In vitro measured leaf-level freezing tolerance predicts photosynthetic impairment during a natural late spring-frost event

Abstract

Climate change has altered the global temperature regimes leading to warmer temperatures occurring earlier in spring in many temperate regions. This has induced an earlier budbreak making trees susceptible to late spring-frost events, however, information on species-specific late spring-frost tolerance is only available from observational studies. Here, we implemented a quantitative study on late spring-frost tolerance determined by in vitro leaf-level measurements of three temperate broad-leaved tree species via the assessment of the maximum quantum yield efficiency of the photosystem II (F_v/F_m). We investigated to what extent in vitro measurements conducted one day before a late spring-frost event can predict the in vivo damage caused by a cold snap. Fraxinus excelsior showed the lowest in vitro tested tolerance to late spring-frost, and the leaves lost 50% of F_v/F_m (LT50) at + 0.60 ± 0.26 °C. The damage induced by the cold snap the following day (minimum temperature of − 3.28 °C) was a fatal decline of F_v/F_m to 5.8% of the maximum. The other two species, namely Fagus sylvatica and Quercus robur, were characterized by LT50 of − 0.17 ± 9.99 °C and − 2.29 ± 1.11 °C, respectively. The cold snap induced less damage, F_v/F_m values declined to 46.9% and 53.5% of the maximum in the two species, respectively. The in vitro measurements precisely predicted the damage caused by the late spring-frost event. We suggest that in vitro estimated LT50 values can be used as a comparative leaf trait as it has high predictive power for tree species performance after late spring-frost.

Keywords

Cold snap / Spring phenology / Leaf unfolding / Frost damage / Temperate trees

References

[1] Augspurger CK. Frost damage and its cascading negative effects on Aesculusglabra. Plant Ecol, 2011, 212(7): 1193-1203

[2] Augspurger CK. Reconstructing patterns of temperature, phenology, and frost damage over 124 years: spring damage risk is increasing. Ecology, 2013, 94(1): 41-50

[3] Barichivich J, Briffa KR, Myneni RB, Osborn TJ, Melvin TM, Ciais P, Piao SL, Tucker C. Large-scale variations in the vegetation growing season and annual cycle of atmospheric CO₂ at high northern latitudes from 1950 to 2011. Glob Chang Biol, 2013, 19(10): 3167-3183

[4] Bigler C, Bugmann H. Climate-induced shifts in leaf unfolding and frost risk of European trees and shrubs. Sci Rep, 2018, 8(1): 9865

[5] Bucher SF, Feiler R, Buchner O, Neuner G, Rosbakh S, Leiterer M, Römermann C. Temporal and spatial trade-offs between resistance and performance traits in herbaceous plant species. Environ Exp Bot, 2019, 157: 187-196

[6] Capdevielle-Vargas R, Estrella N, Menzel A. Multiple-year assessment of phenological plasticity within a beech (Fagus sylvatica L.) stand in southern Germany. Agric for Meteorol, 2015, 211: 13-22

[7] Cunningham SC, Read J. Foliar temperature tolerance of temperate and tropical evergreen rain forest trees of Australia. Tree Physiol, 2006, 26(111435-1443

[8] D’Andrea E, Rezaie N, Battistelli A, Gavrichkova O, Kuhlmann I, Matteucci G, Moscatello S, Proietti S, Scartazza A, Trumbore S, Muhr J. Winter’s bite: beech trees survive complete defoliation due to spring late-frost damage by mobilizing old C reserves. New Phytol, 2019, 224(2): 625-631

[9] Dittmar C, Fricke W, Elling W. Impact of late frost events on radial growth of common beech (Fagus sylvatica L.) in Southern Germany. Eur J Forest Res, 2006, 125(3): 249-259

[10] Düsterhöft E, Burgesmeir B, Mayer J, Nitschky N, Schächer L, Kunert N. Spätfröste – Probleme mit neuen Baumarten. AFZ – der Wald, 2024, 5: 20-23

[11] DWD (2021) Anleitung für die phänologischen Beobachter des Deutschen Wetterdienstes, Vorschriften und Betriebsunterlagen Nr. 17, Deutscher Wetterdienst Offenbach 1991, überarbeitet 2021.

[12] DWD (2025) Deutscher Wetterdienst, Climate Data Center. Available from https://opendata.dwd.de/

[13] Ellenberg H. Vegetation Mitteleuropas mit den Alpen in ökologischer Sicht, 1978, Stuttgart, Verlag Eugen Ulmer82

[14] Fernández M, Alejano R, Domínguez L, Tapias R. Temperature controls cold hardening more effectively than photoperiod in four Mediterranean broadleaf evergreen species. Tree and Forestry Science and Biotechnology, 2008, 2(1): 43-49

[15] Frank A, Pluess AR, Howe GT, Sperisen C, Heiri C. Quantitative genetic differentiation and phenotypic plasticity of European beech in a heterogeneous landscape: indications for past climate adaptation. Perspect Plant Ecol Evol Syst, 2017, 26: 1-13

[16] Inouye DW. The ecological and evolutionary significance of frost in the context of climate change. Ecol Lett, 2000, 3(5): 457-463

[17] IPPC (2019) Summary for Policymakers. In: Climate Change and Land: an IPCC special report on climate change, desertification, land degradation, sustainable land management, food security, and greenhouse gas fluxes in terrestrial ecosystems [P R Shukla, J Skea, E Calvo Buendia, V Masson-Delmotte, H - O Pörtner, D C Roberts, P Zhai, R Slade, S Connors, R van Diemen, M Ferrat, E Haughey, S Luz, S Neogi, M Pathak, J Petzold, J Portugal Pereira, P Vyas, E Huntley, K Kissick, M Belkacemi, J Malley, (eds.)].

[18] Kunert N, Hajek P. Shade-tolerant temperate broad-leaved trees are more sensitive to thermal stress than light-demanding species during a moderate heatwave. Trees, Forests and People, 2022, 9 100282

[19] Kunert N, Hajek P, Hietz P, Morris H, Rosner S, Tholen D. Summer temperatures reach the thermal tolerance threshold of photosynthetic decline in temperate conifers. Plant Biol, 2022, 24(7): 1254-1261

[20] Kunert N, Münchinger IK, Hajek P. Turgor loss point explains climate-driven growth reductions in trees in Central Europe. Plant Biol, 2024

[21] Lamontagne M, Bigras FJ, Margolis HA. Chlorophyll fluorescence and CO₂ assimilation of black spruce seedlings following frost in different temperature and light conditions. Tree Physiol, 2000, 20(4): 249-255

[22] Lenz A, Hoch G, Vitasse Y, Körner C. European deciduous trees exhibit similar safety margins against damage by spring freeze events along elevational gradients. New Phytol, 2013, 200(4): 1166-1175

[23] Menzel A, Fabian P. Growing season extended in Europe. Nature, 1999, 397(6721): 659

[24] Mittelstädt H, Rudolph K. Ice nucleation activity of strains from Pseudomonas syringae pathovars atrofadens and syringae, mainly isolated from cereals. J Phytopathol, 1998, 146(11–12581-586

[25] Mura C, Buttò V, Silvestro R, Deslauriers A, Charrier G, Raymond P, Rossi S. The early bud gets the cold: diverging spring phenology drives exposure to late frost in a Picea mariana [(Mill.) BSP] common garden. Physiol Plant, 2022, 174(6 e13798

[26] Mura C, Charrier G, Buttò V, Delagrange S, Surget-Groba Y, Raymond P, Rossi S, Deslauriers A. Local conditions have greater influence than provenance on sugar maple (Acer saccharum Marsh.) frost hardiness at its northern range limit. Tree Physiol, 2025, 45(1 tpae167

[27] Neuner G, Buchner O. The dose makes the poison: the longer the heat lasts, the lower the temperature for functional impairment and damage. Environ Exp Bot, 2023, 212 105395

[28] Neuner G, Pramsohler M. Freezing and high temperature thresholds of photosystem 2 compared to ice nucleation, frost and heat damage in evergreen subalpine plants. Physiol Plant, 2006, 126(2): 196-204

[29] Neuner G, Erler A, Ladinig U, Hacker J, Wagner J. Frost resistance of reproductive tissues during various stages of development in high mountain plants. Physiol Plant, 2013, 147(1): 88-100

[30] Perks MP, Osborne BA, Mitchell DT. Rapid predictions of cold tolerance in Douglas-fir seedlings using chlorophyll fluorescence after freezing. New for, 2004, 28(1): 49-62

[31] Piao SL, Liu Q, Chen AP, Janssens IA, Fu YS, Dai JH, Liu LL, Lian X, Shen MG, Zhu XL. Plant phenology and global climate change: current progresses and challenges. Glob Change Biol, 2019, 25(6): 1922-1940

[32] Príncipe A, van der Maaten E, van der Maaten-Theunissen M, Struwe T, Wilmking M, Kreyling J. Low resistance but high resilience in growth of a major deciduous forest tree (Fagus sylvatica L.) in response to late spring frost in southern Germany. Trees, 2017, 31(2): 743-751

[33] R Core Team (2023) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.

[34] Ralser M, Stegner M, Neuner G. When water turns to ice: control of ice volume and freezing dynamics as important aspects of cold acclimation. Environ Exp Bot, 2024, 227 105957

[35] Ritz C, Baty F, Streibig JC, Gerhard D. Dose-response analysis using R. PLoS ONE, 2015, 10(12 e0146021

[36] Rosique-Esplugas C, Cottrell JE, Cavers S, Whittet R, Ennos RA. Clinal genetic variation and phenotypic plasticity in leaf phenology, growth and stem form in common ash (Fraxinus excelsior L.). Forestry, 2022, 95(1): 83-94

[37] Rubio-Cuadrado Á, Gómez C, Rodríguez-Calcerrada J, Perea R, Gordaliza GG, Camarero JJ, Montes F, Gil L. Differential response of oak and beech to late frost damage: an integrated analysis from organ to forest. Agric for Meteorol, 2021, 297 108243

[38] Sakai A, Larcher W (1987) Frost Survival of Plants: Responses and Adaptation to Freezing Stress. Ecological Studies 62, Springer-Verlag, New York.

[39] Salmela MJ, Cavers S, Cottrell JE, Iason GR, Ennos RA. Spring phenology shows genetic variation among and within populations in seedlings of Scots pine (Pinus sylvestris L.) in the Scottish Highlands. Plant Ecol Divers, 2013, 6: 523-536

[40] Sangüesa-Barreda G, Villalba R, Rozas V, Christie DA, Olano JM. Detecting Nothofagus pumilio growth reductions induced by past spring frosts at the northern Patagonian Andes. Front Plant Sci, 2019, 10: 1413

[41] Sangüesa-Barreda G, Di Filippo A, Piovesan G, Rozas V, Di Fiore L, García-Hidalgo M, García-Cervigón AI, Muñoz-Garachana D, Baliva M, Olano JM. Warmer springs have increased the frequency and extension of late-frost defoliations in southern European beech forests. Sci Total Environ, 2021, 775 145860

[42] Schulze ED, Hartmann H, Lambert S, Weber U. Folgen von Spätfrösten bei der Buche in montanen Lagen. AFZ der Wald, 2020, 18: 46-48

[43] Švara A, Upchurch D, Goderis B, Colaers M, Deckers T, Keulemans W, Abts W, De Storme N, De Maeyer L. Differential scanning calorimetry: a novel approach to study spring frost tolerance in pome tree floral tissues. Fruit Res, 2025

[44] Taschler D, Neuner G. Summer frost resistance and freezing patterns measured in situ in leaves of major alpine plant growth forms in relation to their upper distribution boundary. Plant Cell Environ, 2004, 27(6): 737-746

[45] Twardosz R, Walanus A, Guzik I. Warming in Europe: recent trends in annual and seasonal temperatures. Pure Appl Geophys, 2021, 178(10): 4021-4032

[46] Ummenhofer CC, Meehl GA. Extreme weather and climate events with ecological relevance: a review. Philos Trans R Soc Lond B Biol Sci, 2017, 372(1723 20160135

[47] van Zuijlen K, Kassel M, Dorrepaal E, Lett S. Frost damage measured by electrolyte leakage in subarctic bryophytes increases with climate warming. J Ecol, 2024, 112(2): 220-232

[48] Vitasse Y, Bresson CC, Kremer A, Michalet R, Delzon S. Quantifying phenological plasticity to temperature in two temperate tree species. Funct Ecol, 2010, 24(6): 1211-1218

[49] Wang JM, Hua H, Guo J, Huang X, Zhang X, Yang YC, Wang DY, Guo XL, Zhang R, Smith NG, Rossi S, Peñuelas J, Ciais P, Wu CY, Chen L. Late spring frost delays tree spring phenology by reducing photosynthetic productivity. Nat Clim Chang, 2025, 15(2): 201-209

[50] Winter K. Are tropical forests approaching critical temperature thresholds?. Plant Biol, 2024, 26(4): 495-498

[51] Winter K, Krüger Nuñez CR, Slot M, Virgo A. In thermotolerance tests of tropical tree leaves, the chlorophyll fluorescence parameter F(v)/F(m) measured soon after heat exposure is not a reliable predictor of tissue necrosis. Plant Biol, 2025, 27(1): 146-153

[52] Wisniewski M, Gusta L, Neuner G. Adaptive mechanisms of freeze avoidance in plants: a brief update. Environ Exp Bot, 2014, 99: 133-140

[53] Wolkovich EM, Cook BI, Allen JM, Crimmins TM, Betancourt JL, Travers SE, Pau S, Regetz J, Davies TJ, Kraft NJB, Ault TR, Bolmgren K, Mazer SJ, McCabe GJ, McGill BJ, Parmesan C, Salamin N, Schwartz MD, Cleland EE. Warming experiments underpredict plant phenological responses to climate change. Nature, 2012, 485(7399): 494-497

[54] Zohner CM, Mo LD, Renner SS, Svenning JC, Vitasse Y, Benito BM, Ordonez A, Baumgarten F, Bastin JF, Sebald V, Reich PB, Liang JJ, Nabuurs GJ, De-Miguel S, Alberti G, Antón-Fernández C, Balazy R, Brändli UB, Chen HYH, Chisholm C, Cienciala E, Dayanandan S, Fayle TM, Frizzera L, Gianelle D, Jagodzinski AM, Jaroszewicz B, Jucker T, Kepfer-Rojas S, Khan ML, Kim HS, Korjus H, Johannsen VK, Laarmann D, Lang M, Zawila-Niedzwiecki T, Niklaus PA, Paquette A, Pretzsch H, Saikia P, Schall P, Šebeň V, Svoboda M, Tikhonova E, Viana H, Zhang CY, Zhao XH, Crowther TW. Late-spring frost risk between 1959 and 2017 decreased in North America but increased in Europe and Asia. Proc Natl Acad Sci U S A, 2020, 117(22): 12192-12200

[55] Zsolnay N, Walentowitz A, Aas G. Impact of climatic conditions on radial growth of non-native Cedrus libani compared to native conifers in Central Europe. PLoS ONE, 2023, 18(5 e0275317

Funding

Universität Bayreuth (3145)

RIGHTS & PERMISSIONS

The Author(s)