Researcher Profile and Settings

Master

Affiliation (Master)

• Research Faculty of Agriculture　Fundamental AgriScience Research　Agrobiology and Bioresources

Affiliation (Master)

• Research Faculty of Agriculture　Fundamental AgriScience Research　Agrobiology and Bioresources

researchmap

Profile and Settings

Profile and Settings

• Name (Japanese)

  Kanazawa

• Name (Kana)

  Akira

• Name

  201301078971521929

Alternate Names

http://kaken.nii.ac.jp/d/r/30281794.ja.html

Achievement

Research Interests

• RNA   ダイズ   メチル化   ジーンサイレンシング   TGS   植物   siRNA   エピジェネティクス   プロモーター   種分化   遺伝子ノックアウト   イネ   雑種不稔   PTGS   遺伝子発現   ペチュニア   転写   テンサイ   葉緑体   遺伝子導入   ウイルス   CHS   交雑   生態型   トランスポゾン   ミトコンドリア   GFP   高等植物   減数分裂   

Research Areas

• Life sciences / Plants: molecular biology and physiology
• Life sciences / Genetics
• Environmental science/Agricultural science / Plant genetics and breeding
• Life sciences / Applied molecular and cellular biology

Published Papers

• OlCHR, encoding a chromatin remodeling factor, is a killer causing hybrid sterility between rice species Oryza sativa and O. longistaminata.

  Zin Mar Myint, Yohei Koide, Wakana Takanishi, Tomohito Ikegaya, Choi Kwan, Kiwamu Hikichi, Yoshiki Tokuyama, Shuhei Okada, Kazumitsu Onishi, Ryo Ishikawa, Daisuke Fujita, Yoshiyuki Yamagata, Hideo Matsumura, Yuji Kishima, Akira Kanazawa

  iScience 27 (5) 109761 - 109761 2024/05/17 

  The genetic mechanisms of reproductive isolation have been widely investigated within Asian cultivated rice (Oryza sativa); however, relevant genes between diverged species have been in sighted rather less. Herein, a gene showing selfish behavior was discovered in hybrids between the distantly related rice species Oryza longistaminata and O. sativa. The selfish allele S13l in the S13 locus impaired male fertility, discriminately eliminating pollens containing the allele S13s from O. sativa in heterozygotes (S13s/S13l). Genetic analysis revealed that a gene encoding a chromatin-remodeling factor (CHR) is involved in this phenomenon and a variety of O. sativa owns the truncated gene OsCHR745, whereas its homologue OlCHR has a complete structure in O. longistaminata. CRISPR-Cas9-mediated loss of function mutants restored fertility in hybrids. African cultivated rice, which naturally lacks the OlCHR homologue, is compatible with both S13s and S13l carriers. These results suggest that OlCHR is a Killer gene, which leads to reproductive isolation.
• Characterization of chlorophyll-deficient soybean [Glycine max (L.) Merr.] mutants obtained by ion-beam irradiation reveals concomitant reduction in isoflavone levels

  Yui Shiroshita, Mashiro Yuhazu, Yoshihiro Hase, Tetsuya Yamada, Jun Abe, Akira Kanazawa

  GENETIC RESOURCES AND CROP EVOLUTION 68 (3) 1213 - 1223 0925-9864 2021/03 [Refereed]
   

  Ion-beam irradiation serves as a powerful tool of mutagenesis for engineering novel traits in a wide range of plants. A previous study indicated that coincidental changes in multiple traits are frequently induced by ion-beam irradiation in soybean [Glycine max (L.) Merr.], a paleopolyploid plant whose genome comprises a large number of duplicated genes. Here we analyzed the levels of isoflavones, major secondary compounds in soybean seeds, in a population of mutants with various degrees of chlorophyll deficiency induced by the mutagenesis. The isoflavones daidzin, genistin, and their respective malonylglucosides were significantly reduced in 4-9 plant lines among 28 lines analyzed. The average levels of chlorophyll in the leaf tissues were correlated with the level of each isoflavone in seeds from multiple lines. In selected mutant lines, the levels of isoflavones were lower than in the wild-type plants throughout seed development. Gene expression profiles indicated that the mRNA levels of multiple genes involved in the isoflavone synthetic pathway were also reduced during seed development in these mutants, consistent with the reduced levels of the compounds. These data suggest that ion-beam irradiation caused changes in multiple pathways of metabolism directly or indirectly and can provide useful plant resources for dissecting isoflavone synthesis and/or accumulation in soybean.
• Disturbance of floral colour pattern by activation of an endogenous pararetrovirus, petunia vein clearing virus, in aged petunia plants.

  Kazunori Kuriyama, Midori Tabara, Hiromitsu Moriyama, Akira Kanazawa, Hisashi Koiwa, Hideki Takahashi, Toshiyuki Fukuhara

  The Plant journal : for cell and molecular biology 103 (2) 497 - 511 2020/07 [Refereed]
   

  White areas of star-type bicolour petals of petunia (Petunia hybrida) are caused by post-transcriptional gene silencing (PTGS) of the key enzyme of anthocyanin biosynthesis. We observed blotched flowers and a vein-clearing symptom in aged petunia plants. To determine the cause of blotched flowers, we focused on an endogenous pararetrovirus, petunia vein clearing virus (PVCV), because this virus may have a suppressor of PTGS (VSR). Transcripts and episomal DNAs derived from proviral PVCVs accumulated in aged plants, indicating that PVCV was activated as the host plant aged. Furthermore, DNA methylation of CG and CHG sites in the promoter region of proviral PVCV decreased in aged plants, suggesting that poor maintenance of DNA methylation activates PVCV. In parallel, de novo DNA methylation of CHH sites in its promoter region was also detected. Therefore, both activation and inactivation of PVCV occurred in aged plants. The accumulation of PVCV transcripts and episomal DNAs in blotched regions and the detection of VSR activity support a mechanism in which suppression of PTGS by PVCV causes blotched flowers.
• A Soybean Deletion Mutant That Moderates the Repression of Flowering by Cool Temperatures.

  Jingyu Zhang, Meilan Xu, Maria Stefanie Dwiyanti, Satoshi Watanabe, Tetsuya Yamada, Yoshihiro Hase, Akira Kanazawa, Takashi Sayama, Masao Ishimoto, Baohui Liu, Jun Abe

  Frontiers in plant science 11 429 - 429 2020 [Refereed]
   

  Ambient growing temperature and photoperiod are major environmental stimuli that summer annual crops use to adjust their reproductive phenology so as to maximize yield. Variation in flowering time among soybean (Glycine max) cultivars results mainly from allelic diversity at loci that control photoperiod sensitivity and FLOWERING LOCUS T (FT) orthologs. However, variation in the thermal regulation of flowering and its underlying mechanisms are poorly understood. In this study, we identified a novel mutant (ef1) that confers altered thermal regulation of flowering in response to cool ambient temperatures. Mapping analysis with simple sequence repeat (SSR) markers located the mutation in the upper part of chromosome 19, where no QTL for flowering has been previously reported. Fine-mapping and re-sequencing revealed that the mutation was caused by deletion of a 214 kbp genomic region that contains 11 annotated genes, including CONSTANS-LIKE 2b (COL2b), a soybean ortholog of Arabidopsis CONSTANS. Comparison of flowering times under different photo-thermal conditions revealed that early flowering in the mutant lines was most distinct under cool ambient temperatures. The expression of two FT orthologs, FT2a and FT5a, was dramatically downregulated by cool temperature, but the magnitude of the downregulation was lower in the mutant lines. Cool temperatures upregulated COL2b expression or delayed peak expression, particularly at the fourth trifoliate-leaf stage. Intriguingly, they also upregulated E1, a soybean-specific repressor of FT orthologs. Our results suggest that the ef1 mutation is involved in thermal regulation of flowering in response to cool ambient temperature, and the lack of COL2b in the mutant likely alleviates the repression of flowering by cool temperature. The ef1 mutant can be used as a novel gene resource in breeding soybean cultivars adapted to cool climate and in research to improve our understanding of thermal regulation of flowering in soybean.
• Loss of Function of the E1-Like-b Gene Associates With Early Flowering Under Long-Day Conditions in Soybean

  Jianghui Zhu, Ryoma Takeshima, Kohei Harigai, Meilan Xu, Fanjiang Kong, Baohui Liu, Akira Kanazawa, Tetsuya Yamada, Jun Abe

  Frontiers in Plant Science Frontiers Media {SA} 9 1867  1664-462X 2019/01 [Refereed][Not invited]
  
• Transcription of soybean retrotransposon SORE-1 is temporally upregulated in developing ovules.

  Kenta Nakashima, Mayumi Tsuchiya, Sae Fukushima, Jun Abe, Akira Kanazawa

  Planta 248 (5) 1331 - 1337 2018/11 [Refereed]
   

  MAIN CONCLUSION: Transcription of soybean retrotransposon SORE-1 was temporally upregulated during ovule development. This transcriptional pattern was under intrinsic control conferred by the long terminal repeat of SORE-1. Transcriptionally active retrotransposons are capable of inducing random disruption of genes, providing a powerful tool for mutagenesis. Activation of retrotransposons in reproductive cells, in particular, can lead to heritable changes. Here, we examined developmental control of transcription of soybean retrotransposon SORE-1. Transgenic Arabidopsis plants that contain β-glucuronidase (GUS) reporter gene fused with the SORE-1 long terminal repeat (LTR) had GUS staining in the ovule. Quantitative analysis of transcripts in plants with this DNA construct and those with the full-length SORE-1 element indicated a temporal upregulation of SORE-1 transcription during ovule development. A comparable phenomenon was also observed in soybean plants that had a recent insertion of this element in the GmphyA2 gene. These results provide evidence that the temporal upregulation of SORE-1 in the reproductive organ is sufficiently controlled by its LTR and indicate that the intrinsic expression pattern of SORE-1 is consistent with its mutagenic property.
• Chromosomal distribution of soybean retrotransposon SORE-1 suggests its recent preferential insertion into euchromatic regions.

  Kenta Nakashima, Jun Abe, Akira Kanazawa

  Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology 26 (3) 199 - 210 2018/09 [Refereed]
   

  Retrotransposons constitute a large portion of plant genomes. The chromosomal distribution of a wide variety of retrotransposons has been analyzed using genome sequencing data in several plants, but the evolutionary profile of transposition has been characterized for a limited number of retrotransposon families. Here, we characterized 96 elements of the SORE-1 family of soybean retrotransposons using genome sequencing data. Insertion time of each SORE-1 element into the genome was estimated on the basis of sequence differences between the 5' and 3' long terminal repeats (LTRs). Combining this estimation with information on the chromosomal location of these elements, we found that the insertion of the existing SORE-1 into gene-rich chromosome arms occurred on average more recently than that into gene-poor pericentromeric regions. In addition, both the number of insertions and the proportion of insertions into chromosome arms profoundly increased after 1 million years ago. Solo LTRs were detected in these regions at a similar frequency, suggesting that elimination of SORE-1 via unequal homologous recombination was unbiased. Taken together, these results suggest the preference of a recent insertion of SORE-1 into chromosome arms comprising euchromatic regions. This notion is contrary to an earlier view deduced from an overall profiling of soybean retrotransposons and suggests that the pattern of chromosomal distribution can be more diverse than previously thought between different families of retrotransposons.
• Lineage-specific gene acquisition or loss is involved in interspecific hybrid sterility in rice.

  Yohei Koide, Atsushi Ogino, Takanori Yoshikawa, Yuki Kitashima, Nozomi Saito, Yoshitaka Kanaoka, Kazumitsu Onishi, Yoshihiro Yoshitake, Takuji Tsukiyama, Hiroki Saito, Masayoshi Teraishi, Yoshiyuki Yamagata, Aiko Uemura, Hiroki Takagi, Yoriko Hayashi, Tomoko Abe, Yoshimichi Fukuta, Yutaka Okumoto, Akira Kanazawa

  Proceedings of the National Academy of Sciences of the United States of America 115 (9) E1955-E1962  2018/02/27 [Refereed]
   

  Understanding the genetic basis of reproductive barriers between species has been a central issue in evolutionary biology. The S1 locus in rice causes hybrid sterility and is a major reproductive barrier between two rice species, Oryza sativa and Oryza glaberrima The O. glaberrima-derived allele (denoted S1g) on the S1 locus causes preferential abortion of gametes with its allelic alternative (denoted S1s) in S1g/S1s heterozygotes. Here, we used mutagenesis and screening of fertile hybrid plants to isolate a mutant with an allele, S1mut, which does not confer sterility in the S1mut/S1g and S1mut/S1s hybrids. We found that the causal mutation of the S1mut allele was a deletion in the peptidase-coding gene (denoted "SSP") in the S1 locus of O. glaberrima No orthologous genes of SSP were found in the O. sativa genome. Transformation experiments indicated that the introduction of SSP in carriers of the S1s allele did not induce sterility. In S1mut/S1s heterozygotes, the insertion of SSP led to sterility, suggesting that SSP complemented the loss of the functional phenotype of the mutant and that multiple factors are involved in the phenomenon. The polymorphisms caused by the lineage-specific acquisition or loss of the SSP gene were implicated in the generation of hybrid sterility. Our results demonstrated that artificial disruption of a single gene for the reproductive barrier creates a "neutral" allele, which facilitates interspecific hybridization for breeding programs.
• Frequent generation of mutants with coincidental changes in multiple traits via ion-beam irradiation in soybean.

  Mikuriya S, Kasai M, Nakashima K, Natasia, Hase Y, Yamada T, Abe J, Kanazawa A

  Genes & genetic systems 92 (3) 153 - 161 1341-7568 2018/01 [Refereed][Not invited]
  
• RNA silencing in the life cycle of soybean: multiple restriction systems and spatiotemporal variation associated with plant architecture

  Ayumi Mori, Hiroshi Sato, Megumi Kasai, Tetsuya Yamada, Akira Kanazawa

  TRANSGENIC RESEARCH 26 (3) 349 - 362 0962-8819 2017/06 [Refereed][Not invited]
   

  The expression of transgenes introduced into a plant genome is sometimes suppressed by RNA silencing. Although local and systemic spread of RNA silencing have been studied, little is known about the mechanisms underlying spatial and temporal variation in transgene silencing between individual plants or between plants of different generations, which occurs seemingly stochastically. Here, we analyzed the occurrence, spread, and transmission of RNA silencing of the green fluorescent protein (GFP) gene over multiple generations of the progeny of a single soybean transformant. Observation of GFP fluorescence in entire plants of the T-3-T-5 generations indicated that the initiation and subsequent spread of GFP silencing varied between individuals, although this GFP silencing most frequently began in the primary leaves. In addition, GFP silencing could spread into the outer layer of seed coat tissues but was hardly detectable in the embryos. These results are consistent with the notion that transgene silencing involves its reset during reproductive phase, initiation after germination, and systemic spread in each generation. GFP silencing was absent in the pulvinus, suggesting that its cortical cells inhibit cell-to-cell spread or induction of RNA silencing. The extent of GFP silencing could differ between the stem and a petiole or between petiolules, which have limited vascular bundles connecting them and thus deter long-distant movement of silencing. Taken together, these observations indicate that the initiation and/or spread of RNA silencing depend on specific features of the architecture of the plant in addition to the mechanisms that can be conserved in higher plants.
• The Non-Mendelian Green Cotyledon Gene in Soybean Encodes a Small Subunit of Photosystem II

  Kaori Kohzuma, Yutaka Sato, Hisashi Ito, Ayako Okuzaki, Mai Watanabe, Hideki Kobayashi, Michiharu Nakano, Hiroshi Yamatani, Yu Masuda, Yumi Nagashima, Hiroyuki Fukuoka, Tetsuya Yamada, Akira Kanazawa, Keisuke Kitamura, Yutaka Tabei, Masahiko Ikeuchi, Wataru Sakamoto, Ayumi Tanaka, Makoto Kusaba

  PLANT PHYSIOLOGY 173 (4) 2138 - 2147 0032-0889 2017/04 [Refereed][Not invited]
   

  Chlorophyll degradation plays important roles in leaf senescence including regulation of degradation of chlorophyll-binding proteins. Although most genes encoding enzymes of the chlorophyll degradation pathway have been identified, the regulation of their activity has not been fully understood. Green cotyledon mutants in legume are stay-green mutants, in which chlorophyll degradation is impaired during leaf senescence and seed maturation. Among them, the soybean (Glycine max) green cotyledon gene cytG is unique because it is maternally inherited. To isolate cytG, we extensively sequenced the soybean chloroplast genome, and detected a 5-bp insertion causing a frame-shift in psbM, which encodes one of the small subunits of photosystem II. Mutant tobacco plants (Nicotiana tabacum) with a disrupted psbM generated using a chloroplast transformation technique had green senescent leaves, confirming that cytG encodes PsbM. The phenotype of cytG was very similar to that of mutant of chlorophyll b reductase catalyzing the first step of chlorophyll b degradation. In fact, chlorophyll b-degrading activity in dark-grown cytG and psbM-knockout seedlings was significantly lower than that of wild-type plants. Our results suggest that PsbM is a unique protein linking photosynthesis in presenescent leaves with chlorophyll degradation during leaf senescence and seed maturation. Additionally, we discuss the origin of cytG, which may have been selected during domestication of soybean.
• Enhancement of RNA-directed DNA methylation of a transgene by simultaneously downregulating a ROSI ortholog using a virus vector in Nicotiana benthamiana (vol 4, 44, 2013)

  Shungo Otagaki, Megumi Kasai, Chikara Masuta, Akira Kanazawa

  FRONTIERS IN GENETICS 7 1664-8021 2016/02 [Refereed][Not invited]
  
• "Size and positional effects of promoter RNA segments on virus-induced RNA-directed DNA methylation and transcriptional gene silencing"

  Shungo Otagaki, Miou Kawai, Chikara Masuta, Akira Kanazawa

  EPIGENETICS 11 (1) 101 - 101 1559-2294 2016/01 [Refereed][Not invited]
  
• A recessive allele for delayed flowering at the soybean maturity locus E9 is a leaky allele of FT2a, a FLOWERING LOCUS T ortholog.

  Zhao C, Takeshima R, Zhu J, Xu M, Sato M, Watanabe S, Kanazawa A, Liu B, Kong F, Yamada T, Abe J

  BMC plant biology 16 20  2016/01 [Refereed][Not invited]
  
• The Soybean-Specific Maturity Gene E1 Family of Floral Repressors Controls Night-Break Responses through Down-Regulation of FLOWERING LOCUS T Orthologs.

  Xu M, Yamagishi N, Zhao C, Takeshima R, Kasai M, Watanabe S, Kanazawa A, Yoshikawa N, Liu B, Yamada T, Abe J

  Plant physiology 168 (4) 1735 - 1746 1532-2548 2015/08 [Refereed][Not invited]
  
• Profiles of embryonic nuclear protein binding to the proximal promoter region of the soybean β-conglycinin α subunit gene

  Yoshino, M, Tsutsumi, K, Kanazawa, A

  Plant Biology 17 147 - 152 2015 [Refereed]
  
• Transposon-mediated mutagenesis and its application to plant breeding

  Kanazawa Akira, Fukai Eigo, Miyao Akio, Saze Hidetoshi, Tsukiyama Takuji

  Breeding Research 日本育種学会 17 (2) 77 - 87 1344-7629 2015 [Refereed][Not invited]
  
• Epigenetic Control of Gene Expression and Engineering Novel Traits in Plants

  KANAZAWA Akira

  KAGAKU TO SEIBUTSU 公益社団法人 日本農芸化学会 52 (4) 241 - 248 0453-073X 2014 [Refereed]
   

  植物においては，核酸の塩基配列特異的に起きるRNA分解，ならびにDNAのメチル化やヒストン修飾の変化を伴うエピジェネティックな変化を誘導することが可能である．本稿では，これらの反応経路が明らかになった経緯を概説するとともに，後者の例として，ウイルスベクターを用いてRNA-directed DNA methylationを誘導し，外来遺伝子をもたずに特定の内在性遺伝子の発現が抑制された植物を作出した研究を紹介する．また，遺伝子特異的，あるいは非特異的にエピジェネティックな変化を誘導する方法，ならびにCRISPR/Cas系などによるゲノム編集を含む，新規な植物育種技術の有効性と特徴を論じる．
• Genetic variation in four maturity genes affects photoperiod insensitivity and PHYA-regulated post-flowering responses of soybean.

  Xu M, Xu Z, Liu B, Kong F, Tsubokura Y, Watanabe S, Xia Z, Harada K, Kanazawa A, Yamada T, Abe J

  BMC plant biology Biomed Central Ltd 13 (June) 91  1471-2229 2013/06/25 [Refereed][Not invited]
   

  Background: Absence of or low sensitivity to photoperiod is necessary for short-day crops, such as rice and soybean, to adapt to high latitudes. Photoperiod insensitivity in soybeans is controlled by two genetic systems and involves three important maturity genes: E1, a repressor for two soybean orthologs of Arabidopsis FLOWERING LOCUS T (GmFT2a and GmFT5a), and E3 and E4, which are phytochrome A genes. To elucidate the diverse mechanisms underlying photoperiod insensitivity in soybean, we assessed the genotypes of four maturity genes (E1 through E4) in early-flowering photoperiod-insensitive cultivars and their association with post-flowering responses. Results: We found two novel dysfunctional alleles in accessions originally considered to have a dominant E3 allele according to known DNA markers. The E3 locus, together with E1 and E4, contained multiple dysfunctional alleles. We identified 15 multi-locus genotypes, which we subdivided into 6 genotypic groups by classifying their alleles by function. Of these, the e1-as/e3/E4 genotypic group required an additional novel gene (different from E1, E3, and E4) to condition photoperiod insensitivity. Despite their common pre-flowering photoperiod insensitivity, accessions with different multi-locus genotypes responded differently to the post-flowering photoperiod. Cultivars carrying E3 or E4 were sensitive to photoperiod for post-flowering characteristics, such as reproductive period and stem growth after flowering. The phytochrome A-regulated expression of the determinate growth habit gene Dt1, an ortholog of Arabidopsis TERMINAL FLOWER1, was involved in the persistence of the vegetative activity at the stem apical meristem of flower-induced plants under long-day conditions. Conclusions: Diverse genetic mechanisms underlie photoperiod insensitivity in soybean. At least three multi-locus genotypes consisting of various allelic combinations at E1, E3, and E4 conferred pre-flowering photoperiod insensitivity to soybean cultivars but led to different responses to photoperiod during post-flowering vegetative and reproductive development. The phyA genes E3 and E4 are major controllers underlying not only pre-flowering but also post-flowering photoperiod responses. The current findings improve our understanding of genetic diversity in pre-flowering photoperiod insensitivity and mechanisms of post-flowering photoperiod responses in soybean.
• Deep sequencing uncovers commonality in small RNA profiles between transgene-induced and naturally occurring RNA silencing of chalcone synthase-A gene in petunia

  Megumi Kasai, Hideo Matsumura, Kentaro Yoshida, Ryohei Terauchi, Akito Taneda, Akira Kanazawa

  BMC genomics 14 63 - 63 2013/01/30 [Refereed]
   

  BACKGROUND: Introduction of a transgene that transcribes RNA homologous to an endogenous gene in the plant genome can induce silencing of both genes, a phenomenon termed cosuppression. Cosuppression was first discovered in transgenic petunia plants transformed with the CHS-A gene encoding chalcone synthase, in which nonpigmented sectors in flowers or completely white flowers are produced. Some of the flower-color patterns observed in transgenic petunias having CHS-A cosuppression resemble those in existing nontransgenic varieties. Although the mechanism by which white sectors are generated in nontransgenic petunia is known to be due to RNA silencing of the CHS-A gene as in cosuppression, whether the same trigger(s) and/or pattern of RNA degradation are involved in these phenomena has not been known. Here, we addressed this question using deep-sequencing and bioinformatic analyses of small RNAs. RESULTS: We analyzed short interfering RNAs (siRNAs) produced in nonpigmented sectors of petal tissues in transgenic petunia plants that have CHS-A cosuppression and a nontransgenic petunia variety Red Star, that has naturally occurring CHS-A RNA silencing. In both silencing systems, 21-nt and 22-nt siRNAs were the most and the second-most abundant size classes, respectively. CHS-A siRNA production was confined to exon 2, indicating that RNA degradation through the RNA silencing pathway occurred in this exon. Common siRNAs were detected in cosuppression and naturally occurring RNA silencing, and their ranks based on the number of siRNAs in these plants were correlated with each other. Noticeably, highly abundant siRNAs were common in these systems. Phased siRNAs were detected in multiple phases at multiple sites, and some of the ends of the regions that produced phased siRNAs were conserved. CONCLUSIONS: The features of siRNA production found to be common to cosuppression and naturally occurring silencing of the CHS-A gene indicate mechanistic similarities between these silencing systems especially in the biosynthetic processes of siRNAs including cleavage of CHS-A transcripts and subsequent production of secondary siRNAs in exon 2. The data also suggest that these events occurred at multiple sites, which can be a feature of these silencing phenomena.
• Induction of RNA-directed DNA methylation and heritable transcriptional gene silencing as a tool to engineer novel traits in plants

  Kasai Megumi, Kanazawa Akira

  Plant Biotechnology 30 (3) 233 - 241 1342-4580 2013 [Refereed]
   

  Gene silencing through transcriptional repression can be induced by double-stranded RNA (dsRNA) that targets a gene promoter. This phenomenon, termed RNA-mediated transcriptional gene silencing (TGS), was first discovered in plants using a transgene that transcribes an inverted repeat of promoter sequence. However, endogenous genes differ from transgenes in the feasibility of TGS induction, by being more resistant to silencing. Heritable, transgenerational silencing of an endogenous gene has been induced by targeting dsRNA to the promoter in petunia and tomato plants, using a vector based on Cucumber mosaic virus. Efficient TGS depends on the function of a viral protein, which can facilitate epigenetic modifications through the transport of short interfering RNA to the nucleus. The efficiency of the TGS also depends on the length and nucleotide composition of the promoter RNA segments. Such epigenetic changes induced by the viral vector results in a novel class of modified plant, a plant that does not carry a transgene but has altered traits. Thus, TGS to modify the epigenetic state of a plant is now a feasible tool to engineer novel traits. Here we review epigenetic changes induced in a particular gene through RNA-directed DNA methylation and those induced randomly on the genome in terms of their use for plant biotechnology.
• An Active CACTA-Family Transposable Element is Responsible for Flower Variegation in Wild Soybean Glycine soja

  Ryoji Takahashi, Yasumasa Morita, Masayoshi Nakayama, Akira Kanazawa, Jun Abe

  PLANT GENOME 5 (2) 62 - 70 1940-3372 2012/07 [Refereed][Not invited]
   

  A plant producing flowers with purple and white variegation was discovered in an accession of Glycine soja Siebold & Zucc. that was introduced from Russia. The mutant line was designated as B00146-m. Lines with white flowers (B00146-w) and purple flowers (B00146-r) were developed from the progeny of B00146-m. The flower color was controlled by the W1 locus encoding a flavonoid 3'5'-hydroxylase (F3'5'H). The allele for variegated flowers was designated as w1-m. The gene symbol was approved by the Soybean Genetics Committee. Polymerase chain reaction (PCR) suggested that a DNA fragment with a molecular size of similar to 3.9 kb was inserted in the first exon of the F3'5'H gene in B00146-m whereas such insertion was not observed in B00146-w and B00146-r. These results suggested that an active mobile element was inserted in the first exon and was responsible for flower variegation. The inserted fragment was identified as a 3883 bp long CACTA-family transposable element and it was designated as Tgs1. Similarity of overall sequence and terminal inverted repeats suggested that Tgs1 and the soybean lectin gene transposable element Tgm1 make up a subgroup. Frequency of germinal reversion was low probably due to the integration into an exon. Tgs1 had a truncated version of the transposase gene and may be a nonautonomous element.
• Complex genetic nature of sex-independent transmission ratio distortion in Asian rice species: the involvement of unlinked modifiers and sex-specific mechanisms

  Y. Koide, Y. Shinya, M. Ikenaga, N. Sawamura, K. Matsubara, K. Onishi, A. Kanazawa, Y. Sano

  HEREDITY 108 (3) 242 - 247 0018-067X 2012/03 [Refereed][Not invited]
   

  Transmission ratio distortion (TRD), in which one allele is transmitted more frequently than the opposite allele, is presumed to act as a driving force in the emergence of a reproductive barrier. TRD acting in a sex-specific manner has been frequently observed in interspecific and intraspecific hybrids across a broad range of organisms. In contrast, sex-independent TRD (SITRD), which results from preferential transmission of one of the two alleles in the heterozygote through both sexes, has been detected in only a few plant species. We previously reported an S-6 locus-mediated SITRD, in which the S-6 allele from an Asian wild rice strain (Oryza rufipogon) was transmitted more frequently than the S-6(a) allele from an Asian cultivated rice strain (O. sativa) through both male and female gametes in heterozygous plants. Here, we report on the effect of a difference in genetic background on S-6 locus-mediated SITRD, based on the analysis using near-isogenic lines and the original wild strain as a parental strain for crossing. We found that the degree of TRD through the male gametes varied depending on the genetic background of the female (pistil) plants. Despite the occurrence of TRD through both male and female gametes, abnormality was detected in ovules, but not in pollen grains, in the heterozygote. These results suggest the involvement of unlinked modifiers and developmentally distinct, sex-specific genetic mechanisms in S-6 locus-mediated SITRD, raising the possibility that SITRD driven by a single locus may be affected by multiple genetic factors harbored in natural populations. Heredity (2012) 108, 242-247; doi:10.1038/hdy.2011.64; published online 27 July 2011
• In planta assays involving epigenetically silenced genes reveal inhibition of cytosine methylation by genistein

  Sachiko Arase, Megumi Kasai, Akira Kanazawa

  PLANT METHODS 8 10  1746-4811 2012/03 [Refereed][Not invited]
   

  Background: Cytosine methylation is involved in epigenetic control of gene expression in a wide range of organisms. An increasing number of examples indicate that changing the frequency of cytosine methylation in the genome is a feasible tool to engineer novel traits in plants. Although demethylating effects of compounds have been analyzed in human cultured cells in terms of suppressing cancer, their effect in plant cells has not been analyzed extensively. Here, we developed in planta assay systems to detect inhibition of cytosine methylation using plants that contain a transgene transcriptionally silenced by an epigenetic mechanism. Results: Seeds of two transgenic plants were used: a petunia line that has been identified as a revertant of the co-suppression of the chalcone synthase-A (CHS-A) gene and contains CHS-A transgenes whose transcription is repressed; Nicotiana benthamiana plants that contain the green fluorescent protein (GFP) reporter gene whose transcription is repressed through virus-induced transcriptional gene silencing. Seeds of these plants were sown on a medium that contained a demethylating agent, either 5-azacytidine or trichostatin A, and the restoration of the transcriptionally active state of the transgene was detected in seedlings. Using these systems, we found that genistein, a major isoflavonoid compound, inhibits cytosine methylation, thus restoring transgene transcription. Genistein also restored the transcription of an epigenetically silenced endogenous gene in Arabidopsis plants. Conclusions: Our assay systems allowed us to assess the inhibition of cytosine methylation, in particular of maintenance of methylation, by compounds in plant cells. These results suggest a novel role of flavonoids in plant cells and that genistein is useful for modifying the epigenetic state of plant genomes.
• Coincident sequence-specific RNA degradation of linked transgenes in the plant genome

  Megumi Kasai, Maiko Koseki, Kazunori Goto, Chikara Masuta, Shiho Ishii, Roger P. Hellens, Akito Taneda, Akira Kanazawa

  PLANT MOLECULAR BIOLOGY 78 (3) 259 - 273 0167-4412 2012/02 [Refereed][Not invited]
   

  The expression of transgenes in plant genomes can be inhibited by either transcriptional gene silencing or posttranscriptional gene silencing (PTGS). Overexpression of the chalcone synthase-A (CHS-A) transgene triggers PTGS of CHS-A and thus results in loss of flower pigmentation in petunia. We previously demonstrated that epigenetic inactivation of CHS-A transgene transcription leads to a reversion of the PTGS phenotype. Although neomycin phosphotransferase II (nptII), a marker gene co-introduced into the genome with the CHS-A transgene, is not normally silenced in petunia, even when CHS-A is silenced, here we found that nptII was silenced in a petunia line in which CHS-A PTGS was induced, but not in the revertant plants that had no PTGS of CHS-A. Transcriptional activity, accumulation of short interfering RNAs, and restoration of mRNA level after infection with viruses that had suppressor proteins of gene silencing indicated that the mechanism for nptII silencing was posttranscriptional. Read-through transcripts of the CHS-A gene toward the nptII gene were detected. Deep-sequencing analysis revealed a striking difference between the predominant size class of small RNAs produced from the read-through transcripts (22 nt) and that from the CHS-A RNAs (21 nt). These results implicate the involvement of read-through transcription and distinct phases of RNA degradation in the coincident PTGS of linked transgenes and provide new insights into the destabilization of transgene expression.
• Establishment of an efficient in vitro culture and particle bombardment-mediated transformation systems in Miscanthus sinensis Anderss., a potential bioenergy crop

  Xun Wang, Tetsuya Yamada, Fan-Jiang Kong, Yuki Abe, Yoichiro Hoshino, Hiroko Sato, Tadashi Takamizo, Akira Kanazawa, Toshihiko Yamada

  GLOBAL CHANGE BIOLOGY BIOENERGY 3 (4) 322 - 332 1757-1693 2011/08 [Refereed][Not invited]
   

  Plants belonging to the genus Miscanthus are considered promising bioenergy crops. Here, we report the establishment of tissue culture system through particle bombardment-mediated transformation in Miscanthus sinensis Anderss. Callus was induced efficiently from mature seeds in a medium containing a combination of a relatively high level of 2,4-dichlorophenoxyacetic acid (5 mg L-1) and a relatively low level of 6-benzyladenine (BA) (0.01 mg L-1). Callus induction potential of 18 accessions of M. sinensis, which were collected from various sites in Japan, was compared. Significant correlation was detected between compact (embryogenic) callus induction frequency and average annual air temperature in collection sites. An accession from Tanegashima Island showed the highest production of compact callus. We found that a higher level of BA causes callus browning; the 2 mg L-1 BA is the optimal concentration for regeneration. Both compact and friable calli were suitable for particle bombardment transformation. Through selection under the presence of 50 mg L-1 hygromycin for 3 weeks and further selection under the presence of 150 mg L-1 for 1 month, hygromycin-resistant calli survived, of which 72.2% had been entirely transformed. Plants were regenerated from calli in the presence of hygromycin; transcripts of the hpt and gfp genes, which were cobombarded to the calli, were detected in the regenerated plants. This is the first report on the establishment of the in vitro culture of M. sinensis using mature seeds, the variation of callus formation among accessions collected from various sites in Japan, and particle bombardment-mediated transformation in the genus Miscanthus.
• Size and positional effects of promoter RNA segments on virus-induced RNA-directed DNA methylation and transcriptional gene silencing

  Shungo Otagaki, Miou Kawai, Chikara Masuta, Akira Kanazawa

  EPIGENETICS 6 (6) 681 - 691 1559-2294 2011/06 [Refereed][Not invited]
   

  DNA methylation at a gene promoter can be triggered by double-stranded RNAs (dsRNAs) through the RNA-directed DNA methylation (RdDM) pathway and induces transcriptional gene silencing (TGS). Although genes involved in the RdDM pathway have been identified, whether dsRNAs of different promoter regions have different extent of effects on RdDM and/or TGS is unknown. Here, we addressed this question by targeting the CaMV 35S promoter in the plant genome using a recombinant Cucumber mosaic virus that contained various portions of the promoter. The efficiency of the induction of TGS depended on the length of the promoter segment triggering the RdDM; the lower size limit for TGS induction was 81-91 nt. TGS was induced when 70-nt fragments were connected in tandem, none of which solely induced TGS. TGS induction did not simply depend on the production of small interfering RNAs corresponding to the promoter. Along with the induction of RdDM, spreading of DNA methylation from the originally targeted site toward the adjacent regions was detected. The maintenance of TGS in the progeny that lacks an RNA trigger depended on the promoter segments triggering the RdDM in the former generation and was correlated with the number of cytosines at symmetrical sites in the targeted region. These results indicate that both the length of dsRNA above the threshold and the frequency of cytosines at symmetric sites in the region targeted by dsRNA are the major factors that allow induction of heritable TGS via RdDM.
• Optimization of ion-beam irradiation for mutagenesis in soybean: effects on plant growth and production of visibly altered mutants

  Sachiko Arase, Yoshihiro Hase, Jun Abe, Megumi Kasai, Tetsuya Yamada, Keisuke Kitamura, Issay Narumi, Atsushi Tanaka, Akira Kanazawa

  PLANT BIOTECHNOLOGY 28 (3) 323 - 329 1342-4580 2011 [Refereed][Not invited]
   

  Ion-beam irradiation is attracting increasing attention as a new mutagen. Here, we describe for the first time the dose response and mutagenic effects of ion-beam irradiation in soybean. We irradiated the hilum side of dried mature soybean seeds with 320-MeV carbon ions within a 0.25-20-Gy range. The growth or seed production of the irradiated plants was profoundly affected. In particular, the number of plants that survived until seed-set decreased with the increase of the irradiation dose and was very low in plants irradiated at doses higher than 5.0 Gy, whereas the frequency distribution of the number of seeds produced by each seed-setting plant was not affected by lower doses of irradiation. Based on these results, we produced plant populations irradiated at 2.5 Gy and 5.0 Gy on a large scale to obtain M 2 seeds. Despite the duplicate composition of the soybean genome, which originated from tetraploids, chlorophyll-deficient mutants were detected with a frequency of 0.47% in the M(2) generation of plants irradiated at 5.0 Gy. These results demonstrate that irradiation of the hilum side of dried soybean seeds with carbon-ion beams at a dose range around 2.5-5.0 Gy induces genetic changes while also allowing the production of a considerable number of seed-setting plants, suggesting that these irradiation conditions are suitable for producing a mutant population potentially useful for breeding and/or identifying gene function.
• Virus-mediated efficient induction of epigenetic modifications of endogenous genes with phenotypic changes in plants

  Akira Kanazawa, Jun-ichi Inaba, Hanako Shimura, Shungo Otagaki, Sayuri Tsukahara, Akihiko Matsuzawa, Bo Min Kim, Kazunori Goto, Chikara Masuta

  PLANT JOURNAL 65 (1) 156 - 168 0960-7412 2011/01 [Refereed][Not invited]
   

  P>Gene silencing through transcriptional repression can be induced by targeting double-stranded RNA (dsRNA) to a gene promoter. It has been reported that a transgene was silenced by targeting dsRNA to the promoter, and the silenced state was inherited to the progeny plant even after removal of the silencing inducer from cells. In contrast, no plant has been produced that harbors silenced endogenous gene after removal of promoter-targeting dsRNA. Here, we show that heritable gene silencing can be induced by targeting dsRNA to the endogenous gene promoters in petunia and tomato plants, using the Cucumber mosaic virus (CMV)-based vector. We found that efficient silencing of endogenous genes depends on the function of the 2b protein encoded in the vector virus, which has the ability to facilitate epigenetic modifications through the transport of short interfering RNA to nucleus. Bisulfite sequencing analyses on the targeted promoter in the virus-infected and its progeny plants revealed that cytosine methylation was found not only at CG or CNG but also at CNN sites. The observed inheritance of asymmetric DNA methylation is quite unique, suggesting that plants have a mechanism to maintain even asymmetric methylation. This CMV-based gene silencing system provides a useful tool to artificially modify DNA methylation in plant genomes and elucidate the mechanism for epigenetic controls.
• RNA-mediated epigenetic modifications of an endogenous gene targeted by a viral vector: A potent gene silencing system to produce a plant that does not carry a transgene but has altered traits

  Akira Kanazawa, Jun-ichi Inaba, Megumi Kasai, Hanako Shimura, Chikara Masuta

  Plant Signaling and Behavior 6 (8) 1090 - 1093 1559-2324 2011 [Refereed][Not invited]
   

  Nucleotide-sequence-specific interactions mediated by double-stranded RN A (dsRN A) can induce gene silencing. Gene silencing through transcriptional repression can be induced by dsRN A targeted to a gene promoter. However, until recently, no plant has been produced that harbors an endogenous gene that remains silenced in the absence of promoter-targeting dsRN A. We have reported for the first time that transcriptional gene silencing can be induced by targeting dsRN A to the endogenous gene promoters in petunia and tomato plants, using a Cucumber mosaic virus (CMV)-based vector and that the induced gene silencing is heritable. Efficient silencing depended on the function of the 2b protein encoded in the vector, which facilitates epigenetic modifications through the transport of short interfering RN A (siRN A) to the nucleus. Here we show that gene silencing that is mediated by targeting dsRN A to a gene promoter via the CMV vector can be as strong as co-suppression in terms of both the extent of mRN A decrease and phenotypic changes. We also show that the expression of genes involved in RN A-directed DNA methylation and in demethylation are upregulated and downregulated, respectively, in Arabidopsis plants infected with CMV. Thus, along with the function of the 2b protein, that transports siRN A to the nucleus, the promoter-targeted silencing system using the CMV vector has some property that facilitates heritable epigenetic changes on endogenous genes, enabling the production of a novel class of modified plants that do not have a transgene. © 2011 Landes Bioscience.
• RNA silencing as a tool to uncover gene function and engineer novel traits in soybean

  Megumi Kasai, Akira Kanazawa

  BREEDING SCIENCE 61 (5) 468 - 479 1344-7610 2011/01 [Refereed][Not invited]
   

  RNA silencing refers collectively to diverse RNA-mediated pathways of nucleotide-sequence-specific inhibition of gene expression. It has been used to analyze gene function and engineer novel traits in various organisms. Here, we review the application of RNA silencing in soybean. To produce soybean lines, in which a particular gene is stably silenced, researchers have frequently used a transgene that transcribes inverted repeats of a target gene segment. Suppression of gene expression in developing soybean embryos has been one of the main focuses of metabolic engineering using transgene-induced silencing. Plants that have enhanced resistance against diseases caused by viruses or cyst nematode have also been produced. Meanwhile, Agrobacterium rhizogenes-mediated transformation has been used to induce RNA silencing in roots, which enabled analysis of the roles of gene products in nodulation or disease resistance. RNA silencing has also been induced using viral vectors, which is particularly useful for gene function analysis. So far, three viral vectors for virus-induced gene silencing have been developed for soybean. One of the features of the soybean genome is the presence of a large number of duplicated genes. Potential use of RNA silencing technology in combination with forward genetic approaches for analyzing duplicated genes is discussed.
• Two Coordinately Regulated Homologs of FLOWERING LOCUS T Are Involved in the Control of Photoperiodic Flowering in Soybean

  Fanjiang Kong, Baohui Liu, Zhengjun Xia, Shusei Sato, Bo Min Kim, Satoshi Watanabe, Tetsuya Yamada, Satoshi Tabata, Akira Kanazawa, Kyuya Harada, Jun Abe

  PLANT PHYSIOLOGY 154 (3) 1220 - 1231 0032-0889 2010/11 [Refereed][Not invited]
   

  FLOWERING LOCUS T (FT) is a key flowering integrator in Arabidopsis (Arabidopsis thaliana), with homologs that encode florigens in many plant species regardless of the type of photoperiodic response. We identified 10 FT homologs, which were arranged as five pairs of linked genes in different homoeologous chromosomal regions, in soybean (Glycine max), a paleopolyploid species. Two of the FT homologs, GmFT2a and GmFT5a, were highly up-regulated under short-day (SD) conditions (inductive for flowering in soybean) and had diurnal expression patterns with the highest expression 4 h after dawn. Under long-day (LD) conditions, expression of GmFT2a and GmFT5a was down-regulated and did not follow a diurnal pattern. Flowering took much longer to initiate under LD than under SD, and only the GmFT5a transcript accumulated late in development under LD. Ectopic expression analysis in Arabidopsis confirmed that both GmFT2a and GmFT5a had the same function as Arabidopsis FT, but the effect of GmFT5a was more prominent. A double-mutant soybean line for two PHYTOCHROME A (PHYA) genes expressed high levels of GmFT2a and GmFT5a under LD, and it flowered slightly earlier under LD than the wild type grown under SD. The expression levels of GmFT2a and GmFT5a were regulated by the PHYA-mediated photoperiodic regulation system, and the GmFT5a expression was also regulated by a photoperiod-independent system in LD. Taken together, our results suggest that GmFT2a and GmFT5a coordinately control flowering and enable the adaptation of soybean to a wide range of photoperiodic environments.
• The Soybean Stem Growth Habit Gene Dt1 Is an Ortholog of Arabidopsis TERMINAL FLOWER1

  Baohui Liu, Satoshi Watanabe, Tomoo Uchiyama, Fanjiang Kong, Akira Kanazawa, Zhengjun Xia, Atsushi Nagamatsu, Maiko Arai, Tetsuya Yamada, Keisuke Kitamura, Chikara Masuta, Kyuya Harada, Jun Abe

  PLANT PHYSIOLOGY 153 (1) 198 - 210 0032-0889 2010/05 [Refereed][Not invited]
   

  Classical genetic analysis has revealed that the determinate habit of soybean (Glycine max) is controlled by a recessive allele at the determinate stem (Dt1) locus. To dissect the molecular basis of the determinate habit, we isolated two orthologs of pea (Pisum sativum) TERMINAL FLOWER1a, GmTFL1a and GmTFL1b, from the soybean genome. Mapping analysis indicated that GmTFL1b is a candidate for Dt1. Despite their high amino acid identity, the two genes had different transcriptional profiles. GmTFL1b was expressed in the root and shoot apical meristems (SAMs), whereas GmTFL1a was mainly expressed in immature seed. The GmTFL1b transcript accumulated in the SAMs during early vegetative growth in both the determinate and indeterminate lines but thereafter was abruptly lost in the determinate line. Introduction of the genomic region of GmTFL1b from the indeterminate line complemented the stem growth habit in the determinate line: more nodes were produced, and flowering in the terminal raceme was delayed. The identity between Dt1 and GmTFL1b was also confirmed with a virus-induced gene silencing experiment. Taken together, our data suggest that Dt1 encodes the GmTFL1b protein and that the stem growth habit is determined by the variation of this gene. The dt1 allele may condition the determinate habit via the earlier loss in GmTFL1b expression concomitant with floral induction, although it functions normally under the noninductive phase of flowering. An association test of DNA polymorphisms with the stem growth habit among 16 cultivars suggested that a single amino acid substitution in exon 4 determines the fate of the SAM after floral induction.
• Fertility restoration by Ifr1 in rice with BT-type cytoplasmic male sterility is associated with a reduced level, but not processing, of atp6-orf79 co-transcribed RNA

  Haruka Ohta, Atsushi Ogino, Megumi Kasai, Yoshio Sano, Akira Kanazawa

  PLANT CELL REPORTS 29 (4) 359 - 369 0721-7714 2010/04 [Refereed][Not invited]
   

  BT-type cytoplasmic male sterility (CMS) in rice is associated with accumulation of unprocessed dicistronic RNA containing a duplicated atp6 (B-atp6) and an unusual open reading frame, orf79, encoding a cytotoxic peptide in mitochondria. The male-sterile state of BT-type CMS is stably maintained by backcrossing the plants with line Taichung 65 (T65) that has no restorer gene and is completely suppressed by the presence of the Rf1 gene through the processing of B-atp6-orf79 RNA. A variant of the T65 line, T65(T), has a weak restoration function conferred by the Ifr1 gene, which is genetically independent of the Rf1 gene. However, not much is known about the mechanism(s). In a study to examine whether the mechanism involved in fertility restoration by Ifr1 is analogous to restoration mediated by Rf1, the transcript profile of B-atp6-orf79 in male-sterile plants was compared with that in fertility restored plants obtained by crossing male-sterile plants with T65(T). The cellular level of unprocessed B-atp6-orf79 RNA was reduced in the restored plants, but no change in processing efficiency or the quantity of B-atp6-orf79 DNA was detected. These results suggest that Ifr1 restores fertility through reducing either the transcription rate of B-atp6-orf79 or the stability of its primary transcripts, a mechanism distinct from that involved in fertility restoration of BT-type CMS by Rf1.
• Adaptive evolution as a consequence of gene duplication and subsequent insertion of a novel Ty1/copia-like retrotransposon in soybean

  Kanazawa Akira, Liu Baohui, Kong Fanjiang, Arase Sachiko, Abe Jun

  GENES & GENETIC SYSTEMS 84 (6) 465  1341-7568 2009/12 [Refereed][Not invited]
  
• Adaptive Evolution Involving Gene Duplication and Insertion of a Novel Ty1/copia-Like Retrotransposon in Soybean

  Akira Kanazawa, Baohui Liu, Fanjiang Kong, Sachiko Arase, Jun Abe

  JOURNAL OF MOLECULAR EVOLUTION 69 (2) 164 - 175 0022-2844 2009/08 [Refereed][Not invited]
   

  Gene duplication is a major force for generating evolutionary novelties that lead to adaptations to environments. We previously identified two paralogs encoding phytochrome A (phyA), GmphyA1 and GmphyA2, in soybean, a paleopolyploid species. GmphyA2 is encoded by the E4 locus responsible for photoperiod sensitivity. In photoperiod insensitive lines, GmphyA2 is inactivated by the insertion of a retrotransposon in exon 1. Here, we describe the detailed characterization of the element and its evolutionary significance inferred from the distribution of the allele that harbors the element. Structural characteristics indicated that the element, designated SORE-1, is a novel Ty1/copia-like retrotransposon in soybean, which was phylogenetically related to the Sto-4, BARE-1, and RIRE1 elements. The element was transcriptionally active, and the transcription was partially repressed by an epigenetic mechanism. Sequences homologous with SORE-1 were detected in a genome sequence database of soybean, most of which appeared silent. GmphyA2 that harbors the SORE-1 insertion was detected only in cultivated soybean lines grown in northern regions of Japan, consistent with the notion that photoperiod insensitivity caused by the dysfunction of GmphyA2 is one of genetic changes that allowed soybean cultivation at high latitudes. Taking into account that genetic redundancy is conferred by the two phyA genes, we propose a novel model for the consequences of gene duplication and transposition of retrotransposons: when the gene is duplicated, retrotransposon insertion that causes the loss of a gene function can lead to adaptive evolution while the organism is sustained by the buffering effect brought about by gene duplication.
• Down-regulation of flavonoid 3′-hydroxylase gene expression by virus-induced gene silencing in soybean reveals the presence of a threshold mRNA level associated with pigmentation in pubescence

  Atsushi Nagamatsu, Chikara Masuta, Hideyuki Matsuura, Keisuke Kitamura, Jun Abe, Akira Kanazawa

  Journal of Plant Physiology Elsevier BV 166 (1) 32 - 39 0176-1617 2009/01 [Refereed]
  
• Spatial and temporal control of transcription of the soybean beta-conglycinin alpha subunit gene is conferred by its proximal promoter region and accounts for the unequal distribution of the protein during embryogenesis

  Yusuke Imoto, Tetsuya Yamada, Keisuke Kitamura, Akira Kanazawa

  GENES & GENETIC SYSTEMS 83 (6) 469 - 476 1341-7568 2008/12 [Refereed][Not invited]
   

  Differentiation into specific embryo cell types correlates with the processes that lead to the accumulation of seed storage proteins in plants. The a subunit of beta-conglycinin, a major component of seed storage proteins in soybean, accumulates at a higher level in cotyledons than in the embryonic axis in developing embryos. To understand the mechanisms underlying this phenomenon, we characterized the upstream region of the a subunit gene in terms of transcriptional control using transgenic Arabidopsis thaliana plants carrying reporter gene constructs comprising the 1357-bp upstream sequence of the a subunit gene and the P-glucuronidase (GUS) gene. Analysis of the time-course-dependent pattern of GUS expression revealed that the expression was first confined to the cotyledons and occurred later in the entire embryo during embryogenesis. The level of GUS expression was higher in cotyledons than in the embryonic axis throughout the period of its expression, coincident with the distribution of the a subunit protein in soybean embryos. By testing progressively shorter promoter fragments, the cis-acting elements responsible for transcriptional activation in the cotyledons and the embryonic axis were both localized to the region spanning -245 to -161 relative to the transcription start site. It is also concluded that the upstream region up to -245 is sufficient to control the spatial and temporal pattern of transcription, while further upstream regions influence transcription rate without affecting the transcriptional pattern. Overall, these results indicate that the unequal distribution of a subunit protein within the embryos is established primarily as a consequence of differential transcriptional activation controlled by a short proximal promoter region of the gene in different embryonic tissues.
• RNA silencing of a gene involved in flavonoid synthesis reveals the presence of a threshold mRNA level associated with pigmentation in plant tissues

  Nagamatsu Atsushi, Masuta Chikara, Matsuura Hideyuki, Kitamura Keisuke, Abe Jun, Kanazawa Akira

  GENES & GENETIC SYSTEMS 83 (6) 515  1341-7568 2008/12 [Refereed][Not invited]
  
• Genetic Redundancy in Soybean Photoresponses Associated With Duplication of the Phytochrome A Gene

  Baohui Liu, Akira Kanazawa, Hisakazu Matsumura, Ryoji Takahashi, Kyuya Harada, Jun Abe

  GENETICS 180 (2) 995 - 1007 0016-6731 2008/10 [Refereed][Not invited]
   

  Gene and genome duplications underlie the origins of evolutionary novelty in plants. Soybean, Glycine max, is considered to be a paleopolyploid species with a complex genome. We found multiple homologs of the phytochrome A gene (phyA) in the soybean genome and determined the DNA sequences of two paralogs designated GmphyA1 and GmphyA2. Analysis of the GmphyA2 gene from the lines carrying a recessive allele at a photoperiod insensitivity locus, E4, revealed that a Ty1/copia-like retrotransposon was inserted in exon 1 of the gene, which resulted in dysfunction of the gene. Mapping studies suggested that GmphyA2 is encoded by E4. The GmphyA1 gene was mapped to a region of linkage group 0, which is homeologous to the region harboring E4 in linkage group I. Plants homozygous for the e4 allele were etiolated under continuous far red light, but the de-etiolation Occurred partially, indicating that the mutation alone did not cause a complete loss of phyA function. The genetic redundancy suggests that the presence of duplicated copies of phyA genes accounts for the generation of photoperiod insensitivity, while protecting against the deleterious effects Of Mutation. Thus, this phenomenon provides a link between gene duplication and establishment of an adaptive response of plants to environments.
• The evolution of sex-independent transmission ratio distortion involving multiple allelic interactions at a single locus in rice

  Yohei Koide, Mitsunobu Ikenaga, Noriko Sawamura, Daisuke Nishimoto, Kazuki Matsubara, Kazumitsu Onishi, Akira Kanazawa, Yoshio Sano

  GENETICS 180 (1) 409 - 420 0016-6731 2008/09 [Refereed][Not invited]
   

  Transmission ratio distortion (TRD) is frequently observed in inter-and intraspecific hybrids of plants, leading to a violation of Mendelian inheritance. Sex-independent TRD (siTRD) was detected in a hybrid between Asian cultivated rice and its wild ancestor. Here we examined how siTRD is controlled by the S-6 locus via a mechanism in which the S-6 allele acts as a gamete eliminator, and both the male and female gametes possessing the opposite allele (S-6(a)) are aborted only in heterozygotes (S-6/S-6(a)). Fine mapping revealed that the S-6 locus is located neaar the centromere of chromosome 6. Tescross experiments using near-isogenic lines (NILs) carrying either the S-6 or S-6(a) alleles revealed that Asian rice strains frequently harbor an additional allele (S-6(n)) the presence of which, in heterozygotic states (S-6/S-6(n) and S-6(a)/S-6(n)), does not result in siTRD. A prominent reduction in the nucleotide diversity of S-6 or S-6(a) carries relative to that of S-6(n) carries was detected in the chromosomal region. These results suggest that the two incompatible alleles (S-6/S-6(a)) arose independently from S-6(n) and established genetically discontinuous relationships between limited constituents of the Asian rice population.
• RNA silencing manifested as visibly altered phenotypes in plants

  Akira Kanazawa

  PLANT BIOTECHNOLOGY 25 (5) 423 - 435 1342-4580 2008 [Refereed][Not invited]
   

  Eukaryotes have a mechanism of RNA-guided regulation of gene expression in which double-stranded RNA inhibits the expression of genes with complementary nucleotide sequences. This mechanism plays a crucial role in many processes including development, stability of the genome, and responses against invading genetic materials. These RNA-guided pathways that control gene expression, collectively termed RNA silencing, are thought to have evolved as a form of innate immunity against viruses. RNA silencing provides a mechanism for downregulating gene expression and a tool that is suitable for analyzing gene function and engineering novel traits in organisms. The phenomenon of RNA silencing was discovered in transgenic petunia plants that had altered patterns of flower color as a consequence of overexpression of the chalcone synthase-A gene responsible for an essential enzyme to biosynthesize anthocyanins. After the "visual" discovery of RNA silencing in petunia, visible phenotypes have played an important role in monitoring the silenced state of a gene in various RNA silencing systems. In particular, a photobleached phenotype in leaf tissues is useful in optimizing a virus-induced gene silencing system. Loss of pigmentation in plant tissues has also led to the detection of naturally occurring RNA-silencing phenomena. Visual changes conferred by endogenous reporter genes provide highly informative assessments of RNA silencing that can be applied to a wide spectrum of plant biotechnology.
• Coordinated expression of functionally diverse fructosyltransferase genes is associated with fructan accumulation in response to low temperature in perennial ryegrass

  Hiroshi Hisano, Akira Kanazawa, Midori Yoshida, Mervyn O. Humphreys, Masaru Iizuka, Keisuke Kitamura, Toshihiko Yamada

  NEW PHYTOLOGIST 178 (4) 766 - 780 0028-646X 2008 [Refereed][Not invited]
   

  Fructan is the major nonstructural carbohydrate reserve in temperate grasses. To understand regulatory mechanisms in fructan synthesis and adaptation to cold environments, the isolation, functional characterization and genetic mapping of fructosyltransferase (FT) genes in perennial ryegrass (Lolium perenne) are described. Six cDNAs (prft1-prft6) encoding FTs were isolated from cold-treated ryegrass plants, and three were positioned on a perennial ryegrass linkage map. Recombinant proteins were produced in Pichia pastoris and enzymatic activity was characterized. Changes in carbohydrate levels and mRNA levels of FT genes during cold treatment were also analysed. One gene encodes sucrose-sucrose 1-fructosyltransferase (1-SST), and two gene encode fructan-fructan 6G-fructosyltransferase (6G-FFT). Protein sequences for the other genes (prfts 1, 2 and 6) were similar to sucrose-fructan 6-fructosyltransferase (6-SFT). The 1-SST and prft1 genes were colocalized with an invertase gene on the ryegrass linkage map. The mRNA levels of prft1 and prft2 increased gradually during cold treatment, while those of the 1-SST and 6G-FFT genes first increased, but then decreased before increasing again during a longer period of cold treatment. Thus at least two different patterns of gene expression have developed during the evolution of functionally diverse FT genes, which are associated in a coordinated way with fructan synthesis in a cold environment.
• Sex-independent transmission ratio distortion system responsible for reproductive barriers between Asian and African rice species

  Yohei Koide, Kazumitsu Onishi, Daisuke Nishimoto, Akhil Ranjan Baruah, Akira Kanazawa, Yoshio Sano

  NEW PHYTOLOGIST 179 (3) 888 - 900 0028-646X 2008 [Refereed][Not invited]
   

  A sex-independent transmission ratio distortion (siTRD) system detected in the interspecific cross in rice was analyzed in order to understand its significance in reproductive barriers. The S(1) gene, derived from African rice Oryza glaberrima, induced preferential abortion of both male and female gametes possessing its allelic alternative (S(1)(a)), from Asian rice O. sativa, only in the heterozygote. The siTRD was characterized by resolving it into mTRD and fTRD occurring through male and female gametes, respectively, cytological analysis of gametophyte development, and mapping of the S(1) locus using near-isogenic lines. The allelic distribution of the S(1) locus in Asian and African rice species complexes was also analyzed. The siTRD system involved at least two components affecting male and female gametogeneses, respectively, including a modifier(s) that enhances fTRD. The chromosomal location of the major component causing the mTRD was delimited within an approx. 40 kb region. The S(1) locus induced hybrid sterility in any pairwise combination between Asian and African rice species complexes. The allelic state of the S(1) locus has diverged between Asian and African rice species complexes, suggesting that the TRD system has a significant role in the reproductive barriers in rice.
• Genetics of speciation in rice

  Yohei Koide, Kazumitsu Onishi, Akira Kanazawa, Yoshio Sano

  Biotechnology in Agriculture and Forestry 62 247 - 259 0934-943X 2008 [Refereed][Not invited]
   

  One of the central issues of evolutionary biology is the origin of the species, although the definition of species is an endlessly debated issue (Coyne and Orr 2004). According to the biological species concept (BSC), a species is a group of an actually or potentially interbreeding natural population, which is reproductively isolated from other such groups (Mayr 1942). No concept of speciation could be complete without a genetic interpretation of the rise of isolating mechanisms. Fitness reduction can range from maladaptation to inviability or sterility. The loci that underlie such reduction in fitness might be considered ‘speciation genes’, which are important in driving the nascent species to become independent genetic entities (Wu and Ting 2004). Therefore, we can analyze the genetic basis of speciation as a more tractable problem by focusing on the genetic basis for reproductive isolation. Recent work on reproductive isolation in Drosophila has advanced our understanding of many fundamental questions about speciation (see review in Coyne and Orr 2004). The BSC can be favorably adopted regarding domesticated plants, and the concept of gene pools based on the degree of their sexual affinities is useful for their classification (Harlan 1975). Any good species are by no means completely isolated. Wild and cultivated complexes in crops are taxonomically distinct but phylogenetically conspecific. Their genetic differentiation is maintained through disruptive selection associated with habitat adaptation, indicating that domestication proceeds at the intra-specific level under human influence.
• Temporal and spatial regulation of the soybean seed storage protein β-conglycinin α subunit gene promoter in transgenic Arabidopsis thaliana

  Imoto Y, Kitamura K, Kanazawa A

  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan 日本作物学会 (48) 105 - 106 2007/12
• Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing

  Atsushi Nagamatsu, Chikara Masuta, Mineo Senda, Hideyuki Matsuura, Atsushi Kasai, Sung Hong, Keisuke Kitamura, Jun Abe, Akira Kanazawa

  PLANT BIOTECHNOLOGY JOURNAL 5 (6) 778 - 790 1467-7644 2007/11 [Refereed][Not invited]
   

  Virus-induced gene silencing (VIGS) is a powerful tool for functional analysis of genes in plants. A wide-host-range VIGS vector, which was developed based on the Cucumber mosaic virus (CMV), was tested for its ability to silence endogenous genes involved in flavonoid biosynthesis in soybean. Symptomless infection was established using a pseudorecombinant virus, which enabled detection of specific changes in metabolite content by VIGS. It has been demonstrated that the yellow seed coat phenotype of various cultivated soybean lines that lack anthocyanin pigmentation is induced by natural degradation of chalcone synthase (CHS) mRNA. When soybean plants with brown seed coats were infected with a virus that contains the CHS gene sequence, the colour of the seed coats changed to yellow, which indicates that the naturally occurring RNA silencing is reproduced by VIGS. In addition, CHS VIGS consequently led to a decrease in isoflavone content in seeds. VIGS was also tested on the putative flavonoid 3'-hydroxylase (F3'H) gene in the pathway. This experiment resulted in a decrease in the content of quercetin relative to kaempferol in the upper leaves after viral infection, which suggests that the putative gene actually encodes the F3'H protein. In both experiments, a marked decrease in the target mRNA and accumulation of short interfering RNAs were detected, indicating that sequence-specific mRNA degradation was induced. The present report is a successful demonstration of the application of VIGS for genes involved in flavonoid biosynthesis in plants; the CMV-based VIGS system provides an efficient tool for functional analysis of soybean genes.
• Epigenetic inactivation of chalcone synthase-A transgene transcription in petunia leads to a reversion of the post-transcriptional gene silencing phenotype

  Akira Kanazawa, Michael O'Dell, Roger P. Hellens

  PLANT AND CELL PHYSIOLOGY 48 (4) 638 - 647 0032-0781 2007/04 [Refereed][Not invited]
   

  Petunia plants that exhibit a white-flowering phenotype as a consequence of chakone synthase transgene-induced silencing occasionally give rise to revertant branches that produce flowers with wild-type pigmentation. Transcription run-on assays confirmed that the production of white flowers is caused by post-transcriptional gene silencing (PTGS), and indicated that transgene transcription is repressed in the revertant plants, providing evidence that induction of PTGS depends on the transcription rate. Transcriptional repression of the transgene was associated with cytosine methylation at CpG, CpNpG and CpNpN sites, and the expression was restored by treatment with either 5-azacytidine or trichostatin A. These results demonstrate that epigenetic changes occurred in the PTGS line, and these changes interfere with the initiation of transgene transcription, leading to a reversion of the PTGS phenotype.
• The binding of nuclear factors to the as-1 element in the CaMV 35S promoter is affected by cytosine methylation in vitro

  Kanazawa, A, O’Dell, M, Hellens, R. P

  Plant Biology 9 435 - 441 2007 [Refereed]
  
• Relationship between methylation status of a transgene promoter and the stability of transcriptional gene silencing in plants

  Otagaki S, Masuta C, Kanazawa A

  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan 日本作物学会 (47) 49 - 50 2006/12
• Gene expression and genetic mapping analyses of a perennial ryegrass glycine-rich RNA-binding protein gene suggest a role in cold adaptation

  H Shinozuka, H Hisano, S Yoneyama, Y Shimamoto, ES Jones, JW Forster, T Yamada, A Kanazawa

  MOLECULAR GENETICS AND GENOMICS 275 (4) 399 - 408 1617-4615 2006/04 [Refereed][Not invited]
   

  A perennial ryegrass cDNA clone encoding a putative glycine-rich RNA binding protein (LpGRP1) was isolated from a cDNA library constructed from crown tissues of cold-treated plants. The deduced polypeptide sequence consists of 107 amino acids with a single N-terminal RNA recognition motif (RRM) and a single C-terminal glycine-rich domain. The sequence showed extensive homology to glycine-rich RNA binding proteins previously identified in other plant species. LpGRP1-specific genomic DNA sequence was isolated by an inverse PCR amplification. A single intron which shows conserved locations in plant genes was detected between the sequence motifs encoding RNP-1 and RNP-2 consensus protein domains. A significant increase in the mRNA level of LpGRP1 was detected in root, crown and leaf tissues during the treatment of plants at 4 degrees C, through which freezing tolerance is attained. The increase in the mRNA level was prominent at least 2 h after the commencement of the cold treatment, and persisted for at least 1 week. Changes in mRNA level induced by cold treatment were more obvious than those due to treatments with abscisic acid (ABA) and drought. The LpGRP1 protein was found to localise in the nucleus in onion epidermal cells, suggesting that it may be involved in pre-mRNA processing. The LpGRP1 gene locus was mapped to linkage group 2. Possible roles for the LpGRP1 protein in adaptation to cold environments are discussed.
• The regulatory function of the upstream sequence of the beta-conglycinin alpha subunit gene in seed-specific transcription is associated with the presence of the RY sequence

  Michiko Yoshino, Atsushi Nagamatsu, Ken-ichi Tsutsumi, Akira Kanazawa

  GENES & GENETIC SYSTEMS 81 (2) 135 - 141 1341-7568 2006/04 [Refereed][Not invited]
   

  beta-conglycinin, a major component of seed-storage proteins in soybean, comprises three subunits: alpha, alpha', and beta. Expression of these genes is spatially regulated in a stringent manner and occurs during seed development. To understand the mechanisms that control expression of the alpha subunit gene, we analyzed the nucleotide sequence of the 2.9-kb region upstream of the gene. The upstream sequence up to -1357 or a series of its 5'-deleted derivatives was fused to the beta-glucuronidase (GUS) gene. These reporter gene constructs were introduced into Arabidopsis thaliana plants via Agrobacterium-mediated gene transfer. Prominent GUS activity was detected in developing seeds of the T3 generation when 245 bp or longer sequences of the upstream region were fused to the GUS gene. We found a clear association of decreased GUS activity with a stepwise deletion of a region containing the RY sequence from the original construct. These results are consistent with the notion that multiple sequence elements including the RY sequences are involved in the seed-specific transcriptional activation of the beta-conglycinin alpha subunit gene in soybean.
• Rapid induction of transcriptional and post-transcriptional gene silencing using a novel Cucumber mosaic virus vector

  Shungo Otagaki, Makoto Arai, Akiko Takahashi, Kazunori Goto, Jin-Sung Hong, Chikara Masuta, Akira Kanazawa

  Plant Biotechnology 23 (3) 259 - 265 1347-6114 2006 [Refereed][Not invited]
   

  We developed a novel RNA virus vector based on the Cucumber mosaic virus (CMV), which is able to efficiently induce gene silencing in plants. We manipulated the RNA 2 of the CMV Y strain, whose genome consists of tripartite components, and introduced restriction sites for cloning a foreign sequence into the vector. To evaluate the vector (designated CMV2-A1) in terms of the ability to induce gene silencing, we cloned portions of the green fluorescent protein (GFP) cDNA or Cauliflower mosaic virus (CaMV) 35S promoter sequences into the vector and inoculated the infectious transcripts into Nicotiana benthamiana plants that express the GFP gene under the control of the CaMV 35S promoter. In both cases, a loss of GFP fluorescence accompanying a reduction in the level of GFP mRNA was induced. The short interfering RNAs (siRNAs) harboring the sequences inserted in the CMV2-A1 vector were detected in the silenced plants. When plants were infected with the virus containing the CaMV 35S promoter sequence, the CaMV 35S promoter sequence in the genomic DNA was heavily methylated. A reduction in the mRNA level of the GFP gene and loss of GFP fluorescence were induced as early as 6 and 12 days post-inoculation, respectively, earlier than the 20 days previously achieved with a Potato virus X vector. These results suggest that the CMV2-A1 vector is suitable for the rapid induction of both transcriptional and post-transcriptional gene silencing. Copyright © 2006 The Japanese Society for Plant Cell and Molecular Biology.
• Induction and maintenance of transcriptional repression of a transgene by RNA-directed DNA methylation depend on the target sequence in the promoter

  Otagaki S, Hong J S, Goto K, Masuta C, Kanazawa A

  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan 日本作物学会 (46) 65 - 66 2005/12
• The star-type color pattern in Petunia hybrida 'Red star' flowers is induced by sequence-specific degradation of chalcone synthase RNA

  M Koseki, K Goto, C Masuta, A Kanazawa

  PLANT AND CELL PHYSIOLOGY 46 (11) 1879 - 1883 0032-0781 2005/11 [Refereed][Not invited]
   

  Petunia hybrida 'Red Star' is a variety whose flowers exhibit a star-type red and white bicolor pattern. We analyzed the mRNA levels of six genes involved in anthocyanin biosynthesis. Only the level of chalcone synthase (CHS) mRNA was depressed in the unpigmented flower sectors. Both transcriptional activity and the accumulation of short interfering RNA of CHS in the unpigmented sectors were detected. Viral infection blocked the generation of CHS-silenced sectors. These results indicate that sequence-specific degradation of CHS RNA is the primary cause of the formation of white sectors in 'Red Star' flowers.
• Molecular analysis and physicochemical properties of electrophoretic variants of wild soybean Glycine soja storage proteins

  T Fukuda, N Maruyama, A Kanazawa, J Abe, Y Shimamoto, M Hiemori, H Tsuji, T Tanisaka, S Utsumi

  JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 53 (9) 3658 - 3665 0021-8561 2005/05 [Refereed][Not invited]
   

  Cultivated soybeans (Glycine max) are derived from wild soybeans (Glycine soja) and can be crossed with them to produce fertile offspring. The latter exhibit greater genetic variation than the former, suggesting a possibility that wild soybeans contain storage proteins with properties different from and better than those of cultivated soybeans. To identify a wild soybean suitable for breeding a new soybean cultivar, we analyzed seed proteins from 390 lines of wild soybeans by electrophoresis. We found some lines containing electrophoretic variants of glycinin and beta-conglycinin subunits: one line containing a small alpha' subunit of beta-conglycinin and two and five lines containing small A3 and large A4 polypeptides of glycinin, respectively. beta-Conglycinin and glycinin containing such variant subunits exhibited solubility and emulsifying ability similar to those of the predominant types of wild and cultivated soybeans. Glycinins containing small A3 and large A4 gave a shoulder derived from the start of denaturation at a temperature 4 degrees C lower than that of glycinin from the predominant types of wild and cultivated soybeans, although their thermal denaturation midpoint temperatures were very similar to each other. Cloning and sequencing of the predominant and variant subunit cDNAs revealed that the small alpha' and the small A3 lacked 24 amino acid residues in the extension region and four amino acid residues in the hypervariable region, respectively, and that the large A4 did not have an insert corresponding to the difference in the electrophoretic mobility but Arg279 and Gln305 were replaced by glutamine and histidine, respectively, in the hypervariable region. These suggest that small differences even in the hypervariable region can affect the thermal stability, as well as the electrophoretic mobilities, of the proteins.
• Induction of transcriptional repression on a transgene promoter using a plant virus vector.

  Otagaki S, Hong JS, Goto K, Masuta C, Kanazawa A

  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan 日本作物学会 (45) 109 - 110 2004/12
• Transgenic perennial ryegrass plants expressing wheat fructosyltransferase genes accumulate increased amounts of fructan and acquire increased tolerance on a cellular level to freezing

  H Hisano, A Kanazawa, A Kawakami, M Yoshida, Y Shimamoto, T Yamada

  PLANT SCIENCE 167 (4) 861 - 868 0168-9452 2004/10 [Refereed][Not invited]
   

  The accumulation of fructan in grasses during autumn is linked to winter hardiness. Genetic manipulation of the accumulation of fructan could be an important molecular breeding strategy for the improvement of winter hardiness in grasses. We produced transgenic perennial ryegrass (Lolium perenne) plants that overexpress wheat fructosyltransferase genes, wft1 and wft2, which encode sucrose-fructan 6-fructosyltransferase (6-SFT) and sucrose-sucrose 1-fructosyltransferase (1-SST), respectively, under the control of CaMV 35S promoter using a particle bombardment-mediated method of transformation. Significant increases in fructan content were detected in the transgenic perennial ryegrass plants. A freezing test using the electrical conductivity method indicated that transgenic plants that accumulated a greater amount of fructan than non-transgenic plants have increased tolerance on a cellular level to freezing. The results suggest that the overexpression of the genes involved in fructan synthesis serves as a novel strategy to produce freezing-tolerant grasses. (C) 2004 Elsevier Ireland Ltd. All rights reserved.
• Changes in the Structural Properties of Chloroplasts and Mitochondria in Response to Low Temperature in Perennial Ryegrass (Lolium perenne L.)

  Tominaga Yoko, Kanazawa Akira, Shimamoto Yoshiya

  Journal of Japanese Society of Grassland Science 日本草地学会 50 (1) 31 - 39 0447-5933 2004/04/15 [Refereed][Not invited]
   

  Changes in the ultrastructural properties of chloro-plasts and mitochondria in response to low temperature were analyzed by transmission electron microscopy in perennial ryegrass (Lolium perenne L.). Specimens from plants treated at 4℃ for 24 hr revealed increases in the sizes of chloroplasts in mesophyll cells and mitochondria in mesophyll and vascular bundle sheath cells. Longer cold treatments caused smaller amount of changes in the sizes of these organelles. The enlargement of chloroplasts in mesophyll cells was accompanied by a proportional decrease in the size of vacuoles. The lamella...
• Marker-assisted analysis for soybean hard seededness with isozyme and simple sequence repeat loci

  SI Sakamoto, J Abe, A Kanazawa, Y Shimamoto

  BREEDING SCIENCE 54 (2) 133 - 139 1344-7610 2004 [Refereed][Not invited]
   

  We determined the genomic regions of wild soybean (Glycine max subsp. soja) carrying quantitative trait loci (QTL) for hard seededness, a distinct character between the wild and cultivated soybeans. The segregation of hard seededness, as evaluated using seed coat permeability (SCP), was examined in the progeny of a cross between wild and cultivated soybeans. The high parent-offspring correlation coefficient between F-2 and F-3 indicated that SCP was a highly heritable character. A marker-assisted analysis of five isozyme and 131 simple sequence repeat (SSR) loci revealed that at least two major and one minor QTL were involved in controlling the trait. The QTL near an SSR marker (Satt459) in molecular linkage group D1b + W had the greatest effect on SCP, and accounted for 23.8 and 38.5% of the phenotypic variance observed in the F2 and F3 mean, respectively. Seed coat color, determined by a locus inhibiting seed coat color (I) and a pubescence color locus (7), was also closely associated with SCP in F-4 to F-6, although seed coat color did not appear to be associated with SCP in the F-2. The results of this and a previous study suggest that different sets of QTL contribute to the phenotypic difference of seed coat permeability in wild and cultivated soybeans to different degrees.
• High frequency Agrobacterium-mediated transformation and plant regeneration via direct shoot formation from leaf explants in Beta vulgaris and Beta maritima

  Hisano, H, Kimoto, Y, Hayakawa, H, Takeichi, J, Domae, T, Hashimoto, R, Abe, J, Asano, S, Kanazawa, A, Shimamoto, Y

  Plant Cell Reports Springer Berlin / Heidelberg 22 (12) 910 - 918 0721-7714 2004 [Refereed][Not invited]
   

  Communicated by H. UchimiyaAbstract We have developed a new procedure for Agrobacterium-mediated transformation of plants in the genus Beta using shoot-base as the material for Agrobacterium infection. The frequency of regeneration from shoot bases was analyzed in seven accessions of sugarbeet (Beta vulgaris) and two accessions of B. maritima to select materials suitable for obtaining transformed plants. The frequency of transformation of the chosen accessions using Agrobacterium strain LBA4404 and selection on 150-mg/l kanamycin was found to be higher than that in previously published methods. Genomic DNA analysis and -glucuronidase reporter assays showed that the transgene was inherited and expressed in subsequent generations. In our method, shoot bases are prepared by a simple procedure, and transformation does not involve the callus phase, thus minimizing the occurrence of somaclonal variations.
• Phylogenetic relationships of the chloroplast genomes in the genus Glycine inferred from four intergenic spacer sequences

  M Sakai, A Kanazawa, A Fujii, FS Thseng, J Abe, Y Shimamoto

  PLANT SYSTEMATICS AND EVOLUTION 239 (1-2) 29 - 54 0378-2697 2003/07 [Refereed][Not invited]
   

  The nucleotide sequences of four intergenic spacer regions of chloroplast DNA, atpB-rbcL, trnS-trnG, rps11-rpl36, and rps3-rpl16, were analyzed in the genus Glycine. Phylogenetic analysis based on the sequence data using Neonotonia wightii as the outgroup generated trees supporting the classification of two subgenera, Soja and Glycine, and three plastome groups in the subgenus Glycine. The results were consistent with the presence of diversified chloroplast genomes within tetraploid plants of G. tabacina and G. tomentella, as well as with a close relationship between G. tomentella and G. dolichocarpa that had been suggested based on morphological analyses. Little sequence variation was found in the subgenus Soja, suggesting that G. soja rapidly expanded its distribution in East Asia. The analysis also showed that the differentiation into three plastome groups in the subgenus Glycine occurred in the early stages of its evolution, after the two subgenera diverged.
• Photoperiod-insensitive Japanese soybean landraces differ at two maturity loci

  Abe, J, Xu, D, Miyano, A, Komatsu, K, Kanazawa, A, Shimamoto, Y

  Crop Science 43 1300 - 1304 2003 [Refereed]
  
• A simple and rapid method to detect plant siRNAs using nonradioactive probes

  Goto, K, Kanazawa, A, Kusaba, M, Masuta, C

  Plant Molecular Biology Reporter 21 51 - 58 2003 [Refereed]
  
• Soybean germplasm pools in Asia revealed by nuclear SSRs

  Abe, J, Xu, D. H, Suzuki, Y, Kanazawa, A, Shimamoto, Y

  Theoretical and Applied Genetics 106 445 - 453 2003 [Refereed]
  
• Structural variation around the gene encoding the alpha subunit of soybean beta-conglycinin and correlation with the expression of the alpha subunit

  M Yoshino, A Kanazawa, K Tsutsumi, Nakamura, I, K Takahashi, Y Shimamoto

  BREEDING SCIENCE 日本育種学会 52 (4) 285 - 292 1344-7610 2002/12 [Refereed][Not invited]
   

  We have identified two genes, located approximately 2.5 kb apart in the soybean genome, that are closely related to the alpha subunit of beta-conglycinin. One of these genes has been shown to encode the alpha subunit and the other is very similar to the a subunit gene (referred to as the "alpha-related gene"). To determine whether the latter alpha-related gene expresses the a subunit, the structure of the chromosomal DNA region that contains the two genes was compared among soybean varieties exhibiting different expression levels of the subunit protein. We observed the presence of deletions of the alpha subunit and alpha-related genes in some varieties. In the naturally occurring variety Keburi, which accumulates the a subunit protein, the entire alpha-related gene was deleted in addition to the previously known deletion of the alpha' subunit gene. On the contrary, the alpha subunit gene was deleted in Moshidou Gong 503 and Kari-kei 434. These structural analyses revealed that the alpha-related gene expresses the a subunit at least in Moshidou Gong 503 and Kari-kei 434. These two varieties do not harbour the alpha subunit gene, but carry the intact alpha-related genes and still express the alpha subunit.
• Identification of Cold-responsive Genes in Perennial Ryegrass (Lolium perenne L.) by a Modified Differential Display Method

  TOMINAGA Yoko, KANAZAWA Akira, SHIMAMOTO Yoshiya

  Journal of Japanese Society of Grassland Science 日本草地学会 47 (5) 516 - 519 0447-5933 2001/12/15 [Refereed][Not invited]
  
• Structure and characterization of the gene encoding alpha subunit of soybean beta-conglycinin

  M Yoshino, A Kanazawa, K Tsutsumi, Nakamura, I, Y Shimamoto

  GENES & GENETIC SYSTEMS 日本遺伝学会 76 (2) 99 - 105 1341-7568 2001/04 [Refereed][Not invited]
   

  beta -conglycinin, a soybean seed storage protein, is comprised of three different subunits, alpha, alpha', and beta. Several candidates for the alpha subunit gene have been isolated, however, the structure olf the a subunit gene has not been completely determined. Accordingly, it was also unknown which of the gene candidates are functionally active. Here, we have determined the nucleotide sequence and transcription start site of the alpha subunit genre, and compared the structural components with those of the other subunits or other seed protein genes. The alpha subunit gene, which is located on a 7.6-kb EcoRI fragment, was composed of six exons that had the same organization as those for the alpha' subunit gene. Within a 400 bp upstream region of the transcription start site, four regions (designated as boxes I, II, III, and IV) were found to be conserved among the alpha, alpha', and other seed protein genes. Genomic Southern blot analysis of soybean varieties lacking the alpha subunit gene candidate indicated that the gene characterized in this paper actually encodes the alpha subunit and is functionally active. In addition, these experiments revealed the presence of an additional gene which is also responsible for the expression of the alpha subunit.
• Identification of sequence variations by PCR-RFLP and its application to the evaluation of cpDNA diversity in wild and cultivated soybeans

  Xu, D. H, Abe, J, Kanazawa, A, Gai, J. Y, Shimamoto, Y

  Theoretical and Applied Genetics 102 683 - 688 2001 [Refereed]
  
• Mini-scale method for nuclear run-on transcription assay in plants

  Kanazawa, A, O’Dell, M, Hellens, R. P, Hitchin, E, Metzlaff, M

  Plant Molecular Biology Reporter 18 377 - 383 2000 [Refereed]
  
• Sequence variation of non-coding regions of chloroplast DNA of soybean and related wild species and its implications for the evolution of different chloroplast haplotypes

  Xu, D, Abe, J, Sakai, M, Kanazawa, A, Shimamoto, Y

  Theoretical and Applied Genetics 101 724 - 732 2000 [Refereed]
  
• Inter- and intra-specific distribution of Stowaway transposable elements in AA-genome species of wild rice

  Kanazawa, A, Akimoto, M, Morishima, H, Shimamoto, Y

  Theoretical and Applied Genetics 101 327 - 335 2000 [Refereed]
  
• Soybean recombination sites are present as dispersed segments in Arabidopsis and liverwort mitochondrial DNA

  Kanazawa, A, Shimamoto, Y

  Plant Molecular Biology Reporter 17 19 - 29 1999 [Refereed]
  
• Phylogenetic relationships of the mitochondrial genomes in the genus Glycine subgenus Soja

  A Kanazawa, A Tozuka, S Akimoto, J Abe, Y Shimamoto

  GENES & GENETIC SYSTEMS 73 (4) 255 - 261 1341-7568 1998/08 [Refereed][Not invited]
   

  Restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA (mtDNA) of wild and cultivated soybeans were analyzed to study their phylogenetic relationships. The observed number of differences in hybridization profiles greatly varied (1 - 17 patterns) with both the mtDNA probes and the restriction enzymes that were used. A cladistic analysis was conducted based on the RFLP data. In the parsimonious tree, four distinct groups appeared among 20 accessions of the subgenus Soja representing 20 mitochondrial genome types. Common features with regard to geographic distributions in natural populations in East Asia were observed among the mitochondrial genome types of wild soybean that belonged to the same group: one clade consisted of genome types IIg and VIIg that are detected with very low frequencies; another clade consisted of genome types Ic, Id, Ie, and Ik whose distributions are highly biased mainly in Japan. The genome types that are widely distributed in East Asia such as IVa, IVb, and Va were not grouped into the same clade. The mitochondrial genome types IIIb, IVb, and IVc, in which two different chloroplast genome types exist, belonged to the same clade. Possible changes in mitochondrial genomes during the expansion of the distribution of wild soybeans in East Asia were discussed.
• Rapid Decrease in DNA Content Induced by Gamma Irradiation in Cultured Cells of Tobacco

  KANAZAWA Akira, HATANAKA Mitsuhiro, TSUTSUMI Nobuhiro, EGUCHI Hoshio, HIRAI Atsushi

  Radioisotopes 日本アイソト-プ協会 47 (5) 399 - 404 0033-8303 1998/05 [Refereed][Not invited]
  
• Sequence variation of chloroplast DNA that involves EcoRI and ClaI restriction site polymorphisms in soybean

  A Kanazawa, A Tozuka, Y Shimamoto

  GENES & GENETIC SYSTEMS 73 (2) 111 - 119 1341-7568 1998/04 [Refereed][Not invited]
   

  Restriction fragment length polymorphisms (RFLPs) of EcoRI- and ClaI-digested chloroplast DNA (cpDNA) within the genus Glycine subgenus Soja were characterized. Two mutations were found to be responsible for the EcoRI and ClaI restriction site polymorphisms, and both were located in a region in which many ribosomal protein genes are clustered. This region is within the large single copy region of cpDNA and is located close to an inverted repeat. The locations of restriction sites of EcoRI and ClaI in the cpDNA region were analyzed by DNA gel-blot analyses and PCR amplification, which were followed by sequencing analyses. The EcoRI site polymorphism was found to have occurred in the intergenic spacer between rps11 and rpl36, while the ClaI site polymorphism was located within the 3' part of the coding region of rps3. The mutations that cause EcoRI and ClaI polymorphisms were both found to be single base substitutions. In addition to these polymorphisms, novel sequence variations in soybean cpDNA were detected near the sites of these mutations. Previously, it was shown that cultivated soybeans could be classified into three groups (I, II, and III) based on their cpDNA RFLPs. A comparison of the cpDNA sequences of soybeans in the present study was consistent with the notion that the cpDNA of group II soybeans is an intermediate between the cpDNAs of groups I and III.
• Phylogenetic relationships in the genus Nelumbo based on polymorphism and quantitative variations in mitochondrial DNA

  A Kanazawa, S Watanabe, T Nakamoto, N Tsutsumi, A Hirai

  GENES & GENETIC SYSTEMS 73 (1) 39 - 44 1341-7568 1998/02 [Refereed][Not invited]
   

  Restriction fragment length polymorphisms (RFLPs) of the mitochondrial DNA (mtDNA) of 15 strains of lotus (Nelumbo nucifera and N. pentapetala) were analyzed using 13 rice mtDNA fragments that contained 16 known mitochondrial genes and open reading frames (ORFs) as probes for a DNA gel blot analysis. Five groups appeared as a result of the phylogenetic analysis based on the RFLP data. Ohgahasu (Ohga lotus) and Chuugokukodaibasu (Chinese ancient lotus), strains that were reared from seeds excavated from different ancient deposits were classified into the same group. In the course of this analysis, a unique quantitative variation of mtDNA that indicates a change in the composition of the population of mtDNAs during the evolution of this plant species was detected. In combination with the phylogenetic analysis, the difference in mtDNA composition was found to have generated after the divergence of five strains of N. nucifera from the other seven strains of N. nucifera analyzed in this study.
• Composite and clinal distribution of Glycine soja in Japan revealed by RFLP analysis of mitochondrial DNA

  A Tozuka, H Fukushi, T Hirata, M Ohara, A Kanazawa, T Mikami, J Abe, Y Shimamoto

  THEORETICAL AND APPLIED GENETICS 96 (2) 170 - 176 0040-5752 1998/02 [Refereed][Not invited]
   

  Wild soybean (Glycine soja Sieb. et Zucc.), regarded as the progenitor of cultivated soybean [G. max (L.) Merr.], is widely distributed in East Asia. We have collected 1097 G. soja plants from all over Japan and analyzed restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA (mtDNA) in them. Based on the RFLPs detected by gel-blot analysis, using coxII and atp6 as probes, the collected plants were divided into 18 groups. Five mtDNA types accounted for 94% of the plants examined. The geographic distribution of mtDNA types revealed that, in many regions, wild soybeans grown in Japan consisted of a mixture of plants with different types of mtDNA, occasionally even within sites. Some of the mtDNA types showed marked geographic dines among the regions. Additionally, some wild soybeans possessed mtDNA types that were identical to those widely detected in cultivated soybeans. Our results suggest that the analysis of mtDNA could resolve the maternal lineage among plants of the genus Glycine subgenus Soja.
• Evolutionary changes in the structures of the cox2 and atp6 loci in the mitochondrial genome of soybean involving recombination across small interspersed sequences

  Kato, S, Kanazawa, A, Mikami, T, Shimamoto, Y

  Current Genetics 34 303 - 312 1998 [Refereed]
  
• Multiple changes in tobacco mitochondrial DNA populations during tissue culture

  Kanazawa, A

  Plant Breeding 117 469 - 472 1998 [Refereed]
  
• RFLPs of chloroplast and mitochondrial DNA in wild soybean, Glycine soja, growing in China

  Shimamoto, Y, Fukushi, H, Abe, J, Kanazawa, A, Gai, J, Gao, Z, Xu, D

  Genetic Resources and Crop Evolution 45 433 - 439 1998 [Refereed]
  
• Differential changes in copy numbers of rice mitochondrial plasmid-like DNAs and main mitochondrial genomic DNAs that depend on temperature

  Kanazawa, A, Tsutsumi, N, Hirai, A

  33 437 - 444 1998 [Refereed]
  
• Small interspersed sequences that serve as recombination sites at the cox2 and atp6 loci in the mitochondrial genome of soybean are widely distributed in higher plants

  Kanazawa, A, Tozuka, A, Kato, S, Mikami, T, Abe, J, Shimamoto, Y

  Current Genetics 33 188 - 198 1998 [Refereed]
  
• Changes in the composition of the population of mtDNAs during callus culture and regeneration in tobacco

  Kanazawa A.

  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan 日本作物学会 (38) 96 - 97 1997/12
• Mitochondrial plasmid-like DNAs of the B1 family in the genus Oryza: Sequence heterogeneity and evolution

  S Miyata, A Kanazawa, N Tsutsumi, Y Sano, A Hirai

  JAPANESE JOURNAL OF GENETICS Genetics Society of Japan 70 (6) 675 - 685 0021-504X 1995/12 [Refereed][Not invited]
   

  Four kinds of circular plasmid-like DNA, designated B1, B2, B3 and B4, have been found in the mitochondria of Oryza sativa L. with an AA genome. Three novel B1-homologous mitochondrial plasmid-like DNAs, designated, M1, M2 and M3, were isolated in the present study from strains with CC and CCDD genomes in the genus Oryza. We cloned and sequenced these DNAs and found that the sequences of these molecules have wide regions of homology. B1, M2 and M3 each lack about 300 bp of a region that is present in M1 and small repeats were found at the sites of deleted sequences. Therefore, we propose the hypothesis that the B1 family differentiated from a common ancient molecule that was similar to M1 via, probably, slipped mispairing during DNA replication at several stages in the evolution in the genus Oryza.
• POLYMORPHIC DISTRIBUTION AND MOLECULAR DIVERSIFICATION OF MITOCHONDRIAL PLASMID-LIKE DNAS IN THE GENUS ORYZA

  S MIYATA, A KANAZAWA, N TSUTSUMI, Y SANO, A HIRAI

  JAPANESE JOURNAL OF GENETICS Genetics Society of Japan 70 (5) 601 - 614 0021-504X 1995/10 [Refereed][Not invited]
   

  Four kinds of circular plasmid-like DNA, designated B1, B2, B3 and B4, have been found in the mitochondria of rice (Oryza sativa L.). We analyzed the distribution of families of plasmid-like DNAs homologous to those of O. sativa in 40 strains of the genus Oryza with AA, BE, BBCC, CC, CCDD and EE genomes. Plasmid-like DNAs were observed only strains having AA, CC and CCDD genomes. The distribution patterns of strains with AA genome were highly polymorphic. We amplified the plasmid-like DNAs from strains with the AA genome by PCR and examined restriction fragments length polymorphisms (RFLPs). RFLPs were detected among families of plasmid-like DNA amplified from different strains. This result indicated that some mutations, such as base substitutions and the insertion or deletion of a small fragment of DNA, had occurred and had accumulated during the differentiation of strains with an AA genome.
• Reversible changes in the composition of the population of mtDNAs during dedifferentiation and regeneration in tobacco

  Kanazawa, A, Tsutsumi, N, Hirai, A

  Genetics 138 865 - 870 1994 [Refereed]
  
• イネ・ミトコンドリアのプラスミド様DNA

  金沢 章, 平井 篤志

  バイオサイエンスとインダストリ- バイオインダストリ-協会 51 (5) p404 - 406 0914-8981 1993/05 [Refereed][Not invited]
  
• Restriction fragments homologous to mitochondrial plasmid-like DNAs are located within limited chromosomal regions on the rice nuclear genome

  Kanazawa, A, Kishimoto, N, Sakamoto, W, Ohsawa, R, Ukai, Y, Tsutsumi, N, Hirai, A, Saito, A

  Theoretical and Applied Genetics 87 577 - 586 1993 [Refereed]
  
• Extraction of restrictable DNA from plants of the genusNelumbo

  Akira Kanazawa, Nobuhiro Tsutsumi

  Plant Molecular Biology Reporter Springer Science and Business Media LLC 10 (4) 316 - 318 0735-9640 1992/11 [Refereed]
  
• DISTRIBUTION AND QUANTITATIVE VARIATION OF MITOCHONDRIAL PLASMID-LIKE DNAS IN CULTIVATED RICE (ORYZA-SATIVA L)

  A KANAZAWA, W SAKAMOTO, M NAKAGAHRA, K KADOWAKI, N TSUTSUMI, S TANO

  JAPANESE JOURNAL OF GENETICS 日本遺伝学会 67 (4) 309 - 319 0021-504X 1992/08 [Refereed][Not invited]
   

  Distribution of four kinds of mitochondrial plasmid-like DNAs, B1, B2, B3 and B4 was investigated by Southern analysis using 85 accessions of cultivated rice (Oryza sativa L.). The frequencies of these molecules found in cultivars differed from each other. The mitochondrial genome were categorized into nine types according to their presence or absence. They were found mostly in ecospecies Indica, a few in Javanica, and not found in Japonica. The result indicated the polymorphic and monomorphic patterns of the mitochondrial genomic organization within Indica and Japonica cultivars, respectively. Quantitative variation was found among four kinds of mitochondiral plasmid-like DNAs, suggesting that these molecules were unequally distributed or replicated during the process of mitochondrial division.
• Linkage analysis of the nuclear homologues of mitochondrial plasmid-like DNAs in rice

  KANAZAWA Akira, SAKAMOTO Wataru, KISHIMOTO Naoki, YANO Masahiro, TSUTSUMI Nobuhiro, SAITO Akira, TANO Shigemitsu

  The Japanese Journal of Genetics 日本遺伝学会 66 (5) 597 - 607 0021-504X 1991 [Refereed]
   

  A genetical study on the nucleotide sequences of the nuclear DNAs which share homology with rice mitochondrial plasmid-like DNAs, B1, B2, B3 and B4 was carried out. Restriction fragments of the nuclear DNAs hybridized with these plasmid-like DNAs showed polymorphisms in their length between Indica and Japonica rice cultivars. The hybridized signals found specifically in Indica or Japonica cultivars segregated in the F₂ population derived from a cross between these two subspecies. The observed ratio of the nuclear homologues in the F₂ population demonstrated that they were transmitted according to the Mendelian inheritance. The co-segregation of homologues was examined and the linkage was detected between the B1-nuclear homologue of Japonica and the B4-nuclear homologue of Indica, and also between the nuclear homologues of B2 and B3 of Indica. The linkage between the B1-nuclear homologue of Japonica and the B4-nuclear homologue of Indica was conserved in the different rice cultivars.
  

MISC

• Virus-mediated efficient induction of epigenetic modifications of endogenous genes with phenotypic changes in plants (vol 65, pg 156, 2011)

  Akira Kanazawa, J. Inaba, H. Shimura, S. Otagaki, S. Tsukahara, A. Matsuzawa, B. M. Kim, K. Goto, C. Masuta  PLANT JOURNAL  90-  (6)  1208  -1211  2017/06  [Not refereed][Not invited]
  
• Deep sequencing analysis reveals commonality in small RNA production between co-suppression and naturally occurring RNA silencing in petunia

  Megumi Kasai, Hideo Matsumura, Kentaro Yoshida, Ryohei Terauchi, Akito Taneda, Akira Kanazawa  GENES & GENETIC SYSTEMS  87-  (6)  412  -412  2012/12  [Not refereed][Not invited]
  
• Tissue-specific promoter activity of the SORE-1 retrotransposon in soybean.

  Tsuchiya M, Abe J, Kanazawa A  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan  0-  (52)  5  -6  2011/12  [Not refereed][Not invited]
  
• P-63 Development of Novel Miccanthus Breeding Materials through Genetic Modification for Highly Effident Biofuel Production

  UKAJI Norifumi, WANG Xun, LI Xiaoyu, KANAZAWA Akira, YAMADA Toshihiko  Proceedings of the Conference on Biomass Science  0-  (6)  178  -179  2011/01/12  [Not refereed][Not invited]
   

  For control of global warning, it is proposed the utilizaiion of liquid biofuel produced from plant biomass. Perennial herbaceous plant, Miscanthus is a suitabis for the mateiial for biofuel because of a potential of massive-biomass production. However, it is necessaxy the further improvement of Miscanthus variety for efficient production of biofuel. Especially, low lignin contents Miscanthus cultiver will be greatly useful biomass materials that easily converted to biofuel. In the present study, we are attempting to reduce the lignin contents in Miscanthus by suppression of lignin synthesi...
• Modification of soybean seed components by gene silencing

  金澤 章, 永松 敦, 井元 勇介  Soy protein research,Japan  12-  (30)  33  -39  2009/10
• A sterility between rice species S_<13^n>, a novel allele at the S_<13> locus that induces hybrid sterility between rice species

  Ikegaya T, Koide Y, Sano Y, Kanazawa  日本育種学会・日本作物学会北海道談話会会報  (49)  7  -8  2008/12  [Not refereed][Not invited]
  
• Modification of soybean components through gene expression control by the function of RNA

  永松 敦, 金澤 章  Soy protein research,Japan  11-  (29)  26  -31  2008/10
• (19)Induction of Methylation on the Promoter of an Endogene in Petunia Infected with Cucumber mosaic virus Vector(Abstracts Presented at the Meeting of the Hokkaido Division,Abstracts of Papers Presented at the Division Meetings of the Phytopathological Society of Japan, 2007)

  Inaba J., Tukahara S., Kanazawa A., Masuta C.  Annals of the Phytopathological Society of Japan  74-  (1)  83  -83  2008/02/20
• Hybrid male sterility induced by S13 found in a distantly related rice species

  Yohei Koide, Tomohito Ikegaya, Daisuke Nishimoto, Akira Kanazawa, Yoshio Sano  GENES & GENETIC SYSTEMS  82-  (6)  522  -522  2007/12  [Not refereed][Not invited]
  
• The mRNA level of F3'H gene is associated with pigmentation in pubescence in soybean

  Nagamatsu A, Masuta C, Matsuura H, Kitamura K, Abe J, Kanazawa A  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan  0-  (48)  103  -104  2007/12  [Not refereed][Not invited]
  
• Gene expression control of β-conglycinin in molecular breeding of soybean seed storage protein

  井元 勇介, 永松 敦, 金澤 章  Soy protein research,Japan  10-  (28)  12  -17  2007/10
• Analysis of the gene expression control of soybean seed storage protein β-conglycinin α subunit

  永松 敦, 金澤 章  Soy protein research,Japan  9-  (27)  30  -35  2006/10
• Analysis of the seed-specific transcriptional regulation of the soybean β-conglycinin α subunit gene

  Nagamatsu A, Yoshino M, Tsutsumi K, Kanazawa A  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan  0-  (46)  45  -46  2005/12  [Not refereed][Not invited]
  
• Mechanisms for epigenetic regulation of pattern formation in petunia flower.

  Koseki M, Goto K, Masuta C, Kanazawa A  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan  0-  (45)  107  -108  2004/12  [Not refereed][Not invited]
  
• ペレニアルライグラス (Lolium perenne L) におけるフルクタンネオシリーズおよびイヌリンを合成する酵素遺伝子の単離と同定

  久野 裕, 金澤 章, 吉田 みどり, 中嶋 博, 喜多村 啓介, 山田 敏彦  日本草地学会誌  50-  (0)  274  -275  2004/03/26  [Not refereed][Not invited]
  
• Characterization of transgenic perennial ryegrass (Lolium perenne L.) carrying wheat (Triticum aestivum L.) genes encoding enzymes involved in fructan synthesis

  H Hisano, A Kanazawa, A Kawakami, M Yoshida, T Yamada, Y Shimamoto  PLANT AND CELL PHYSIOLOGY  44-  S100  -S100  2003  [Not refereed][Not invited]
  
• ダイズβ-コングリシニンのαサブユニットをコードする遺伝子の多様性とαサブユニットの発現(英文誌和文摘要)

  吉野 道子, 金澤 章, 堤 賢一, 中村 郁郎, 高橋 浩司, 島本 義也  育種学研究  4-  (4)  2002/12/01  [Not refereed][Not invited]
  
• 6-24 コムギ由来のフルクタン合成酵素をコードする遺伝子を導入したペレニアルライグラス (Lolium perenne L.) の特性

  久野 裕, 金澤 章, 川上 顕, 吉田 みどり, 山田 敏彦, 中嶋 博, 阿部 純, 島本 義也  日本草地学会誌  48-  (0)  350  -351  2002/09/21  [Not refereed][Not invited]
  
• DNA methylation and phenotypes of the progenies derived from crossing between PTGS line and TGS line in petunia

  TAKAHASHI A., KANAZAWA A.  育種学研究 = Breeding research  4-  118  -118  2002/08/26
• Production of transgenic perennial ryegrass (Lolium perenne L. ) carrying wheat genes encoding enzymes involved in fructan synthesis

  HISANO H, KANAZAWA A, KAWAKAMI K, YOSHIDA M, YAMADA T, NAKASHIMA H, ABE J, SHIMAMOTO Y  育種学研究 = Breeding research  4-  (0)  214  -214  2002/08/26  [Not refereed][Not invited]
  
• Mapping of cold-induced genes in Lolium perenne L.

  SHINOZUKA H, HISANO H, YONEYAMA S, KANAZAWA A, JONES E. S, FORSTER J. W, SHIMAMOTO Y, YAMADA T  育種学研究 = Breeding research  4-  (0)  247  -247  2002/08/26  [Not refereed][Not invited]
  
• Evolutionary dynamics of hybrid swarms between wild and cultivated soybeans

  SAKAMOTO S, ABE J, KANAZAWA A, SHIMAMOTO Y  育種学研究 = Breeding research  4-  (0)  420  -420  2002/08/26  [Not refereed][Not invited]
  
• Induction of RNA silencing in limited cells of petals in petunia.

  KANAZAWA A.  育種学研究 = Breeding research  4-  (1)  56  -56  2002/03/30
• Wild and Cultivated soybeans with common cytoplasmic DNA type differs in an allelic constitutions of nuclear SSRs.

  SAKAMOTO S, ABE J, KANAZAWA A, SHIMAMOTO Y  育種学研究 = Breeding research  4-  (1)  118  -118  2002/03/30  [Not refereed][Not invited]
  
• Traits and origin of wild soybean having dominant cytoplasmic type of cultivated soybean

  Sakamoto S, Abe J, Kanazawa A, Shimamoto Y  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan  0-  (42)  161  -162  2001/12  [Not refereed][Not invited]
  
• Genetic relationship of soybean landraces assesd by SSR markers located on the same linkage groups

  Suzuki Y, Abe J, Kanazawa A, Shimamoto Y  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan  0-  (41)  107  -108  2000/12  [Not refereed][Not invited]
  
• Phylogenetic relationships of the genus Glycine inferred from sequence variations of chloroplast DNA

  SAKAI M, XU D. H, KANAZAWA A, ABE J, SHIMAMOTO Y  育種学研究 = Breeding research  2-  (2)  215  -215  2000/09/25  [Not refereed][Not invited]
  
• Screening of markers linked with hard seedednessin soybean.

  SAKAMOTO S, KANAZAWA A, ABE J, SHIMAMOTO Y  育種学研究 = Breeding research  2-  (1)  43  -43  2000/04  [Not refereed][Not invited]
  
• Variation of nuclear RFLP markers between Chinese and Japanese Soybean populations.

  SUZUKI Y, KANAZAWA A, ABE J, SHIMAMOTO Y  育種学研究 = Breeding research  2-  (1)  256  -256  2000/04  [Not refereed][Not invited]
  
• Genetic diversity of soybean (G. max) germplasm in SSR markers

  XU D. H, ABE J, KANAZAWA A, SHIMAMOTO Y  育種学研究 = Breeding research  2-  (1)  257  -257  2000/04  [Not refereed][Not invited]
  
• Variation at four cpDNA mutation sites in wild and cultivated soybeans and its implication to the establishment of soybean.

  ABE J, XU D. H, KANAZAWA A, SHIMAMOTO Y  育種学研究 = Breeding research  2-  (1)  258  -258  2000/04  [Not refereed][Not invited]
  
• Genetic analysis of wild traits with wild soybean

  Sakamoto S, Abe J, Kanazawa A, Shimamoto Y  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan  0-  (40)  93  -94  1999/12  [Not refereed][Not invited]
  
• Genetic variation in Chinese and Japanese soybean (G. max) assessed by SSR markers

  Xu D. H, Kanazawa A, Abe J, Shimamoto Y  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan  0-  (40)  95  -96  1999/12  [Not refereed][Not invited]
  
• Genetic difference among soybean landraces in China and Japan based on nuclear DNA polymorphisms

  Suzuki Y, Abe J, Kanazawa A, Shimamoto Y  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan  0-  (40)  97  -98  1999/12  [Not refereed][Not invited]
  
• Phylogenetic relationships in the genus Glycine based on sequence variations of chloroplast DNA

  Sakai M, Kanazawa A, Abe J, Shimamoto Y  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan  0-  (40)  99  -100  1999/12  [Not refereed][Not invited]
  
• 3-2 ペレニアルライグラスにおける低温応答遺伝子群の発現および構造解析

  富永 陽子, 金澤 章, 島本 義也  日本草地学会誌  45-  (0)  110  -111  1999/03/20  [Not refereed][Not invited]
  
• Characterization of the seed storage proteins of the wild soybeans in Far East Russia

  Yoshino M, Kanazawa A, Abe J, Shimamoto Y  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan  0-  (39)  103  -104  1998/12  [Not refereed][Not invited]
  
• Genetic analysis of characters on evolution in wild soybean with hybrids distributed

  Sakamoto S, Abe J, Kanazawa A, Shimamoto Y  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan  0-  (39)  105  -106  1998/12  [Not refereed][Not invited]
  
• 3-9 3'-RACE法の併用によるdifferential display法を利用したペレニアルライグラスにおける低温応答遺伝子の検出

  富永 陽子, 金澤 章, 島本 義也  日本草地学会誌  44-  (0)  122  -123  1998/08/25  [Not refereed][Not invited]
  
• Evoluationary relationships of genes for subunit of β-conglycinin in soybean based on the sequences of non-coding region

  Yoshino M, Kanazawa A, Nakamura I, Shimamoto Y  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan  0-  (38)  106  -107  1997/12  [Not refereed][Not invited]
  
• Soybean breeding and wild soybeans in Russia

  Abe J, Kanazawa A, Komatsu K, Shimamoto Y, Shavrukov Y. N  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan  0-  (38)  116  -117  1997/12  [Not refereed][Not invited]
  
• Structure of the gene for e subunit of rice mitochondrial F_1-ATP synthase.

  Kanazawa A, Shimamoto Y  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan  0-  (37)  16  -17  1996/12  [Not refereed][Not invited]
  
• Rearrangement of the mitochondrial genome during evolutionary process of the genus Glycine

  Tozuka A, Kato S, Kanazawa A, Abe J, Shimamoto Y  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan  0-  (37)  146  -147  1996/12  [Not refereed][Not invited]
  

Research Projects

• 植物におけるエピジェネティックな形質変化の動態に関する階層的理解とその制御

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2017/04 -2021/03 

  Author : 金澤 章

   

  本研究課題では、植物におけるエピジェネティックな機構を介した遺伝子発現および形質の変化の実態を明らかにする目的で研究を行ってきた。これまでの研究過程では、主として遺伝子内におけるシトシンのメチル化の誘導およびその誘導後の動態について、コサプレッションを起こした遺伝子を持つ植物系統、ならびに、それに由来し、エピジェネティックな変化を起こした外来遺伝子のエピアレルを持つ植物系統を研究材料として用い、解析を行った。対立遺伝子間、とりわけ、エピアレル間での相互作用を介した変化の可能性を検討する目的から、異なるエピアレルを持つ系統間での交配後代を解析したところ、親世代とは異なる表現型を持つ個体を見出した。この知見に基づき、更なる遺伝学的解析を進めた。すなわち、両親由来のエピアレルを持つ個体と野生型との戻し交雑を行い、次世代の集団における表現型とシトシンメチル化の状態に関する分離を調べた。その結果、両親由来のエピアレルが存在する際に、それらの間の相互作用を介してパラミューテーション様の現象が起きたことが示唆された。一方、これまでの解析で組織特異的な発現変化を見出していた転移因子に関して、その発現制御とシトシンのメチル化の関連性を調べる目的から、シロイヌナズナに導入した転移因子ならびに内在性の転移因子を対象として解析を行った。その結果、検出された組織特異的な発現制御が、転移因子の顕著なメチル化頻度の変化を伴わずに起きていることが示唆された。
• Toward the development of an RNA silencing control system allowing quantitative modification of plant metabolites

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2014/04 -2018/03 

  Author : KANAZAWA Akira

   

  Spatial and temporal dynamics of RNA silencing in the life cycle of plants were analyzed in terms of exploiting quantitative modification of plant metabolites through gene expression control mediated by RNA silencing. Using transgenic soybean plants that carry a reporter gene, the following points were revealed: the induction and spread of RNA silencing during plant growth and its reset during reproduction occur in each generation; specific features of the architecture of the plant, in addition to the mechanisms conserved in plants, are involved in the diversity of these phenomena.
• Development of a method of molecular breeding involving epigenetic control of gene expression

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2013/04 -2017/03 

  Author : KANAZAWA Akira

   

  This work was aimed to understand the factors affecting the efficient induction and stabilization of epigenetic changes from the view point of establishing modification of plant traits via epigenetic control of gene expression as a tool for molecular breeding. Epigenetic changes that occurred under a specific condition in plants that had RNA silencing were analyzed. The patterns of dynamic changes in small RNA production and cytosine methylation, together with the stability of the altered state, were uncovered.