bioRxiv preprint doi: https://doi.org/10.1101/2020.09.21.306886; this version posted September 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 A high-throughput method for measuring critical thermal limits of 2 leaves by chlorophyll imaging fluorescence 3 4 Short title: Thermal limits and chlorophyll fluorescence 5 6 Authors: 7 Pieter A. Arnold1,*, Verónica F. Briceño1, Kelli M. Gowland1, Alexandra A. Catling1, 8 León A. Bravo2, Adrienne B. Nicotra1 9 10 1 Division of Ecology and Evolution, Research School of Biology, The Australian National 11 University, Canberra, ACT, Australia 12 2 Department of Agronomical Sciences and Natural Resources, Faculty of Agropecuary and 13 Forestry Sciences & Center of Plant, Soil Interaction and Natural Resources Biotechnology, 14 Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Casilla 54D, 15 Temuco, Chile 16 17 *Corresponding author: Pieter A. Arnold, address: 46 Sullivans Creek Rd, Acton, ACT 2600, 18 Australia, email: [email protected], phone: +61 2 6125 2543 19 20 One sentence summary: 21 There are species-specific effects of experimental conditions during the measurement of the 22 critical thermal limits of leaf photosynthetic performance. 23 24 Author contribution statement: 25 PAA, KMG, AAC, and ABN designed the experiments. PAA, KMG, AAC performed the 26 experiments and collected the data. PAA curated the data and performed the data analyses and 27 visualisation. PAA, VFB, LAB, and ABN interpreted the results and wrote the manuscript with 28 input from all authors. 29 30 Funding information: 31 This research was supported by the Australian Research Council (DP170101681). 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.21.306886; this version posted September 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 32 Abstract 33 Plant thermal tolerance is a crucial research area as the climate warms and extreme weather 34 events become more frequent. We developed and tested a high-throughput method for 35 measuring photosynthetic critical thermal limits at low (CTMIN) and high (CTMAX) temperatures 36 to achieve pragmatic and robust measures of thermal tolerance limits using a Maxi-Imaging 37 fluorimeter and a thermoelectric Peltier plate temperature ramping system. Leaves exposed to 38 temperature extremes accumulate damage to photosystem II (PSII). Temperature-dependent 39 changes in basal chlorophyll fluorescence (T-F0) can be used to identify the critical temperature 40 at which PSII is damaged. We examined how experimental conditions: wet vs dry surfaces for 41 leaves and temperature ramp rate, affect CTMIN and CTMAX across four species. CTMAX estimates 42 were not different whether measured on wet or dry surfaces, but leaves were apparently less 43 cold tolerant when on wet surfaces. Temperature ramp rate had a strong effect on both CTMAX 44 and CTMIN that was species-specific. We discuss potential mechanisms for these results and 45 recommend settings for researchers to use when measuring T-F0. The system described and 46 tested here allows high-throughput measurement of critical temperature thresholds of leaf 47 photosynthetic performance for characterising plant function in response to thermal extremes. 48 49 Introduction 50 Understanding both vulnerability and tolerance of plants to thermal extremes is a priority for 51 plant biology research as the Earth’s climate continues to change, thereby exposing these sessile 52 organisms to increased thermal stress (O'Sullivan et al., 2017; IPCC, 2018; Geange et al., under 53 review). Thermal stress disrupts and inhibits physiological processes (Goraya et al., 2017), 54 induces protective and repair mechanisms (Sung et al., 2003; Goh et al., 2012), leads to declines 55 in plant performance, and threatens survival (Zinn et al., 2010; Bita and Gerats, 2013). Plant 56 photosynthesis is highly sensitive to thermal stress and has distinct thermal limits beyond which 57 tissue damage begins to accumulate (Neuner and Pramsohler, 2006). The temperature sensitivity 58 of photosynthesis mostly derives from the thermally-dependent stability of protein-pigment 59 complexes in the light harvesting complex II (LHCII) of photosystem II (PSII) of the thylakoid 60 membrane of chloroplasts (Ilík et al., 2003), which is integral to the photosynthetic electron 61 transport chain (Berry and Björkman, 1980; Mathur et al., 2014). 62 Chlorophyll fluorimetry has become a widely used tool for assessing the thermal limits 63 of photosynthesis for both cold and heat tolerance (Geange et al., under review). Chlorophyll 64 can dissipate absorbed light energy via photochemistry, or re-emit it as heat energy or 65 fluorescence (Baker, 2008; Murchie and Lawson, 2013). A dark-adapted leaf exposed to a low- 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.21.306886; this version posted September 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 66 intensity modulated measuring light, which does not induce electron transport, emits a minimal 67 amount of chlorophyll-a fluorescence from LHCII, called F0. Under more intense or actinic 68 light, processes that are highly dynamic and sensitive to other factors but not well correlated 69 with the viability of the photosynthetic tissue cannot be isolated from the measurement of the 70 temperature dependence (thermal stability) of chlorophyll fluorescence. To assess the thermal 71 stability limits of LHCII, plant ecophysiologists typically measure the temperature-dependent 72 change in basal chlorophyll-a fluorescence (T-F0) to determine the critical temperature threshold 73 (Tcrit), denoted by a sudden increased in F0 at which PSII undergoes temperature-dependent 74 conformational changes and begins to incur thermal damage (e.g., Schreiber and Berry, 1977; 75 Berry and Björkman, 1980; Knight and Ackerly, 2002; Ilík et al., 2003; Hüve et al., 2006; 76 Neuner and Pramsohler, 2006; O'Sullivan et al., 2013; O'Sullivan et al., 2017; Zhu et al., 2018). 77 F0 is a fluorescence parameter that can be measured rapidly and continuously throughout 78 heating or cooling in darkness, without the need of a saturating pulse and re-dark adaptation as 79 for FV/FM measurements that are commonly used to detect photosynthetic damage. 80 One critique of T-F0 measurements and Tcrit determination is that they are conducted on 81 detached leaves. Detaching leaves to expose them to a precisely controlled and measured 82 thermal surface is usually, but not always, a necessary component of this trait measurement. 83 Leaf detachment can affect leaf hydration and fluorescence through reduced PSII activity, ionic 84 leakage, and oxidations compared to attached leaves (Potvin, 1985; Smillie et al., 1987). Leaf 85 dehydration could be problematic for certain species if leaves are sampled long before they are 86 assessed for Tcrit or if they are measured as leaf sections or discs. To avoid dehydration during 87 the T-F0 measurement, a wet surface (e.g., damp paper surface as in Knight and Ackerly (2002)) 88 could physically impair evaporation by saturating the atmosphere surrounding the leaf. 89 However, it is not clear whether a wet surface interferes with the T-F0 measurement or how it 90 might affect the Tcrit value compared to using a dry surface. 91 A great advantage of using temperature-dependent changes in chlorophyll fluorescence 92 and a thermoelectric plate is that both cold and heat tolerance of leaves can be measured with 93 much of the same equipment. However, the protocol may need to be altered slightly because 94 cold transitions in nature occur much more slowly than heat transitions, which may induce 95 different mechanisms in response to thermal stress. For example, leaf temperature can rapidly 96 increase during a lull in wind speed, far exceeding ambient temperature on a hot and sunny day 97 (Vogel, 2009; Leigh et al., 2012). On a cold frosty night, even considering air temperature 98 stratification, the rate of leaf temperature cooling rarely exceeds 5°C h-1, especially below 99 freezing (Sakai and Larcher, 1987). Therefore, the ‘standard’ protocols for measuring Tcrit 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.21.306886; this version posted September 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 100 typically ramp temperature changes much faster for heat tolerance than for cold tolerance. While 101 this approach is justified by rates observed in natural systems, the first published application of 102 the T-F0 technique (Schreiber and Berry, 1977) used an apparently arbitrary ‘slow’ heating rate 103 of 1°C min-1 (i.e., 60°C h-1). Subsequently, while many studies followed suit, a vast range of 104 ramp rates have been applied (see Table S1), often with little justification. We have known for 105 decades that different rates of heating and cooling can affect the T-F0 curve and shift the Tcrit 106 value by at least 2°C (Bilger et al., 1984; Frolec et al., 2008). Therefore, studies employing T-F0 107 methods for measuring thermal tolerance limits that use different temperature ramp rates might 108 not be directly comparable, even within a given species.