Abstract
Fertilization comprises a complex series of cellular processes leading to the fusion of a male and female gamete. Many studies have been carried out to investigate each step of fertilization in plants; however, our comprehensive understanding of all the sequential events during fertilization is still limited. This is largely due to difficulty in investigating events in the female gametophyte, which is deeply embedded in the maternal tissue. Recent advances in confocal microcopy assisted by fluorescent marker lines have contributed to visualizing subcellular dynamics in real time during fertilization in vivo. In this chapter, we describe a method focusing on the investigation of F-actin dynamics in the central cell during male gamete nuclear migration. This method also allows the study of a wide range of early sexual reproduction events, from pollen tube guidance to the early stage of seed development.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kawashima T, Berger F (2011) Green love talks; cell-cell communication during double fertilization in flowering plants. AoB Plants 2011:plr015
Dresselhaus T, Sprunck S, Wessel GM (2016) Fertilization mechanisms in flowering plants. Curr Biol 26:R125–R139
Higashiyama T, Takeuchi H (2015) The mechanism and key molecules involved in pollen tube guidance. Annu Rev Plant Biol 66:393–413
Palanivelu R, Preuss D (2006) Distinct short-range ovule signals attract or repel Arabidopsis thaliana pollen tubes in vitro. BMC Plant Biol 6:7
Jeong S, Lukowitz W (2015) Embryos, camera, laser, action. Dev Cell 34:137–138
Hamamura Y, Saito C, Awai C et al (2011) Live-cell imaging reveals the dynamics of two sperm cells during double fertilization in Arabidopsis thaliana. Curr Biol 21:497–502
Rotman N, Rozier F, Boavida L et al (2003) Female control of male gamete delivery during fertilization in Arabidopsis thaliana. Curr Biol 13:432–436
Ngo QA, Vogler H, Lituiev DS et al (2014) A calcium dialog mediated by the FERONIA signal transduction pathway controls plant sperm delivery. Dev Cell 29:491–500
Iwano M, Ngo QA, Entani T et al (2012) Cytoplasmic Ca2+ changes dynamically during the interaction of the pollen tube with synergid cells. Development 139:4202–4209
Hamamura Y, Nishimaki M, Takeuchi H et al (2014) Live imaging of calcium spikes during double fertilization in Arabidopsis. Nat Commun 5:4722
Denninger P, Bleckmann A, Lausser A et al (2014) Male-female communication triggers calcium signatures during fertilization in Arabidopsis. Nat Commun 5:4645
Kawashima T, Maruyama D, Shagirov M et al (2014) Dynamic F-actin movement is essential for fertilization in Arabidopsis thaliana. Elife 3:e04501
Kimata Y, Higaki T, Kawashima T et al (2016) Cytoskeleton dynamics control the first asymmetric cell division in Arabidopsis zygote. Proc Natl Acad Sci U S A 113:14157–14162
Gooh K, Ueda M, Aruga K et al (2015) Live-cell imaging and optical manipulation of Arabidopsis early embryogenesis. Dev Cell 34:242–251
Mizuta Y, Kurihara D, Higashiyama T (2015) Two-photon imaging with longer wavelength excitation in intact Arabidopsis tissues. Protoplasma 252:1231–1240
Riedl J, Crevenna AH, Kessenbrock K et al (2008) Lifeact: a versatile marker to visualize F-actin. Nat Methods 5:605–607
Ingouff M, Hamamura Y, Gourgues M et al (2007) Distinct dynamics of HISTONE3 variants between the two fertilization products in plants. Curr Biol 17:1032–1037
Kawashima T, Berger F (2015) The central cell nuclear position at the micropylar end is maintained by the balance of F-actin dynamics, but dispensable for karyogamy in Arabidopsis. Plant Reprod 28:103–110
Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682
Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675
Acknowledgments
D.M. is supported by Toyoaki Scholarship Foundation and JSPS KAKENHI grant, number 15K14541 (Grant-in-Aid for Challenging Exploratory Research) and 16H06173 (Grant-in-Aid for Young Scientists).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Susaki, D., Maruyama, D., Yelagandula, R., Berger, F., Kawashima, T. (2017). Live-Cell Imaging of F-Actin Dynamics During Fertilization in Arabidopsis thaliana . In: Schmidt, A. (eds) Plant Germline Development. Methods in Molecular Biology, vol 1669. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7286-9_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7286-9_4
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7285-2
Online ISBN: 978-1-4939-7286-9
eBook Packages: Springer Protocols