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      Live imaging of calcium spikes during double fertilization in Arabidopsis

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          Abstract

          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.

          Abstract

          Intracellular calcium waves are key signalling elements during the fertilization process of animals, involved in egg activation. Here the authors image calcium oscillations during the fertilization process in flowering plants, revealing specific signatures involved in the success of this process.

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          Most cited references44

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          The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation.

          The new pPZP Agrobacterium binary vectors are versatile, relatively small, stable and are fully sequenced. The vectors utilize the pTiT37 T-DNA border regions, the pBR322 bom site for mobilization from Escherichia coli to Agrobacterium, and the ColE1 and pVS1 plasmid origins for replication in E. coli and in Agrobacterium, respectively. Bacterial marker genes in the vectors confer resistance to chloramphenicol (pPZP100 series) or spectinomycin (pPZP200 series), allowing their use in Agrobacterium strains with different drug resistance markers. Plant marker genes in the binary vectors confer resistance to kanamycin or to gentamycin, and are adjacent to the left border (LB) of the transferred region. A lacZ alpha-peptide, with the pUC18 multiple cloning site (MCS), lies between the plant marker gene and the right border (RB). Since the RB is transferred first, drug resistance is obtained only if the passenger gene is present in the transgenic plants.
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            Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins.

            Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for various cellular functions. Although most indicators have cyan- and yellow-emitting fluorescent proteins (CFP and YFP) as FRET donor and acceptor, their poor dynamic range often prevents detection of subtle but significant signals. Here, we optimized the relative orientation of the two chromophores in the Ca(2+) indicator, yellow cameleon (YC), by fusing YFP at different angles. We generated circularly permuted YFPs (cpYFPs) that showed efficient maturation and acid stability. One of the cpYFPs incorporated in YC absorbs a great amount of excited energy from CFP in its Ca(2+)-saturated form, thereby increasing the Ca(2+)-dependent change in the ratio of YFP/CFP by nearly 600%. Both in cultured cells and in the nervous system of transgenic mice, the new YC enables visualization of subcellular Ca(2+) dynamics with better spatial and temporal resolution than before. Our study provides an important guide for the development and improvement of indicators using GFP-based FRET.
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              Two-photon absorption properties of fluorescent proteins.

              Two-photon excitation of fluorescent proteins is an attractive approach for imaging living systems. Today researchers are eager to know which proteins are the brightest and what the best excitation wavelengths are. Here we review the two-photon absorption properties of a wide variety of fluorescent proteins, including new far-red variants, to produce a comprehensive guide to choosing the right fluorescent protein and excitation wavelength for two-photon applications.
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                Author and article information

                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Pub. Group
                2041-1723
                22 August 2014
                : 5
                : 4722
                Affiliations
                [1 ]Division of Biological Sciences, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku , Nagoya, Aichi 464-8602, Japan
                [2 ]JST ERATO Higashiyama Live-Holonics Project, Nagoya University, Furo-cho, Chikusa-ku , Nagoya, Aichi 464-8602, Japan
                [3 ]Institut de recherche en biologie végétale, Département de sciences biologiques, Université de Montréal , 4101 Rue Sherbrooke est, Montréal, Québec, Canada H1X 2B2
                [4 ]Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo-cho, Chikusa-ku , Nagoya, Aichi 464-8602, Japan
                [5 ]Present address: Institut de recherche en biologie végétale, Département de sciences biologiques, Université de Montréal, 4101 Rue Sherbrooke est, Montréal, Québec, Canada H1X 2B2
                Author notes
                Article
                ncomms5722
                10.1038/ncomms5722
                4143913
                25146889
                7ba53977-f552-4217-9542-c5930cfaaa67
                Copyright © 2014, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

                This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/

                History
                : 28 November 2013
                : 17 July 2014
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