Ryutaro Aida

National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan

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Publications (28)45.54 Total impact

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    ABSTRACT: The class B genes DEFICIENS (DEF)/APETALA3 (AP3) and GLOBOSA (GLO)/PISTILLATA (PI), encoding MADS-box transcription factors, and their functions in petal and stamen development have been intensely studied in Arabidopsis and Antirrhinum. However, the functions of class B genes in other plants, including ornamental species exhibiting floral morphology different from these model plants, have not received nearly as much attention. Here, we examine the cooperative functions of TfDEF and TfGLO on floral organ development in the ornamental plant torenia (Torenia fournieri Lind.). Torenia plants co-overexpressing TfDEF and TfGLO showed a morphological alteration of sepals to petaloid organs. Phenotypically, these petaloid sepals were nearly identical to petals but had no stamens or yellow patches like those of wild-type petals. Furthermore, the inflorescence architecture in the co-overexpressing torenias showed a characteristic change in which, unlike the wild-types, their flowers developed without peduncles. Evaluation of the petaloid sepals showed that these attained a petal-like nature in terms of floral organ phenotype, cell shape, pigment composition, and the expression patterns of anthocyanin biosynthesis-related genes. In contrast, torenias in which TfDEF and TfGLO were co-suppressed exhibited sepaloid petals in the second whorl. The sepaloid petals also attained a sepal-like nature, in the same way as the petaloid sepals. The results clearly demonstrate that TfDEF and TfGLO play important cooperative roles in petal development in torenia. Furthermore, the unique transgenic phenotypes produced create a valuable new way through which characteristics of petal development and inflorescence architecture can be investigated in torenia.
    Plant molecular biology. 08/2014;
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    ABSTRACT: Chrysanthemums (Chrysanthemum morifolium Ramat.) have no purple-, violet-, or blue-flowered cultivars because they lack delphinidin-based anthocyanins. This deficiency is due to the absence of the flavonoid 3',5'-hydroxylase gene (F3'5'H), which encodes the key enzyme for delphinidin biosynthesis. In F3'5'H-transformed chrysanthemums, unpredictable and unstable expression levels have hampered successful production of delphinidin and reduced desired changes in flower color. With the aim of achieving delphinidin production in chrysanthemum petals, we found that anthocyanin biosynthetic gene promoters combined with a translational enhancer increased expression of some F3'5'H genes and accompanying delphinidin-based anthocyanin accumulation in transgenic chrysanthemums. Dramatic accumulation of delphinidin (up to 95%) was achieved by simple overexpression of Campanula F3'5'H controlled by a petal-specific flavanone 3-hydroxylase promoter from chrysanthemum combined with the 5'-untranslated region of the alcohol dehydrogenase gene as a translational enhancer. The flower colors of transgenic lines producing delphinidin-based anthocyanins changed from red-purple to purple/violet hue in the Royal Horticultural Society Colour Charts. This result represents a promising step toward molecular breeding of blue chrysanthemums.
    Plant and Cell Physiology 08/2013; · 4.98 Impact Factor
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    ABSTRACT: Chrysanthemum is globally the second most important ornamental in terms of socioeconomic importance. Even though the vast range of flower colors, shapes and forms were initially created using conventional and mutation breeding, transgenic strategies are now more frequently used with Agrobacterium-mediated transformation being the most popular form of introducing foreign genes into chrysanthemums. Even so, transformation efficiency remains dependent on cultivar and regeneration procedure. Transgenic molecular breeding has seen the introduction of important traits such as novel flower color and form and plant architecture, prolonged cut-flower vase-life, resistance to biotic stresses such as viruses/viroids, pathogens and insects. However, chimerism and transgene silencing continue to be limiting factors. Transgenic strategies, despite opening up new avenues for creating new cultivars with improved agronomic and horticultural traits, may be limited due to the risk of transgenic pollen escaping into the wild.
    Critical Reviews in Plant Sciences 01/2013; 32(1):21-52. · 4.36 Impact Factor
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    ABSTRACT: We identified a Torenia fournieri Lind. mutant (no. 252) that exhibited a sepaloid phenotype in which the second whorls were changed to sepal-like organs. This mutant had no stamens, and the floral organs consisted of sepals and carpels. Although the expression of a torenia class B MADS-box gene, GLOBOSA (TfGLO), was abolished in the 252 mutant, no mutation of TfGLO was found. Among torenia homologs such as APETALA1 (AP1), LEAFY (LFY), and UNUSUAL FLORAL ORGANS (UFO), which regulate expression of class B genes in Arabidopsis, only accumulation of the TfUFO transcript was diminished in the 252 mutant. Furthermore, a missense mutation was found in the coding region of the mutant TfUFO. Intact TfUFO complemented the mutant phenotype whereas mutated TfUFO did not; in addition, the transgenic phenotype of TfUFO-knockdown torenias coincided with the mutant phenotype. Yeast two-hybrid analysis revealed that the mutated TfUFO lost its ability to interact with TfLFY protein. In situ hybridization analysis indicated that the transcripts of TfUFO and TfLFY were partially accumulated in the same region. These results clearly demonstrate that the defect in TfUFO caused the sepaloid phenotype in the 252 mutant due to the loss of interaction with TfLFY.
    The Plant Journal 05/2012; 71(6):1002-14. · 6.58 Impact Factor
  • Ryutaro Aida
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    ABSTRACT: This chapter describes an Agrobacterium tumefaciens-mediated transformation protocol for torenia, a plant that has several useful characteristics and is primarily used for ornamental and experimental purposes. Leaf segments of torenia were co-cultured with A. tumefaciens containing a vector plasmid for 7 days at 22°C under dark conditions on Murashige and Skoog (MS) medium containing 1 mg/L benzyladenine, 1 mg/L indoleacetic acid, and 100 μM acetosyringone. Subsequent culturing at 25°C under a 16-h photoperiod with fluorescent light on MS medium containing 1 mg/L benzyladenine, 300 mg/L carbenicillin, and selection agent (300 mg/L kanamycin or 20 mg/L hygromycin) allowed for transformant selection. Transgenic shoots were obtained from green compact calli after 2-3 months of culture in the selection medium. This method can achieve a transformation rate of approximately 5% (transformants/explant).
    Methods in molecular biology (Clifton, N.J.) 01/2012; 847:267-74. · 1.29 Impact Factor
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    ABSTRACT: While heavy-ion beam irradiation is becoming popular technology for mutation breeding in Japan, the combination with genetic manipulation makes it more convenient to create greater variation in plant phenotypes. We have succeeded in producing over 200 varieties of transgenic torenia (Torenia fournieri Lind.) from over 2,400 regenerated plants by this procedure in only 2 years. Mutant phenotypes were observed mainly in flowers and showed wide variation in colour and shape. Higher mutation rates in the transgenics compared to those in wild type indicate the synergistic effect of genetic manipulation and heavy-ion beam irradiation, which might be advantageous to create greater variation in floral traits.
    Methods in molecular biology (Clifton, N.J.) 01/2012; 847:275-89. · 1.29 Impact Factor
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    ABSTRACT: It has long been proposed that white-flowered chrysanthemums (Chrysanthemum morifolium Ramat.) have a single dominant gene that inhibits carotenoid formation or accumulation in ray petals. However, the precise function of the proposed gene was unknown. We previously isolated a gene encoding carotenoid cleavage dioxygenase 4, designated CmCCD4a, which is specifically expressed in the ray petals of white-flowered chrysanthemums. Because CmCCD4a was a strong candidate for the single dominant gene, we analyzed the relationship between CmCCD4a expression and carotenoid content in two sets of petal color mutants. Here, we show that CmCCD4a represents a small gene family containing at least four members. Two of them, CmCCD4a-1 and CmCCD4a-2, were highly expressed in ray petals of two taxa with low carotenoid levels. In petal color mutants derived from these taxa, increases in carotenoid levels accompanied decreases in CmCCD4a expression levels in ray petals. Two different circumstances reduced the levels of CmCCD4a expression in the mutants: either a CmCCD4a gene was lost from the genome or the expression of a CmCCD4a gene was suppressed. In the latter case, suppression may be caused by the loss of a function that normally enhances CmCCD4a transcription. A stepwise decrease in the amount of CmCCD4a expression in either L1 or L2 resulted in a corresponding stepwise increase in the carotenoid content in ray petals. From these results, we propose that CmCCD4a expression is the key factor that controls the carotenoid content in ray petals of chrysanthemum.
    Euphytica 01/2012; 184(3). · 1.64 Impact Factor
  • Journal of the Japanese Society for Horticultural Science. 01/2011; 80(1):113-120.
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    ABSTRACT: Homeotic class B genes GLOBOSA (GLO)/PISTILLATA (PI) and DEFICIENS (DEF)/APETALA3 (AP3) are involved in the development of petals and stamens in Arabidopsis. However, functions of these genes in the development of floral organs in torenia are less well known. Here, we demonstrate the unique floral phenotypes of transgenic torenia formed due to the modification of class B genes, TfGLO and TfDEF. TfGLO-overexpressing plants showed purple-stained sepals that accumulated anthocyanins in a manner similar to that of petals. TfGLO-suppressed plants showed serrated petals and TfDEF-suppressed plants showed partially decolorized petals. In TfGLO-overexpressing plants, cell shapes on the surfaces of sepals were altered to petal-like cell shapes. Furthermore, TfGLO- and TfDEF-suppressed plants partially had sepal-like cells on the surfaces of their petals. We isolated putative class B gene-regulated genes and examined their expression in transgenic plants. Three xyloglucan endo-1,4-beta-D: -glucanase genes were up-regulated in TfGLO- and TfDEF-overexpressing plants and down-regulated in TfGLO- and TfDEF-suppressed plants. In addition, 10 anthocyanin biosynthesis-related genes, including anthocyanin synthase and chalcone isomerase, were up-regulated in TfGLO-overexpressing plants and down-regulated in TfGLO-suppressed plants. The expression patterns of these 10 genes in TfDEF transgenic plants were diverse and classified into several groups. HPLC analysis indicated that sepals of TfGLO-overexpressing plants accumulate the same type of anthocyanins and flavones as wild-type plants. The difference in phenotypes and expression patterns of the 10 anthocyanin biosynthesis-related genes between TfGLO and TfDEF transgenic plants indicated that TfGLO and TfDEF have partial functional divergence, while they basically work synergistically in torenia.
    MGG Molecular & General Genetics 11/2010; 284(5):399-414. · 2.58 Impact Factor
  • Journal of The Japanese Society for Horticultural Science - J JPN SOC HORT SCI. 01/2009; 78(4):450-455.
  • Plant Biotechnology 03/2008; 25:37-43. · 0.94 Impact Factor
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    Plant Biotechnology 03/2008; 25:45-53. · 0.94 Impact Factor
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    ABSTRACT: The white petals of chrysanthemum (Chrysanthemum morifolium Ramat.) are believed to contain a factor that inhibits the accumulation of carotenoids. To find this factor, we performed polymerase chain reaction-Select subtraction screening and obtained a clone expressed differentially in white and yellow petals. The deduced amino acid sequence of the protein (designated CmCCD4a) encoded by the clone was highly homologous to the sequence of carotenoid cleavage dioxygenase. All the white-flowered chrysanthemum cultivars tested showed high levels of CmCCD4a transcript in their petals, whereas most of the yellow-flowered cultivars showed extremely low levels. Expression of CmCCD4a was strictly limited to flower petals and was not detected in other organs, such as the root, stem, or leaf. White petals turned yellow after the RNAi construct of CmCCD4a was introduced. These results indicate that in white petals of chrysanthemums, carotenoids are synthesized but are subsequently degraded into colorless compounds, which results in the white color.
    Plant physiology 12/2006; 142(3):1193-201. · 6.56 Impact Factor
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    ABSTRACT: We generated mutated ethylene receptor genes (mDG-ERS1s) from the chrysanthemum ethylene receptor (DG-ERS1) cDNA by introducing one-nucleotide substitutions corresponding to those present in Arabidopsis etr1-1, etr1-2, etr1-3, and etr1-4 and tomato Nr. The promoter of a tobacco elongation factor 1α (EF1α) gene was fused to DG-ERS1 cDNA or one of the mDG-ERS1s. The resulting constructs were named EF1α∷mDG-ERS1(etr1-1), -ERS1(etr1-2) and so on, and introduced into chrysanthemum cv. Sei-Marine. We obtained putative transformants resistant to an antibiotic paromomycin with a yield of 2.4–6.2% depending on the construct. The mDG-ERS1(etr1-4) construct tended to be more effective in conferring reduced ethylene sensitivity in chrysanthemum than the others. PCR analysis gave amplification corresponding to a partial sequence of EF1α∷mDG-ERS1 transgenes. Southern blot analysis showed that, in the mDG-ERS1(etr1-4) transformant, not only the lines with reduced sensitivity to ethylene but also those sensitive to ethylene harbored the mDG-ERS1(etr1-4) transgene. The present results showed the usefulness of mutated ethylene receptor genes mDG-ERS1s for generation of transgenic chrysanthemums with reduced ethylene sensitivity.
    Postharvest Biology and Technology 08/2005; 37(2):101-110. · 2.45 Impact Factor
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    ABSTRACT: We investigated the usefulness of the promoter of a gene for tobacco elongation factor 1α protein (EF1 α) for transgene expression in the chrysanthemum (Chrysanthemum morifolium Ramat.). The EF1 α promoter was fused to the β-glucuronidase gene (gus) and introduced into the chrysanthemum. We obtained 238 putative transformants and GUS assay of the leaves of the in vitro plants revealed that 29.0% (69/238) of the putative plants were GUS-positive. The plants in the greenhouse that were 20 months after regeneration still showed a GUS activity in their leaves and petals. The tobacco EF1 α promoter expressed the transgene more efficiently than the 35S promoter of Cauliflower mosaic virus and could be used for transgene expression in chrysanthemum.
    Japan Agricultural Research Quarterly 01/2005; 39(4). · 0.47 Impact Factor
  • Breeding Science - BREEDING SCI. 01/2004; 54(1):51-58.
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    ABSTRACT: Previously, we isolated AGAMOUS (AG) homologues MASAKO C1 and D1 from the wild rose, Rosa rugosa Thunb. ex Murray. To understand the functional difference between MASAKO C1 and D1, the effect on the floral and vegetative development of each gene was investigated using transgenic Arabidopsis thaliana and Torenia fournieri Lind. The transgenic plants with ectopically expressed MASAKO C1 or D1 under the control of a cauliflower mosaic virus (CaMV) 35S promoter produced flowers with sepal-to-carpel and petal-to-stamen transformation in Arabidopsis and sepal-to-carpel transformation in Torenia.These results suggest that both genes have similar function and are involved in stamen and carpel identification in the rose.
    Plant Science. 01/2004;
  • Engei Gakkai zasshi 01/2003; 72(3):197-204.
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    ABSTRACT: The function of a 1.1-kb transcript of WAG, a wheat AGAMOUS homolog, has not been clarified. To analyze its function, it was fused with the cauliflower mosaic virus 35S promoter and introduced into torenia (Torenia fournieri). Torenia transformants grew normally with regular plant height, size and shape of leaves, and development of inflorescence. However, the corolla was significantly smaller in 10 transformants out of 21. Generally, the vertical and horizontal diameters of the lip part of the corolla, as well as the length of the corolla (including the tube part), were all reduced in the smaller flowers, suggesting that the size of the corolla was reduced equally in all dimensions. Southern blot analysis showed the existence of the WAG gene in the genomes of these plants. We report that WAG reduces the size of the corolla in torenia. Summary
    01/2003;
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    ABSTRACT: We report a method for Agrobacterium-mediated transformation of Elatior Begonia (Begonia×hiemalis Fotsch). Young leaf discs were infected with Agrobacteriumtumefaciens strains AGL0 and LBA4404. Each strain has a binary vector plasmid, pIG121Hm that includes the β-glucuronidase (GUS) gene with an intron as a reporter gene, and both the neomycin phosphotransferase II and the hygromycin phosphotransferase genes as selection markers. Explants were cultured on modified MS medium supplemented with 1.0 mg/l BA, 0.5 mg/l IAA, 300 mg/l ticarcillin, and either 100 mg/l kanamycin and 5 mg/l hygromycin, or 300 mg/l kanamycin for selection and regeneration. Out of 500 explants infected with AGL0, 16 plantlets were regenerated, and out of 628 explants infected with LBA4404, two plantlets were regenerated after 4 months of culture. Transformation was confirmed by Southern blot analysis of the GUS gene and by histochemical assays of GUS activity in plant tissues. Ten in vitro transgenic plants were obtained from AGL0 infected explants only.
    Plant Science - PLANT SCI. 01/2002; 162(5):697-703.