The Experts below are selected from a list of 318 Experts worldwide ranked by ideXlab platform
Tetsuya Higashiyama - One of the best experts on this subject based on the ideXlab platform.
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a homolog of the alog family controls corolla tube differentiation in Torenia fournieri
Development, 2019Co-Authors: Wei Xiao, Shihao Su, Tetsuya HigashiyamaAbstract:ABSTRACT Flowers of honey plants (Torenia) face various abiotic stressors, including rain, that can damage pollens and dilute nectar. Many Torenia species are thought to have evolved a modified corolla base termed the corolla neck to prevent raindrops from contacting the nectar. Although this hypothesis was postulated long ago, direct validation is lacking. Here, we have evaluated Torenia fournieri, the corolla tube of which differentiates into distinct regions: a conical tube above that connects to an inflated base through a constriction. This constriction and inflated base are collectively referred to as the corolla neck. Using transcriptomic sequencing and genome-editing approaches, we have characterized an ALOG gene, TfALOG3, that is involved in formation of the corolla neck. TfALOG3 was found expressed in the epidermis of the corolla neck. Cells in the corolla bottom differentiated and expanded in wild-type T. fournieri, whereas such cells in TfALOG3 loss-of-function mutants failed to develop into a corolla neck. Water easily contacted the nectary in the absence of the corolla neck. Taken together, our study unveils a novel gene that controls corolla tube differentiation and demonstrates a hypothetical property of the corolla neck.
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Quantitative assessment of chemotropism in pollen tubes using microslit channel filters
Biomicrofluidics, 2018Co-Authors: Naoki Yanagisawa, Tetsuya HigashiyamaAbstract:We present a semi-in vitro chemotropism assay that can be used to evaluate the chemoattractant effect of diffusible plant signaling molecules on growing pollen tubes. We constructed an array of microslit channels in a microfluidic device that prevented the passage of randomly growing pollen tubes but permitted ones that are responsive to the chemoattractant. Depending on the microslit channel size, 80%–100% of the randomly growing Torenia fournieri pollen tubes were excluded from reaching the source of the attractant. Thus, the selection of pollen tubes that are capable of responding to chemoattractants from a mixed population can be realized using this platform.
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live imaging and laser disruption reveal the dynamics and cell cell communication during Torenia fournieri female gametophyte development
Plant and Cell Physiology, 2015Co-Authors: Daichi Susaki, Hidenori Takeuchi, Hiroki Tsutsui, Daisuke Kurihara, Tetsuya HigashiyamaAbstract:: The female gametophytes of many flowering plants contain one egg cell, one central cell, two synergid cells and three antipodal cells with respective morphological characteristics and functions. These cells are formed by cellularization of a multinuclear female gametophyte. However, the dynamics and mechanisms of female gametophyte development remain largely unknown due to the lack of a system to visualize directly and manipulate female gametophytes in living material. Here, we established an in vitro ovule culture system to examine female gametophyte development in Torenia fournieri, a unique plant species with a protruding female gametophyte. The four-nucleate female gametophyte became eight nucleate by the final (third) mitosis and successively cellularized and matured to attract a pollen tube. The duration of final mitosis was 28 ± 6.5 min, and cellularization was completed in 54 ± 20 min after the end of the third mitosis. Fusion of polar nuclei in the central cell occurred in 13.1 ± 1.1 h, and onset of expression of LURE2, a pollen tube attractant gene, was visualized by a green fluorescent protein reporter 10.7 ± 2.3 h after cellularization. Laser disruption analysis demonstrated that the egg and central cells were required for synergid cells to acquire the pollen tube attraction function. Moreover, aberrant nuclear positioning and down-regulation of LURE2 were observed in one of the two synergid cells after disrupting an immature egg cell, suggesting that cell specification was affected. Our system provides insights into the precise dynamics and mechanisms of female gametophyte development in T. fournieri.
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Live Imaging and Laser Disruption Reveal the Dynamics and Cell–Cell Communication During Torenia fournieri Female Gametophyte Development
Plant and Cell Physiology, 2015Co-Authors: Daichi Susaki, Hidenori Takeuchi, Hiroki Tsutsui, Daisuke Kurihara, Tetsuya HigashiyamaAbstract:The female gametophytes of many flowering plants contain one egg cell, one central cell, two synergid cells and three antipodal cells with respective morphological characteristics and functions. These cells are formed by cellularization of a multinuclear female gametophyte. However, the dynamics and mechanisms of female gametophyte development remain largely unknown due to the lack of a system to visualize directly and manipulate female gametophytes in living material. Here, we established an in vitro ovule culture system to examine female gametophyte development in Torenia fournieri, a unique plant species with a protruding female gametophyte. The four-nucleate female gametophyte became eight nucleate by the final (third) mitosis and successively cellularized and matured to attract a pollen tube. The duration of final mitosis was 28± 6.5 min, and cellularization was completed in 54± 20 min after the end of the third mitosis. Fusion of polar nuclei in the central cell occurred in 13.1± 1.1 h, and onset of expression of LURE2, a pollen tube attractant gene, was visualized by a green fluorescent protein reporter 10.7± 2.3 h after cellularization. Laser disruption analysis demonstrated that the egg and central cells were required for synergid cells to acquire the pollen tube attraction function. Moreover, aberrant nuclear positioning and down-regulation of LURE2 were observed in one of the two synergid cells after disrupting an immature egg cell, suggesting that cell specification was affected. Our system provides insights into the precise dynamics and mechanisms of female gametophyte development in T. fournieri.
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acquisition of lure binding activity at the pollen tube tip of Torenia fournieri
Molecular Plant, 2013Co-Authors: Satohiro Okuda, Takamasa Suzuki, Masahiro M Kanaoka, Hitoshi Mori, Narie Sasaki, Tetsuya HigashiyamaAbstract:ABSTRACT Pollen tube guidance is controlled by multiple complex interactions with the female tissues. Here, we show that pollen tubes of Torenia fournieri are regulated by a stylar tissue in a length-dependent manner to receive and respond to attractant LURE peptides secreted from synergid cells. We developed an immunostaining method to visualize LURE peptides bound at the plasma membrane of the tip region of the pollen tube. Using this method, we found that LURE peptides bound specifically to pollen tubes growing through a cut style. The peptides also bound to pollen tubes growing through a shorter style, which were not competent to respond to these peptides. These observations suggested a possibility that acquisition of the LURE peptide reception ability and acquisition of full competency are separable processes. RNA-Seq suggested that the transcription profile of pollen tubes was affected by both the length of the style and the cultivation period, consistently with physiological changes in binding activity and LURE response ability. The database generated from de novo RNA-Seq of Torenia pollen tubes was shown to be useful to identify pollen tube proteins by mass spectrometry. Our studies provide insight and an effective platform for protein identification to understand pollen tube guidance.
Norihiro Ohtsubo - One of the best experts on this subject based on the ideXlab platform.
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production of multi petaled Torenia fournieri flowers by functional disruption of two class c mads box genes
Planta, 2020Co-Authors: Katsutomo Sasaki, Norihiro OhtsuboAbstract:MAIN CONCLUSION: Simultaneous knockdown or knockout of Torenia fournieri PLENA (TfPLE) and FALINELLI (TfFAR) genes with RNAi or genome-editing technologies generated a multi-petal phenotype in Torenia. The MADS-box gene AGAMOUS (AG) is well known to play important roles in the development of stamens and carpels in Arabidopsis. Mutations in AG cause the morphological transformation of stamens and carpels into petaloid organs. In contrast, Torenia (Torenia fournieri Lind.) has two types of class-C MADS-box genes, PLENA (PLE) and FALINELLI (FAR); however, their functions were previously undetermined. To examine the function of TfPLE and TfFAR in Torenia, we used RNAi to knockdown expression of these two genes. TfPLE and TfFAR double-knockdown transgenic Torenia plants had morphologically altered stamens and carpels that developed into petaloid organs. TfPLE knockdown transgenic plants also exhibited morphological transformations that included shortened styles, enlarged ovaries, and absent stigmata. Furthermore, simultaneous disruption of TfPLE and TfFAR genes by CRISPR/Cas9-mediated genome editing also resulted in the conversion of stamens and carpels into petaloid organs as was observed in the double-knockdown transgenic plants mediated by RNAi. In addition, the carpels of one TfPLE knockout mutant had the same morphological abnormalities as TfPLE knockdown transgenic plants. TfFAR knockdown genome-edited mutants had no morphological changes in their floral organs. These results clearly show that TfPLE and TfFAR cooperatively play important roles in the development of stamens and carpels. Simultaneous disruption of TfPLE and TfFAR functions caused a multi-petal phenotype, which is expected to be a highly valuable commercial floral trait in horticultural flowers.
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Generation of Novel Floral Traits Using a Combination of Floral Organ-Specific Promoters and a Chimeric Repressor in Torenia fournieri Lind.
Plant and Cell Physiology, 2016Co-Authors: Katsutomo Sasaki, Hiroyasu Yamaguchi, Takako Narumi, Ichiro Kasajima, Norihiro OhtsuboAbstract:In this study, we attempted to develop a new biotechnological method for the efficient modification of floral traits. Because transcription factors play an important role in determining floral traits, chimeric repressors, which are generated by attaching a short transcriptional repressor domain to transcription factors, have been widely used as effective tools for modifying floral traits in many plant species. However, the overexpression of these chimeric repressors by the Cauliflower mosaic virus 35S promoter sometimes causes undesirable morphological alterations to other organs. We attempted simultaneously to generate new floral traits and avoid such quality loss by examining five additional floral organ-specific promoters, one Arabidopsis thaliana promoter and four Torenia fournieri promoters, for the expression of the chimeric repressor of Arabidopsis TCP3 (AtTCP3), whose overexpression drastically alters floral traits but also generates dwarf phenotypes and deformed leaves. We found that the four Torenia promoters exhibited particularly strong activity in the petals but not in the leaves, and that the combination of these floral organ-specific promoters with the chimeric repressor of AtTCP3 caused changes in the color, color patterns and cell shapes of petals, whilst avoiding other unfavorable phenotypes. Interestingly, each promoter that we used in this study generated characteristic and distinguishable floral traits. Thus, the use of different floral organ-specific promoters with different properties enables us to generate diverse floral traits using a single chimeric repressor without changing the phenotypes of other organs.
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Co-modification of class B genes TfDEF and TfGLO in Torenia fournieri Lind. alters both flower morphology and inflorescence architecture
Plant Molecular Biology, 2014Co-Authors: Katsutomo Sasaki, R Aida, Hiroyasu Yamaguchi, Masayoshi Nakayama, Norihiro OhtsuboAbstract: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.
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MIXTA-Like Transcription Factors and WAX INDUCER1/SHINE1 Coordinately Regulate Cuticle Development in Arabidopsis and Torenia fournieri
The Plant Cell, 2013Co-Authors: Yoshimi Oshima, Norihiro Ohtsubo, Masahito Shikata, Tomotsugu Koyama, Nobutaka Mitsuda, Masaru Ohme-takagiAbstract:The waxy plant cuticle protects cells from dehydration, repels pathogen attack, and prevents organ fusion during development. The transcription factor WAX INDUCER1/SHINE1 (WIN1/SHN1) regulates the biosynthesis of waxy substances in Arabidopsis thaliana. Here, we show that the MIXTA-like MYB transcription factors MYB106 and MYB16, which regulate epidermal cell morphology, also regulate cuticle development coordinately with WIN1/SHN1 in Arabidopsis and Torenia fournieri. Expression of a MYB106 chimeric repressor fusion (35S:MYB106-SRDX) and knockout/down of MYB106 and MYB16 induced cuticle deficiencies characterized by organ adhesion and reduction of epicuticular wax crystals and cutin nanoridges. A similar organ fusion phenotype was produced by expression of a WIN1/SHN1 chimeric repressor. Conversely, the dominant active form of MYB106 (35S:MYB106-VP16) induced ectopic production of cutin nanoridges and increased expression of WIN1/SHN1 and wax biosynthetic genes. Microarray experiments revealed that MYB106 and WIN1/SHN1 regulate similar sets of genes, predominantly those involved in wax and cutin biosynthesis. Furthermore, WIN1/SHN1 expression was induced by MYB106-VP16 and repressed by MYB106-SRDX. These results indicate that the regulatory cascade of MIXTA-like proteins and WIN1/SHN1 coordinately regulate cutin biosynthesis and wax accumulation. This study reveals an additional key aspect of MIXTA-like protein function and suggests a unique relationship between cuticle development and epidermal cell differentiation.
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mixta like transcription factors and wax inducer1 shine1 coordinately regulate cuticle development in arabidopsis and Torenia fournieri
The Plant Cell, 2013Co-Authors: Yoshimi Oshima, Norihiro Ohtsubo, Masahito Shikata, Tomotsugu Koyama, Nobutaka Mitsuda, Masaru OhmetakagiAbstract:The waxy plant cuticle protects cells from dehydration, repels pathogen attack, and prevents organ fusion during development. The transcription factor WAX INDUCER1/SHINE1 (WIN1/SHN1) regulates the biosynthesis of waxy substances in Arabidopsis thaliana. Here, we show that the MIXTA-like MYB transcription factors MYB106 and MYB16, which regulate epidermal cell morphology, also regulate cuticle development coordinately with WIN1/SHN1 in Arabidopsis and Torenia fournieri. Expression of a MYB106 chimeric repressor fusion (35S:MYB106-SRDX) and knockout/down of MYB106 and MYB16 induced cuticle deficiencies characterized by organ adhesion and reduction of epicuticular wax crystals and cutin nanoridges. A similar organ fusion phenotype was produced by expression of a WIN1/SHN1 chimeric repressor. Conversely, the dominant active form of MYB106 (35S:MYB106-VP16) induced ectopic production of cutin nanoridges and increased expression of WIN1/SHN1 and wax biosynthetic genes. Microarray experiments revealed that MYB106 and WIN1/SHN1 regulate similar sets of genes, predominantly those involved in wax and cutin biosynthesis. Furthermore, WIN1/SHN1 expression was induced by MYB106-VP16 and repressed by MYB106-SRDX. These results indicate that the regulatory cascade of MIXTA-like proteins and WIN1/SHN1 coordinately regulate cutin biosynthesis and wax accumulation. This study reveals an additional key aspect of MIXTA-like protein function and suggests a unique relationship between cuticle development and epidermal cell differentiation.
Tsuneyoshi Kuroiwa - One of the best experts on this subject based on the ideXlab platform.
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species preferentiality of the pollen tube attractant derived from the synergid cell of Torenia fournieri
Plant Physiology, 2006Co-Authors: Tetsuya Higashiyama, Narie Sasaki, Haruko Kuroiwa, Tsuneyoshi Kuroiwa, Rie Inatsugi, Sachio Sakamoto, Toshiyuki Mori, Takashi Nakada, Hisayoshi Nozaki, Akihiko NakanoAbstract:The synergid cell of Torenia fournieri attracts pollen tubes by a diffusible but yet unknown chemical attractant. Here we investigated the species difference of the attractant using five closely related species in two genera, namely T. fournieri, Torenia baillonii, Torenia concolor, Lindernia (Vandellia) crustacea, and Lindernia micrantha. These five species have an exserted embryo sac, and ablation experiments confirmed that their synergid cells attracted the pollen tube. When ovules of T. fournieri and one of the other species were cultivated together with pollen tubes of each species, pollen tubes were significantly more attracted to synergid cells of the corresponding species. The attraction was not affected by the close proximity of embryo sacs of different species. This suggests that the attractant is a species-preferential molecule that is likely synthesized in the synergid cell. The calcium ion, long considered a potential attractant, could not serve as the sole attractant in these species, because elevation of the calcium ion concentration did not affect the observed attraction. In vivo crossing experiments also showed that the attraction of the pollen tube to the embryo sac was impaired when pollen tubes of different species arrived around the embryo sac, suggesting that the species preferentiality of the attractant may serve as a reproductive barrier in the final step of directional control of the pollen tube.
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explosive discharge of pollen tube contents in Torenia fournieri
Plant Physiology, 2000Co-Authors: Tetsuya Higashiyama, Haruko Kuroiwa, Shigeyuki Kawano, Tsuneyoshi KuroiwaAbstract:When animals copulate, the male organ penetrates the female. Similarly, the pollen tube of a flowering plant (the male gametophyte) penetrates the embryo sac (the female gametophyte) and then discharges its contents, which include male gametes. Details of the interaction between the pollen tube and
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guidance in vitro of the pollen tube to the naked embryo sac of Torenia fournieri
The Plant Cell, 1998Co-Authors: Tetsuya Higashiyama, Haruko Kuroiwa, Shigeyuki Kawano, Tsuneyoshi KuroiwaAbstract:The precise guidance of the pollen tube to the embryo sac is critical to the successful sexual reproduction of flowering plants. We demonstrate here the guidance of the pollen tube to the embryo sac in vitro by using the naked embryo sac of Torenia fournieri , which protrudes from the micropyle of the ovule. We developed a medium for culture of both the ovule and the pollen tube of T. fournieri and cocultivated them in a thin layer of solid medium. Although pollen tubes that had germinated in vitro passed naked embryo sacs, some pollen tubes that grew semi–in vitro through a cut style arrived precisely at the site of entry into the embryo sac, namely, the filiform apparatus of the synergids. When pollen tubes were unable to enter the embryo sac, they continuously grew toward the same filiform apparatus, forming narrow coils. Pollen tubes selectively arrived at complete, unfertilized embryo sacs but did not arrive at those of heat-treated ovules or those with disrupted synergids. These results convincingly demonstrate that pollen tubes are specifically attracted to the region of the filiform apparatus of living synergids in vitro.
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Kinetics of double fertilization in Torenia fournieri based on direct observations of the naked embryo sac
Planta, 1997Co-Authors: Tetsuya Higashiyama, Haruko Kuroiwa, Shigeyuki Kawano, Tsuneyoshi KuroiwaAbstract:Torenia fournieri Lind. has a naked embryo sac that protrudes from the micropyle. The precise time course of the entire process of double fertilization and the kinetics of fertilization events were determined in this species by the following methods: (i) without squashing, pollen tubes on the torn stylar canal were observed by fluorescence microscopy after staining with both 4′,6-diamidino-2-phenylindole (DAPI) and aniline blue; and (ii) large numbers of living embryo sacs were observed directly by differential interference microscopy before and after fertilization. The pollen began to germinate 5 min after pollination and extruded pollen tubes which elongated at a constant rate of 2.3 mm · h^−1. At 4.0 h after pollination, the mitotic index of the generative cell within the pollen tube reached 88% and the two sperm cells were formed. Pollen tubes began to arrive at ovules 8.9 h after pollination and directly entered one of two synergids in the naked embryo sac. The time required for transport of sperm cells in the degenerated synergid was estimated statistically to be 1.9 ± 1.8 min for transport of the first cell and 7.4 ± 1.6 min for the second. In the nucleus of the fertilized egg cell, the male nucleolus began to emerge 10 h after pollination and the female nucleolus often decreased in size. The two nucleoli fused together prior to elongation of the zygote, which began 28 h after pollination. In the central cell, the secondary nucleus migrated to a region adjacent to the egg apparatus after pollination but prior to the arrival of the pollen tube. The primary endosperm nucleus rapidly returned to the inner region after fertilization. Prior to embryogenesis, the first division of the primary endosperm began about 15 h after pollination, at a defined site, to form the chalazal haustorium.
Daichi Susaki - One of the best experts on this subject based on the ideXlab platform.
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live imaging and laser disruption reveal the dynamics and cell cell communication during Torenia fournieri female gametophyte development
Plant and Cell Physiology, 2015Co-Authors: Daichi Susaki, Hidenori Takeuchi, Hiroki Tsutsui, Daisuke Kurihara, Tetsuya HigashiyamaAbstract:: The female gametophytes of many flowering plants contain one egg cell, one central cell, two synergid cells and three antipodal cells with respective morphological characteristics and functions. These cells are formed by cellularization of a multinuclear female gametophyte. However, the dynamics and mechanisms of female gametophyte development remain largely unknown due to the lack of a system to visualize directly and manipulate female gametophytes in living material. Here, we established an in vitro ovule culture system to examine female gametophyte development in Torenia fournieri, a unique plant species with a protruding female gametophyte. The four-nucleate female gametophyte became eight nucleate by the final (third) mitosis and successively cellularized and matured to attract a pollen tube. The duration of final mitosis was 28 ± 6.5 min, and cellularization was completed in 54 ± 20 min after the end of the third mitosis. Fusion of polar nuclei in the central cell occurred in 13.1 ± 1.1 h, and onset of expression of LURE2, a pollen tube attractant gene, was visualized by a green fluorescent protein reporter 10.7 ± 2.3 h after cellularization. Laser disruption analysis demonstrated that the egg and central cells were required for synergid cells to acquire the pollen tube attraction function. Moreover, aberrant nuclear positioning and down-regulation of LURE2 were observed in one of the two synergid cells after disrupting an immature egg cell, suggesting that cell specification was affected. Our system provides insights into the precise dynamics and mechanisms of female gametophyte development in T. fournieri.
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Live Imaging and Laser Disruption Reveal the Dynamics and Cell–Cell Communication During Torenia fournieri Female Gametophyte Development
Plant and Cell Physiology, 2015Co-Authors: Daichi Susaki, Hidenori Takeuchi, Hiroki Tsutsui, Daisuke Kurihara, Tetsuya HigashiyamaAbstract:The female gametophytes of many flowering plants contain one egg cell, one central cell, two synergid cells and three antipodal cells with respective morphological characteristics and functions. These cells are formed by cellularization of a multinuclear female gametophyte. However, the dynamics and mechanisms of female gametophyte development remain largely unknown due to the lack of a system to visualize directly and manipulate female gametophytes in living material. Here, we established an in vitro ovule culture system to examine female gametophyte development in Torenia fournieri, a unique plant species with a protruding female gametophyte. The four-nucleate female gametophyte became eight nucleate by the final (third) mitosis and successively cellularized and matured to attract a pollen tube. The duration of final mitosis was 28± 6.5 min, and cellularization was completed in 54± 20 min after the end of the third mitosis. Fusion of polar nuclei in the central cell occurred in 13.1± 1.1 h, and onset of expression of LURE2, a pollen tube attractant gene, was visualized by a green fluorescent protein reporter 10.7± 2.3 h after cellularization. Laser disruption analysis demonstrated that the egg and central cells were required for synergid cells to acquire the pollen tube attraction function. Moreover, aberrant nuclear positioning and down-regulation of LURE2 were observed in one of the two synergid cells after disrupting an immature egg cell, suggesting that cell specification was affected. Our system provides insights into the precise dynamics and mechanisms of female gametophyte development in T. fournieri.
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Identification and characterization of TcCRP1, a pollen tube attractant from Torenia concolor
Annals of Botany, 2011Co-Authors: Masahiro M Kanaoka, Satohiro Okuda, Narie Sasaki, Daichi Susaki, Nao Kawano, Yoshiyuki Matsubara, Tetsuya HigashiyamaAbstract:Background and Aims During sexual reproduction in higher angiosperms, the pollen tubes are directed to the ovules in the pistil to deliver sperm cells. This pollen tube attraction is highly species specific, and a group of small secreted proteins, TfCRPs, are necessary for this process in Torenia fournieri.
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defensin like polypeptide lures are pollen tube attractants secreted from synergid cells
Nature, 2009Co-Authors: Satohiro Okuda, Daichi Susaki, Hidenori Takeuchi, Hiroki Tsutsui, Keiko Shiina, Stefanie Sprunck, Ryushiro D Kasahara, Yuki Hamamura, Akane Mizukami, Nao KawanoAbstract:Precise pollen tube guidance is critical for the successful fertilization of flowering plants. The concept of a pollen tube attractant was proposed in the late nineteenth century when pollen tubes were found to grow towards excised pistil tissues on medium. In 2001 the synergid cells, situated next to the egg, were shown to secrete a soluble factor that guides the growing pollen tube towards the embryo sac. Now at long last, these pollen tube attractants have been identified. Synergid cells were isolated from Torenia fournieri ('wishbone flower') plants, which are unique in having a protruding embryo sac. The secreted guidance factors were identified as cysteine-rich polypeptides belonging to the sub group of defensin-like proteins, and named LUREs. In this study, Higashiyama and colleagues examine pollen tube guidance in Torenia fournieri and identify the secreted guidance factors. These are cysteine-rich polypeptides belonging to the subgroup of defensin-like proteins (designated as LUREs), which are predominantly expressed in synergid cells and are required for pollen tube guidance. For more than 140 years, pollen tube guidance in flowering plants has been thought to be mediated by chemoattractants derived from target ovules1. However, there has been no convincing evidence of any particular molecule being the true attractant that actually controls the navigation of pollen tubes towards ovules. Emerging data indicate that two synergid cells on the side of the egg cell emit a diffusible, species-specific signal to attract the pollen tube at the last step of pollen tube guidance1,2,3. Here we report that secreted, cysteine-rich polypeptides (CRPs) in a subgroup of defensin-like proteins are attractants derived from the synergid cells. We isolated synergid cells of Torenia fournieri, a unique plant with a protruding embryo sac, to identify transcripts encoding secreted proteins as candidate molecules for the chemoattractant(s). We found two CRPs, abundantly and predominantly expressed in the synergid cell, which are secreted to the surface of the egg apparatus. Moreover, they showed activity in vitro to attract competent pollen tubes of their own species and were named as LUREs. Injection of morpholino antisense oligomers against the LUREs impaired pollen tube attraction, supporting the finding that LUREs are the attractants derived from the synergid cells of T. fournieri.
Katsutomo Sasaki - One of the best experts on this subject based on the ideXlab platform.
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production of multi petaled Torenia fournieri flowers by functional disruption of two class c mads box genes
Planta, 2020Co-Authors: Katsutomo Sasaki, Norihiro OhtsuboAbstract:MAIN CONCLUSION: Simultaneous knockdown or knockout of Torenia fournieri PLENA (TfPLE) and FALINELLI (TfFAR) genes with RNAi or genome-editing technologies generated a multi-petal phenotype in Torenia. The MADS-box gene AGAMOUS (AG) is well known to play important roles in the development of stamens and carpels in Arabidopsis. Mutations in AG cause the morphological transformation of stamens and carpels into petaloid organs. In contrast, Torenia (Torenia fournieri Lind.) has two types of class-C MADS-box genes, PLENA (PLE) and FALINELLI (FAR); however, their functions were previously undetermined. To examine the function of TfPLE and TfFAR in Torenia, we used RNAi to knockdown expression of these two genes. TfPLE and TfFAR double-knockdown transgenic Torenia plants had morphologically altered stamens and carpels that developed into petaloid organs. TfPLE knockdown transgenic plants also exhibited morphological transformations that included shortened styles, enlarged ovaries, and absent stigmata. Furthermore, simultaneous disruption of TfPLE and TfFAR genes by CRISPR/Cas9-mediated genome editing also resulted in the conversion of stamens and carpels into petaloid organs as was observed in the double-knockdown transgenic plants mediated by RNAi. In addition, the carpels of one TfPLE knockout mutant had the same morphological abnormalities as TfPLE knockdown transgenic plants. TfFAR knockdown genome-edited mutants had no morphological changes in their floral organs. These results clearly show that TfPLE and TfFAR cooperatively play important roles in the development of stamens and carpels. Simultaneous disruption of TfPLE and TfFAR functions caused a multi-petal phenotype, which is expected to be a highly valuable commercial floral trait in horticultural flowers.
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Generation of Novel Floral Traits Using a Combination of Floral Organ-Specific Promoters and a Chimeric Repressor in Torenia fournieri Lind.
Plant and Cell Physiology, 2016Co-Authors: Katsutomo Sasaki, Hiroyasu Yamaguchi, Takako Narumi, Ichiro Kasajima, Norihiro OhtsuboAbstract:In this study, we attempted to develop a new biotechnological method for the efficient modification of floral traits. Because transcription factors play an important role in determining floral traits, chimeric repressors, which are generated by attaching a short transcriptional repressor domain to transcription factors, have been widely used as effective tools for modifying floral traits in many plant species. However, the overexpression of these chimeric repressors by the Cauliflower mosaic virus 35S promoter sometimes causes undesirable morphological alterations to other organs. We attempted simultaneously to generate new floral traits and avoid such quality loss by examining five additional floral organ-specific promoters, one Arabidopsis thaliana promoter and four Torenia fournieri promoters, for the expression of the chimeric repressor of Arabidopsis TCP3 (AtTCP3), whose overexpression drastically alters floral traits but also generates dwarf phenotypes and deformed leaves. We found that the four Torenia promoters exhibited particularly strong activity in the petals but not in the leaves, and that the combination of these floral organ-specific promoters with the chimeric repressor of AtTCP3 caused changes in the color, color patterns and cell shapes of petals, whilst avoiding other unfavorable phenotypes. Interestingly, each promoter that we used in this study generated characteristic and distinguishable floral traits. Thus, the use of different floral organ-specific promoters with different properties enables us to generate diverse floral traits using a single chimeric repressor without changing the phenotypes of other organs.
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Co-modification of class B genes TfDEF and TfGLO in Torenia fournieri Lind. alters both flower morphology and inflorescence architecture
Plant Molecular Biology, 2014Co-Authors: Katsutomo Sasaki, R Aida, Hiroyasu Yamaguchi, Masayoshi Nakayama, Norihiro OhtsuboAbstract: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.
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mutation in Torenia fournieri lind ufo homolog confers loss of tflfy interaction and results in a petal to sepal transformation
Plant Journal, 2012Co-Authors: Katsutomo Sasaki, R Aida, Masahito Shikata, Hiroyasu Yamaguchi, Norihiro OhtsuboAbstract:Summary 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.
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overexpression of arabidopsis mir157b induces bushy architecture and delayed phase transition in Torenia fournieri
Planta, 2012Co-Authors: Masahito Shikata, Katsutomo Sasaki, Hiroyasu Yamaguchi, Norihiro OhtsuboAbstract:miR156/157 is a small RNA molecule that is highly conserved among various plant species. Overexpression of miR156/157 has been reported to induce bushy architecture and delayed phase transition in several plant species. To investigate the effect of miR157 overexpression in a horticultural plant, and to explore the applicability of miRNA to molecular breeding, we introduced Arabidopsis MIR157b (AtMIR157b) into Torenia (Torenia fournieri). The resulting 35S:AtMIR157b plants showed a high degree of branching along with small leaves, which resembled miR156/157-overexpressing plants of other species. We also isolated Torenia SBP-box genes with target miR156/157 sequences and confirmed that their expression was selectively downregulated in 35S:AtMIR157b plants. The reduced accumulation of mRNA was probably due to sequence specificity. Moreover, expression of Torenia homologs of the SBP-box protein-regulated genes TfLFY and TfMIR172 was also reduced by AtmiR157 overexpression. These findings suggest that the molecular mechanisms of miR156/157 regulation are conserved between Arabidopsis and Torenia. The bushy architecture and small leaves of 35S:AtMIR157b Torenia plants could be applied in molecular breeding of various horticultural plants as well as for increasing biomass and crop production.
