Language：English   Publishing type：Research paper (scientific journal)  

Sexual reproduction between males and females of the same species is essential for species maintenance. Ovular micropylar guidance, the last step of pollen tube guidance in angiosperms, contributes to species-preferential reproduction. Previous studies using semi-in vivo attraction assays showed that species-preferential attractant peptides are secreted from the ovule through its micropyle. However, conventional semi-in vivo assays usually depend on transgenic pollen tubes expressing a fluorescent protein to determine whether the tubes are attracted to the ovule to precisely penetrate the micropyle. Here, we found that fluorescein diacetate (FDA) staining was suitable for evaluating the micropylar guidance rate of non-transgenic pollen tubes in semi-in vivo conditions. Micropylar guidance was quantified for ovules and pollen tubes of Arabidopsis thaliana and Arabidopsis lyrata by combining FDA staining with modified semi-in vivo assays. Our results using the simple staining method showed that the ovules of each species secrete species-preferential attractants, and that pollen tubes respond more strongly to attractants of their own species compared with those of closely related species. LURE-type CRP810 attractant peptides were shown to be responsible for micropylar attraction of A. thaliana in the semi-in vivo assay. The POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 6 (PRK6) receptor for LURE1, as well as an unidentified receptor for other LURE-type attractants, are involved in the species-preferential response of these two Arabidopsis species.

DOI： 10.3390/ijms23052722

Web of Science

PubMed

CiNii Research

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

In angiosperm sexual reproduction, the male pollen tube undergoes a series of interactions with female tissues. For efficient growth and precise guidance, the pollen tube perceives extracellular ligands. In recent decades, various types of secreted cysteine-rich peptides (CRPs) have been identified as peptide ligands that regulate diverse angiosperm reproduction processes, including pollen tube germination, growth, guidance, and rupture. Notably, in two distant core eudicot plants, multiple LURE-type CRPs were found to be secreted from egg-accompanying synergid cells, and these CRPs act as a cocktail of pollen tube attractants for the final step of pollen tube guidance. LURE-type CRPs have species-preferential activity, even among close relatives, and exhibit remarkably divergent molecular evolution with conserved cysteine frameworks, demonstrating that they play a key role in species recognition in pollen tube guidance. In this review, I focus on "reproductive CRPs," particularly LURE-type CRPs, which underlie common but species-specific mechanisms in angiosperm sexual reproduction, and discuss their action, functional regulation, receptors, and evolution.

DOI： 10.1016/j.peptides.2021.170572

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

During the double fertilization process, pollen tubes deliver two sperm cells to an ovule containing the female gametes. In the pollen tube, the vegetative nucleus and sperm cells move together to the apical region where the vegetative nucleus is thought to play a crucial role in controlling the direction and growth of the pollen tube. Here, we report the generation of pollen tubes in Arabidopsis thaliana whose vegetative nucleus and sperm cells are isolated and sealed by callose plugs in the basal region due to apical transport defects induced by mutations in the WPP domain-interacting tail-anchored proteins (WITs) and sperm cell-specific expression of a dominant mutant of the CALLOSE SYNTHASE 3 protein. Through pollen-tube guidance assays, we show that the physiologically anuclear mutant pollen tubes maintain the ability to grow and enter ovules. Our findings provide insight into the sperm cell delivery mechanism and illustrate the independence of the tip-localized vegetative nucleus from directional growth control of the pollen tube.

DOI： 10.1038/s41467-021-22661-8

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi.org/10.3389/fpls.2020.588700

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi.org/10.1104/pp.20.00344

Language：English   Publishing type：Research paper (scientific journal)  

Development of pollen, the male gametophyte of flowering plants, is tightly controlled by dynamic changes in gene expression. Recent research to clarify the molecular aspects of pollen development has revealed the involvement of several transcription factors in the induction of gene expression. However, limited information is available about the factors involved in the negative regulation of gene expression to eliminate unnecessary transcripts during pollen development. In this study, we revealed that AtNOT1 is an essential protein for proper pollen development and germination capacity. AtNOT1 is a scaffold protein of the AtCCR4-NOT complex, which includes multiple components related to mRNA turnover control in Arabidopsis. Phenotypic analysis using atnot1 heterozygote mutant pollen showed that the mature mutant pollen failed to germinate and also revealed abnormal localization of nuclei and a specific protein at the tricellular pollen stage. Furthermore, transcriptome analysis of atnot1 heterozygote mutant pollen showed that the downregulation of a large number of transcripts, along with the upregulation of specific transcripts required for pollen tube germination by AtNOT1 during late microgametogenesis, is important for proper pollen development and germination. Overall, our findings provide new insights into the negative regulation of gene expression during pollen development, by showing the severely defective phonotype of atnot1 heterozygote mutant pollen.

DOI： 10.1093/pcp/pcz235

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41467-017-01323-8

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BioMed Central Ltd.  

BACKGROUND: Gene bodies of vertebrates and flowering plants are occupied by the histone variant H3.3 and DNA methylation. The origin and significance of these profiles remain largely unknown. DNA methylation and H3.3 enrichment profiles over gene bodies are correlated and both have a similar dependence on gene transcription levels. This suggests a mechanistic link between H3.3 and gene body methylation. RESULTS: We engineered an H3.3 knockdown in Arabidopsis thaliana and observed transcription reduction that predominantly affects genes responsive to environmental cues. When H3.3 levels are reduced, gene bodies show a loss of DNA methylation correlated with transcription levels. To study the origin of changes in DNA methylation profiles when H3.3 levels are reduced, we examined genome-wide distributions of several histone H3 marks, H2A.Z, and linker histone H1. We report that in the absence of H3.3, H1 distribution increases in gene bodies in a transcription-dependent manner. CONCLUSIONS: We propose that H3.3 prevents recruitment of H1, inhibiting H1's promotion of chromatin folding that restricts access to DNA methyltransferases responsible for gene body methylation. Thus, gene body methylation is likely shaped by H3.3 dynamics in conjunction with transcriptional activity.

DOI： 10.1186/s13059-017-1221-3

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.cell.2015.03.018

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

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.

DOI： 10.1093/pcp/pcv031

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Key message: New gametic homozygous mutants. In angiosperms, a haploid male gamete (sperm cell) fuses with a haploid female gamete (egg cell) during fertilization to form a zygote carrying paternally and maternally derived chromosomes. Several fertilization-defective mutants in Arabidopsis thaliana, including a generative cell-specific 1 (gcs1)/hapless 2 mutant, the sperm cells of which are unable to fuse with female gametes, can only be maintained as heterozygous lines due to the infertile male or female gametes. Here, we report successful generation of a gcs1 homozygous mutant by heat-inducible removal of the GCS1 transgene. Using the gcs1 homozygous mutant as male, the defect in gamete fusion was observed with great frequency; in our direct observation by semi-in vivo fertilization assay using ovules, 100 % of discharged sperm cells in culture failed to show gamete fusion. More than 70 % of ovules in the pistil received a second pollen tube as attempted fertilization recovery. Moreover, gcs1 mutant sperm cells could fertilize female gametes at a low frequency in the pistil. This strategy to generate homozygous fertilization-defective mutants will facilitate novel approaches in plant reproduction research.

DOI： 10.1007/s00497-015-0256-4

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ANNUAL REVIEWS  

During sexual reproduction of flowering plants, pollen tube guidance by pistil tissue is critical for the delivery of nonmotile sperm cells to female gametes. Multistep controls of pollen tube guidance can be divided into two phases: preovular guidance and ovular guidance. During preovular guidance, various female molecules, including stimulants for pollen germination and pollen tube growth, are provided to support tube growth toward the ovary, where the ovules are located. After entering the ovary, pollen tubes receive directional cues from their respective target ovules, including attractant peptides for precise, species-preferential attraction. Successful pollen tube guidance in the pistil requires not only nutritional and directional controls but also competency controls to make pollen tubes responsive to guidance cues, regulation to terminate growth once a pollen tube arrives at the target, and strategies to stop ovular attraction depending on the fertilization of female gametes.

DOI： 10.1146/annurev-arplant-043014-115635

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Ca(2+) waves and oscillation are key signalling elements during the fertilization process of animals, and are involved, for example, in egg activation. In the unique double fertilization process in flowering plants, both the egg cell and the neighbouring central cell fuse with a sperm cell each. Here we succeeded in imaging cytosolic Ca(2+) in these two cells, and in the two synergid cells that accompany the gametes during semi-in vivo double fertilization. Following pollen tube discharge and plasmogamy, the egg and central cells displayed transient Ca(2+) spikes, but not oscillations. Only the events in the egg cell correlated with the plasmogamy. In contrast, the synergid cells displayed Ca(2+) oscillations on pollen tube arrival. The two synergid cells showed distinct Ca(2+) dynamics depending on their respective roles in tube reception. These Ca(2+) dynamics in the female gametophyte seem to represent highly specific signatures that coordinate successful double fertilization in the flowering plants.

DOI： 10.1038/ncomms5722

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

In flowering plants, double fertilization is normally accomplished by the first pollen tube, with the fertilized ovule subsequently inhibiting the attraction of a second pollen tube. However, the mechanism of second-pollen-tube avoidance remains unknown. We discovered that failure to fertilize either the egg cell or the central cell compromised second-pollen-tube avoidance in Arabidopsis thaliana. A similar disturbance was caused by disrupting the fertilization-independent seed (FIS) class polycomb-repressive complex 2 (FIS-PRC2), a central cell- and endosperm-specific chromatin-modifying complex for gene silencing. Therefore, the two female gametes have evolved their own signaling pathways. Intriguingly, second-pollen-tube attraction induced by half-successful fertilization allowed the ovules to complete double fertilization, producing a genetically distinct embryo and endosperm. We thus propose that each female gamete independently determines second-pollen-tube avoidance to maximize reproductive fitness in flowering plants.

DOI： 10.1016/j.devcel.2013.03.013

Web of Science

PubMed

Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：The Japanese Society for Chemical Regulation of Plants  

Pollen tube guidance is a crucial mechanism for successful fertilization in angiosperms, which precisely controls directional growth of pollen tubes of own species to deliver immotile sperm cells to an embryo sac. Since pollen tubes were observed to grow toward excised pistil tissues on a medium in the late nineteenth century, plant biologists have conducted studies to reveal mechanisms of pollen tube growth and guidance. Recently, pollen tube attractants, LURE peptides, have been identified in a unique dicot, Torenia fournieri. However, universal molecular mechanisms involving species-specific pollen tube attraction are still unclear. In this review, we present identification of pollen tube attractants of Arabidopsis thaliana, AtLURE1 peptides, and discuss universality, species specificity, and expression mechanism of the attractant molecules.

DOI： 10.18978/jscrp.48.1_73

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)   Publisher：CURRENT BIOLOGY LTD  

Pollen tube guidance is the mechanism whereby the direction of pollen tube growth is controlled by female cells of the pistil. Some key genes and molecules have recently been identified as being involved in pollen tube guidance. In this review article, we discuss the molecular basis of pollen tube guidance, especially in Arabidopsis thaliana, by summarizing recent progress in various plant species. Attractant molecules and receptors for gametophytic pollen tube guidance are the focus of this article.

DOI： 10.1016/j.pbi.2011.07.010

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

For more than 140 years, pollen tube guidance in flowering plants has been thought to be mediated by chemoattractants derived from target ovules. 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 guidance. 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.

DOI： 10.1038/nature07882

Web of Science

PubMed

Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

DOI： 10.1271/kagakutoseibutsu.47.617

J-GLOBAL

Authorship：Lead author,　Corresponding author  

Authorship：Lead author,　Corresponding author  

Authorship：Corresponding author  

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DOI： 10.14841/jspp.2011.0.0024.0

J-GLOBAL

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Language：Japanese  

DOI： 10.14841/jspp.2010.0.0261.0

J-GLOBAL

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DOI： 10.14841/jspp.2009.0.0112.0

J-GLOBAL

Event date： 2022.3

Language：Japanese   Presentation type：Poster presentation  

Event date： 2020.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：名古屋・オンライン開催  

Event date： 2020.3

Language：Japanese  

Country：Japan  

Event date： 2019.9

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2019.9

Language：Japanese   Presentation type：Poster presentation  

Country：Japan  

Event date： 2019.3

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2018.9

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Event date： 2018.9

Language：Japanese   Presentation type：Poster presentation  

Country：Japan  

Event date： 2018.9

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2018.6

Language：English   Presentation type：Poster presentation  

Country：Japan  

Event date： 2018.6

Language：English   Presentation type：Poster presentation  

Country：Japan  

Event date： 2018.3

Language：Japanese   Presentation type：Poster presentation  

Country：Japan  

Event date： 2017.9

Language：English   Presentation type：Poster presentation  

Country：Germany  

Event date： 2013

Language：Japanese   Presentation type：Oral presentation (general)  

Pollen tube guidance is a crucial mechanism for successful fertilization in angiosperms, which precisely controls directional growth of pollen tubes of own species to deliver immotile sperm cells to an embryo sac. Since pollen tubes were observed to grow toward excised pistil tissues on a medium in the late nineteenth century, plant biologists have conducted studies to reveal mechanisms of pollen tube growth and guidance. Recently, pollen tube attractants, LURE peptides, have been identified in a unique dicot, Torenia fournieri. However, universal molecular mechanisms involving species-specific pollen tube attraction are still unclear. In this review, we present identification of pollen tube attractants of Arabidopsis thaliana, AtLURE1 peptides, and discuss universality, species specificity, and expression mechanism of the attractant molecules.

DOI： 10.18978/jscrp.48.1_73

Event date： 2009.9

Language：Japanese   Presentation type：Oral presentation (general)  

DOI： 10.1271/kagakutoseibutsu.47.617

Language：Japanese   Presentation type：Oral presentation (general)  

Language：Japanese   Presentation type：Oral presentation (general)  

Language：Japanese   Presentation type：Oral presentation (general)  

Language：English   Presentation type：Oral presentation (general)