Abstract
Quantitative assessment of freezing tolerance is essential to unravel plant adaptations to cold temperatures. Not only the survival of whole plants, but also impairment of detached leaves or small rosettes after a freeze–thaw cycle can be used to accurately quantify plant freezing tolerance in terms of LT50 values. Here we describe two methods to determine the freezing tolerance of detached leaves or rosettes using a full or selected set of freezing temperatures and an additional method using chlorophyll fluorescence as a different physiological parameter. Firstly, we illustrate how to assess the integrity of (predominantly) the plasma membrane during freezing using an electrolyte leakage assay. Secondly, we provide a chlorophyll fluorescence imaging protocol to determine the freezing tolerance of the photosynthetic apparatus.
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
Steponkus PL (1984) Role of the plasma membrane in freezing injury and cold acclimation. Annu Rev Plant Physiol 35:543–584
Thalhammer A, Bryant G, Sulpice R et al (2014) Disordered cold regulated15 proteins protect chloroplast membranes during freezing through binding and folding, but do not stabilize chloroplast enzymes in vivo. Plant Physiol 166:190–201
Rohde P, Hincha DK, Heyer AG (2004) Heterosis in the freezing tolerance of crosses between two Arabidopsis thaliana accessions (Columbia-0 and C24) that show differences in non-acclimated and acclimated freezing tolerance. Plant J 38:790–799
Hannah MA, Wiese D, Freund S et al (2006) Natural genetic variation of freezing tolerance in Arabidopsis. Plant Physiol 142:98–112
Korn M, Peterek S, Mock HP et al (2008) Heterosis in the freezing tolerance, and sugar and flavonoid contents of crosses between Arabidopsis thaliana accessions of widely varying freezing tolerance. Plant Cell Environ 31:813–827
Zuther E, Schulz E, Childs LH et al (2012) Clinal variation in the non-acclimated and cold-acclimated freezing tolerance of Arabidopsis thaliana accessions. Plant Cell Environ 35:1860–1878
Zuther E, Juszczak I, Lee YP et al (2015) Time-dependent deacclimation after cold acclimation in Arabidopsis thaliana accessions. Sci Rep 5:12199
Le MQ, Pagter M, Hincha DK (2015) Global changes in gene expression, assayed by microarray hybridization and quantitative RT-PCR, during acclimation of three Arabidopsis thaliana accessions to sub-zero temperatures after cold acclimation. Plant Mol Biol 87:1–15
Takahashi D, Gorka M, Erban A et al (2019) Both cold and sub-zero acclimation induce cell wall modification and changes in the extracellular proteome in Arabidopsis thaliana. Sci Rep 9:2289
Lee YP, Babakov A, de Boer B et al (2012) Comparison of freezing tolerance, compatible solutes and polyamines in geographically diverse collections of Thellungiella sp. and Arabidopsis thaliana accessions. BMC Plant Biol 12:131
Ristic Z, Ashworth EN (1993) Changes in leaf ultrastructure and carbohydrates in Arabidopsis thaliana L. (Heyn) cv. Columbia during rapid cold acclimation. Protoplasma 172:111–123
Ehlert B, Hincha DK (2008) Chlorophyll fluorescence imaging accurately quantifies freezing damage and cold acclimation responses in Arabidopsis leaves. Plant Methods 4:12
Krause GH, Grafflage S, Rumich-Bayer S et al (1988) Effects of freezing on plant mesophyll cells. Symp Soc Exp Biol 42:311–327
Woo NS, Badger MR, Pogson BJ (2008) A rapid, non-invasive procedure for quantitative assessment of drought survival using chlorophyll fluorescence. Plant Methods 4:27
Maxwell K, Johnson GN (2000) Chlorophyll fluorescence-a practical guide. J Exp Bot 51:659–668
Lawson T, Vialet-Chabrand S (2018) Chlorophyll fluorescence imaging. Methods Mol Biol 1770:121–140
Oxborough K (2004) Imaging of chlorophyll a fluorescence: theoretical and practical aspects of an emerging technique for the monitoring of photosynthetic performance. J Exp Bot 55:1195–1205
Lichtenthaler HK, Miehé JA (1997) Fluorescence imaging as a diagnostic tool for plant stress. Trend Plant Sci 2:316–320
McKhann HI, Gery C, Bérard A et al (2008) Natural variation in CBF gene sequence, gene expression and freezing tolerance in the Versailles core collection of Arabidopsis thaliana. BMC Plant Biol 8:105
Zuther E, Schaarschmidt S, Fischer A et al (2019) Molecular signatures associated with increased freezing tolerance due to low temperature memory in Arabidopsis. Plant Cell Environ 42:854–873
Ritz C, Streibig JC (2005) Bioassay analysis using R. J Stat Software 12:1–22
Schreiber U, Bilger W (1987) Rapid assessment of stress effects on plant leaves by chlorophyll fluorescence measurements. In: Plant response to stress. Springer, Berlin, pp 27–53
Hincha DK, Pfüller U, Schmitt JM (1997) The concentration of cryoprotective lectins in mistletoe (Viscum album L.) leaves is correlated with leaf frost hardiness. Planta 203:140–144
Hunt S (2003) Measurements of photosynthesis and respiration in plants. Physiol Plant 117:314–325
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Thalhammer, A., Pagter, M., Hincha, D.K., Zuther, E. (2020). Measuring Freezing Tolerance of Leaves and Rosettes: Electrolyte Leakage and Chlorophyll Fluorescence Assays. In: Hincha, D., Zuther, E. (eds) Plant Cold Acclimation. Methods in Molecular Biology, vol 2156. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0660-5_2
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0660-5_2
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0659-9
Online ISBN: 978-1-0716-0660-5
eBook Packages: Springer Protocols