Biology subjects

Mizuta, Y.

Publications and source records attributed to Mizuta, Y..

7 recordsLinked to original sources

bioRxiv2026

Calcium Dynamics During Pollen Tube Reception in Arabidopsis Ovules

In flowering plants, pollen tubes communicate with ovular cells to achieve precise one-to-one pollen tube reception. The final step of this communication between the pollen tube and synergid cells has been extensively investigated and visualized by calcium imaging. Synergid cells exhibit characteristic cytoplasmic calcium concentration oscillations, which are thought to play a critical role in pollen tube reception. However, their significance and relationship with calcium dynamics in the entire ovule remain unclear. Here, we show, using the calcium sensor GCaMP6s, that proteins involved in asparagine-linked glycosylation (N-linked glycosylation) are required for normal calcium oscillations in synergid cells but are not essential for pollen tube reception. Using a semi-in vivo assay in Arabidopsis thaliana, we found that the amplitude of these oscillations prior to rapid pollen tube growth across the filiform apparatus was reduced in mutants lacking the oligosaccharyltransferase (OST) 3/6 subunit or alpha1,2-glucosyltransferase (ALG) 10, both of which are involved in N-linked glycosylation. Notably, these mutants did not exhibit reduced fertility attributable to defects in the female gametophyte but instead showed a polytubey phenotype due to a sporophytic defect. These findings suggest that N-linked glycans mediate communication between synergid cells and the pollen tube and indicate that the typical pattern of calcium oscillations in synergid cells is not essential for triggering pollen tube rupture. Furthermore, we show that sporophytic tissues of the ovule exhibit calcium waves that propagate toward the funiculus in correlation with pollen tube contact and rupture, implying that ovular tissues can potentially transmit these signals distantly beyond the ovule. Together, these findings reveal previously unrecognized intercellular calcium signaling and its significance in pollen tube reception by the ovule.

plant biology↗

bioRxiv2026

Extracellular calcium modulates pollen tube growth and guidance in Arabidopsis thaliana

In angiosperms, pollen tubes deliver sperm cells to the ovule and communicate with the external environment as they elongate through the pistils. Although pollination alters Ca2+ conditions within the pistil, the effects of extracellular Ca2+ fluctuations on pollen tube growth and guidance remain largely unknown. In this study, we visualized intracellular Ca2+ dynamics using a semi-in vivo assay with the Ca2+-sensitive fluorescent protein GCaMP6s to investigate how pollen tubes respond to changes in extracellular Ca2+ levels. We found that the Ca2+ levels in the apical region of the pollen tubes reflected the extracellular Ca2+ concentrations. The pollen tube growth rate increased depending on the Ca2+ concentration in the growth medium. However, excessive Ca2+ affected the polar growth of pollen tubes. At elevated Ca2+ concentrations of 10 mM, the pollen tube exhibited coiling behavior and failed to maintain directional growth toward the ovule. Moreover, we provided the first evidence that Ca2+ oscillations are not restricted to the apical region but propagate as a wave, reaching 30-50 m from the apex toward the basal regions. As the pollen tube approached the ovule, it coincided with a substantial elevation in Ca2+ levels, which appeared to drive the accelerated nuclear migration toward the tube apex. Our findings demonstrate that the extracellular Ca2+ environment directly regulates intracellular Ca2+ levels in pollen tubes, thereby influencing their growth and guidance.

plant biology↗

bioRxiv2025

In vitro live cell imaging reveals nuclear dynamics and role of the cytoskeleton during asymmetric division of pollen mitosis I in Nicotiana benthamiana

Pollen is a male gametophyte of angiosperms. Following meiosis, the microspore undergoes an asymmetric division called pollen mitosis I (PMI), which produces two cells of different sizes: a large vegetative cell and a small generative cell. Polarized nuclear migration and positioning during PMI are important for successful pollen development and cell differentiation. However, analyzing the pollen development process in real-time is challenging in many model plants with tricellular pollen, including Arabidopsis and rice. In this study, we established a method for live confocal imaging of microtubule and actin dynamics using suspension cultures with biolistic delivery of plasmid DNAs during PMI in Nicotiana benthamiana (Benthams tobacco), containing bicellular pollen. Pharmacological studies have indicated that actin filaments are crucial for microspore nuclear positioning before PMI, cell plate expansion during cytokinesis, and chromatin dispersion in vegetative cell nucleus after PMI. By contrast, inhibition of microtubule assembly resulted in abnormal chromosome segregation and nuclear behavior after PMI, although nuclear positioning and asymmetric division were observed. Our in vitro live cell imaging system for PMI provides insights into the importance of cytoskeletal regulation in asymmetric division and differentiation during pollen development.

plant biology↗

bioRxiv2023

Blue light irradiation induces pollen tube rupture in various flowering plants

Pollen tubes exhibit one of the fastest apical growth rates among plant cells. Maintaining the proper balance between turgor pressure and cell wall synthesis at the pollen tube tip is crucial for this rapid growth, and any disruption can result in pollen tube rupture. In our study, we reveal that exposure to short-wavelength visible light, specifically blue light, induces pollen tube rupture. The frequency of pollen tube rupture increases in an intensity-dependent manner. Additionally, we observed Ca2+ influx after blue light irradiation, accompanying with either pollen tube rupture or a temporary halt in elongation. These findings offer insights into the interplay between pollen tube integrity maintenance and Ca2+ influx at the pollen tube tip, presenting a novel and efficient method to control pollen tube burst. Subject Areas(1) growth and development (11) new methodology

plant biology↗

bioRxiv2023

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

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.

plant biology↗

bioRxiv2021

Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube

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 is generally applicable to a limited range of plants, such as model species. To overcome this limitation, we developed a method to genetically modify male germ cells without the need for Agrobacterium-mediated transfection and tissue culture, by 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 for driving expression of the delivered gene in pollen tubes. We also evaluated 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 on the stigma and produced two sperm cells within the pistil. We also observed ovules showing fluorescence derived from bombarded pollen. The findings of this study provide important insights into the editing of pollen tube genomes and the delivery of genome-modified male germ cells for seed production.

plant biology↗

bioRxiv2019

A peptide pair coordinates regular ovule initiation patterns with seed number and fruit size

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, how a regular spacing of ovules is achieved is not known. Using natural variation analysis combined with quantitative trait locus analysis, we found that the spacing of ovules in the developing 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 signalling 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 signalling results in shorter fruits and irregular spacing of ovules or even ovule twinning. The EPFL2 expression pattern between ovules is under negative-feedback regulation by auxin, which accumulates in the arising ovule primordia. We propose that the auxin-EPFL2 signalling module evolved to control the initiation and regular, equidistant spacing of ovule primordia, which serves to minimise competition between developing seeds. Together, EPFL2 and EPFL9 coordinate ovule patterning and thereby seed number with fruit growth through a set of shared receptors.

plant biology↗