Springer Science and Business Media LLC Acta Medica Okayama 2399-3642 9 1 2026 Structural study of monomeric and dimeric photosystem I-LHCI supercomplexes from a bryophyte 146 EN Pi-Cheng Tsai Research Institute for Interdisciplinary Science, Advanced Research Field, Okayama University Romain La Rocca Research Institute for Interdisciplinary Science, Advanced Research Field, Okayama University Hiroyasu Motose Department of Biology, Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Jian-Ren Shen Research Institute for Interdisciplinary Science, Advanced Research Field, Okayama University Fusamichi Akita Research Institute for Interdisciplinary Science, Advanced Research Field, Okayama University Photosystem I (PSI) is one of the two photosystems conserved from cyanobacteria to vascular plants, and associates with multiple light-harvesting complexes (LHCs) that capture and transfer solar energy. Liverworts such as Marchantia polymorpha occupy an early evolutionary position among land plants and faced major challenges during terrestrial adaptation, including desiccation, strong light, and UV radiation. We reveal the cryo-electron microscopic structures of PSI-LHCI monomer and homodimer from the liverwort M. polymorpha at resolutions of 1.94 and 2.52 Å, respectively. The high-resolution map allows identification of the cofactors of the monomer and reveal differences between the liverwort and moss, another clade of bryophytes. The PSI-LHCI monomer-monomer is stabilized by PsaG and PsaH interactions on the stromal side, which causes the bending and twisting of the homodimer. PsaM interacts with PsaB tightly, indicating a key role of PsaM in mediating the dimerization. No potential conflict of interest relevant to this article was reported.

Frontiers Media SA Acta Medica Okayama 1664-462X 16 2025 The response to thermospermine is fine-tuned by the balance between SAC51 and LHW family proteins in Arabidopsis thaliana EN Yao Xu Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Mitsuru Saraumi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Tomohiko Toyoshima Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Hiroyasu Motose Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Taku Takahashi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Thermospermine negatively regulates xylem formation. In Arabidopsis, SAC51 and SACL3, members of the SAC51 gene family encoding basic loop-helix-loop (bHLH) proteins play a key role in this regulation. These mRNAs contain an upstream open-reading-frame (uORF) that is highly conserved across species, and its inhibitory effect on the main ORF translation is alleviated by thermospermine. A double knockout of SAC51 and SACL3 results in thermospermine insensitivity at high concentrations that normally inhibit xylem formation and shoot growth in the wild type. Conversely, uORF mutants of SAC51, SACL3, and SACL1 suppress the excessive xylem formation and dwarf phenotype of acl5, a mutant defective in thermospermine biosynthesis. In this study, we generated genome-edited uORF mutants of SACL2 and confirmed that they partially recover the acl5 phenotype. All uORF mutants exhibited increased sensitivity to thermospermine. SACL3 represses the function of LHW, a key bHLH transcription factor required for xylem proliferation, through direct interaction. We found that the lhw mutant is also hypersensitive to thermospermine, while this sensitivity was suppressed by the sac51 sacl3 double knockout. Yeast two-hybrid assays demonstrated that all four SAC51 family members interact with LHW and its family members. These findings suggest that overaccumulation of SAC51 family proteins leads to thermospermine hypersensitivity by repressing the function of LHW family proteins, whose activity must be fine-tuned to ensure proper xylem development. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0960-7412 123 5 2025 RNA processing/modifying enzymes play key roles in the response to thermospermine in Arabidopsis thaliana e70476 EN Mitsuru Saraumi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Takahiro Tanaka Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Daiki Koyama Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yoshitaka Nishi Graduate School of Engineering, Kyushu Sangyo University Yoshihiro Takahashi Department of Life Science, Faculty of Life Science, Kyushu Sangyo University Hiroyasu Motose Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Taku Takahashi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Thermospermine is involved in negative regulation of xylem differentiation by enhancing the translation of mRNAs of the SAC51 gene family in Arabidopsis (Arabidopsis thaliana). These mRNAs contain conserved upstream open reading frames (uORFs) that interfere with the translation of the main ORF. To investigate the mechanism by which thermospermine acts in this process, we isolated mutants insensitive to thermospermine, named ‘its’. We show that the four genes responsible for these mutants, its1 to its4, encode: (i) a homolog of SPOUT RNA methyltransferase, (ii) an rRNA pseudouridine synthase CBF5/NAP57, (iii) a putative spliceosome disassembly factor STIPL1/NTR1, and (iv) a plant-specific RNA-binding protein PHIP1. These four mutants were found to have much higher levels of thermospermine than the wild-type. While all these mutants except its1 appear almost normal, they enhance the dwarf phenotype of a mutant of ACL5, which encodes thermospermine synthase, resulting in tiny plants resembling a double knockout of ACL5 and SACL3, a member of the SAC51 family. Reporter assays revealed that GUS activity from the CaMV 35S promoter-SAC51 5′-GUS fusion construct was significantly reduced in its1 and its4 or not affected in its2 and its3, while it was slightly increased in its1, its3, and its4, or not changed in its2 by thermospermine. These findings underscore the critical role of RNA processing and modification in the thermospermine-dependent translational regulation of uORF-containing transcripts. No potential conflict of interest relevant to this article was reported.

Taylor and Francis Group Acta Medica Okayama 1559-2316 20 1 2025 Specific enhancement of the translation of thermospermine-responsive uORF-containing mRNAs by ribosomal mutations in Arabidopsis thaliana 2480231 EN Koki Mutsuda Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yuichi Nishii Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Tomohiko Toyoshima Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Hiroko Fukushima Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Hiroyasu Motose Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Taku Takahashi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Auxin-induced xylem formation in angiosperms is negatively regulated by thermospermine, whose biosynthesis is also induced by auxin. In Arabidopsis thaliana, loss-of-function mutants of ACL5, which encodes thermospermine synthase, exhibit a dwarf phenotype accompanied by excessive xylem formation. Studies of suppressor mutants that recover from the acl5 dwarf phenotype suggest that thermospermine alleviates the inhibitory effect of an upstream open-reading frame (uORF) on the main ORF translation of SAC51 mRNA. Many suppressor mutations for acl5 have been mapped to the uORF conserved in the SAC51 family or to ribosomal protein genes, such as RPL10A, RPL4A, and RACK1A. In this study, we identified newly isolated acl5 suppressors, sac501, sac504, and sac506, which are additional alleles of RPL10A and the uORFs of SAC51 family members, SACL1 and SACL3, respectively. To investigate whether acl5-suppressor alleles of ribosomal genes broadly affect translation of uORF-containing mRNAs, we examined GUS activity in several 5'-GUS fusion constructs. Our results showed that these alleles enhanced GUS activity in SAC51 and SACL3 5'-fusion constructs but had no effect on other 5'-fusion constructs unrelated to thermospermine response. This suggests that these ribosomal proteins are specifically involved in the thermospermine-mediated regulation of mRNA translation. No potential conflict of interest relevant to this article was reported.

Taylor and Francis Acta Medica Okayama 1559-2316 18 1 2023 Microtubule-associated proteins WDL5 and WDL6 play a critical role in pollen tube growth in Arabidopsis thaliana 2281159 EN Takashi Okamoto Department of Biological Science, Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Hiroyasu Motose Department of Biological Science, Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Taku Takahashi Department of Biological Science, Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Morphological response of cells to environment involves concerted rearrangements of microtubules and actin microfilaments. A mutant of WAVE-DAMPENED2-LIKE5 (WDL5), which encodes an ethylene-regulated microtubule-associated protein belonging to the WVD2/WDL family in Arabidopsis thaliana, shows attenuation in the temporal root growth reduction in response to mechanical stress. We found that a T-DNA knockout of WDL6, the closest homolog of WDL5, oppositely shows an enhancement of the response. To know the functional relationship between WDL5 and WDL6, we attempted to generate the double mutant by crosses but failed in isolation. Close examination of gametophytes in plants that are homozygous for one and heterozygous for the other revealed that these plants produce pollen grains with a reduced rate of germination and tube growth. Reciprocal cross experiments of these plants with the wild type confirmed that the double mutation is not inherited paternally. These results suggest a critical and cooperative function of WDL5 and WDL6 in pollen tube growth. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0014-5793 596 23 2022 Loss of function of an Arabidopsis homologue of JMJD6 suppresses the dwarf phenotype of acl5, a mutant defective in thermospermine biosynthesis 3005 3014 EN Hirotoshi Matsuo Graduate School of Natural Science and Technology, Okayama University Hiroko Fukushima Graduate School of Natural Science and Technology, Okayama University Shinpei Kurokawa Graduate School of Natural Science and Technology, Okayama University Eri Kawano Graduate School of Natural Science and Technology, Okayama University Takashi Okamoto Graduate School of Natural Science and Technology, Okayama University Hiroyasu Motose Graduate School of Natural Science and Technology, Okayama University Taku Takahashi Graduate School of Natural Science and Technology, Okayama University In Arabidopsis thaliana, the ACL5 gene encodes thermospermine synthase and its mutant, acl5, exhibits a dwarf phenotype with excessive xylem formation. Studies of suppressor mutants of acl5 reveal the involvement of thermospermine in enhancing mRNA translation of the SAC51 gene family. We show here that a mutant, sac59, which partially suppresses the acl5 phenotype, has a point mutation in JMJ22 encoding a D6-class Jumonji C protein (JMJD6). A T-DNA insertion allele, jmj22-2, also partially suppressed the acl5 phenotype while mutants of its closest two homologs JMJ21 and JMJ20 had no such effects, suggesting a unique role for JMJ22 in plant development. We found that mRNAs of the SAC51 family are more stabilized in acl5 jmj22-2 than in acl5. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 1434193X 2020 18 2020 Chemical Synthesis and Biological Effect on Xylem Formation of Xylemin and Its Analogues 2745 2753 EN Hiroyoshi Takamura Department of Biological Science, Graduate School of Natural Science and Technology, Okayama University Hiroyasu Motose Graduate School of Natural Science and Technology, Okayama University Taichi Otsu Department of Chemistry, Graduate School of Natural Science and Technology, Okayama University Shiori Shinohara Department of Biological Science, Graduate School of Natural Science and Technology, Okayama University Ryugo Kouno Department of Biological Science, Graduate School of Natural Science and Technology, Okayama University Isao Kadota Department of Chemistry, Graduate School of Natural Science and Technology, Okayama University Taku Takahashi Department of Biological Science, Graduate School of Natural Science and Technology, Okayama University 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. No potential conflict of interest relevant to this article was reported.

Japanese Society of Plant Physiologists Acta Medica Okayama 0032-0781 57 8 2016 The SAC51 Family Plays a Central Role in Thermospermine Responses in Arabidopsis 1583 1592 EN Qingqing Cai Graduate School of Natural Science and Technology, Okayama University Hiroko Fukushima Graduate School of Natural Science and Technology, Okayama University Mai Yamamoto Graduate School of Natural Science and Technology, Okayama University Nami Ishii Graduate School of Natural Science and Technology, Okayama University Tomoaki Sakamoto Graduate School of Biological Sciences, Nara Institute of Science and Technology Tetsuya Kurata Graduate School of Biological Sciences, Nara Institute of Science and Technology Hiroyasu Motose Graduate School of Natural Science and Technology, Okayama University Taku Takahashi Graduate School of Natural Science and Technology, Okayama University 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 (β-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. No potential conflict of interest relevant to this article was reported.

Nature Publishing Group Acta Medica Okayama 20452322 7 2017 Directional cell expansion requires NIMA-related kinase 6 (NEK6)-mediated cortical microtubule destabilization; 7826 EN Shogo Takatani Department of Biological Science, Graduate School of Natural Science and Technology, Okayama University Shinichiro Ozawa Department of Biological Science, Graduate School of Natural Science and Technology, Okayama University Noriyoshi Yagi Graduate School of Biological Science, Nara Institute of Science and Technology Takashi Hotta Graduate School of Biological Science, Nara Institute of Science and Technology Takashi Hashimoto Graduate School of Biological Science, Nara Institute of Science and Technology Yuichiro Takahashi Graduate School of Natural Science and Technology/Faculty of Science, Okayama University Taku Takahashi Department of Biological Science, Graduate School of Natural Science and Technology, Okayama University Hiroyasu Motose Department of Biological Science, Graduate School of Natural Science and Technology, Okayama University 　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 β-tubulin that serve as substrates for NEK6 in vitro. Alanine substitution of the phosphorylation site Thr166 promoted incorporation of mutant β-tubulin into microtubules. Taken together, these results suggest that NEK6 promotes directional cell growth through phosphorylation of β-tubulin and the resulting destabilization of cortical microtubules. No potential conflict of interest relevant to this article was reported.

Nature Publishing Acta Medica Okayama 2045-2322 6 2016 Chemical control of xylem differentiation by thermospermine, xylemin and auxin 21487 EN Kaori Yoshimoto Department of Biological Science, Graduate School of Natural Science and Technology, Okayama University Hiroyoshi Takamura Department of Chemistry, Graduate School of Natural Science and Technology, Okayama University Isao Kadota Department of Chemistry, Graduate School of Natural Science and Technology, Okayama University Hiroyasu Motose Department of Biological Science, Graduate School of Natural Science and Technology, Okayama University Taku Takahashi Department of Biological Science, Graduate School of Natural Science and Technology, Okayama University 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. No potential conflict of interest relevant to this article was reported.

Macmillan Publishers Limited. Acta Medica Okayama 2045-2322 6 2016 Chemical control of xylem differentiation by thermospermine, xylemin, and auxin 21487 EN Kaori Yoshimoto Hiroyoshi Takamura Isao Kadota Hiroyasu Motose Department of Biological Science, Graduate School of Natural Science and Technology, Okayama University Taku Takahashi 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. No potential conflict of interest relevant to this article was reported.

Springer Japan Acta Medica Okayama 0918-9440 128 6 2015 Structure, function, and evolution of plant NIMA-related kinases: implication for phosphorylation-dependent microtubule regulation 875 891 EN Shogo Takatani Kento Otani Mai Kanazawa Taku Takahashi Division of Bioscience, Graduate School of Natural Science and Technology, Okayama University Hiroyasu Motose Department of Biology, Faculty of Science, Okayama University icrotubules 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 β-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. No potential conflict of interest relevant to this article was reported.

Nature Publishing Group Acta Medica Okayama 2045-2322 5 2015 Abscisic acid induces ectopic outgrowth in epidermal cells through cortical microtubule reorganization in Arabidopsis thaliana 11364 EN Shogo Takatani Takashi Hirayama Takashi Hashimoto Taku Takahashi Hiroyasu Motose 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. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1559-2316 7 12 2012 NIMA-related kinases regulate directional cell growth and organ development through microtubule function in Arabidopsis thaliana. 1552 1555 EN Hiroyasu Motose Shogo Takatani Tatsuya Ikeda Taku Takahashi 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. No potential conflict of interest relevant to this article was reported.

RSC Publishing Acta Medica Okayama 1759-9660 5 11 2013 Determination of polyamines in Arabidopsis thaliana by capillary electrophoresis using salicylaldehyde-5-sulfonate as a derivatizing reagent 2854 2859 EN Genki Inoue Takashi Kaneta Toshio Takayanagi Junichi Kakehi Hiroyasu Motose Taku Takahashi 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-μM 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 μM and correlation coefficients (R2) were greater than 0.998. The proposed method was applied to the determination of spermidine in a plant sample, Arabidopsis thaliana. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 0032-0781 53 4 2012 A chemical biology approach reveals an opposite action between thermospermine and auxin in xylem development in Arabidopsis thaliana 635 645 EN Kaori Yoshimoto Division of Bioscience, Graduate School of Natural Science and Technology, Okayama University Yoshiteru Noutoshi Ken-ichiro Hayashi Ken Shirasu Taku Takahashi Hiroyasu Motose 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. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1559-2316 7 8 2012 Thermospermine suppresses auxin-inducible xylem differentiation in Arabidopsis thaliana 937 939 EN Kaori Yoshimoto Yoshiteru Noutoshi Ken-ichiro Hayashi Ken Shirasu Taku Takahashi Hiroyasu Motose 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 xylem-inducing 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. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 0960-7412 67 6 2011 NIMA-related kinases 6, 4, and 5 interact with each other to regulate microtubule organization during epidermal cell expansion in Arabidopsis thaliana 993 1005 EN Hiroyasu Motose Takahiro Hamada Kaori Yoshimoto Takashi Murata Mitsuyasu Hasebe Yuichiro Watanabe Takashi Hashimoto Tatsuya Sakai Taku Takahashi 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. No potential conflict of interest relevant to this article was reported.