研究活動情報

• Stomatal closing compounds dissect the signaling pathway for light-induced stomatal opening and confer drought tolerance in plants.

  Kwang-Chul Shin, Yusuke Aihara, Kohei Fukatsu, Shigeo Toh, Kei Murakami, Yuki Hayashi, Ayato Sato, Toshinori Kinoshita

  Stomata in the plant epidermis open in response to light, but the molecular mechanisms underlying this process remain incompletely understood. In this study, we screened for compounds that affect light-induced stomatal opening and identified two stomatal closing compounds (SCLs), SCL10 and SCL11 (clorgyline). Both compounds completely suppressed light-induced stomatal opening in Commelina benghalensis but did not affect fungal toxin fusicoccin (FC)-induced opening. The half-maximal inhibitory concentrations (IC50) of SCL10 and SCL11 on light-induced opening were 1.40 and 38.25 μM, respectively. Consistently, both compounds fully inhibited blue-light-induced phosphorylation of the penultimate residue, a threonine (Thr948; numbering according to Arabidopsis AHA1) of plasma membrane (PM) H+-ATPase, which is essential for stomatal opening, but did not interfere with FC-induced phosphorylation in Arabidopsis thaliana guard cells. Notably, red- and blue-light-induced phosphorylation of Thr881 in PM H+-ATPase, another key step in stomatal opening, was inhibited by SCL11 but not by SCL10 in guard cells. Additionally, stomatal opening induced by PP242, a compound that promotes stomatal opening partly by suppressing steady-state abscisic acid signaling, was inhibited by SCL11 but not by SCL10. These results imply that SCL10 inhibits blue-light-induced phosphorylation of Thr948 and stomatal opening upstream of the site of action of PP242, whereas SCL11 inhibits the phosphorylation of both Thr948 and Thr881, and acts downstream of the site of action of PP242. Finally, the compounds were sprayed onto rose leaves, and we observed that wilting was suppressed only in leaves sprayed with SCL10, for up to 5 h.

  2026年01月, Plant & cell physiology, 英語, 国内誌

  [査読有り]

  研究論文（学術雑誌）


• The Protein Kinase Inhibitor, Tyrphostin AG126, and Its Derivatives Suppress Phosphorylation of Plasma Membrane H+-ATPase and Light-Induced Stomatal Opening.

  Shogo Kuwayama, Maki Hayashi, Koji Takahashi, Yuki Hayashi, Kohei Fukatsu, Kei Murakami, Yusuke Aihara, Ayato Sato, Toshinori Kinoshita

  Phosphorylation of the penultimate residue, threonine (pen-Thr), of plasma membrane (PM) H+-ATPase is essential for its activation and blue light (BL)-induced stomatal opening. However, the regulatory mechanism of action of PM H+-ATPase pen-Thr phosphorylation is not completely understood. Here, we performed screening using a protein kinase inhibitor library and found that tyrphostin AG126 inhibited phosphorylation of PM H+-ATPase pen-Thr in guard cells in response to light and fungal toxin fusicoccin (FC), in addition to inhibition of light- and FC-induced stomatal opening. Analysis of the structure-activity relationship using AG126 derivatives revealed the hydroxyl group at the C-5 position of the compound to be essential for its activity. We further characterized one AG126 derivative, AGD-1, which effectively suppressed BL-induced stomatal opening with a half-inhibitory concentration of 2.0 μM. AGD-1 inhibited PM H+-ATPase pen-Thr phosphorylation in guard cells in response to BL and FC. In addition, AGD-1 suppressed FC-induced PM H+-ATPase pen-Thr phosphorylation in mesophyll cell protoplasts, implying that the effect of AGD-1 is not specific to guard cells. Furthermore, to improve the permeability of AGD-1, we synthesized acetylated AGD-1 (AcAGD-1), which was found to suppress BL- and FC-induced stomatal opening. AcAGD-1 suppressed light-induced PM H+-ATPase pen-Thr phosphorylation, but not Thr881 phosphorylation, in leaf discs, which is important for guard cell PM H+-ATPase activation in addition to pen-Thr phosphorylation. This study identified a novel stomatal opening inhibitor capable of specifically inhibiting PM H+-ATPase pen-Thr phosphorylation.

  2025年05月, Plant & cell physiology, 英語, 国内誌

  [査読有り]

  研究論文（学術雑誌）


• Chemical inhibition of stomatal differentiation by perturbation of the master-regulatory bHLH heterodimer via an ACT-Like domain.

  Ayami Nakagawa, Krishna Mohan Sepuru, Shu Jan Yip, Hyemin Seo, Calvin M Coffin, Kota Hashimoto, Zixuan Li, Yasutomo Segawa, Rie Iwasaki, Hiroe Kato, Daisuke Kurihara, Yusuke Aihara, Stephanie Kim, Toshinori Kinoshita, Kenichiro Itami, Soon-Ki Han, Kei Murakami, Keiko U Torii

  Selective perturbation of protein interactions with chemical compounds enables dissection and control of developmental processes. Differentiation of stomata, cellular valves vital for plant growth and survival, is specified by the basic-helix-loop-helix (bHLH) heterodimers. Harnessing a new amination reaction, we here report a synthesis, derivatization, target identification, and mode of action of an atypical doubly-sulfonylated imidazolone, Stomidazolone, which triggers stomatal stem cell arrest. Our forward chemical genetics followed by biophysical analyses elucidates that Stomidazolone directly binds to the C-terminal ACT-Like (ACTL) domain of MUTE, a master regulator of stomatal differentiation, and perturbs its heterodimerization with a partner bHLH, SCREAM in vitro and in plant cells. On the other hand, Stomidazolone analogs that are biologically inactive do not bind to MUTE or disrupt the SCREAM-MUTE heterodimers. Guided by structural docking modeling, we rationally design MUTE with reduced Stomidazolone binding. These engineered MUTE proteins are fully functional and confer Stomidazolone resistance in vivo. Our study identifies doubly-sulfonylated imidazolone as a direct inhibitor of the stomatal master regulator, further expanding the chemical space for perturbing bHLH-ACTL proteins to manipulate plant development.

  2024年10月, Nature communications, 15(1) (1), 8996 - 8996, 英語, 国際誌

  研究論文（学術雑誌）


• Discovery of a Plant 14-3-3 Inhibitor Possessing Isoform Selectivity and In Planta Activity.

  Kotaro Nishiyama, Yusuke Aihara, Takehiro Suzuki, Koji Takahashi, Toshinori Kinoshita, Naoshi Dohmae, Ayato Sato, Shinya Hagihara

  Synthetic modulators for plant 14-3-3s are promising chemical tools both for understanding the 14-3-3-related signaling pathways and controlling plant physiology. Here, we describe a novel small-molecule inhibitor for 14-3-3 proteins ofArabidopsis thaliana. The inhibitor was identified from unexpected products in DMSO stock solution of an in-house chemical library. Mass spectroscopy, mutant-based analyses, fluorescence polarization assays, and thermal shift assaysrevealed that the inhibitor covalently binds to an allosteric site of 14-3-3 with isoform selectivity. Moreover, infiltration of the inhibitor to Arabidopsis leaves suppressed the stomatal aperture. The inhibitor should provide a new insight into the design of potent and isoform-selective 14-3-3 modulators.

  2024年04月, Angewandte Chemie (International ed. in English), e202400218, 英語, 国際誌

  研究論文（学術雑誌）


• Identification and improvement of isothiocyanate-based inhibitors on stomatal opening to act as drought tolerance-conferring agrochemicals

  Yusuke Aihara, Bumpei Maeda, Kanna Goto, Koji Takahashi, Mika Nomoto, Shigeo Toh, Wenxiu Ye, Yosuke Toda, Mami Uchida, Eri Asai, Yasuomi Tada, Kenichiro Itami, Ayato Sato, Kei Murakami, Toshinori Kinoshita

  Abstract Stomatal pores in the plant epidermis open and close to regulate gas exchange between leaves and the atmosphere. Upon light stimulation, the plasma membrane (PM) H⁺-ATPase is phosphorylated and activated via an intracellular signal transduction pathway in stomatal guard cells, providing a primary driving force for the opening movement. To uncover and manipulate this stomatal opening pathway, we screened a chemical library and identified benzyl isothiocyanate (BITC), a Brassicales-specific metabolite, as a potent stomatal-opening inhibitor that suppresses PM H⁺-ATPase phosphorylation. We further developed BITC derivatives with multiple isothiocyanate groups (multi-ITCs), which demonstrate inhibitory activity on stomatal opening up to 66 times stronger, as well as a longer duration of the effect and negligible toxicity. The multi-ITC treatment inhibits plant leaf wilting in both short (1.5 h) and long-term (24 h) periods. Our research elucidates the biological function of BITC and its use as an agrochemical that confers drought tolerance on plants by suppressing stomatal opening.

  Springer Science and Business Media LLC, 2023年05月, Nature Communications, 14(1) (1)

  研究論文（学術雑誌）


• Image-based quantification of Arabidopsis thaliana stomatal aperture from leaf images.

  Momoko Takagi, Rikako Hirata, Yusuke Aihara, Yuki Hayashi, Miya Mizutani Aihara, Eigo Ando, Megumi Yoshimura Kono, Masakazu Tomiyama, Toshinori Kinoshita, Akira Mine, Yosuke Toda

  The quantification of stomatal pore size has long been a fundamental approach to understand the physiological response of plants in the context of environmental adaptation. Automation of such methodologies not only alleviates human labor and bias, but also realizes new experimental research methods through massive analysis. Here, we present an image analysis pipeline that automatically quantifies stomatal aperture of Arabidopsis thaliana leaves from brightfield microscopy images containing mesophyll tissue as noisy backgrounds. By combining a YOLOX-based stomatal detection submodule and a U-Net-based pore segmentation submodule, we achieved 0.875 mAP50 (mean average precision; stomata detection performance) and 0.745 IoU (intersection of union; pore segmentation performance) against images of leaf discs taken with a brightfield microscope. Moreover, we designed a portable imaging device that allows easy acquisition of stomatal images from detached/undetached intact leaves on-site. We demonstrated that this device in combination with fine-tuned models of the pipeline we generated here provides robust measurements that can substitute for manual measurement of stomatal responses against pathogen inoculation. Utilization of our hardware and pipeline for automated stomatal aperture measurements is expected to accelerate research on stomatal biology of model dicots.

  2023年03月, Plant & cell physiology, 英語, 国内誌

  研究論文（学術雑誌）


• Discovery of 2,6-Dihalopurines as Stomata Opening Inhibitors: Implication of an LRX-Mediated H+-ATPase Phosphorylation Pathway

  Ayaka Ueda, Yusuke Aihara, Shinya Sato, Keiko Kano, Emi Mishiro-Sato, Hiroyuki Kitano, Ayato Sato, Kazuhiro J. Fujimoto, Takeshi Yanai, Kazuma Amaike, Toshinori Kinoshita, Kenichiro Itami

  2023年02月, ACS Chemical Biology

  研究論文（学術雑誌）


• Photoinduced Synthesis of Thiocyanates through Hydrogen Atom Transfer and One-Pot Derivatization to Isothiocyanates.

  Bumpei Maeda, Yusuke Aihara, Ayato Sato, Toshinori Kinoshita, Kei Murakami

  Photoinduced benzylic C-H thiocyanation is described. A series of alkyl thiocyanates were efficiently obtained by using Selectfluor as the oxidant. Moreover, we accomplished the one-pot isothiocyanation following the C-H thiocyanation. The thiocyanates and isothiocyanates were applied to the divergent transformation of pharmaceuticals.

  2022年10月, Organic letters, 24(40) (40), 7366 - 7371, 英語, 国際誌

  研究論文（学術雑誌）


• Identification of stomatal-regulating molecules from de novo arylamine collection through aromatic C-H amination.

  Yosuke Toda, Gregory J P Perry, Shimpei Inoue, Eri Ito, Takahiro Kawakami, Mina R Narouz, Koji Takahashi, Yusuke Aihara, Bumpei Maeda, Toshinori Kinoshita, Kenichiro Itami, Kei Murakami

  Stomata-small pores generally found on the leaves of plants-control gas exchange between plant and the atmosphere. Elucidating the mechanism that underlies such control through the regulation of stomatal opening/closing is important to understand how plants regulate photosynthesis and tolerate against drought. However, up-to-date, molecular components and their function involved in stomatal regulation are not fully understood. We challenged such problem through a chemical genetic approach by isolating and characterizing synthetic molecules that influence stomatal movement. Here, we describe that a small chemical collection, prepared during the development of C-H amination reactions, lead to the discovery of a Stomata Influencing Molecule (SIM); namely, a sulfonimidated oxazole that inhibits stomatal opening. The starting molecule SIM1 was initially isolated from screening of compounds that inhibit light induced opening of dayflower stomata. A range of SIM molecules were rapidly accessed using our state-of-the-art C-H amination technologies. This enabled an efficient structure-activity relationship (SAR) study, culminating in the discovery of a sulfonamidated oxazole derivative (SIM*) having higher activity and enhanced specificity against stomatal regulation. Biological assay results have shed some light on the mode of action of SIM molecules within the cell, which may ultimately lead to drought tolerance-conferring agrochemicals through the control of stomatal movement.

  2022年01月, Scientific reports, 12(1) (1), 949 - 949, 英語, 国際誌

  研究論文（学術雑誌）


• Protease Inhibitor-Dependent Inhibition of Light-Induced Stomatal Opening.

  Tenghua Wang, Wenxiu Ye, Yin Wang, Maoxing Zhang, Yusuke Aihara, Toshinori Kinoshita

  Stomata in the epidermis of plants play essential roles in the regulation of photosynthesis and transpiration. Stomata open in response to blue light (BL) by phosphorylation-dependent activation of the plasma membrane (PM) H+-ATPase in guard cells. Under water stress, the plant hormone abscisic acid (ABA) promotes stomatal closure via the ABA-signaling pathway to reduce water loss. We established a chemical screening method to identify compounds that affect stomatal movements in Commelina benghalensis. We performed chemical screening using a protease inhibitor (PI) library of 130 inhibitors to identify inhibitors of stomatal movement. We discovered 17 PIs that inhibited light-induced stomatal opening by more than 50%. Further analysis of the top three inhibitors (PI1, PI2, and PI3; inhibitors of ubiquitin-specific protease 1, membrane type-1 matrix metalloproteinase, and matrix metalloproteinase-2, respectively) revealed that these inhibitors suppressed BL-induced phosphorylation of the PM H+-ATPase but had no effect on the activity of phototropins or ABA-dependent responses. The results suggest that these PIs suppress BL-induced stomatal opening at least in part by inhibiting PM H+-ATPase activity but not the ABA-signaling pathway. The targets of PI1, PI2, and PI3 were predicted by bioinformatics analyses, which provided insight into factors involved in BL-induced stomatal opening.

  2021年, Frontiers in plant science, 12, 735328 - 735328, 英語, 国際誌

  研究論文（学術雑誌）


• Effect of nanocarbon molecules on theArabidopsis thalianatranscriptome

  Norihito Nakamichi, Ayato Sato, Yusuke Aihara, Toshinori Kinoshita, Yasutomo Segawa, Kazuma Amaike, Kenichiro Itami

  Abstract Nanocarbons, such as fullerenes, carbon nanotubes, and graphene have attracted a great deal of attention as next-generation materials because of their unprecedented structures and unique physicochemical properties; however, almost all nanocarbons reported previously were used as mixtures. Thus, there are still many unsolved issues about their biological functions at the molecular level. Our synthetic campaign in the last decade has synthesized structurally uniform and atomically precise nanocarbons, leading to the preparation of a library consisting of eighty structurally diverse nanocarbon molecules. This resource motivated us to explore the as yet uncovered biological functions of these nanocarbon molecules in organisms. Recently, nanotubes were used to deliver genes to plants; however, the effects of the molecules on plants are not well known. To monitor the effects of nanocarbon molecules on plants, we analyzed the transcriptome ofArabidopsis thalianaseedlings treated with [9]cycloparaphenylene (CPP), decaborylated warped nanographene (WNG), and dimethoxyhexabenzotetracene (HBT). Clustering analysis indicated few effects of nanocarbon molecules on the transcriptome, perhaps suggesting a low toxicity of nanocarbon molecules on plants. We found thatAT1G05880(ARIADNE 12) gene, categorized into ‘response to hypoxia’ genes, was up-regulated by nanocarbon molecules, suggesting that this gene is usable as maker for treatment of nanocarbon molecules.

  Cold Spring Harbor Laboratory, 2020年05月


• Global Shifts in Gene Expression Profiles Accompanied with Environmental Changes in Cnidarian-Dinoflagellate Endosymbiosis.

  Yuu Ishii, Shinichiro Maruyama, Hiroki Takahashi, Yusuke Aihara, Takeshi Yamaguchi, Katsushi Yamaguchi, Shuji Shigenobu, Masakado Kawata, Naoto Ueno, Jun Minagawa

  Stable endosymbiotic relationships between cnidarian animals and dinoflagellate algae are vital for sustaining coral reef ecosystems. Recent studies have shown that elevated seawater temperatures can cause the collapse of their endosymbiosis, known as 'bleaching', and result in mass mortality. However, the molecular interplay between temperature responses and symbiotic states still remains unclear. To identify candidate genes relevant to the symbiotic stability, we performed transcriptomic analyses under multiple conditions using the symbiotic and apo-symbiotic (symbiont free) Exaiptasia diaphana, an emerging model sea anemone. Gene expression patterns showed that large parts of differentially expressed genes in response to heat stress were specific to the symbiotic state, suggesting that the host sea anemone could react to environmental changes in a symbiotic state-dependent manner. Comparative analysis of expression profiles under multiple conditions highlighted candidate genes potentially important in the symbiotic state transition under heat-induced bleaching. Many of these genes were functionally associated with carbohydrate and protein metabolisms in lysosomes. Symbiont algal genes differentially expressed in hospite encode proteins related to heat shock response, calcium signaling, organellar protein transport, and sugar metabolism. Our data suggest that heat stress alters gene expression in both the hosts and symbionts. In particular, heat stress may affect the lysosome-mediated degradation and transportation of substrates such as carbohydrates through the symbiosome (phagosome-derived organelle harboring symbiont) membrane, which potentially might attenuate the stability of symbiosis and lead to bleaching-associated symbiotic state transition.

  2019年07月, G3 (Bethesda, Md.), 9(7) (7), 2337 - 2347, 英語, 国際誌

  研究論文（学術雑誌）


• Green fluorescence from cnidarian hosts attracts symbiotic algae.

  Yusuke Aihara, Shinichiro Maruyama, Andrew H Baird, Akira Iguchi, Shunichi Takahashi, Jun Minagawa

  Reef-building corals thrive in nutrient-poor marine environments because of an obligate symbiosis with photosynthetic dinoflagellates of the genus Symbiodinium Symbiosis is established in most corals through the uptake of Symbiodinium from the environment. Corals are sessile for most of their life history, whereas free-living Symbiodinium are motile; hence, a mechanism to attract Symbiodinium would greatly increase the probability of encounter between host and symbiont. Here, we examined whether corals can attract free-living motile Symbiodinium by their green fluorescence, emitted by the excitation of endogenous GFP by purple-blue light. We found that Symbiodinium have positive and negative phototaxis toward weak green and strong purple-blue light, respectively. Under light conditions that cause corals to emit green fluorescence, (e.g., strong blue light), Symbiodinium were attracted toward live coral fragments. Symbiodinium were also attracted toward an artificial green fluorescence dye with similar excitation and emission spectra to coral-GFP. In the field, more Symbiodinium were found in traps painted with a green fluorescence dye than in controls. Our results revealed a biological signaling mechanism between the coral host and its potential symbionts.

  2019年02月, Proceedings of the National Academy of Sciences of the United States of America, 116(6) (6), 2118 - 2123, 英語, 国際誌

  研究論文（学術雑誌）


• Algal photoprotection is regulated by the E3 ligase CUL4-DDB1DET1.

  Yusuke Aihara, Konomi Fujimura-Kamada, Tomohito Yamasaki, Jun Minagawa

  Light is essential for photosynthesis, but the amounts of light that exceed an organism's assimilation capacity can cause serious damage1. Photosynthetic organisms minimize such potential harm through protection mechanisms collectively referred to as non-photochemical quenching2. One mechanism of non-photochemical quenching called energy-dependent quenching (qE quenching) is readily activated under high-light conditions and dissipates excess energy as heat. LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEINS 1 and 3 (LHCSR1 and LHCSR3) have been proposed to mediate qE quenching in the green alga Chlamydomonas reinhardtii when grown under high-light conditions3. LHCSR3 induction requires a blue-light photoreceptor, PHOTOTROPIN (PHOT)4, although the signal transduction pathway between PHOT and LHCSR3 is not yet clear. Here, we identify two phot suppressor loci involved in qE quenching: de-etiolated 1 (det1)5 and damaged DNA-binding 1 (ddb1)6. Using a yeast two-hybrid analysis and an inhibitor assay, we determined that these two genetic elements are part of a protein complex containing CULLIN 4 (CUL4). These findings suggest a photoprotective role for the putative E3 ubiquitin ligase CUL4-DDB1DET1 in unicellular photosynthetic organisms that may mediate blue-light signals to LHCSR1 and LHCSR3 gene expression.

  2019年01月, Nature plants, 5(1) (1), 34 - 40, 英語, 国際誌

  研究論文（学術雑誌）


• Acceptable symbiont cell size differs among cnidarian species and may limit symbiont diversity.

  Elise Biquand, Nami Okubo, Yusuke Aihara, Vivien Rolland, David C Hayward, Masayuki Hatta, Jun Minagawa, Tadashi Maruyama, Shunichi Takahashi

  Reef-building corals form symbiotic relationships with dinoflagellates of the genus Symbiodinium. Symbiodinium are genetically and physiologically diverse, and corals may be able to adapt to different environments by altering their dominant Symbiodinium phylotype. Notably, each coral species associates only with specific Symbiodinium phylotypes, and consequently the diversity of symbionts available to the host is limited by the species specificity. Currently, it is widely presumed that species specificity is determined by the combination of cell-surface molecules on the host and symbiont. Here we show experimental evidence supporting a new model to explain at least part of the specificity in coral-Symbiodinium symbiosis. Using the laboratory model Aiptasia-Symbiodinium system, we found that symbiont infectivity is related to cell size; larger Symbiodinium phylotypes are less likely to establish a symbiotic relationship with the host Aiptasia. This size dependency is further supported by experiments where symbionts were replaced by artificial fluorescent microspheres. Finally, experiments using two different coral species demonstrate that our size-dependent-infection model can be expanded to coral-Symbiodinium symbiosis, with the acceptability of large-sized Symbiodinium phylotypes differing between two coral species. Thus the selectivity of the host for symbiont cell size can affect the diversity of symbionts in corals.

  2017年07月, The ISME journal, 11(7) (7), 1702 - 1712, 英語, 国際誌

  研究論文（学術雑誌）


• Heat Induction of Cyclic Electron Flow around Photosystem I in the Symbiotic Dinoflagellate Symbiodinium.

  Yusuke Aihara, Shunichi Takahashi, Jun Minagawa

  Increases in seawater temperature impair photosynthesis (photoinhibition) in the symbiotic dinoflagellate Symbiodinium within cnidarian hosts, such as corals and sea anemones, and may destroy their symbiotic relationship. Although the degree of photoinhibition in Symbiodinium under heat stress differs among strains, the differences in their responses to increased temperatures, including cyclic electron flow (CEF), which sustains photoprotective thermal energy dissipation, have not been investigated. Here, we examined CEF in cultured Symbiodinium cells or those in an endosymbiotic relationship within a cnidarian host. The light-dependent reduction of the primary electron donor photosystem I, i.e. P700(+), was enhanced in any Symbiodinium cell by increasing temperatures, indicating CEF was induced by heat, which was accompanied by thermal energy dissipation activation. The critical temperatures for inducing CEF were different among Symbiodinium strains. The clade A strains with greater susceptibility to photoinhibition, OTcH-1 and Y106, exhibited higher CEF activities under moderate heat stress than a more phototolerant clade B strain Mf1.05b, suggesting that the observed CEF induction was not a preventive measure but a stress response in Symbiodinium.

  2016年05月, Plant physiology, 171(1) (1), 522 - 9, 英語, 国際誌

  研究論文（学術雑誌）


• The phototropic response is locally regulated within the topmost light-responsive region of the Arabidopsis thaliana seedling.

  Kazuhiko Yamamoto, Tomomi Suzuki, Yusuke Aihara, Ken Haga, Tatsuya Sakai, Akira Nagatani

  Phototropism is caused by differential cell elongation between the irradiated and shaded sides of plant organs, such as the stem. It is widely accepted that an uneven auxin distribution between the two sides crucially participates in this response. Plant-specific blue-light photoreceptors, phototropins (phot1 and phot2), mediate this response. In grass coleoptiles, the sites of light perception and phototropic bending are spatially separated. However, these sites are less clearly distinguished in dicots. Furthermore, the exact placement of the action of each phototropic signaling factor remains unknown. Here, we investigated the spatial aspects of phototropism using spotlight irradiation with etiolated Arabidopsis seedlings. The results demonstrated that the topmost part of about 1.1 mm of the hypocotyl constituted the light-responsive region in which both light perception and actual bending occurred. In addition, cotyledons and the shoot apex were dispensable for the response. Hence, the response was more region autonomous in dicots than in monocots. We next examined the elongation rates, the levels of phot1 and the auxin-reporter gene expression along the hypocotyl during the phototropic response. The light-responsive region was more active than the non-responsive region with respect to all of those parameters.

  2014年03月, Plant & cell physiology, 55(3) (3), 497 - 506, 英語, 国内誌

  研究論文（学術雑誌）


• Light-induced conformational changes of LOV1 (light oxygen voltage-sensing domain 1) and LOV2 relative to the kinase domain and regulation of kinase activity in Chlamydomonas phototropin.

  Koji Okajima, Yusuke Aihara, Yuki Takayama, Mihoko Nakajima, Sachiko Kashojiya, Takaaki Hikima, Tomotaka Oroguchi, Amane Kobayashi, Yuki Sekiguchi, Masaki Yamamoto, Tomomi Suzuki, Akira Nagatani, Masayoshi Nakasako, Satoru Tokutomi

  Phototropin (phot), a blue light (BL) receptor in plants, has two photoreceptive domains named LOV1 and LOV2 as well as a Ser/Thr kinase domain (KD) and acts as a BL-regulated protein kinase. A LOV domain harbors a flavin mononucleotide that undergoes a cyclic photoreaction upon BL excitation via a signaling state in which the inhibition of the kinase activity by LOV2 is negated. To understand the molecular mechanism underlying the BL-dependent activation of the kinase, the photochemistry, kinase activity, and molecular structure were studied with the phot of Chlamydomonas reinhardtii. Full-length and LOV2-KD samples of C. reinhardtii phot showed cyclic photoreaction characteristics with the activation of LOV- and BL-dependent kinase. Truncation of LOV1 decreased the photosensitivity of the kinase activation, which was well explained by the fact that the signaling state lasted for a shorter period of time compared with that of the phot. Small angle x-ray scattering revealed monomeric forms of the proteins in solution and detected BL-dependent conformational changes, suggesting an extension of the global molecular shapes of both samples. Constructed molecular model of full-length phot based on the small angle x-ray scattering data proved the arrangement of LOV1, LOV2, and KD for the first time that showed a tandem arrangement both in the dark and under BL irradiation. The models suggest that LOV1 alters its position relative to LOV2-KD under BL irradiation. This finding demonstrates that LOV1 may interact with LOV2 and modify the photosensitivity of the kinase activation through alteration of the duration of the signaling state in LOV2.

  2014年01月, The Journal of biological chemistry, 289(1) (1), 413 - 22, 英語, 国際誌

  研究論文（学術雑誌）


• Mutations in N-terminal flanking region of blue light-sensing light-oxygen and voltage 2 (LOV2) domain disrupt its repressive activity on kinase domain in the Chlamydomonas phototropin.

  Yusuke Aihara, Takaharu Yamamoto, Koji Okajima, Kazuhiko Yamamoto, Tomomi Suzuki, Satoru Tokutomi, Kazuma Tanaka, Akira Nagatani

  Phototropin is a light-regulated kinase that mediates a variety of photoresponses such as phototropism, chloroplast positioning, and stomata opening in plants to increase the photosynthetic efficiency. Blue light stimulus first induces local conformational changes in the chromophore-bearing light-oxygen and voltage 2 (LOV2) domain of phototropin, which in turn activates the serine/threonine (Ser/Thr) kinase domain in the C terminus. To examine the kinase activity of full-length phototropin conventionally, we employed the budding yeast Saccharomyces cerevisiae. In this organism, Ser/Thr kinases (Fpk1p and Fpk2p) that show high sequence similarity to the kinase domain of phototropins exist. First, we demonstrated that the phototropin from Chlamydomonas reinhardtii (CrPHOT) could complement loss of Fpk1p and Fpk2p to allow cell growth in yeast. Furthermore, this reaction was blue light-dependent, indicating that CrPHOT was indeed light-activated in yeast cells. We applied this system to a large scale screening for amino acid substitutions in CrPHOT that elevated the kinase activity in darkness. Consequently, we identified a cluster of mutations located in the N-terminal flanking region of LOV2 (R199C, L202L, D203N/G/V, L204P, T207I, and R210H). An in vitro phosphorylation assay confirmed that these mutations substantially reduced the repressive activity of LOV2 on the kinase domain in darkness. Furthermore, biochemical analyses of the representative T207I mutant demonstrated that the mutation affected neither spectral nor multimerization properties of CrPHOT. Hence, the N-terminal flanking region of LOV2, as is the case with the C-terminal flanking Jα region, appears to play a crucial role in the regulation of kinase activity in phototropin.

  2012年03月, The Journal of biological chemistry, 287(13) (13), 9901 - 9909, 英語, 国際誌

  研究論文（学術雑誌）


• Molecular basis of the functional specificities of phototropin 1 and 2.

  Yusuke Aihara, Ryohei Tabata, Tomomi Suzuki, Ken-Ichiro Shimazaki, Akira Nagatani

  A blue-light photoreceptor in plants, phototropin, mediates phototropism, chloroplast relocation, stomatal opening, and leaf-flattening responses. Phototropin is divided into two functional moieties, the N-terminal photosensory and the C-terminal signaling moieties. Phototropin perceives light stimuli by the light, oxygen or voltage (LOV) domain in the N-terminus; the signal is then transduced intramolecularly to the C-terminal kinase domain. Two phototropins, phot1 and phot2, which have overlapping and distinct functions, exist in Arabidopsis thaliana. Phot1 mediates responses with higher sensitivity than phot2. Phot2 mediates specific responses, such as the chloroplast avoidance response and chloroplast dark positioning. To elucidate the molecular basis for the functional specificities of phot1 and phot2, we exchanged the N- and C-terminal moieties of phot1 and phot2, fused them to GFP and expressed them under the PHOT2 promoter in the phot1 phot2 mutant background. With respect to phototropism and other responses, the chimeric phototropin consisting of phot1 N-terminal and phot2 C-terminal moieties (P1n/2cG) was almost as sensitive as phot1; whereas the reverse combination (P2n/1cG) functioned with lower sensitivity. Hence, the N-terminal moiety mainly determined the sensitivity of the phototropins. Unexpectedly, both P1n/2cG and P2n/1cG mediated the chloroplast avoidance response, which is specific to phot2. Hence, chloroplast avoidance activity appeared to be suppressed specifically in the combination of N- and C-terminal moieties of phot1. Unlike the chloroplast avoidance response, chloroplast dark positioning was observed for P2G and P2n/1cG but not for P1G or P1n/2cG, suggesting that a specific structure in the N-terminal moiety of phot2 is required for this activity.

  2008年11月, The Plant journal : for cell and molecular biology, 56(3) (3), 364 - 75, 英語, 国際誌

  研究論文（学術雑誌）

• 植物を操る機能分子の創生と活用 4 気孔開閉を制御する分子とその先

  相原悠介, 相原悠介

  2025年, アグリバイオ, 9(6) (6)


• 「標的タンパク質をゲットしたい!」植物研究者によるプロテオーム解析事始め

  相原悠介

  2025年, 日本プロテオーム学会大会プログラム・抄録集, 2025


• イソチオシアネートを基盤とした気孔開口阻害剤の機能解析と分子改良

  相原悠介, 相原悠介, 村上慧, 村上慧, 佐藤綾人, 木下俊則, 木下俊則

  2024年, 植物の生長調節, 59(1) (1)


• 植物修飾分子による多面的機能の解明とテイラーメイド制御を目指して

  相原悠介

  2024年, 日本化学会春季年会講演予稿集(Web), 104th


• 植物の気孔開口を抑えてしおれを防ぐケミカルツール その作用・分子改良・応用展開まで

  相原悠介, 相原悠介

  2024年, 化学と生物, 62(11) (11)


• 気孔発生を特異的に阻害するイミダゾロン化合物Stomidazoloneの同定

  中川彩美, MOHAN SEPURU Krishna, MOHAN SEPURU Krishna, SHU JAN Yip, SEO Hyemin, SEO Hyemin, COFFIN Calvin, COFFIN Calvin, 瀬川泰知, 岩崎理恵, 加藤弘恵, KIM Stephanie, 相原悠介, 相原悠介, 木下俊則, 伊丹健一郎, HAN Soon-ki, HAN Soon-ki, 村上慧, 村上慧, 村上慧, 鳥居啓子, 鳥居啓子, 鳥居啓子

  2024年, 日本植物生理学会年会(Web), 65th


• 植物修飾分子イソチオシアネートの植物細胞内標的タンパク質を同定するためのプロテオミクス解析

  相原悠介, 相原悠介, 前田文平, 加納圭子, 三城恵美, 佐藤綾人, 木下俊則, 木下俊則, 村上慧, 村上慧

  2024年, 日本プロテオーム学会大会プログラム・抄録集, 2024 (Web)


• イソチオシアネートを基盤とした植物の乾燥耐性付与剤の発見と改良

  相原悠介, 前田文平, 後藤栞奈, 藤茂雄, 藤茂雄, 叶文秀, 叶文秀, 戸田陽介, 村上慧, 村上慧, 佐藤綾人, 伊丹健一郎, 伊丹健一郎, 木下俊則, 木下俊則

  2022年, 日本植物学会大会研究発表記録(CD-ROM), 86th


• サンゴの緑色蛍光は共生藻類の誘引にはたらく

  相原悠介, 相原悠介

  2019年, 光合成研究, 29(2) (2)


• CUL4-DDB1^DET1複合体は,緑藻クラミドモナスにおけるNPQ誘導の制御因子である

  相原悠介, 鎌田このみ, 山崎朋人, 皆川純

  2018年, 日本植物生理学会年会(Web), 59th


• フォトトロピンのLOV2領域・N末端側近傍の変異によりキナーゼ活性の抑制機能が失われる

  相原悠介, 山本隆晴, 岡島公司, 山本和彦, 鈴木友美, 徳富哲, 田中一馬, 長谷あきら

  2012年, 日本植物生理学会年会要旨集, 53rd


• 青色光受容体フォトトロピンの"Flippase-kinase"としての役割の解明

  相原 悠介, 山本 隆晴, 武宮 淳史, 鈴木 友美, 加藤 詩子, 梅田 真郷, 田中 一馬, 島崎 研一郎, 長谷 あきら

  フォトトロピン(phot)は光屈性・葉緑体運動・気孔開口等を制御する青色光受容体である。photは青色光依存的にキナーゼ活性を示すが、どのような分子のリン酸化／機能制御を担うのか不明である。我々は、photとキナーゼ領域において高い相同性を示す、出芽酵母のFpk1 (Flippase kinase 1)およびFpk2に着目した。Fpkはリン脂質トランスロケース”Flippase”をリン酸化により活性化すると示唆されている。Flippaseは、生体膜脂質二重層間におけるリン脂質の非対称分布を形成・調節することで、多彩な細胞機能に寄与する。
  本研究ではまず、photが出芽酵母におけるFpkの機能を相補するか調べた。出芽酵母のfpk1fpk2欠損変異株は、アミノリン脂質結合ペプチドによる致死作用に対し高感受性を示す。またFlippaseのサブユニットをコードするCDC50遺伝子の欠損変異と合成致死を示す。これらの表現型は、シロイヌナズナのphotを導入することで相補された。一方、シロイヌナズナにおいてはFlippase遺伝子としてALA1～12が報告されている。そこでこれらの欠損変異体の幾つかを調べたところ、ala3変異体において青色光による気孔開口が見られなかった。これらの結果から、photがALA3の機能を制御することで気孔開口シグナルが伝達されると仮説を立て、解析を進めている。

  日本植物生理学会, 2010年, 日本植物生理学会年会およびシンポジウム 講演要旨集, 2010, 0350 - 0350


• シロイヌナズナphot1・phot2の生理応答特異性に関するドメイン機能解析

  相原 悠介, 鈴木 友美, 長谷 あきら

  シロイヌナズナの青色光受容体phot1とphot2はいずれもN末端側に光感受性（LOV）領域、C末端側にSer/Thrキナーゼ領域を持つ。phot1とphot2は構造上、非常に良く似ているにもかかわらず、異なる生理機能を持つ。すなわち、光屈性などにおいてphot1はphot2よりも高い光感受性を示す一方、葉緑体の逃避反応はphot2でのみ起こる。これらの差は両分子の構造の違いに由来すると推測される。そこで本研究ではLOVとキナーゼ領域をphot1、phot2間で交換し、GFPを融合したキメラ遺伝子（P1n/2cG、P2n/1cG）を構築した。これらキメラ遺伝子およびPHOT1-GFP、PHOT2-GFPをPHOT2プロモーター制御下で発現させた形質転換植物（phot1/phot2二重変異体背景）を作出し、生理応答を観察した。光屈性の解析の結果から、光感受性の違いは主にN末端領域により決定されることが示された。また葉緑体定位運動の観察ではP1n/2cG、P2n/1cGともに葉緑体の集合・逃避両反応を引き起こした。すなわち、逃避反応はphot2に特有の機能ではなく、むしろ、phot1(N)/phot1(C)の組み合わせ（すなわち内在phot1）でのみ逃避反応が抑制されると考えられる。これらキメラ分子を含めた、phototropinの細胞内局在についても報告する。

  日本植物生理学会, 2008年, 日本植物生理学会年会およびシンポジウム 講演要旨集, 2008, 0205 - 0205

• シロイヌナズナを用いた揮発性有機物のリアルタイム検出プラットフォーム

  関本奏子, 杉山龍介, 福山大輔, 呂行子, 野元美佳, 多田安臣, 相原悠介

  日本植物生理学会年会, 2026年03月, 日本語

  口頭発表（一般）


• 気孔運動を制御する植物分子イソチオシアネートの多面的機能の解明を目指して

  相原悠介

  日本植物学会年会, 2025年09月, 日本語

  [招待有り]

  シンポジウム・ワークショップパネル（指名）

• 天然物生物学：植物二次代謝産物が有する生物学的意義の体系化

  瀬戸 義哉, 佐藤 道大, 杉山 龍介, 白川 一, 相原 悠介

  日本学術振興会, 科学研究費助成事業, 学術変革領域研究(B), 明治大学, 2024年04月01日 - 2027年03月31日


• 一細胞レベルでの高解像度発現解析と代謝産物の標的同定による天然物生物学

  白川 一, 相原 悠介

  日本学術振興会, 科学研究費助成事業, 学術変革領域研究(B), 奈良先端科学技術大学院大学, 2024年04月01日 - 2027年03月31日


• 植物修飾分子による多面的機能のテイラーメイド制御

  科学技術振興機構(JST), 戦略的創造研究推進事業 さきがけ 植物分子の機能と制御, 名古屋大学, 2022年10月 - 2026年03月, 研究代表者


• 植物の特化代謝物による新規の翻訳後修飾機構

  科学技術振興機構(JST), 戦略的創造研究推進事業 ACT-X 生命と化学, 名古屋大学, 2019年10月 - 2023年03月, 研究代表者


• サンゴの蛍光タンパク質は褐虫藻の誘引に働く

  相原 悠介

  日本学術振興会, 科学研究費助成事業 挑戦的萌芽研究, 挑戦的萌芽研究, 基礎生物学研究所, 2016年04月01日 - 2018年03月31日

  造礁サンゴは緑色蛍光タンパク質（GFP）とその類縁の蛍光タンパク質を大量に産生し鮮やかに呈色する。しかしながら蛍光タンパク質がサンゴにとってどのような役割を持つかはいまだに明らかでない。本研究では「サンゴの蛍光タンパク質が共生相手の褐虫藻を誘引する」ことを検証した。褐虫藻の正と負の走光性は、それぞれ緑色光と青色光に対して最大となった。さらに、緑色蛍光を発するサンゴ及び人工物が褐虫藻を誘引することを確かめた。実際にサンゴ礁にて、蛍光塗料を塗布したトラップに自然海水中の褐虫藻が誘引される傾向を見出した。以上により、サンゴ礁光環境において、褐虫藻がサンゴの蛍光タンパク質に誘引されうることを実証した。


• 青色光受容体フォトトロピンによる生体膜リン脂質の非対称性分布制御

  相原 悠介

  日本学術振興会, 科学研究費助成事業 特別研究員奨励費, 特別研究員奨励費, 京都大学, 2010年 - 2011年

  フォトトロピンは青色光を受容して自身を活性化するプロテインキナーゼであり、植物の様々な生理応答を制御する。その光受容機構としては、発色団を結合するLOV2領域が青色光を吸収して局所的に構造変化し、周辺領域の更なる変化がキナーゼ領域を活性化すると考えられている。本研究では出芽酵母を利用し、全長フォトトロピンのキナーゼ活性化を簡便に検定する実験系を確立した。出芽酵母はフォトトロピンと高い配列相同性を示すキナーゼ、Fpk1pおよびFpk2pを持つ。まず、酵母菌の変異株において、クラミドモナス由来のフォトトロピン(CrPHOT)がFpk1p・Fpk2pの欠損を相補し、青色光依存的に細胞増殖を促進することを明らかにした。次にこの実験系を分子内変異のスクリーニングに応用し、暗所でキナーゼ活性が上昇してしまう変異型CrPHOTを多数取得した。得られた変異は、すべてこれまで知られていない新奇のものであった。中でも、LOV2のN末端側近傍に新奇の変異が集中することを見出した。更にin vitroリン酸化実験により、これらの変異がCrPHOTのキナーゼ活性を暗所で上昇させることを生化学的に確かめた。一方、少なくとも代表として解析したT207I変異では、分光学的性質、多量体化のいずれにも変化はなかった。以上より、LOV2のN末端側近傍領域が、これまで報告されていたC末端側近傍(Jα)領域と同様に、フォトトロピンのキナーゼ活性化に決定的な役割を果たすことが初めて示された。