Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Toyoaki Anai Last modified date:2023.11.27

Professor / Agrobiological Science / University Farm,Kyushu University / Faculty of Agriculture


Papers
1. Sakae Agarie, Makiko Umemoto, Haruki Sunagawa, Toyoaki Anai, John C. Cushman, An Agrobacterium-mediated transformation via organogenesis regeneration of a facultative CAM plant, the common ice plant Mesembryanthemum crystallinum L, Plant Production Science, 10.1080/1343943X.2020.1730700, 23, 3, 343-349, 2020.07, The common ice plant, Mesembryanthemum crystallinum L. provides a useful model for the study of environmentally induced photosynthetic conversion and abiotic stresses tolerance. However, a procedure for the production of transgenic ice plant, which is essential for functional genomics, has not been fully established. Here we tested the factors on the transformation of cotyledonary nodes excised from the ice plant seedlings such as thidiazuron (TDZ), NaCl and phytosulfokine (PSK), a peptidyl plant growth factor using Agrobacterium tumefaciens strains EHA101 and EHA105 harboring binary vector plasmids pBI7EGFP and pCAMBIA1302, respectively. The established procedure is as follows: the explants (cotyledonary nodes) were co-cultivated with Agrobacterium for 3 days, and the explants were cultured in the medium with 0.5 mg l−1 kinetin and 100 mg l−1 carbenicillin for 72 h, and they were cultured in the medium with 0.5 mg l−1 kinetin and 100 nM PSKfor 4 weeks. Thidiazuron and NaCl enhanced the production of multiple adventitious shoot formation during regeneration but reduced the transformation efficiency due to the vitrification of adventitious shoots. PSK was effective in the production of healthy adventitious shoots. The transformation frequency at the stage of whole plants was 0.6% and 4.6% per inoculated cotyledonary nodes using the Agrobacterium strain EHA101 (pBI7EGFP) and EHA105 (pCAMBIA1302), respectively..
2. Shin Kato, Yuko Yokota, Rintaro Suzuki, Yukiko Fujisawa, Takashi Sayama, Akito Kaga, Toyoaki Anai, Kunihiko Komatsu, Nobuhiko Oki, Akio Kikuchi, Masao Ishimoto, Identification of a cytochrome P450 hydroxylase, CYP81E22, as a causative gene for the high sensitivity of soybean to herbicide bentazon, Theoretical and Applied Genetics, 10.1007/s00122-020-03580-6, 133, 7, 2105-2115, 2020.07, Key message: A frame shift invoked by a single-base deletion in the gene encoding a cytochrome P450 hydroxylase, CYP81E22, causes the loss of bentazon detoxification function in soybean. Abstract: Bentazon is an effective herbicide in soybean cultivation applied at post-emergence stages for control of several broadleaf weeds. However, some soybean cultivars are highly sensitive to bentazon and are killed upon application. In this study, the gene related to the high sensitivity of soybean cultivars to bentazon was mapped to chromosome 16, and its location was narrowed down to a 257-kb region where three cytochrome P450 genes were located. In these genes, a single-base deletion of cytosine was detected in the coding region of Glyma.16G149300, CYP81E22, at + 1465 bp downstream from the translation start codon, leading to a frame shift in the open reading frame and creating a premature stop codon. This stop codon resulted in the loss of more than half of the P450, and consequently, the remaining molecule failed to form a functioning protein. This single-base deletion was common among the highly sensitive cultivars screened from the soybean mini-core collection and other previously reported highly sensitive cultivars. Furthermore, we screened plant lines from the targeting-induced local lesions in genomes library of the soybean cultivar Enrei based on a modelled 3D structure of CYP81E22. The lines with mutations in Glyma.16G149300 were highly sensitive to bentazon, which provides strong evidence that Glyma.16G149300 is the gene responsible for high sensitivity to bentazon..
3. Sakae Agarie, Makiko Umemoto, Haruki Sunagawa, Toyoaki Anai, John C. Cushman, An Agrobacterium-mediated transformation via organogenesis regeneration of a facultative CAM plant, the common ice plant Mesembryanthemum crystallinum L, Plant Production Science, 10.1080/1343943X.2020.1730700, 23, 3, 343-349, 2020.07.
4. Shin Kato, Yuko Yokota, Rintaro Suzuki, Yukiko Fujisawa, Takashi Sayama, Akito Kaga, Toyoaki Anai, Kunihiko Komatsu, Nobuhiko Oki, Akio Kikuchi, Masao Ishimoto, Identification of a cytochrome P450 hydroxylase, CYP81E22, as a causative gene for the high sensitivity of soybean to herbicide bentazon, Theoretical and Applied Genetics, 10.1007/s00122-020-03580-6, 133, 7, 2105-2115, 2020.07.
5. Yuki Nishida, Reona Hiraoka, Satomi Kawano, Norio Suganuma, Shusei Sato, Satoshi Watanabe, Toyoaki Anai, Susumu Arima, Akiyoshi Tominaga, Akihiro Suzuki, SEN1 gene from Lotus japonicus MG20 improves nitrogen fixation and plant growth, SOIL SCIENCE AND PLANT NUTRITION, 10.1080/00380768.2020.1834829, 66, 6, 864-869, 2020.07.
6. Sarkar, Md Abdur Rauf; Otsu, Wakana; Suzuki, Akihiro; Hashimoto, Fumio; Anai, Toyoaki; Watanabe, Satoshi, Single-base deletion in GmCHR5 increases the genistein-to-daidzein ratio in soybean seed, BREEDING SCIENCE, 10.1270/jsbbs.19134, 70, 3, 265-276, 2020.06.
7. Yamamoto, Akiko; Matsunaga, Ken-ichiro; Anai, Toyoaki; Kawano, Hitoshi; Ueda, Toshihisa; Matsumoto, Toshihiko; Ando, Shoji, Characterization of an Intermediate Filament Protein from the Platyhelminth, Dugesia japonica, PROTEIN AND PEPTIDE LETTERS, 10.2174/0929866526666191025102902, 27, 5, 432-446, 2020.05.
8. Akiko Yamamoto, Ken Ichiro Matsunaga, Toyoaki Anai, Hitoshi Kawano, Toshihisa Ueda, Toshihiko Matsumoto, Shoji Ando, Characterization of an intermediate filament protein from the platyhelminth, Dugesia japonica, Protein and Peptide Letters, 10.2174/0929866526666191025102902, 27, 5, 432-446, 2020.05, Background: Intermediate filaments (IFs) are major constituents of the cytoskeletal systems in animal cells. Objective: To gain insights into the structure-function relationship of invertebrate cytoplasmic IF proteins, we characterized an IF protein from the platyhelminth, Dugesia japonica, termed Dif-1. Methods: cDNA cloning, in situ hybridization, immunohistochemical analysis, and IF assembly experiments in vitro using recombinant Dif-1, were performed for protein characterization. Results: The structure deduced from the cDNA sequence showed that Djf-1 comprises 568 amino acids and has a tripartite domain structure (N-terminal head, central rod, and C-terminal tail) that is characteristic of IF proteins. Similar to nuclear IF lamins, Djf-1 contains an extra 42 residues in the coil 1b subdomain of the rod domain that is absent from vertebrate cytoplasmic IF proteins and a nuclear lamin-homology segment of approximately 105 residues in the tail domain; however, it contains no nuclear localization signal. In situ hybridization analysis showed that Djf-1 mRNA is specifically expressed in cells located within the marginal region encircling the worm body. Immunohistochemical analysis showed that Djf-1 protein forms cytoplasmic IFs located close to the microvilli of the cells. In vitro IF assembly experiments using recombinant proteins showed that Djf-1 alone polymerizes into IFs. Deletion of the extra 42 residues in the coil 1b subdomain resulted in the failure of IF formation. Conclusion: Together with data from other histological studies, our results suggest that Djf-1 is expressed specifically in anchor cells within the glandular adhesive organs of the worm and that Djf-1 IFs may play a role in protecting the cells from mechanical stress..
9. Tomoki Hoshino, Nobushige Iijima, Masakazu Hata, Anri Watanabe, Tamae Kawakami, Toyoaki Anai, Molecular characterization of high stearic acid soybean mutants and post-transcriptional control of GmSACPD genes in the mutant with a single nucleotide deletion, Plant Gene, 10.1016/j.plgene.2019.100207, 21, 2020.03.
10. Yu Takahashi, Hiroaki Sakai, Yuki Yoshitsu, Chiaki Muto, Toyoaki Anai, Muthaiyan Pandiyan, Natesan Senthil, Norihiko Tomooka, Ken Naito, Domesticating Vigna Stipulacea: A Potential Legume Crop With Broad Resistance to Biotic Stresses, Frontiers in Plant Science, 10.3389/fpls.2019.01607, 10, 1607-1607, 2019.12, Though crossing wild relatives to modern cultivars is a usual means to introduce alleles of stress tolerance, an alternative is de novo domesticating wild species that are already tolerant to various kinds of stresses. As a test case, we chose Vigna stipulacea Kuntze, which has fast growth, short vegetative stage, and broad resistance to pests and diseases. We developed an ethyl methanesulfonate–mutagenized population and obtained three mutants with reduced seed dormancy and one with reduced pod shattering. We crossed one of the mutants of less seed dormancy to the wild type and confirmed that the phenotype was inherited in a Mendelian manner. De novo assembly of V. stipulacea genome, and the following resequencing of the F2 progenies successfully identified a Single Nucleotide Polymorphism (SNP) associated with seed dormancy. By crossing and pyramiding the mutant phenotypes, we will be able to turn V. stipulacea into a crop which is yet primitive but can be cultivated without pesticides..
11. Kazuhiro Ishibashi, Masayasu Saruta, Takehiko Shimizu, Miao Shu, Toyoaki Anai, Kunihiko Komatsu, Naohiro Yamada, Yuichi Katayose, Masayuki Ishikawa, Masao Ishimoto, Akito Kaga, Soybean antiviral immunity conferred by dsRNase targets the viral replication complex, Nature Communications, 10.1038/s41467-019-12052-5, 10, 1, 2019.12, Eukaryotic positive-strand RNA viruses replicate their genomes in membranous compartments formed in a host cell, which sequesters the dsRNA replication intermediate from antiviral immune surveillance. Here, we find that soybean has developed a way to overcome this sequestration. We report the positional cloning of the broad-spectrum soybean mosaic virus resistance gene Rsv4, which encodes an RNase H family protein with dsRNA-degrading activity. An active-site mutant of Rsv4 is incapable of inhibiting virus multiplication and is associated with an active viral RNA polymerase complex in infected cells. These results suggest that Rsv4 enters the viral replication compartment and degrades viral dsRNA. Inspired by this model, we design three plant-gene-derived dsRNases that can inhibit the multiplication of the respective target viruses. These findings suggest a method for developing crops resistant to any target positive-strand RNA virus by fusion of endogenous host genes..
12. Md. Abdur Rauf Sarkar, Satoshi Watanabe, Akihiro Suzuki, Fumio Hashimoto, Toyoaki Anai, Identification of novel MYB transcription factors involved in the isoflavone biosynthetic pathway by using the combination screening system with agroinfiltration and hairy root transformation, PLANT BIOTECHNOLOGY, 10.5511/plantbiotechnology.19.1025a, 36, 4, 241-251, 2019.12.
13. Kyoko Takagi, Ryoichi Yano, Saeko Tochigi, Yukiko Fujisawa, Hiroki Tsuchinaga, Yuya Takahashi, Yoshitake Takada, Akito Kaga, Toyoaki Anai, Chigen Tsukamoto, Hikaru Seki, Toshiya Muranaka, Masao Ishimoto, Genetic and functional characterization of Sg-4 glycosyltransferase involved in the formation of sugar chain structure at the C-3 position of soybean saponins, Phytochemistry, 10.1016/j.phytochem.2018.09.002, 156, 96-105, 2018.12, Triterpenoid saponins are specialized metabolites, which are abundant in soybean seeds. They have a wide variety of effects on human health and physiology. The composition of sugar chain attached to the aglycone moiety of saponins can be controlled by genetic loci, such as Sg-1, 3, and 4. Among these, the homozygous recessive sg-4 impairs the accumulation of saponins that have an arabinose moiety at the second position of the C-3 sugar chain (i.e., saponins Ad and βa) in the hypocotyls. In this study, we found that sg-4 cultivars are disabled in Glyma.01G046300 expression in hypocotyls. This gene encodes a putative glycosyltransferase (UGT73P10) and is a homolog of GmSGT2 (UGT73P2) whose recombinant protein has been previously shown, in vitro, to conjugate the second galactose moiety at the C-3 position of soyasapogenol B monoglucuronide (SBMG). The sg-4 phenotype (absence of saponins Ad and βa in hypocotyls) was restored by introducing the Glyma.01G046300 genomic DNA fragment that was obtained from the Sg-4 cultivar ‘Ibarakimame 7’. Although Glyma.01G046300 is expressed in the cotyledons even in the sg-4 cultivars such as ‘Enrei’ the induced premature stop codon mutation (W244*) resulted in impaired accumulation of saponin βa in this tissue also in the ‘Enrei’ genetic background. Furthermore, the recombinant Glyma.01G046300 protein was shown to conjugate the second Ara moiety at the C-3 position of SBMG using UDP-Ara as a sugar donor. These results demonstrate that Sg-4 is responsible for conjugation of the second Ara moiety at the C-3 position of soybean saponins..
14. Md. Harun-Ur-Rashid, Hironori Iwasaki, Shahanaz Parveen, Shigeki Oogai, Masakazu Fukuta, Md Amzad Hossain, Toyoaki Anai, Hirosuke Oku, Cytosolic Cysteine Synthase Switch Cysteine and Mimosine Production in Leucaena leucocephala, Applied Biochemistry and Biotechnology, 10.1007/s12010-018-2745-z, 186, 3, 613-632, 2018.11, In higher plants, multiple copies of the cysteine synthase gene are present for cysteine biosynthesis. Some of these genes also have the potential to produce various kinds of β-substitute alanine. In the present study, we cloned a 1275-bp cDNA for cytosolic O-acetylserine(thiol)lyase (cysteine synthase) (Cy-OASTL) from Leucaena leucocephala. The purified protein product showed a dual function of cysteine and mimosine synthesis. Kinetics studies showed pH optima of 7.5 and 8.0, while temperature optima of 40 and 35 °C, respectively, for cysteine and mimosine synthesis. The kinetic parameters such as apparent Km, kcat were determined for both cysteine and mimosine synthesis with substrates O-acetylserine (OAS) and Na2S or 3-hydroxy-4-pyridone (3H4P). From the in vitro results with the common substrate OAS, the apparent kcat for Cys production is over sixfold higher than mimosine synthesis and the apparent Km is 3.7 times lower, suggesting Cys synthesis is the favored pathway..
15. Ryoichi Yano, Kyoko Takagi, Saeko Tochigi, Yukiko Fujisawa, Yuhta Nomura, Hiroki Tsuchinaga, Yuya Takahashi, Yoshitake Takada, Akito Kaga, Toyoaki Anai, Chigen Tsukamoto, Hikaru Seki, Toshiya Muranaka, Masao Ishimoto, Isolation and Characterization of the Soybean Sg-3 Gene that is Involved in Genetic Variation in Sugar Chain Composition at the C-3 Position in Soyasaponins, Plant and Cell Physiology, 10.1093/pcp/pcy019, 59, 4, 792-805, 2018.04, Soyasaponins are specialized metabolites present in soybean seeds that affect the taste and quality of soy-based foods. The composition of the sugar chains attached to the aglycone moiety of soyasaponins is regulated by genetic loci such as sg-1, sg-3 and sg-4. Here, we report the cloning and characterization of the Sg-3 gene, which is responsible for conjugating the terminal (third) glucose (Glc) at the C-3 sugar chain of soyasaponins. The gene Glyma.10G104700 is disabled in the sg-3 cultivar, € Mikuriya-ao', due to the deletion of genomic DNA that results in the absence of a terminal Glc residue on the C-3 sugar chain. Sg-3 encodes a putative glycosyltransferase (UGT91H9), and its predicted protein sequence has a high homology with that of the product of GmSGT3 (Glyma.08G181000; UGT91H4), which conjugates rhamnose (Rha) to the third position of the C-3 sugar chain in vitro. A recombinant Glyma.10G104700 protein could utilize UDP-Glc as a substrate to conjugate the third Glc to the C-3 sugar chain, and introducing a functional Glyma.10G104700 transgene into the mutant complemented the sg-3 phenotype. Conversely, induction of a premature stop codon mutation in Glyma.10G104700 (W270∗) resulted in the sg-3 phenotype, suggesting that Glyma.10G104700 was Sg-3. The gmsgt3 (R339H) mutant failed to accumulate soyasaponins with the third Rha at the C-3 sugar chain, and the third Glc and Rha conjugations were both disabled in the sg-3 gmsgt3 double mutant. These results demonstrated that Sg-3 and GmSGT3 are non-redundantly involved in conjugation of the third Glc and Rha at the C-3 sugar chain of soyasaponins, respectively..
16. Yano, Ryoichi, Takagi, Kyoko, Saeko, Tochigi, Fujisawa, Yukiko, Nomura, Yuhta, Tsuchinaga, Hiroki, Takahashi, Yuya, Takada, Yoshitake, Kaga, Akito, Anai, Toyoaki, Tsukamoto, Chigen, Seki, Hikaru, Muranaka, Toshiya, Ishimoto, Masao, Isolation and characterization of the soybean Sg-3 gene that is involved in genetic variation in sugar chain composition at the C-3 position in soyasaponins, Plant and Cell Physiology, 10.1093/pcp/pcy019, 59, 4, 792-805, 2018.04.
17. Constantine Busungu, Satoru Taura, Jun Ichi Sakagami, Toyoaki Anai, Katsuyuki Ichitani, High-resolution mapping and characterization of xa42, a resistance gene against multiple xanthomonas oryzae pv. Oryzae races in rice (oryza sativa l.), Breeding Science, 10.1270/jsbbs.17094, 68, 2, 188-199, 2018.04, Improvement of resistance against rice bacterial blight (BB) disease is an important breeding strategy in breeding programs across the world, especially in Africa and southern Asia where BB is more prevalent. This report describes a high-resolution map and characterization of xa42 at XA42 locus, a rice BB resistance gene in XM14, a mutant line originating from IR24. The candidate gene region was narrowed down from 582 kb, which had been obtained in our previous study, to 57 kb. XM14 shows brown spots in its leaves like lesion mimic mutants. This line also shows a shorter stature than the original cultivar IR24. In XA42 gene segregating populations, homozygotes of xa42 allele were consistently resistant to the six Japanese Xanthomonas oryzae pv. oryzae races used for this study. They also showed brown spots and markedly short stature compared with the other genotypes, suggesting that xa42 gene exhibits pleiotropic effects..
18. Md Harun Ur Rashid, Hironori Iwasaki, Shigeki Oogai, Masakazu Fukuta, Shahanaz Parveen, Md Amzad Hossain, Toyoaki Anai, Hirosuke Oku, Molecular characterization of cytosolic cysteine synthase in Mimosa pudica, Journal of Plant Research, 10.1007/s10265-017-0986-5, 131, 2, 319-329, 2018.03, In the cysteine and mimosine biosynthesis process, O-acetyl-l-serine (OAS) is the common substrate. In the presence of O-acetylserine (thiol) lyase (OASTL, cysteine synthase) the reaction of OAS with sulfide produces cysteine, while with 3-hydroxy-4-pyridone (3H4P) produces mimosine. The enzyme OASTL can either catalyze Cys synthesis or both Cys and mimosine. A cDNA for cytosolic OASTL was cloned from M. pudica for the first time containing 1,410 bp nucleotides. The purified protein product from overexpressed bacterial cells produced Cys only, but not mimosine, indicating it is Cys specific. Kinetic studies revealed that pH and temperature optima for Cys production were 6.5 and 50 °C, respectively. The measured Km, Kcat, and Kcat Km−1 values were 159 ± 21 µM, 33.56 s−1, and 211.07 mM−1s−1 for OAS and 252 ± 25 µM, 32.99 s−1, and 130.91 mM−1s−1 for Na2S according to the in vitro Cys assay. The Cy-OASTL of Mimosa pudica is specific to Cys production, although it contains sensory roles in sulfur assimilation and the reduction network in the intracellular environment of M. pudica..
19. Masafumi Kadowaki, Yuki Fujimaru, Seiga Taguchi, Jannatul Ferdouse, Kazutaka Sawada, Yuta Kimura, Yohei Terasawa, Gennaro Agrimi, Toyoaki Anai, Hideki Noguchi, Atsushi Toyoda, Asao Fujiyama, Takeshi Akao, Hiroshi Kitagaki, Chromosomal Aneuploidy Improves the Brewing Characteristics of Sake Yeast, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 10.1128/AEM.01620-17, 83, 24, e01620, 2017.12, The effect of chromosomal aneuploidy on the brewing characteristics of brewery yeasts has not been studied. Here we report that chromosomal aneuploidy in sake brewery yeast (Saccharomyces cerevisiae) leads to the development of favorable brewing characteristics. We found that pyruvate-underproducing sake yeast, which produces less off-flavor diacetyl, is aneuploid and trisomic for chromosomes XI and XIV. To confirm that this phenotype is due to aneuploidy, we obtained 45 haploids with various chromosomal additions and investigated their brewing profiles. A greater number of chromosomes correlated with a decrease in pyruvate production. Especially, sake yeast haploids with extra chromosomes in addition to chromosome XI produced less pyruvate than euploids. Mitochondrion-related metabolites and intracellular oxygen species in chromosome XI aneuploids were higher than those in euploids, and this effect was canceled in their "petite" strains, suggesting that an increase in chromosomes upregulated mitochondrial activity and decreased pyruvate levels. These findings suggested that an increase in chromosome number, including chromosome XI, in sake yeast haploids leads to pyruvate underproduction through the augmentation of mitochondrial activity. This is the first report proposing that aneuploidy in brewery yeasts improves their brewing profile.IMPORTANCE Chromosomal aneuploidy has not been evaluated in development of sake brewing yeast strains. This study shows the relationship between chromosomal aneuploidy and brewing characteristics of brewery yeast strains. High concentrations of pyruvate during sake storage give rise to alpha-acetolactate and, in turn, to high concentrations of diacetyl, which is considered an off-flavor. It was demonstrated that pyruvate-underproducing sake yeast is trisomic for chromosome XI and XIV. Furthermore, sake yeast haploids with extra chromosomes produced reduced levels of pyruvate and showed metabolic processes characteristic of increased mitochondrial activity. This novel discovery will enable the selection of favorable brewery yeasts by monitoring the copy numbers of specific chromosomes through a process that does not involve generation/use of genetically modified organisms..
20. Masafumi Kadowaki, Yuki Fujimaru, Seiga Taguchi, Jannatul Ferdouse, Kazutaka Sawada, Yuta Kimura, Yohei Terasawa, Gennaro Agrimi, Toyoaki Anai, Hideki Noguchi, Atsushi Toyod, Asao Fujiyama, Takeshi Akao, Hiroshi Kitagaki, Chromosomal aneuploidy improves the brewing characteristics of sake yeast, Applied and Environmental Microbiology, 10.1128/AEM.01620-17, 83, 24, 2017.12, The effect of chromosomal aneuploidy on the brewing characteristics of brewery yeasts has not been studied. Here we report that chromosomal aneuploidy in sake brewery yeast (Saccharomyces cerevisiae) leads to the development of favorable brewing characteristics. We found that pyruvate-underproducing sake yeast, which produces less off-flavor diacetyl, is aneuploid and trisomic for chromosomes XI and XIV. To confirm that this phenotype is due to aneuploidy, we obtained 45 haploids with various chromosomal additions and investigated their brewing profiles. A greater number of chromosomes correlated with a decrease in pyruvate production. Especially, sake yeast haploids with extra chromosomes in addition to chromosome XI produced less pyruvate than euploids. Mitochondrion-related metabolites and intracellular oxygen species in chromosome XI aneuploids were higher than those in euploids, and this effect was canceled in their "petite" strains, suggesting that an increase in chromosomes upregulated mitochondrial activity and decreased pyruvate levels. These findings suggested that an increase in chromosome number, including chromosome XI, in sake yeast haploids leads to pyruvate underproduction through the augmentation of mitochondrial activity. This is the first report proposing that aneuploidy in brewery yeasts improves their brewing profile..
21. Watanabe S, Tsukamoto C, Oshita T, Yamada T, Anai T, Kaga A, Identification of quantitative trait loci for owering time by a combination of restriction site–associated DNA sequencing and bulked segregant analysis in soybean, Breeding Science, 10.1270/jsbbs.17013, 67, 3, 277-285, 2017.04.
22. Takashi Sayama, Takanari Tanabata, Masayasu Saruta, Testsuya Yamada, Toyoaki Anai, Akito Kaga, Masao Ishimoto, Confirmation of the pleiotropic control of leaflet shape and number of seeds per pod by the Ln gene in induced soybean mutants, Breeding Science, 10.1270/jsbbs.16201, 67, 4, 363-369, 2017.03, Most soybean cultivars possess broad leaflets; however, a recessive allele on the Ln locus is known to cause the alteration of broad to narrow leaflets. The recessive allele ln has also been considered to increase the number of seeds per pod (NSP) and has the potential to improve yield. Recently, Gm-JAG1 (Glyma20g25000), a gene controlling Ln, has been shown to complement leaf shape and silique length in Arabidopsis mutants. However, whether Gm-JAG1 is responsible for those traits in soybean is not yet known. In this study, we investigated the pleiotropic effect of soybean Ln gene on leaflet shape and NSP by using two independent soybean Gm-jag1 mutants and four ln near isogenic lines (NILs). The leaflet shape was evaluated using a leaf image analysis software, SmartLeaf, which was customized from SmartGrain. The leaflets of both the Gmjag1 mutants were longer and narrower than those of the wild-type plants. Interestingly, the image analysis results clarified that the perimeter of the mutant leaflets did not change, although their leaflet area decreased. Furthermore, one mutant line with narrow leaflets showed significantly higher NSP than that in the wild (or Ln) genotype, indicating that soybean Ln gene pleiotropically controls leaflet shape and NSP..
23. Ryoichi Yano, Kyoko Takagi, Yoshitake Takada, Kyosuke Mukaiyama, Chigen Tsukamoto, Takashi Sayama, Akito Kaga, Toyoaki Anai, Satoru Sawai, Kiyoshi Ohyama, Kazuki Saito, Masao Ishimoto, Metabolic switching of astringent and beneficial triterpenoid saponins in soybean is achieved by a loss-of-function mutation in cytochrome P450 72A69, Plant Journal, 10.1111/tpj.13403, 89, 3, 527-539, 2017.02, Triterpenoid saponins are major components of secondary metabolites in soybean seeds and are divided into two groups: group A saponins, and 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) saponins. The aglycone moiety of group A saponins consists of soyasapogenol A (SA), which is an oxidized β-amyrin product, and the aglycone moiety of the DDMP saponins consists of soyasapogenol B (SB). Group A saponins produce a bitter and astringent aftertaste in soy products, whereas DDMP saponins have known health benefits for humans. We completed map-based cloning and characterization of the gene Sg-5, which is responsible for SA biosynthesis. The naturally occurring sg-5 mutant lacks group A saponins and has a loss-of-function mutation (L164*) in Glyma15g39090, which encodes the cytochrome P450 enzyme, CYP72A69. An enzyme assay indicated the hydroxylase activity of recombinant CYP72A69 against SB, which also suggested the production of SA. Additionally, induced Glyma15g39090 mutants (R44* or S348P) lacked group A saponins similar to the sg-5 mutant, indicating that Glyma15g39090 corresponds to Sg-5. Endogenous levels of DDMP saponins were higher in the sg-5 mutant than in the wild-type lines due to the loss of the enzyme activity that converts SB to SA. Interestingly, the genomes of palaeopolyploid soybean and the closely related common bean carry multiple Sg-5 paralogs in a genomic region syntenic to the soybean Sg-5 region. However, SA did not accumulate in common bean samples, suggesting that Sg-5 activity evolved after gene duplication event(s). Our results demonstrate that metabolic switching of undesirable saponins with beneficial saponins can be achieved in soybean by disabling Sg-5..
24. Ryoichi Yano, Kyoko Takagi, Yoshitake Takada, Kyosuke Mukaiyama, Chigen Tsukamoto, Takashi Sayama, Akito Kaga, Toyoaki Anai, Satoru Sawai, Kiyoshi Ohyama, Kazuki Saito, Masao Ishimoto, Metabolic switching of astringent and beneficial triterpenoid saponins in soybean is achieved by a loss-of-function mutation in cytochrome P450 72A69, PLANT JOURNAL, 10.1111/tpj.13403, 89, 3, 527-539, 2017.02, Triterpenoid saponins are major components of secondary metabolites in soybean seeds and are divided into two groups: group A saponins, and 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) saponins. The aglycone moiety of group A saponins consists of soyasapogenol A (SA), which is an oxidized -amyrin product, and the aglycone moiety of the DDMP saponins consists of soyasapogenol B (SB). Group A saponins produce a bitter and astringent aftertaste in soy products, whereas DDMP saponins have known health benefits for humans. We completed map-based cloning and characterization of the gene Sg-5, which is responsible for SA biosynthesis. The naturally occurring sg-5 mutant lacks group A saponins and has a loss-of-function mutation (L164*) in Glyma15g39090, which encodes the cytochrome P450 enzyme, CYP72A69. An enzyme assay indicated the hydroxylase activity of recombinant CYP72A69 against SB, which also suggested the production of SA. Additionally, induced Glyma15g39090 mutants (R44* or S348P) lacked group A saponins similar to the sg-5 mutant, indicating that Glyma15g39090 corresponds to Sg-5. Endogenous levels of DDMP saponins were higher in the sg-5 mutant than in the wild-type lines due to the loss of the enzyme activity that converts SB to SA. Interestingly, the genomes of palaeopolyploid soybean and the closely related common bean carry multiple Sg-5 paralogs in a genomic region syntenic to the soybean Sg-5 region. However, SA did not accumulate in common bean samples, suggesting that Sg-5 activity evolved after gene duplication event(s). Our results demonstrate that metabolic switching of undesirable saponins with beneficial saponins can be achieved in soybean by disabling Sg-5..
25. 有用作物の作成.
26. Yuta Kimura, Mari Naeshiro, Yuri Tominaga, Toyoaki Anai, Fuminori Komai, Metabolite composition of grapefruit (Citrus paradisi) grown in Japan depends on the growing environment and harvest period, Horticulture Journal, 10.2503/hortj.MI-139, 86, 4, 543-551, 2017.01, ‘Sagan-Ruby’ is the first grapefruit (Citrus paradisi) cultivar to be developed in Japan and is used for food, cosmetics, and other purposes owing to its favorable characteristics, such as the absence of harmful pesticides and its long shelf life. The desired qualities of grapefruit depend on the specific use, and these qualities are influenced by the metabolite composition of the fruits. However, little is known about the influence of the growing environment or harvest period on the metabolite composition of the ‘Sagan-Ruby’ grapefruit. Therefore, we harvested fruits that were grown either in a plastic house without artificial heating or outdoors with rain cover from December, 2014 to April, 2015, on a monthly basis, and we investigated the composition of the primary metabolites such as sugars, organic acids, and amino acids, in the juice and peel of the fruit using gas chromatography mass spectrometry (GC/MS). We detected a total of 53 and 68 compounds in the juice and peel, respectively, and the first and second components of the principal component analyses of the detected metabolites of both juice and peel were associated with the growing environment and harvest period, respectively. Since we observed that glucose, fructose, sucrose, and citric acid were more concentrated in the juice of outdoor-grown fruits than in that of the house-grown fruits, especially in March and April, it is likely that the sweetness and acidity of the fruits are dependent on the growing environment. Similarly, the primary metabolite contents, including succinic acid and other organic acids, were higher in peels from outdoor-grown fruits. In addition, we also observed that the contents of proline, phenylalanine, and other amino acids in the juice increased continuously from December to April, and many sugars, including glucose and fructose, gradually decreased in peels from December to February and were lower from February to April. These results indicated that quality of the ‘Sagan-Ruby’ grapefruit varies with the harvest period..
27. Development and utilization of soybean mutant resources..
28. Yan Fan, Di Shaokang, Murai Yoshinori, Iwashina Tsukasa, Anai Toyoaki, Takahashi Ryoji, New Allelic Variant Discovered at Soybean Flower Color Locus W1 Encoding Flavonoid 3′5′-hydroxylase., Crop Science, 10.2135/cropsci2015.07.0413, 56, 4, 1506-1513, 2016.07.
29. Toyoaki Anai, Mutant-based reverse genetics for functional genomics of non-model crops, Advances in Plant Breeding Strategies: Breeding, Biotechnology and Molecular Tools, 10.1007/978-3-319-22521-0_16, 1, 473-487, 2016.01, In the past decade, innovations in high-throughput sequencing (nextgeneration sequencing) technologies have accelerated whole-genome sequencing of various non-model crop species. Taking advantage of huge polymorphic sequence data provided by the results of whole-genome sequencing, we can easily develop novel molecular markers. It may boost the use of forward genetics approach to isolating the corresponding genes for QTLs in non-model crops. Furthermore, this forward genetics approach is a steady and robust method but it is still difficult to increase its throughput. The sequenced genes have been annotated on the basis of sequence similarity
however, the functions of most genes (and the resulting phenotypes) are still obscure. Although we can easily obtain multiple crop genomic sequences from public databases, it is necessary to increase the throughput of functional genomics. Reverse genetics, which uses mutants or transgenic lines for the genes of interest, is an attractive approach to determine gene function. Mutant-based reverse genetics has several advantages over transgene-based reverse genetics: (a) its higher throughput, (b) the absence of restrictions for growing non-transgenic mutants in the field and (c) the possibility to use the mutants directly for traditional cross-breeding programs as valuable genetic resources of non-model crops. This chapter describes recent advances in functional genomics research on non-model crops, with a focus on mutant-based reverse genetics approaches..
30. Dong Cao, Ying Li, Sijia Lu, Jialin Wang, Haiyang Nan, Xiaoming Li, Danning Shi, Chao Fang, Hong Zhai, Xiaohui Yuan, Toyoaki Anai, Zhengjun Xia, Baohui Liu, Fanjiang Kong, GmCOL1a and GmCOL1b Function as Flowering Repressors in Soybean Under Long-Day Conditions, PLANT AND CELL PHYSIOLOGY, 10.1093/pcp/pcv152, 56, 12, 2409-2422, 2015.12, CONSTANS (CO) has a central role in the photoperiod response mechanism in Arabidopsis. However, the functions of legume CO genes in controlling flowering remain unknown. Here, we analyze the expression patterns of E1, E2 and GmCOL1a/1b using near-isogenic lines (NILs), and we further analyze flowering-related genes in gmcol1b mutants and GmCOL1a-overexpressing plants. Our data showed that both E3 and E4 up-regulate E1 expression, with the effect of E3 on E1 being greater than the effect of E4 on E1. E2 was up-regulated by E3 and E4 but down-regulated by E1. GmCOL1a/1b were up-regulated by E1, E2, E3 and E4. Although the spatial and temporal patterns of GmCOL1a/1b expression were more similar to those of AtCOL2 than to those of AtCO, gmcol1b mutants flowered earlier than wild-type plants under long-day (LD) conditions, and the overexpression of GmCOL1a caused late flowering under LD or natural conditions. In addition, GmFT2a/5a, E1 and E2 were down-regulated in GmCOL1a-overexpressing plants under LD conditions. Because E1/2 influences the expression of GmCOL1a, and vice versa, we conclude that these genes may function as part of a negative feedback loop, and GmCOL1a/b genes may serve as suppressors in photoperiodic flowering in soybean under LD conditions..
31. Mai Tsuda, Akito Kaga, Toyoaki Anai, Takehiko Shimizu, Takashi Sayama, Kyoko Takagi, Kayo Machita, Satoshi Watanabe, Minoru Nishimura, Naohiro Yamada, Satomi Mori, Harumi Sasaki, Hiroyuki Kanamori, Yuichi Katayose, Masao Ishimoto, Construction of a high-density mutant library in soybean and development of a mutant retrieval method using amplicon sequencing, BMC GENOMICS, 10.1186/s12864-015-2079-y, 16, 1014, 2015.11, Background: Functions of most genes predicted in the soybean genome have not been clarified. A mutant library with a high mutation density would be helpful for functional studies and for identification of novel alleles useful for breeding. Development of cost-effective and high-throughput protocols using next generation sequencing (NGS) technologies is expected to simplify the retrieval of mutants with mutations in genes of interest.
Results: To increase the mutation density, seeds of the Japanese elite soybean cultivar Enrei were treated with the chemical mutagen ethyl methanesulfonate (EMS); M2 seeds produced by M1 plants were treated with EMS once again. The resultant library, which consisted of DNA and seeds from 1536 plants, revealed large morphological and physiological variations. Based on whole-genome re-sequencing analysis of 12 mutant lines, the average number of base changes was 12,796 per line. On average, 691 and 35 per line were missense and nonsense mutations, respectively. Two screening strategies for high resolution melting (HRM) analysis and indexed amplicon sequencing were designed to retrieve the mutants; the mutations were confirmed by Sanger sequencing as the final step. In comparison with HRM screening of several genes, indexed amplicon sequencing allows one to scan a longer sequence range and skip screening steps and to know the sequence information of mutation because it uses systematic DNA pooling and the index of NGS reads, which simplifies the discovery of mutants with amino acid substitutions.
Conclusions: A soybean mutant library with a high mutation density was developed. A high mutation density (1 mutation/74 kb) was achieved by repeating the EMS treatment. The mutation density of our library is sufficiently high to obtain a plant in which a gene is nonsense mutated. Thus, our mutant library and the indexed amplicon sequencing will be useful for functional studies of soybean genes and have a potential to yield useful mutant alleles for soybean breeding..
32. Maki Nagata, Naoya Yamamoto, Tamaki Shigeyama, Yohei Terasawa, Toyoaki Anai, Tatsuya Sakai, Sayaka Inada, Susumu Arima, Masatsugu Hashiguchi, Ryo Akashi, Hideyuki Nakayama, Daisuke Ueno, Ann M. Hirsch, Akihiro Suzuki, Red/Far Red Light Controls Arbuscular Mycorrhizal Colonization via Jasmonic Acid and Strigolactone Signaling, PLANT AND CELL PHYSIOLOGY, 10.1093/pcp/pcv135, 56, 11, 2100-2109, 2015.11, Establishment of a nitrogen-fixing symbiosis between legumes and rhizobia not only requires sufficient photosynthate, but also the sensing of the ratio of red to far red (R/FR) light. Here, we show that R/FR light sensing also positively influences the arbuscular mycorrhizal (AM) symbiosis of a legume and a non-legume through jasmonic acid (JA) and strigolactone (SL) signaling. The level of AM colonization in high R/FR light-grown tomato and Lotus japonicus significantly increased compared with that determined for low R/FR light-grown plants. Transcripts for JA-related genes were also elevated under high R/FR conditions. The root exudates derived from high R/FR light-grown plants contained more (+)-5-deoxystrigol, an AM-fungal hyphal branching inducer, than those from low R/FR light-grown plants. In summary, high R/FR light changes not only the levels of JA and SL synthesis, but also the composition of plant root exudates released into the rhizosphere, in this way augmenting the AM symbiosis..
33. Hoshino T, Watanabe S, Takagi Y, Anai T, A novel GmFAD3-2a mutant allele developed through TILLING reduces α-linolenic acid content in soybean seed oil, Breeding Science, 10.1270/jsbbs.64.371, 64, 4, 371-377, 2014.12.
34. Fan Yan, Shaokang Di, Felipe Rojas Rodas, Tito Rodriguez Torrico, Yoshinori Murai, Tsukasa Iwashina, Toyoaki Anai, Ryoji Takahashi, Allelic variation of soybean flower color gene W4 encoding dihydroflavonol 4-reductase 2, BMC PLANT BIOLOGY, 10.1186/1471-2229-14-58, 14, 58, 2014.03, Background: Flower color of soybean is primarily controlled by six genes, viz., W1, W2, W3, W4, Wm and Wp. This study was conducted to investigate the genetic and chemical basis of newly-identified flower color variants including two soybean mutant lines, 222-A-3 (near white flower) and E30-D-1 (light purple flower), a near-isogenic line (Clark-w4), flower color variants (T321 and T369) descended from the w4-mutable line and kw4 (near white flower, Glycine soja).
Results: Complementation tests revealed that the flower color of 222-A-3 and kw4 was controlled by the recessive allele (w4) of the W4 locus encoding dihydroflavonol 4-reductase 2 (DFR2). In 222-A-3, a single base was deleted in the first exon resulting in a truncated polypeptide consisting of 24 amino acids. In Clark-w4, base substitution of the first nucleotide of the fourth intron abolished the 5' splice site, resulting in the retention of the intron. The DFR2 gene of kw4 was not expressed. The above results suggest that complete loss-of-function of DFR2 gene leads to near white flowers. Light purple flower of E30-D-1 was controlled by a new allele at the W4 locus, w4-lp. The gene symbol was approved by the Soybean Genetics Committee. In E30-D-1, a single-base substitution changed an amino acid at position 39 from arginine to histidine. Pale flowers of T369 had higher expression levels of the DFR2 gene. These flower petals contained unique dihydroflavonols that have not yet been reported to occur in soybean and G. soja.
Conclusions: Complete loss-of-function of DFR2 gene leads to near white flowers. A new allele of the W4 locus, w4-lp regulates light purple flowers. Single amino acid substitution was associated with light purple flowers. Flower petals of T369 had higher levels of DFR2 gene expression and contained unique dihydroflavonols that are absent in soybean and G. soja. Thus, mutants of the DFR2 gene have unique flavonoid compositions and display a wide variety of flower color patterns in soybean, from near white, light purple, dilute purple to pale..
35. Fan Yan, Shaokang Di, Felipe Rojas Rodas, Tito Rodriguez Torrico, Yoshinori Murai, Tsukasa Iwashina, Toyoaki Anai, Ryoji Takahashi, Allelic variation of soybean flower color gene W4 encoding dihydroflavonol 4-reductase 2, BMC PLANT BIOLOGY, 10.1186/1471-2229-14-58, 14, 58, 2014.03, Background: Flower color of soybean is primarily controlled by six genes, viz., W1, W2, W3, W4, Wm and Wp. This study was conducted to investigate the genetic and chemical basis of newly-identified flower color variants including two soybean mutant lines, 222-A-3 (near white flower) and E30-D-1 (light purple flower), a near-isogenic line (Clark-w4), flower color variants (T321 and T369) descended from the w4-mutable line and kw4 (near white flower, Glycine soja).
Results: Complementation tests revealed that the flower color of 222-A-3 and kw4 was controlled by the recessive allele (w4) of the W4 locus encoding dihydroflavonol 4-reductase 2 (DFR2). In 222-A-3, a single base was deleted in the first exon resulting in a truncated polypeptide consisting of 24 amino acids. In Clark-w4, base substitution of the first nucleotide of the fourth intron abolished the 5' splice site, resulting in the retention of the intron. The DFR2 gene of kw4 was not expressed. The above results suggest that complete loss-of-function of DFR2 gene leads to near white flowers. Light purple flower of E30-D-1 was controlled by a new allele at the W4 locus, w4-lp. The gene symbol was approved by the Soybean Genetics Committee. In E30-D-1, a single-base substitution changed an amino acid at position 39 from arginine to histidine. Pale flowers of T369 had higher expression levels of the DFR2 gene. These flower petals contained unique dihydroflavonols that have not yet been reported to occur in soybean and G. soja.
Conclusions: Complete loss-of-function of DFR2 gene leads to near white flowers. A new allele of the W4 locus, w4-lp regulates light purple flowers. Single amino acid substitution was associated with light purple flowers. Flower petals of T369 had higher levels of DFR2 gene expression and contained unique dihydroflavonols that are absent in soybean and G. soja. Thus, mutants of the DFR2 gene have unique flavonoid compositions and display a wide variety of flower color patterns in soybean, from near white, light purple, dilute purple to pale..
36. Yohei Terasawa, Kanenori Takata, Toyoaki Anai, Tatsuya M. Ikeda, Identification and distribution of Puroindoline b-2 variant gene homologs in Hordeum, GENETICA, 10.1007/s10709-013-9735-4, 141, 7-9, 359-368, 2013.09, The barley hordoindoline genes (Hina and Hinb) are homologous to the wheat puroindoline genes (Pina and Pinb). These genes are involved in grain hardness, which is an important quality for barley processing. We identified novel variants of Hina and Hinb in 10 wild Hordeum species (H. bogdanii, H. brachyantherum, H. bulbosum, H. chilense, H. comosum, H. marinum, H. murinum, H. patagonicum, H. pusillum, and H. roshevitzii) covering all Hordeum genomes and preliminarily named them Hinc. These nucleotide sequences were highly similar to those of Puroindoline b-2 variant genes (Pinb-2v) and were located on chromosome 7I in H. chilense. The Hinc genes in H. bogdanii, H. bulbosum, H. patagonicum, and H. roshevitzii were pseudogenes possessing in-frame stop codons. We also found a partial Hinc sequence in H. murinum. This gene was not found in cultivated barley and H. vulgare subsp. spontaneum. The phylogenetic tree of Gsp-1, Hin, and Pin genes demonstrates that Hinc and Pinb-2v genes formed one cluster. Therefore, we considered that Hinc and Pinb-2v genes shared a common ancestral gene and were homologous to each other. We also studied the evolutional process of Gsp-1, Hin, and Pin genes. Our results suggested that Gsp-1 might be the most closely related to a putative ancestral gene on Ha locus..
37. Mycorrhizal infection is controlled by the light quality and quantity in higher plants.
38. ダイズ変異体ライブラリーの作出と利用.
39. ダイズ突然変異体リソースの開発と利用.
40. Zhengjun Xia, Satoshi Watanabe, Tetsuya Yamada, Yasutaka Tsubokura, Hiroko Nakashima, Hong Zhai, Toyoaki Anai, Shusei Sato, Toshimasa Yamazaki, Shixiang Lu, Hongyan Wu, Satoshi Tabata, Kyuya Harada, Positional cloning and characterization reveal the molecular basis for soybean maturity locus E1 that regulates photoperiodic flowering, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.1117982109, 109, 32, E2155-E2164, 2012.08, The complex and coordinated regulation of flowering has high ecological and agricultural significance. The maturity locus E1 has a large impact on flowering time in soybean, but the molecular basis for the E1 locus is largely unknown. Through positional cloning, we delimited the E1 locus to a 17.4-kb region containing an intron-free gene (E1). The E1 protein contains a putative bipartite nuclear localization signal and a region distantly related to B3 domain. In the recessive allele, a nonsynonymous substitution occurred in the putative nuclear localization signal, leading to the loss of localization specificity of the E1 protein and earlier flowering. The early-flowering phenotype was consistently observed in three ethylmethanesulfonate-induced mutants and two natural mutations that harbored a premature stop codon or a deletion of the entire E1 gene. E1 expression was significantly suppressed under short-day conditions and showed a bimodal diurnal pattern under long-day conditions, suggesting its response to photoperiod and its dominant effect induced by long day length. When a functional E1 gene was transformed into the early-flowering cultivar Kariyutaka with low E1 expression, transgenic plants carrying exogenous E1 displayed late flowering. Furthermore, the transcript abundance of E1 was negatively correlated with that of GmFT2a and GmFT5a, homologues of FLOWERING LOCUS T that promote flowering. These findings demonstrated the key role of E1 in repressing flowering and delaying maturity in soybean. The molecular identification of the maturity locus E1 will contribute to our understanding of the molecular mechanisms by which a short-day plant regulates flowering time and maturity..
41. NBRPミヤコグサ・ダイズにおけるダイズリソースの概要について.
42. ANAI T., Potential of a mutant-based reverse genetic approach for functional genomics and molecular breeding in soybean, Breed. Sci., 61, 1, 462-467, 2012.01.
43. Hashiguchi Masatsugu, Abe Jun, Aoki Toshio, ANAI Toyoaki, SUZUKI Akihiro, AKASHI Ryo, The National BioResource Project (NBRP) Lotus and Glycine in Japan, Breeding science, 61, 1, 453-461, 2012.01.
44. TILLING法によるダイズ青色光受容体CRY1突然変異体の単離.
45. TILLING法によるダイズGmPhyA1突然変異体の単離とその開花に及ぼす影響.
46. 大豆品種「トヨシロメ」に見出された高ショ糖遺伝子の特性.
47. Tomoki Hoshino, Nobuhisa Kawashita, Yutaka Takagi, Toyoaki Anai, Molecular characterization and marker development of mid-oleic-acid mutant M23 for the development of high-oleic cultivars of soybean, PLANT BREEDING, 10.1111/j.1439-0523.2011.01871.x, 130, 5, 544-550, 2011.10, The mid-oleic-acid soybean mutant M23 has been frequently used to develop lines with elevated oleic acid content. M23, which was produced by X-ray irradiation, was previously shown to contain a large deletion including GmFAD2-1a, one of the genes encoding microsomal omega-6 fatty acid desaturase (GmFAD2). However, it was not possible then to develop a codominant marker for the selection of the M23 mutation because the extent of the deletion was unknown. Here, we report that PCR analysis of M23 using published soybean genome sequences as a reference revealed a 164 014-bp deletion including GmFAD2-1a and nineteen other predicted genes. We generated a codominant PCR-based marker that can distinguish wild-type/M23 heterozygotes from both homozygous classes. We genotyped F(2) plants segregating for the M23 mutation and confirmed the association of genotype and phenotype. This information will be useful in developing new commercial cultivars of high-oleic-acid soybean. We discuss the possible functions of the genes in the deleted region of M23 in relation to reduced seed yield, which occurs frequently in soybean lines containing the M23 mutation..
48. Tomoki Hoshino, Nobuhisa Kawashita, Yutaka Takagi, Toyoaki Anai, Molecular characterization and marker development of mid-oleic-acid mutant M23 for the development of high-oleic cultivars of soybean, PLANT BREEDING, 10.1111/j.1439-0523.2011.01871.x, 130, 5, 544-550, 2011.10, The mid-oleic-acid soybean mutant M23 has been frequently used to develop lines with elevated oleic acid content. M23, which was produced by X-ray irradiation, was previously shown to contain a large deletion including GmFAD2-1a, one of the genes encoding microsomal omega-6 fatty acid desaturase (GmFAD2). However, it was not possible then to develop a codominant marker for the selection of the M23 mutation because the extent of the deletion was unknown. Here, we report that PCR analysis of M23 using published soybean genome sequences as a reference revealed a 164 014-bp deletion including GmFAD2-1a and nineteen other predicted genes. We generated a codominant PCR-based marker that can distinguish wild-type/M23 heterozygotes from both homozygous classes. We genotyped F(2) plants segregating for the M23 mutation and confirmed the association of genotype and phenotype. This information will be useful in developing new commercial cultivars of high-oleic-acid soybean. We discuss the possible functions of the genes in the deleted region of M23 in relation to reduced seed yield, which occurs frequently in soybean lines containing the M23 mutation..
49. 植物生理化学研究と遺伝子解析法.
50. 烏骨鶏と白色レグホーンにおけるプロラクチン遺伝子領域周辺の連鎖不平衡ブロックについて.
51. TILLING法に利用できるダイズ突然変異体リソース.
52. ダイズ品種「トヨシロメ」に見出された高ショ糖遺伝子の特性.
53. Satoshi Watanabe, Zhengjun Xia, Rumiko Hideshima, Yasutaka Tsubokura, Shusei Sato, Naoki Yamanaka, Ryoji Takahashi, Toyoaki Anai, Satoshi Tabata, Keisuke Kitamura, Kyuya Harada, A Map-Based Cloning Strategy Employing a Residual Heterozygous Line Reveals that the GIGANTEA Gene Is Involved in Soybean Maturity and Flowering, GENETICS, 10.1534/genetics.110.125062, 188, 2, 395-U260, 2011.06, Flowering is indicative of the transition from vegetative to reproductive phase, a critical event in the life cycle of plants. In soybean (Glycine max), a flowering quantitative trait locus, FT2, corresponding to the maturity locus E2, was detected in recombinant inbred lines (RILs) derived from the varieties "Misuzudaizu" (ft2/ft2; JP28856) and "Moshidou Gong 503" (FT2/FT2; JP27603). A map-based cloning strategy using the progeny of a residual heterozygous line (RHL) from the RIL was employed to isolate the gene responsible for this quantitative trait locus. A GIGANTEA ortholog, GmGIa (Glyma10g36600), was identified as a candidate gene. A common premature stop codon at the 10th exon was present in the Misuzudaizu allele and in other near isogenic lines (NILs) originating from Harosoy (e2/e2; PI548573). Furthermore, a mutant line harboring another premature stop codon showed an earlier flowering phenotype than the original variety, Bay (E2/E2; PI553043). The e2/e2 genotype exhibited elevated expression of GmFT2a, one of the florigen genes that leads to early flowering. The effects of the E2 allele on flowering time were similar among NILs and constant under high (43 degrees N) and middle (36 degrees N) latitudinal regions in Japan. These results indicate that GmGIa is the gene responsible for the E2 locus and that a null mutation in GmGIa may contribute to the geographic adaptation of soybean..
54. Toyoaki Anai, Tomoki Hoshino, Naoko Imai, Yutaka Takagi, Molecular characterization of two high-palmitic-acid mutant loci induced by X-ray irradiation in soybean, BREEDING SCIENCE, 10.1270/jsbbs.61.631, 61, 5, 631-638, 2011.01, Palmitic acid is the most abundant (approx. 11% of total fatty acids) saturated fatty acid in conventional soybean seed oil. Increasing the saturated acid content of soybean oil improves its oxidative stability and plasticity. We have developed three soybean mutants with high palmitic acid content by X-ray irradiation. In this study, we successfully identified the mutated sites of two of these high-palmitic-acid mutants, J10 and M22. PCR-based mutant analysis revealed that J10 has a 206,203-bp-long deletion that includes the GmKASIIA gene and 16 other predicted genes, and M22 has a 26-bp-long deletion in the sixth intron of GmKASIIB. The small deletion in M22 causes mis-splicing of GmKASIIB transcripts, which should result in nonfunctional products. In addition, we designed co-dominant marker sets for these mutant alleles and confirmed the association of genotypes and palmitic acid contents in F-2 seeds of J10 X M22. This information will be useful in breeding programs to develop novel soybean cultivars with improved palmitic acid content. However, in the third mutant, KK7, we found no polymorphism in either GmKASIIA or GmKASIIB, which suggests that several unknown genes in addition to GmKASIIA and GmKASIIB may be involved in elevating the palmitic acid content of soybean seed oil..
55. Toyoaki Anai, Potential of a mutant-based reverse genetic approach for functional genomics and molecular breeding in soybean, BREEDING SCIENCE, 10.1270/jsbbs.61.462, 61, 5, 462-467, 2011.01, Mutant-based reverse genetics offers a powerful way to create novel mutant alleles at a selected locus. This approach makes it possible to directly identify plants that carry a specific modified gene from the nucleotide sequence data. Soybean [Glycine max (L.) Merr.] has a highly redundant paleopolyploid genome (approx. 1.1 Gb), which was completely sequenced in 2010. Using reverse genetics to support functional genomics studies designed to predict gene function would accelerate post-genomics research in soybean. Furthermore, the novel mutant alleles created by this approach would be useful genetic resources for improving various traits in soybean. A reverse genetic screening platform in soybean has been developed that combines more than 40,000 mutant lines with a high-throughput method, Targeting Local Lesions IN Genome (TILLING). In this review, the mutant-based reverse genetic approach based on this platform is described, and the likely evolution of this approach in the near future..
56. Masatsugu Hashiguchi, Jun Abe, Toshio Aoki, Toyoaki Anai, Akihiro Suzuki, Ryo Akashi, The National BioResource Project (NBRP) Lotus and Glycine in Japan, BREEDING SCIENCE, 10.1270/jsbbs.61.453, 61, 5, 453-461, 2011.01, The objective of the National BioResource Project (NBRP) in Japan is to collect, conserve and distribute biological materials for life sciences research. The project consists of twenty-eight bioresources, including animal, plant, microorganism and DNA resources. NBRP Lotus and Glycine aims to support the development of legume research through the collection, conservation, and distribution of these bioresources. Lotus japonicus is a perennial legume that grows naturally throughout Japan and is widely used as a model plant for legumes because of such advantages as its small genome size and short life cycle. Soybean (Glycine max) has been cultivated as an important crop since ancient times, and numerous research programs have generated a large amount of basic research information and valuable bioresources for this crop. We have also developed a "LegumeBase" a specialized database for the genera Lotus and Glycine, and are maintaining this database as a part of the NBRP. In this paper we will provide an overview of the resources available from the NBRP Lotus and Glycine database site, called "LegumeBase"..
57. Tomoki Hoshino, Yutaka Takagi, Toyoaki Anai, Novel GmFAD2-1b mutant alleles created by reverse genetics induce marked elevation of oleic acid content in soybean seeds in combination with GmFAD2-1a mutant alleles, BREEDING SCIENCE, 10.1270/jsbbs.60.419, 60, 4, 419-425, 2010.12, The generation of useful mutant alleles of specific genes would accelerate conventional breeding programs in various commercially important crops. Common soybean oil is easily oxidized because it is rich in polyunsaturated fatty acids (PUFAs). Microsomal omega-6 fatty acid desaturase (FAD2), which introduces a second unsaturated bond into oleic acid, is a primary target for elevating oleic acid levels and reducing PUFA levels. The paleopolyploid soybean genome contains five FAD2 gene homologues, at least three of which (GmFAD2-1a, 2-1b, and 2-2a) are functional. In spite of their importance, very little genetic variation has been identified in these genes except in GmFAD2-1a, because fatty acid content is easily affected by environmental conditions such as temperature. Here we isolated novel mutant alleles of GmFAD2-1b from ethyl methanesulfonate-treated soybean mutant populations through Targeting Induced Local Lesions In Genomes (TILLING), a reverse genetic method. Evaluation of enzyme activity in a yeast heterologous expression system suggested that two mutant lines, 'B12' and 'E11', contain near-null and null alleles, respectively, of GmFAD2-1b. Furthermore, by combining GmFAD2-1a and GmFAD2-1b mutant alleles, we successfully generated soybean lines with > 80% oleic acid content. TILLING could provide a practical method for expanding the genetic diversity of polyploid crops..
58. Tshering Penjor, Toyoaki Anai, Yukio Nagano, Ryoji Matsumoto, Masashi Yamamoto, Phylogenetic relationships of Citrus and its relatives based on rbcL gene sequences, TREE GENETICS & GENOMES, 10.1007/s11295-010-0302-1, 6, 6, 931-939, 2010.12, We sequenced the rbcL genes of 64 accessions from 24 genera of Citrus relatives and analyzed them by neighbor-joining and maximum parsimony methods. Both trees supported Swingle and Reece's (1967) treatment of the subfamily Aurantioideae as monophyletic. However, the trees did not support Swingle and Reece's treatment of tribes and subtribes. The subgenera Citrus and Papeda were not clustered clearly. The analysis associated the Fortunella group with mandarin, Poncirus with Citrus ichangensis, Severinia buxifolia with Atalantia ceylanica, Microcitrus with Eremocitrus and Citrus micrantha, and Hesperethusa crenulata with Citropsis. Furthermore, Atalantia species showed polytomy. The classification of Swingle and Reece should be reviewed..
59. TILLING法を用いたダイズ品種「フクユタカ」からのFT3変異体の単離.
60. X線照射によって誘発されたダイズ脂肪酸突然変異体におけるDNA変異形態の特徴.
61. ダイズの矮性突然変異遺伝子についての解析.
62. Hidenobu Uchida, Hirofumi Yamashita, Toyoaki Anai, Toshiya Muranaka, Kanji Ohyama, Agrobacterium-Mediated Transformation of Euphorbia tirucalli Callus, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 10.1271/bbb.90783, 74, 4, 851-853, 2010.04, In order to establish a basis for transformation technology in the petroleum plant Euphorbia the callus of the plant was infected with Agrobacterium, washed with distilled water, sterilized with distilled water containing 100 mg/l of carbenicillin, selected on solidified B5 medium containing 13 mg/l of G418 and 100 mg/l of carbenicillin. and then on solidified B5 medium containing 25 mg/l of G418 and 100 mg/l of carbenicillin for the transgenic calli, and then the callus lines were subcultured successively on solidified B5 medium containing 50 mg/l of G418. We performed PCR analysis of sterilized G418-resistant callus line DNA and concluded that the gene introduced was integrated into the callus genome..
63. ダイズ突然変異体ライブラリーの増幅を目的としたMultiple Displacement Amplification(MDA)法の最適化.
64. MDA法によって増幅を行ったダイズ突然変異体ライブラリーを鋳型としたTILLING法による変異の検出.
65. ダイズ種子イソフラボン含量の改変を目指したUDP-グルコース:イソフラボン7-O-グルコシルトランスフェラーゼ突然変異体のスクリーニング.
66. 新規ダイズ突然変異体リソースとTILLINGによる変異体スクリーニング.
67. TILLING法を用いたダイズ(Glycine max)種子脂肪酸組成の代謝工学的改変.
68. MDA法によって増幅を行ったダイズ突然変異体ライブラリーを鋳型としたTILLING法による変異の検出.
69. TILLING法によるダイズ突然変異体ライブラリーからのGmFAD3-2a変異体の単離.
70. TILLING法による超高オレイン酸ダイズ突然変異系統の開発.
71. Screening and evaluation of nodulation mutant of soybean.
72. ダイズ開花期関連遺伝子座FT2のマップベースクローニング.
73. Satoshi Watanabe, Rumiko Hideshima, Zhengjun Xia, Yasutaka Tsubokura, Shusei Sato, Yumi Nakamoto, Naoki Yamanaka, Ryoji Takahashi, Masao Ishimoto, Toyoaki Anai, Satoshi Tabata, Kyuya Harada, Map-Based Cloning of the Gene Associated With the Soybean Maturity Locus E3, GENETICS, 10.1534/genetics.108.098772, 182, 4, 1251-1262, 2009.08, Photosensitivity plays an essential role in the response of plants to their changing environments throughout life cycle. In soybean [Glycine max (L.) Merrill], several associations between photosensitivity and maturity loci are known, but only limited information at the molecular level is available. The FT3 locus is one of the quantitative trait loci (QTL) for flowering time that corresponds to the maturity locus E3. To identify the gene responsible for this QTL, a map-based cloning strategy was undertaken. One phytochrome A gene (GmPhyA3) was considered a strong candidate for the FT3 locus. Allelism tests and gene sequence comparisions showed that alleles of Misuzudaizu (FT3/FT3; JP28856) and L62-667 (e3/e3; PI547716) showed weak or complete loss of function, respectively. High red/far-red (R/FR) long-day conditions enhanced the effects of the E3/FT3 alleles in various genetic backgrounds. Moreover, a mutant line harboring the nonfunctional GmPhyA3 flowered earlier than the original Bay (E3/E3; PI553043) under similar conditions. These results suggest that the variation in phytochrome A may contribute to the complex systems of soybean flowering response and geogrphic adaptation..
74. ダイズ油脂中のオレイン酸含量を増加させる突然変異体及びその原因遺伝子.
75. ダイズ遺伝資源中に見出されたFAD2遺伝子ファミリーの多様性.
76. 突然変異体を活用したダイズ油脂組成の代謝工学的改良.
77. Taishi Umezawa, Tetsuya Sakurai, Yasushi Totoki, Atsushi Toyoda, Motoaki Seki, Atsushi Ishiwata, Kenji Akiyama, Atsushi Kurotani, Takuhiro Yoshida, Keiichi Mochida, Mie Kasuga, Daisuke Todaka, Kyonoshin Maruyama, Kazuo Nakashima, Akiko Enju, Saho Mizukado, Selina Ahmed, Kyoko Yoshiwara, Kyuya Harada, Yasutaka Tsubokura, Masaki Hayashi, Shusei Sato, Toyoaki Anai, Masao Ishimoto, Hideyuki Funatsuki, Masayoshi Teraishi, Mitsuru Osaki, Takuro Shinano, Ryo Akashi, Yoshiyuki Sakaki, Kazuko Yamaguchi-Shinozaki, Kazuo Shinozaki, Sequencing and Analysis of Approximately 40 000 Soybean cDNA Clones from a Full-Length-Enriched cDNA Library, DNA RESEARCH, 10.1093/dnares/dsn024, 15, 6, 333-346, 2008.12, A large collection of full-length cDNAs is essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. We obtained a total of 39 936 soybean cDNA clones (GMFL01 and GMFL02 clone sets) in a full-length-enriched cDNA library which was constructed from soybean plants that were grown under various developmental and environmental conditions. Sequencing from 5' and 3' ends of the clones generated 68 661 expressed sequence tags (ESTs). The EST sequences were clustered into 22 674 scaffolds involving 2580 full-length sequences. In addition, we sequenced 4712 full-length cDNAs. After removing overlaps, we obtained 6570 new full-length sequences of soybean cDNAs so far. Our data indicated that 87.7% of the soybean cDNA clones contain complete coding sequences in addition to 5'- and 3'-untranslated regions. All of the obtained data confirmed that our collection of soybean full-length cDNAs covers a wide variety of genes. Comparative analysis between the derived sequences from soybean and Arabidopsis, rice or other legumes data revealed that some specific genes were involved in our collection and a large art of them could be annotated to unknown functions. A large set of soybean full-length cDNA clones reported in this study will serve as a useful resource for gene discovery from soybean and will also aid a precise annotation of the soybean genome..
78. Taishi Umezawa, Tetsuya Sakurai, Yasushi Totoki, Atsushi Toyoda, Motoaki Seki, Atsushi Ishiwata, Kenji Akiyama, Atsushi Kurotani, Takuhiro Yoshida, Keiichi Mochida, Mie Kasuga, Daisuke Todaka, Kyonoshin Maruyama, Kazuo Nakashima, Akiko Enju, Saho Mizukado, Selina Ahmed, Kyoko Yoshiwara, Kyuya Harada, Yasutaka Tsubokura, Masaki Hayashi, Shusei Sato, Toyoaki Anai, Masao Ishimoto, Hideyuki Funatsuki, Masayoshi Teraishi, Mitsuru Osaki, Takuro Shinano, Ryo Akashi, Yoshiyuki Sakaki, Kazuko Yamaguchi-Shinozaki, Kazuo Shinozaki, Sequencing and Analysis of Approximately 40 000 Soybean cDNA Clones from a Full-Length-Enriched cDNA Library, DNA RESEARCH, 10.1093/dnares/dsn024, 15, 6, 333-346, 2008.12, A large collection of full-length cDNAs is essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. We obtained a total of 39 936 soybean cDNA clones (GMFL01 and GMFL02 clone sets) in a full-length-enriched cDNA library which was constructed from soybean plants that were grown under various developmental and environmental conditions. Sequencing from 5' and 3' ends of the clones generated 68 661 expressed sequence tags (ESTs). The EST sequences were clustered into 22 674 scaffolds involving 2580 full-length sequences. In addition, we sequenced 4712 full-length cDNAs. After removing overlaps, we obtained 6570 new full-length sequences of soybean cDNAs so far. Our data indicated that 87.7% of the soybean cDNA clones contain complete coding sequences in addition to 5'- and 3'-untranslated regions. All of the obtained data confirmed that our collection of soybean full-length cDNAs covers a wide variety of genes. Comparative analysis between the derived sequences from soybean and Arabidopsis, rice or other legumes data revealed that some specific genes were involved in our collection and a large art of them could be annotated to unknown functions. A large set of soybean full-length cDNA clones reported in this study will serve as a useful resource for gene discovery from soybean and will also aid a precise annotation of the soybean genome..
79. Toyoaki Anai, Tomoko Yamada, Rumiko Hideshima, Takehito Kinoshita, Shaikh M. Rahman, Yutaka Takagi, Two high-oleic-acid soybean mutants, M23 and KK21, have disrupted microsomal omega-6 fatty acid desaturase, encoded by GmFAD2-1a, BREEDING SCIENCE, 58, 4, 447-452, 2008.12, Elevating the oleic acid content of soybean (Glycine max (L.) Merr.) is a Major focus or breeding programs. Previously, we created two high-oleic-acid soybean Mutants, M23 and KK21, by X-irradiation. We expected them to have modifications in genes encoding microsomal omega-6 fatty acid desaturase. The objectives of this study were to evaluate which members of the GmFAD2 gene family contribute to oleic acid production during seed maturation, to characterize the mutant genes, and to establish molecular markers for breeding of high-oleic-acid soybeans. Three GmFAD2 genes were expressed in developing seeds; the gene products of GmFAD2-1a and GmFAD2-1b were more active than that of GmFAD2-2a during seed development. We identified different nucleotide modifications in GmFAD2-1a in M23 and KK21. Using nuclease-cleaved DNA fragment-length polymorphisms, we developed a novel molecular marker to distinguish between KK21 mutant and wild-type alleles. This information Could be Useful for improving soybean oil quality by using the mutant genes from M23 or KK21, and for screening novel high-oleic-acid soybean mutants..
80. Toyoaki Anai, Tomoko Yamada, Rumiko Hideshima, Takehito Kinoshita, Shaikh M. Rahman, Yutaka Takagi, Two high-oleic-acid soybean mutants, M23 and KK21, have disrupted microsomal omega-6 fatty acid desaturase, encoded by GmFAD2-1a, BREEDING SCIENCE, 10.1270/jsbbs.58.447, 58, 4, 447-452, 2008.12, Elevating the oleic acid content of soybean (Glycine max (L.) Merr.) is a Major focus or breeding programs. Previously, we created two high-oleic-acid soybean Mutants, M23 and KK21, by X-irradiation. We expected them to have modifications in genes encoding microsomal omega-6 fatty acid desaturase. The objectives of this study were to evaluate which members of the GmFAD2 gene family contribute to oleic acid production during seed maturation, to characterize the mutant genes, and to establish molecular markers for breeding of high-oleic-acid soybeans. Three GmFAD2 genes were expressed in developing seeds; the gene products of GmFAD2-1a and GmFAD2-1b were more active than that of GmFAD2-2a during seed development. We identified different nucleotide modifications in GmFAD2-1a in M23 and KK21. Using nuclease-cleaved DNA fragment-length polymorphisms, we developed a novel molecular marker to distinguish between KK21 mutant and wild-type alleles. This information Could be Useful for improving soybean oil quality by using the mutant genes from M23 or KK21, and for screening novel high-oleic-acid soybean mutants..
81. MDA法によって増幅したDNAからの変異の検出.
82. TILLING法を用いたダイズミュータントライブラリーからのFT3突然変異系統の単離.
83. 突然変異体を利用したダイズ貯蔵脂質の改良.
84. TILLING法によるダイズ突然変異体ライブラリーのスクリーニング.
85. ダイズ開花期関連遺伝子座FT3のマップベースクローニング.
86. 大豆 オレリッチ50.
87. Zhengjun Xia, Yasutaka Tsubokura, Masako Hoshi, Masayoshi Hanawa, Chizuru Yano, Kayo Okamura, Talaat A. Ahmed, Toyoaki Anai, Satoshi Watanabe, Masaki Hayashi, Takashi Kawai, Khwaja G. Hossain, Hirokazu Masaki, Kazumi Asai, Naoki Yamanaka, Nakao Kubo, Koh-ichi Kadowaki, Yoshiaki Nagamura, Masahiro Yano, Takuji Sasaki, Kyuya Harada, An integrated high-density linkage map of soybean with RFLP, SSR, STS, and AFLP markers using a single F-2 population, DNA RESEARCH, 10.1093/dnares/dsm027, 14, 6, 257-269, 2007.12, Soybean [Glycine max (L.) Merrill] is the most important leguminous crop in the world due to its high contents of high-quality protein and oil for human and animal consumption as well as for industrial uses. An accurate and saturated genetic linkage map of soybean is an essential tool for studies on modern soybean genomics. In order to update the linkage map of a F-2 population derived from a cross between Misuzudaizu and Moshidou Gong 503 and to make it more informative and useful to the soybean genome research community, a total of 318 AFLP, 121 SSR, 108 RFLP, and 126 STS markers were newly developed and integrated into the framework of the previously described linkage map. The updated genetic map is composed of 509 RFLP, 318 SSR, 318 AFLP, 97 AFLP-derived STS, 29 BAC-end or EST-derived STS, 1 RAPD, and five morphological markers, covering a map distance of 3080 cM (Kosambi function) in 20 linkage groups (LGs). To our knowledge, this is presently the densest linkage map developed from a single F2 population in soybean. The average intermarker distance was reduced to 2.41 from 5.78 cM in the earlier version of the linkage map. Most SSR and RFLP markers were relatively evenly distributed among different LGs in contrast to the moderately clustered AFLP markers. The number of gaps of more than 25 cM was reduced to 6 from 19 in the earlier version of the linkage map. The coverage of the linkage map was extended since 17 markers were mapped beyond the distal ends of the previous linkage map. In particular, 17 markers were tagged in a 5.7 cM interval between CE47M5a and Satt100 on LG C2, where several important QTLs were clustered. This newly updated soybean linkage map will enable to streamline positional cloning of agronomically important trait locus genes, and promote the development of physical maps, genome sequencing, and other genomic research activities..
88. Yukio Nagano, Syoko Takao, Takahiro Kudo, Ei'ichi Iizasa, Toyoaki Anai, Yeast-based recombineering of DNA fragments into plant transformation vectors by one-step transformation, PLANT CELL REPORTS, 26, 12, 2111-2117, 2007.12, T-DNA binary vectors are often used in plant transformation experiments. Because they are usually very large and have few restriction sites suitable for DNA ligation reactions, cloning DNA fragments into these vectors is difficult. We provide herein an alternative to cloning DNA fragments into very large vectors. Our yeast-based recombineering method enables DNA fragments to be cloned into certain types of T-DNA binary vectors by one-step transformation without the requirement of specific recombination sites or precisely positioned restriction ends, thus making the cloning process more flexible. Moreover, this method is inexpensive and is applicable to multifragment cloning..
89. X-線照射によるダイズ突然変異体ライブラリーの開発.
90. 組換え型CEL1エンドヌクレアーゼの発現.
91. Tissue cultures and production of secondary metabolites in Hemiphragma heterophyllum.
Shoot culture of Hemiphragma heterophyllum (Scrophulariaceae) was established for the first time. Six known compounds, plantamajoside, hemiphroside B, 2', 6"-O-diacetylplantamajoside, globularin, iso-scrophurarioside and cinnamic acid were isolated from the shoot tissues of H. heterophyllum. 2', 6"-O-diacetylplantamajoside, which was isolated from this genius for the first time, is important chemotaxonomic marker suggesting the relationship between Hemiphragma and Wulfenia. Hairy root culture of H. heterophyllum was also established. In the hairy root cultures, high concentration of plantamajoside and hemiphroside B was observed. A phenolic compound p-tyrosol, which was not detected in the parent plantlets, was also isolated from the hairy roots. H. heterophyllum has a potential to become a new useful material for functional secondary metabolites in the field of food ingredients..
92. T Anai, T Yamada, T Kinoshita, SM Rahman, Y Takagi, Identification of corresponding genes for three low-alpha-linolenic acid mutants and elucidation of their contribution to fatty acid biosynthesis in soybean seed, PLANT SCIENCE, 10.1016/j.plantsci.2005.02.016, 168, 6, 1615-1623, 2005.06, In order to keep the quality of vegetable oil stable, it is important to reduce the a.-linolenic acid content. Previously, we developed three low-alpha-linonenic acid soybean mutants, 'J18', 'M5', and 'M24'. In this study, we obtained and characterized four cDNAs, GmFAD3-1a, GmFAD3-1b, GmFAD3-2a, and GniFAD3-2b, encoding microsomal omega-3 fatty acid desaturase from soybean developing seeds. The GmFAD3-1b transcript does not accumulate in the J18' mutant, whereas 'M5' and 'M24' contain a 19-base pair and a single-base pair deletion in the coding region of the GmFAD3-1b and GmFAD3-1a genes, respectively. Furthermore, heterologous expression of these two mutant genes in Saccharomyces cerevisiae revealed that the COOH-terminal region of both mutant gene products is essential for their enzymatic activity. In this report, we also discuss the contribution of each microsomal omega-3 fatty acid desaturase gene for a-linolenic acid biosynthesis in soybean seeds. (c) 2005 Elsevier Ireland Ltd. All rights reserved..
93. SM Rahman, T Anai, T Kinoshita, S Arima, Y Takagi, Three novel soybean germplasms with unique fatty acid composition using multiple mutant alleles, BREEDING SCIENCE, 54, 3, 225-229, 2004.09, Three soybean [Glycine max (L.) Merr.] germplasms were previously developed for unique fatty acid content: LPKKC-3 has fap1 and sop1 alleles for reduced palmitic acid, HPKKC-7 has fap2 and fapx alleles for elevated palmitic acid and DHL has the ol allele for elevated oleic acid, and fan and fanx(a) alleles for reduced linolenic acid. If these loci are independently inherited, soybean germplasms with useful combinations of these fatty acids could be developed that would result in a novel oil quality of great value for the food industry. Crosses were made between DHL and LPKKC-3, and DHL and HPKKC-7. The data from F-2 seed and F-2 progeny of DHL X LPKKC-3 indicated that the loci controlling reduced palmitic acid (fap1 and sop1) were independently inherited from the locus controlling elevated oleic acid (ol), and the loci controlling reduced linolenic acid (fan and fanx(a)). Thus, the germplasm (LPDHL) with 4.0% palmitic, 51.0% oleic and 2.9% linolenic acids was easily developed. The data from F-2 seeds and F-2 progeny of DHL X HPKKC-7 indicated that the loci controlling elevated palmitic acid (fap2 and fapx), and reduced linolenic acid (fan and fanx(a)) were independently inherited. The locus controlling elevated oleic acid (ol) was also distinctly segregated but the contents of oleic acid were found to be reduced due to the presence of loci for elevated palmitic acid. Thus, one germplasm (HPLL) with 22.5% palmitic acid and 2.7% linolenic acid, and another germplasm (MHPDHL) with 17.1% palmitic acid, 41.8% oleic and 2.9% linolenic acids were developed..
94. T Anai, M Koga, H Tanaka, T Kinoshita, SM Rahman, Y Takagi, Improvement of rice (Oryza sativa L.) seed oil quality through introduction of a soybean microsomal omega-3 fatty acid desaturase gene, PLANT CELL REPORTS, 10.1007/s00299-003-0609-6, 21, 10, 988-992, 2003.06, Microsomal omega-3 fatty acid desaturase is an essential enzyme in the production of the n-3 polyunsaturated fatty acid alpha-linolenic acid during the seed developing stage. We have constructed a chimeric gene consisting of a maize Ubi1-P-int and a soybean GmFAD3 cDNA, which was introduced into rice plants by Agrobacterium-mediated transformation. Ten transformants containing the chimeric gene were established and expression subsequently confirmed by Northern blotting. Furthermore, alpha-linolenic acid content of the T-1 seeds increased dramatically up to tenfold that of the control, and this phenotype was also stably inherited in the T-2 and T-3 progenies. These results demonstrate that the alpha-linolenic acid content of rice seed oil can easily be altered using the combination of a high-activity promoter and a GmFAD3 gene..
95. SM Rahman, T Anai, T Kinoshita, Y Takagi, A novel soybean germplasm with elevated saturated fatty acids, CROP SCIENCE, 43, 2, 527-531, 2003.03, Two soybean [Glycine mar (L.) Merr.] germplasm lines have been identified for unique fatty acid content. The very high content of palmitic acid in HPKKJ10 is controlled by the fap2 and fapx loci, and the very high content of stearic acid in M25 is controlled by the st(2) locus. If the fap2 andfapx loci are independently inherited from the st(2) locus, soybean germplasm could be developed with more unique and useful combinations of these fatty acids. The objectives of this study were to combine the loci of high palmitic and stearic acids, and determine the effects of altered contents of palmitic and stearic acids on other fatty acids. HPKKJ10 was reciprocally crossed to M25. The data from F-2 seed indicated that the fap2 and fapx loci controlling high palmitic acid were independently inherited from the st(2) locus controlling high stearic acid. Thus, the germplasm (HPS) with the very high palmitic acid trait from HPKKJ10, and very high stearic acid trait from M25, was easily developed. The increases in palmitic acid due to the fap2 andfapx loci in HPKKJ10, and the increases in stearic acid due to the st(2) locus in M25 were individually associated with changes in oleic acid. The combined increases in these two fatty acids in HPS were associated with decreases in oleic and linoleic acids, and increases in linolenic acid. The development of HPS with high content of saturated fatty acids could open new markets for soybean oil..
96. To Explore the Gibberellic Acid-regulated Genes in Rice Seedlings.
97. 7.1 生理活性物質(分子・構造・作用・受容体・分布・容量など).
98. SM Rahman, T Kinoshita, T Anai, Y Takagi, Combining ability in loci for high oleic and low linolenic acids in soybean, CROP SCIENCE, 10.2135/cropsci2001.41126x, 41, 1, 26-29, 2001.01, Two soybean [Glycine mar (L.) Merr.] germplasm lines have been identified for unique fatty acrid content. The contents of oleic and linolenic acids in HOLL are controlled by ol and fan led, respectively, and the very low content of linolenic add in LOLL is controlled by fan and fanx(a) loci combined. The fan locus is identical for both HOLL and LOLL, Therefore, if ol and fanx(a) loci are independent, soybean germplasm could be developed with more unique and useful combinations of these fatty acids. The objectives of this study were to combine the loci of high oleic and low linolenic acids, and determine the effects of altered contents of oleic and Linolenic acids on other fatty acids. HOLL was reciprocally crossed to LOLL. The data from S seed indicated that the ol locus controlling high oleic acid was independently inherited from the fanx(a) locus controlling low linolenic acid, Thus, the germplasm (DHL) with the high oleic acid trait from MOLL, and very low linolenic acid trait from LOLL, was easily developed. The increases in oleic acid due to ol was associated completely with changes in Linoleic acids, indicating a pleiotropic effect of the ol locus. Decreases in linolenic acid due to fan and fanx(a) were associated primarily with increases in linoleic acid. The development of DHL with increased contents of oleic acid and decreased contents of polyunsaturated fatty acids could open new markets for soybean oil..
99. H Uchida, L Suzuki, T Anai, K Doi, H Takano, H Yamashita, T Oka, S Kawano, KI Tomizawa, T Kawazu, H Kuroiwa, T Kuroiwa, A pair of invertedly repeated genes in Chlamydomonas reinhardtii encodes a zygote-specific protein whose expression is UV-sensitive, CURRENT GENETICS, 36, 4, 232-240, 1999.10, Uniparental inheritance of the chloroplast genome has been observed in a wide variety of green plants. In Chlamydomonas this phenomenon, which can be selectively inhibited by UV irradiation of mrt gametes, has been shown cytologically to be due to the preferential degradation of mt(-)-derived chloroplast nucleoids in young zygotes. The zygote-specific pair of zys1 genes, zys1A and zys1B, is expressed earliest among five genes isolated from a "10-min" zygote library. We report here that the ZYS1 protein, which is encoded by the invertedly duplicated zys1 gene, accumulates in zygotes and is localized in nuclei. In addition, when mtf gametes (but not mt(-) gametes) are UV-irradiated before mating, only very limited accumulation of ZYS1 protein can be detected in the resulting zygotes..
100. SM Rahman, T Kinoshita, T Anai, Y Takagi, Genetic relationships between loci for palmitate contents in soybean mutants, JOURNAL OF HEREDITY, 90, 3, 423-428, 1999.05.
101. Y Takagi, SM Rahman, T Anai, SK Wasala, T Kinoshita, M Khalekuzzaman, Development of a reduced linolenate soy mutant by Re-irradiation and its genetic analysis, BREEDING SCIENCE, 49, 1, 1-5, 1999.03, A high content of linolenic acid leads to reduction of keeping quality and frying stability of cooking oil. The present study was conducted to obtain a further reduction of linolenic acid in soybean [Glycine max (L.) Merr.] oil by re-irradiation of M-5 and determine its genetic system. M-5 is a mutant with 4.5% linolenic acid content, derived from the cultivar Bay (8.0% linolenic acid). A seed lot from line M-5 was treated with X-ray irradiation and M-2 plants were obtained from randomly selected seeds of M-1 plants. The M-2 plants were screened for reduced linolenic acid. One plant was found with 3.0% linolenic acid content and was named MS382. The M-3 and M-4 generations of this line proved that the character was fixed and significantly lower than the M-5 control. For inheritance studies, MS382 was reciprocally crossed with M-5 (fan) and LOLL [fanfanx(a), a recombinant of M-5 (fan) x M24 (fanx(a))]. The F-2 segregation ratio and the segregation of F-3 seeds from F-2 plants of MS382 x M-5 indicated that reduced linolenic acid in MS382 was conbindly controlled by fan (M-5) and an additional gene. To determine if this additional gene was similar with the fanx(a) gene in LOLL, F-2 seeds and F-3 seeds from each F-2 plant of MS382 x LOLL were evaluated. No transgressive segregation for linolenic acid was found in this cross, indicating the genes for reduced linolenic acid content in MS382 and LOLL were identical. However, the mutant MS382 was developed by reirradiation which indicates the practicability of this technique to develop new gene for further reduction of linolenic acid in soybean oil..
102. D Shibata, M Seki, N Mitsukawa, N Hayashida, T Ito, T Taji, T Tsuge, M Matsui, T Anai, YG Liu, RF Whittier, K Shinozaki, Establishment of framework P1 clones for map-based cloning and genome sequencing: direct RFLP mapping of large clones, GENE, 10.1016/S0378-1119(98)00534-4, 225, 1-2, 31-38, 1998.12, Large insert capacity, clone stability and convenient propagation in Escherichia coli have made bacterial artificial chromosome and phage P1 vector-based libraries the first choice for large-scale sequencing projects, and these libraries have also proven useful for chromosome walking. The application of these libraries for either purpose is greatly facilitated by the establishment of a set of framework clones distributed across the genome. Using a Pi-based library of Arabidopsis thaliana with genomic inserts of 70-90 kb (Liu, Y.-G., Mitsukawa, N., Vazquez-Tello, A., Whittler, R.F., 1995. Generation of a high-quality P1 library of Arabidopsis suitable for chromosome walking. Plant J. 7, 351-358), we have now established such a set of framework clones. To date, such clones have usually been identified by hybridization to smaller, previously mapped clones that detect restriction fragment length polymorphisms (RFLPs). In order to establish framework clones more efficiently, we refined protocols for PI clone DNA isolation and RFLP detection in order to employ whole P1 clones directly as probes. This strategy enabled a very high rate of RFLP detection, and obviated the need to screen the P1 library with smaller RFLP probes. Altogether 95 clones were mapped providing a framework into which further clones can be integrated by physical overlap. (C) 1998 Elsevier Science B.V. All rights reserved..
103. T Kinoshita, SM Rahman, T Anai, Y Takagi, Inter-locus relationship between genes controlling palmitic acid contents in soybean mutants, BREEDING SCIENCE, 48, 4, 377-381, 1998.12, Palmitic acid is one of the two major saturated fatty acids of soybean [Glycine max (L.) Merr.] oil that is closely related with physical, chemical and nutritional qualities, The average palmitic acid content in the seed oil of common cultivars is 11.0%. Soybean mutants with both reduced and elevated palmitic acid have been developed. Previous studies had shown that reduced palmitic acid in a mutant C1726 and elevated palmitic acid in a mutant C1727 were respectively controlled by fap1 and fap2 alleles, and reduced palmitic acid in a mutant J3 was controlled by sop1 allele. The objective of this study is to determine the genetic relationships of sop1 allele in J3 with fap1 and fap2 alleles in C1726 and C1727, respectively, Reciprocal crosses were conducted for J3 x Bay, C1726 x Bay, J3 x C1727 and J3 x C1726, No maternal effect for palmitic acid content was observed from the analysis of reciprocal F-1 seeds in any of the crosses The data for palmitic acid content in F-2 seeds of J3 x Bay and C1726 x Bay fitted a 1:2:1 ratio. The segregation observed from the analysis of F-2 seeds of J3 x C1727 fitted the ratio of 1:14:1 and of J3 x C1726 fitted the ratio of 3:10:3. The F-2 segregation ratio and the segregation of F-3 seeds from F-2 plants of these crosses indicated that allele for palmitic acid in J3 was at a different locus from the alleles in C1726 and C1727. The segregant with the genotype sop1sop1fap1fap1 from J3 : C1726 has an average 3.5% palmitic acid and therefore, it is considered as an important germplasm that would be an advantage for soybean oil with better physical, chemical and nutritional qualities..
104. H Uchimiya, T Anai, ET Aspuria, M Matsui, A Nakano, T Ueda, The biological roles of small GTPases and interacting proteins in plants, JOURNAL OF PLANT RESEARCH, 111, 1102, 257-260, 1998.06, Extensive studies on the molecular mechanisms of vesicular trafficking have revealed that molecules involved in this cellular function are remarkably well conserved from yeast to higher plants. However, it is not clear at all how a variety of organisms maintain the individual divergent systems using the common machinery of vesicular traffic. We have been attempting to understand the roles and regulatory mechanisms of vesicular traffic in plants through the study of Rab/Ypt GTPases. Ara proteins are Rab/Ypt homologues of Arabidopsis, which are implicated in the regulation of vesicular traffic. Their biochemical properties are similar to those of the Rab/Ypt proteins from animal and yeast cells. The overexpression of ARA2 or ARA4 causes pleiotropic morphological abnormalities in the transgenic tobacco plants. The GTPase cycle of Ara proteins has to be strictly controlled for their proper functions. We have identified two classes of regulator molecules of Ara2 and Ara4. One is the GTPase activating protein (GAP), and the other is the GDP dissociation inhibitor (GDI). GAP has been identified as an activity accelerating the hydrolysis of GTP by Ara2 or Ara4. GDI (AtGDI1) has been isolated as a molecule interacting with Ara4 using a novel method for detecting interactions between foreign molecules in yeast. Further studies on the interacting molecules should unveil the regulatory system of and signal transduction pathway via Ara proteins..
105. SM Rahman, T Kinoshita, T Anai, S Arima, Y Takagi, Genetic relationships of soybean mutants for different linolenic acid contents, CROP SCIENCE, 10.2135/cropsci1998.0011183X003800030014x, 38, 3, 702-706, 1998.05, The normal content (80-90 g kg(-1)) of linolenic acid in soybean [Glycine max (L,) Merr.] oil adversely affects oil flavor and stability. A new mutant (M-24) with lower linolenic acid content (62 g kg(-1)) was developed by x-ray irradiation. Our objective was to determine the inheritance of linolenic acid content in M-24 and to determine the genetic relationship of this trait with the fan and fanx loci known to control linolenic acid in M-5 and KL-8 mutants, respectively. Reciprocal crosses were made between each mutant and 'Bay', and among the three mutants. No maternal or cytoplasmic effects were observed in any of the crosses. Data from F-2 seeds of the cross M-24 x Bay indicated that linolenic acid content in M-W was controlled by an allele at a single locus with no dominance effects. In the cross of M-24 x KL-8, F-2 segregation indicated that linolenic acid content in M-24 and KL-8 was controlled by two different alleles at the same Locus. For the M-5 x M-24 cross, I; segregation patterns and the segregation of F-3 seeds from individual F-2 plants indicated that M-5 and M-24 mutants have alleles at different independent loci that control linolenic acid content. therefore, the allele in M-24 is designated as fanx(a) (M-24) to distinguish it from those of fan (M-5) and fanx (KL-8). The segregate with the fanfanfanx(a)fanx(a) genotype can be considered as an important germplasm that can reduce the linolenic acid content in soybean oil..
106. T Ueda, T Yoshizumi, T Anai, M Matsui, H Uchimiya, A Nakano, AtGDI2, a novel Arabidopsis gene encoding a Rab GDP dissociation inhibitor, GENE, 206, 1, 137-143, 1998.01, The GTPase cycle of Rab/Ypt proteins is strictly controlled by several classes of regulators to ensure their proper roles in membrane traffic. GDP dissociation inhibitor (GDI) is known to play essential roles in regulating nucleotide states and subcellular localizations of Rab/Ypt proteins. To obtain further knowledge on this regulator molecule in plants, we isolated and characterized two genes of Arabidopsis thaliana that encode different GDIs. AtGDI1 has been identified by a novel functional cloning in yeast [Ueda et al. (1996) Plant Cell, 8, 2079-2091] and AtGDI2 was isolated by cross-hybridization in this study. AtGDI2, as well as AtGDI1, complements the yeast sec19/gdi1 mutant, indicating that they can replace the function of yeast GDI. Evidence is shown that both AtGDI1 and AtGDI2 can interact with Ara4, an Arabidopsis Rab protein, in the yeast ypt1 mutant cells. AtGDI2 is ubiquitously expressed in Arabidopsis tissues with some difference from AtGDI1 in expression level. Genomic DNA hybridization using specific probes reveals the presence of one more GDI gene in Arabidopsis. This may imply differentiated roles of GDI in higher plants. (C) 1997 Elsevier Science B.V..
107. T Anai, R Takai, M Miyata, H Uchida, S Kosemura, S Yamamura, R Ishizaki, K Hasegawa, Isolation and characterization of an auxin-binding protein gene from radish, and its expression in insect cells, PHYSIOLOGIA PLANTARUM, 10.1034/j.1399-3054.1997.1010322.x, 101, 3, 606-611, 1997.11, Auxin-binding protein (ABP1) is a putative receptor for auxin in the plasma membrane. We isolated a full-length cDNA encoding ABP1 from radish by screening a cDNA library with its partial cDNA fragment generated by the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Radish abp1 mRNA was highly expressed in cotyledons, hooks and hypocotyls, and less so in roots of radish seedlings. The deduced amino acid sequence of radish abp1 consisted of conserved auxin-binding motifs, a signal peptide and ER-retention signal, and contained two potentially N-linked glycosylation signals. To analyze the biochemical properties of the radish abp1 product, this cDNA was expressed in insect cells by the baculovirus vector system. The result of tunicamycin-treatment showed that the signal peptide was cleaved and that the radish abp1 product was glycosylated at both target sites in insect cells..
108. T Anai, M Miyata, S Kosemura, S Yamamura, T Tsuge, M Matsui, H Uchida, K Hasegawa, Comparison of abp1 primary sequences from monocotyledonous and dicotyledonous species, JOURNAL OF PLANT PHYSIOLOGY, 151, 4, 446-449, 1997.10, The cDNA fragments of auxin-binding protein (ABP1) were isolated by degenerated-primer mediated reverse transcription-polymerase chain reaction (RT-PCR) from dicotyledonous plant species, i.e. cress, mung bean, pea, radish and soybean, and monocotyledonous species, i.e, oat and rice. Cloned abp1 cDNA fragments were sequenced and compared with known abp1 clones from A. thaliana, maize, strawberry and tobacco at the amino acid level. In all plant species studied, the amino acid sequences of newly isolated abp1 clones were highly conserved at two regions (region A: Thr-Pro-Ile-His-Arg-His-Ser-Cys-Glu-Glu-Ile/Val-Phe-Ile /Thr/Val-Val-Leu/Pro/Val-Lys-Gly-Xaa-Gly-Thr-Leu/Val; region B: His-Glu-Asp-Leu-Gln-Phe/Val-Leu-Asp/Val-Ile/Val-Ile-Ser-Arg-Pro-Pro), which were previously reported to be important for auxin-binding. On the other hand, some putative residues of amino acids in the region between regions A and B could be found to be specific in dicot and monocot species, respectively. Southern-blot analysis indicated a small abp1 gene family in all species studied. Northern-blot analysis indicated that the size of abp1 mRNA transcripts in all species studied was conserved at approximately 850 bp. The phylogenetic tree of ABP1 was analyzed by the UPGMA method..
109. Seiji Kosemura, Hideyuki Emori, Shosuke Yamamura, Toyoaki Anai, Kaori Tomita, Koji Hasegawa, Design of photoaffinity reagents for labeling the auxin receptor in maize, Tetrahedron Letters, 10.1016/S0040-4039(97)00323-7, 38, 12, 2125-2128, 1997.03, In order to isolate the auxin receptor, we have successfully synthesized two analogues of benzoxazolinones with a trifluoromethyldiazirine group as a photoaffinity probe. These compounds inhibited the auxin-induced growth of etiolated Avena coleoptile segments. Photolyses of these compounds in methanol gave intermolecular O-H insertion products in moderate yields, respectively..
110. UEDA Takashi, MATSUDA Noriyuki, ANAI Toyoaki, TSUKAYA Hirokazu, UCHIMIYA Hirofumi, NAKANO Akihiko, Ara4 GTPase AND ITS INTERACTANTS, Plant and cell physiology, 38, s122, 1997.03.
111. T Ueda, N Matsuda, T Anai, H Tsukaya, H Uchimiya, A Nakano, An arabidopsis gene isolated by a novel method for detecting genetic interaction in yeast encodes the GDP dissociation inhibitor of Ara4 GTPase, PLANT CELL, 8, 11, 2079-2091, 1996.11, The Arabidopsis Ara proteins belong to the Rab/Ypt family of small GTPases, which are implicated in intracellular vesicular traffic. To understand their specific roles in the cell, it is imperative to identify molecules that regulate the GTPase cycle. Such molecules have been found and characterized in animals and yeasts but not in plants. Using a yeast system, we developed a novel method of functional screening to detect interactions between foreign genes and identified this Rab regulator in plants. We found that the expression of the ARA4 gene in yeast ypt mutants causes exaggeration of the mutant phenotype. By introducing an Arabidopsis cDNA library into the ypt1 mutant, we isolated a clone whose coexpression overcame the deleterious effect of ARA4. This gene encodes an Arabidopsis homolog of the nab GDP dissociation inhibitor (GDI) and was named AtGDI1. The expression of AtGDI1 complemented the yeast sec19-1 (gdi1) mutation. AtGDI1 is expressed almost ubiquitously in Arabidopsis tissues. The method described here indicates the physiological interaction of two plant molecules, Ara4 and GDI, in yeast and should be applicable to other foreign genes..
112. T Ueda, H Tsukaya, T Anai, A Hirata, K Hasegawa, H Uchimiya, Brefeldin A induces the accumulation of unusual membrane structures in elongating pollen tubes of Nicotiana tabacum L., JOURNAL OF PLANT PHYSIOLOGY, 149, 6, 683-689, 1996.11, To investigate the effects of a specific inhibitor of the post-Golgi secretion on the secretory pathway in the plant cell, the growing pollen tubes were treated with Brefeldin A (BFA) and examined at the ultrastructural level. BFA-treated pollens were processed by rapid-freezing and freeze-substitution for the preparation of samples for electron microscopy. BFA completely inhibited the elongation of pollen tubes and the secretion of vesicles in the tip region, and dictyosomes disappeared in BFA-treated pollen tubes. Unusual membrane structures, filled with various types of electron-dense matrix, were observed in both the pollen tubes and grains after exposure to BFA. Some of the structures contained cell-wall-like fibrous structures, and the results of cytochemical analysis indicated that the membranes were similar to plasma membranes of control pollen tubes. These membranes were ribosome-free, and clathrin-like coats were observed on the surface of the membranes on the cytosolic side. From these observations, it seems that processing of glycoproteins and membranes can proceed in BFA-treated pollen tubes..
113. Takashi Ueda, Noriyuki Matsuda, Toyoaki Anai, Hirokazu Tsukaya, Hirofumi Uchimiya, Akihiko Nakano, An Arabidopsis gene isolated by a novel method for detecting genetic interaction in yeast encodes the GDP dissociation inhibitor of Ara4 GTPase, Plant Cell, 10.1105/tpc.8.11.2079, 8, 11, 2079-2091, 1996.11, The Arabidopsis Ara proteins belong to the Reb/Ypt family of small GTPases, which are implicated in intracellular vesicular traffic. To understand their specific roles in the cell, it is imperative to identify molecules that regulate the GTPase cycle. Such molecules have been found and characterized in animals and yeasts but not in plants. Using a yeast system, we developed a novel method of functional screening to detect interactions between foreign genes and identified this Rab regulator in plants. We found that the expression of the ARA4 gene in yeast ypt mutants causes exaggeration of the mutant phenotype. By introducing an Arabidopsis cDNA library into the ypt1 mutant, we isolated e clone whose coexpression overcame the deleterious effect of ARA4. This gene encodes an Arabidopsis homolog of the Rab GDP dissociation inhibitor (GDI) and was named AtGDI1. The expression of AtGDI1 complemented the yeast sec19-1 (gdl1) mutation. AtGDI1 is expressed almost ubiquitously in Arabidopsis tissues. The method described here indicates the physiological interaction of two plant molecules, Ara4 and GDI, in yeast and should be applicable to other foreign genes..
114. T Anai, H Aizawa, N Ohtake, S Kosemura, S Yamamura, K Hasegawa, A new auxin-inhibiting substance, 4-Cl-6,7-dimethoxy-2-benzoxazolinone, from light-grown maize shoots, PHYTOCHEMISTRY, 42, 2, 273-275, 1996.05, Two auxin-inhibiting substances were isolated from light-grown maize shoots. One was a new compound and determined from its spectral data as 4-Cl-6,7-dimethoxy-2-benzoxazolinone (Cl-DMBOA). Another was identified from its spectral data as a known compound, 6,7-dimethoxy-2-benzoxazolinone (DMBOA). Above concentrations of 10(-5) M, Cl-DMBOA inhibited the auxin-inducing elongation in the Avena coleoptile section test. Its inhibitory activity was almost comparable to that of DMBOA and higher than that of 6-methoxy-2-benzoxazolinone (MBOA) which we previously isolated..
115. T Ueda, T Anai, H Tsukaya, A Hirata, H Uchimiya, Characterization and subcellular localization of a small GTP binding protein (Ara-4) from Arabidopsis: Conditional expression under control of the promoter of the gene for heat-shock protein HSP81-1, MOLECULAR & GENERAL GENETICS, 10.1007/s004380050106, 250, 5, 533-539, 1996.03, Small GTP-binding proteins belonging to the rab/YPT family play key roles at various steps in intracellular transport pathways in yeast and mammalian cells. Many members of rab/YPT family have been isolated from plants to date. However, detailed information about the localization and function of the gene products remains limited, even though intracellular transport is likely to be involved in important phenomena such as cell elongation, transport of storage proteins, determination and maintenance of cell polarity and intercellular signal transduction. We have attempted to establish transgenic Arabidopsis plants that overexpress ARA-4, a rab/YPT homologue in order to analyze the function and the localization of the gene product. For overexpression and also for regulation of the expression of this gene, the promoter of the gene for HSP81-1 was employed to drive the transcription of ARA-4 in transgenic plants. The response of the introduced genes to heat shock was analyzed. Upon heat-shock treatment, the ARA-4 gene was efficiently transcribed and translated. The induction of ARA-4 by heat shock was transient, and at least two distinct forms of this protein were found in membrane and cytosolic fractions from transgenic plants. Prolonged incubation after heat shock reduced the amount of the cytosolic form of the induced protein, and the cytosolic form of the protein thus probably represents the unprocessed precursor. Using transgenic plants, we determined the subcellular localization of the product of ARA-4. The protein was predominantly localized on Golgi-derived vesicles, Golgi cisternae and the trans-Golgi network..
116. K Yamada, T Anai, S Kosemura, S Yamamura, K Hasegawa, Structure activity relationship of lepidimoide and its analogues, PHYTOCHEMISTRY, 41, 3, 671-673, 1996.02, The structure-activity relationship of lepidimoide and its analogues was investigated by means of the Amaranthus caudatus L. hypocotyl elongation test. In addition, the activities of alpha-D-galacturonic acid and L-(+)-rhamnose, which are component sugars of lepidimoide, were also studied. The carboxylic acid free type of lepidimoide showed growth-promoting activity as high as the original lepidimoide (sodium type). The acetylated compound showed considerably higher activity than lepidimoide, whereas the methylated lepidimoide did not show any activity. The hydroxylated lepidimoide without a double bond in the C-4,5 position showed lower activity. The sugar alcohol type of lepidimoide [2-O-(alpha-D-glucopyranosyl)-L-rhamnose] showed the highest activity in all the compounds studied. alpha-D-Galacturonic acid, L-(+)-rhamnose and their mixtures, which are component sugars of lepidimoide, exhibited only slight or no activity, respectively. D-Glucose and the mixture of D-glucose and L-(+)-rhamnose were also slightly active or inactive. These data suggest that the active sites in the chemical structure of the lepidimoide are the uronic acid derivative bearing an alpha,beta-unsaturated carboxylate bonded to rhamnose via an alpha-glucoside linkage and a double bond in the C-4,5 position in the uronic acid..
117. S KOSEMURA, H EMORI, S YAMAMURA, T ANAI, H AIZAWA, N OHTAKE, K HASEGAWA, ISOLATION AND CHARACTERIZATION OF 4-CHLORO-6,7-DIMETHOXYBENZOXAZOLIN-2-ONE A NEW AUXIN-INHIBITING BENZOXAZOLINONE FROM ZEA-MAYS, CHEMISTRY LETTERS, 1995, 11, 1053-1054, 1995.11, A new auxin-inhibiting substance was isolated from light-grown maize shoots. The structural determination was performed by spectroscopic methods and synthesis of 4-chloro-6,7-dimethoxybenzoxazolin-2-one..
118. PK Samarajeewa, M Kawai, T Anai, A Hirai, H Uchimiya, Sodium chloride stimulates adenylate kinase level in seedlings of salt-sensitive rice varieties, JOURNAL OF PLANT PHYSIOLOGY, 147, 2, 277-280, 1995.11, Comparison of adenylate kinase activities in rice seedlings (Oryza sativa L. cv. Yamahoushi) that had been grown in the absence or presence of NaCl indicated that NaCl apparently stimulated enzyme activities in roots of this salt-sensitive japonica rice cultivar. Such stimulation of enzyme activities is not limited to a specific portion of root tissues. Furthermore, NaCl-induced adenylate kinase activation was confirmed in the indica rice cultivar IR 28 susceptible to salinity-stress, but not in the NaCl-tolerant indica cultivar Nona Bokra. Thus in salt sensitive rice plants, adenylate homeostatis at the early stages of rice seedling growth subjected to salt-stress..
119. T ANAI, ET ASPURIA, N FUJII, T UEDA, M MATSUI, K HASEGAWA, H UCHIMIYA, IMMUNOLOGICAL ANALYSIS OF A SMALL GTP-BINDING PROTEIN IN HIGHER-PLANT CELLS, JOURNAL OF PLANT PHYSIOLOGY, 147, 1, 48-52, 1995.10, ara genes encoding YPT/RAB like small GTP-binding proteins, which are involved in the vesicular transport system in various eukaryotic cells, have been isolated from Arabidopsis thaliana. To determine the subcellular localization of ARA-4 protein, suspensions of cultured transgenic tobacco cells were analyzed using monoclonal antibody to Arabidopsis thaliana ARA-4 protein. ARA-4 protein was found in the microsomal membrane fraction. Similar results were obtained with Arabidopsis thaliana cells. The ARA-4 protein was readily released by detergents, such as 0.5% sodium deoxycholate and NP-40. The results of immune-blot analysis with extracts of different organs show that the ARA-4 protein is present in most organs of Arabidopsis thaliana plants, e. g. root, stem, flower and young fruit, at the same level in all organs except for root, where the level is lower..
120. K YAMADA, T ANAI, K HASEGAWA, LEPIDIMOIDE, AN ALLELOPATHIC SUBSTANCE IN THE EXUDATES FROM GERMINATED SEEDS, PHYTOCHEMISTRY, 39, 5, 1031-1032, 1995.07, The occurrence in the plant kingdom of the allelopathic substance, lepidimoide, was studied. Lepidimoide is widespread in the exudates from all plant species studied. Lepidimoide occurred in especially large amounts in the exudates of germinated seeds of sunflower and buckwheat, but it was also detected in those of rice, lettuce, slender smaranth, leek and persian speedwell. Lepidimoide amounts did not differ greatly among genera or families..
121. ET ASPURIA, T ANAI, N FUJII, T UEDA, M MIYOSHI, M MATSUI, H UCHIMIYA, PHENOTYPIC INSTABILITY OF TRANSGENIC TOBACCO PLANTS AND THEIR PROGENIES EXPRESSING ARABIDOPSIS-THALIANA SMALL GTP-BINDING PROTEIN GENES, MOLECULAR & GENERAL GENETICS, 246, 4, 509-513, 1995.02, Chimeric genes consisting of the cauliflower mosaic virus 35S promoter, a cDNA encoding a small GTP-binding protein from Arabidopsis thaliana (ara-2 or ara-4) and the terminator of the nopaline synthase gene were cloned into a binary vector. Tobacco leaf tissues were transformed with this plasmid via Agrobacterium-mediated transformation. Transgenic plants possessing either ara-2 or ara-4 occasionally showed morphological abnormalities in leaves and other organs. However, such alterations were not always associated with co-transferred characters, such as kanamycin tolerance, and they arose in no more than 10% of the transgenic plants. Such phenomena were also observed in the progenies of the primary transgenic plants. Despite such unusual inheritance of the phenotypic abnormalities, GTP-binding activity of the inserted ara gene products was detected in all plants tested..
122. 植物のGタンパク質と細胞内情報伝達.
123. S KOSEMURA, S YAMAMURA, T ANAI, K HASEGAWA, CHEMICAL STUDIES ON 2,4-DIHYDROXY-7-METHOXY-2H-1,4-BENZORAZIN-3(4H)-ONE IN CONNECTION WITH 6-METHOXY-2-BENZOXAZOLINONE, AN AUXIN-INHIBITING SUBSTANCE OF ZEA-MAYS L, TETRAHEDRON LETTERS, 35, 44, 8221-8224, 1994.10, Some chemical studies on 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA, 1) and related compounds from Zea mays L. have been carried out, where DIMBOA (1) has been very rapidly converted into 6-methoxy-2-benzoxazolinone (MBOA, 2), an auxin-inhibiting substanse of maize, in the presence of acetic anhydride at room temperature. Therefore, such an enzymatic acylation of N-OH group of DIMBOA (1) as in vitro presumably plays an important role on phototropism..
124. T ANAI, M MATSUI, N NOMURA, R ISHIZAKI, H UCHIMIYA, IN-VITRO MUTATION ANALYSIS OF ARABIDOPSIS-THALIANA SMALL GTP-BINDING PROTEINS AND DETECTION OF GAP-LIKE ACTIVITIES IN PLANT-CELLS, FEBS LETTERS, 346, 2-3, 175-180, 1994.06, Previously, we have reported the molecular cloning of ara genes encoding a small GTP-binding protein from Arabidopsis thaliana. The criterion based on amino acid sequences suggest that such an ara gene family can be classified to be of the YPT/rab type. To examine the biochemical properties of ARA proteins, several deletions and point mutations were introduced into nra cDNAs. Mutant proteins were expressed in E. coli as GST-chimeric molecules and analyzed in terms of their GTP-binding or GTP-hydrolysing ability in vitro. The results indicate that four conserved amino acid sequence regions of ARA proteins are necessary for GTP-binding. A point mutation of Asn at position 72 for ARA-2 or 71 for ARA-4, to Ile decreased GTP-binding and a point mutation of Gin at position 126 for ARA-2, or 125 for ARA-4 to Leu suppressed GTP-hydrolysis activity. Furthermore, certain factors associated with the membrane fraction accelerated GTPase activities of ARA proteins, suggesting the presence of GTPase activating protein(s) (GAP(s)) in the vesicular transport system of higher plant cells..
125. S KIDOU, T ANAI, M UMEDA, S AOTSUKA, T TSUGE, A KATO, H UCHIMIYA, MOLECULAR-STRUCTURE OF RAS-RELATED SMALL GTP-BINDING PROTEIN GENES OF RICE PLANTS AND GTPASE ACTIVITIES OF GENE-PRODUCTS IN ESCHERICHIA-COLI, FEBS LETTERS, 332, 3, 282-286, 1993.10, We isolated two rice cDNA clones (ric1 and ric2) encoding proteins homologous to the ras-related small GTP-binding protein. The amino acid sequences of ric1 and ric2 are conserved in four regions involved in GTP binding and hydrolysis which are characteristic in the ras and ras-related small GTP-binding protein genes. In addition, two consecutive cysteine residues near the carboxyl-terminal end required for membrane anchoring are also present in ric1 and ric2. The ric1 and ric2 proteins synthesized in Escherichia coli possessed GTPase activity (i.e. hydrolysis of GTP to GDP)..
126. Toyoaki Anai, Koji Hasegawa, Yuichiro Watanabe, Hirofumi Uchimiya, Ryotaro Ishizaki, Minami Matsui, Isolation and analysis of cDNAs encoding small GTP-binding proteins of Arabidopsis thaliana, GENE, 10.1016/0378-1119(91)90442-E, 108, 2, 259-264, 1991.12, We previously isolated a DNA fragment from Arabidopsis thaliana homologous to the mammalian ras gene and named it ara [Matsui et al., Gene 76 (1989) 313–319]. Screening of cDNA clones homologous to ara in A. thaliana resulted in the isolation of four homologous genes. The products of these genes, ARA-2, ARA-3, ARA-4 and ARA-5, showed conservation of amino acids (aa) in four regions, all of which are present in small GTP-binding proteins, and are important for GTPase/GTP-binding activities. These products were highly homologous to those of the YPT genes of taccharomyces cerevisiae and the ypt gene of Schizosaccharomyces pombe in the regions around aa 45, which is thought to be the site interacting with effector molecules. The products of these four genes showed characteristic aa sequence at their C termini, Cys-Cys-Xaa-Xaa. Another characteristic of this family is presence of Ser in place of Gly in the first conserved region (Gly12 of mammalian GTP-binding Ras protein)..
127. T HIRAYAMA, Y IMAJUKU, T ANAI, M MATSUI, A OKA, IDENTIFICATION OF 2 CELL-CYCLE-CONTROLLING CDC2 GENE HOMOLOGS IN ARABIDOPSIS-THALIANA, GENE, 105, 2, 159-165, 1991.09, The cdc2 gene product (p34cdc2) has been thought to play a central role in control of the mitotic cell cycle of yeasts and animals. To approach an understanding of the cell-cycle-control system in higher plants, we isolated, from an Arabidopsis thaliana cDNA library, two clones (CDC2a and CDC2b) similar to the Schizosaccharomyces pombe cdc2 gene. Genomic Southern-blot analysis with the CDC2a and CDC2b cDNA probes suggested that the A. thaliana genome contains several additional cdc2-like genes, which together with the CDC2a and CDC2b genes may constitute at CDC2 gene family. The CDC2a cDNA expressed in Sc. pombe corrected the elongated morphology, caused by the temperature-sensitive cdc2-33 mutation, to the normal shapes, indicating that the A. thaliana CDC2a gene product resembles Sc. pombe p34cdc2 functionally as well as structurally. These results support the view that the cell cycle of higher plants is controlled by an analogue of a p34cdc2 -centered regulatory system like that of yeasts and animals..
128. T HIRAYAMA, Y IMAJUKU, T ANAI, M MATSUI, A OKA, IDENTIFICATION OF 2 CELL-CYCLE-CONTROLLING CDC2 GENE HOMOLOGS IN ARABIDOPSIS-THALIANA, GENE, 105, 2, 159-165, 1991.09, The cdc2 gene product (p34cdc2) has been thought to play a central role in control of the mitotic cell cycle of yeasts and animals. To approach an understanding of the cell-cycle-control system in higher plants, we isolated, from an Arabidopsis thaliana cDNA library, two clones (CDC2a and CDC2b) similar to the Schizosaccharomyces pombe cdc2 gene. Genomic Southern-blot analysis with the CDC2a and CDC2b cDNA probes suggested that the A. thaliana genome contains several additional cdc2-like genes, which together with the CDC2a and CDC2b genes may constitute at CDC2 gene family. The CDC2a cDNA expressed in Sc. pombe corrected the elongated morphology, caused by the temperature-sensitive cdc2-33 mutation, to the normal shapes, indicating that the A. thaliana CDC2a gene product resembles Sc. pombe p34cdc2 functionally as well as structurally. These results support the view that the cell cycle of higher plants is controlled by an analogue of a p34cdc2 -centered regulatory system like that of yeasts and animals..
129. Satoshi Watanabe, Risa Yamada, Hazuki Kanetake, Akito Kaga, Toyoaki Anai, Identification and characterization of a major QTL underlying soybean isoflavone malonylglycitin content, BREEDING SCIENCE, 10.1270/jsbbs.19027, 69, 4, 564-572.
130. Kaori Hirata, Kyoko Takagi, Tetsuya Yamada, Takashi Sayama, Toyoaki Anai, Akio Kikuchi, Masao Ishimoto, Isolation and characterization of induced mutants in the gene associated with seed cadmium accumulation in soybean, BREEDING SCIENCE, 10.1270/jsbbs.18091, 69, 2, 345-351.