Abstract
The deposition and turnover of callose (beta-1,3 glucan polymer) in the cell wall surrounding the neck regions of plasmodesmata (PD) controls the cell-to-cell diffusion rate of molecules and, therefore, plays an important role in the regulation of intercellular communication in plants.
Here we describe a simple and fast in vivo staining procedure for the imaging and quantification of callose at PD. We also introduce calloseQuant, a plug-in for semiautomated image analysis and non-biased quantification of callose levels at PD using ImageJ.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
De Storme N, Geelen D (2014) Callose homeostasis at plasmodesmata: molecular regulators and developmental relevance. Front Plant Sci 5:138
Lim GH, Shine MB, de Lorenzo L et al (2016) Plasmodesmata Localizing proteins regulate transport and signaling during systemic acquired immunity in plants. Cell Host Microbe 19:541–549
Cui W, Lee JY (2016) Arabidopsis callose synthases CalS1/8 regulate plasmodesmal permeability during stress. Nat Plants 2:16034
Vaten A, Dettmer J, Wu S et al (2011) Callose biosynthesis regulates symplastic trafficking during root development. Dev Cell 21:1144–1155
Simpson C, Thomas C, Findlay K et al (2009) An Arabidopsis GPI-anchor plasmodesmal neck protein with callose binding activity and potential to regulate cell-to-cell trafficking. Plant Cell 21:581–594
Levy A, Erlanger M, Rosenthal M et al (2007) A plasmodesmata-associated beta-1,3-glucanase in Arabidopsis. Plant J 49:669–682
Zavaliev R, Ueki S, Epel BL et al (2011) Biology of callose (beta-1,3-glucan) turnover at plasmodesmata. Protoplasma 248:117–130
Benitez-Alfonso Y, Faulkner C, Pendle A et al (2013) Symplastic intercellular connectivity regulates lateral root patterning. Dev Cell 26:136–147
Stahl Y, Faulkner C (2016) Receptor complex mediated regulation of symplastic traffic. Trends Plant Sci 21:450–459
Lee JY (2015) Plasmodesmata: a signaling hub at the cellular boundary. Curr Opin Plant Biol 27:133–140
Faulkner C, Petutschnig E, Benitez-Alfonso Y et al (2013) LYM2-dependent chitin perception limits molecular flux via plasmodesmata. Proc Natl Acad Sci U S A 110:9166–9170
Lee JY, Lu H (2011) Plasmodesmata: the battleground against intruders. Trends Plant Sci 16:201–210
Oparka KJ, Roberts AG, Boevink P et al (1999) Simple, but not branched, plasmodesmata allow the nonspecific trafficking of proteins in developing tobacco leaves. Cell 97:743–754
Brunkard JO, Zambryski P (2019) Plant cell-cell transport via plasmodesmata is regulated by light and the circadian clock. Plant Physiol 181:1459–1467
Brunkard JO, Xu M, Scarpin MR et al (2020) TOR dynamically regulates plant cell-cell transport. Proc Natl Acad Sci U S A 117:5049–5058
Brunkard JO, Runkel AM, Zambryski PC (2013) Plasmodesmata dynamics are coordinated by intracellular signaling pathways. Curr Opin Plant Biol 16:614–620
Liang D (2018) A salutary role of reactive oxygen species in intercellular tunnel-mediated communication. Front Cell Dev Biol 6:2
Xu B, Cheval C, Laohavisit A et al (2017) A calmodulin-like protein regulates plasmodesmal closure during bacterial immune responses. New Phytol 215:77–84
Han X, Hyun TK, Zhang M et al (2014) Auxin-callose-mediated plasmodesmal gating is essential for tropic auxin gradient formation and signaling. Dev Cell 28:132–146
Wang X, Sager R, Cui W et al (2013) Salicylic acid regulates plasmodesmata closure during innate immune responses in Arabidopsis. Plant Cell 25:2315–2329
Rinne PL, van der Schoot C (1998) Symplasmic fields in the tunica of the shoot apical meristem coordinate morphological events. Development 125:1477–1485
Rinne PL, Welling A, Vahala J et al (2011) Chilling of dormant buds hyperinduces FLOWERING LOCUS T and recruits GA-inducible 1,3-beta-glucanases to reopen signal conduits and release dormancy in Populus. Plant Cell 23:130–146
Ruan YL, Llewellyn DJ, Furbank RT (2001) The control of single-celled cotton fiber elongation by developmentally reversible gating of plasmodesmata and coordinated expression of sucrose and K+ transporters and expansin. Plant Cell 13:47–60
Zavaliev R, Levy A, Gera A et al (2013) Subcellular dynamics and role of Arabidopsis beta-1,3-glucanases in cell-to-cell movement of tobamoviruses. Mol Plant-Microbe Interact 26:1016–1030
Gaudioso-Pedraza R, Beck M, Frances L et al (2018) Callose-regulated symplastic communication coordinates symbiotic root nodule development. Curr Biol 28:3562–3577.e6
Currier HB (1957) Callose substance in plant cells. Am J Bot 44:478–488
Thistlewhite P, Porter I, Evans N (1986) Photophysics of the aniline blue fluorophore: a fluorescent probe showing specificity toward (1->3)-beta-D-glycans. J Phys Chem 90:5058–5063
Smith MM, McCully ME (1978) A critical evaluation of the specificity of aniline blue induced fluorescence. Protoplasma 95:229–254
Zavaliev R, Epel BL (2015) Imaging callose at plasmodesmata using aniline blue: quantitative confocal microscopy. Methods Mol Biol 1217:105–119
Acknowledgments
We thank the Chinese Scholarship Council (CSC) for a PhD fellowship to C.H.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Huang, C., Mutterer, J., Heinlein, M. (2022). In Vivo Aniline Blue Staining and Semiautomated Quantification of Callose Deposition at Plasmodesmata. In: Benitez-Alfonso, Y., Heinlein, M. (eds) Plasmodesmata. Methods in Molecular Biology, vol 2457. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2132-5_9
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2132-5_9
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2131-8
Online ISBN: 978-1-0716-2132-5
eBook Packages: Springer Protocols