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      Dynamic F-actin movement is essential for fertilization in Arabidopsis thaliana

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          Abstract

          In animals, microtubules and centrosomes direct the migration of gamete pronuclei for fertilization. By contrast, flowering plants have lost essential components of the centrosome, raising the question of how flowering plants control gamete nuclei migration during fertilization. Here, we use Arabidopsis thaliana to document a novel mechanism that regulates F-actin dynamics in the female gametes and is essential for fertilization. Live imaging shows that F-actin structures assist the male nucleus during its migration towards the female nucleus. We identify a female gamete-specific Rho-GTPase that regulates F-actin dynamics and further show that actin–myosin interactions are also involved in male gamete nucleus migration. Genetic analyses and imaging indicate that microtubules are dispensable for migration and fusion of male and female gamete nuclei. The innovation of a novel actin-based mechanism of fertilization during plant evolution might account for the complete loss of the centrosome in flowering plants.

          DOI: http://dx.doi.org/10.7554/eLife.04501.001

          eLife digest

          Sexual reproduction involves combining the genetic material from two parents to create an offspring. The genetic material in the male sperm cell and the female egg cell is contained in the nucleus of each cell. Once these two cells fuse at fertilization, their nuclei must then navigate towards each other and fuse.

          When an animal egg cell is fertilized, cable-like protein filaments called microtubules guide the two nuclei into contact. These microtubules are organized by a cellular structure called a centrosome. However, flowering plants do not have centrosomes; as such, it was unclear how genetic material from the sperm and egg cells is brought together after fertilization in flowering plants.

          To investigate this, Kawashima et al. turned to a flowering plant commonly used in research, called Arabidopsis thaliana, and found that microtubules are not needed to guide the nuclei of the sperm and the egg cell after fertilization. Instead, another cable-forming protein—called F-actin—fulfills a similar role in Arabidopsis cells.

          F-actin filaments often connect together to form a network; and when Kawashima et al. disrupted the F-actin in Arabidopsis egg cells, the nucleus of the sperm cell failed to fuse with that of the female. Pollen from Arabidopsis plants actually contains two sperm cells. One sperm cell fertilizes the egg cell; the other fertilizes the so-called ‘central cell’, which develops into a tissue that nourishes the plant embryo. Kawashima et al. found that the fertilization of both of these cells requires an intact F-actin network.

          By looking more closely at F-actin networks in the larger central cell, Kawashima et al. discovered that the sperm nucleus becomes surrounded by a star-shaped structure of F-actin cables and that this F-actin structure migrates together with the sperm nucleus. The F-actin network constantly moves inward, from the edges of the cell towards the nucleus, prior to fertilization. This movement is essential for guiding the sperm nucleus towards the central cell nucleus.

          Kawashima et al. also found that this continual movement of the F-actin network depends on a small signaling protein found in the central cell, called ROP8. It also involves a motor protein that normally transports “cargo”, such as proteins and other molecules, inside cells by walking along the F-actin networks. However, rather than transporting the sperm nucleus as cargo, Kawashima et al. believe that the motor protein instead helps to maintain the inward movement of the F-actin network. One of the next challenges will be to investigate the molecular mechanism that underlies this motor protein's involvement in this dynamic F-actin network.

          DOI: http://dx.doi.org/10.7554/eLife.04501.002

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          Most cited references52

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          Nonequilibrium mechanics of active cytoskeletal networks.

          Cells both actively generate and sensitively react to forces through their mechanical framework, the cytoskeleton, which is a nonequilibrium composite material including polymers and motor proteins. We measured the dynamics and mechanical properties of a simple three-component model system consisting of myosin II, actin filaments, and cross-linkers. In this system, stresses arising from motor activity controlled the cytoskeletal network mechanics, increasing stiffness by a factor of nearly 100 and qualitatively changing the viscoelastic response of the network in an adenosine triphosphate-dependent manner. We present a quantitative theoretical model connecting the large-scale properties of this active gel to molecular force generation.
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            Global analysis of gene activity during Arabidopsis seed development and identification of seed-specific transcription factors.

            Most of the transcription factors (TFs) responsible for controlling seed development are not yet known. To identify TF genes expressed at specific stages of seed development, including those unique to seeds, we used Affymetrix GeneChips to profile Arabidopsis genes active in seeds from fertilization through maturation and at other times of the plant life cycle. Seed gene sets were compared with those expressed in prefertilization ovules, germinating seedlings, and leaves, roots, stems, and floral buds of the mature plant. Most genes active in seeds are shared by all stages of seed development, although significant quantitative changes in gene activity occur. Each stage of seed development has a small gene set that is either specific at the level of the GeneChip or up-regulated with respect to genes active at other stages, including those that encode TFs. We identified 289 seed-specific genes, including 48 that encode TFs. Seven of the seed-specific TF genes are known regulators of seed development and include the LEAFY COTYLEDON (LEC) genes LEC1, LEC1-LIKE, LEC2, and FUS3. The rest represent different classes of TFs with unknown roles in seed development. Promoter-beta-glucuronidase (GUS) fusion experiments and seed mRNA localization GeneChip datasets showed that the seed-specific TF genes are active in different compartments and tissues of the seed at unique times of development. Collectively, these seed-specific TF genes should facilitate the identification of regulatory networks that are important for programming seed development.
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              Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells.

              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.
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                Author and article information

                Contributors
                Role: Reviewing editor
                Journal
                eLife
                Elife
                eLife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                10 October 2014
                2014
                : 3
                : e04501
                Affiliations
                [1 ]Temasek Life Sciences Laboratory, National University of Singapore , Singapore, Singapore
                [2 ]Nagoya Institute of Transformative Bio-Molecules, Nagoya University , Nagoya, Japan
                [3 ]Department of Biological Sciences, National University of Singapore , Singapore, Singapore
                [4 ]Division of Biological Sciences, Nagoya University Graduate School of Science , Nagoya, Japan
                [5 ]Mechanobiology Institute, National University of Singapore , Singapore, Singapore
                University of California-Berkeley & USDA Agricultural Research Service , United States
                University of California-Berkeley & USDA Agricultural Research Service , United States
                Author notes
                [* ]For correspondence: fred@ 123456tll.org.sg
                [†]

                Gregor Mendel Institute, Vienna, Austria.

                Author information
                http://orcid.org/0000-0003-3803-3070
                http://orcid.org/0000-0002-0989-7622
                Article
                04501
                10.7554/eLife.04501
                4221737
                25303363
                bfb70fda-c217-485c-bd9b-13fb4cb98130
                Copyright © 2014, Kawashima et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                History
                : 26 August 2014
                : 09 October 2014
                Funding
                Funded by: Temasek Life Sciences Laboratory
                Award Recipient :
                Funded by: National University of Singapore FundRef identification ID: http://dx.doi.org/10.13039/501100001352
                Award ID: Department of Biological Sciences
                Award Recipient :
                Funded by: National University of Singapore FundRef identification ID: http://dx.doi.org/10.13039/501100001352
                Award ID: Mechanobiology Institute
                Award Recipient :
                Funded by: Japan Society for the Promotion of Science FundRef identification ID: http://dx.doi.org/10.13039/501100001691
                Award ID: 6526
                Award Recipient :
                Funded by: Nagoya University FundRef identification ID: http://dx.doi.org/10.13039/501100004823
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Categories
                Research Article
                Plant Biology
                Custom metadata
                0.8
                The movement of F-actin filaments, which is regulated by actin-myosin interactions together with a female gamete-specific Rho-GTPase, enables migration of the Arabidopsis sperm cell nucleus towards the female nucleus; which might account for the loss of centrosomes in flowering plants.

                Life sciences
                fertilization,cytoskeleton,gamete nuclear migration,rho-gtpase,f-actin,reproduction,arabidopsis

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