研究者詳細 - 水多陽子

Blue light irradiation induces pollen tube rupture in various flowering plants

その他リンク： https://www.nagoya-u.ac.jp/researchinfo/result/2024/05/post-665.html

Letter to the Editor: Blue Light Irradiation Induces Pollen Tube Rupture in Various Flowering Plants 査読有り Open Access

Sugi, N; Susaki, D; Mizuta, Y; Kinoshita, T; Maruyama, D

PLANT AND CELL PHYSIOLOGY 65 巻 ( 5 ) 頁： 704 - 707 2024年5月

Naoya Sugi, Daichi Susaki, Yoko Mizuta, Tetsu Kinoshita, Daisuke Maruyama

bioRxiv 2023年12月

Deep imaging revealed dynamics and signaling in one-to-one pollen tube guidance

Yoko Mizuta, Daigo Sakakibara, Shiori Nagahara, Ikuma Kaneshiro, Takuya T. Nagae, Daisuke Kurihara, Tetsuya Higashiyama

bioRxiv 2023年4月

担当区分：筆頭著者,　責任著者出版者・発行元：Cold Spring Harbor Laboratory

ABSTRACT

In angiosperms, pollen tube guidance allows sperm cell delivery to the female gametes within the ovule, which are deeply embedded in a flower. However, when an ovary includes multiple pollen tubes and ovules, it is unclear how each ovule is fertilized one-to-one by a pollen tube. Here, our two-photon imaging revealed the pollen tube dynamics in living ovaries. The number of pollen tubes and ovule maturity affected the target selection among multiple ovules. On the inner surface of the septum epidermis within the transmitting tract, pollen tube behavior and emergence were regulated by the ovular sporophytic signals. In funicular guidance, the second pollen tube was strictly repelled by the FERONIA and LORELEI-dependent gametophytic signal, especially more than 45 minutes after the first pollen tube had passed. Such highly spatiotemporal regulation mechanisms in the one-to-one pollen tube guidance may allow angiosperms to produce more offspring in nature.

DOI： 10.1101/2023.04.19.537439

一過的導入による花粉の可視化とゲノム編集査読有り

水多陽子

植物科学の最前線（BSJ-Review） 14 巻 ( A ) 頁： 21 - 29 2023年3月

植物細胞の分化運命の制御と可塑性査読有り

丸山大輔, 水多陽子, 山岡尚平

植物科学の最前線（BSJ-Review） 14 巻 ( A ) 頁： 1 - 2 2023年

Target pollen isolation using automated infrared laser-mediated cell disruption 査読有り Open Access

Kaneshiro, I; Igarashi, M; Higashiyama, T; Mizuta, Y

QUANTITATIVE PLANT BIOLOGY 3 巻頁： e30 2022年12月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Quantitative Plant Biology

Single-cell analysis is important to understand how individual cells work and respond at the cell population level. Experimental single-cell isolation techniques, including dilution, fluorescence-activated cell sorting, microfluidics, and micromanipulation, have been developed in recent decades. However, such applications typically require large cell populations and skilled professionals. Additionally, these methods are unsuitable for sequential analysis before and after cell isolation. In this study, we propose a method for target cell isolation using automated infrared laser-mediated disruption of pollen grains in pollen populations. Germination of the target pollen was observed at the same location as that before laser irradiation, and germinated pollen grains were enriched in the cell population. Pollination of laser-irradiated bulk pollen populations also showed that the target pollen preferentially germinated on the stigma. This method is expected to facilitate physiological analyses of target cells at the single-cell level and effectively produce seeds derived from target pollen.

DOI： 10.1017/qpb.2022.24

その他リンク： https://www.microscope.healthcare.nikon.com/ja_JP/resources/application-notes/automatic-isolation-of-pollen-using-nis-elements-general-analysis-ga-and-jobs-imaging-workflow-tools

Deep Fluorescence Observation in Rice Shoots via Clearing Technology 査読有り国際誌 Open Access

Niimi, Y; Nagai, K; Ashikari, M; Mizuta, Y

JOVE-JOURNAL OF VISUALIZED EXPERIMENTS 2022 巻 ( 184 ) 2022年6月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Visualized Experiments

The recently developed clearing technology that eliminates refractive index mismatches and diminishes auto-fluorescent material has made it possible to observe plant tissues in three dimensions (3D) while preserving their internal structures. In rice (Oryza sativa L.), a monocot model plant and a globally important crop, clearing technology has been reported in organs that are relatively easy to observe, such as the roots and leaves. Applications of clearing technology in shoot apical meristem (SAM) and stems have also been reported, but only to a limited degree because of the poor penetration of the clearing solution (CS) in these tissues. The limited efficiency of the clearing solutions in these tissues has been attributed to auto-fluorescence, thickening, and hardening of the tissues in the stem as the vascular bundles and epidermis develop and layering of the SAM with water-repellent leaves. The present protocol reports the optimization of a clearing approach for continuous and 3D observation of gene expression from the SAM/young panicle to the base of the shoots during development. Fixed tissue samples expressing a fluorescent protein reporter were trimmed into sections using a vibrating micro-slicer. When an appropriate thickness was achieved, the CS was applied. By specifically targeting the central tissue, the penetration rate and uniformity of the CS increased, and the time required to make the tissue transparent decreased. Additionally, clearing of the trimmed sections enabled the observation of the internal structure of the whole shoot from a macro perspective. This method has potential applications in deep imaging of tissues of other plant species that are difficult to clear.

DOI： 10.3791/64116

Optical Clearing of Plant Tissues for Fluorescence Imaging 査読有り Open Access

Kurihara, D; Mizuta, Y; Nagahara, S; Sato, Y; Higashiyama, T

JOVE-JOURNAL OF VISUALIZED EXPERIMENTS 2021 巻 ( 179 ) 2022年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Visualized Experiments

It is challenging to directly observe the internal structure of multi-layered and opaque plant specimens, without dissection, under a microscope. In addition, autofluorescence attributed to chlorophyll hampers the observation of fluorescent proteins in plants. For a long time, various clearing reagents have been used to make plants transparent. However, conventional clearing reagents diminish fluorescent signals; therefore, it has not been possible to observe the cellular and intracellular structures with fluorescent proteins. Reagents were developed that can clear plant tissues by removing chlorophyll while maintaining fluorescent protein stability. A detailed protocol is provided here for the optical clearing of plant tissues using clearing reagents, ClearSee (CS) or ClearSeeAlpha (CSA). The preparation of cleared plant tissues involves three steps: fixation, washing, and clearing. Fixation is a crucial step in maintaining the cellular structures and intracellular stability of fluorescent proteins. The incubation time for clearing depends on the tissue type and species. In Arabidopsis thaliana, the time required for clearing with CS was 4 days for leaves and roots, 7 days for seedlings, and 1 month for pistils. CS also required a relatively short time of 4 days to make the gametophytic leaves of Physcomitrium patens transparent. In contrast, pistils in tobacco and torenia produced brown pigment due to oxidation during CS treatment. CSA reduced the brown pigment by preventing oxidation and could make tobacco and torenia pistils transparent, although it took a relatively long time (1 or 2 months). CS and CSA were also compatible with staining using chemical dyes, such as DAPI (4',6-diamidino-2-phenylindole) and Hoechst 33342 for DNA and Calcofluor White, SR2200, and Direct Red 23 for the cell wall. This method can be useful for whole-plant imaging to reveal intact morphology, developmental processes, plant-microbe interactions, and nematode infections.

DOI： 10.3791/63428

Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube 査読有り Open Access

Nagahara, S; Higashiyama, T; Mizuta, Y

PLANT REPRODUCTION 34 巻 ( 3 ) 頁： 191 - 205 2021年9月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Plant Reproduction

Key message: Biolistic delivery into pollen. Abstract: In recent years, genome editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding. Agrobacterium-mediated transformation has been widely utilized to generate transgenic plants by introducing plasmid DNA containing CRISPR/Cas9 into plant cells. However, this method has general limitations, such as the limited host range of Agrobacterium and difficulties in tissue culture, including callus induction and regeneration. To avoid these issues, we developed a method to genetically modify germ cells without the need for Agrobacterium-mediated transfection and tissue culture using tobacco as a model. In this study, plasmid DNA containing sequences of Cas9, guide RNA, and fluorescent reporter was introduced into pollen using a biolistic delivery system. Based on the transient expression of fluorescent reporters, the Arabidopsis UBQ10 promoter was found to be the most suitable promoter for driving the expression of the delivered gene in pollen tubes. We also evaluated the delivery efficiency in male germ cells in the pollen by expression of the introduced fluorescent marker. Mutations were detected in the target gene in the genomic DNA extracted from CRISPR/Cas9-introduced pollen tubes, but were not detected in the negative control. Bombarded pollen germinated pollen tubes and delivered their contents into the ovules in vivo. Although it is necessary to improve biolistic delivery efficiency and establish a method for the screening of genome-modified seeds, our findings provide important insights for the detection and production of genome-modified seeds by pollen biolistic delivery.

DOI： 10.1007/s00497-021-00418-z

その他リンク： https://link.springer.com/article/10.1007/s00497-021-00418-z/fulltext.html

ClearSeeAlpha: Advanced Optical Clearing for Whole-Plant Imaging 査読有り Open Access

Kurihara, D; Mizuta, Y; Nagahara, S; Higashiyama, T

PLANT AND CELL PHYSIOLOGY 62 巻 ( 8 ) 頁： 1302 - 1310 2021年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Plant and Cell Physiology

To understand how the body of plants is made, it is essential to observe the morphology, structure and arrangement of constituent cells. However, the opaque nature of the plant body makes it difficult to observe the internal structures directly under a microscope. To overcome this problem, we developed a reagent, ClearSee, that makes plants transparent, allowing direct observation of the inside of a plant body without inflicting damage on it, e.g. through physical cutting. However, because ClearSee is not effective in making some plant species and tissues transparent, in this study, we further improved its composition to prevent oxidation, and have developed ClearSeeAlpha, which can be applied to a broader range of plant species and tissues. Sodium sulfite, one of the reductants, prevented brown pigmentation due to oxidation during clearing treatment. Using ClearSeeAlpha, we show that it is possible to obtain clear chrysanthemum leaves, tobacco and Torenia pistils and fertilized Arabidopsis thaliana fruits - tissues that have hitherto been challenging to clear. Moreover, we show that the fluorescence intensity of purified fluorescent proteins emitting light of various colors was unaffected in the ClearSeeAlpha solution; only the fluorescence intensity of TagRFP was reduced by about half. ClearSeeAlpha should be useful in the discovery and analysis of biological phenomena occurring deep inside the plant tissues.

DOI： 10.1093/pcp/pcab033

その他リンク： https://academic.oup.com/pcp/article-pdf/62/8/1302/41119229/pcab033.pdf

Advances in Two-Photon Imaging in Plants 査読有り Open Access

Mizuta, Y

PLANT AND CELL PHYSIOLOGY 62 巻 ( 8 ) 頁： 1224 - 1230 2021年8月

担当区分：筆頭著者,　最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Plant and Cell Physiology

Live and deep imaging play a significant role in the physiological and biological study of organisms. Two-photon excitation microscopy (2PEM), also known as multiphoton excitation microscopy, is a fluorescent imaging technique that allows deep imaging of living tissues. Two-photon lasers use near-infrared (NIR) pulse lasers that are less invasive and permit deep tissue penetration. In this review, recent advances in two-photon imaging and their applications in plant studies are discussed. Compared to confocal microscopy, NIR 2PEM exhibits reduced plant-specific autofluorescence, thereby achieving greater depth and high-resolution imaging in plant tissues. Fluorescent proteins with long emission wavelengths, such as orange-red fluorescent proteins, are particularly suitable for two-photon live imaging in plants. Furthermore, deep- and high-resolution imaging was achieved using plant-specific clearing methods. In addition to imaging, optical cell manipulations can be performed using femtosecond pulsed lasers at the single cell or organelle level. Optical surgery and manipulation can reveal cellular communication during development. Advances in in vivo imaging using 2PEM will greatly benefit biological studies in plant sciences.

DOI： 10.1093/pcp/pcab062

その他リンク： https://academic.oup.com/pcp/article-pdf/62/8/1224/41119175/pcab062.pdf

A Peptide Pair Coordinates Regular Ovule Initiation Patterns with Seed Number and Fruit Size 査読有り国際誌 Open Access

Kawamoto, N; Del Carpio, DP; Hofmann, A; Mizuta, Y; Kurihara, D; Higashiyama, T; Uchida, N; Torii, KU; Colombo, L; Groth, G; Simon, R

CURRENT BIOLOGY 30 巻 ( 22 ) 頁： 4352 - + 2020年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Current Biology

Ovule development in Arabidopsis thaliana involves pattern formation, which ensures that ovules are regularly arranged in the pistils to reduce competition for nutrients and space. Mechanisms underlying pattern formation in plants, such as phyllotaxis, flower morphogenesis, or lateral root initiation, have been extensively studied, and genes controlling the initiation of ovules have been identified. However, the fundamental patterning mechanism that determines the spacing of ovule anlagen within the placenta remained unexplored. Using natural variation analysis combined with quantitative trait locus analysis, we found that the spacing of ovules in the developing gynoecium and fruits is controlled by two secreted peptides, EPFL2 and EPFL9 (also known as Stomagen), and their receptors from the ERECTA (ER) family that act from the carpel wall and the placental tissue. We found that a signaling pathway controlled by EPFL9 acting from the carpel wall through the LRR-receptor kinases ER, ERL1, and ERL2 promotes fruit growth. Regular spacing of ovules depends on EPFL2 expression in the carpel wall and in the inter-ovule spaces, where it acts through ERL1 and ERL2. Loss of EPFL2 signaling results in shorter gynoecia and fruits and irregular spacing of ovules or even ovule twinning. We propose that the EPFL2 signaling module evolved to control the initiation and regular, equidistant spacing of ovule primordia, which may serve to minimize competition between seeds or facilitate equal resource allocation. Together, EPFL2 and EPFL9 help to coordinate ovule patterning and thereby seed number with gynoecium and fruit growth through a set of shared receptors.

DOI： 10.1016/j.cub.2020.08.050

フィールドにおける生命現象の解明とその制御に向けた基盤技術の創出招待有り査読有り

山内卓樹, 藤井壮太, 岡本昌憲, 田中佑, 水多陽子, 晝間敬, 吉田健太郎, 山本英司, 大西孝幸, 犬飼義明

育種学研究 22 巻 ( 1 ) 頁： 75 - 82 2020年

Chemical signaling for pollen tube guidance at a glance 招待有り査読有り Open Access

Mizuta, Y; Higashiyama, T

JOURNAL OF CELL SCIENCE 131 巻 ( 2 ) 頁： jcs208447 2018年1月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Cell Science

Pollen tube guidance is a unique navigating system that is required for the successful sexual reproduction of plants. As plant sperm cells are non-motile and egg cells are embedded deep inside the female tissues, a pollen tube delivers the two sperm cells that it contains by growing towards the ovule, in which the egg cell resides. Pollen tube growth towards the ovule is precisely controlled and divided into two stages, preovular and ovular guidance. In this Cell Science at a Glance article and accompanying poster, we provide a comprehensive overview of pollen tube guidance and highlight some of the attractant peptides used during ovular guidance. We further discuss the precise one-to-one guidance system that exists in multi-ovular plants. The pollen tube-blocking system, which is mediated by male-female crosstalk communication, to avoid attraction of multiple pollen tubes, is also reviewed.

DOI： 10.1242/jcs.208447

Three-Dimensional Multiphoton Imaging of Transcription Factor by ClearSee 査読有り

Mizuta, Y; Tsuda, K

PLANT TRANSCRIPTION FACTORS: METHODS AND PROTOCOLS 1830 巻頁： 257 - 268 2018年

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Methods in Molecular Biology

In plants, transcription factors often act as cell-to-cell trafficking mobile proteins and specify cell fate. Thus, to visualize spatiotemporal expression pattern and localization of transcription factors are essential to understand their functions during development. Several protocols have been developed to observe fluorescent protein. However, plant-specific autofluorescent compounds and various tissue components with different refractive indexes interfere with detection of fluorescent signals of your interest. Furthermore, cell fate specification often occurs in a limited number of cells covered by lateral/layers of organs. To overcome those issues, the plant clearing method, ClearSee, was recently developed for high-resolution imaging inside tissues by making background transparent. In this chapter, we provide three-dimensional imaging of fluorescent-protein-fused transcription factors by two-photon excitation microscopy in Arabidopsis and rice. Complex cell patterning with gene expression could be observed from any direction three-dimensionally. This method could be applicable to visualize any protein of your interest or it can readily be adapted in various other plants.

DOI： 10.1007/978-1-4939-8657-6_15