Standardization of electrolyte leakage data and a novel liquid nitrogen control improve measurements of cold hardiness in woody tissue

Abstract

Background A variety of basic and applied research programs in plant biology require the accurate and reliable determination of plant tissue cold hardiness. Over the past 50 years, the electrolyte leakage method has emerged as a popular and practical method for quantifying the amount of damage inflicted on plant tissue by exposure to freezing temperatures. Numerous approaches for carrying out this method and analyzing the resultant data have emerged. These include multiple systems for standardizing and modeling raw electrolyte leakage data and multiple protocols for boiling or autoclaving samples in order to maximize leakage as a positive control. We compare four different routines for standardization of leakage data and assess a novel control method—immersion in liquid nitrogen in lieu of traditional autoclaving—and apply them to woody twigs collected from 12 maple (Acer) species in early spring. We compare leakage data from these samples using each of four previously published forms of data analysis and autoclaving vs. liquid nitrogen controls and validate each of these approaches against visual estimates of freezing damage and differential thermal analysis. Results Through presentation of our own data and re-analysis of previously published findings, we show that standardization of raw data against estimates of both minimum and maximum attainable freezing damage allows for reliable estimation of cold hardiness at the species level and across studies in diverse systems. Furthermore, use of our novel liquid nitrogen control produces data commensurate across studies and enhances the consistency and realism of the electrolyte leakage method, especially for very cold hardy samples. Conclusion Future leakage studies that relativize data against minimum and maximum leakage and that employ our updated liquid nitrogen control will contribute generalizable, repeatable, and realistic data to the existing body of cold hardiness research in woody plants. Data from studies conducted using a liquid nitrogen (and not an autoclaving) control can still be compared to previously published data, especially when raw data are standardized using the best-performing approach among those we assessed. Electrolyte leakage of woody twigs emerges as a useful technique for quickly assessing the probability of tissue death in response to freezing in dormant plants. Differential thermal analysis may provide different and complementary information on cold hardiness.

Phenological physiology: seasonal patterns of plant stress tolerance in a changing climate

“…Typically, a critical leakage value (LT 50 , °C) is extracted from EL analysis, reflecting the temperature at which leakage has reached 50% of the maximum estimated for a particular sample. Estimates of LT 50 predict visually estimated tissue damage (Palonen & Lind en, 1999;Savage & Cavender-Bares, 2013) and generally align with LTEs (Ani sko & Lindstrom, 1995; Mancuso & Fiorino, 2000), although the two metrics do capture different aspects of cold (Kovaleski & Grossman, 2021).…”

“…A second approach, electrolyte leakage (EL) analysis, complements DTA by elucidating the temperature threshold at which cellular membranes become unstable, allowing symplastic solutes to leave the cell (Fig. 3c,d; Flint et al, 1967;Lim et al, 1998;Kovaleski & Grossman, 2021). In an EL measurement, sampled tissue (buds, leaves, or stems are most common) is incubated in water to a range of temperatures, after which conductivity is measured.…”

“…Cold hardiness is gained through acclimation in response to progressively lower temperatures and/or shorter days (hardening) and lost through exposure to higher temperatures and/or longer days (dehardening). As for thermotolerance, cold hardiness is estimated using diverse phenotypic measurements (Kovaleski & Grossman, 2021), including observation of tissue necrosis (Deans et al ., 1995; Lindén et al ., 2000). Here, I focus on two metrics that, like the calculation of T c from chlorophyll fluorescence analysis, have been shown to predict plant performance and can be carried out across diverse clades in a high‐throughput approach: differential thermal analysis (DTA), which yields estimates of low‐temperature exotherms (LTE), and electrolyte leakage (EL), which yields estimates of critical leakage values (LT 50 ).…”

“…Maximum water-deficit tolerance is shown for leaf tissue of both evergreen and deciduous plants. Curves synthesized from patterns reported in Hinckley et al (1980), Seemann et al (1986, Grossnickle (1992), Kolb & Sperry (1999), Froux et al (2004), Lenz et al (2013), Deacon et al (2019), Kovaleski &Grossman (2021), andNorthing (2022).…”

Dehydrins and Soluble Sugars Involved in Cold Acclimation of Rosa wichurana and Rose Cultivar ‘Yesterday’

“…Cold hardiness has a seasonal dynamic characterized by three phases: cold acclimation during autumn-winter, midwinter hardiness (maximum level of cold hardiness), and deacclimation (loss of tolerance) during winter-spring. Evaluation of cold hardiness using electrolyte leakage analysis is a reliable method for the stems of woody plants [1,2]. Low temperatures can lead to the loss of membrane integrity, resulting in cellular damage and solute leakage across the membrane.…”