Performance information

• The deficiency of methylglyoxal synthase promotes cell proliferation in Synechocystis sp. PCC 6803 under mixotrophic conditions　　　　　　　　　　　　　　　
  Aikeranmu Kadeer; Yuuma Ishikawa; Kaushalya Dayarathne; Atsuko Miyagi; Toshiki Ishikawa; Masatoshi Yamaguchi; Maki Kawai-Yamada
  Plant Biotechnology, Volume：41, Number：4, First page：393, Last page：399, Dec. 2024, [Reviewed]
  English, Scientific journal
  DOI：https://doi.org/10.5511/plantbiotechnology.24.0718a
  DOI ID：10.5511/plantbiotechnology.24.0718a
• Accumulation of acyl plastoquinol and triacylglycerol in six cyanobacterial species with different sets of genes encoding type-2 diacylglycerol acyltransferase-like proteins
  Riko Tanikawa; Haruna Sakaguchi; Toshiki Ishikawa; Yukako Hihara
  Plant And Cell Physiology, Volume：66, Number：1, First page：15, Last page：22, Nov. 2024, [Reviewed]
  Abstract
  
  Recently, acyl plastoquinol (APQ) and plastoquinone-B (PQ-B), which are fatty acid esters of plastoquinol and plastoquinone-C respectively, have been identified as the major neutral lipids in cyanobacteria. In Synechocystis sp. PCC 6803, Slr2103 having homology with the eukaryotic enzyme for triacylglycerol (TAG) synthesis, diacylglycerol acyltransferase 2 (DGAT2), was identified as responsible for the synthesis of these plastoquinone-related lipids. On the other hand, TAG synthesis in cyanobacteria remains controversial due to the low accumulation level within cyanobacterial cells together with the high contamination level from the environment. In this study, to quantify more precisely and elucidate the relationship between the accumulation of neutral lipids and the presence or absence of DGAT2-like genes, plastoquinone-related lipids and TAG were analyzed directly from total lipids of six cyanobacterial species with different sets of genes encoding DGAT2-like proteins belonging to two distinct subclades. The results showed that the synthesis of these neutral lipids is highly dependent on clade A DGAT2-like proteins under the culture conditions used in this study, although accumulation level of TAG was quite low. In contrast to APQ highly abundant in saturated fatty acids, the fatty acid composition of TAG was species-specific and partly reflected the total lipid composition. Gloeobacter violaceus PCC 7421, which lacks a DGAT2-like gene, accumulated APQ with a high proportion of C18:0, suggesting APQ synthesis by an unidentified acyltransferase.
  Oxford University Press (OUP), Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcae137
  DOI ID：10.1093/pcp/pcae137, ISSN：0032-0781, eISSN：1471-9053
• Arabidopsis DREB26/ERF12 and its close relatives regulate cuticular wax biosynthesis under drought stress condition.　　　　　　　　　　　　　　　
  Kaoru Urano; Yoshimi Oshima; Toshiki Ishikawa; Takuma Kajino; Shingo Sakamoto; Mayuko Sato; Kiminori Toyooka; Miki Fujita; Maki Kawai-Yamada; Teruaki Taji; Kyonoshin Maruyama; Kazuko Yamaguchi-Shinozaki; Kazuo Shinozaki
  The Plant journal : for cell and molecular biology, Oct. 2024, [Reviewed], ［International magazine］
  Land plants have evolved a hydrophobic cuticle on the surface of aerial organs as an adaptation to ensure survival in terrestrial environments. Cuticle is mainly composed of lipids, namely cutin and intracuticular wax, with epicuticular wax deposited on plant surface. The composition and permeability of cuticle have a large influence on its ability to protect plants against drought stress. However, the regulatory mechanisms underlying cuticular wax biosynthesis in response to drought stress have not been fully elucidated. Here, we identified three AP2/ERF transcription factors (DREB26/ERF12, ERF13 and ERF14) involved in the regulation of water permeability of the plant surface. Transmission electron microscopy revealed thicker cuticle on the leaves of DREB26-overexpressing (DREB26OX) plants, and thinner cuticle on the leaves of transgenic plants expressing SRDX repression domain-fused DREB26 (DREB26SR). Genes involved in cuticular wax formation were upregulated in DREB26OX and downregulated in DREB26SR. The levels of very-long chain (VLC) alkanes, which are a major wax component, increased in DREB26OX leaves and decreased in DREB26SR leaves. Under dehydration stress, water loss was reduced in DREB26OX and increased in DREB26SR. The erf12/13/14 triple mutant showed delayed growth, decreased leaf water content, and reduced drought-inducible VLC alkane accumulation. Taken together, our results indicate that the DREB26/ERF12 and its closed family members, ERF13 and ERF14, play an important role in cuticular wax biosynthesis in response to drought stress. The complex transcriptional cascade involved in the regulation of cuticular wax biosynthesis under drought stress conditions is discussed.
  English, Scientific journal
  DOI：https://doi.org/10.1111/tpj.17100
  DOI ID：10.1111/tpj.17100, PubMed ID：39466828
• The effect of light availability and light wavelength on growth, 2-MIB biosynthesis, and 2-MIB-related gene expression in Pseudanabaena foetida var. intermedia.　　　　　　　　　　　　　　　
  Kaushalya Dayarathne; Toshiki Ishikawa; Aikeranmu Kadeer; Masatoshi Yamaguchi; Maki Kawai-Yamada
  Archives of microbiology, Volume：206, Number：9, First page：367, Last page：367, Aug. 2024, [Reviewed], ［International magazine］
  2-methylisoborneol (2-MIB) is an odiferous metabolite mainly produced by cyanobacteria, contributing to taste and odor problems in drinking water. The mechanisms involved in 2-MIB biosynthesis in cyanobacteria are not yet completely understood. This study investigated the effect of light availability and wavelength on growth, 2-MIB synthesis, and related gene expression in Pseudanabaena foetida var. intermedia. A significantly lower 2-MIB production was observed in P. foetida var. intermedia during the dark period of a 12-h photoperiod. Exposure to green light resulted in a significant decrease in 2-MIB production compared to white light and red light. The relative expression levels of 2-MIB-related genes in P. foetida var. intermedia were significantly lower during the dark period of a 12-h photoperiod and when cultured under green light. The expression of 2-MIB-related genes in cyanobacteria appears to be light-dependent. This study suggests that the demand for photopigment synthesis under unfavorable light conditions affects the 2-MIB synthesis in cyanobacteria.
  English, Scientific journal
  DOI：https://doi.org/10.1007/s00203-024-04099-w
  DOI ID：10.1007/s00203-024-04099-w, PubMed ID：39105810
• Inhibitor of cardiolipin biosynthesis‐related enzyme MoGep4 confers broad‐spectrum anti‐fungal activity　　　　　　　　　　　　　　　
  Peng Sun; Juan Zhao; Gan Sha; Yaru Zhou; Mengfei Zhao; Renjian Li; Xiaojing Kong; Qiping Sun; Yun Li; Ke Li; Ruiqing Bi; Lei Yang; Ziting Qin; Wenzheng Huang; Yin Wang; Jie Gao; Guang Chen; Haifeng Zhang; Muhammad Adnan; Long Yang; Lu Zheng; Xiao‐Lin Chen; Guanghui Wang; Toshiki Ishikawa; Qiang Li; Jin‐Rong Xu; Guotian Li
  Plant, Cell & Environment, Volume：47, Number：11, First page：4259, Last page：4274, Jul. 2024, [Reviewed]
  Abstract
  
  Plant pathogens cause devastating diseases, leading to serious losses to agriculture. Mechanistic understanding of pathogenesis of plant pathogens lays the foundation for the development of fungicides for disease control. Mitophagy, a specific form of autophagy, is important for fungal virulence. The role of cardiolipin, mitochondrial signature phospholipid, in mitophagy and pathogenesis is largely unknown in plant pathogenic fungi. The functions of enzymes involved in cardiolipin biosynthesis and relevant inhibitors were assessed using a set of assays, including genetic deletion, plant infection, lipidomics, chemical‐protein interaction, chemical inhibition, and field trials. Our results showed that the cardiolipin biosynthesis‐related gene MoGEP4 of the rice blast fungus Magnaporthe oryzae regulates growth, conidiation, cardiolipin biosynthesis, and virulence. Mechanistically, MoGep4 regulated mitophagy and Mps1‐MAPK phosphorylation, which are required for virulence. Chemical alexidine dihydrochloride (AXD) inhibited the enzyme activity of MoGep4, cardiolipin biosynthesis and mitophagy. Importantly, AXD efficiently inhibited the growth of 10 plant pathogens and controlled rice blast and Fusarium head blight in the field. Our study demonstrated that MoGep4 regulates mitophagy, Mps1 phosphorylation and pathogenesis in M. oryzae. In addition, we found that the MoGep4 inhibitor, AXD, displays broad‐spectrum antifungal activity and is a promising candidate for fungicide development.
  Wiley, Scientific journal
  DOI：https://doi.org/10.1111/pce.15021
  DOI ID：10.1111/pce.15021, ISSN：0140-7791, eISSN：1365-3040, PubMed ID：38946254
• Characterization of Unique Eukaryotic Sphingolipids with Temperature-Dependent Δ8-Unsaturation from the Picoalga Ostreococcus tauri.　　　　　　　　　　　　　　　
  Toshiki Ishikawa; Frédéric Domergue; Alberto Amato; Florence Corellou
  Plant & cell physiology, Volume：65, Number：6, First page：1029, Last page：1046, Jun. 2024, [Reviewed], ［Domestic magazine］
  Sphingolipids (SLs) are ubiquitous components of eukaryotic cell membranes and are found in some prokaryotic organisms and viruses. They are composed of a sphingoid backbone that may be acylated and glycosylated. Assembly of various sphingoid base, fatty acyl and glycosyl moieties results in highly diverse structures. The functional significance of variations in SL chemical diversity and abundance is still in the early stages of investigation. Among SL modifications, Δ8-desaturation of the sphingoid base occurs only in plants and fungi. In plants, SL Δ8-unsaturation is involved in cold hardiness. Our knowledge of the structure and functions of SLs in microalgae lags far behind that of animals, plants and fungi. Original SL structures have been reported from microalgae. However, functional studies are still missing. Ostreococcus tauri is a minimal microalga at the base of the green lineage and is therefore a key organism for understanding lipid evolution. In the present work, we achieved the detailed characterization of O. tauri SLs and unveiled unique glycosylceramides as sole complex SLs. The head groups are reminiscent of bacterial SLs, as they contain hexuronic acid residues and can be polyglycosylated. Ceramide backbones show a limited variety, and SL modification is restricted to Δ8-unsaturation. The Δ8-SL desaturase from O. tauri only produced E isomers. Expression of both Δ8-SL desaturase and Δ8-unsaturation of sphingolipids varied with temperature, with lower levels at 24°C than at 14°C. Overexpression of the Δ8-SL desaturase dramatically increases the level of Δ8 unsaturation at 24°C and is paralleled by a failure to increase cell size. Our work provides the first characterization of O. tauri SLs and functional evidence for the involvement of SL Δ8-unsaturation for temperature acclimation in microalgae, suggesting that this function is an ancestral feature in the green lineage.
  English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcae007
  DOI ID：10.1093/pcp/pcae007, PubMed ID：38252418
• High Myristic Acid in Glycerolipids Enhances the Repair of Photodamaged Photosystem II under Strong Light.　　　　　　　　　　　　　　　
  Kazuki Kurima; Haruhiko Jimbo; Takashi Fujihara; Masakazu Saito; Toshiki Ishikawa; Hajime Wada
  Plant & cell physiology, Volume：65, Number：5, First page：790, Last page：797, May 2024, [Reviewed], ［Domestic magazine］
  Cyanobacteria inhabit areas with a broad range of light, temperature and nutrient conditions. The robustness of cyanobacterial cells, which can survive under different conditions, may depend on the resilience of photosynthetic activity. Cyanothece sp. PCC 8801 (Cyanothece), a freshwater cyanobacterium isolated from a Taiwanese rice field, had a higher repair activity of photodamaged photosystem II (PSII) under intense light than Synechocystis sp. PCC 6803 (Synechocystis), another freshwater cyanobacterium. Cyanothece contains myristic acid (14:0) as the major fatty acid at the sn-2 position of the glycerolipids. To investigate the role of 14:0 in the repair of photodamaged PSII, we used a Synechocystis transformant expressing a T-1274 encoding a lysophosphatidic acid acyltransferase (LPAAT) from Cyanothece. The wild-type and transformant cells contained 0.2 and 20.1 mol% of 14:0 in glycerolipids, respectively. The higher content of 14:0 in the transformants increased the fluidity of the thylakoid membrane. In the transformants, PSII repair was accelerated due to an enhancement in the de novo synthesis of D1 protein, and the production of singlet oxygen (1O2), which inhibited protein synthesis, was suppressed. The high content of 14:0 increased transfer of light energy received by phycobilisomes to PSI and CP47 in PSII and the content of carotenoids. These results indicated that an increase in 14:0 reduced 1O2 formation and enhanced PSII repair. The higher content of 14:0 in the glycerolipids may be required as a survival strategy for Cyanothece inhabiting a rice field under direct sunlight.
  English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcae021
  DOI ID：10.1093/pcp/pcae021, PubMed ID：38441322, PubMed Central ID：PMC11138363
• The phosphorylated pathway of serine biosynthesis affects sperm, embryo, and sporophyte development, and metabolism in Marchantia polymorpha　　　　　　　　　　　　　　　
  Mengyao Wang; Hiromitsu Tabeta; Kinuka Ohtaka; Ayuko Kuwahara; Ryuichi Nishihama; Toshiki Ishikawa; Kiminori Toyooka; Mayuko Sato; Mayumi Wakazaki; Hiromichi Akashi; Hiroshi Tsugawa; Tsubasa Shoji; Yozo Okazaki; Keisuke Yoshida; Ryoichi Sato; Ali Ferjani; Takayuki Kohchi; Masami Yokota Hirai
  Communications Biology, Volume：7, Number：1, First page：102, Last page：102, Jan. 2024, [Reviewed], ［International magazine］
  Abstract
  
  Serine metabolism is involved in various biological processes. Here we investigate primary functions of the phosphorylated pathway of serine biosynthesis in a non-vascular plant Marchantia polymorpha by analyzing knockout mutants of MpPGDH encoding 3-phosphoglycerate dehydrogenase in this pathway. Growth phenotypes indicate that serine from the phosphorylated pathway in the dark is crucial for thallus growth. Sperm development requires serine from the phosphorylated pathway, while egg formation does not. Functional MpPGDH in the maternal genome is necessary for embryo and sporophyte development. Under high CO₂ where the glycolate pathway of serine biosynthesis is inhibited, suppressed thallus growth of the mutants is not fully recovered by exogenously-supplemented serine, suggesting the importance of serine homeostasis involving the phosphorylated and glycolate pathways. Metabolomic phenotypes indicate that the phosphorylated pathway mainly influences the tricarboxylic acid cycle, the amino acid and nucleotide metabolism, and lipid metabolism. These results indicate the importance of the phosphorylated pathway of serine biosynthesis in the dark, in the development of sperm, embryo, and sporophyte, and metabolism in M. polymorpha.
  Springer Science and Business Media LLC, English, Scientific journal
  DOI：https://doi.org/10.1038/s42003-023-05746-6
  DOI ID：10.1038/s42003-023-05746-6, eISSN：2399-3642, PubMed ID：38267515, PubMed Central ID：PMC10808223
• Degradation of glycosylinositol phosphoceramide during plant tissue homogenization.　　　　　　　　　　　　　　　
  Yoshimichi Takai; Rumana Yesmin Hasi; Naoko Matsumoto; Chiho Fujita; Hanif Ali; Junji Hayashi; Ryushi Kawakami; Mutsumi Aihara; Toshiki Ishikawa; Hiroyuki Imai; Mayuko Wakida; Kazuya Ando; Tamotsu Tanaka
  Journal of biochemistry, Volume：175, Number：1, First page：115, Last page：124, Dec. 2023, ［International magazine］
  A convenient method for the determination of plant sphingolipids (glycosylinositol phosphoceramide, GIPC; glucosylceramide, GluCer; phytoceramide 1-phosphate, PC1P and phytoceramide, PCer) was developed. This method includes the extraction of lipids using 1-butanol, alkali hydrolysis with methylamine and separation by TLC. The amounts of sphingolipids in the sample were determined based on the relative intensities of standard sphingolipids visualized by primulin/UV on TLC. Using this method, we found that almost all GIPCs were degraded in response to tissue homogenization in cruciferous plants (cabbage, broccoli and Arabidopsis thaliana). The decrease in GIPCs was compensated for by increases in PC1P and PCer, indicating that GIPC was degraded by hydrolysis at the D and C positions of GIPC, respectively. In carrot roots and leaves, most of GIPC degradation was compensated for by an increase in PCer. In rice roots, the decrease in GIPCs was not fully explained by the increases in PC1P and PCer, indicating that enzymes other than phospholipase C and D activities operated. As the visualization of lipids on TLC is useful for detecting the appearance or disappearance of lipids, this method will be available for the characterization of metabolism of sphingolipids in plants.
  English, Scientific journal
  DOI：https://doi.org/10.1093/jb/mvad080
  DOI ID：10.1093/jb/mvad080, PubMed ID：37827526
• MHP1 and MHL generate odd-chain fatty acids from 2-hydroxy fatty acids in sphingolipids and are related to immunity in Arabidopsis thaliana　　　　　　　　　　　　　　　
  Marina Ushio; Toshiki Ishikawa; Takakazu Matsuura; Izumi C. Mori; Maki Kawai-Yamada; Yoichiro Fukao; Minoru Nagano
  Plant Science, Volume：336, First page：111840, Last page：111840, Nov. 2023, ［International magazine］
  In plants, the 2-hydroxy fatty acids (HFAs) of sphingolipids are important for plant growth and stress responses. Although the synthetic pathway of HFAs is well understood, their degradation has not yet been elucidated. In Saccharomyces cerevisiae, Mpo1 has been identified as a dioxygenase that degrades HFAs. This study examined the functions of two homologs of yeast Mpo1, MHP1 and MHL, in Arabidopsis thaliana. The mhp1 and mhp1mhl mutants showed a dwarf phenotype compared to that of the wild type. Lipid analysis of the mutants revealed the involvement of MHP1 and MHL in synthesizing odd-chain fatty acids (OCFAs), possibly by the degradation of HFAs. OCFAs are present in trace amounts in plants; however, their physiological significance is largely unknown. RNA sequence analysis of the mhp1mhl mutant revealed that growth-related genes decreased, whereas genes involved in stress response increased. Additionally, the mhp1mhl mutant had increased expression of defense-related genes and increased resistance to infection by Pseudomonas syringae pv. tomato DC3000 (Pto), and Pto carrying the effector AvrRpt2. Phytohormone analysis demonstrated that jasmonic acid in mhp1mhl was higher than that in the wild type. These results indicate that MHP1 and MHL are involved in synthesizing OCFAs and immunity in Arabidopsis.
  Elsevier BV, English, Scientific journal
  DOI：https://doi.org/10.1016/j.plantsci.2023.111840
  DOI ID：10.1016/j.plantsci.2023.111840, ISSN：0168-9452, PubMed ID：37619867
• Heterologous expression of mtf and mtc genes of Pseudanabaena foetida var. intermedia is sufficient to produce 2-methylisoborneol in Escherichia coli.　　　　　　　　　　　　　　　
  Kaushalya Dayarathne; Toshiki Ishikawa; Satoru Watanabe; Yuuma Ishikawa; Kadeer Aikeranmu; Hina Kitagawaa; Natsumi Komatsubara; Masatoshi Yamaguchi; Maki Kawai-Yamada
  Microbiology spectrum, Volume：11, Number：5, First page：e0256123, Sep. 2023, ［International magazine］
  Microbial volatile metabolite 2-methylisoborneol (2-MIB) causes odor and taste issues in drinking water, making it unappealing for human consumption. It has been suggested that 2-MIB biosynthesis consists of two main steps, namely, methylation of geranyl diphosphate into 2-methyl geranyl diphosphate by geranyl diphosphate methyl transferase (GPPMT) and subsequent cyclization into 2-MIB by 2-MIB synthase (MIBS). Pseudanabaena foetida var. intermedia is a 2-MIB-producing cyanobacterium whose GPPMT and MIBS enzymes are encoded by adjacent mtf and mtc genes. The present study identified a 2-MIB-related gene cluster composed of cnbA, mtf, mtc, and cnbB genes in P. foetida var. intermedia. The two homologous cyclic nucleotide-binding protein genes, cnbA and cnbB, were detected adjacent to the mtf and mtc genes, respectively. The nucleotide sequence of the cnbA-mtf-mtc-cnbB gene cluster showed 99.55% identity with 2-MIB synthesis-associated gene cluster of Pseudanabaena sp. dqh15. RT-PCR results revealed that mtf and mtc genes are co-expressed, while cnbA and cnbB genes are expressed independently in P. foetida var. intermedia. To investigate whether only mtf and mtc genes are sufficient for 2-MIB synthesis, the two-gene unit (mtf-mtc) was introduced into Escherichia coli strain JM109 via overexpression vector pYS1C. Gas chromatograph-mass spectrometry results showed that the E. coli strain transformed with mtf-mtc was able to produce 2-MIB. The intracellular 2-MIB level in P. foetida var. intermedia was higher than the extracellular 2-MIB level, while the transformed E. coli strain showed an opposite trend. Growth inhibition was observed in the 2-MIB-producing transformed E. coli strain. IMPORTANCE Contamination of drinking water with odiferous microbial metabolite 2-MIB is a worldwide concern. Removal of 2-MIB from drinking water burdens the water purification process. Therefore, it is important to search for alternative methods, such as suppressing the production of 2-MIB by aquatic microorganisms. For that, it is necessary to expand the current knowledge about the mechanism of 2-MIB synthesis at the genetic level. This study revealed that mtf and mtc genes of the 2-MIB-related gene cluster are transcribed as a single unit in P. foetida var. intermedia, and the expression of both mtf and mtc genes is essential and sufficient for 2-MIB synthesis in E. coli heterologous gene expression system.
  English, Scientific journal
  DOI：https://doi.org/10.1128/spectrum.02561-23
  DOI ID：10.1128/spectrum.02561-23, PubMed ID：37732762, PubMed Central ID：PMC10580876
• NAC domain transcription factors VNI2 and ATAF2 form protein complexes and regulate leaf senescence　　　　　　　　　　　　　　　
  Isura Sumeda Priyadarshana Nagahage; Kohei Matsuda; Kyoko Miyashita; Sumire Fujiwara; Chanaka Mannapperuma; Takuya Yamada; Shingo Sakamoto; Toshiki Ishikawa; Minoru Nagano; Misato Ohtani; Ko Kato; Hirofumi Uchimiya; Nobutaka Mitsuda; Maki Kawai‐Yamada; Taku Demura; Masatoshi Yamaguchi
  Plant Direct, Volume：7, Number：9, First page：e529, Sep. 2023, [Reviewed], ［International magazine］
  Abstract
  
  The NAM, ATAF1/2, and CUC2 (NAC) domain transcription factor VND‐INTERACTING2 (VNI2) negatively regulates xylem vessel formation by interacting with another NAC domain transcription factor, VASCULAR‐RELATED NAC‐DOMAIN7 (VND7), a master regulator of xylem vessel formation. Here, we screened interacting proteins with VNI2 using yeast two‐hybrid assay and isolated two NAC domain transcription factors, Arabidopsis thaliana ACTIVATION FACTOR 2 (ATAF2) and NAC DOMAIN CONTAINING PROTEIN 102 (ANAC102). A transient gene expression assay showed that ATAF2 upregulates the expression of genes involved in leaf senescence, and VNI2 effectively inhibits the transcriptional activation activity of ATAF2. vni2 mutants accelerate leaf senescence, whereas ataf2 mutants delay leaf senescence. In addition, the accelerated leaf senescence phenotype of the vni2 mutant is recovered by simultaneous mutation of ATAF2. Our findings strongly suggest that VNI2 interacts with and inhibits ATAF2, resulting in negatively regulating leaf senescence.
  Wiley, English, Scientific journal
  DOI：https://doi.org/10.1002/pld3.529
  DOI ID：10.1002/pld3.529, ISSN：2475-4455, eISSN：2475-4455, PubMed ID：37731912, PubMed Central ID：PMC10507225
• Metabolic changes associated with dark-induced leaf senescence in Arabidopsis nadk2 mutants　　　　　　　　　　　　　　　
  Chaomurilege; Atsuko Miyagi; Toshiki Ishikawa; Masatoshi Yamaguchi; Hideki Murayama; Maki Kawai-Yamada
  Plant Signaling and Behavior, Volume：18, Number：1, First page：e2215618, Last page：2215618, May 2023, [Reviewed], ［International magazine］
  Arabidopsis NADK2 (NAD kinase 2) is a chloroplast-localized enzyme involved in NADP+ synthesis, which acts as the final electron acceptor in the photosynthetic electron transfer chain. The NADK2-deficient mutant (nadk2) was used to analyze the effect of NAD(P)(H) unbalance in the dark-induced leaf senescence. During senescence, WT plants and nadk2 mutants showed a similar reduction in chlorophyll content. NAD(P)(H) quantification showed that the amount of total NAD(P)(H) decreased on the day 7 in WT but on the day 3 in nadk2. The phosphorylation ratio (i.e. NADP(H)/NAD(H)) decreased on day 1 in WT. In contrast, the nadk2 showed lower phosphorylation ratio at 0 day and no change throughout the aging process. Metabolome analysis showed that the metabolic profiles of both WT plants and nadk2 mutants subjected to dark-induced senescence adopted similar patterns as the senescence progressed. However, the changes in individual metabolites in the nadk2 mutants were different from those of the WT during dark-induced senescence.
  English, Scientific journal
  DOI：https://doi.org/10.1080/15592324.2023.2215618
  DOI ID：10.1080/15592324.2023.2215618, PubMed ID：37272565, PubMed Central ID：PMC10324968
• Functions of serine from the phosphorylated pathway on growth, male gametogenesis, and metabolism in Marchantia polymorpha
  Masami Hirai; Mengyao Wang; Hiromitsu Tabeta; Kinuka Ohtaka; Ayuko Kuwahara; Ryuichi Nishihama; Toshiki Ishikawa; Kiminori Toyooka; Mayuko Sato; Mayumi Wakazaki; Hiromichi Akashi; Hiroshi Tsugawa; Tsubasa Shoji; Yozo Okazaki; Keisuke Yoshida; Ryoichi Sato; Ali Ferjani; Takayuki Kohchi
  Apr. 2023
  Abstract
  
  Serine is an important precursor of various biomolecules. Here, we investigated the role of the phosphorylated pathway of serine biosynthesis in a non-vascular plant Marchantia polymorpha by analyzing knockout mutants of MpPGDH, a single gene encoding the first committed enzyme 3-phosphoglycerate dehydrogenase (PGDH), to assess functions of this pathway in relation to those of the other two pathways. Growth phenotypes of the mutants indicated that serine supply from the phosphorylated pathway in the dark was crucial for vegetative growth. Sperm formation required serine from this pathway, while egg formation did not depend on it. Knockout of MpPGDH in the maternal genome disrupted sporophyte development. When the mutants were grown in high CO₂ where the photorespiratory glycolate pathway for serine biosynthesis is inhibited, thallus growth was suppressed and not fully recovered to wild-type level by exogenous serine supplement, suggesting that serine homeostasis involving both the phosphorylated and glycolate pathways was essential. Metabolome and lipidome analyses indicated that the phosphorylated pathway mainly influenced the tricarboxylic acid cycle, the amino acid and nucleotide metabolism, and lack of serine significantly perturbed lipid metabolism. Our results indicate the importance of serine from the phosphorylated pathway for sperm formation, sporophyte development, and metabolism in M. polymorpha.
  Research Square Platform LLC
  DOI：https://doi.org/10.21203/rs.3.rs-2663856/v1
  DOI ID：10.21203/rs.3.rs-2663856/v1
• Chloroplastic Sec14-like proteins modulate growth and phosphate deficiency responses in Arabidopsis and rice　　　　　　　　　　　　　　　
  Mailun Yang; Yasuhito Sakruaba; Toshiki Ishikawa; Namie Ohtsuki; Maki Kawai-Yamada; Shuichi Yanagisawa
  Plant Physiology, Volume：192, Number：4, First page：3030, Last page：3048, Apr. 2023, [Reviewed], ［International magazine］
  Abstract
  
  Phosphorus is an essential nutrient acquired from soil as phosphate (Pi), and its deficiency severely reduces plant growth and crop yield. Here, we show that single nucleotide polymorphisms (SNPs) at the PHOSPHATIDYLINOSITOL TRANSFER PROTEIN7 (AtPITP7) locus, which encodes a chloroplastic Sec14-like protein, are associated with genetic diversity regarding Pi uptake activity in Arabidopsis (Arabidopsis thaliana). Inactivation of AtPITP7 and its rice (Oryza sativa) homolog (OsPITP6) through T-DNA insertion and CRISPR/Cas9-mediated gene editing, respectively, decreased Pi uptake and plant growth, regardless of Pi availability. By contrast, overexpression of AtPITP7 and OsPITP6 enhanced Pi uptake and plant growth, especially under limited Pi supply. Importantly, overexpression of OsPITP6 increased the tiller number and grain yield in rice. Targeted metabolome analysis of glycerolipids in leaves and chloroplasts revealed that inactivation of OsPITP6 alters phospholipid contents, independent of Pi availability, diminishing the reduction in phospholipid content and increase in glycolipid content induced by Pi deficiency; meanwhile, overexpression of OsPITP6 enhanced Pi deficiency-induced metabolic alterations. Together with transcriptome analysis of ospitp6 rice plants and phenotypic analysis of grafted Arabidopsis chimeras, these results suggest that chloroplastic Sec14-like proteins play an essential role in growth modulations in response to changes in Pi availability, although their function is critical for plant growth under any Pi condition. The superior traits of OsPITP6-overexpressing rice plants also highlight the potential of OsPITP6 and its homologs in other crops as additional tools for improving Pi uptake and plant growth in low Pi environments.
  Oxford University Press (OUP), English, Scientific journal
  DOI：https://doi.org/10.1093/plphys/kiad212
  DOI ID：10.1093/plphys/kiad212, ISSN：0032-0889, eISSN：1532-2548, PubMed ID：37021761, PubMed Central ID：PMC10400038
• Acylated plastoquinone is a novel neutral lipid accumulated in cyanobacteria　　　　　　　　　　　　　　　
  Toshiki Ishikawa; Shunya Takano; Riko Tanikawa; Takashi Fujihara; Kimie Atsuzawa; Yasuko Kaneko; Yukako Hihara
  PNAS Nexus, Volume：2, Number：5, First page：pgad092, Mar. 2023, [Reviewed], [Lead], ［International magazine］
  Abstract
  
  Although cyanobacteria do not possess bacterial triacylglycerol (TAG) synthesizing enzymes, accumulation of TAGs and/or lipid droplets has been repeatedly reported in a wide range of species. In most cases, identification of TAG has been based on the detection of the spot showing the mobility similar to the TAG standard in thin-layer chromatography of neutral lipids. In this study, we identified monoacyl plastoquinol (acyl PQH) as the predominant molecular species in the TAG-like spot from the unicellular Synechocystis sp. PCC 6803 (S.6803) as well as the filamentous Nostocales species, Nostoc punctiforme PCC 73102 and Anabaena sp. PCC 7120. In S.6803, the accumulation level of acyl PQH but not TAG was affected by deletion or overexpression of slr2103, indicating that acyl PQH is the physiological product of Slr2103 having homology with the eukaryotic diacylglycerol acyltransferase-2 (DGAT2). Electron microscopy revealed that cyanobacterial strains used in this study do not accumulate lipid droplet structures such as those observed in oleaginous microorganisms. Instead, they accumulate polyhydroxybutyrate (PHB) granules and/or aggregates of alkane, free C16 and C18 saturated fatty acids and low amounts of TAG in the cytoplasmic area, which can be detected by staining with a fluorescent dye specific to neutral lipids. Unlike these lipophilic materials, acyl PQH is exclusively localized in the membrane fraction. There must be DGAT2-like enzymatic activity esterifying de novo-synthesized C16 and C18 fatty acids to PQH2 in the thylakoid membranes.
  Oxford University Press (OUP), English, Scientific journal
  DOI：https://doi.org/10.1093/pnasnexus/pgad092
  DOI ID：10.1093/pnasnexus/pgad092, eISSN：2752-6542, PubMed ID：37152674, PubMed Central ID：PMC10156143
• Loss of peroxisomal NAD kinase 3 (NADK3) affects photorespiration metabolism in Arabidopsis　　　　　　　　　　　　　　　
  Shota Suzuki; Daimu Tanaka; Atsuko Miyagi; Kentaro Takahara; Masaru Kono; Chaomurilege; Ko Noguchi; Toshiki Ishikawa; Minoru Nagano; Masatoshi Yamaguchi; Maki Kawai-Yamada
  Journal of Plant Physiology, Volume：283, First page：153950, Last page：153950, Mar. 2023, [Reviewed], ［International magazine］
  Nicotinamide adenine dinucleotides (NAD+ and NADP+) are electron mediators involved in various metabolic pathways. NADP(H) are produced by NAD kinase (NADK) through the phosphorylation of NAD(H). The Arabidopsis NADK3 (AtNADK3) is reported to preferentially phosphorylate NADH to NADPH and is localized in the peroxisome. To elucidate the biological function of AtNADK3 in Arabidopsis, we compared metabolites of nadk1, nadk2 and nadk3 Arabidopsis T-DNA inserted mutants. Metabolome analysis revealed that glycine and serine, which are intermediate metabolites of photorespiration, both increased in the nadk3 mutants. Plants grown for 6 weeks under short-day conditions showed increased NAD(H), indicating a decrease in the phosphorylation ratio in the NAD(P)(H) equilibrium. Furthermore, high CO2 (0.15%) treatment induced a decrease in glycine and serine in nadk3 mutants. The nadk3 showed a significant decrease in post-illumination CO2 burst, suggesting that the photorespiratory flux was disrupted in the nadk3 mutant. In addition, an increase in CO2 compensation points and a decrease in CO2 assimilation rate were observed in the nadk3 mutants. These results indicate that the lack of AtNADK3 causes a disruption in the intracellular metabolism, such as in amino acid synthesis and photorespiration.
  English, Scientific journal
  DOI：https://doi.org/10.1016/j.jplph.2023.153950
  DOI ID：10.1016/j.jplph.2023.153950, PubMed ID：36889102
• Metabolomic analysis of rice brittle culm mutants reveals each mutant- specific metabolic pattern in each organ　　　　　　　　　　　　　　　
  Atsuko Miyagi; Kazuhisa Mori; Toshiki Ishikawa; Satoshi Ohkubo; Shunsuke Adachi; Masatoshi Yamaguchi; Taiichiro Ookawa; Toshihisa Kotake; Maki Kawai-Yamada
  Metabolomics, Volume：18, Number：12, First page：95, Last page：95, Nov. 2022, [Reviewed], ［International magazine］
  INTRODUCTION: Plant cell walls play an important role in providing physical strength and defence against abiotic stress. Rice brittle culm (bc) mutants are a strength-decreased mutant because of abnormal cell walls, and it has been reported that the causative genes of bc mutants affect cell wall composition. However, the metabolic alterations in each organ of bc mutants have remained unknown. OBJECTIVES: To evaluate the metabolic changes in rice bc mutants, comparative analysis of the primary metabolites was conducted. METHODS: The primary metabolites in leaves, internodes, and nodes of rice bc mutants and wild-type control were measured using CE- and LC-MS/MS. Multivariate analyses using metabolomic data was performed. RESULTS: We found that mutations in each bc mutant had different effects on metabolism. For example, higher oxalate content was observed in bc3 and bc1 bc3 mutants, suggesting that surplus carbon that was not used for cell wall components might be used for oxalate synthesis. In addition, common metabolic alterations such as a decrease of sugar nucleotides in nodes were found in bc1 and Bc6, in which the causative genes are involved in cellulose accumulation. CONCLUSION: These results suggest that metabolic analysis of the bc mutants could elucidate the functions of causative gene and improve the cell wall components for livestock feed or bioethanol production.
  Springer Science and Business Media LLC, English, Scientific journal
  DOI：https://doi.org/10.1007/s11306-022-01958-9
  DOI ID：10.1007/s11306-022-01958-9, eISSN：1573-3890, PubMed ID：36409428
• Loss of chloroplast-localized NAD kinase causes ROS stress in Arabidopsis thaliana　　　　　　　　　　　　　　　
  Chaomurilege; Yanhui Zu; Atsuko Miyagi; Shin-Nosuke Hashida; Toshiki Ishikawa; Masatoshi Yamaguchi; Maki Kawai-Yamada
  Journal of Plant Research, Volume：in press, Number：1, First page：97, Last page：106, Nov. 2022, [Reviewed], ［International magazine］
  Chloroplast-localized NAD kinase (NADK2) is responsible for the production of NADP+, which is an electron acceptor in the linear electron flow of photosynthesis. The Arabidopsis T-DNA-inserted mutant of NADK2 (nadk2) showed delayed growth and pale-green leaves under continuous light conditions. Under short-day conditions (8 h light / 16 h dark), the nadk2 mutant showed more severe growth inhibition.The genomic fragment containing the promoter and coding region of NADK2 complemented the phenotypes of nadk2 obtained under continuous light and short-day conditions. The nadk2 mutant produced higher amounts of H2O2 and O2-, which were reduced in the complementary line. Under short-day conditions, the nadk2 mutant accumulated more H2O2 than under continuous light conditions. The accumulation of ascorbate and up-regulation of the PDF1.2 and PR1 genes indicated that the nadk2 mutant is under ROS stress and responding to keep its living activities.
  English, Scientific journal
  DOI：https://doi.org/10.1007/s10265-022-01420-w
  DOI ID：10.1007/s10265-022-01420-w, PubMed ID：36367584
• Nonspecific phospholipase C3 of radish has phospholipase D activity towards glycosylinositol phosphoceramide　　　　　　　　　　　　　　　
  Rumana Yesmin Hasi; Toshiki Ishikawa; Keigo Sunagawa; Yoshimichi Takai; Hanif Ali; Junji Hayashi; Ryushi Kawakami; Keizo Yuasa; Mutsumi Aihara; Kaori Kanemaru; Hiroyuki Imai; Tamotsu Tanaka
  FEBS Letters, Volume：596, Number：23, First page：3024, Last page：3036, Oct. 2022, [Reviewed], ［International magazine］
  Glycosylinositol phosphoceramide (GIPC) is a major sphingolipid in the plasma membranes of plants. Previously, we found an enzyme activity that produces phytoceramide 1-phosphate (PC1P) by hydrolysis of the D position of GIPC in cabbage and named this activity as GIPC-phospholipase D (PLD). Here, we purified GIPC-PLD by sequential chromatography from radish roots. Peptide mass fingerprinting analysis revealed that the potential candidate for GIPC-PLD protein was nonspecific phospholipase C3 (NPC3), which has not been characterized as a PLD. The recombinant NPC3 protein obtained by heterologous expression system in Escherichia coli produced PC1P from GIPC and showed essentially the same enzymatic properties as those we characterized as GIPC-PLD in cabbage, radish and Arabidopsis thaliana. From these results, we conclude that NPC3 is one of the enzymes that degrade GIPC.
  Wiley, English, Scientific journal
  DOI：https://doi.org/10.1002/1873-3468.14520
  DOI ID：10.1002/1873-3468.14520, ISSN：0014-5793, eISSN：1873-3468, PubMed ID：36266963
• Sphingolipids with 2-hydroxy fatty acids aid in plasma membrane nanodomain organization and oxidative burst　　　　　　　　　　　　　　　
  Tomomi Ukawa; Fumihiko Banno; Toshiki Ishikawa; Kota Kasahara; Yuuta Nishina; Rika Inoue; Keigo Tsujii; Masatoshi Yamaguchi; Takuya Takahashi; Yoichiro Fukao; Maki Kawai-Yamada; Minoru Nagano
  Plant Physiology, Volume：189, Number：2, First page：839, Last page：857, Jun. 2022, [Reviewed], ［International magazine］
  Abstract
  
  Plant sphingolipids mostly possess 2-hydroxy fatty acids (HFA), the synthesis of which is catalyzed by FA 2-hydroxylases (FAHs). In Arabidopsis (Arabidopsis thaliana), two FAHs (FAH1 and FAH2) have been identified. However, the functions of FAHs and sphingolipids with HFAs (2-hydroxy sphingolipids) are still unknown because of the lack of Arabidopsis lines with the complete deletion of FAH1. In this study, we generated a FAH1 mutant (fah1c) using CRISPR/Cas9-based genome editing. Sphingolipid analysis of fah1c, fah2, and fah1cfah2 mutants revealed that FAH1 hydroxylates very long-chain FAs (VLCFAs), whereas the substrates of FAH2 are VLCFAs and palmitic acid. However, 2-hydroxy sphingolipids are not completely lost in the fah1cfah2 double mutant, suggesting the existence of other enzymes catalyzing the hydroxylation of sphingolipid FAs. Plasma membrane (PM) analysis and molecular dynamics simulations revealed that hydroxyl groups of sphingolipid acyl chains play a crucial role in the organization of nanodomains, which are nanoscale liquid-ordered domains mainly formed by sphingolipids and sterols in the PM, through hydrogen bonds. In the PM of the fah1cfah2 mutant, the expression levels of 26.7% of the proteins, including defense-related proteins such as the pattern recognition receptors (PRRs) brassinosteroid insensitive 1-associated receptor kinase 1 and chitin elicitor receptor kinase 1, NADPH oxidase respiratory burst oxidase homolog D (RBOHD), and heterotrimeric G proteins, were lower than that in the wild-type. In addition, reactive oxygen species (ROS) burst was suppressed in the fah1cfah2 mutant after treatment with the pathogen-associated molecular patterns flg22 and chitin. These results indicated that 2-hydroxy sphingolipids are necessary for the organization of PM nanodomains and ROS burst through RBOHD and PRRs during pattern-triggered immunity.
  Oxford University Press (OUP), English, Scientific journal
  DOI：https://doi.org/10.1093/plphys/kiac134
  DOI ID：10.1093/plphys/kiac134, ISSN：0032-0889, eISSN：1532-2548, PubMed ID：35312013, PubMed Central ID：PMC9157162
• VNI2 effectively inhibits transcriptional activities of VND7 through a conserved sequence　　　　　　　　　　　　　　　
  Aili Ailizati; Isura Sumeda Priyadarshana Nagahage; Atsuko Miyagi; Toshiki Ishikawa; Maki Kawai-Yamada; Taku Demura; Masatoshi Yamaguchi
  Plant Biotechnology, Volume：39, Number：2, First page：147, Last page：153, Jun. 2022, [Reviewed], ［Internationally co-authored］, ［Domestic magazine］
  An Arabidopsis NAC domain transcription factor VND-INTERACTING2 (VNI2) was originally isolated as an interacting protein with another NAC domain transcription factor, VASCULAR-RELATED NAC-DOMAIN7 (VND7), a master regulator of xylem vessel element differentiation. VNI2 inhibits transcriptional activation activity of VND7 by forming a protein complex. Here, to obtain insights into how VNI2 regulates VND7, we tried to identify the amino acid region of VNI2 required for inhibition of VND7. VNI2 has an amino acid sequence similar to the ETHYLENE-RESPONSIVE ELEMENT BINDING FACTOR (ERF)-associated amphiphilic repression (EAR) motif, conserved in transcriptional repressors, at the C-terminus. A transient expression assay showed that the EAR-like motif of VNI2 was not required for inhibition of VND7. The C-terminal deletion series of VNI2 revealed that 10 amino acid residues, highly conserved in the VNI2 orthologs contributed to effective repression of the transcriptional activation activity of VND7. Observation of transgenic plants ectopically expressing VNI2 showed that the identified 10 amino acid sequence strongly affected xylem vessel formation and plant growth. These data indicated that the 10 amino acid sequence of VNI2 has an important role in its transcriptional repression activity and negative regulation of xylem vessel formation.
  English, Scientific journal
  DOI：https://doi.org/10.5511/plantbiotechnology.22.0122a
  DOI ID：10.5511/plantbiotechnology.22.0122a, PubMed ID：35937523, PubMed Central ID：PMC9300430
• An Arabidopsis NAC domain transcriptional activator VND7 negatively regulates VNI2 expression　　　　　　　　　　　　　　　
  Aili Ailizati; Isura Sumeda Priyadarshana Nagahage; Atsuko Miyagi; Toshiki Ishikawa; Maki Kawai-Yamada; Taku Demura; Masatoshi Yamaguchi
  Plant Biotechnology, Volume：38, Number：4, First page：415, Last page：420, Dec. 2021, [Reviewed], ［Internationally co-authored］
  English, Scientific journal
  DOI：https://doi.org/10.5511/plantbiotechnology.21.1013a
  DOI ID：10.5511/plantbiotechnology.21.1013a
• Change in expression levels of NAD kinase-encoding genes in Flaveria species　　　　　　　　　　　　　　　
  Masami Tanaka; Yuuma Ishikawa; Sayaka Suzuki; Takako Ogawa; Yukimi Y. Taniguchi; Atsuko Miyagi; Toshiki Ishikawa; Masatoshi Yamaguchi; Yuri N. Munekage; Maki Kawai-Yamada
  Journal of Plant Physiology, Volume：265, First page：153495, Last page：153495, Oct. 2021, [Reviewed]
  Elsevier BV, Scientific journal
  DOI：https://doi.org/10.1016/j.jplph.2021.153495
  DOI ID：10.1016/j.jplph.2021.153495, ISSN：0176-1617
• The NAD Kinase Slr0400 Functions as a Growth Repressor in Synechocystis sp. PCC 6803　　　　　　　　　　　　　　　
  Yuuma Ishikawa; Cedric Cassan; Aikeranmu Kadeer; Koki Yuasa; Nozomu Sato; Kintake  Sonoike; Yasuko Kaneko; Atsuko Miyagi; Hiroko Takahashi; Toshiki Ishikawa; Masatoshi Yamaguchi; Yoshitaka Nishiyama; Yukako Hihara; Yves Gibon; Maki Kawai-Yamada
  Plant and Cell Physiology, Volume：62, Number：4, First page：668, Last page：677, Sep. 2021, [Reviewed]
  
  NADP+, the phosphorylated form of nicotinamide adenine dinucleotide (NAD), plays an essential role in many cellular processes. NAD kinase (NADK), which is conserved in all living organisms, catalyzes the phosphorylation of NAD+ to NADP+. However, the physiological role of phosphorylation of NAD+ to NADP+ in the cyanobacterium Synechocystis remains unclear. In this study, we report that slr0400, an NADK-encoding gene in Synechocystis, functions as a growth repressor under light-activated heterotrophic growth conditions and light and dark cycle conditions in the presence of glucose. We show, via characterization of NAD(P)(H) content and enzyme activity, that NAD+ accumulation in slr0400-deficient mutant results in the unsuppressed activity of glycolysis and tricarboxylic acid (TCA) cycle enzymes. In determining whether Slr0400 functions as a typical NADK, we found that constitutive expression of slr0400 in an Arabidopsis nadk2-mutant background complements the pale-green phenotype. Moreover, to determine the physiological background behind the growth advantage of mutants lacking slr04000, we investigated the photobleaching phenotype of slr0400-deficient mutant under high-light conditions. Photosynthetic analysis found in the slr0400-deficient mutant resulted from malfunctions in the Photosystem II (PSII) photosynthetic machinery. Overall, our results suggest that NADP(H)/NAD(H) maintenance by slr0400 plays a significant role in modulating glycolysis and the TCA cycle to repress the growth rate and maintain the photosynthetic capacity.
  Oxford University Press (OUP), Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcab023
  DOI ID：10.1093/pcp/pcab023, ISSN：0032-0781, eISSN：1471-9053, PubMed ID：33210985, PubMed Central ID：PMC7781788
• Reprogramming sphingolipid glycosylation is required for endosymbiont persistence in Medicago truncatula　　　　　　　　　　　　　　　
  William M. Moore; Candace Chan; Toshiki Ishikawa; Emilie A. Rennie; Heidi M.-L. Wipf; Veronica Benites; Maki Kawai-Yamada; Jenny C. Mortimer; Henrik V. Scheller
  Current Biology, Volume：31, Number：11, First page：2374, Last page：2385.e4, Jun. 2021
  Plant endosymbiosis relies on the development of specialized membranes that encapsulate the endosymbiont and facilitate nutrient exchange. However, the identity and function of lipids within these membrane interfaces is largely unknown. Here, we identify GLUCOSAMINE INOSITOL PHOSPHORYLCERAMIDE TRANSFERASE1 (GINT1) as a sphingolipid glycosyltransferase highly expressed in Medicago truncatula root nodules and roots colonized by arbuscular mycorrhizal (AM) fungi and further demonstrate that this enzyme functions in the synthesis of N-acetyl-glucosamine-decorated glycosyl inositol phosphoryl ceramides (GIPCs) in planta. MtGINT1 expression was developmentally regulated in symbiotic tissues associated with the development of symbiosome and periarbuscular membranes. RNAi silencing of MtGINT1 did not affect overall root growth but strongly impaired nodulation and AM symbiosis, resulting in the senescence of symbiosomes and arbuscules. Our results indicate that, although M. truncatula root sphingolipidome predominantly consists of hexose-decorated GIPCs, local reprogramming of GIPC glycosylation by MtGINT1 is required for the persistence of endosymbionts within the plant cell.
  Cell Press, English, Scientific journal
  DOI：https://doi.org/10.1016/j.cub.2021.03.067
  DOI ID：10.1016/j.cub.2021.03.067, ISSN：1879-0445, PubMed ID：33857428, SCOPUS ID：85104939324
• The Arabidopsis thaliana nucleotide sugar transporter GONST2 is a functional homolog of GONST1.　　　　　　　　　　　　　　　
  Beibei Jing; Toshiki Ishikawa; Nicole Soltis; Noriko Inada; Yan Liang; Gosia Murawska; Lin Fang; Fekadu Andeberhan; Ramana Pidatala; Xiaolan Yu; Edward Baidoo; Maki Kawai-Yamada; Dominique Loque; Daniel J Kliebenstein; Paul Dupree; Jenny C Mortimer
  Plant direct, Volume：5, Number：3, First page：e00309, Mar. 2021, [Reviewed], ［International magazine］
  Glycosylinositolphosphorylceramides (GIPCs) are the predominant lipid in the outer leaflet of the plasma membrane. Characterized GIPC glycosylation mutants have severe or lethal plant phenotypes. However, the function of the glycosylation is unclear. Previously, we characterized Arabidopsis thaliana GONST1 and showed that it was a nucleotide sugar transporter which provides GDP-mannose for GIPC glycosylation. gonst1 has a severe growth phenotype, as well as a constitutive defense response. Here, we characterize a mutant in GONST1's closest homolog, GONST2. The gonst2-1 allele has a minor change to GIPC headgroup glycosylation. Like other reported GIPC glycosylation mutants, gonst1-1gonst2-1 has reduced cellulose, a cell wall polymer that is synthesized at the plasma membrane. The gonst2-1 allele has increased resistance to a biotrophic pathogen Golovinomyces orontii but not the necrotrophic pathogen Botrytis cinerea. Expression of GONST2 under the GONST1 promoter can rescue the gonst1 phenotype, indicating that GONST2 has a similar function to GONST1 in providing GDP-D-Man for GIPC mannosylation.
  English, Scientific journal
  DOI：https://doi.org/10.1002/pld3.309
  DOI ID：10.1002/pld3.309, PubMed ID：33763627, PubMed Central ID：PMC7980081
• The NAD kinase Slr0400 functions as a growth repressor in Synechocystis sp. PCC 6803.　　　　　　　　　　　　　　　
  Yuuma Ishikawa; Cedric Cassan; Aikeranmu Kadeer; Koki Yuasa; Nozomu Sato; Kintake Sonoike; Yasuko Kaneko; Atsuko Miyagi; Hiroko Takahashi; Toshiki Ishikawa; Masatoshi Yamaguchi; Yoshitaka Nishiyama; Yukako Hihara; Yves Gibon; Maki Kawai-Yamada
  Plant & cell physiology, Feb. 2021, [Reviewed], ［Domestic magazine］
  NADP+, the phosphorylated form of nicotinamide adenine dinucleotide (NAD), plays an essential role in many cellular processes. NAD kinase (NADK), which is conserved in all living organisms, catalyzes the phosphorylation of NAD+ to NADP+. However, the physiological role of phosphorylation of NAD+ to NADP+ in the cyanobacterium Synechocystis remains unclear. In this study, we report that slr0400, an NADK-encoding gene in Synechocystis, functions as a growth repressor under light-activated heterotrophic growth conditions and light and dark cycle conditions in the presence of glucose. We show, via characterization of NAD(P)(H) content and enzyme activity, that NAD+ accumulation in slr0400-deficient mutant results in unsuppressed activity of glycolysis and tricarboxylic acid (TCA) cycle enzymes. In determining whether Slr0400 functions as a typical NADK, we found that constitutive expression of slr0400 in an Arabidopsis nadk2-mutant background complements the pale-green phenotype. Moreover, to determine the physiological background behind the growth advantage of mutants lacking slr04000, we investigated the photobleaching phenotype of slr0400-deficient mutant under high-light conditions. Photosynthetic analysis found in the slr0400-deficient mutant resulted from malfunctions in the PSII photosynthetic machinery. Overall, our results suggest that NADP(H)/NAD(H) maintenance by slr0400 plays a significant role in modulating glycolysis and the TCA cycle to repress the growth rate and maintain photosynthetic capacity.
  English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcab023
  DOI ID：10.1093/pcp/pcab023, PubMed ID：33560438
• Ceramides mediate positional signals in Arabidopsis thaliana protoderm differentiation.　　　　　　　　　　　　　　　
  Kenji Nagata; Toshiki Ishikawa; Maki Kawai-Yamada; Taku Takahashi; Mitsutomo Abe
  Development (Cambridge, England), Volume：148, Number：2, Jan. 2021, [Reviewed], ［International magazine］
  The differentiation of distinct cell types in appropriate patterns is a fundamental process in the development of multicellular organisms. In Arabidopsis thaliana, protoderm/epidermis differentiates as a single cell layer at the outermost position. However, little is known about the molecular nature of the positional signals that achieve correct epidermal cell differentiation. Here, we propose that very-long-chain fatty acid-containing ceramides (VLCFA-Cers) mediate positional signals by stimulating the function of ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1), a master regulator of protoderm/epidermis differentiation, during lateral root development. We show that VLCFA-Cers, which are synthesized predominantly in the outermost cells, bind to the lipid-binding domain of ATML1. Importantly, this cell type-specific protein-lipid association alters the activity of ATML1 protein and consequently restricts its expression to the protoderm/epidermis through a transcriptional feedback loop. Furthermore, establishment of a compartment, enriched with VLCFA-containing sphingolipids, at the outer lateral membrane facing the external environment may function as a determinant of protodermal cell fate. Taken together, our results indicate that VLCFA-Cers play a pivotal role in directing protoderm/epidermis differentiation by mediating positional signals to ATML1.This article has an associated 'The people behind the papers' interview.
  English, Scientific journal
  DOI：https://doi.org/10.1242/dev.194969
  DOI ID：10.1242/dev.194969, PubMed ID：33495212
• Altered metabolism of chloroplastic NAD kinase-overexpressing Arabidopsis in response to magnesium sulfate supplementation.　　　　　　　　　　　　　　　
  Maki Kawai-Yamada; Atsuko Miyagi; Yuki Sato; Yuki Hosoi; Shin-Nosuke Hashida; Toshiki Ishikawa; Masatoshi Yamaguchi
  Plant signaling & behavior, Volume：16, Number：1, First page：1844509, Last page：1844509, Jan. 2021, [Reviewed], ［International magazine］
  Nicotinamide adenine dinucleotide (NAD)/NAD phosphate (NADPH) is essential for numerous redox reactions and serve as co-factors in multiple metabolic processes in all organisms. NAD kinase (NADK) is an enzyme involved in the synthesis of NADP+ from NAD+ and ATP. Arabidopsis NADK2 (AtNADK2) is a chloroplast-localizing enzyme that provides recipients of reducing power in photosynthetic electron transfer. When Arabidopsis plants were grown on MS medium supplemented with 5 mM MgSO4, an AtNADK2-overexpressing line exhibited higher glutathione and total sulfur accumulation than control plants. Metabolomic analysis of major amino acids and organic acids using capillary electrophoresis-mass spectrometry demonstrated that overexpression of AtNADK2 affected a range of metabolic processes in response to MgSO4 supplementation.
  English, Scientific journal
  DOI：https://doi.org/10.1080/15592324.2020.1844509
  DOI ID：10.1080/15592324.2020.1844509, PubMed ID：33210985, PubMed Central ID：PMC7781788
• An Arabidopsis NAC domain transcription factor, ATAF2, promotes age-dependent and dark-induced leaf senescence.　　　　　　　　　　　　　　　
  Isura Sumeda Priyadarshana Nagahage; Shingo Sakamoto; Minoru Nagano; Toshiki Ishikawa; Nobutaka Mitsuda; Maki Kawai-Yamada; Masatoshi Yamaguchi
  Physiologia plantarum, Volume：170, Number：2, First page：299, Last page：308, Oct. 2020, [Reviewed], ［International magazine］
  Leaf senescence is controlled developmentally and environmentally and is affected by numerous genes, including transcription factors. An Arabidopsis NAC domain transcription factor, ATAF2, is known to regulate biotic stress responses. Recently, we have demonstrated that ATAF2 upregulates ORE1, a key regulator of leaf senescence. Here, to investigate the function of ATAF2 in leaf senescence further, we generated and analyzed overexpressing transgenic and T-DNA inserted mutant lines. Transient expression analysis indicated that ATAF2 upregulates several NAC domain transcription factors that regulate senescence. Indeed, ATAF2 overexpression induced the expression of senescence-related genes, thereby accelerating leaf senescence, whereas the expression of such genes in ataf2 mutants was lower than that of wild-type plants. Furthermore, the ataf2 mutants exhibited significant delays in dark-induced leaf senescence. It was also found that ATAF2 induces the expression of transcription factors, which both promotes and represses leaf senescence. The present study demonstrates that ATAF2 promotes leaf senescence in response to developmental and environmental signals.
  English, Scientific journal
  DOI：https://doi.org/10.1111/ppl.13156
  DOI ID：10.1111/ppl.13156, PubMed ID：32579231
• Quantitative and Qualitative Analyses of Triacylglycerol Production in the Wild-Type Cyanobacterium Synechocystis sp. PCC 6803 and the Strain Expressing AtfA from Acinetobacter baylyi ADP1.　　　　　　　　　　　　　　　
  Motoki Tanaka; Toshiki Ishikawa; So Tamura; Yujiro Saito; Maki Kawai-Yamada; Yukako Hihara
  Plant & cell physiology, Volume：61, Number：9, First page：1537, Last page：1547, Sep. 2020, [Reviewed], ［Domestic magazine］
  Although cyanobacteria do not possess wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT), the bacterial enzyme for triacylglycerol (TAG) production, there have been several studies reporting the accumulation of TAG-like compounds in cyanobacteria. In this study, we aimed to evaluate TAG productivity of the ΔrecJ::atfA strain of Synechocystis sp. PCC 6803 generated by inserting atfA encoding WS/DGAT from Acinetobacter baylyi ADP1 into recJ (sll1354), together with the wild type (WT) and the gene-disrupted strain of slr2103 having homology with eukaryotic DGAT2 gene family (Δ2103). Thin-layer chromatography (TLC) of neutral lipids or isolation of the neutral lipid-enriched fraction followed by gas chromatography or liquid chromatography-tandem mass spectrometry was employed for analyses. The ΔrecJ::atfA strain accumulated 0.508 nmol ml-1OD730-1 of TAG after a week of incubation at 100 μmol photons m-2 s-1. The saturated fatty acids C16:0 and C18:0 accounted for about 50% and 20% of the TAG fatty acids, respectively, suggesting that de novo-synthesized fatty acids were preferentially incorporated into TAG molecules. When the neutral lipid profile of the lipid extracts was examined by TLC, a spot located in a slightly lower position compared with the TAG standard was detected in WT but not in the Δ2103 strain. TAG accumulation levels of both strains was only 0.01-0.03 nmol ml-1OD730-1, but the fatty acid composition was substantially different from that of the background. These results suggest that trace amounts of TAG can be produced in Synechocystis cells by enzymes other than Slr2103, and major constituents of the TAG-like spot are unknown lipid species produced by Slr2103.
  English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcaa069
  DOI ID：10.1093/pcp/pcaa069, PubMed ID：32433767
• Plant-unique cis/trans isomerism of long-chain base unsaturation is selectively required for aluminum tolerance resulting from glucosylceramide-dependent plasma membrane fluidity　　　　　　　　　　　　　　　
  Masaya Sato; Minoru Nagano; Song Jin; Atsuko Miyagi; Masatoshi Yamaguchi; Maki Kawai-Yamada; Toshiki Ishikawa
  Plants, Volume：9, Number：1, First page：19, Jan. 2020, [Reviewed], ［International magazine］
  Cis/trans isomerism of the Δ8 unsaturation of long-chain base (LCB) is found only in plant sphingolipids. This unique geometry is generated by sphingolipid LCB Δ8 desaturase SLD which produces both isomers at various ratios, resulting in diverse cis/trans ratios in plants. However, the biological significance of this isomeric diversity remains controversial. Here, we show that the plant-specific cis unsaturation of LCB selectively contributes to glucosylceramide (GlcCer)-dependent tolerance to aluminum toxicity. We established three transgenic rice lines with altered LCB unsaturation profiles. Overexpression of SLD from rice (OsSLD-OX), which preferentially exhibits cis-activity, or Arabidopsis (AtSLD-OX), showing preference for trans-activity, facilitated Δ8 unsaturation in different manners: a slight increase of cis-unsaturated glycosylinositolphosphoceramide (GIPC) in OsSLD-OX, and a drastic increase of trans-unsaturated GlcCer and GIPC in AtSLD-OX. Disruption of LCB Δ4 desaturase (des) significantly decreased the content of GlcCer. Fluorescence imaging analysis revealed that OsSLD-OX and AtSLD-OX showed increased plasma membrane fluidity, whereas des had less fluidity, demonstrating that the isomers universally contributed to increasing membrane fluidity. However, the results of a hydroponic assay showed decreased aluminum tolerance in AtSLD-OX and des compared to OsSLD-OX and the control plants, which did not correlate with membrane fluidity. These results suggest that cis-unsaturated GlcCer, not GIPC, selectively serves to maintain the membrane fluidity specifically associated with aluminum tolerance.
  English, Scientific journal
  DOI：https://doi.org/10.3390/plants9010019
  DOI ID：10.3390/plants9010019, PubMed ID：31877922, PubMed Central ID：PMC7020186
• Glycosylinositol phosphoceramide-specific phospholipase D activity catalyzes transphosphatidylation.　　　　　　　　　　　　　　　
  Rumana Yesmin Hasi; Makoto Miyagi; Katsuya Morito; Toshiki Ishikawa; Maki Kawai-Yamada; Hiroyuki Imai; Tatsuya Fukuta; Kentaro Kogure; Kaori Kanemaru; Junji Hayashi; Ryushi Kawakami; Tamotsu Tanaka
  Journal of biochemistry, Volume：166, Number：5, First page：441, Last page：448, Nov. 2019, [Reviewed], ［International magazine］
  Glycosylinositol phosphoceramide (GIPC) is the most abundant sphingolipid in plants and fungi. Recently, we detected GIPC-specific phospholipase D (GIPC-PLD) activity in plants. Here, we found that GIPC-PLD activity in young cabbage leaves catalyzes transphosphatidylation. The available alcohol for this reaction is a primary alcohol with a chain length below C4. Neither secondary alcohol, tertiary alcohol, choline, serine nor glycerol serves as an acceptor for transphosphatidylation of GIPC-PLD. We also found that cabbage GIPC-PLD prefers GIPC containing two sugars. Neither inositol phosphoceramide, mannosylinositol phosphoceramide nor GIPC with three sugar chains served as substrate. GIPC-PLD will become a useful catalyst for modification of polar head group of sphingophospholipid.
  English, Scientific journal
  DOI：https://doi.org/10.1093/jb/mvz056
  DOI ID：10.1093/jb/mvz056, PubMed ID：31504617
• One of the NAD kinases, sll1415, is required for the glucose metabolism of Synechocystis sp. PCC 6803.　　　　　　　　　　　　　　　
  Yuuma Ishikawa; Atsuko Miyagi; Toshiki Ishikawa; Minoru Nagano; Masatoshi Yamaguchi; Yukako Hihara; Yasuko Kaneko; Maki Kawai-Yamada
  The Plant journal : for cell and molecular biology, Volume：98, Number：4, First page：654, Last page：666, May 2019, [Reviewed], ［International magazine］
  Pyridine nucleotides (NAD(P)(H)) are electron carriers that are the driving forces in various metabolic pathways. Phosphorylation of NAD(H) to NADP(H) is performed by the enzyme NAD kinase (NADK). Synechocystis sp. PCC 6803 harbors two genes (sll1415 and slr0400) that encode proteins with NADK homology. When genetic mutants for sll1415 and slr0400 (Δ1415 and Δ0400, respectively) were cultured under photoheterotrophic growth conditions only the Δ1415 cells showed a growth defect. In wild-type cells, the sll1415 transcript accumulated after the cells were transferred to photoheterotrophic conditions. Furthermore, NAD(P)(H) measurements demonstrated that a dynamic metabolic conversion was implemented during the adaptation from photoautotrophic to photoheterotrophic conditions. Electron microscopy observation and biochemistry quantification demonstrated the accumulation of glycogen in the Δ1415 cells under photoheterotrophic conditions at 96 h. Quantitative real-time reverse transcription PCR (qRT-PCR) demonstrated the accumulation of mRNAs that encoded glycogen biosynthesis-related enzymes in photoheterotrophic Δ1415 cells. At 96 h, enzyme activity measurement in the photoheterotrophic Δ1415 cells demonstrated that the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were decreased, but the activities of glucose dehydrogenase were increased. Furthermore, metabolomics analysis demonstrated that the Δ1415 cells showed increased glucose-6-phosphate and 6-phosphogluconate content at 96 h. Therefore, sll1415 has a significant function in the oxidative pentose phosphate (OPP) pathway for catabolism of glucose under photoheterotrophic conditions. Additionally, it is presumed that the slr0400 had a different role in glucose catabolism during growth. These results suggest that the two Synechocystis sp. PCC 6803 NADKs (Sll1415 and Slr0400) have distinct functions in photoheterotrophic cyanobacterial metabolism.
  English, Scientific journal
  DOI：https://doi.org/10.1111/tpj.14262
  DOI ID：10.1111/tpj.14262, PubMed ID：30693583
• DSH5, a dihydrosphingosine C4 hydroxylase gene family member, shows spatially restricted expression in rice and is lethal when expressed ectopically.　　　　　　　　　　　　　　　
  Imamura T; Obata C; Yoneyama K; Ichikawa M; Ikura A; Mutsuro-Aoki H; Ishikawa T; Kawai-Yamada M; Sasaki T; Kusano H; Shimada H
  Genes Genet. Syst., Volume：93, Number：4, First page：135, Last page：142, 2018, [Reviewed], ［Domestic magazine］
  Dihydrosphingosine C4 hydroxylase (DSH), a diiron-binding membrane enzyme, catalyzes the hydration of dihydrosphingosine and acyl-sphinganine to produce phytosphingosine and phytoceramide, respectively. Rice has two types of DSH homologs: general DSHs, namely DSH1, DSH2 and DSH4, and others that show spatial expression profiles, namely DSH3 and DSH5. The general DSHs exist in many plant species. These DSHs showed similarity in their functions and complemented the yeast sur2D mutation. In contrast, homologs of DSH3 and DSH5 were found only in monocot plants. Phylogenetic analysis placed these DSHs in different clades that are evolutionarily divergent from those of the general DSHs. DSH3 and DSH5 showed low-level expression. DSH5 expression was specifically in vascular bundle tissues. Ectopic expression of DSH5 induced a dwarf phenotype characterized by severe growth inhibition and an increase in the thickness of the leaf body caused by enlargement of bulliform cells in the leaves. However, no significant difference was observed in the amount of sphingolipid species. DSH5 did not complement the yeast sur2D mutation, implying that DSH5 has little effect on sphingolipid metabolism. These findings suggested that DSH3 and DSH5 originated and diverged in monocot plants.
  English, Scientific journal
  DOI：https://doi.org/10.1266/ggs.17-00054
  DOI ID：10.1266/ggs.17-00054, PubMed ID：30185720
• An NAC domain transcription factor ATAF2 acts as transcriptional activator or repressor dependent on promoter context.　　　　　　　　　　　　　　　
  Nagahage ISP; Sakamoto S; Nagano M; Ishikawa T; Kawai-Yamada M; Mitsuda N; Yamaguchi M
  Plant Biotech., Volume：35, First page：285, Last page：289, 2018, [Reviewed]
  English, Scientific journal
  DOI：https://doi.org/10.5511/plantbiotechnology.18.0507a
  DOI ID：10.5511/plantbiotechnology.18.0507a
• Effects of inactivation of the cyAbrB2 transcription factor together with glycogen synthesis on cellular metabolism and free fatty acid production in the cyanobacterium Synechocystis sp. PCC 6803.　　　　　　　　　　　　　　　
  Kodama Y; Kawahara A; Miyagi A; Ishikawa T; Kawai-Yamada M; Kaneko Y; Takimura Y; Hihara Y
  Biotech. Bioeng., Volume：115, First page：2974, Last page：2985, 2018, [Reviewed]
  English, Scientific journal
  DOI：https://doi.org/10.1002/bit.26842
  DOI ID：10.1002/bit.26842
• Suppressing Arabidopsis GGLT1 affects growth by reducing the L-galactose content and borate cross-linking of rhamnogalacturonan II.　　　　　　　　　　　　　　　
  Sechet J; Htwe S; Urbanowicz B; Agyeman A; Feng W; Ishikawa T; Dinneny J; Colomes M; Satish Kumar K; Kawai-Yamada M; O'Neill M; Mortimer JC
  Plant J., Volume：96, First page：1036, Last page：1050, 2018, [Reviewed]
  English, Scientific journal
  DOI：https://doi.org/10.1111/tpj.14088
  DOI ID：10.1111/tpj.14088
• GLUCOSAMINE INOSITOLPHOSPHORYLCERAMIDE TRANSFERASE1 (GINT1) is a GlcNAc-containing glycosylinositol phosphorylceramide glycosyltransferase.　　　　　　　　　　　　　　　
  Ishikawa T; Fang L; Rennie EA; Sechet J; Yan J; Jing B; Moore W; Cahoon EB; Scheller HV; Kawai-Yamada M; Mortimer JC
  Plant Physiol., Volume：177, First page：938, Last page：952, 2018, [Reviewed]
  English, Scientific journal
  DOI：https://doi.org/10.1104/pp.18.00396
  DOI ID：10.1104/pp.18.00396
• Synthesis and degradation of long-chain base phosphates affect fumonisin B-1-induced cell death in Arabidopsis thaliana　　　　　　　　　　　　　　　
  Daiki Yanagawa; Toshiki Ishikawa; Hiroyuki Imai
  JOURNAL OF PLANT RESEARCH, Volume：130, Number：3, First page：571, Last page：585, May 2017, [Reviewed]
  Fumonisin B-1 (FB1), an inducer of cell death, disrupts sphingolipid metabolism; large accumulations of de novo synthesized free long-chain bases (LCBs) are observed. However, it remains unclear whether tolerance to FB1 toxicity in plants is connected with preventing the accumulation of free LCBs through their phosphorylation. Here a workflow for the extraction, detection and quantification of LCB phosphates (LCBPs) in Arabidopsis thaliana was developed. We studied the effect of expression of genes for three enzymes involved in the synthesis and degradation of LCBPs, LCB kinase (LCBK1), LCBP phosphatase (SPP1) and lyase (DPL1) on FB1-induced cell death. As expected, large accumulations of saturated free LCBs, dihydrosphingosine and phytosphingosine, were observed in the FB1-treated leaves. On the other hand, a high level of sphingenine phosphate was found in the FB1-treated leaves even though free sphingenine was found in low amounts in these leaves. In comparison of WT and spp1 plants, the LCBP/LCB ratio is likely to be correlated with the degree of FB1-induced cell death determined by trypan blue staining. The FB1-treated leaves in dpl1 plants showed severe cell death and the elevation of free LCBs and LCBPs. LCBK1-OX and -KD plants showed resistance and sensitivity to FB1, respectively, whereas free LCB and LCBP levels in FB1-treated LCBK1-OX and -KD plants were moderately different to those in FB1-treated WT plants. Overall, the findings described here suggest that LCBP/LCB homeostasis is an important topic that participates in the tolerance of plant cells to FB1.
  SPRINGER JAPAN KK, English, Scientific journal
  DOI：https://doi.org/10.1007/s10265-017-0923-7
  DOI ID：10.1007/s10265-017-0923-7, ISSN：0918-9440, eISSN：1618-0860, Web of Science ID：WOS:000400234200018
• Loss of Inositol Phosphorylceramide Sphingolipid Mannosylation Induces Plant Immune Responses and Reduces Cellulose Content in Arabidopsis　　　　　　　　　　　　　　　
  Lin Fang; Toshiki Ishikawa; Emilie A. Rennie; Gosia M. Murawska; Jeemeng Lao; Jingwei Yan; Alex Yi-Lin Tsai; Edward E. K. Baidoo; Jun Xu; Jay D. Keasling; Taku Demura; Maki Kawai-Yamada; Henrik V. Scheller; Jenny C. Mortimer
  PLANT CELL, Volume：28, Number：12, First page：2991, Last page：3004, Dec. 2016, [Reviewed]
  Glycosylinositol phosphorylceramides (GIPCs) are a class of glycosylated sphingolipids found in plants, fungi, and protozoa. These lipids are abundant in the plant plasma membrane, forming; 25% of total plasma membrane lipids. Little is known about the function of the glycosylated headgroup, but two recent studies have indicated that they play a key role in plant signaling and defense. Here, we show that a member of glycosyltransferase family 64, previously named ECTOPICALLY PARTING CELLS1, is likely a Golgi-localized GIPC-specific mannosyl-transferase, which we renamed GIPC MANNOSYL-TRANSFERASE1 (GMT1). Sphingolipid analysis revealed that the Arabidopsis thaliana gmt1 mutant almost completely lacks mannose-carrying GIPCs. Heterologous expression of GMT1 in Saccharomyces cerevisiae and tobacco (Nicotiana tabacum) cv Bright Yellow 2 resulted in the production of non-native mannosylated GIPCs. gmt1 displays a severe dwarfed phenotype and a constitutive hypersensitive response characterized by elevated salicylic acid and hydrogen peroxide levels, similar to that we previously reported for the Golgi-localized, GIPC-specific, GDP-Man transporter GONST1 (Mortimer et al., 2013). Unexpectedly, we show that gmt1 cell walls have a reduction in cellulose content, although other matrix polysaccharides are unchanged.
  AMER SOC PLANT BIOLOGISTS, English, Scientific journal
  DOI：https://doi.org/10.1105/tpc.16.00186
  DOI ID：10.1105/tpc.16.00186, ISSN：1040-4651, eISSN：1532-298X, Web of Science ID：WOS:000393167800009
• Molecular characterization and targeted quantitative profiling of the sphingolipidome in rice　　　　　　　　　　　　　　　
  Toshiki Ishikawa; Yukihiro Ito; Maki Kawai-Yamada
  PLANT JOURNAL, Volume：88, Number：4, First page：681, Last page：693, Nov. 2016, [Reviewed]
  Recent advances in comprehensive metabolite profiling techniques, the foundation of metabolomics, is facilitating our understanding of the functions, regulation and complex networks of various metabolites in organisms. Here, we report a quantitative metabolomics technique for complex plant sphingolipids, composed of various polar head groups as well as structural isomers of hydrophobic ceramide moieties. Rice (Oryza sativa L.) was used as an experimental model of monocotyledonous plants and has been demonstrated to possess a highly complex sphingolipidome including hundreds of molecular species with a wide range of abundance. We established a high-throughput scheme for lipid preparation and mass spectrometry-based characterization of complex sphingolipid structures, which provided basic information to create a comprehensive theoretical library for targeted quantitative profiling of complex sphingolipids in rice. The established sphingolipidomic approach combined with multivariate analyses of the large dataset obtained clearly showed that different classes of rice sphingolipids, particularly including subclasses of glycosylinositol phosphoceramide with various sugar-chain head groups, are distributed with distinct quantitative profiles in various rice tissues, indicating tissue-dependent metabolism and biological functions of the lipid classes and subclasses. The sphingolipidomic analysis also highlighted that disruption of a lipid-associated gene causes a typical sphingolipidomic change in a gene-dependent manner. These results clearly support the utility of the sphingolipidomic approach in application to wide screening of sphingolipid-metabolic phenotypes as well as deeper investigation of metabolism and biological functions of complex sphingolipid species in plants.
  Significance Statement Sphingolipids play vital roles in plants, but the biological implications of their structural diversity remains to be addressed. Here we detail methodological advances for characterizing complex sphingolipid species quantitatively, and demonstrate its utility for profiling tissue-specific sphingolipids and for metabolic fingerprinting of sphingolipid-associated mutants.
  WILEY-BLACKWELL, English, Scientific journal
  DOI：https://doi.org/10.1111/tpj.13281
  DOI ID：10.1111/tpj.13281, ISSN：0960-7412, eISSN：1365-313X, Web of Science ID：WOS:000389928200013
• Metabolomic analysis of NAD kinase-deficient mutants of the cyanobacterium Synechocystis sp PCC 6803　　　　　　　　　　　　　　　
  Yuuma Ishikawa; Atsuko Miyagi; Yuto Haishima; Toshiki Ishikawa; Minoru Nagano; Masatoshi Yamaguchi; Yukako Hihara; Maki Kawai-Yamada
  JOURNAL OF PLANT PHYSIOLOGY, Volume：205, First page：105, Last page：112, Oct. 2016, [Reviewed]
  NAD kinase (NADK) phosphorylates NAD(H) to NADP(H). The enzyme has a crucial role in the regulation of the NADP(H)/NAD(H) ratio in various organisms. The unicellular cyanobacterium Synechocystis sp. PCC 6803 possesses two NADK-encoding genes, sll1415 and slr0400. To elucidate the metabolic change in NADK-deficient mutants growing under photoautotrophic conditions, we conducted metabolomic analysis using capillary electrophoresis mass spectrometry (CE-MS). The growth curves of the wild type parent (WT) and NADK-deficient mutants (Delta 1415 and Delta 0400) did not show any differences under photoautotrophic conditions. The NAD(P)(H) balance showed abnormality in both mutants. However, only the metabolite pattern of Delta 0400 showed differences compared to WT. These results indicated that the two NADK isoforms have distinct functions in cyanobacterial metabolism. (C) 2016 Elsevier GmbH. All rights reserved.
  ELSEVIER GMBH, URBAN & FISCHER VERLAG, English, Scientific journal
  DOI：https://doi.org/10.1016/j.jplph.2016.09.002
  DOI ID：10.1016/j.jplph.2016.09.002, ISSN：0176-1617, eISSN：1618-1328, Web of Science ID：WOS:000385858600014
• Plasma Membrane Microdomains Are Essential for Rac1-RbohB/H-Mediated Immunity in Rice　　　　　　　　　　　　　　　
  Minoru Nagano; Toshiki Ishikawa; Masayuki Fujiwara; Yoichiro Fukao; Yoji Kawano; Maki Kawai-Yamada; Ko Shimamoto
  PLANT CELL, Volume：28, Number：8, First page：1966, Last page：1983, Aug. 2016, [Reviewed]
  Numerous plant defense-related proteins are thought to congregate in plasma membrane microdomains, which consist mainly of sphingolipids and sterols. However, the extent to which microdomains contribute to defense responses in plants is unclear. To elucidate the relationship between microdomains and innate immunity in rice (Oryza sativa), we established lines in which the levels of sphingolipids containing 2-hydroxy fatty acids were decreased by knocking down two genes encoding fatty acid 2-hydroxylases (FAH1 and FAH2) and demonstrated that microdomains were less abundant in these lines. By testing these lines in a pathogen infection assay, we revealed that microdomains play an important role in the resistance to rice blast fungus infection. To illuminate the mechanism by which microdomains regulate immunity, we evaluated changes in protein composition, revealing that microdomains are required for the dynamics of the Rac/ROP small GTPase Rac1 and respiratory burst oxidase homologs (Rbohs) in response to chitin elicitor. Furthermore, FAHs are essential for the production of reactive oxygen species (ROS) after chitin treatment. Together with the observation that RbohB, a defense-related NADPH oxidase that interacts with Rac1, is localized in microdomains, our data indicate that microdomains are required for chitin-induced immunity through ROS signaling mediated by the Rac1-RbohB pathway.
  AMER SOC PLANT BIOLOGISTS, English, Scientific journal
  DOI：https://doi.org/10.1105/tpc.16.00201
  DOI ID：10.1105/tpc.16.00201, ISSN：1040-4651, eISSN：1532-298X, Web of Science ID：WOS:000386169200016
• KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect L-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis　　　　　　　　　　　　　　　
  Shota Sawake; Noriaki Tajima; Jenny C. Mortimer; Jeemeng Lao; Toshiki Ishikawa; Xiaolan Yu; Yukiko Yamanashi; Yoshihisa Yoshimi; Maki Kawai-Yamada; Paul Dupree; Yoichi Tsumuraya; Toshihisa Kotake
  PLANT CELL, Volume：27, Number：12, First page：3397, Last page：3409, Dec. 2015, [Reviewed]
  Humans are unable to synthesize L-ascorbic acid (AsA), yet it is required as a cofactor in many critical biochemical reactions. The majority of human dietary AsA is obtained from plants. In Arabidopsis thaliana, a GDP-mannose pyrophosphorylase (GMPP), VITAMIN C DEFECTIVE1 (VTC1), catalyzes a rate-limiting step in AsA synthesis: the formation of GDP-Man. In this study, we identified two nucleotide sugar pyrophosphorylase-like proteins, KONJAC1 (KJC1) and KJC2, which stimulate the activity of VTC1. The kjc1kjc2 double mutant exhibited severe dwarfism, indicating that KJC proteins are important for growth and development. The kjc1 mutation reduced GMPP activity to 10% of wild-type levels, leading to a 60% reduction in AsA levels. On the contrary, overexpression of KJC1 significantly increased GMPP activity. The kjc1 and kjc1kjc2 mutants also exhibited significantly reduced levels of glucomannan, which is also synthesized from GDP-Man. Recombinant KJC1 and KJC2 enhanced the GMPP activity of recombinant VTC1 in vitro, while KJCs did not show GMPP activity. Yeast two-hybrid assays suggested that the stimulation of GMPP activity occurs via interaction of KJCs with VTC1. These results suggest that KJCs are key factors for the generation of GDP-Man and affect AsA level and glucomannan accumulation through the stimulation of VTC1 GMPP activity.
  AMER SOC PLANT BIOLOGISTS, English, Scientific journal
  DOI：https://doi.org/10.1105/tpc.15.00379
  DOI ID：10.1105/tpc.15.00379, ISSN：1040-4651, eISSN：1532-298X, Web of Science ID：WOS:000368297100010
• Glycosylated sphingolipid biosynthesis and function in Arabidopsis
  Jenny C. Mortimer; Toshiki Ishikawa; Lin Fang; Beibei Jing; Emilie Rennie; Noriko Inada; Xiaolan Yu; Jeemeng Lao; Taku Demura; Maki Kawai-Yamada; Henrik Scheller; Paul Dupree
  GLYCOBIOLOGY, Volume：25, Number：11, First page：1266, Last page：1267, Nov. 2015, [Reviewed]
  OXFORD UNIV PRESS INC, English
  ISSN：0959-6658, eISSN：1460-2423, ORCID：45646360, Web of Science ID：WOS:000362991500108
• Overexpression of BAX INHIBITOR-1 Links Plasma Membrane Microdomain Proteins to Stress　　　　　　　　　　　　　　　
  Toshiki Ishikawa; Toshihiko Aki; Shuichi Yanagisawa; Hirofumi Uchimiya; Maki Kawai-Yamada
  PLANT PHYSIOLOGY, Volume：169, Number：2, First page：1333, Last page：1343, Oct. 2015, [Reviewed]
  BAX INHIBITOR-1 (BI-1) is a cell death suppressor widely conserved in plants and animals. Overexpression of BI-1 enhances tolerance to stress-induced cell death in plant cells, although the molecular mechanism behind this enhancement is unclear. We recently found that Arabidopsis (Arabidopsis thaliana) BI-1 is involved in the metabolism of sphingolipids, such as the synthesis of 2-hydroxy fatty acids, suggesting the involvement of sphingolipids in the cell death regulatory mechanism downstream of BI-1. Here, we show that BI-1 affects cell death-associated components localized in sphingolipid-enriched microdomains of the plasma membrane in rice (Oryza sativa) cells. The amount of 2-hydroxy fatty acid-containing glucosylceramide increased in the detergent-resistant membrane (DRM; a biochemical counterpart of plasma membrane microdomains) fraction obtained from BI-1-overexpressing rice cells. Comparative proteomics analysis showed quantitative changes of DRM proteins in BI-1-overexpressing cells. In particular, the protein abundance of FLOTILLIN HOMOLOG (FLOT) and HYPERSENSITIVE-INDUCED REACTION PROTEIN3 (HIR3) markedly decreased in DRM of BI-1-overexpressing cells. Loss-of-function analysis demonstrated that FLOT and HIR3 are required for cell death by oxidative stress and salicylic acid, suggesting that the decreased levels of these proteins directly contribute to the stress-tolerant phenotypes in BI-1-overexpressing rice cells. These findings provide a novel biological implication of plant membrane microdomains in stress-induced cell death, which is negatively modulated by BI-1 overexpression via decreasing the abundance of a set of key proteins involved in cell death.
  AMER SOC PLANT BIOLOGISTS, English, Scientific journal
  DOI：https://doi.org/10.1104/pp.15.00445
  DOI ID：10.1104/pp.15.00445, ISSN：0032-0889, eISSN：1532-2548, Web of Science ID：WOS:000365401000038
• Arabidopsis NAC domain proteins VND-INTERACTING1 and ANAC103 interact with multiple NAC domain proteins　　　　　　　　　　　　　　　
  Masatoshi Yamaguchi; Isura Sumeda Priyadarshana Nagahage; Misato Ohtani; Toshiki Ishikawa; Hirofumi Uchimiya; Maki Kawai-Yamada; Taku Demura
  PLANT BIOTECHNOLOGY, Volume：32, Number：2, First page：119, Last page：U14, Jun. 2015, [Reviewed]
  The Arabidopsis thaliana NAM, ATAF1/2 and CUC2 (NAC) domain transcription factor VND-INTERACTING1 (VNI1) was previously isolated as an interacting factor of VASCULAR-RELATED NAC-DOMAIN PROTEIN7 (VND7), a key regulator of xylem vessel differentiation, in a yeast two-hybrid screening. Here, we characterized VNI1 and its closest homolog, ANAC103, at the molecular level. Both VNI1 and ANAC103 interacted in vitro not only with VND proteins but also with other NAC domain proteins, such as NAC1 and CUC2. A transient expression assay showed that both VNI1 and ANAC103 are transcriptional activators. ANAC103 promoter activity was detected in vascular tissues, as well as in the trichomes, guard cells, and margins of young leaves. These data suggest that VNI1 and ANAC103 promote the differentiation of various types of cells by modulating the transcriptional activities of a wide range of NAC domain transcription factors.
  JAPANESE SOC PLANT CELL & MOLECULAR BIOLOGY, English, Scientific journal
  DOI：https://doi.org/10.5511/plantbiotechnology.15.0208a
  DOI ID：10.5511/plantbiotechnology.15.0208a, ISSN：1342-4580, Web of Science ID：WOS:000356970600002
• Does the Upstream Region Possessing MULE-Like Sequence in Rice Upregulate PsbS1 Gene Expression?　　　　　　　　　　　　　　　
  Mohammed Nuruzzaman; Tatsuo Kanno; Rika Amada; Yoshiki Habu; Ichiro Kasajima; Toshiki Ishikawa; Maki Kawai-Yamada; Hirofumi Uchimiya
  PLOS ONE, Volume：9, Number：9, First page：e102742, Sep. 2014, [Reviewed]
  The genomic nucleotide sequences of japonica rice (Sasanishiki and Nipponbare) contained about 2.7-kb unique region at the point of 0.4-kb upstream of the OsPsbS1 gene. In this study, we found that japonica rice with a few exceptions possessing such DNA sequences [denoted to OsMULE-japonica specific sequence (JSS)] is distinct by the presence of Mutator-like-element (MULE). Such sequence was absent in most of indica cultivars and Oryza glaberrima. In OsMULE-JSS1, we noted the presence of possible target site duplication (TSD; CTTTTCCAG) and about 80-bp terminal inverted repeat (TIR) near TSD. We also found the enhancement ofOsPsbS1 mRNA accumulation by intensified light, which was not associated with the DNA methylation status in OsMULE/JSS. In addition, O. rufipogon, possible ancestor of modern rice cultivars was found to compose PsbS gene of either japonica (minor) or indica (major) type. Transient gene expression assay showed that the japonica type promoter elevated a reporter gene activity than indica type.
  PUBLIC LIBRARY SCIENCE, English, Scientific journal
  DOI：https://doi.org/10.1371/journal.pone.0102742
  DOI ID：10.1371/journal.pone.0102742, ISSN：1932-6203, Web of Science ID：WOS:000342685600002
• Arabidopsis Bax inhibitor-1 promotes sphingolipid synthesis during cold stress by interacting with ceramide-modifying enzymes　　　　　　　　　　　　　　　
  Minoru Nagano; Toshiki Ishikawa; Yoshie Ogawa; Mitsuru Iwabuchi; Akari Nakasone; Ko Shimamoto; Hirofumi Uchimiya; Maki Kawai-Yamada
  PLANTA, Volume：240, Number：1, First page：77, Last page：89, Jul. 2014, [Reviewed]
  Bax inhibitor-1 (BI-1) is a widely conserved cell death suppressor localized in the endoplasmic reticulum membrane. Our previous results revealed that Arabidopsis BI-1 (AtBI-1) interacts with not only Arabidopsis cytochrome b (5) (Cb5), an electron transfer protein, but also a Cb5-like domain (Cb5LD)-containing protein, Saccharomyces cerevisiae fatty acid 2-hydroxylase 1, which 2-hydroxylates sphingolipid fatty acids. We have now found that AtBI-1 binds Arabidopsis sphingolipid Delta 8 long-chain base (LCB) desaturases AtSLD1 and AtSLD2, which are Cb5LD-containing proteins. The expression of both AtBI-1 and AtSLD1 was increased by cold exposure. However, different phenotypes were observed in response to cold treatment between an atbi-1 mutant and a sld1sld2 double mutant. To elucidate the reasons behind the difference, we analyzed sphingolipids and found that unsaturated LCBs in atbi-1 were not altered compared to wild type, whereas almost all LCBs in sld1sld2 were saturated, suggesting that AtBI-1 may not be necessary for the desaturation of LCBs. On the other hand, the sphingolipid content in wild type increased in response to low temperature, whereas total sphingolipid levels in atbi-1 were unaltered. In addition, the ceramide-modifying enzymes AtFAH1, sphingolipid base hydroxylase 2 (AtSBH2), acyl lipid desaturase 2 (AtADS2) and AtSLD1 were highly expressed under cold stress, and all are likely to be related to AtBI-1 function. These findings suggest that AtBI-1 contributes to synthesis of sphingolipids during cold stress by interacting with AtSLD1, AtFAH1, AtSBH2 and AtADS2.
  SPRINGER, English, Scientific journal
  DOI：https://doi.org/10.1007/s00425-014-2065-7
  DOI ID：10.1007/s00425-014-2065-7, ISSN：0032-0935, eISSN：1432-2048, Web of Science ID：WOS:000338318300005
• Development of an LC-MS/MS Method for the Analysis of Free Sphingoid Bases Using 4-Fluoro-7-nitrobenzofurazan (NBD-F)　　　　　　　　　　　　　　　
  Toshiki Ishikawa; Hiroyuki Imai; Maki Kawai-Yamada
  LIPIDS, Volume：49, Number：3, First page：295, Last page：304, Mar. 2014, [Reviewed]
  The molecular species of sphingoid bases were tagged with the fluorescent amino group reagent, 4-fluoro-7-nitrobenzofurazan (NBD-F). The NBD-sphingoid bases were analyzed by a highly selective and sensitive liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) technique capable of reliable detection of several fmol of the derivatives. Lipid extracts from plant samples were derivatized with NBD-F, and all nine species of free sphingoid bases present in plant sphingolipids were separated and quantified for the first time; a complete baseline resolution was achieved for cis-8 and trans-8 isomers of sphingoid bases by reversed phase HPLC on a C-18 column. The extraction and derivatization procedures and LC-MS/MS method can facilitate the progress of the studies for seeking the active components of sphingoid bases species in response to biological challenges.
  SPRINGER HEIDELBERG, English, Scientific journal
  DOI：https://doi.org/10.1007/s11745-013-3871-6
  DOI ID：10.1007/s11745-013-3871-6, ISSN：0024-4201, eISSN：1558-9307, Web of Science ID：WOS:000332665600009
• Culture temperature affects gene expression and metabolic pathways in the 2-methylisoborneol-producing cyanobacterium Pseudanabaena galeata　　　　　　　　　　　　　　　
  Masayuki Kakimoto; Toshiki Ishikawa; Atsuko Miyagi; Kazuaki Saito; Motonobu Miyazaki; Takashi Asaeda; Masatoshi Yamaguchi; Hirofumi Uchimiya; Maki Kawai-Yamada
  JOURNAL OF PLANT PHYSIOLOGY, Volume：171, Number：3-4, First page：292, Last page：300, Feb. 2014, [Reviewed]
  A volatile metabolite, 2-methylisoborneol (2-MIB), causes an unpleasant taste and odor in tap water. Some filamentous cyanobacteria produce 2-MIB via a two-step biosynthetic pathway: methylation of geranyl diphosphate (GPP) by methyl transferase (GPPMT), followed by the cyclization of methyl-GPP by monoterpene cyclase (MIBS). We isolated the genes encoding GPPMT and MIBS from Pseudanabaena galeata, a filamentous cyanobacterium known to be a major causal organism of 2-MIB production in Japanese lakes. The predicted amino acid sequence showed high similarity with that of Pseudanabaena limnetica (96% identity in GPPMT and 97% identity in MIBS). P. galeata was cultured at different temperatures to examine the effect of growth conditions on the production of 2-MIB and major metabolites. Gas chromatograph-mass spectrometry (GC-MS) measurements showed higher accumulation of 2-MIB at 30 degrees C than at 4 degrees C or 20 degrees C after 24 h of culture. Real-time-RT PCR analysis showed that the expression levels of the genes encoding GPPMT and MIBS decreased at 4 degrees C and increased at 30 degrees C, compared with at 20 degrees C. Furthermore, metabolite analysis showed dramatic changes in primary metabolite concentrations in cyanobacteria grown at different temperatures. The data indicate that changes in carbon flow in the TCA cycle affect 2-MIB biosynthesis at higher temperatures. (C) 2013 Elsevier GmbH. All rights reserved.
  ELSEVIER GMBH, URBAN & FISCHER VERLAG, English, Scientific journal
  DOI：https://doi.org/10.1016/j.jplph.2013.09.005
  DOI ID：10.1016/j.jplph.2013.09.005, ISSN：0176-1617, Web of Science ID：WOS:000332052200013
• Advanced LC-MS/MS techniques dissecting diverse isomers of plant sphingolipid species.　　　　　　　　　　　　　　　
  Ishikawa T; Yanagawa D; Kawai-Yamada M; Imai H
  J. Anal. Bioanal. Tech., Volume：S5, First page：7, 2014, [Reviewed]
  English, Scientific journal
  DOI：https://doi.org/10.4172/2155-9872.S5-007
  DOI ID：10.4172/2155-9872.S5-007
• Arabidopsis glycerol-3-phosphate permease 4 is localized in the plastids and involved in the accumulation of seed oil　　　　　　　　　　　　　　　
  Hiromitsu Kawai; Toshiki Ishikawa; Toshiaki Mitsui; Shin Kore-eda; Maki Kawai-Yamada; Jun-ichi Ohnishi
  PLANT BIOTECHNOLOGY, Volume：31, Number：2, First page：159, Last page：U80, 2014, [Reviewed]
  In plant cells, glycerol 3-phosphate (G3P) is apparently able to permeate the plastid envelope, but no specific transporter has been characterized so far. The Arabidopsis five G3Pp proteins have been predicted as putative G3P permeases because of their high homologies with the prokaryotic G3P/phosphate antiporter GlpT. In the present study, G3Pp4 was characterized in detail utilizing reverse genetic approaches. Promoter analysis using GUS expression revealed that G3Pp4 was expressed strongly throughout the embryos during late developmental stages, and the seed lipid contents decreased in two g3pp4 knockout mutants. An enhanced yellow fluorescent protein-fused G3Pp4 was localized in the plastids, functioned physiologically in A. thaliana, and had G3P-transport activity in E. coli. These results suggest that Arabidopsis G3Pp4 is a plastid envelope-localized G3P transporter and involved in accumulation of storage lipids in late embryogenesis.
  JAPANESE SOC PLANT CELL & MOLECULAR BIOLOGY, English, Scientific journal
  DOI：https://doi.org/10.5511/plantbiotechnology.14.0222a
  DOI ID：10.5511/plantbiotechnology.14.0222a, ISSN：1342-4580, Web of Science ID：WOS:000339501900009
• Rapid Induction of Lipid Droplets in Chlamydomonas reinhardtii and Chlorella vulgaris by Brefeldin A　　　　　　　　　　　　　　　
  Sangwoo Kim; Hanul Kim; Donghwi Ko; Yasuyo Yamaoka; Masumi Otsuru; Maki Kawai-Yamada; Toshiki Ishikawa; Hee-Mock Oh; Ikuo Nishida; Yonghua Li-Beisson; Youngsook Lee
  PLOS ONE, Volume：8, Number：12, First page：e81978, Dec. 2013, [Reviewed]
  Algal lipids are the focus of intensive research because they are potential sources of biodiesel. However, most algae produce neutral lipids only under stress conditions. Here, we report that treatment with Brefeldin A (BFA), a chemical inducer of ER stress, rapidly triggers lipid droplet (LD) formation in two different microalgal species, Chlamydomonas reinhardtii and Chlorella vulgaris. LD staining using Nile red revealed that BFA-treated algal cells exhibited many more fluorescent bodies than control cells. Lipid analyses based on thin layer chromatography and gas chromatography revealed that the additional lipids formed upon BFA treatment were mainly triacylglycerols (TAGs). The increase in TAG accumulation was accompanied by a decrease in the betaine lipid diacylglyceryl N,N,N-trimethylhomoserine (DGTS), a major component of the extraplastidic membrane lipids in Chlamydomonas, suggesting that at least some of the TAGs were assembled from the degradation products of membrane lipids. Interestingly, BFA induced TAG accumulation in the Chlamydomonas cells regardless of the presence or absence of an acetate or nitrogen source in the medium. This effect of BFA in Chlamydomonas cells seems to be due to BFA-induced ER stress, as supported by the induction of three homologs of ER stress marker genes by the drug. Together, these results suggest that ER stress rapidly triggers TAG accumulation in two green microalgae, C. reinhardtii and C. vulgaris. A further investigation of the link between ER stress and TAG synthesis may yield an efficient means of producing biofuel from algae.
  PUBLIC LIBRARY SCIENCE, English, Scientific journal
  DOI：https://doi.org/10.1371/journal.pone.0081978
  DOI ID：10.1371/journal.pone.0081978, ISSN：1932-6203, Web of Science ID：WOS:000328734200021
• The role of plant bax inhibitor-1 in suppressing H2O 2-induced cell death　　　　　　　　　　　　　　　
  Toshiki Ishikawa; Hirofumi Uchimiya; Maki Kawai-Yamada
  Methods in Enzymology, Volume：527, First page：239, Last page：256, 2013, [Reviewed]
  Hydrogen peroxide (H2O2) is known to be a typical endogenous signaling molecule that triggers programmed cell death in plants and metazoan. In this respect, they seem to share the mechanism of cell death caused by H2O2 and other reactive oxygen species (ROS). Bax inhibitor-1 (BI-1) is a well-conserved protein in plants and animals that serves as the inhibitor of mammalian proapoptotic proteins as well as plant ROS-induced cell death. As a target of H2O2, mitochondrion is considered to be an organelle of the primary ROS generation and perception. Thus, analysis of mitochondrial behavior in relation to functional roles of regulatory proteins (e.g., BI-1) will lead us to understand the core mechanisms of cell death regulation conserved in eukaryotes. In this chapter, we first introduce techniques of analyzing H2O2- (and ROS-) mediated changes in mitochondrial behavior. Next, we describe our understanding of the functions of plant BI-1 in regulation of ROS-induced cell death, with a technical basis for assessment of tolerance to ROS-mediated cell death in model plant systems. © 2013 Elsevier Inc.
  English, Scientific journal
  DOI：https://doi.org/10.1016/B978-0-12-405882-8.00013-1
  DOI ID：10.1016/B978-0-12-405882-8.00013-1, ISSN：0076-6879, ORCID：45646377, PubMed ID：23830635, SCOPUS ID：84879857652, Web of Science ID：WOS:000322846400014
• Nitrate Addition Alleviates Ammonium Toxicity Without Lessening Ammonium Accumulation, Organic Acid Depletion and Inorganic Cation Depletion in Arabidopsis thaliana Shoots　　　　　　　　　　　　　　　
  Takushi Hachiya; Chihiro K. Watanabe; Masaru Fujimoto; Toshiki Ishikawa; Kentaro Takahara; Maki Kawai-Yamada; Hirofumi Uchimiya; Yukifumi Uesono; Ichiro Terashima; Ko Noguchi
  PLANT AND CELL PHYSIOLOGY, Volume：53, Number：3, First page：577, Last page：591, Mar. 2012, [Reviewed]
  When ammonium is the sole nitrogen (N) source, plant growth is suppressed compared with the situation where nitrate is the N source. This is commonly referred to as ammonium toxicity. It is widely known that a combination of nitrate and ammonium as N source alleviates this ammonium toxicity (nitrate-dependent alleviation of ammonium toxicity), but the underlying mechanisms are still not completely understood. In plants, ammonium toxicity is often accompanied by a depletion of organic acids and inorganic cations, and by an accumulation of ammonium. All these factors have been considered as possible causes for ammonium toxicity. Thus, we hypothesized that nitrate could alleviate ammonium toxicity by lessening these symptoms. We analyzed growth, inorganic N and cation content and various primary metabolites in shoots of Arabidopsis thaliana seedlings grown on media containing various concentrations of nitrate and/or ammonium. Nitrate-dependent alleviation of ammonium toxicity was not accompanied by less depletion of organic acids and inorganic cations, and showed no reduction in ammonium accumulation. On the other hand, shoot growth was significantly correlated with the nitrate concentration in the shoots. This suggests that nitrate-dependent alleviation of ammonium toxicity is related to physiological processes that are closely linked to nitrate signaling, uptake and reduction. Based on transcript analyses of various genes related to nitrate signaling, uptake and reduction, possible underlying mechanisms for the nitrate-dependent alleviation are discussed.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcs012
  DOI ID：10.1093/pcp/pcs012, ISSN：0032-0781, eISSN：1471-9053, Web of Science ID：WOS:000301359500008
• Midgut juice of Plutella xylostella highly resistant to Bacillus thuringiensis Cry1Ac contains a three times larger amount of glucosinolate sulfatase which binds to Cry1Ac compared to that of susceptible strain　　　　　　　　　　　　　　　
  Takanori Yamazaki; Toshiki Ishikawa; Ganesh N. Pandian; Keiichi Okazaki; Kohsuke Haginoya; Yuka Tachikawa; Toshiaki Mitsui; Kazuhisa Miyamoto; Chanan Angusthanasombat; Hidetaka Hori
  PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY, Volume：101, Number：2, First page：125, Last page：131, Oct. 2011, [Reviewed]
  Midgut juice of Plutella xylostella strain PXR which is resistant to Cry1Ac was biochemically characterized relative to the susceptible PXS strain. The midgut juice of PXR (PXR-Juice) was shown to process Cry1Ac protoxin to 60 kDa active toxin with the same processing pattern as that of juice from PXS (PXS-Juice) in SOS-PAGE. PXS larvae which were given the Cry1Ac toxin pre-processed with PXR-Juice were killed with the same rate as that with Cry1Ac pre-activated by trypsin. PXR-Juice was found to contain three times larger amount of 66 kDa protein (P66) than PXS-Juice and the N-terminal amino acid sequence of P66 was matched to that of glucosinolate sulfatase in data base search. The protein band of P66 was coincided with the band of p-nitro phenyl sulfatase activity in zymogram. P66 purified to homogeneity in SOS-PAGE bound to Cry1Ac and soybean agglutinin, and K-D for Cry1Ac was estimated to be 718 nM with surface plasmon resonance analysis. Using purified sulfatase, K-m and V-max were estimated and involvement of the enzyme in the PXR resistance was discussed. (C) 2011 Elsevier Inc. All rights reserved.
  ACADEMIC PRESS INC ELSEVIER SCIENCE, English, Scientific journal
  DOI：https://doi.org/10.1016/j.pestbp.2011.09.001
  DOI ID：10.1016/j.pestbp.2011.09.001, ISSN：0048-3575, eISSN：1095-9939, Web of Science ID：WOS:000296401900010
• Bax inhibitor-1: a highly conserved endoplasmic reticulum-resident cell death suppressor　　　　　　　　　　　　　　　
  T. Ishikawa; N. Watanabe; M. Nagano; M. Kawai-Yamada; E. Lam
  CELL DEATH AND DIFFERENTIATION, Volume：18, Number：8, First page：1271, Last page：1278, Aug. 2011, [Reviewed]
  In spite of fundamental differences between plant and animal cells, it is remarkable that some cell death regulators that were identified to control cell death in metazoans can also function in plants. The fact that most of these proteins do not have structural homologs in plant genomes suggests that they may be targeting a highly conserved 'core' mechanism with conserved functions that is present in all eukaryotes. The ubiquitous Bax inhibitor-1 (BI-1) is a common cell death suppressor in eukaryotes that has provided a potential portal to this cell death core. In this review, we will update the current status of our understanding on the function and activities of this intriguing protein. Genetic, molecular and biochemical studies have so far suggested a consistent view that BI-1 is an endoplasmic reticulum (ER)-resident transmembrane protein that can interact with multiple partners to alter intracellular Ca(2+) flux control and lipid dynamics. Functionally, the level of BI-1 protein has been hypothesized to have the role of a rheostat to regulate the threshold of ER-stress inducible cell death. Further, delineation of the cell death suppression mechanism by BI-1 should shed light on an ancient cell death core-control pathway in eukaryotes, as well as novel ways to improve stress tolerance. Cell Death and Differentiation (2011) 18, 1271-1278; doi:10.1038/cdd.2011.59; published online 20 May 2011
  NATURE PUBLISHING GROUP, English, Scientific journal
  DOI：https://doi.org/10.1038/cdd.2011.59
  DOI ID：10.1038/cdd.2011.59, ISSN：1350-9047, Web of Science ID：WOS:000292634000006
• Roles of Plasma Membrane Microdomain in Cell Death Regulation Modulated by Bax Inhibitor-1　　　　　　　　　　　　　　　
  Ishikawa Toshiki; Aki Toshihiko; Yanagisawa Syuichi; Uchimiya Hirofumi; Kawai-Yamada Maki
  Plant and Cell Physiology Supplement, Volume：2011, First page：0404, Last page：0404, 2011
  Bax inhibitor-1 (BI-1) is an endoplasmic reticulum-localized cell death suppressor widely conserved in animals and plants. Our recent studies have revealed that Arabidopsis BI-1 (AtBI-1) interacts with sphingolipid-metabolic enzymes, leading us to further analyses to elucidate roles of BI-1-modulated sphingolipids on cell death regulation. Sphingolipids are well-known to play an important role for formation of liquid-ordered membrane microdomain in plants as well as animals. Here we show compositional alterations of plasma membrane microdomain in AtBI-1 overexpressing rice cells. Glycosphingolipid composition in plasma membrane microdomain was elevated in AtBI-1 overexpressor. Furthermore, shotgun proteomics combined with label-free comparative analysis revealed significant decreases of microdomain-localized proteins possible to participate in cell death regulation, such as hypersensitive cell death-associated proteins and scaffolds for protein complexes in microdomain. Based on these results, we will discuss functional roles of microdomain remodeling in the cell death regulatory mechanism of BI-1.
  The Japanese Society of Plant Physiologists
  DOI：https://doi.org/10.14841/jspp.2011.0.0404.0
  DOI ID：10.14841/jspp.2011.0.0404.0, CiNii Articles ID：130006997013
• A shotgun proteomics-based approach to identify membrane microdomain proteins involved in regulation of stress-induced cell death　　　　　　　　　　　　　　　
  Ishikawa Toshiki; Aki Toshihiko; Yanagisawa Shuichi; Nagano Minoru; Uchimiya Hirofumi; Kawai-Yamada Maki
  Abstracts for Annual Meeting of Japanese Proteomics Society, Volume：2011, First page：215, Last page：215, 2011
  Japanese Proteomics Society (Japan Human Proteome Organisation), Japanese
  DOI：https://doi.org/10.14889/jhupo.2011.0.215.0
  DOI ID：10.14889/jhupo.2011.0.215.0, CiNii Articles ID：130005454359
• Formation of Macromolecule Complex with Bacillus thuringiensis Cry1A Toxins and Chlorophyllide Binding 252-kDa Lipocalin-Like Protein Locating on Bombyx mori Midgut Membrane　　　　　　　　　　　　　　　
  Ganesh N. Pandian; Toshiki Ishikawa; Thangavel Vaijayanthi; Delwar M. Hossain; Shuhei Yamamoto; Tadayuki Nishiumi; Chanan Angsuthanasombat; Kohsuke Haginoya; Toshiaki Mitsui; Hidetaka Hori
  JOURNAL OF MEMBRANE BIOLOGY, Volume：237, Number：2-3, First page：125, Last page：136, Oct. 2010, [Reviewed]
  P252, a 252-kDa Bombyx mori protein located on the larval midgut membrane, has been shown to bind strongly with Bacillus thuringiensis Cry1A toxins (Hossain et al. Appl Environ Microbiol 70:4604-4612, 2004). P252 was also shown to bind chlorophyllide (Chlide) to form red fluorescence-emitting complex Bm252RFP with significant antimicrobial activity (Pandian et al. Appl Environ Microbiol 74:1324-1331, 2008). In this article, we show that Cry1A toxin bound with Bm252RFP and Bm252RFP-Cry1A macrocomplex, with both antimicrobial and insecticidal activities, was formed. The insecticidal activity of Bm252RFP-Cry1Ab was reduced from an LD(50) of 1.62 to 5.05 mu g, but Bm252RFP-Cry1Aa and Bm252RFP-Cry1Ac did not show such reduction. On the other hand, the antimicrobial activity of Bm252RFP-Cry1Ab was shown to retain almost the same activity as Bm252RFP, while the other two complexes lost around 30% activity. The intensity of photo absorbance and fluorescence emission of Bm252RFP-Cry1Ab were significantly reduced compared to those of the other two complexes. Circular dichroism showed that the contents of Cry1Ab alpha-helix was significantly decreased in Bm252RFP-Cry1Ab but not in the other two toxins. These data suggested that the reduction of contents of alpha-helix in Cry1Ab affected the insecticidal activity of the macrocomplex but did not alter the antimicrobial moiety in the macrocomplex of Bm252RFP-Cry1Ab.
  SPRINGER, English, Scientific journal
  DOI：https://doi.org/10.1007/s00232-010-9314-x
  DOI ID：10.1007/s00232-010-9314-x, ISSN：0022-2631, Web of Science ID：WOS:000284542200007
• Isoform-Specific Localization of Brassica rapa Nitrilases in Root Infected with Plasmodiophora brassicae Revealed Using In Situ Hybridization Probes Improved with Locked Nucleic Acids　　　　　　　　　　　　　　　
  Toshiki Ishikawa; Keiichi Okazaki; Tomohiko Nagaoka; Kimiko Itoh; Toshiaki Mitsui; Hidetaka Hori
  JOURNAL OF PLANT GROWTH REGULATION, Volume：29, Number：2, First page：210, Last page：222, Jun. 2010, [Reviewed]
  We established an in situ hybridization (ISH) technique by modification of hybridization probes with locked nucleic acids (LNAs) and demonstrated isoform-specific localization of transcripts of Brassica rapa nitrilase (BrNIT-T) genes in clubroot tissue infected with Plasmodiophora brassicae. Chimeric oligo DNA probes containing LNAs demonstrated highly improved specificities and could discriminate between BrNIT-T1 and BrNIT-T2. These LNA-containing probes were applied to ISH. BrNIT-T1 was strongly expressed in cells containing expanding secondary plasmodia of P. brassicae, but not in cells containing resting spores. On the other hand, BrNIT-T2 transcripts were localized in noninfected cells rather than infected cells during the clubroot growth phase but coexisted with mature resting spores at a later phase of clubroot development. Immunostaining for indole-3-acetic acid (IAA) revealed IAA accumulation in cells containing growing plasmodia. IAA immunostaining in infected cells was reduced as the pathogen formed resting spores, but the signal was again enhanced in cells containing mature resting spores at a later phase of infection, suggesting that IAA is involved in both the early growth and the latest maturation phase of clubroot development. Expression of BrNIT-T1 and BrNIT-T2 in turnip roots was upregulated by exogenous treatment with cytokinin and jasmonic acid, respectively. Thus, these two phytohormones are possible triggers of abnormal IAA production in clubroot tissue via induction of the respective nitrilase. Given these results, we propose a model for isoform-specific roles of B. rapa nitrilases in auxin biosynthesis involved in phytohormone crosstalk during development of clubroot disease.
  SPRINGER, English, Scientific journal
  DOI：https://doi.org/10.1007/s00344-009-9131-6
  DOI ID：10.1007/s00344-009-9131-6, ISSN：0721-7595, Web of Science ID：WOS:000277939500008
• Identification of QTLs that control clubroot resistance in Brassica oleracea and comparative analysis of clubroot resistance genes between B. rapa and B. oleracea　　　　　　　　　　　　　　　
  T. Nagaoka; M. A. U. Doullah; S. Matsumoto; S. Kawasaki; T. Ishikawa; H. Hori; K. Okazaki
  THEORETICAL AND APPLIED GENETICS, Volume：120, Number：7, First page：1335, Last page：1346, May 2010, [Reviewed]
  To perform comparative studies of CR (clubroot resistance) loci in Brassica oleracea and Brassica rapa and to develop marker-assisted selection in B. oleracea, we constructed a B. oleracea map, including specific markers linked to CR genes of B. rapa. We also analyzed CR-QTLs using the mean phenotypes of F(3) progenies from the cross of a resistant double-haploid line (Anju) with a susceptible double-haploid line (GC). In the nine linkage groups obtained (O1-O9), the major QTL, pb-Bo(Anju)1, was derived from Anju with a maximum LOD score (13.7) in O2. The QTL (LOD 5.1) located in O5, pb-Bo(GC)1, was derived from the susceptible GC. Other QTLs with smaller effects were found in O2, O3, and O7. Based on common markers, it was possible to compare our finding CR-QTLs with the B. oleracea CR loci reported by previous authors; pb-Bo(GC)1 may be identical to the CR-QTL reported previously or a different member contained in the same CR gene cluster. In total, the markers linked to seven B. rapa CR genes were mapped on the B. oleracea map. Based on the mapping position and markers of the CR genes, informative comparative studies of CR loci between B. oleracea and B. rapa were performed. Our map discloses specific primer sequences linked to CR genes and includes public SSR markers that will promote pyramiding CR genes in intra- and inter-specific crosses in Brassica crops. Five genes involved in glucosinolates biosynthesis were also mapped, and GSL-BoELONG and GSL-BoPro were found to be linked to the pb-Bo(Anju)1 and Bo(GC)1 loci, respectively. The linkage drag associated with the CR-QTLs is briefly discussed.
  SPRINGER, English, Scientific journal
  DOI：https://doi.org/10.1007/s00122-010-1259-z
  DOI ID：10.1007/s00122-010-1259-z, ISSN：0040-5752, Web of Science ID：WOS:000276674500005
• Metabolome Analysis of Response to Oxidative Stress in Rice Suspension Cells Overexpressing Cell Death Suppressor Bax Inhibitor-1　　　　　　　　　　　　　　　
  Toshiki Ishikawa; Kentaro Takahara; Takayuki Hirabayashi; Hideo Matsumura; Shizuko Fujisawa; Ryohei Terauchi; Hirofumi Uchimiya; Maki Kawai-Yamada
  PLANT AND CELL PHYSIOLOGY, Volume：51, Number：1, First page：9, Last page：20, Jan. 2010, [Reviewed]
  Bax inhibitor-1 (BI-1) is a cell death suppression factor widely conserved in higher plants and animals. Overexpression of Arabidopsis BI-1 (AtBI-1) in plants confers tolerance to various cell death-inducible stresses. However, apart from the cell death-suppressing activity, little is known about the physiological roles of BI-1-overexpressing plants. In this study, we evaluated the effects of AtBI-1 overexpression on the rice metabolome in response to oxidative stress. AtBI-1-overexpressing rice cells in suspension displayed enhanced tolerance to menadione-induced oxidative stress compared with vector control cells, whereas AtBI-1 overexpression did not influence the increase of intracellular H2O2 concentration or inhibition of oxidative stress-sensitive aconitase activity. Capillary electrophoresismass spectrometry (CE-MS)-based metabolome analysis revealed dynamic metabolic changes in oxidatively stressed rice cells, e.g. depletion of the central metabolic pathway, imbalance of the redox state and energy charge, and accumulation of amino acids. Furthermore, comparative metabolome analysis demonstrated that AtBI-1 overexpression did not affect primary metabolism in rice cells under normal growth conditions but significantly altered metabolite composition within several distinct pathways under cell death-inducible oxidative stress. The AtBI-1-mediated metabolic alteration included recovery of the redox state and energy charge, which are known as important factors for metabolic defense against oxidative stress. These observations suggest that although AtBI-1 does not affect rice metabolism directly, its cell death suppression activity leads to enhanced capacity to acclimate oxidative stress.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcp162
  DOI ID：10.1093/pcp/pcp162, ISSN：0032-0781, eISSN：1471-9053, Web of Science ID：WOS:000273704500002
• Loss of Calmodulin Binding to Bax Inhibitor-1 Affects Pseudomonas-mediated Hypersensitive Response-associated Cell Death in Arabidopsis thaliana　　　　　　　　　　　　　　　
  Maki Kawai-Yamada; Zenta Hori; Taro Ogawa; Yuri Ihara-Ohori; Katsunori Tamura; Minoru Nagano; Toshiki Ishikawa; Hirofumi Uchimiya
  JOURNAL OF BIOLOGICAL CHEMISTRY, Volume：284, Number：41, First page：27998, Last page：28003, Oct. 2009, [Reviewed]
  Bax inhibitor-1 (BI-1) is a cell death suppressor protein conserved across a variety of organisms. The Arabidopsis atbi1-1 plant is a mutant in which the C-terminal 6 amino acids of the expressed BI-1 protein have been replaced by T-DNA insertion. This mutant BI-1 protein (AtBI-CM) produced in Escherichia coli can no longer bind to calmodulin. A promoter-reporter assay demonstrated compartmentalized expression of BI-1 during hypersensitive response, introduced by the inoculation of Pseudomonas syringae possessing the avrRTP2 gene, Pst(avr-RPT2). In addition, both BI-1 knockdown plants and atbi1-1 showed increased sensitivity to Pst(avrRPT2)-induced cell death. The results indicated that the loss of calmodulin binding reduces the cell death suppressor activity of BI-1 in planta.
  AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, English, Scientific journal
  DOI：https://doi.org/10.1074/jbc.M109.037234
  DOI ID：10.1074/jbc.M109.037234, ISSN：0021-9258, Web of Science ID：WOS:000270676300028
• Bombyx mori midgut membrane protein P252, which binds to Bacillus thuringiensis Cry1A, is a chlorophyllide-binding protein, and the resulting complex has antimicrobial activity　　　　　　　　　　　　　　　
  Ganesh N. Pandian; Toshiki Ishikawa; Makoto Togashi; Yasuyuki Shitomi; Kohsuke Haginoya; Shuhei Yamamoto; Tadayuki Nishiumi; Hidetaka Hori
  APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Volume：74, Number：5, First page：1324, Last page：1331, Mar. 2008, [Reviewed]
  The epithelial cell membrane 252-kDa protein (P252) isolated in our laboratory from Bombyx mori midgut was shown to bind strongly with Cry1Aa, Cry1Ab, and Cry1Ac toxins of Bacillus thuringiensis (15). In the current paper, P252 was shown to bind with chlorophyllide (Chlide) to form red fluorescent protein (RFP) complex, termed Bm252RFP, with absorbance and fluorescence emission peaks at 600 nm and 620 nm, respectively. P252 at a concentration of 1 mu M is shown to bind with about 50 mu M Chlide in a positively cooperative reaction to form Bm252RFP under aerobic conditions and in the presence of light at 37 degrees C. Various parameters influencing this reaction have been optimized for efficient in vitro chemical synthesis of Bm252RFP. Circular dichroism spectra revealed that P252 is composed of a P-structure (39.8% +/- 2.2%, based on 5 samples) with negligible contribution of alpha-helix structure. When bound to Chlide, the P-structure content in the complex is reduced to 21.6% +/- 3.1% (n = 5). Since Chlide had no secondary structure, the observed reduction suggests significant conformational changes of P252 during the formation of Bm252RFP complex. Bm252RFP had antimicrobial activity against Escherichia coli, Serratia marcescens, B. thuringiensis, and Saccharomyces cerevisiae with 50% effective concentrations of 2.82, 2.94, 5.88 mu M, and 21.6 mu M, respectively. This is the first report ever to show clear, concrete binding characteristics of the midgut protein to form an RFP having significant antimicrobial activity.
  AMER SOC MICROBIOLOGY, English, Scientific journal
  DOI：https://doi.org/10.1128/AEM.01901-07
  DOI ID：10.1128/AEM.01901-07, ISSN：0099-2240, eISSN：1098-5336, Web of Science ID：WOS:000253792700004
• Molecular cloning of Brassica rapa nitrilases and their expression during clubroot development　　　　　　　　　　　　　　　
  Toshiki Ishikawa; Keiichi Okazaki; Haruka Kuroda; Kimiko Itoh; Toshiaki Mitsui; Hidetaka Hori
  MOLECULAR PLANT PATHOLOGY, Volume：8, Number：5, First page：623, Last page：637, Sep. 2007, [Reviewed]
  Three isoforms of nitrilase were cloned from turnip, Brassica rapa L., and their expression during clubroot development caused by Plasmodiophora brassicae was investigated. The isoforms were designated BrNIT-T1, BrNIT-T2 and BrNIT-T4 based on homology to known nitrilases. BrNIT-T1 and BrNIT-T2 have 80% homology to three nitrilases from Arabidopsis thaliana (AtNIT1, AtNIT2 and AtNIT3). BrNIT-T4 showed 90% homology to AtNIT4. To confirm their enzyme activity, the recombinant proteins were expressed in Escherichia coli. The recombinant BrNIT-T1 and BrNIT-T2 but not BrNIT-T4 converted indole-3-acetonitrile to indole-3-acetic acid, an endogenous plant auxin, although kinetic analysis showed that indole-3-acetonitrile is a poor substrate compared with various aliphatic and aromatic nitriles. By contrast, the recombinant BrNIT-T4 specifically converted beta-cyano-L-alanine to aspartic acid and asparagine and these findings agree with the idea that it is involved in the cyanide detoxification pathway. Real-time PCR analysis clearly showed that these isoforms were differentially expressed during clubroot development. BrNIT-T1 transcripts were very low in non-infected roots but were enhanced up to 100-fold in infected roots exhibiting club growth. By contrast, BrNIT-T2 transcripts remained at a very low level during clubroot formation. All these results clearly indicate the specific involvement of BrNIT-T1 in clubroot formation. The BrNIT-T4 transcripts were substantially reduced in the clubroot-growing phase, but thereafter they increased rapidly to a level found in non-infected roots as the clubroot growth reached a plateau. These findings suggest the specific involvement of BrNIT-T4 in clubroot maturation. In fully developed clubs, the BrNIT-T1 and BrNIT-T2 transcripts also increased. Free indole-3-acetic acid (IAA) content increased in the early and the latest phase of infected roots compared with noninfected roots, but decreased substantially at the middle phase. Thus, free IAA may play a role in the initiation and maturation of clubroot. Total IAA content was significantly higher in infected roots than in non-infected roots throughout clubroot development and IAA conjugation/conjugate hydrolysis system as well as BrNIT-Ts appear to be involved in clubroot development.
  WILEY-BLACKWELL PUBLISHING, INC, English, Scientific journal
  DOI：https://doi.org/10.1111/J.1364-3703.2007.00414.X
  DOI ID：10.1111/J.1364-3703.2007.00414.X, ISSN：1464-6722, Web of Science ID：WOS:000249160800007
• Plasmodiophora brassicae-induced cell death and medium alkalization in clubroot-resistant cultured roots of Brassica rapa　　　　　　　　　　　　　　　
  H Takahashi; T Ishikawa; M Kaido; K Takita; T Hayakawa; K Okazaki; K Itoh; T Mitsui; H Hori
  JOURNAL OF PHYTOPATHOLOGY, Volume：154, Number：3, First page：156, Last page：162, Mar. 2006, [Reviewed]
  Plasmodiophora brassicae causes clubroot in the turnip, Brassica rapa L. We used organ cultures of adventitious roots from B. rapa seedlings to investigate the initial response of resistant and susceptible cultivars to P. brassicae infection. Primary plasmodia of P. brassicae were observed in root hairs of both susceptible and resistant cultured roots. On the other hand, secondary plasmodia were able to proliferate only in the susceptible root culture but not in the resistant one. Root cultures from the susceptible cultivar all developed clubroot 4 weeks after treatment with 10(4), 10(5) or 10(6) spores/ml, but roots from the resistant cultivar did not develop clubroot under the same conditions. Cell death, as measured by Evans blue and TTC dye methods, was observed in cultured roots from the resistant cultivar but did not occur in roots from the susceptible cultivar after exposure to P. brassicae spores. Cell death was inhibited almost completely by EGTA and verapamil but not by the calmodulin antagonist W7. These results suggest the involvement of Ca2+ in P. brassicae-induced cell death. Alkalization of the root culture medium of the resistant cultivar was observed 2 days after treatment with P. brassicae spores but was not observed in root culture medium from the susceptible strain. We conclude that our root culture system must be a useful tool for further studies of the molecular mechanism of clubroot resistance.
  BLACKWELL PUBLISHING, English, Scientific journal
  DOI：https://doi.org/10.1111/j.1439-0434.2006.01076.x
  DOI ID：10.1111/j.1439-0434.2006.01076.x, ISSN：0931-1785, Web of Science ID：WOS:000235357600005

• Metabolome analysis revealed each brittle culm mutant-specific metabolism in rice　　　　　　　　　　　　　　　
  宮城敦子; 宮城敦子; 森和久; 石川寿樹; 大久保智司; 安達俊輔; 大川泰一郎; 山口雅利; 小竹敬久; 川合真紀
  日本植物生理学会年会(Web), Volume：64th, 2023
  J-Global ID：202302234679567567
• イネのカマイラズ変異体における代謝変動　　　　　　　　　　　　　　　
  宮城敦子; 森和久; 大久保智司; 安達俊輔; 大川泰一郎; 石川寿樹; 山口雅利; 小竹敬久; 川合真紀
  Volume：85th, 2021
  J-Global ID：202302210880983493
• シロイヌナズナVND遺伝子群の遺伝学的解析
  伊藤敦也; 久保稔; 大谷美沙都; 石川寿樹; 川合真紀; 出村拓; 山口雅利
  日本植物細胞分子生物学会大会・シンポジウム講演要旨集, Volume：36th, First page：153, 15 Aug. 2018
  Japanese
  J-Global ID：201802264094945466
• 植物に見出されたグリコシルイノシトールホスホセラミド特異的ホスホリパーゼDの性質　　　　　　　　　　　　　　　
  田中保; 宮城諒; 辻和樹; 藤原美奈; 森戸克弥; 石川寿樹; 今井博之; 川合真紀; 福田達也; 小暮健太朗
  Volume：51st, 2018
  J-Global ID：201802215895962288
• 植物に見出されたグリコシルイノシトールホスホセラミド特異的ホスホリパーゼDの性質　　　　　　　　　　　　　　　
  宮城諒; 辻和樹; 藤原美奈; 森戸克弥; 石川寿樹; 今井博之; 川合真紀; 福田達也; 小暮健太朗; 田中保
  Volume：91st, 2018
  J-Global ID：201902296826129470
• NAD(P)代謝改変シロイヌナズナの作出および代謝解析　　　　　　　　　　　　　　　
  鈴木渉太; 宮城敦子; 石川寿樹; 刑部敬史; 長野稔; 山口雅利; 川合真紀
  日本植物細胞分子生物学会大会・シンポジウム講演要旨集, Volume：33rd, First page：140, 20 Jul. 2015
  Japanese
  J-Global ID：201502202919376033
• Ethylene production during clubroot development in turnip　　　　　　　　　　　　　　　
  Ishikawa Toshiki; Okazaki Keiichi; Itoh Kimiko; Mitsui Toshiaki; Hori Hidetaka
  Bulletin of the Faculty of Agriculture, Niigata University, Volume：63, Number：2, First page：83, Last page：87, Mar. 2011
  Auxin and cytokinin play a crucial role for initiation stage of clubroot development in Plasmodiophora brassicaeinfectedcruciferous plants. On the other hand, the roles of phytohormones in a later maturation stage of clubroot remainunclear, regardless of significant accumulation of endogenous auxin indole-3-acetic acid (IAA) in fully developed clubrootgetting deteriorated. Here we analyzed the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) contents andACC oxidase (ACO) activity during clubroot development in turnip (Brassica rapa) to evaluate involvement of ethylene inclubroot development. Ethylene biosynthesis capacity estimated from endogenous ACC contents and ACO activity wasnot significantly affected in early growing clubroot but was elevated in later maturating tissues. This result suggests thatethylene biosynthesis is affected by P. brassicae infection and its level was coordinated with IAA during the maturationphase. Real-time PCR analysis of nitrilase, an IAA biosynthetic enzyme, demonstrated that treatment of turnip roots withACC inhibits nitrilase expression, whereas ACC-dependent ethylene biosynthesis is well-known to be upregulated by IAA.These insights imply that IAA biosynthesis via nitrilase precedes and induces ethylene production in the maturatingclubroot. This is the first report that ethylene could be involved in the maturation and deterioration of clubroot.Plasmodiophora brassicae 感染による根こぶ組織形成の初期段階におけるオーキシンやサイトカイニンといった植物ホルモンの関与がよく知られる一方で、根こぶ組織の成熟、腐熟段階における植物ホルモンの関与はほとんどわかっていない。本研究では、根こぶ組織成熟過程へのエチレンの関与に着目し、根こぶ病の進行に伴うエチレン前駆体1- アミノシクロプロパン-1- カルボン酸（ACC）含量とACC 酸化酵素（ACO）活性の変動を解析した。ACC 含量とACO 活性から推定されるエチレン生合成活性は、肥大初期の根こぶ組織で大きな変化は無かったが、肥大後期から腐熟期にある根こぶ組織では大きく増加していることが示された。リアルタイム定量PCR 解析の結果、ACC はオーキシン合成酵素ニトリラーゼのmRNA 発現を強く抑制することが明らかとなり、根こぶ成熟期におけるオーキシンの増加はエチレンの蓄積に先立って起こることが示唆された。
  新潟大学農学部, English
  ISSN：0385-8634, CiNii Articles ID：120005068686, CiNii Books ID：AN00183393
• Synthesis of high quality probe using locked nucleic acid (LNA) and exploitation for ISH　　　　　　　　　　　　　　　
  Ishikawa Toshiki; Okazaki Keiichi; Itoh Kimiko; Mitsui Toshiaki; Hori Hidetaka
  Bulletin of the Faculty of Agriculture, Niigata University, Volume：63, Number：1, First page：35, Last page：39, Aug. 2010
  高度に保存された塩基配列を有するmRNA のin situ hybridization（ISH）による特異的検出を目的として、locked nucleicacid（LNA）を利用したキメラオリゴヌクレオチドプローブを作製し、その実用性を評価した。塩基配列で90％以上の相同性を示すカブのニトリラーゼアイソフォームBrNIT-T1及びBrNIT-T2との間で相同性の低い24～25塩基の領域に相補的なオリゴヌクレオチドを設計し、両者間で異なる塩基（９～10塩基）をLNA に置換したLNA-DNA キメラオリゴヌクレオチドを作製した。配列中にLNA を部分的に導入することにより、DNA のみでは55～57℃であったオリゴヌクレオチドのTm 値は78～82℃に上昇し、ISH 解析に十分な値を示した。BrNIT-T1及びBrNIT-T2の全長転写産物に対する結合性をドットブロットノザンハイブリダイゼーションにより調査したところ、BrNIT-T1及びBrNIT-T2のそれぞれの配列に対応するLNA-DNA キメラプローブは互いの転写産物に非特異的に結合することなく、目的の転写産物のみを検出できることが確認された。さらにこれらのキメラプローブをカブ根こぶ病組織のISH 解析に応用し、従来のcRNA プローブを用いた解析では得られなかった、BrNIT-T1とBrNIT-T2の時空間的な発現特異性を明らかにすることに成功した。To achieve specific detection of highly-conserved sequences by in situ hybridization (ISH), locked nucleic acid(LNA)-containing oligonucleotide probes were designed and assessed. Two isoforms of turnip nitrilases, BrNIT-T1 andBrNIT-T2, that possess more than 90% homology at the nucleotide level, were targeted in this study. Oligonucleotidescorresponding to a relatively different region between BrNIT-T1 and BrNIT-T2 were generated to contain LNAs in placeof DNA bases different between the two sequences. The partial incorporation of LNA into DNA oligonucleotides providedsufficient Tm values for ISH analysis. Dot-blot hybridization assay confirmed that the two LNA-modified oligonucleotideprobes corresponding to sequences of BrNIT-T1 and BrNIT-T2 specifically hybridized with the desired RNA sequencebut not with another one. Advantages of the LNA-modified probes in ISH analysis were also observed as specific andsensitive detection of BrNIT-T localization. Based on these results, we here report that partial incorporation of LNA intooligonucleotide probe is useful for highly specific detection of similar genes in ISH analysis.
  新潟大学農学部, Japanese
  ISSN：0385-8634, CiNii Articles ID：120005067423, CiNii Books ID：AN00183393
• Roles of nitrilase in auxin biosynthesis and phytohormone crosstalk in turnip clubroot　　　　　　　　　　　　　　　
  ISHIKAWA Toshiki; OKAZAKI Keiichi; HORI Hidetaka
  Volume：42, First page：25, Last page：25, 05 Oct. 2007
  Plasmodiophora brassicae parasitizes cruciferous plant roots and manipulates indole-3-acetic acid (IAA) biosynthesis system of host plants to induce hypertrophy, called clubroot. We isolated three cDNAs encoding nitrilase from Brassica rapa (turnip), i.e. BrNIT-T1, BrNIT-T2 and BrNIT-T4. Enzyme analysis of recombinant proteins showed that BrNIT-T1 and BrNIT-T2 possess IAA synthetic activity, but BrNIT-T4 is involved in the plant cyanide detoxification pathway downstream of ethylene production. Realtime PCR analysis revealed specific involvement of BrNIT-T1 in clubroot initiation phase: in early-growing clubroot, BrNIT-T1 was strongly up-regulated but BrNIT-T2 and BrNIT-T4 were down-regulated. In maturing clubroot, not only BrNIT-T1 but also BrNIT-T2 and BrNIT-T4 were up-regulated. Free IAA level was transiently elevated at the early and later phases, despite a high IAA conjugation activity throughout disease development. Ethylene-forming activity was reduced during clubroot formation but subsequently increased, just like BrNIT-T4 expression. These results indicate a close relationship of the nitrilases to auxin and ethylene biosynthesis. Treatment of roots with cytokinin or jasmonic acid activated BrNIT-T1 or BrNIT-T2 expression, respectively. Thus, the two phytohormones may trigger IAA production in clubroot. Based on these observations, we propose a scheme regarding individual roles of the nitrilases in hormone crosstalk during clubroot initiation and maturation in turnip.
  The Japanese Society for Chemical Regulation of Plants, Japanese
  ISSN：1346-5406, CiNii Articles ID：110006474705, CiNii Books ID：AA11550064
• Expression of Bombyx mori Aminopeptidase N on Plasma Membrane of Cultured Insect Cells and Analysis of Its Receptor Function for Insecticidal Cry Toxins　　　　　　　　　　　　　　　
  Noguchi Rieko; Ishikawa Toshiki; Haginoya Kohsuke; Shitomi Yasuyuki; Sato Ryoichi; Hayakawa Tohru; Hori Hidetaka
  Bulletin of the Faculty of Agriculture, Niigata University, Volume：60, Number：1, First page：73, Last page：81, Aug. 2007
  Aminopeptidase N(APN) localizing on the apical membrane of midgut epithelial cells is one of the plausible candidate of the receptor proteins for Bacillus thuringiensis Cry1Aa toxin which specifically kills lepidopteran insects. However, comprehensive evidences to address above possibility have not been established yet. We tried to express the APN1, BmAPN1, onto plasma membrane of cultured insect cells to investigate the interaction with Cry1Aa. Gene expression system was constructed with High five cultured insect cells and baculovirus expression system. On 48 h after the infection of the cells with gene modified baculovirus, 110 kDa BmAPN1 was expressed on the cell membranes. Aminopeptidase activity was ten times higher than that of non-infected cultured cells. The recombinant BmAPN1 was the same size as native one localizing on the brush border membrane of midgut epithelial cells. The transformed cells expressing BmAPN1 is a good system for the investigation of interaction between APN and Cry1Aa toxin.
  Niigata University, Japanese
  ISSN：0385-8634, CiNii Articles ID：110006387670, CiNii Books ID：AN00183393
• Comparative Proteomic Analysis of the Resistant Response in Brassica rapa Root Culture to the Clubroot Disease Agent Plasmodiophora brassicae　　　　　　　　　　　　　　　
  Kaido Masakazu; Ishikawa Toshiki; Hori Hidetaka
  Bulletin of the Faculty of Agriculture, Niigata University, Volume：60, Number：1, First page：67, Last page：71, Aug. 2007
  Comparative proteome analysis of the resistance responses in cultured roots of Brassica rapa (turnip) on the treatment with resting spore of Plasmodiophora brassicae, which is the causal agent of clubroot disease, was performed by 2-D PAGE. Using cultured roots from a susceptible and a resistant cultivar, 251 protein spots were clearly observed in 2D-PAGE and protein profiles were compared between resistant and susceptible roots as well as spore-treated and non-treated roots. In comparison between resistant and susceptible non-treated roots, 11 and 8 spots were significantly higher in intensity in resistant and susceptible roots, respectively. The treatment with resting spores of P. brassicae affected protein profiles of 2D-PAGE in a different manner between susceptible and resistant roots. In spore-treated susceptible roots, concentration of 6 protein spots increased but 7 were reduced compared to those in untreated control. These proteins are possibly involved in infectious events necessary for root hair infection or for defense responses in non-pathogen-specific stresses. On the other hand, in the spore treated resistant roots, 10 and 13 spots were up- and down-regulated, respectively. The protein profile of the resistant roots treated with the spore was clearly different from that of the susceptible ones, suggested that the fluctuated proteins of resistant roots should include the one(s) specific to resistant responses.
  Niigata University, English
  ISSN：0385-8634, CiNii Articles ID：110006387666, CiNii Books ID：AN00183393
• Evaluation of roles amidase which converts indole-3-acetamide to indole-3-acetic acid, in formation of clubroot in turnip　　　　　　　　　　　　　　　
  Ishikawa Toshiki; Kuroda Haruka; Okazaki Keiichi; Itoh Kimiko; Mitsui Toshiaki; Hori Hidetaka
  Bulletin of the Faculty of Agriculture,Niigata University, Volume：60, Number：1, First page：53, Last page：60, Aug. 2007
  Amidase was investigated if it was one of auxin-producing enzymes of Brassica rapa. We found amidase activity to convert indole-3-acetamide to indole-3-acetic acid in soluble protein extracts from B. rapa. Optimum condition for the enzyme activity was searched and two optimum temperatures were obtained at 45 and 55℃. Search for the heat stability of the enzyme strongly suggested that the two summits of the optimum temperatures were resulted from occurrence of variant amidases showing different temperature stabilities. The possibility of the presence of several amidase isoforms was supported by the following two observations, i.e., firstly, amidase activities at 45 and 55℃ had different pH optimums, 8.5 and 7.5, respectively. Secondly, the enzyme activities at the two temperatures were differentially fluctuated during the vegetative growth of turnip and clubroot development. Fluctuation of the amidase activity and IAA contents observed in various turnip tissues such as healthy turnip leaf, hypocotyl and roots suggested that the enzyme had a constitutive role for keeping IAA homeostasis in those tissues. Interestingly, the amidase in turnip was shown to have high activity in hypocotyl and root rather than leaf, unlike Arabidopsis one. Changes of the enzyme activity during development of B. rapa was analyzed using the tissues of clubrootdiseased turnips. The activities fluctuated differently from each other in the temperature at 45 and 55℃ in infected tissues. In addition, activity at 45℃ was specifically enhanced in a later phase of clubroot development, whereas one at 55℃ increased only in an early phase in the infected root tissues. These results indicated that the amidase played an important role in turnip growth and clubroot development.
  Niigata University, English
  ISSN：0385-8634, CiNii Articles ID：110006387205, CiNii Books ID：AN00183393
• Loading of Fura-2 into Liquid Organ Cultured Adventitious Root of Turnip (Brassica rapa L.) Resistant to Clubroot Pathogen Plasmodiophora brassicae and Determination of [Ca2+]cyt　　　　　　　　　　　　　　　
  Takahashi Hideyuki; Ishikawa Toshiki; Hayakawa Tohru; Itoh Kimiko; Mitsui Toshiaki; Hori Hidetaka
  Bulletin of the Faculty of Agriculture, Niigata University, Volume：60, Number：1, First page：61, Last page：66, Aug. 2007
  Our previous study using liquid organ cultured adventitious roots from turnip (Brassica rapa L.) showed that Ca2+ is required for induction of defense responses in the cultured roots on the treatment with Plasmodiophora brassicae resting spores (Takahashi et al., 2006). To evaluate change in [Ca2+]cyt in cultured root cells on contact with the spores, acetoxymethyl ester derivative of Fura-2 (Fura-2/AM) was loaded into the roots. When ionophore A23187 was treated with Ca2+ simultaneously, the Fura-2 fluorescence ratio that represents relative [Ca2+]cyt increased promptly, showing that the Fura-2/AM system is suitable to evaluate [Ca2+]cyt change in cultured root cells in a second range. Applicability of this method for studies on Ca2+ fluctuation against various extracellular stimuli was supported by observations that treatment with mannitol or NaCl also immediately increased the Fura-2 ratio. When the Fura-2/AM loaded roots were treated with resting spores, no [Ca2+]cyt change was observed during 500 second but when treated with spores pre-incubated with germination-enhancing suspension (GES), a slight but reproducible increase in [Ca2+]cyt was observed. We conclude that although further analysis is needed, the Fura-2/AM system will contribute to revealing the Ca2+ involvement in clubroot-resistance response.
  Niigata University, English
  ISSN：0385-8634, CiNii Articles ID：110006387665, CiNii Books ID：AN00183393

• セラミド研究の新展開　　　　　　　　　　　　　　　
  石川 寿樹; 今井 博之, [Joint work]
  Jun. 2019
• The role of plant Bax inhibitor-1 in suppressing H2O2-induced cell death.　　　　　　　　　　　　　　　
  石川 寿樹, [Joint work]
  2013

• 植物GIPCの構造多様性と代謝機構：リピドミクスからのアプローチ　　　　　　　　　　　　　　　
  Oct. 2022, [Invited]
  Invited oral presentation
  共同研究・競争的資金等ID：13207319;12237085
• 植物スフィンゴ脂質のターゲットメタボロミクス　　　　　　　　　　　　　　　
  石川寿樹
  Sep. 2022, [Invited]
  Invited oral presentation
  共同研究・競争的資金等ID：12237085;13207319
• 分子進化から読み解く植物独自のスフィンゴ脂質多様化戦略　　　　　　　　　　　　　　　
  石川寿樹
  Sep. 2020, [Invited]
  Sep. 2020 - Sep. 2020, Japanese, Invited oral presentation
• The Evolutionary Journey of Plant-Unique Long-Chain Base Unsaturation　　　　　　　　　　　　　　　
  Toshiki Ishikawa; Maki Kawai-Yamada
  Jul. 2018, [International conference]
  English, Oral presentation
• New insights into molecular evolution of sphingolipid unsaturation in plants　　　　　　　　　　　　　　　
  Toshiki Ishikawa; Maki Kawai-Yamada
  7th Asian Symposium on Plant Lipid, Nov. 2017
  Nov. 2017 - Dec. 2017, English, Oral presentation
• 長鎖塩基不飽和化を介したスフィンゴ脂質合成制御とストレス耐性　　　　　　　　　　　　　　　
  石川 寿樹; 川合真紀
  Sep. 2014, [Invited], [Domestic conference]
  Japanese, Nominated symposium
• ショットガンプロテオミクスによる植物のストレス誘導性細胞死制御に関わる細胞膜マイクロドメインタンパク質の探索　　　　　　　　　　　　　　　
  石川寿樹; 秋利彦; 柳澤修一; 長野稔; 内宮博文; 川合真紀
  Jul. 2011, [Invited], [Domestic conference]
  Japanese, Nominated symposium

• Apr. 2022 - Present
  Basic Biochemistry, Saitama University
• Apr. 2021 - Present
  Lipid Biochemistry, Saitama University

• THE BOTANICAL SOCIETY OF JAPAN
• JAPANESE SOCIETY FOR PLANT CELL AND MOLECULAR BIOLOGY
• THE JAPANESE SOCIETY OF PLANT PHYSIOLOGISTS
• THE JAPANESE CONFERENCE ON THE BIOCHEMISTRY OF LIPIDS
• THE JAPANESE BIOCHEMICAL SOCIETY

• Elucidation of a plant immune signaling pathway triggered by hydrolysis of plant sphingolipid GIPC　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Challenging Research (Exploratory), 28 Jun. 2024 - 31 Mar. 2026
  Gifu University
  Grant amount(Total)：6500000, Direct funding：5000000, Indirect funding：1500000
  Grant number：24K21848
• 食品成分として未開拓なホスホイノシトール含有スフィンゴ脂質の評価　　　　　　　　　　　　　　　
  01 Apr. 2023 - 31 Mar. 2026
  Grant amount(Total)：19110000, Direct funding：14700000, Indirect funding：4410000
  Grant number：23H02158
• Characterization of seed size regulation driven by various head groups of sphingolipids aimed for genetic breeding of seed crop plants　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Apr. 2022 - Mar. 2025
  Saitama University
  Grant amount(Total)：4290000, Direct funding：3300000, Indirect funding：990000
  Grant number：22K05553
• 植物独自のスフィンゴ脂質糖鎖構造多様性が担う生物学的意義の解明　　　　　　　　　　　　　　　
  Apr. 2019 - Mar. 2022
  Principal investigator
  Competitive research funding
  講演・口頭発表等ID：42182940
• 植物固有なスフィンゴ脂質糖鎖の形成機構と生物学的機能の解明　　　　　　　　　　　　　　　
  Apr. 2017 - Mar. 2019
  Principal investigator
  Competitive research funding
  講演・口頭発表等ID：42182940
• 植物スフィンゴ脂質の分子機能解明と代謝改変技術の確立　　　　　　　　　　　　　　　
  Apr. 2015 - Mar. 2017
  Principal investigator
  Competitive research funding
• 植物スフィンゴ脂質代謝酵素の機能同定と分子育種への利用　　　　　　　　　　　　　　　
  Apr. 2012 - Mar. 2014
  Principal investigator
  Competitive research funding
• リピドミクスを基盤とした植物スフィンゴ脂質分子種の機能解析と育種利用　　　　　　　　　　　　　　　
  01 Apr. 2012 - 31 Mar. 2013
  Grant amount(Total)：4550000, Direct funding：3500000, Indirect funding：1050000
  Grant number：24780328