML1. Importantly, this cell type-specific protein-lipid association alters the activity of ATML1 protein and consequently restricts its expression to the protoderm/epidermis through a transcriptional feedback loop. Furthermore, establishment of a compartment, enriched with VLCFA-containing sphingolipids, at the outer lateral membrane facing the external environment may function as a determinant of protodermal cell fate. Taken together, our results indicate that VLCFA-Cers play a pivotal role in directing protoderm/epidermis differentiation by mediating positional signals to ATML1.This article has an associated 'The people behind the papers' interview.

DOI： 10.1242/dev.194969

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

Map-based gene cloning has played a key role in many genetic studies using the model plant, Arabidopsis thaliana. In the post- next generation sequencing era, identification of point mutations and their corresponding genes is increasingly becoming a powerful and important approach to define plant gene function. To perform initial mapping experiments efficiently on Arabidopsis mutants, enrichment of easy-to-use and reliable polymorphic DNA markers would be desirable. We present here a list of InDel polymorphic markers between Col-0 and Ler-0 accessions that can be detected in standard agarose gel electrophoresis.

DOI： 10.3390/plants9060779

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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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Polyamines are small organic compounds found in all living organisms. According to the high degree of positive charge at physiological pH, they interact with negatively charged macromolecules, such as DNA, RNA, and proteins, and modulate their activities. In plants, polyamines, some of which are presented as a conjugated form with cinnamic acids and proteins, are involved in a variety of physiological processes. In recent years, the study of plant polyamines, such as their biosynthetic and catabolic pathways and the roles they play in cellular processes, has flourished, becoming an exciting field of research. There is accumulating evidence that polyamine oxidation, the main catabolic pathway of polyamines, may have a potential role as a source of hydrogen peroxide. The papers in this Special Issue highlight new discoveries and research in the field of plant polyamine biology. The information will help to stimulate further research and make readers aware of the link between their own work and topics related to polyamines.

DOI： 10.3390/plants9040511

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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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DOI： 10.1111/tpj.14448

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DOI： 10.1080/15592324.2019.1669417

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

<jats:p>A mutant defective in the biosynthesis of thermospermine, acaulis5 (acl5), shows a dwarf phenotype with excess xylem vessels in Arabidopsis thaliana. Exogenous supply of thermospermine remarkably represses xylem differentiation in the root of seedlings, indicating the role of thermospermine in proper repression of xylem differentiation. However, the effect of thermospermine has rarely been investigated in other plant species. In this paper, we examined its effect on the growth and gene expression in rice seedlings. When grown with thermospermine, rice seedlings had no clearly enlarged metaxylem vessels in the root. Expression of OsACL5 was reduced in response to thermospermine, suggesting a negative feedback control of thermospermine biosynthesis like in Arabidopsis. Unlike Arabidopsis, however, rice showed up-regulation of phloem-expressed genes, OsHB5 and OsYSL16, by one-day treatment with thermospermine. Furthermore, expression of OsPAO2 and OsPAO6, encoding extracellular polyamine oxidase whose orthologs are not present in Arabidopsis, was induced by both thermospermine and spermine. These results suggest that thermospermine affects the expression of a subset of genes in rice different from those affected in Arabidopsis.</jats:p>

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：Part of collection (book)   Publisher：Humana Press Inc.  

Themospermine is a structural isomer of spermine and is present in some bacteria and most of plants. An Arabidopsis mutant, acaulis5 (acl5), that is defective in the biosynthesis of thermospermine displays excessive proliferation of xylem vessels with dwarfed growth. Recent studies using acl5 and its suppressor mutants that recover the growth without thermospermine have revealed that thermospermine plays a key role in the negative control of the proliferation of xylem vessels through enhancing translation of specific mRNAs that contain a conserved upstream open-reading-frame (uORF) in the 50 leader region.

DOI： 10.1007/978-1-4939-7398-9_4

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Language：English   Publishing type：Part of collection (book)   Publisher：Humana Press Inc.  

Thermospermine, a structural isomer of spermine, is widely spread in the plant kingdom and has recently been shown to play a key role in the repression of xylem differentiation in vascular plants. However, a standard high-performance liquid chromatography (HPLC) protocol for detecting polyamines as their dansyl derivative cannot distinguish themospermine from spermine. These isomers become separated from each other after benzoylation. In this chapter, we describe a simple protocol for extraction, benzoylation, and HPLC detection of thermospermine and spermine with other polyamines from plant material.

DOI： 10.1007/978-1-4939-7398-9_6

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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   Publisher：FRONTIERS MEDIA SA  

DOI： 10.3389/fpls.2017.00014

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

The excessive xylem phenotype of acaulis5 (acl5), an Arabidopsis mutant defective in the synthesis of thermospermine, indicates that thermospermine is required for negative regulation of xylem differentiation. SAC51 was identified from a dominant suppressor of acl5, sac51-d, and encodes a basic helix-loop-helix (bHLH) protein. sac51-d has a premature termination codon in one of upstream open-reading frames (uORFs) of the SAC51 mRNA that is conserved among the SAC51 family members. Thermospermine may act to bypass the inhibitory effect of the uORF on main ORF translation. Another suppressor, sac57-d, also has a mutation in the conserved uORF of SACL3, a member of the SAC51 family. On the other hand, the double knockout of SAC51 and SACL3 is insensitive to thermospermine, suggesting their key role in the response to thermospermine. However, we found that thermospermine enhances mRNA translation of SAC51 and SACL1 but not of SACL2 and SACL3. Taken together with recent findings from other groups, we propose a mechanism by which thermospermine diffused from xylem precursor cells acts non-cell-autonomously to restrict their proliferation.

DOI： 10.1080/15592324.2016.1276685

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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：OXFORD UNIV PRESS  

The epidermis of shoot organs in plants develops from the outermost layer (L1) of the shoot apical meristem. In Arabidopsis, a pair of homeobox genes, ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1) and PROTODERMAL FACTOR2 (PDF2), play a role in regulating the expression of L1-specific genes. atml1aEuroE1 pdf2aEuroE1 double mutants show striking defects in the differentiation of shoot epidermal cells. However, because atml1aEuroE1 and pdf2aEuroE1 have a T-DNA inserted downstream of the respective homeobox sequences, these alleles may not represent null mutations. Here we characterized additional mutant alleles that have a T-DNA insertion at different positions of each gene. Double mutants of a strong atml1aEuroE3 allele with each pdf2 allele were found to cause embryonic arrest at the globular stage. Although with low frequency, all double mutant combinations of a weak atml1aEuroE1 allele with each pdf2 allele germinated and showed phenotypes defective in shoot epidermal cell differentiation. We further confirmed that transgenic induction of PDF2 fused to the Drosophila Engrailed repressor domain temporarily interferes with epidermal cell differentiation in the wild-type background. These results indicate that ATML1 and PDF2 act redundantly as a positive regulator of shoot epidermal cell differentiation and at least one copy of these genes is essential for embryo development.

DOI： 10.1093/pcp/pcv045

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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：Part of collection (book)   Publisher：Springer Japan  

In higher plants, the main route for putrescine synthesis may be from arginine. Higher accumulation of putrescine under biotic and abiotic stress conditions is attributed at least in part to stress-inducible expression of arginine decarboxylase genes. Regulation of spermidine synthesis involves translational control of the S -adenosylmethionine decarboxylase mRNA, which is responsive to cellular polyamine levels. Genes encoding S -adenosylmethionine decarboxylase and spermine synthase are also responsive to environmental stimuli or show preferential expression in certain organs. Stress-inducible expression of these genes involves plant hormone signaling of abscisic acid (ABA) or methyl jasmonate (MeJA). Thermospermine synthase is widely distributed in the plant kingdom but has a unique role in the repression control of xylem differentiation in higher plants. Expression of the gene for thermospermine synthase is under negative feedback control by thermospermine. Putrescine, spermidine, and cadaverine are also essential precursors for alkaloid biosynthesis in some plant species. Polyamines occur in various aspects of plant growth as a conjugated form with cinnamic acids and proteins. Genes responsible for these conjugations have been increasingly identified.

DOI： 10.1007/978-4-431-55212-3_3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Thermospermine acts in negative regulation of xylem differentiation and its deficient mutant of Arabidopsis thaliana, acaulis5 (acl5), shows excessive xylem formation and severe dwarfism. Studies of two dominant suppressors of acl5, sac51-d and sac52-d, have revealed that SAC51 and SAC52 encode a transcription factor and a ribosomal protein L10 (RPL10), respectively, and these mutations enhance translation of the SAC51 mRNA, which contains conserved upstream open reading frames in the 5' leader. Here we report identification of SAC53 and SAC56 responsible for additional suppressors of acl5. sac53-d is a semi-dominant allele of the gene encoding a receptor for activated C kinase 1 (RACK1) homolog, a component of the 40S ribosomal subunit. sac56-d represents a semi-dominant allele of the gene for RPL4. We show that the GUS reporter activity driven by the CaMV 35S promoter plus the SAC51 5' leader is reduced in acl5 and restored by sac52-d, sac53-d, and sac56-d as well as thermospermine. Furthermore, the SAC51 mRNA, which may be a target of nonsense-mediated mRNA decay, was found to be stabilized in these ribosomal mutants and by thermospermine. These ribosomal proteins are suggested to act in the control of uORF-mediated translation repression of SAC51, which is derepressed by thermospermine.

DOI： 10.1371/journal.pone.0117309

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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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The class IV Homeodomain-leucine zipper (HD-ZIP IV) gene family includes several genes that are functionally significant in epidermal development. Our recent study revealed that double mutants of the epidermis-expressed HD-ZIP IV members, PROTODERMAL FACTOR2 (PDF2) in combination with some HOMEODOMAIN GLABROUS (HDG, pronounced "hedge") genes, affect stamen development and specification of petal and stamen identity, possibly in a non cell-autonomous manner. However, the effect of the pdf2 mutations on the floral development was largely different depending on T-DNA insertion locations: pdf2-1 hdg flowers exhibited homeotic conversion of petals and stamens, while pdf2-2 hdg flowers had only a reduced number of stamens. Here, we used 2 additional pdf2 alleles to make double mutants and found that their floral phenotypes were rather similar to those of pdf2-2 hdg. The allele-specific effect caused by pdf2-1, which carries a T-DNA in a steroidogenic acute regulatory protein-related lipid transfer (START) domainencoding region, suggests the importance of the START domain in proper function of HD-ZIP IV proteins. © 2014 Landes Bioscience.

DOI： 10.4161/psb.27417

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Development of the epidermis involves members of the class-IV homeodomain-leucine zipper (HD-ZIP IV) transcription factors. The Arabidopsis HD-ZIP IV family consists of 16 members, among which PROTODERMAL FACTOR 2 (PDF2) and ARABIDOPSIS THALIANA MERISTEM LAYER 1 (ATML1) play an indispensable role in the differentiation of shoot epidermal cells
however, the functions of other HD-ZIP IV genes that are also expressed specifically in the shoot epidermis remain to be fully elucidated. We constructed double mutant combinations of these HD-ZIP IV mutant alleles and found that the double mutants of pdf2-1 with homeodomain glabrous1-1 (hdg1-1), hdg2-3, hdg5-1 and hdg12-2 produced abnormal flowers with sepaloid petals and carpelloid stamens in association with the reduced expression of the petal and stamen identity gene APETALA 3 (AP3). Expression of another petal and stamen identity gene PISTILATA (PI) was less affected in these mutants. We confirmed that AP3 expression in pdf2-1 hdg2-3 was normally induced at the initial stages of flower development, but was attenuated both in the epidermis and internal cell layers of developing flowers. As the expression of PDF2 and these HD-ZIP IV genes during floral organ formation is exclusively limited to the epidermal cell layer, these double mutations may have non-cell-autonomous effects on AP3 expression in the internal cell layers. Our results suggest that cooperative functions of PDF2 and other members of the HD-ZIP IV family in the epidermis are crucial for normal development of floral organs in Arabidopsis. © 2013 The Authors The Plant Journal © 2013 John Wiley &amp
Sons Ltd.

DOI： 10.1111/tpj.12211

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

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

Thermospermine is a structural isomer of spermine, which is one of the polyamines studied extensively in the past, and is produced from spermidine by the action of thermospermine synthase encoded by a gene named ACAULIS5 (ACL5) in plants. According to recent genome sequencing analyses, ACL5-like genes are widely distributed throughout the plant kingdom. In Arabidopsis, ACL5 is expressed specifically during xylem formation from procambial cells to differentiating xylem vessels. Loss-of-function mutants of ACL5 display overproliferation of xylem vessels along with severe dwarfism, suggesting that thermospermine plays a role in the repression of xylem differentiation. Studies of suppressor mutants of acl5 that recover the wild-type phenotype in the absence of thermospermine suggest that thermospermine acts on the translation of specific mRNAs containing upstream open reading frames (uORFs). Thermospermine is a novel type of plant growth regulator and may also serve in the control of wood biomass production.

DOI： 10.1093/pcp/pcs019

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

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

Leaves are the most important, fundamental units of organogenesis in plants. Although the basic form of a leaf is clearly divided into the leaf blade and leaf petiole, no study has yet revealed how these are differentiated from a leaf primordium. We analyzed the spatiotemporal pattern of mitotic activity in leaf primordia of Arabidopsis (Arabidopsis thaliana) in detail using molecular markers in combination with clonal analysis. We found that the proliferative zone is established after a short interval following the occurrence of a rod-shaped early leaf primordium; it is separated spatially from the shoot apical meristem and seen at the junction region between the leaf blade and leaf petiole and produces both leaf-blade and leaf-petiole cells. This proliferative region in leaf primordia is marked by activity of the ANGUSTIFOLIA3 (AN3) promoter as a whole and seems to be differentiated into several spatial compartments: activities of the CYCLIN D4;2 promoter and SPATULA enhancer mark parts of it specifically. Detailed analyses of the an3 and blade-on-petiole mutations further support the idea that organogenesis of the leaf blade and leaf petiole is critically dependent on the correct spatial regulation of the proliferative region of leaf primordia. Thus, the proliferative zone of leaf primordia is spatially differentiated and supplies both the leaf-blade and leaf-petiole cells.

DOI： 10.1104/pp.111.185066

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

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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P&gt;SYP2 proteins are a sub-family of Qa-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) that may be responsible for protein trafficking between pre-vacuolar compartments (PVC) and vacuoles. Arabidopsis thaliana SYP22/VAM3/SGR3 and SYP21/PEP12 proteins function independently, but are both reported to be essential for male gametophytic viability. Here, we systematically examined the redundancy of three SYP2 paralogs (i.e. SYP21, 22 and 23) using a Col-0 ecotype harboring a SYP2 paralog (SYP23/PLP) that lacked a transmembrane domain. Surprisingly, no visible phenotypes were observed, even in the double knockout syp21/pep12 syp23/plp. Deficiency of either SYP21/PEP12 or SYP23/PLP in the syp22 background resulted in a defect in vacuolar protein sorting, characterized by abnormal accumulation of protein precursors in seeds. SYP21/PEP12 knockdown enhanced the syp22 phenotype (i.e. semi-dwarfism, poor leaf vein development and abnormal development of myrosin cells), and additional knockout of SYP23/PLP further aggravated the phenotype. A GFP-SYP23/PLP fusion localized to the cytosol, but not to the PVC or vacuolar membrane, where SYP21/PEP12 or SYP22/VAM3, respectively, were localized. Immunoprecipitation analysis showed that SYP23/PLP interacted with the vacuolar Qb- and Qc-SNAREs, VTI11 and SYP5, respectively, suggesting that SYP23/PLP is able to form a SNARE complex anchoring the membrane. Unexpectedly, we found that expression of multiple copies of a genomic fragment of SYP23/PLP suppressed the abnormal syp22-3 phenotype. Thus, SYP2 proteins, including cytosolic SYP23/PLP, appear to function redundantly in vacuolar trafficking and plant development.

DOI： 10.1111/j.1365-313X.2010.04394.x

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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：OXFORD UNIV PRESS  

Polyamines are small polycationic molecules found ubiquitously in all organisms and function in a wide variety of biological processes. In the past decade, molecular and genetic studies using mutants and transgenic plants with an altered activity of enzymes involved in polyamine biosynthesis have contributed much to a better understanding of the biological functions of polyamines in plants.
Spermidine is essential for survival of Arabidopsis embryos. One of the reasons may lie in the fact that spermidine serves as a substrate for the lysine -&gt; hypusine post-translational modification of the eukaryotic translation initiation factor 5A, which is essential in all eukaryotic cells. Spermine is not essential but plays a role in stress responses, probably through the modulation of cation channel activities, and as a source of hydrogen peroxide during pathogen infection. Thermospermine, an isomer of spermine, is involved in stem elongation, possibly by acting on the regulation of upstream open reading frame-mediated translation.
The mechanisms of action of polyamines differ greatly from those of plant hormones. There remain numerous unanswered questions regarding polyamines in plants, such as transport systems and polyamine-responsive genes. Further studies on the action of polyamines will undoubtedly provide a new understanding of plant growth regulation and stress responses.

DOI： 10.1093/aob/mcp259

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Publisher：The Japanese Society of Plant Physiologists  

Arabidopsis VAM3/SYP22, one of the SYP2 family SNARE proteins, is classified as Q_a-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and interacts with AtVTI11 (Q_b-SNARE) and SYP5 (Q_c-SNARE). Previously, we reported that vam3 mutants exhibited the phenotypes of wavy leaves, delayed growth, semi-dwarfism and abnormal distribution of myrosin cells (1). Although PEP12/SYP21 and PLP/SYP23 are homologues to VAM3/SYP22, the physiological function of PEP12/SYP21 and PLP/SYP23 in plants remains unknown. To elucidate a role of PEP12/SYP21 and PLP/SYP23 in protein trafficking and development of plant cells, we generated double and triple mutants of the SYP2 family SNARE proteins of Arabidopsis thaliana. By analyzing the phenotypes of these mutants, we found the functional redundancy between these three SYP2 family SNARE proteins. We will discuss about the functions of SYP2 family SNARE proteins in higher plants. <br>(1) Shirakawa, M. et al, Plant Cell Physiol., 50, 1319-1328 (2009).

DOI： 10.14841/jspp.2010.0.0127.0

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Heat shock promoters are powerful tools for the precise control of exogenous gene induction in living organisms. In addition to the temporal control of gene expression, the analysis of gene function can also require spatial restriction. Recently, we reported a new method for in vivo, single-cell gene induction using an infrared laser-evoked gene operator (IR-LEGO) system in living nematodes (Caenorhabditis elegans). It was demonstrated that infrared (IR) irradiation could induce gene expression in single cells without incurring cellular damage. Here, we report the application of IR-LEGO to the small fish, medaka (Japanese killifish
Oryzias latipes) and zebrafish (Danio rerio), and a higher plant (Arabidopsis thaliana). Using easily observable reporter genes, we successfully induced gene expression in various tissues in these living organisms. IR-LEGO has the potential to be a useful tool in extensive research fields for cell/tissue marking or targeted gene expression in local tissues of small fish and plants. © 2009 Japanese Society of Developmental Biologists.

DOI： 10.1111/j.1440-169X.2009.01135.x

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

Disruption of the Arabidopsis thaliana ACAULIS5 (ACL5) gene, which has recently been shown to encode thermospermine synthase, results in a severe dwarf phenotype. A previous study showed that sac51-d, a dominant suppressor mutant of acl5-1, has a premature termination codon in an upstream open reading frame (ORF) of SAC51, which encodes a putative transcription factor, and suggested the involvement of upstream ORF-mediated translational control in ACL5-dependent stem elongation. Here we report the identification of a gene responsible for sac52-d, another semi-dominant suppressor mutant of acl5-1. SAC52 encodes ribosomal protein L10 (RPL10A), which is highly conserved among eukaryotes and implicated in translational regulation. Transformation of acl5-1 mutants with a genomic fragment containing the sac52-d allele rescued the dwarf phenotype of acl5-1. GUS reporter activity under the control of a SAC51 promoter with its upstream ORF was higher in acl5-1 sac52-d than in acl5-1, suggesting that suppression of the acl5-1 phenotype by sac52-d is attributable, in part, to enhanced translation of certain transcripts including SAC51. We also found that a T-DNA insertion allele of SAC52/RPL10A causes lethality in the female gametophyte.

DOI： 10.1111/j.1365-313X.2008.03647.x

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

Loss-of-function mutants of the ACAULIS5 (ACL5) gene in Arabidopsis thaliana have severe defects in stem elongation. ACL5 was previously reported as encoding a spermine synthase. A more recent study, however, showed that the bacterial expressed recombinant ACL5 protein catalyzes the conversion of spermidine to thermospermine, a structural isomer of spermine, rather than to spermine. In the present study, we found that thermospermine was detected in wild-type seedlings but was not detectable in the acl5-1 mutant. We further examined the effect of exogenous application of these isomers on the growth of acl5-1. Daily application of 0.1 mM thermospermine onto the shoot apex partially rescued the dwarf phenotype of acl5-1, while that of spermine had no effects on the morphology of the mutant. The acl5-1 transcript level in acl5-1 seedlings, which is much higher than the ACL5 transcript level in wild-type seedlings, was reduced by exogenous thermospermine. Thus we conclude that thermospermine is indeed produced through the action of ACL5 and required for stem elongation in Arabidopsis.

DOI： 10.1093/pcp/pcn109

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Publisher：The Japanese Society of Plant Physiologists  

Myrosin cells are idioblasts specific for Capparales plants, and accumulate large amounts of myrosinases, which hydrolyze glucosinolates to produce toxic compounds for repelling pests. Previously, we showed that myrosin cells were scattered along leaf veins in wild-type leaves, while they were abnormally distributed in atvam3leaves. The mutants developed a network of myrosin cells throughout the leaves: myrosin cells were not only distributed continuously along leaf veins, but were also observed independent of leaf veins. The result indicates that AtVAM3 is involved in development of myrosin cells. AtVAM3 is a SNARE protein belonging to SYP2 family, which is localized to vacuoles and prevacuolar compartments. In this study, we analyzed the involvement of other SYP2 members, AtPEP12 and AtPLP, in development of myrosin cells. We will show the functional differentiation of SYP2 proteins in development of myrosin cells.

DOI： 10.14841/jspp.2008.0.0794.0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Cellular polyamine content often changes in response to abiotic stresses. However, its physiological relevance is unknown. We found that an Arabidopsis mutant plant (acl5lspms), which cannot produce spermine, is hypersensitive to high salt. Examination of drought sensitivity of the mutant and comparison with wild type plants indicated hypersensitivity to drought. This phenotype was cured by spermine pretreatment but not by the other polyamines putrescine and spermidine, suggesting that drought-hypersensitivity exhibited by the mutant is due to spermine deficiency. The water loss rate of wild type and mutant plants were similar until 20 min after onset of dehydration stress, but after a longer exposure the rate in mutant plants was higher than in wild type plants. Consistent with this result, the stomata of the mutant leaves remained open while in wild type leaves they closed. Based on the collected data, we discuss a role for spermine in response to drought stress. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2006.11.041

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

It is well known that changes in abiotic conditions such as the concentration of ions, temperature and humidity lead to modulation of polyamine contents in plants. However, little is known about the relevant parts these polyamines play in abiotic stress responses. Here we addressed a specific role of spermine during high salt stress using an Arabidopsis double knockoutmutant plant (acl5lspnrs) which cannot produce spermine. The mutant showed higher sensitivity to high salt than wild type plants. This phenotype was cured by exogenous spermine but not by the other polyamines putrescine and spermidine, suggesting a strong link between spermine-deficiency and NaCl-hypersensitivity. The mutant was also hypersensitive to high levels of KCl but not to MgCl2 or to high osmoticum. NaCl-hypersensitivity of the mutant was compromised by treatment with Ca2+ channel blockers. Moreover, the mutant showed poor growth on Ca2+-depleted Murashige-Skoog agar media. The data suggest that the absence of spermine causes an imbalance in Ca2+ homeostasis in the mutant plant. Based on the data obtained, we propose a model for a role of spermine in high salt stress responses. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2006.10.078

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

Loss-of-function mutants of the Arabidopsis thaliana ACAULIS 5 (ACL5) gene, which encodes spermine synthase, exhibit a severe dwarf phenotype. To elucidate the ACL5-mediated regulatory pathways of stem internode elongation, we isolated four suppressor of acaulis (sac) mutants that reverse the acl5 dwarf phenotype. Because these mutants do not rescue the dwarfism of known phytohormone-related mutants, the SAC genes appear to act specifically on the ACL5 pathways. We identify the gene responsible for the dominant sac51-d mutant, which almost completely suppresses the acl5 phenotype. sac51-d disrupts a short upstream open reading frame (uORF) of SAC51, which encodes a bHLH-type transcription factor. Our results indicate that premature termination of the uORF in sac51-d results in an increase in its own transcript level, probably as a result of an increased translation of the main ORF. We suggest a model in which ACL5 plays a role in the translational activation of SAC51, which may lead to the expression of a subset of genes required for stem elongation.

DOI： 10.1242/dev.02535

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

The Arabidopsis ( Arabidopsis thaliana) genome contains 16 genes belonging to the class IV homeodomain-Leucine zipper gene family. These include GLABRA2, ANTHOCYANINLESS2, FWA, ARABIDOPSIS THALIANA MERISTEM LAYER1 ( ATML1), and PROTODERMAL FACTOR2 (PDF2). Our previous study revealed that atml1 pdf2 double mutants have severe defects in the shoot epidermal cell differentiation. Here, we have characterized additional members of this gene family, which we designated HOMEODOMAIN GLABROUS1 (HDG1) through HDG12. Analyses of transgenic Arabidopsis plants carrying the gene-specific promoter fused to the bacterial beta-glucuronidase reporter gene revealed that some of the promoters have high activities in the epidermal layer of the shoot apical meristem and developing shoot organs, while others are temporarily active during reproductive organ development. Expression profiles of highly conserved paralogous gene pairs within the family were found to be not necessarily overlapping. Analyses of T-DNA insertion mutants of these HDG genes revealed that all mutants except hdg11 alleles exhibit no abnormal phenotypes. hdg11 mutants show excess branching of the trichome. This phenotype is enhanced in hdg11 hdg12 double mutants. Double mutants were constructed for other paralogous gene pairs and genes within the same subfamily. However, novel phenotypes were observed only for hdg3 atml1 and hdg3 pdf2 mutants that both exhibited defects in cotyledon development. These observations suggest that some of the class IV homeodomain-Leucine zipper members act redundantly with other members of the family during various aspects of cell differentiation. DNA-binding sites were determined for two of the family members using polymerase chain reaction-assisted DNA selection from random oligonucleotides with their recombinant proteins. The binding sites were found to be similar to those previously identified for ATML1 and PDF2, which correspond to the pseudopalindromic sequence 5'-GCATTAAATGC-3' as the preferential binding site.

DOI： 10.1104/pp.106.077388

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

Myrosin cells in Capparales plants are idioblasts that accumulate thioglucoside glucohydrolase (TGG, also called myrosinase), which hydrolyzes glucosinolates to produce toxic compounds for repelling pests. Here, we show that AtVAM3 is involved in development of myrosin cells. It has been shown that yeast VAM3 is a Q.-SNARE that is involved in vesicle transport of vacuolar proteins and vacuolar assembly. We found that two Arabidopsis atvam3 alleles, atvam3-3 and atvain3-4/ssin, accumulate large amounts of TGG1 and TGG2 that are enzymatically active. An immunogold analysis revealed that TGGs were specifically localized in the vacuole of myrosin cells in atvam3 mutants. This result indicates that TGGs are normally transported to vacuoles in these mutants and that AtVAM3 is not essential for vacuolar transport of the proteins. We developed a staining method with Coomassie brilliant blue that detects myrosin cells in whole leaves by their high TGG content. This method showed that atvam3 leaves have a larger number of myrosin cells than do wild-type leaves. Myrosin cells were scattered along leaf veins in wild-type leaves, while they were abnormally distributed in atvam3 leaves. The mutants developed a network of myrosin cells throughout the leaves: myrosin cells were not only distributed continuously along leaf veins, but were also observe independent of leaf veins. The excess of myrosin cells in atvam3 mutants might be responsible for the abnormal abundance of TGGs and the reduction of elongation of inflorescence stems and leaves in these mutants. Our results suggest that AtVAM3 has a plant-specific function in development of myrosin cells.

DOI： 10.1093/pcp/pci232

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

The short stem and midrib (ssm) mutants of Arabidopsis thaliana show both semi-dwarf and wavy leaf phenotypes due to defects in the elongation of the stem internodes and leaves. Moreover, these abnormalities cannot be recovered by exogenous phytohormones. ssm was originally identified as a single recessive mutant of the ecotype Columbia (Col-0), but genetic crossing experiments have revealed that this mutant phenotype is restored by another gene that is functional in the ecotype Landsberg erecta (Ler) and not in Col-0. Map-based cloning of the gene that is defective in ssm mutants has uncovered a small deletion in the sixth intron of a gene encoding a syntaxin, VAM3/SYP22, which has been implicated in vesicle transport to the vacuole. This mutation appears to cause a peptide insertion in the deduced VAM3/SYP22 polypeptide sequence due to defective splicing of the shortened sixth intron. Significantly, when compared with the wild-type Ler genome, the wild-type Col-0 genome has a single base pair deletion causing a frameshift mutation in SYP23, a gene with the highest known homology to VAM3/SYP22. These findings suggest that VAM3/SYP22 and SYP23 have overlapping functions and that the vesicle transport mediated by these syntaxins is important for shoot morphogenesis.

DOI： 10.1093/pcp/pci146

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

Xyloglucan endotransglucosylases/hydrolases (XTHs) are a class of enzymes capable of catalyzing the molecular grafting between xyloglucans and/or the endotype hydrolysis of a xyloglucan molecule. They are encoded by 33 genes in Arabidopsis. Whereas recent studies have revealed temporally and spatially specific expression profiles for individual members of this family in plants, their biological roles are still to be clarified. To identify the role of each member of this gene family, we examined phenotypes of mutants in which each of the Arabidopsis XTH genes was disrupted. This was undertaken using a reverse genetic approach, and disclosed two loss-of-function mutants for the AtXTH27 gene, xth27-1 and xth27-2. These exhibited short-shaped tracheary elements in tertiary veins, and reduced the number of tertiary veins in the first leaf. In mature rosette leaves of the mutant, yellow lesion-mimic spots were also observed. Upon genetic complementation by introducing the wild-type XTH27 gene into xth27-1 mutant plants, the number of tertiary veins was restored, and the lesions disappeared completely. Extensive expression of the pXTH27::GUS fusion gene was observed in immature tracheary elements in the rosette leaves. The highest level of AtXTH27 mRNA expression in the rosette leaves was observed during leaf expansion, when the tracheary elements were elongating. These findings indicate that AtXTH27 plays an essential role during the generation of tracheary elements in the rosette leaves of Arabidopsis.

DOI： 10.1111/j.1365-313X.2005.02395.x

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

We have developed a new tool, named the plant exon finder (PEF), for identifying exons in plant genome sequences as an applied technique of T-DNA insertional mutagenesis. The T-DNA constructs contain a heat-shock gene promoter or the cauliflower mosaic virus (CaMV) 35S promoter, followed by the first exon of an Arabidopsis gene with its start codon and the intron donor sequence facing the TDNA left border (LB) in order to trap exons in the genome. The constructs were used to make transgenic Arabidopsis plants. We generated 280 transgenic lines and identified 156 T-DNA-tagged readthrough transcripts by reverse transcriptase-polymerase chain reaction (RT-PCR) using an oligo(dT)-linker primer and a T-DNA-specific primer. Sequence analysis of the RT-PCR products revealed that 18 of them carried cDNAs processed by the use of an intron acceptor sequence adjacent to T-DNA insertion sites and 11 of them were in-frame fusions. In one case, the readthrough transcript trapped an exon located 1.6 kb downstream of the site of the insertion. (C) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.gene.2004.06.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Controlling gene expression during plant development is an efficient tool to explore gene function. In this paper, we describe a gene expression system driven by a heat-shock gene promoter (HSP18.2), to trigger the expression of an intron-containing inverted-repeat. RNA interference became a powerful way for gene functional analysis by reverse genetic approaches. However, constitutive gene silencing cannot be used with genes involved in fundamental processes such as embryo viability. Inducible promoters provide an alternative approach for temporal and spatial gene expression control and we described here a new system, complementary to those using chemical gene inducers. To evaluate the efficiency of this system, RNA corresponding to the phytoene desaturase gene of Arabidopsis thaliana was used as a reporter gene in transgenic plants and a comparative study was performed using either the CaMV35S constitutive promoter or the HSP18.2 inducible promoter. (C) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2004.06.154

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

The cellular polyamines putrescine, spermidine, and spermine are ubiquitous in nature and have been implicated in a wide range of growth and developmental processes. There is little information, however, on mutant plants or animals defective in the synthesis of polyamines. The Arabidopsis genome has two genes encoding spermidine synthase, SPDS1 and SPDS2. In this paper, we describe T-DNA insertion mutants of both of these genes. While each mutant allele shows normal growth, spds1-1 spds2-1 double-mutant seeds are abnormally shrunken and they have embryos that are arrested morphologically at the heart-torpedo transition stage. These seeds contain significantly reduced levels of spermidine and high levels of its precursor, putrescine. The embryo lethal phenotype of spds1-1 spds2-1 is complemented by the wild-type SPDS1 gene. In addition, we observed a nearly identical seed phenotype among an F, seed population from the cross between the spds2-1. allele and SPDS1 RNA interference transgenic lines. These data provide the first genetic evidence indicating a critical role of the spermidine synthase in plant embryo development.

DOI： 10.1104/pp.104.041699

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

Spermine is the final product of the polyamine biosynthetic pathway and is ubiquitously present in most organisms. The genome of Arabidopsis thaliana has two genes encoding spermine synthase: ACAULIS5 (ACL5), whose loss-of-function mutants show a severe defect in stem elongation, and SPMS. In order to elucidate the function of spermine in plants, we isolated a T-DNA insertion mutant of the SPMS gene. Free and conjugated spermine levels in the mutant, designated spms-1, were significantly decreased compared with those in the wildtype, but no obvious morphological phenotype was observed in spms-1 plants. We further confirmed that acl5-1 spms-1 double mutants contained no spermine. Surprisingly, acl5-1 spms-1 was fully as viable as the wild-type and showed no phenotype except for the reduced stem growth due to acl5-1. These results indicate that spermine is not essential for survival of Arabidopsis, at least under normal growth conditions. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/S0014-5793(03)01395-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

In higher plants, the outermost cell layer (L1) of the shoot apex gives rise to the epidermis of shoot organs. Our previous study demonstrated that an 8-bp motif named the L1 box functions as a cis-regulatory element for L1-specific gene expression in the shoot system of Arabidopsis. We show here that PROTODERMAL FACTOR2 (PDF2), a member of the HD-GL2 class of homeobox genes, is expressed exclusively in the L1 of shoot meristems and that recombinant PDF2 protein specifically binds to the L1 box in vitro. Although knockout mutants of PDF2 and ATML1, another L1-specific HD-GL2 class gene sharing the highest homology with PDF2, display normal shoot development, the double mutant results in severe defects in shoot epidermal cell differentiation. This suggests that PDF2 and ATML1 are functionally interchangeable and play a critical role in maintaining the identity of L1 cells, possibly by interacting with their L1 box and those of downstream target-gene promoters.

DOI： 10.1242/dev.00292

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The Arabidopsis genome contains four genes that encode proteins similar to both spermidine synthase and spermine synthase of other organisms. Our previous study revealed that one of these genes, designated ACAULIS5 (ACL5), encodes spermine synthase and that its null mutation results in a severe defect in the elongation of stem internodes. Here we report the characterization of the other three genes, designated SPDS1, SPDS2 and SPDS3. Our results showed that SPDS1 and SPDS2 possess spermidine synthase activity in yeast spermidine synthase-deficient mutants, but the enzyme activity of SPDS3 remained to be determined. RNA gel blot analysis revealed that all of these genes are expressed in all plant organs but show different responses to exogenous plant hormones, suggesting that they are involved in different aspects of growth by modulating the contents of polyamines in plant cells. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0014-5793(02)03217-9

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

The genesis of phyllotaxis, which often is associated with the Fibonacci series of numbers, is an old unsolved puzzle in plant morphogenesis. Here, we show that disruption of an Arabidopsis topoisomerase (topo) I gene named TOP1alpha affects phyllotaxis and plant architecture. The divergence angles and internode lengths between two successive flowers were more random in the top1alpha mutant than in the wild type. The top1alpha plants sporadically produced multiple flowers from one node, and the number of floral organ primordia often was different. The mutation also caused the twisting of inflorescences; and individual flowers and the serration of leaf margins. These morphological abnormalities indicate that TOP1alpha may play a critical role in the maintenance of a regular pattern of organ initiation. The top1alpha mutant transformed with the RNA interference construct for TOP1beta, another topo I gene arrayed tandemly with TOP1alpha, was found to be lethal at young seedling stages, suggesting that topo I activity is essential in plants.

DOI： 10.1105/tpc.001925

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

Among the wild-type ecotypes of Arabidopsis thaliana vegetative shoot meristem to an inflorescence meristem is whose shape of inflorescence is categorized as raceme, the ecotype Landsberg harboring the erecta (er) mutation shows a corymb-like inflorescence, namely, a compact inflorescence with a flattened arrangement of flower buds at the tip. The fact that the ER gene encodes a receptor-like protein kinase implies the presence of a signaling cascade responsible for the inflorescence morphology of flowering plants. We report here the characterization of another mutant with a corymb-like inflorescence, named corymbosa2 (crm2), and the isolation of the CRM2 gene. While the er mutation causes a severe reduction in the length of pedicels, the crm2 mutation results in a significant delay in the initiation of internode elongation and in the development of flowers, despite having little effect on the timing of floral induction. Consequently, the number of flower buds is apparently increased at the tip of crm2 inflorescence. The crm2 er double mutant shows an additive phenotype. These results suggest that CRM2 and ER may act in different ways to generate wild-type inflorescence. The CRM2 gene was isolated by positional cloning and appears to encode a polypeptide with no significant homology to known sequences.

DOI： 10.1093/pcp/pcf036

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE LTD  

The Arabidopsis thaliana PROTODERMAL FACTOR1 (PDF1) gene encoding a putative extracellular proline-rich protein is exclusively expressed in the L1 layer of shoot apices and the protoderm of organ primordia. In order to identify essential cis-regulatory sequences required for the L1 layer-specific expression, a series of 5' deletions of the PDF1 promoter were fused to the beta -glucronidase (GUS) gene and introduced into Arabidopsis plants. Our analysis revealed that the minimum region necessary to confer L1-specific expression of PDF1 is confined within a 260-bp fragment upstream of the transcription start site. We identified an 8-bp motif in this region that is conserved between promoter regions of all the L1-specific genes so far cloned, and we designated it the L1 box. Electrophoretic mobility shift assays demonstrated that the L1-specific homeodomain protein ATML1 can bind to the L1 box sequence in vitro. The GUS expression in transgenic plants disappeared when a mutation that abolishes binding of ATML1 was introduced into the PDF1 /1 box sequence of the construct. These results suggest that the L1 box plays a crucial role in the regulation of PDF1 expression in L1 cells and that ATML1 could cooperate to drive L1-specific expression.

DOI： 10.1046/j.1365-313X.2001.01047.x

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Polyamines have been implicated in a wide range of biological processes, including growth and development in bacteria and animals, but their function in higher plants is unclear. Here we show that the Arabidopsis ACAULIS5 (ACL5) gene, whose inactivation causes a defect in the elongation of stem internodes by reducing cell expansion, encodes a protein that shares sequence similarity with the polyamine biosynthetic enzymes spermidine synthase and spermine synthase, Expression of the recombinant ACL5 protein in Escherichia coli showed that ACL5 possesses spermine synthase activity. Restoration of the acl5 mutant phenotype by somatic reversion of a transposon-induced allele suggests a non-cell-autonomous function for the ACL5 gene product, We also found that expression of the ACL5 cDNA under the control of a heat shock gene promoter in acl5 mutant plants restores the phenotype in a heat shock-dependent manner. The results of the experiments showed that polyamines play an essential role in promotion of internode elongation through cell expansion in Arabidopsis, We discuss the relationships to plant growth regulators such as auxin and gibberellins that have related functions.

DOI： 10.1093/emboj/19.16.4248

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This paper describes expression profiles of the Arabidopsis HSP18.2 heat-shock gene promoter by using three different reporter genes, and the application of this promoter to a method we have developed to drive heat-shock-dependent transcription of plant genome DNA flanked by T DNA insertions. We show that, irrespective of the location of the T-DNA insertion, an HSP18.2 promoter towards the left border of the T-DNA effectively induces transcription of flanking genome sequences in response to heat shock, if polyadenylated, tagged transcripts can be easily identified by RT-PCR.

DOI： 10.1046/j.1365-313X.2000.00716.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Plant Physiologists  

Receptor-like protein kinases (RLKs) are encoded by a divergent multigene family and their functions have been implicated in a wide range of signal transduction pathways. In this study, we examined the effect of salicylic acid (SA) on the expression of RLK genes in Arabidopsis thaliana. RNA gel blot analysis revealed that transcripts of RKC1 and a number of its homologs, whose translation products contain C-X8-C-X2-C motifs in the putative extracellular domain, accumulated to a higher level in response to SA treatment of plants. The chimeric fusion between the RKC1 5'-upstream region and the β-glucuronidase (GUS) reporter gene reproduced the SA responsiveness in transgenic plants. In addition, some of RLK genes of the leucine-rich repeat (LRR) class and those of the S-domain class were also induced by SA. We found that the upstream regions of these SA-responsive RLK genes contain the TTGAC sequence, which has been suggested to be important for induced expression of many plant defense genes. These results suggest the involvement of a number of RLKs in SA-mediated defense responses.

DOI： 10.1093/pcp/pcd028

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A mutant of Arabidopsis with reduced internodal cell length, acaulis5 (acl5), has recently been shown to have reduced transcript levels of a gene for endoxyloglucan transferase, EXGT-A1 (Y. Hanzawa, T. Takahashi, Y. Komeda [1997] Plant J 12: 863-874). In the present study, we cloned genomic fragments of five members of the EXGT gene family, EXGT-A1, EXGT-A3, EXGT-A4, XTR2, and XTR3, and examined their expression in the wild type and in a series of acl mutants. In wild-type plants, the EXGT-A3 gene showed higher expression in lower internodes (internodes between nodes bearing axillary shoots) than in upper and young internodes, in which EXGT-A1 was highly expressed. EXGT-A4 was preferentially expressed in roots and XTR3 in siliques. The XTR2 gene was constitutively expressed. In acl1, acl3, and acl4 mutants, which have a severe defect in leaf expansion as well as in internode elongation, the EXGT-A1 gene showed reduced levels of expression before bolting of plants. In contrast, XTR3 was increased in these mutant seedlings. Reduction of EXGT-A1 expression was also detected after bolting of all acl mutants except acl2, whose growth defect is restricted to lower internodes. These results suggest the involvement of each EXGT in different aspects of organ development.

DOI： 10.1104/pp.121.3.715

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The shoot apical meristem unknown. In order to identify the genes that function in the SAM, we performed cDNA subtraction experiments between wild-type and terminal flower1 shoot apices. Here, we describe the cloning of a gene designated PDF1 (PROTODERMAL FACTOR1). In situ hybridization revealed that the expression of PDF1 is exclusively limited to the L1 layer of vegetative, inflorescence and floral meristems and to the protoderm of organ primordia. By contrast, P (SAM) of Arabidopsis thaliana constitutes the tunica of L1 and L2 and the corpus represented by L3 cells. Regulatory networks involved in establishing and maintaining this structure of shoot meristems remain largelyDF1 shows no detectable level of expression in the epidermis of mature organs. Specific expression of the PDF1 gene in protodermal cells is also observed during embryogenesis. The deduced amino acid sequence of PDF1 shares no significant homology with that of other known proteins but contains a putative signal peptide and novel proline-rich repeat motifs, suggesting a cell-wall protein. Possible roles of the PDF1 gene in the SAM are discussed.

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In Arabidopsis thaliana (L.) Heynh, the mutation in ERECTA is known to confer a compact inflorescence by a reduction in the lengths of internodes and pedicels. We analyzed the expression pattern of this gene during plant development. In situ hybridization and histochemical analysis using transgenic plants carrying chimeric gene fusions, with the ERECTA promoter fused to the β-glucuronidase (GUS) gene, showed that ERECTA was predominantly expressed in the shoot apical meristems and organ primordia. ERECTA expression in the shoot apical meristem was weak early in plant development but increased with the transition from the vegetative to the reproductive growth phase. ERECTA was also strongly expressed in organ primordia and immature organs but weakly in mature organs. Thus, ERECTA was expressed in a cell-specific and developmentally regulated manner. In order to identify the regulatory mechanism responsible for the expression pattern of ERECTA, the cis-acting regions in the ERECTA promoter were defined by study of the expression of the chimeric genes that consist of the 5'- or internal deleted promoter and a GUS reporter gene in transgenic plants. The results showed that the essential cis-regulatory elements governing the spatially and temporally specific expression of ERECTA are located between positions -462 and -228 bp and between positions -228 and -153 bp with respect to the transcriptional initiation site.

DOI： 10.1046/j.1365-313X.1998.00203.x

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We have isolated three receptor-like kinase cDNAs from an Arabidopsis flower cDNA library by PCR using degenerate oligonucleotide primers for conserved domains of protein kinases. Cloning and sequencing of the full-length cDNAs, designated RKF1 to 3 (receptor-like kinase in flowers), showed that the putative extracellular domain of the RKF1 protein contains 13 tandem repeats of leucine-rich sequences and those of RKF2 and RKF3 have no significant homology with other plant sequences. RNA blot analysis revealed that the RKF1 mRNA is highly expressed in stamens while RKF2 and RKF3 mRNAs are present at low levels in all organs examined. In situ localization experiments indicated that the RKF1 mRNA is detectable in early flower primordia and during stamen development. In addition, when fused to a GUS reporter gene, the RKF1 promoter directed high GUS expression in pollen grains. Recombinant RKF1, produced in Escherichia coli, was found to have kinase activity with serine/threonine specificity in vitro.

DOI： 10.1023/A:1005924817190

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

In rosette plants, the formation of initial flowers is closely linked to the lengthening of internodes (bolting). In order to clarify the molecular basis of bolting, mutants with reduced lengths of internodes were screened. This paper presents the identification and characterization of recessive mutations in ACAULIS5 (ACL5), a gene required for internodal growth in Arabidopsis thaliana. Unlike previously described mutants with reduced size of organs, the acl5 mutant has a severe defect that is restricted to the process of cell elongation after transition to the reproductive stage and shows no phenotype before floral induction. The results of RNA blot hybridizations showed that the acl5 mutation causes a striking reduction in the transcript levels of genes encoding the tonoplast intrinsic protein (γ-TIP) and the endoxyloglucan transferase (EXGT-A1), both of which have recently been suggested to be important for cell elongation. Furthermore, our morphological study indicates that the mutation also causes proliferative arrest of the apical inflorescence meristem. These results strongly suggest that, during the reproductive phase, the wild-type ACL5 gene product has a critical function not only in the control of elongation growth of organs but also in the continued maintenance of the proliferative activity of flower-producing meristems.

DOI： 10.1046/j.1365-313X.1997.12040863.x

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

We have isolated a recessive mutation named diminuto (dim) from T-DNA transformed lines of Arabidopsis thaliana. Under normal growth conditions, the dim mutant has very short hypocotyls, petioles, stems, and roots because of the reduced size of cells along the longitudinal axes of these organs. In addition, dim results in the development of open cotyledons and primary leaves in dark-grown seedlings. The gene for DIM was cloned by T-DNA tagging. DIM encodes a novel protein of 561 amino acids that possesses bipartite sequence domains characteristic of nuclear localization signals. Molecular and physiological studies indicate that the loss-of-function mutant allele does not abolish the response of seedlings to light or phytohormones, although the inhibitory effect of light on hypocotyl elongation is greater in the mutant than in wild type. Moreover, the dim mutation affects the expression of a beta-tubulin gene, TUB1, which is thought to be important for plant cell growth. Our results suggest that the DIM gene product plays a critical role in the general process of plant cell elongation.

DOI： 10.1101/gad.9.1.97

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We have isolated three HSP90-family genes from Arabidopsis: HSP81-1 which is heat-inducible, and HSP81-2 and -3 which are highly expressed under normal growth temperatures. Northern blot analysis and RNase protection analysis, using gene specific probes, showed that HSP81-2 and -3 mRNA were present in all tissues and abundant in roots, floral bud clusters, and flowers at 22 degrees C. A small amount of HSP81-1 mRNA was detected only in roots. In situ hybridization and histochemical analysis using transgenic plants carrying chimeric gene fusions, with an HSP81 promotor region fused to a beta-glucuronidase (GUS) gene, confirmed these results. At 22 degrees C, high GUS activity was observed in the root apical meristems, pollen and tapeta in HSP81-2::GUS and HSP81-3::GUS transgenic plants, while only branches of the root in HSP81-1::GUS transgenic plants expressed high GUS activity. After 2 hours of 35 degrees C treatment, extensively high GUS activity was observed in all tissues in HSP81-1::GUS transgenic plants, while elevated but tissue specific expression was observed in HSP81-2 and -3 transgenic plants.
Exogenous application of various chemicals such as ABA, GA(3), kinetin, IAA, NaCl, and mannitol revealed that 10 mM IAA and 0.1 M NaCl significantly enhanced the accumulation of HSP81-2 and -3 transcripts. Only a slight response to IAA was observed in HSP81-1 mRNA accumulation at 22 degrees C; the increase was possibly caused by a novel pathway other than heat-shock-response pathway.

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Production of a functional β-glucuronidase (GUS) protein was induced by exposure of exponentially growing yeast cells to heat shock after transformation of the GUS gene under the control of the promoter of the heat-shock gene, HSP18.2, from Arabidopsis. Yeast cyr and bcy mutations appeared to have essentially no effect.

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A detailed study of the expression of the promoter of the HSP18.2 gene from Arabidopsis fused to the bacterial gene for β‐glucuronidase (GUS) in transgenic Arabidopsis plants is described. High levels of GUS activity were induced in all organs of transformants except for seeds during heat shock. The optimum temperature for expression of GUS in Arabidopsis was 35°C regardless of the plant growth temperature. Heat shock of 40°C did not induce any detectable levels of GUS activity. Pre‐incubation at 35°C was found to have a protective effect on the induction of GUS activity at 40°C. GUS activity was also increased in response to a gradual increase in temperature. Histochemical analysis revealed that basal levels of GUS activity were induced in the vascular tissue of leaves and sepals, as well as at the tips of carpels, at the normal growth temperature. Heat treatment of a limited part of the plant tissue did not appear to cause systemic induction of GUS activity. To extend the analysis of the plant heat‐shock response, we attempted to screen mutations in genes involved in the regulation of the induction of heat‐shock protein (HSP) genes, using the GUS gene as a selection marker in transgenic Arabidopsis plants, and the results of this analysis are described. 1992 BIOS Scientific Publishers Ltd

DOI： 10.1111/j.1365-313X.1992.tb00144.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society of Plant Biologists  

We have cloned and characterized two members of the family of genes for the 81-kilodalton heat-shock proteins from Arabidopsis thaliana, HSP81-1 and HSP81-2. Comparison of the entire genomic sequence of the HSP81-1 gene with the corresponding full-length cDNA previously reported as AtHS83 (TW Conner, PR LaFayette, RT Nagao, JL Key [1990] Plant Physiol 94: 1689-1695) reveals the presence of three introns of 315, 83, and 88 base pairs. By contrast, analysis of the HSP81-2 genomic and partial cDNA sequences suggests that the HSP81-2 gene is interrupted by only two introns of 304 and 106 base pairs. The 5′-initiation sites of the two corresponding mRNAs were mapped from results of experiments with S1 nuclease. The deduced amino acid sequences of the proteins encoded by these two genes show 88% identity, and they also show striking similarities to the hsp90 family of proteins in yeast and animal cells. From the results of northern blot analysis of transcripts, it appears that the expression of the HSP81-1 gene occurs at only very low levels in the absence of heat shock and is strongly induced by heat (35°C). The HSP81-2 gene is constitutively expressed at much higher levels, and its expression is moderately enhanced by elevated temperatures. Severe heat shock appears to block the splicing of the pre-mRNA transcribed from HSP81-1. We also examined the effects of arsenite and cadmium on the expression of the HSP81 genes, as well as on other groups of hsp genes in Arabidopsis. Treatment with cadmium was marginally effective in inducing hsp genes, whereas arsenite stress strongly stimulated the accumulation of each mRNA in a coordinated fashion.

DOI： 10.1104/pp.99.2.383

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A cDNA clone containing a 1.2-kb fragment derived from the ribosomal DNA (rDNA) intergenic spacer (IGS) of Arabidopsis was characterized. Isolation of this clone was achieved in a previous study by differential screening of a cDNA library prepared from heat-shocked Arabidopsis seedlings (Takahashi and Komeda 1989). Nucleotide sequence analysis revealed that the cDNA had an oligo(dA) sequence that corresponded to the sequence found in the IGS. Northern blot analysis using the cloned cDNA as probe demonstrated that heat-shock treatment of seedlings induced the accumulation of extremely long transcripts with apparent lengths of more than 7 kb. Treatment with arsenite and cadmium ions also led to the accumulation of the RNA, a phenomenon very similar to that observed in the case of expression of genes for heat-shock proteins (HSPs). Although the possibility remains to be excluded that these transcripts might be derived from,a region homologous to the IGS Iocated outside the rDNA clusters in Arabidopsis, it is most likely that processing of precursor rRNA and/or termination of transcription of rRNA are greatly impaired under stressed conditions in plants, as reported in the case of animal genes for rRNA (Labhart and Reeder 1987, Parker and Bond 1989).

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DOI： 10.1007/BF00259608

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DOI： 10.14841/jspp.2011.0.0110.0

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DOI： 10.14841/jspp.2006.0.053.0

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DOI： 10.14841/jspp.2005.0.515.0

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Language：English   Publisher：SPRINGER VERLAG  

Organ-specific and constitutive expression of the Arabidosis HSP18.2 gene under normal growth conditions (22-degrees-C) was observed in transgenic A. thaliana, which carried a fusion gene composed of the promoter region of HSP18.2, one of the genes for low molecular weight heat-shock proteins in Arabidopsis, and the gene for beta-glucuronidase (GUS) from Escherichia coli. In order to clarify the organ-specific nature of promoter expression, various mutations that affect flower morphology were introduced into the transgenic Arabidopsis line, AHS9. The results show that the pattern of expression observed in sepals, filaments, and styles is regulated in a structure-dependent manner, and suggest that the HSP18.2 gene might have an important role in the process of differentiation of flower buds, as do several other stress-related genes.

DOI： 10.1007/BF00280197

Web of Science

PubMed

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