Abstract
Fluorescent protein fusions are widely used for visualizing the subcellular localization and mobility of intercellular proteins. There is now a variety of colors, expression vectors, and photoactivated molecules to choose from, each with their own strengths and limitations. In this chapter, the methodologies for expressing and quantifying protein secretion with fluorescent protein fusion constructs using two separate protocols—one in which the retention of a transiently expressed fluorescent marker is measured in seedling roots to quantify a block in secretion, and one in which the secretion of a fluorescent marker into the space of the apoplast is measured to quantify secretion in plant leaves—are described. In the first protocol, seedling roots are transiently transformed with multicistronic constructs; and in the second protocol, markers can be stably expressed and controlled under an inducible promoter in mature plants. Both methods provide tools for quantifying protein secretion and visualizing defects in secretion pathways in Arabidopsis.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bolte S, Talbot C, Boutte Y, Catrice O, Read ND, Satiat-Jeunemaitre B (2004) FM-dyes as experimental probes for dissecting vesicle trafficking in living plant cells. J Microsc 214(2):159–173
Blatt M, Grefen C (2014) Applications of fluorescent marker proteins in plant cell biology. In: Sanchez-Serrano JJ, Salinas J (eds) Arabidopsis protocols [Internet]. Methods in molecular biology. Humana Press, New York, pp 487–507. Available from: http://dx.doi.org/10.1007/978-1-62703-580-4_26. Cited 28 Nov 2016
Bücherl CA, Bader A, Westphal AH, Laptenok SP, Borst JW (2014) FRET-FLIM applications in plant systems. Protoplasma 251(2):383–394
Hecker A, Wallmeroth N, Peter S, Blatt MR, Harter K, Grefen C (2015) Binary 2in1 vectors improve in planta (co)localization and dynamic protein interaction studies. Plant Physiol 168(3):776–787
Wachsmuth M (2014) Molecular diffusion and binding analyzed with FRAP. Protoplasma 251(2):373–382
Grefen C, Karnik R, Larson E, Lefoulon C, Wang Y, Waghmare S et al (2015) A vesicle-trafficking protein commandeers Kv channel voltage sensors for voltage-dependent secretion. Nat Plants 1(8):15108
Karnik R, Grefen C, Bayne R, Honsbein A, Köhler T, Kioumourtzoglou D et al (2013) Arabidopsis Sec1/Munc18 protein SEC11 is a competitive and dynamic modulator of SNARE binding and SYP121-dependent vesicle traffic. Plant Cell 25(4):1368–1382
Grefen C, Donald N, Hashimoto K, Kudla J, Schumacher K, Blatt MR (2010) A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies. Plant J Cell Mol Biol 64(2):355–365
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
Larson, E.R. (2017). Measuring Plant Protein Secretion. In: Jiang, L. (eds) Plant Protein Secretion. Methods in Molecular Biology, vol 1662. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7262-3_18
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7262-3_18
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7261-6
Online ISBN: 978-1-4939-7262-3
eBook Packages: Springer Protocols