Abstract
While cytosolic calcium (Ca2+) plays a central role in a myriad of signaling pathways as a secondary messenger, how dynamic changes of cytosolic calcium relate to cell growth control remains poorly understood. The engineering and continuous improvements of genetically encoded calcium sensors such as the Yellow Cameleon (YC) sensors combined with advances in microscopy have allowed imaging with great resolution of the spatiotemporal characteristics of cytosolic [Ca2+]cyt in individual cells. An exciting new step consists therefore in cautiously studying calcium dynamics in mutant backgrounds that display disturbed cellular growth behavior to further enhance our understanding on growth-related processes. Here, we describe methods to perform imaging of [Ca2+]cyt dynamics in growing Arabidopsis thaliana wild-type and NADPH-oxidase deficient rbohH rbohJ pollen tubes stably expressing YC3.6 using confocal laser scanning microscopy. We also present different ways to extract meaningful qualitative and quantitative information about calcium dynamics during growth.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Permyakov EA, Kretsinger RH (2009) Cell signaling, beyond cytosolic calcium in eukaryotes. J Inorg Biochem 103:77–86
Pierson ES, Miller DD, Callaham DA, van Aken J, Hackett G, Hepler PK (1996) Tip-localized calcium entry fluctuates during pollen tube growth. Dev Biol 174:160–173
Michard E, Alves F, FeijĂ³ JA (2009) The role of ion fluxes in polarized cell growth and morphogenesis: the pollen tube as an experimental paradigm. Int J Dev Biol 53:1609–1622
Iwano M, Entani T, Shiba H, Kakita M, Nagai T, Mizuno H et al (2009) Fine-tuning of the cytoplasmic Ca2+ concentration is essential for pollen tube growth. Plant Physiol 150:1322–1334
Steinhorst L, Kudla J (2012) Calcium - a central regulator of pollen germination and tube growth. Biochim Biophys Acta 1833:1573–1581
Reiss HD, Herth W (1985) Nifedipine-sensitive calcium channels are involved in polar growth of lily pollen tubes. J Cell Sci 76:247–254
CĂ¡rdenas L, Lovy-Wheeler A, Kunkel JGG, Hepler PKK, Cardenas L, Lovy-Wheeler A et al (2008) Pollen tube growth oscillations and intracellular calcium levels are reversibly modulated by actin polymerization. Plant Physiol 146:1611–1621
Boisson-Dernier A, Lituiev DS, Nestorova A, Franck CM, Thirugnanarajah S, Grossniklaus U (2013) ANXUR receptor-like kinases coordinate cell wall integrity with growth at the pollen tube tip via NADPH oxidases. PLoS Biol 11:e1001719
Lassig R, Gutermuth T, Bey TD, Konrad KR, Romeis T (2014) Pollen tube NAD(P)H oxidases act as a speed control to dampen growth rate oscillations during polarized cell growth. Plant J 78:94–106
Kong SK, Lee CY (1995) The use of fura 2 for measurement of free calcium concentration. Biochemist 23:97–98
LĂ¼ckhoff A (1986) Measuring cytosolic free calcium concentration in endothelial cells with indo-1: the pitfall of using the ratio of two fluorescence intensities recorded at different wavelengths. Cell Calcium 7:233–248
Gee KR, Brown KA, Chen W-NU, Bishop-Stewart J, Gray D, Johnson I (2000) Chemical and physiological characterization of fluo-4 Ca2+-indicator dyes. Cell Calcium 27:97–106
Lee S-K, Lee J-Y, Lee M-Y, Chung S-M, Chung J-H (1999) Advantages of calcium green-1 over other fluorescent dyes in measuring cytosolic calcium in platelets. Anal Biochem 273:186–191
Swanson SJ, Choi W-G, Chanoca A, Gilroy S (2011) In vivo imaging of Ca2+, pH, and reactive oxygen species using fluorescent probes in plants. Annu Rev Plant Biol 62:273–297
Loro G, Drago I, Pozzan T, Lo SF, Zottini M, Costa A (2012) Targeting of Cameleons to various subcellular compartments reveals a strict cytoplasmic/mitochondrial Ca2+ handling relationship in plant cells. Plant J 71:1–13
Krebs M, Held K, Binder A, Hashimoto K, Den Herder G, Parniske M et al (2012) FRET-based genetically encoded sensors allow high-resolution live cell imaging of Ca2+ dynamics. Plant J 69:181–192
Nagai T, Yamada S, Tominaga T, Ichikawa M, Miyawaki A (2004) Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc Natl Acad Sci U S A 101:10554–10559
Schiøtt M, Romanowsky SMM, Bækgaard L, Jakobsen MKK, Palmgren MGG, Harper JFF (2004) A plant plasma membrane Ca2+ pump is required for normal pollen tube growth and fertilization. Proc Natl Acad Sci U S A 101:9502–9507
Twell D, Yamaguchi J, Wing RA, Ushiba J, McCormick S (1991) Promoter analysis of genes that are coordinately expressed during pollen development reveals pollen-specific enhancer sequences and shared regulatory elements. Genes Dev 5:496–507
Boavida LC, McCormick S (2007) Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana. Plant J 52:570–582
Monshausen GB, Messerli MA, Gilroy S (2008) Imaging of the yellow cameleon 3.6 indicator reveals that elevations in cytosolic ca2+ follow oscillating increases in growth in root hairs of Arabidopsis. Plant Physiol 147:1690–1698
Acknowledgments
We are grateful to Megumi Iwano (Osaka University, Japan) for gifting us seeds of the pACT1-YC3.60 line. We thank all members of Martin HĂ¼lskamp’s group (University of Cologne, Germany) for sharing their facilities and CEPLAS for access to the Leica SP8 confocal microscope. This work was supported by the University of Cologne, the Deutsche Forschungsgemeinschaft Grant BO 4470/1-1, and a grant from the University of Cologne Centre of Excellence in Plant Sciences to A.B.D.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
1 Electronic Supplementary Material
Below is the link to the electronic supplementary material.
Two minute long time course of a steady growing WT PT with FRET, CFP, ratio and DIC channels. Scale bar is 10 μm (AVI 69125 kb)
Two minute long time course of an unsteady growing rbohH-3 rbohJ-3 PT with FRET, CFP, ratio and DIC channels. Scale bar is 10 μm (AVI 69125 kb)
Two minute long time course of a bursting rbohH-3 rbohJ-3 PT with FRET, CFP, ratio and DIC channels. Scale bar is 10 μm (AVI 69125 kb)
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Franck, C.M., Westermann, J., Boisson-Dernier, A. (2017). Imaging Ca2+ Dynamics in Wild-Type and NADPH Oxidase-Deficient Mutant Pollen Tubes with Yellow Cameleon and Confocal Laser Scanning Microscopy. 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_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7286-9_10
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