Watahiki Masaaki

Faculty of Science　Biological Sciences　Cell Structure and FunctionAssociate Professor
Last Updated :2025/03/17

■Researcher basic information

Researchmap personal page

Home Page URL

J-Global ID

Research Keyword

  • Aux
  • オーキシン
  • 伸長成長
  • 偏差成長
  • タンパク質分解調節
  • 受粉
  • オーキシン応答因子
  • 蛍光相関分光法
  • イメージング
  • 組織特異性
  • 屈地性
  • 花糸
  • 側根形成
  • 化学発光
  • 根端分裂組織
  • 転写調節
  • IAAタンパク質
  • 細胞核-細胞質移行
  • タンパク質分解
  • 転写調節ネットワーク
  • 分子遺伝学
  • IAA
  • 信号統御
  • 稔性
  • 雄蘂
  • ルシフェラーゼ融合タンパク質
  • 屈性

Research Field

  • Life sciences, Plants: molecular biology and physiology

Educational Organization

■Career

Career

  • Apr. 2010 - Present
    Hokkaido University, Faculty of Science, Associate professor
  • Oct. 2009 - Mar. 2010
    Hokkaido University, Advanced Life science, Associate professor
  • Apr. 2006 - Sep. 2009
    Hokkaido University, Fafulty of Science, Assistant professor
  • Oct. 2004 - Mar. 2006
    Hokkaido University, Faculty of Science, Assistant professor
  • Aug. 1998 - Sep. 2004
    University of Edinburgh, ICMB, PDRA
  • Apr. 1996 - Mar. 1998
    JSPP, fellow

Educational Background

  • Apr. 1994 - Mar. 1997, Graduate School of Hokkaido University, Environmental Earth Science, Environmental molecular Biology
  • Apr. 1992 - Mar. 1994, Graduate School of Hokkaido University, Faculty of Science, Plant biology course
  • Hokkaido University, Faculty of Science, Plant ｂiology course

■Research activity information

Papers

  • Exogenous application of the apocarotenoid retinaldehyde negatively regulates auxin-mediated root growth.
    Kang Xu, Haoran Zeng, Feiyang Lin, Emi Yumoto, Masashi Asahina, Ken-Ichiro Hayashi, Hidehiro Fukaki, Hisashi Ito, Masaaki K Watahiki
    Plant physiology, 09 Aug. 2024, [Peer-reviewed], [Corresponding author], [International Magazine]
    English, Scientific journal, Root development is essential for plant survival. The lack of carotenoid biosynthesis in the phytoene desaturase 3 (pds3) mutant results in short primary roots (PR) and reduced lateral root (LR) formation. In this study, we showed that short-term inhibition of PDS by fluridone suppresses PR growth in WT, but to a lesser extent in auxin mutants of Arabidopsis (Arabidopsis thaliana). Such an inhibition of PDS activity increased endogenous indole-3-acetic acid (IAA) levels, promoted auxin signaling, and partially complemented the PR growth of an auxin deficient mutant, the YUCCA 3 5 7 8 9 quadruple mutant (yucQ). The exogenous application of retinaldehyde (retinal), an apocarotenoid derived from β-carotene, complemented the fluridone-induced suppression of root growth, as well as the short roots of the pds3 mutant. Retinal also partially complemented the auxin-induced suppression of root growth. These results suggest that retinal may play a role in regulating root growth by modulating endogenous auxin levels.
  • Periodic cytokinin responses in Lotus japonicus rhizobium infection and nodule development.
    Takashi Soyano, Akira Akamatsu, Naoya Takeda, Masaaki K Watahiki, Tatsuaki Goh, Nao Okuma, Norio Suganuma, Mikiko Kojima, Yumiko Takebayashi, Hitoshi Sakakibara, Keiji Nakajima, Masayoshi Kawaguchi
    Science (New York, N.Y.), 385, 6706, 288, 294, 19 Jul. 2024, [Peer-reviewed], [International Magazine]
    English, Scientific journal, Host plants benefit from legume root nodule symbiosis with nitrogen-fixing bacteria under nitrogen-limiting conditions. In this interaction, the hosts must regulate nodule numbers and distribution patterns to control the degree of symbiosis and maintain root growth functions. The host response to symbiotic bacteria occurs discontinuously but repeatedly at the region behind the tip of the growing roots. Here, live-imaging and transcriptome analyses revealed oscillating host gene expression with approximately 6-hour intervals upon bacterial inoculation. Cytokinin response also exhibited a similar oscillation pattern. Cytokinin signaling is crucial to maintaining the periodicity, as observed in cytokinin receptor mutants displaying altered infection foci distribution. This periodic regulation influences the size of the root region responsive to bacteria, as well as the nodulation process progression.
  • Carotenoid metabolism negatively regulates auxin-mediated root growth
    Kang Xu, Haoran Zeng, Emi Yumoto, Masashi Asahina, Ken-ichiro Hayashi, Hidehiro Fukaki, Hisashi Ito, Masaaki K. Watahiki
    12 Sep. 2023
  • Circadian Clock Controls Root Hair Elongation through Long-Distance Communication
    Hikari Ikeda, Taiga Uchikawa, Yohei Kondo, Nozomu Takahashi, Takuma Shishikui, Masaaki K. Watahiki, Akane Kubota, Motomu Endo
    PLANT AND CELL PHYSIOLOGY, OXFORD UNIV PRESS, Jul. 2023, [Peer-reviewed]
    English, Scientific journal, Plants adapt to periodic environmental changes, such as day and night, by using circadian clocks. Cell division and elongation are primary steps to adjust plant development according to their environments. In Arabidopsis, hypocotyl elongation has been studied as a representative model to understand how the circadian clock regulates cell elongation. However, it remains unknown whether similar phenomena exist in other organs, such as roots, where circadian clocks regulate physiological responses. Here, we show that root hair elongation is controlled by both light and the circadian clock. By developing machine-learning models to automatically analyze the images of root hairs, we found that genes encoding major components of the central oscillator, such as TIMING OF CAB EXPRESSION1 (TOC1) or CIRCADIAN CLOCK ASSOCIATED1 (CCA1), regulate the rhythmicity of root hair length. The partial illumination of light to either shoots or roots suggested that light received in shoots is mainly responsible for the generation of root hair rhythmicity. Furthermore, grafting experiments between wild-type (WT) and toc1 plants demonstrated that TOC1 in shoots is responsible for the generation of root hair rhythmicity. Our results illustrate the combinational effects of long-distance signaling and the circadian clock on the regulation of root hair length.
  • P-MIRU, a polarized-spectral imaging system, reveals reflection information on the biological surface
    Alfonso Balandra, Yuki Doll, Shogo Hirose, Tomoaki Kajiwara, Zendai Kashino, Masahiko Inami, Shizuka Koshimizu, Hidehiro Fukaki, Masaaki K Watahiki
    Plant And Cell Physiology, Oxford University Press (OUP), 22 May 2023, [Peer-reviewed], [Corresponding author]
    Scientific journal, Abstract

    The reflection light forms the core of our visual perception of the world. We can obtain vast information by examining reflection light from biological surfaces, including pigment composition and distribution, tissue structure, and surface microstructure. However, because of the limitations in our visual system, the complete information in reflection light, which we term “reflectome,” cannot be fully exploited. For example, we may miss reflection light information outside our visible wavelengths. In addition, unlike insects, we have virtually no sensitivity to light polarization. We can detect non-chromatic information lurking in reflection light only with appropriate devices. Though previous studies have designed and developed systems for specialized uses supporting our visual systems, we still do not have a versatile, rapid, convenient, and affordable system for analyzing broad aspects of reflection from biological surfaces. To overcome this situation, we developed P-MIRU, a novel multi-spectral and polarization imaging system for reflecting light from biological surfaces. The hardware and software of P-MIRU are open-source and customizable and thus can be applied for virtually any research on biological surfaces. Furthermore, P-MIRU is a user-friendly system for biologists with no specialized programming or engineering knowledge. P-MIRU successfully visualized multi-spectral reflection in visible/non-visible wavelengths and simultaneously detected various surface phenotypes of spectral polarization. P-MIRU system extends our visual ability and unveils information on biological surfaces. (217/250 words)
  • Conjugates of 3-phenyllactic acid and tryptophan enhance root-promoting activity without adverse effects in Vigna angularis.
    Yuko Maki, Hiroshi Soejima, Tamizi Sugiyama, Takeo Sato, Junji Yamaguchi, Masaaki K Watahiki
    Plant biotechnology (Tokyo, Japan), 39, 2, 173, 177, 25 Jun. 2022, [Peer-reviewed], [Corresponding author], [Domestic magazines]
    English, Scientific journal, 3-Phenyllactic acid (PLA) is a common secondary product of Lactobacillus sp. and promotes adventitious-root formation in Azuki beans (Vigna angularis). Root promotion activity of PLA is synergistically enhanced by tryptophan (Trp). In this study, stereoisomers of PLA and Trp amide conjugates and their alkyl esters were synthesized to investigate the structure-activity relationships on root-promotion activity. The rooting activity of D-PLA-L-Trp conjugate shows more than 40 times higher than that of the mixture of D-PLA and L-Trp. Modification of PLA-Trp with ethyl ester showed the highest activity at 3,400 times of a mixture of D-PLA and L-Trp. However, L-or D-PLA-D-Trp conjugate and the isopropyl ester of PLA-Trp conjugates, both lost the root promotion activity and implicated that a requirement for steric structure for PLA related root promotion mechanism. Unlike auxin substances, which are commonly used as rooting agents that displayed high activity in low concentrations, PLA-Trp ethyl ester exhibited far less phytotoxicity at high concentration of 1 mM, despite its high rooting activity. Innovation of PLA-Trp ethyl ester may be expected for agricultural aspects with low environmental impact.
  • 3-Phenyllactic acid is converted to phenylacetic acid and induces auxin-responsive root growth in Arabidopsis plants.
    Yuko Maki, Hiroshi Soejima, Tamizi Sugiyama, Masaaki K Watahiki, Takeo Sato, Junji Yamaguchi
    Plant biotechnology (Tokyo, Japan), 39, 2, 111, 117, 25 Jun. 2022, [Peer-reviewed], [Domestic magazines]
    English, Scientific journal, Many microorganisms have been reported to produce compounds that promote plant growth and are thought to be involved in the establishment and maintenance of symbiotic relationships. 3-Phenyllactic acid (PLA) produced by lactic acid bacteria was previously shown to promote root growth in adzuki cuttings. However, the mode of action of PLA as a root-promoting substance had not been clarified. The present study therefore investigated the relationship between PLA and auxin. PLA was found to inhibit primary root elongation and to increase lateral root density in wild-type Arabidopsis, but not in an auxin signaling mutant. In addition, PLA induced IAA19 promoter fused β-glucuronidase gene expression, suggesting that PLA exhibits auxin-like activity. The inability of PLA to promote degradation of Auxin/Indole-3-Acetic Acid protein in a yeast heterologous reconstitution system indicated that PLA may not a ligand of auxin receptor. Using of a synthetic PLA labeled with stable isotope showed that exogenously applied PLA was converted to phenylacetic acid (PAA), an endogenous auxin, in both adzuki and Arabidopsis. Taken together, these results suggest that exogenous PLA promotes auxin signaling by conversion to PAA, thereby regulating root growth in plants.
  • A PSTAIRE-type cyclin-dependent kinase controls light responses in land plants.
    Liang Bao, Natsumi Inoue, Masaki Ishikawa, Eiji Gotoh, Ooi-Kock Teh, Takeshi Higa, Tomoro Morimoto, Eggie Febrianto Ginanjar, Hirofumi Harashima, Natsumi Noda, Masaaki Watahiki, Yuji Hiwatashi, Masami Sekine, Mitsuyasu Hasebe, Masamitsu Wada, Tomomichi Fujita
    Science Advances, 8, 4, eabk2116, American Association for the Advancement of Science (AAAS), 28 Jan. 2022, [Peer-reviewed]
    Scientific journal, Light is a critical signal perceived by plants to adapt their growth rate and direction. Although many signaling components have been studied, how plants respond to constantly fluctuating light remains underexplored. Here, we showed that in the moss
    Physcomitrium
    (
    Physcomitrella
    )
    patens
    , the PSTAIRE-type cyclin-dependent kinase PpCDKA is dispensable for growth. Instead, PpCDKA and its homolog in
    Arabidopsis thaliana
    control light-induced tropisms and chloroplast movements by probably influencing the cytoskeleton organization independently of the cell cycle. In addition, lower PpCDKA kinase activity was required to elicit light responses relative to cell cycle regulation. Thus, our study suggests that plant CDKAs may have been co-opted to control multiple light responses, and owing to the bistable switch properties of PSTAIRE-type CDKs, the noncanonical functions are widely conserved for eukaryotic environmental adaptation.
  • 3-Phenyllactic acid, a root-promoting substance isolated from Bokashi fertilizer, exhibits synergistic effects with tryptophan
    Yuko Maki, Hiroshi Soejima, Toru Kitamura, Tamizi Sugiyama, Takeo Sato, Masaaki K. Watahiki, Junji Yamaguchi
    Plant Biotechnology, 38, 1, 9, 16, Japanese Society for Plant Cell and Molecular Biology, 25 Mar. 2021, [Peer-reviewed], [Domestic magazines]
    English, Scientific journal, Bokashi fertilizer, an organic fertilizer made of plant residue, has been used in Japan not only to fertilize plants but to regulate their growth. Lactic acid bacteria have been found to play an important role in the fermentation process of Bokashi, but the relationship between these bacteria and plant growth activity has not been clarified. Using the adzuki rooting assay, this study identified 3-phenyllactic acid (PLA) produced by lactic acid bacteria as a root promoting compound in Bokashi. PLA showed synergistic effect with tryptophan, but no stem elongation activity. Lactic acid bacteria produced equal quantities of the L- and D-forms of PLA, which have similar root promoting activity. PLA did not significantly affect the amount of endogenous indole-3-acetic acid (IAA), although the chemical structure of PLA is highly similar to that of L-2-aminooxy-3-phenypropionic acid (L-AOPP), which inhibits IAA biosynthesis. These results indicate that the root promoting activity of PLA is not simply due to its increase in the amount of active auxin.
  • ZEITLUPE enhances expression of PIF4 and YUC8 in the upper aerial parts of Arabidopsis seedlings to positively regulate hypocotyl elongation
    Aya Saitoh, Tomoyuki Takase, Hiroshi Abe, Masaaki Watahiki, Yuki Hirakawa, Tomohiro Kiyosue
    Plant Cell Reports, 40, 3, 479, 489, Springer Science and Business Media LLC, Mar. 2021, [Peer-reviewed], [International Magazine]
    English, Scientific journal, KEY MESSAGE: Microarray and genetic analyses reveal that ZTL induces the expression of genes related to auxin synthesis, thereby promoting hypocotyl elongation. ZTL is a blue-light receptor that possesses a light-oxygen-voltage-sensing (LOV) domain, an F-box motif, and a kelch repeat domain. ZTL promotes hypocotyl elongation under high temperature (28 °C) in Arabidopsis thaliana; however, the mechanism of this regulation is unknown. Here, we divided seedlings into hypocotyls and upper aerial parts, and performed microarray analyses. In hypocotyl, 1062 genes were down-regulated in ztl mutants (ztl-3 and ztl-105) compared with wild type; some of these genes encoded enzymes involved in cell wall modification, consistent with reduced hypocotyl elongation. In upper aerial parts, 1038 genes were down-regulated in the ztl mutants compared with wild type; these included genes involved in auxin synthesis and auxin response. Furthermore, the expression of the PHYTOCHROME INTERACTING FACTOR 4 (PIF4) gene, which encodes a transcription factor known to positively regulate YUCCA genes (YUCs), was also decreased in the ztl mutants. Genetic analysis revealed that overexpression of PIF4 and YUC8 could restore the suppressed hypocotyl length in the ztl mutants. Our results suggest that ZTL induces expression of YUC8 via PIF4 in upper aerial parts and promotes hypocotyl elongation.
  • Systems, variation, individuality and plant hormones.
    Watahiki M, Trewavas A
    Progress in biophysics and molecular biology, 146, 3, 22, Oct. 2018, [Peer-reviewed], [Lead author], [International Magazine]
    English, Scientific journal, Inter-individual variation in plants and particularly in hormone content, figures strongly in evolution and behaviour. Homo sapiens and Arabidopsis exhibit similar and substantial phenotypic and molecular variation. Whereas there is a very substantial degree of hormone variation in mankind, reports of inter-individual variation in plant hormone content are virtually absent but are likely to be as large if not larger than that in mankind. Reasons for this absence are discussed. Using an example of inter-individual variation in ethylene content in ripening, the article shows how biological time is compressed by hormones. It further resolves an old issue of very wide hormone dose response that result directly from negative regulation in hormone (and light) transduction. Negative regulation is used because of inter-individual variability in hormone synthesis, receptors and ancillary proteins, a consequence of substantial genomic and environmental variation. Somatic mosaics have been reported for several plant tissues and these too contribute to tissue variation and wide variation in hormone response. The article concludes by examining what variation exists in gravitropic responses. There are multiple sensing systems of gravity vectors and multiple routes towards curvature. These are an aspect of the need for reliability in both inter-individual variation and unpredictable environments. Plant hormone inter-individuality is a new area for research and is likely to change appreciation of the mechanisms that underpin individual behaviour.
  • Correction to: Space-time analysis of gravitropism in etiolated Arabidopsis hypocotyls using bioluminescence imaging of the IAA19 promoter fusion with a destabilized luciferase reporter.
    Yamamoto KT, Watahiki MK, Matsuzaki J, Satoh S, Shimizu H
    Journal of plant research, 131, 5, 889, 889, Sep. 2018, [Peer-reviewed], [Lead author], [Domestic magazines]
    English, The article Space-time analysis of gravitropism in etiolated Arabidopsis hypocotyls using bioluminescence imaging of the IAA19 promoter fusion with a destabilized luciferase reporter, written by Kotaro T. Yamamoto, Masaaki K. Watahiki, Jun Matsuzaki, Soichirou Satoh and Hisayo Shimizu, was originally published electronically on the publisher's internet portal (currently SpringerLink) on 10 April 2017 without open access.
  • YUCCA9-Mediated Auxin Biosynthesis and Polar Auxin Transport Synergistically Regulate Regeneration of Root Systems Following Root Cutting
    Dongyang Xu, Jiahang Miao, Emi Yumoto, Takao Yokota, Masashi Asahina, Masaaki Watahiki
    PLANT AND CELL PHYSIOLOGY, 58, 10, 1710, 1723, OXFORD UNIV PRESS, Oct. 2017, [Peer-reviewed], [Corresponding author]
    English, Scientific journal, Recovery of the root system following physical damage is an essential issue for plant survival. An injured root system is able to regenerate by increases in lateral root (LR) number and acceleration of root growth. The horticultural technique of root pruning (root cutting) is an application of this response and is a common garden technique for controlling plant growth. Although root pruning is widely used, the molecular mechanisms underlying the subsequent changes in the root system are poorly understood. In this study, root pruning was employed as a model system to study the molecular mechanisms of root system regeneration. Notably, LR defects in wild-type plants treated with inhibitors of polar auxin transport (PAT) or in the auxin signaling mutant auxin/indole-3-acetic acid19/massugu2 were recovered by root pruning. Induction of IAA19 following root pruning indicates an enhancement of auxin signaling by root pruning. Endogenous levels of IAA increased after root pruning, and YUCCA9 was identified as the primary gene responsible. PAT-related genes were induced after root pruning, and the YUCCA inhibitor yucasin suppressed root regeneration in PAT-related mutants. Therefore, we demonstrate the crucial role of YUCCA9, along with other redundant YUCCA family genes, in the enhancement of auxin biosynthesis following root pruning. This further enhances auxin transport and activates downstream auxin signaling genes, and thus increases LR number.
  • Space-time analysis of gravitropism in etiolated Arabidopsis hypocotyls using bioluminescence imaging of the IAA19 promoter fusion with a destabilized luciferase reporter
    Kotaro T. Yamamoto, Masaaki K. Watahiki, Jun Matsuzaki, Soichirou Satoh, Hisayo Shimizu
    JOURNAL OF PLANT RESEARCH, 130, 4, 765, 777, SPRINGER JAPAN KK, Jul. 2017, [Peer-reviewed], [Lead author]
    English, Scientific journal, Imaging analysis was carried out during the gravitropic response of etiolated Arabidopsis hypocotyls, using an IAA19 promoter fusion of destabilized luciferase as a probe. From the bright-field images we obtained the local deflection angle to the vertical, A, local curvature, C, and the partial derivative of C with respect to time, . These were determined every 19.9 A mu m along the curvilinear length of the hypocotyl, at similar to 10 min intervals over a period of similar to 6 h after turning hypocotyls through 90A degrees to the horizontal. Similarly from the luminescence images we measured the luminescence intensity of the convex and concave flanks of the hypocotyl as well as along the median of the hypocotyl, to determine differential expression of auxin-inducible IAA19. Comparison of these parameters as a function of time and curvilinear length shows that the gravitropic response is composed of three successive elements: the first and second curving responses and a decurving response (autostraightening). The maximum of the first curving response occurs when A is 76A degrees along the entire length of the hypocotyl, suggesting that A is the sole determinant in this response; in contrast, the decurving response is a function of both A and C, as predicted by the newly-proposed graviproprioception model (Bastien et al., Proc Natl Acad Sci USA 110:755-760, 2013). Further, differential expression of IAA19, with higher expression in the convex flank, is observed at A = 44A degrees, and follows the Sachs' sine law. This also suggests that IAA19 is not involved in the first curving response. In summary, the gravitropic response of Arabidopsis hypocotyls consists of multiple elements that are each determined by separate principles.
  • UV-B-Induced CPD Photolyase Gene Expression is Regulated by UVR8-Dependent and -Independent Pathways in Arabidopsis
    Nan Li, Mika Teranishi, Hiroko Yamaguchi, Tomonao Matsushita, Masaaki K. Watahiki, Tomohiko Tsuge, Shao-Shan Li, Jun Hidema
    PLANT AND CELL PHYSIOLOGY, 56, 10, 2014, 2023, OXFORD UNIV PRESS, Oct. 2015, [Peer-reviewed]
    English, Scientific journal, Plants have evolved various mechanisms that protect against the harmful effects of UV-B radiation (280-315 nm) on growth and development. Cyclobutane pyrimidine dimer (CPD) photolyase, the repair enzyme for UV-B-induced CPDs, is essential for protecting cells from UV-B radiation. Expression of the CPD photolyase gene (PHR) is controlled by light with various wavelengths including UV-B, but the mechanisms of this regulation remain poorly understood. In this study, we investigated the regulation of PHR expression by light with various wavelengths, in particular low-fluence UV-B radiation (280 nm, 0.2 mu mol m(-2) s(-1)), in Arabidopsis thaliana seedlings grown under light-dark cycles for 7 d and then adapted to the dark for 3 d. Low-fluence UV-B radiation induced CPDs but not reactive oxygen species. AtPHR expression was effectively induced by UV-B, UV-A (375 nm) and blue light. Expression induced by UV-A and blue light was predominantly regulated by the cryptochrome-dependent pathway, whereas phytochromes A and B played a minor but noticeable role. Expression induced by UV-B was predominantly regulated by the UVR8-dependent pathway. AtPHR expression was also mediated by a UVR8-independent pathway, which is correlated with CPD accumulation induced by UV-B radiation. These results indicate that Arabidopsis has evolved diverse mechanisms to regulate CPD photolyase expression by multiple photoreceptor signaling pathways, including UVR8-dependent and -independent pathways, as protection against harmful effects of UV-B radiation.
  • Reduced phototropism in pks mutants may be due to altered auxin-regulated gene expression or reduced lateral auxin transport
    Chitose Kami, Laure Allenbach, Melina Zourelidou, Karin Ljung, Frederic Schuetz, Erika Isono, Masaaki K. Watahiki, Kotaro T. Yamamoto, Claus Schwechheimer, Christian Fankhauser
    PLANT JOURNAL, 77, 3, 393, 403, WILEY-BLACKWELL, Feb. 2014, [Peer-reviewed]
    English, Scientific journal, Phototropism allows plants to orient their photosynthetic organs towards the light. In Arabidopsis, phototropins 1 and 2 sense directional blue light such that phot1 triggers phototropism in response to low fluence rates, while both phot1 and phot2 mediate this response under higher light conditions. Phototropism results from asymmetric growth in the hypocotyl elongation zone that depends on an auxin gradient across the embryonic stem. How phototropin activation leads to this growth response is still poorly understood. Members of the phytochrome kinase substrate (PKS) family may act early in this pathway, because PKS1, PKS2 and PKS4 are needed for a normal phototropic response and they associate with phot1 in vivo. Here we show that PKS proteins are needed both for phot1- and phot2-mediated phototropism. The phototropic response is conditioned by the developmental asymmetry of dicotyledonous seedlings, such that there is a faster growth reorientation when cotyledons face away from the light compared with seedlings whose cotyledons face the light. The molecular basis for this developmental effect on phototropism is unknown; here we show that PKS proteins play a role at the interface between development and phototropism. Moreover, we present evidence for a role of PKS genes in hypocotyl gravi-reorientation that is independent of photoreceptors. pks mutants have normal levels of auxin and normal polar auxin transport, however they show altered expression patterns of auxin marker genes. This situation suggests that PKS proteins are involved in auxin signaling and/or lateral auxin redistribution.
  • Arabidopsis RPT2a, 19S Proteasome Subunit, Regulates Gene Silencing via DNA Methylation
    Kaori Sako, Yuko Maki, Tomoyuki Kanai, Eriko Kato, Shugo Maekawa, Shigetaka Yasuda, Takeo Sato, Masaaki K. Watahiki, Junji Yamaguchi
    PLOS ONE, 7, 5, PUBLIC LIBRARY SCIENCE, May 2012, [Peer-reviewed]
    English, Scientific journal, The ubiquitin/proteasome pathway plays a crucial role in many biological processes. Here we report a novel role for the Arabidopsis 19S proteasome subunit RPT2a in regulating gene activity at the transcriptional level via DNA methylation. Knockout mutation of the RPT2a gene did not alter global protein levels; however, the transcriptional activities of reporter transgenes were severely reduced compared to those in the wild type. This transcriptional gene silencing (TGS) was observed for transgenes under control of either the constitutive CaMV 35S promoter or the cold-inducible RD29A promoter. Bisulfite sequencing analysis revealed that both the transgene and endogenous RD29A promoter regions were hypermethylated at CG and non-CG contexts in the rpt2a mutant. Moreover, the TGS of transgenes driven by the CaMV 35S promoters was released by treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine, but not by application of the inhibitor of histone deacetylase Trichostatin A. Genetic crosses with the DNA methyltransferase met1 single or drm1drm2cmt3 triple mutants also resulted in a release of CaMV 35S transgene TGS in the rpt2a mutant background. Increased methylation was also found at transposon sequences, suggesting that the 19S proteasome containing AtRPT2a negatively regulates TGS at transgenes and at specific endogenous genes through DNA methylation.
  • Changes in growth kinetics of stamen filaments cause inefficient pollination in massugu2, an auxin insensitive, dominant mutant of Arabidopsis thaliana
    Satoko Tashiro, Chang-en Tian, Masaaki K. Watahiki, Kotaro T. Yamamoto
    PHYSIOLOGIA PLANTARUM, 137, 2, 175, 187, WILEY-BLACKWELL PUBLISHING, INC, Oct. 2009, [Peer-reviewed]
    English, Scientific journal, We investigated the physiological and molecular basis of lower fecundity of massugu2 (msg2), which is a dominant mutant of an auxin primary response gene, IAA19, in Arabidopsis thaliana. By measuring the length of all stamens and pistils in inflorescences and the reference growth rate of pistils, we constructed growth curves of pistils and stamens between stages 12 and 15 of flower development. Pistil growth was found to consist of a single exponential growth, while stamen growth consisted of three exponential phases. During the second exponential phase, the growth rate of stamen filaments was similar to 10 times greater than the growth rates in the other two phases. Consequently, stamens whose growth was initially retarded grew longer than the pistil, putting pollen grains on the stigma. msg2-1 stamens, on the other hand, exhibited a less obvious growth increase, resulting in less frequent contact between anthers and stigma. MSG2 was expressed in the stamen filaments and its expression almost coincided with the second growth phase. Stamen filaments appeared to elongate by cell elongation rather than cell division in the epidermal cell file. Considering that MSG2 is likely to be a direct target of the auxin F-box receptors, MSG2 may be one of the master genes that control the transient growth increase of stamen filaments.
  • Specificity and similarity of functions of the Aux/IAA genes in auxin signaling of arabidopsis revealed by promoter-exchange experiments among MSG2 IAA19, AXR2/IAA7, and SLR/IAA141
    Hideki Muto, Masaaki K. Watahiki, Daisuke Nakamoto, Masataka Kinjo, Kotaro T. Yamamoto
    PLANT PHYSIOLOGY, 144, 1, 187, 196, AMER SOC PLANT BIOLOGISTS, May 2007, [Peer-reviewed]
    English, Scientific journal, As indicated by various and some overlapped phenotypes of the dominant mutants, the Aux/IAA genes of Arabidopsis ( Arabidopsis thaliana) concomitantly exhibit a functional similarity and differentiation. To evaluate the contributions of their expression patterns determined by promoter activity and molecular properties of their gene products to Aux/IAA function, we examined phenotypes of transgenic plants expressing the green fluorescent protein (GFP)-tagged msg2-1/iaa19, axr2-1/iaa7, or slr-1/iaa14 cDNA by the MSG2 or AXR2 promoter. When driven by the MSG2 promoter (pMSG2), each GFP-tagged cDNA caused the msg2-1 phenotype, that is, the wild-type stature in the mature-plant stage, long and straight hypocotyls in the dark, reduced lateral root formation, relatively mild agravitropic traits in hypocotyls, and a normal gravitropic response in roots. However, development of one or two cotyledonary primordia was often arrested in embryogenesis of the pMSG2::axr2-1::GFP and pMSG2::slr-1::GFP plants, resulting in monocotyledonary or no cotyledonary seedlings. Such defects in embryogenesis were never seen in pMSG2::msg2-1GFP or the msg2-1, axr2-1, or slr-1 mutant. The MSG2 promoter-GUS staining showed that expression of MSG2 started specifically in cotyledonary primordia of the triangular-stage embryos. When driven by the AXR2 promoter (pAXR2), each GFP-tagged mutant cDNA caused, in principle, aberrant aboveground phenotypes of the corresponding dominant mutant. However, either the axr2-1::GFP or slr-1::GFP cDNA brought about dwarf, agravitropic stems almost identical to those of axr2-1, and the pAXR2::msg2-1::GFP and pAXR2::slr-1::GFP hypocotyls exhibited complete loss of gravitropism as did axr2-1. These results showed functional differences among the msg2-1, axr2-1, and slr-1 proteins, though some phenotypes were determined by the promoter activity.
  • Differential expression of the auxin primary response gene MASSUGU2/IAA19 during tropic responses of Arabidopsis hypocotyls
    Kyoko Saito, Masaaki K. Watahiki, Kotaro T. Yamamoto
    PHYSIOLOGIA PLANTARUM, 130, 1, 148, 156, WILEY-BLACKWELL, May 2007, [Peer-reviewed]
    English, Scientific journal, We have examined the expression pattern of an auxin primary response gene, MSG2/IAA19, during photo- and gravitropic responses of hypocotyls using a transgenic Arabidopsis harboring MSG2/IAA19 promoter::GUS. The upper portion of most etiolated hypocotyls showed uniform beta-glucuronidase (GUS) staining with the strongest activity in the pericycle. When hypocotyls were irradiated with unilateral blue light, GUS activity on the concave side of hypocotyls was decreased, resulting in differential GUS staining with a stronger signal on the convex side. The number of differentially stained hypocotyls peaked at 24 h after the onset of the phototropic stimuli, while hypocotyl curvature continued to increase for the entire 36-h experimental period. This result suggests that the MSG2/IAA19 expression precedes the phototropic responses. When seedlings were grown under dim white light, their hypocotyls displayed almost no GUS activity. The light-grown hypocotyls also showed differential GUS staining after phototropic stimuli as result of the increase in GUS activity on the convex side of hypocotyls, especially in the epidermis, the outer cortex and pericycle, although GUS activity was much weaker than that observed in etiolated hypocotyls. Similar but less obvious differential staining was obtained for gravitropic response of hypocotyls. Considering the recent finding that Aux/IAA proteins are immediate targets of the auxin F box receptors, MSG2/IAA19 is likely to act as one of master genes for tropic responses.
  • What makes each Aux/IAA gene unique in its gene family, expression pattern or properties of the gene product?
    Hideki Muto, Masaaki K. Watahiki, Kotaro T. Yamamoto
    Plant Signaling and Behavior, 2, 5, 390, 392, Landes Bioscience, 2007, [Peer-reviewed]
    English, Scientific journal
  • Spatial and temporal analysis of MSG2/IAA 19 gene of arabidopsis by the use of firefly luciferase gene fusion
    Soichirou Satoh, Masaaki Watahiki, Kotaro Yamamoto
    PLANT AND CELL PHYSIOLOGY, 48, S195, S195, OXFORD UNIV PRESS, 2007
    English
  • Measuring molecular interaction between Aux/IAA proteins and auxin response factors of Arabidopsis by fluorescence cross-correlation analyses
    MUTO Hideki, NAGAO Issei, WATAHIKI Masaaki, KINJO Masataka, YAMAMOTO Kotaro
    Journal of plant research, 119, 0, 179, 179, 01 Dec. 2006, [Peer-reviewed]
    English
  • Analysis of degradative activity of the MSG2/IAA19 protein by the use of MSG2-fused luciferase in Arabidopsis
    SATOH Soichirou, WATAHIKI Masaaki, TASHIRO Satoko, YAMAMOTO Kotaro
    Journal of plant research, 119, 0, 174, 175, 01 Dec. 2006, [Peer-reviewed]
    English
  • Overexpression of the RADICAL-INDUCED CELL DEATH1 (RCD1) gene of arabidopsis causes weak rcd1 phenotype with compromised oxidative-stress responses
    Takahiro Fujibe, Hikaru Saji, Masaaki K. Watahiki, Kotaro T. Yamamoto
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 70, 8, 1827, 1831, TAYLOR & FRANCIS LTD, Aug. 2006, [Peer-reviewed]
    English, Scientific journal, rcd1 is a mutant of Arabidopsis thaliana that is more resistant to methyl viologen, but more sensitive to ozone than the wild type. rcdl-2 is caused by a single nucleotide substitution that results in a premature stop codon at Trp-332. The rcdl-2 mRNA level does not change significantly with the mutation. Since overexpression of rcd1-1 cDNA has been shown to bring about an rcd1-like phenotype, we created and examined the overexpression lines of RCD1 by the use of the cauliflower mosaic virus 35S promoter. The transgenic lines exhibited a weak rcd1-like phenotype, although no resistance to methyl viologen was observed. Further, they fully complemented the aberrant rcdl-2 phenotype. Subcellular localization of RCD1 was examined by transiently expressing green fluorescent protein (GFP) fused with RCD1 in onion epidermal cells. GFP signals are observed as aggregated foci in the inner nuclear matrix-like region.
  • Fluctuations in the pollen tube tip-focused calcium gradient are not reflected in nuclear calcium level: a comparative analysis using recombinant yellow cameleon calcium reporter
    MK Watahiki, AJ Trewavas, RM Parton
    SEXUAL PLANT REPRODUCTION, 17, 3, 125, 130, SPRINGER, Sep. 2004, [Peer-reviewed], [Lead author]
    English, Scientific journal, For the first time in pollen tubes, both cytoplasmic and nuclear calcium have been imaged to allow comparative analysis of calcium dynamics in these two compartments with high spatial and temporal dynamics. An improved cameleon (YC2.1) calcium reporter was expressed cytoplasmically in both Lilium longiflorum and Nicotiana tabacum pollen tubes and the periodically fluctuating tip-focused calcium gradient typical of normal growth was recorded by ratio image analysis. The micleoplasmin targeting sequence was then used to localise expressed YC2.1 to the vegetative nucleus of N. tabacum pollen tubes to permit imaging of nuclear location, shape and calcium dynamics. Nuclear-targeted YC2.1 (NupYC2.1) showed an absence of any obvious regular fluctuations in nuclear calcium levels during tube extension in vitro with typical growth rate fluctuation. The use of targeted cameleons to study subcellular calcium dynamics in pollen tubes is discussed.
  • MASSUGU2 encodes Aux/IAA19, an auxin-regulated protein that functions together with the transcriptional activator NPH4/ARF7 to regulate differential growth responses of hypocotyl and formation of lateral roots in Arabidopsis thaliana
    K Tatematsu, S Kumagai, H Muto, A Sato, MK Watahiki, RM Harper, E Liscum, KT Yamamoto
    PLANT CELL, 16, 2, 379, 393, AMER SOC PLANT BIOLOGISTS, Feb. 2004, [Peer-reviewed]
    English, Scientific journal, We have isolated a dominant, auxin-insensitive mutant of Arabidopsis thaliana, massugu2 (msg2), that displays neither hypocotyl gravitropism nor phototropism, fails to maintain an apical hook as an etiolated seedling, and is defective in lateral root formation. Yet other aspects of growth and development of msg2 plants are almost normal. These characteristics of msg2 are similar to those of another auxin-insensitive mutant, non-phototropic hypocotyl4 (nph4), which is a loss-of-function mutant of AUXIN RESPONSE FACTOR7 (ARF7) (Harper et al., 2000). Map-based cloning of the MSG2 locus reveals that all four mutant alleles result in amino acid substitutions in the conserved domain 11 of an Auxin/Indole-3-Acetic Acid protein, IAA19. Interestingly, auxin inducibility of MSG2/IAA19 gene expression is reduced by 65% in nph4/arf7. Moreover, MSG2/IAA19 protein binds to the C-terminal domain of NPH4/ARF7 in a Saccharomyces cerevisiae (yeast) two-hybrid assay and to the whole latter protein in vitro by pull-down assay. These results suggest that MSG2/IAA19 and NPH4/ARF7 may constitute a negative feedback loop to regulate differential growth responses of hypocotyls and lateral root formation.
  • Pollen tubes exhibit regular periodic membrane trafficking events in the absence of apical extension
    RM Parton, S Fischer-Parton, AJ Trewavas, MK Watahiki
    JOURNAL OF CELL SCIENCE, 116, 13, 2707, 2719, COMPANY OF BIOLOGISTS LTD, Jul. 2003, [Peer-reviewed], [Corresponding author]
    English, Scientific journal, The growing pollen tube provides an excellent single cell model system in which to study the mechanisms determining growth regulation, polarity and periodic behaviour. Previously, using FM4-64, we identified periodic movements within the apical vesicle accumulation that were related to the period of oscillatory growth. This suggested a more complex interdependence between membrane traffic, apical extension and periodicity than previously thought. To investigate this a comparison was made between normally growing and Brefeldin-A-treated, non-growing, tubes. Brefeldin-A treatment established an intriguing, stable yet dynamic system of membrane aggregations in the pollen tube tip that exhibited regular movements of material with a 5-7 second period compared with the normal similar to30 second periodicity observed in growing tubes. Heat treatment was found to reduce period length in both cases. After BFA treatment membrane was demonstrated to flow from the extreme pollen tube apex back through a distinct subapical Brefeldin-A-induced membrane accumulation. The effects of Brefeldin-A on the distribution of ER- and Golgi-targeted fluorescent proteins revealed that ER did not contribute directly to the system of membrane aggregations while only certain compartments of the Golgi might be involved. The involvement of membrane derived from the apical vesicle accumulation was strongly implicated. Calcium measurements revealed that Brefeldin-A abolished the typical tip-focused calcium gradient associated with growth and there were no obvious periodic fluctuations in apical calcium associated with the continued periodic Brefeldin-A membrane aggregation associated movements. Our experiments reveal an underlying periodicity in the pollen tube that is independent of secretion, apical extension and the oscillating tip-focused calcium gradient normally associated with growth, but requires an active actin cytoskeleton.
  • Localization of pollen tube NpCDPK7 protein in generative cell membrane mediated by amino terminal myristoylation.
    MK Watahiki, RM Parton, AJ Trewavas
    ADVANCED RESEARCH ON PLANT LIPIDS, 327, 330, SPRINGER, 2003, [Peer-reviewed], [Lead author]
    English, International conference proceedings, Calcium dependent protein kinases (CDPKs) are a group of protein kinases whose primary function is the interpretation of Ca2+ signals. We have investigated the localization of a calcium dependent protein kinase (CDPK) in the germinating pollen grain and growing pollen tubes. Pollen specific CDPK of Nicotiana plumbaginifolia, NpCDPK7, has 52 N-terminal amino acid residues containing a putative myristoylation site and 34 amino acids in the C-terminal domain which includes a putative microbody targeting sequence. Green florescent protein (GFP) was introduced between the N-terminal domain and kinase domain of NpCDPK7 and pollen grains were transformed with this construct by biolistic gun. In pollen tubes, GFP florescence was observed on the surface of the generative cell (containing the two sperm cells), on the plasma membrane and in granules in the ungerminated pollen grain. Mutation or deletion analysis of the N-terminal domain supports the localization of NpCDPK7 protein as determined by the N-terminal region myristoylation. NpCDPK7 may be involved in localized calcium signaling, sensing calcium transients directly under the plasma membrane.
  • Dynamics of the apical vesicle accumulation and the rate of growth are related in individual pollen tubes
    RM Parton, S Fischer-Parton, MK Watahiki, AJ Trewavas
    JOURNAL OF CELL SCIENCE, 114, 14, 2685, 2695, COMPANY OF BIOLOGISTS LTD, Jul. 2001, [Peer-reviewed]
    English, Scientific journal, Regulated secretory vesicle delivery, vesicle fusion and rapid membrane recycling are all contentious issues with respect to tip growth in plant, fungal and animal cells. To examine the organisation and dynamics of membrane movements at the growing pollen tube apex and address the question of their relationship to growth, we have used the membrane stain FM4-64 both as a structural marker and as a quantitative assay. Labelling of living Lilium Longiflorum pollen tubes by FM4-64 resulted in a distinct staining pattern in the tube apex, which corresponds spatially to the previously identified cone-shaped 'apical clear zone' containing secretory vesicles. Dye uptake could be inhibited by sodium azide and followed a strict temporal sequence from the plasma membrane to a population of small (1-2 mum diameter) discrete internal structures, with subsequent appearance of dye in the apical region and ultimately in vacuolar membranes. Washout of the dye rapidly removed the plasma membrane staining, which was followed by a gradual decline in the apical fluorescence over more than an hour. Injected aqueous FM4-64 solution showed a relatively even distribution within the pollen tube. Association of FM4-64 with apical secretory vesicles was supported by the effects of the inhibitors Brefeldin-A and Cytochalasin-D, which are known to affect the localisation and number of such vesicles, on the FM4-64 staining pattern. Examination of the dynamics of FM4-64 labelling in the pollen tube tip by time-lapse observation, supported by fluoreseence-recovery-after-photobleaching (FRAP) analysis, suggested the possibility of distinct pathways of bulk membrane movement both towards and, significantly, away from the apex. Quantitative analysis of FM4-64 distribution in the apex revealed that fluctuations in fluorescence 5 to 10 mum subapically, and to a lesser extent the apical 3 mum, could be related to the periodic oscillation in pollen tube growth rate. This data reveals a quantitative relationship between FM4-64 staining and growth rate within an individual tube.
  • An agravitropic mutant of Arabidopsis, endodermal-amyloplast less 1, that lacks amyloplasts in hypocotyl endodermal cell layer
    K Fujihira, T Kurata, MK Watahiki, Karahara, I, KT Yamamoto
    PLANT AND CELL PHYSIOLOGY, 41, 11, 1193, 1199, OXFORD UNIV PRESS, Nov. 2000, [Peer-reviewed]
    English, Scientific journal, We have isolated a new recessive mutant of Arabidopsis thaliana for gravitropism, endodermal-amyloplast less1 (eal1), eal1 shows reduced gravitropism in hypocotyl, and completely lacks gravitropism in inflorescence stems; root gravitropism is not affected. Starch staining with I-KI solution reveals almost no amyloplasts in call hypocotyls when grown on a sucrose-free medium, though the root columella cells contain as many amyloplasts as wild type, On a medium containing 1% sucrose, call hypocotyls contain as many starch granules as those of wild type, suggesting that starch synthesis is not affected in eal1. The endodermal cell layer which is thought to function as statocytes in hypocotyls is present in call. These results suggest that differentiation or development of gravity-responsive amyloplasts are affected in eal1 hypocotyls.
  • The NPH4 locus encodes the auxin response factor ARF7, a conditional regulator of differential growth in aerial Arabidopsis tissue
    RM Harper, EL Stowe-Evans, DR Luesse, H Muto, K Tatematsu, MK Watahiki, K Yamamoto, E Liscum
    PLANT CELL, 12, 5, 757, 770, AMER SOC PLANT PHYSIOLOGISTS, May 2000, [Peer-reviewed]
    English, Scientific journal, Organ bending through differential growth represents a major mechanism by which plants are able to adaptively alter their morphology in response to local changes in the environment. Two plant hormones, auxin and ethylene, have been implicated as regulators of differential growth responses; however, the mechanisms by which they elicit their effects remain largely unknown. Here, we describe isolation of the NPH4 gene of Arabidopsis, which is conditionally required for differential growth responses of aerial tissues, and we report that NPH4 encodes the auxin-regulated transcriptional activator ARF7. The phenotypes of nph4 mutants, which include multiple differential growth defects associated with reduced auxin responsiveness, including impaired auxin-induced gene expression, are consistent with the predicted loss of function of a transcriptional activator, and these phenotypes indicate that auxin-dependent changes in gene transcription are prerequisite for proper organ bending responses. Although NPH4/ARF7 appears to be a major regulator of differential growth, it is not the sole regulator because phenotypes of nph4 null mutants were suppressed by application of ethylene. This latter finding illustrates the intimate connection between auxin and ethylene in the control of growth in higher plants.
  • A NOVEL ARABIDOPSIS MUTANT, eall DEFECTIVE IN GRAVITY PERCEPTION IN HYPOCOTYLS AND STEMS
    FUJIHIRA Kenichiro, KURATA Tetsuya, WATAHIKI Masaaki K, YAMAMOTO Kotaro T
    Plant and cell physiology, 40, 0, s145, s145, Mar. 1999
    English
  • The MSG1 and AXR1 genes of Arabidopsis are likely to act independently in growth-curvature responses of hypocotyls
    MK Watahiki, K Tatematsu, K Fujihira, M Yamamoto, KT Yamamoto
    PLANTA, 207, 3, 362, 369, SPRINGER VERLAG, Jan. 1999, [Peer-reviewed], [Lead author]
    English, Scientific journal, Growth-curvature responses of hypocotyls of Arabidopsis thaliana (L.) Heynh. were measured in double mutants between msg1 and axr1, both of which are auxin-resistant and defective in hypocotyl growth curvature induced upon unilateral application of auxin. The msg1 axr1 double mutants showed no auxin-induced growth curvature, that is, they exhibited the msg1 phenotype, though the axr1 defects were partial. Hypocotyls of both the msg1 and axil mutants were partially defective in second-positive phototropism, whereas the double mutants lost the response completely. When grown on vertically held agar plates, the axr1 mutant showed normal hypocotyl gravitropism and the mutation did not affect the reduced hypocotyl gravitropism of msg1. Hypocotyls of msg1 and axr1 mutants grew upward like wild-type ones when grown along an agar surface, while they grew more randomly when grown without an agar support, suggesting that axr1 hypocotyls are not completely normal in gravitropism. The extent of defects in growth orientation increased in the order: msg1 axr1 double mutants > msg1 > axr1 > wild type. The hypocotyls of these mutants showed auxin resistance in the order: msg1 axr1 > axr1 > msg1 > wild type. The msg1 mutant had epinastic leaves and axr1 had wrinkled leaves; leaves of the msg1 axr1 double mutants were epinastic and wrinkled. These results suggest that MSG1 and AXR1 act independently in separate pathways of the reactions tested in the present study. In contrast, the phenotype of the msg1 aux1 double mutants shows that AUX1 is not significantly involved in these phenomena.
  • The massugu1 mutation of Arabidopsis identified with failure of auxin-induced growth curvature of hypocotyl confers auxin insensitivity to hypocotyl and leaf
    MK Watahiki, KT Yamamoto
    PLANT PHYSIOLOGY, 115, 2, 419, 426, AMER SOC PLANT PHYSIOLOGISTS, Oct. 1997, [Peer-reviewed], [Lead author]
    English, Scientific journal, Unilateral application of indole-3-acetic acid (IAA) in a lanolin base to hypocotyls of partially etiolated seedlings of wild-type Arabidopsis thaliana induced growth curvature in a dose-dependent manner. The effects of IAA in concentrations from 1 to 1000 mu M were studied, with maximum IAA-induced curvature at 100 mu M. Three IAA-insensitive mutants were isolated and are all in the same locus, massugu1 (msg1). They did not undergo hypocotyl growth curvature at any of the IAA concentrations tested. msg1 is recessive and is located on chromosome 5. msg1 hypocotyl growth is resistant to 2,4-dichlorophenoxyacetic acid (2,4-D), but the roots are as sensitive to 2,4-D as the wild type. Growth of the hypocotyl was inhibited to essentially the same extent as the wild type by 6-benzylaminopurine, abscisic acid, and 1-aminocyclopropane-1-carboxylate, an ethylene precursor. The msg1 leaves were also resistant to 2,4-D-induced chlorosis. The gravitropic response of the msg1 hypocotyl takes much more time to initiate and achieve the wild-type degree of curvature, whereas the msg1 roots responded normally to gravity. The mature plants and the etiolated seedlings of msg1 were generally wild type in appearance, except that their rosette leaves were either epinastic or hyponastic. msg1 is the first auxin-insensitive mutant in which its effects are mostly restricted to the hypocotyl and leaf, and msg1 also appears to be auxin specific.
  • Arabidopsis mutants that have defects in hypocotyl growth curvature induced by unilateral application of auxin.
    KT Yamamoto, K Tatematsu, K Fujihira, M Yamamoto, MK Watahiki
    PLANT PHYSIOLOGY, 114, 3, 771, 771, AMER SOC PLANT PHYSIOLOGISTS, Jul. 1997
    English
  • INHIBITORY EFFECTS OF AUXINS AND RELATED SUBSTANCES ON THE ACTIVITY OF AN ARABIDOPSIS GLUTATHIONE-S-TRANSFERASE ISOZYME EXPRESSED IN ESCHERICHIA-COLI
    MK WATAHIKI, H MORI, KT YAMAMOTO
    PHYSIOLOGIA PLANTARUM, 94, 4, 566, 574, MUNKSGAARD INT PUBL LTD, Aug. 1995, [Peer-reviewed], [Lead author]
    English, Scientific journal, Glutathione S-transferases (GSTs; EC 2.5.1.18) are encoded by a gene family. Some GSTs have the capacity to bind to indole-3-acetic acid (IAA), whereas the gene expression of other GSTs is regulated by auxin. In order to assess a possible physiological significance of the auxin binding of GST, we investigated effects of auxins on the activity of GST expressed in Escherichia coli. cDNA cloning was carried out for the fifth gene (GST5) of GST in Arabidopsis. Although the deduced amino acid sequence of GST5 was remotely related to that of the other Arabidopsis GSTs (less than 20% identical), the GST5 protein (GST5) expressed in E. coli showed GST activity. Apparent K-m values of GST5 are 0.86 and 1.29 mM for glutathione (GSH) and 1-chloro-2,4-dinitrobenzene, respectively. IAA, 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphthaleneacetic acid (1-NAA) and 2-NAA inhibited the enzyme activity competitively with respect to GSH. The apparent K-i of IAA is 1.56 mM. Salicylic acid inhibited GST activity in a noncompetitive manner. 2,4-D was the most inhibitory among the tested chemicals. GST5 bound to GSH-immobilized agarose gel was effectively eluted by IAA. These results indicate that IAA and the related substances bind to GST5 at the GSH-binding site, and exclude the possibility that the compounds could be substrates for GST5. Although the K-i value of IAA is too high for any physiological consequences, it might be assumed that GST activity is modulated in vivo by an auxin-related substance(s). The steady-state level of the GST5 mRNA was increased by wounding, hear shock, and spraying buffer on the plant, but was not influenced by auxin treatment.
  • A NEW ISOZYME OF PLASTID OMEGA-3-FATTY-ACID DESATURASE IN ARABIDOPSIS-THALIANA
    MK WATAHIKI, KT YAMAMOTO
    PLANT PHYSIOLOGY, 105, 4, 1451, 1452, AMER SOC PLANT PHYSIOLOGISTS, Aug. 1994, [Peer-reviewed], [Lead author]
    English

Other Activities and Achievements

Research Themes

  • 側根新生の時空間的周期性と可塑性を実現する仕組みの解明
    Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
    Jun. 2019 - Mar. 2024
    深城 英弘, 綿引 雅昭
    維管束植物の根は，内生・外生の要因に応じた側根形成を通じて根系構造を可塑的に構築する。側根は一次根に沿って一定の間隔で作られるが，その周期性は根端付近における数千遺伝子の発現振動の総体として観測されるRoot clockに相関している。この時間周期が一次根軸に沿って一定の間隔でオーキシン応答極大を形成し，これが予定分岐部位（プレブランチサイト：PBS）へと運命づけられる。根系構造の可塑性は，Root clock の時間振動が根に沿ったPBS の空間周期に変換され，さらにPBS から側根原基が形成される過程に何らかの変調作用が入力されることで生み出されるが，この機構は不明である。本研究では，側根新生の長時間広域発光・蛍光イメージング系や変異体・マーカー系統を画像解析，数理モデル解析，人間拡張工学と融合させ，Root clock に依存した側根形成の空間的周期性の制御とその変調機構を明らかにする。2019年度の主な成果を以下に示す。
    １）Root clock に応答して周期的に発現変動する発光・蛍光レポーター系統や，PBS・側根創始細胞・側根原基で発現するレポーター系統を用いた高精度長時間広域イメージングによる観察測定系を確立するため，根の成長を維持しながら観察できる横倒し（水平型）発光・蛍光顕微鏡の構築を行った。
    ２）DR5:LUCレポーター系統を用いた解析を行い，側根形成開始を制御するSLR/IAA14-ARF7-ARF19を介したオーキシン応答が， 側根創始細胞の形成以前のPBS形成過程に関与することが示唆された。また，変異原処理したDR5:LUC系統から，側根新生の時空間的周期性など，側根プレパターニングに異常を示す変異体や，側根創始細胞間の距離を調節するTOLS2ペプチドへの応答性が異常な変異体の選抜を進め，変異体候補を複数系統単離した。
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), Kobe University, Coinvestigator, 19H05673
  • 新規オーキシンレポーターによるオーキシン応答再編メカニズムの解明
    科学研究費補助金(新学術領域研究(研究領域提案型))
    2013 - 2014
    綿引 雅昭
    本研究は領域研究前半で明らかになった「オーキシン応答の再編メカニズム」を分子レベルで明らかにし，オーキシン遺伝子発現のプロファイリング（将来予測）を行うための技術開発を本研究の第一目標とした。さらにオーキシン応答の至適濃度と器官特異性の関係もオーキシン応答機構の再編（記憶）が関わっていると考えられるので，本新技術の適用によってそのメカニズムを明らかにすることが第2の目標である。１．多色ルシフェラーゼ発光の色分離；プロモーターの状態をリアルタイムで計測できるようにPEST配列を持つRLucPESTを作成し，pIAA19-ElucPEST，RLucPESTを両方持つシロイヌナズナを作出し，色分離に成功した。２．新奇IAA19突然変異体の作出；AUX/IAA19の優性突然変異体であるmassugu2はオーキシン応答能が低下しているだけではなく，「オーキシン応答の再編」過程に異常があることがわかっている。そこでmsg2-1およびMSG2のcDNAにランダム変異を誘発し，pIAA19-ElucPESTをもつシロイヌナズナに導入した個体の作出と解析を行う。３．多色オーキシンレポーターの作出；異なるオーキシン応答遺伝子間の「オーキシン応答の再編」を観察することで，オーキシン応答再編がスッテップワイズに進行する様子が明らかになると考えられる。そこでDR5人工プロモーターの下流にRLucPESTを融合したコンストラクトをシロイヌナズナに導入し，「オーキシン応答の再編」が起こることを確認する。；４．光応答とオーキシン関連突然変異体におけるオーキシン応答の再編オーキシン合成，光応答変異体におけるオーキシン応答の再編を調べるため，掛け合わせによりpIAA19-ElucPESTを導入し，解析を行う。
    文部科学省, 新学術領域研究(研究領域提案型), 北海道大学, Principal investigator, Competitive research funding, 25120701
  • 発光を用いた植物環境センサーとイメージング技術の開発
    科学研究費補助金(新学術領域研究(研究領域提案型))
    2011 - 2012
    綿引 雅昭
    本研究では短寿命型ルシフェラーゼを使って，早期オーキシン誘導性AUX/IAA19の発現挙動を詳細に解析することで，形態形成の時間的空間的制御過程を明らかにし，オーキシン応答性遺伝子の挙動を可視化する技術の開発と検証を行った。
    pIAA19:ELuc-PESTシロイヌナズナの根切片を用い，pIAA19:ELuc-PEST活性の基底状態からの外性オーキシンに対するオーキシン応答キネティクスを計測した。その結果，１）AUX/IAA19のIAAに対する応答は一過的である，２）オーキシン応答は濃度依存的であるが，至適値以上では阻害される，３）一過的な応答の後，さらに高濃度のオーキシン投与によって一過的な応答を誘導できるが，初回の応答とは異なったキネティクスを示す。４）オーキシン投与から応答までの時間差に濃度依存性は見られない。これらのことから，オーキシン基底状態を活性化させる因子の性質は濃度依存性という観点から同一であるが，活性の進行にかかわる因子に濃度依存性があることが示唆された。さらにオーキシンレセプターであるTir1/AFBs阻害剤PEO-IAAで前処理した場合，１）一過的な発現は維持されているが，発現速度においてPEO-IAAは外性IAAと競合している，２）同レベルのIAA濃度で発現速度を比較したとき，PEO-IAA投与時のほうが総発光量が低い。ドミナント突然変異体aux/iaa19/msg2-1，ARF7, ARF19，ARF7/ARF19背景では１）野生型に比べて発現速度が低下していたが，高濃度のオーキシンで活性化できる，２）総発光量は野生型msg2-1arf7arf7/arf19の順に低下していた。
    以上のことから，オーキシン初期応答はAUX/IAA-ARF-Tir1/AFBsのコンビネーション変化による次空間的な制御を受けていることが示唆された。
    文部科学省, 新学術領域研究(研究領域提案型), 北海道大学, Principal investigator, Competitive research funding, 23120501
  • Integrative regulatory system by a plant hormone, auxin
    Grants-in-Aid for Scientific Research(特定領域研究)
    2007 - 2012
    Kotaro YAMAMOTO, 綿引 雅昭
    シロイヌナズナで花茎の成長方向を調節している遺伝子LAZY1の機能を調べた。同遺伝子の機能欠損変異体は、花茎が明所で水平方向に傾斜して成長する。同遺伝子のプロモーターGUS融合遺伝子を用いて発現部位を調べると、側枝が主茎から分岐する部位では上側(向軸側)の内皮細胞層に偏差的に発現していた。一方、オーキシン誘導性遺伝子MSG2/IAA19の発現は、側枝の下側に偏差的に発現していた。また、LAZY1発現はオーキシン抑制性だった。LAZY1は細胞膜周辺に存在するので、以上の結果は、LAZY1がオーキシンの偏差分布形成を助ける機能を持っていることを示唆しているだろう。上述のMSG2は根に発現しているにも関わらず、その優性突然変異体msg2は根の屈地性が異常とはならない。MSG2発現のオーキシン応答をmsg2変異体でルシフェラーゼ融合タンパク質を用いて調べると、オーキシン添加後約1時間の間は正常で、その後応答が起こりにくくなることが分かった。そこで、根の成長方向に障害物を置いて、根が障害物を避けるには長期間の屈地性応答が必要になるようにしてやると、野生型に比べてmsg2変異体では屈地性応答が起こりにくくなることが分かった。つまり、MSG2タンパク質の蓄積量には閾値があって、長いオーキシン応答によってMSG2が閾値以上蓄積するようになると、オーキシンによる屈地性応答に関わるオーキシン応答...
    Ministry of Education, Culture, Sports, Science and Technology, 特定領域研究, 北海道大学, Coinvestigator not use grants, Competitive research funding, 19060008
  • オーキシン情報伝達系の分子機構研究
    科学研究費補助金(特定領域研究)
    2002 - 2006
    山本 興太朗, 綿引 雅昭
    MSG2タンパク質を検出するために、ルシフェラーゼ融合タンパク質として検出することを試みた。その結果、シロイヌナズナ黄化芽生えでのMSG2 promoter (pMSG2)::MSG2::LUC活性は、pMSG2::LUCの1/10程度だった。この差がMSG2タンパク質にかかる分解調節だと考えられる。このMSG2::LUC活性はプロモーター活性同様、オーキシン処理によって早期に増加した。明所で栽培した芽生えでは、プラシノステロイドによってMSG2のメッセージが増加することが報告されているが、MSG2::LUCを用いて調べたところ、MSG2タンパク質レベルも同様に増加することが分かった。Aux/IAAとARFとの相互作用を物理的な蛍光相関分光法(fluorescence correlation spectroscopy)を用いて測定した。Aux/IAAとしては、MSG2とAXR2とSLR、 ARFのタンパク質問相互作用ドメインとしてはNPH4/ARF7とMP/ARF5のドメインIIIとドメインIV(CTD)を選んで、両者をHeLa細胞に一過的に発現させ、蛍光強度の相関係数を測定した。その結果、Aux/IAAとARF-CTDの相互作用は強く、両者はほぼ100%結合すること、一方Aux/IAA同士、またはARF-CTD同士の結合は比較的弱く、この場合は20%程度しか結合していないこ...
    文部科学省, 特定領域研究, 北海道大学, Coinvestigator not use grants, Competitive research funding, 14036201