Impacts of thermal fluctuations on heat tolerance and its metabolomic basis across plant and animal species

Temperature varies on a daily and seasonal scale and thermal fluctuations are likely to become even more pronounced under future climate changes. Studies suggest that plastic responses are crucial for species’ ability to cope with thermal stress, but traditionally laboratory studies on ectotherms are performed at constant temperatures and often limited to a few model species and thus not representative for the natural environment. We argue that thermoregulatory behavior and microhabitat shape the response exerted by different organisms to fluctuating temperatures. Thus, a sessile organism incapable of significant behavioral temperature avoidance will be more plastic and exert greater physiological response to thermal fluctuations than mobile organisms that can quickly evade temperature stress. Here we investigate how acclimation to fluctuating (13.2-26.9°C) and constant (20.4°C) temperatures impact heat stress tolerance across a plant (Arabidopsis thaliana) and two animal species (Orchesella cincta and Drosophila melanogaster) inhabiting widely different thermal microhabitats and selective pasts. Moreover, we investigate the underlying metabolic responses of acclimation using an NMR metabolomics approach. We find increased heat tolerance for all species exposed to fluctuating acclimation temperatures; most pronounced for A. thaliana which also showed a strong metabolic response to thermal fluctuations. Generally, sugars were more abundant across A. thaliana and D. melanogaster when exposed to fluctuating compared to constant temperatures, whereas amino acids were less abundant. However, we do not find much evidence for similar metabolomics responses to fluctuating temperature acclimation across species. Differences between the investigated species’ ecology, their distinct selective past and different ability to behaviorally thermoregulate may have shaped their physiological response to thermal fluctuations.


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species (Orchesella cincta and Drosophila melanogaster) inhabiting widely different thermal 24 microhabitats and selective pasts. Moreover, we investigate the underlying metabolic responses of 25 acclimation using an NMR metabolomics approach. We find increased heat tolerance for all species 26 exposed to fluctuating acclimation temperatures; most pronounced for A. thaliana which also showed a 27 strong metabolic response to thermal fluctuations. Generally, sugars were more abundant across A.
28 thaliana and D. melanogaster when exposed to fluctuating compared to constant temperatures, 29 whereas amino acids were less abundant. However, we do not find much evidence for similar   Fig. 2 and S2). An inspection of the PCA scores plotted for PC1 and PC2 shows a distinct 237 separation of metabolites associated with species and this was substantiated by a test for differential 238 metabolite response on PCA scores (MANOVA; P < .001). PC1 further separates clusters associated with 239 thermal regime ( Fig. 2 and 3), but this effect was not significant (MANOVA; P = 0.113).

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Because of large variation caused by species-specific metabolite differences, an OPLS-DA was performed 241 to focus the analysis towards differences in acclimation treatment while diminishing variation caused by 242 species. The OPLS-DA model was composed of one predictive component and three orthogonal 243 components (Table S1). Thermal regime accounted for merely 3% of the total metabolite variation in the 244 samples, but the predictive ability of the model to correctly group a sample into constant or fluctuating 245 acclimation treatment based on the metabolite content in the sample was significant (predictability Q 2 Y 246 = 0.5, Table S1).

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In addition, OPLS-DA models were performed on individual species (Table S1). All models were of good 248 quality and showed a high correlation between metabolite content and thermal regime. For A. thaliana 249 60% of the total metabolite variation was explained by the acclimation treatment, while the 250 corresponding number was 18% for both D. melanogaster and O. cincta (Table S1).

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The OPLS-DA loadings from each individual OPLS-DA model was used to identify metabolites that 252 differed significantly between individuals acclimated to fluctuating and constant thermal regimes ( Fig. 4  and S3). The analysis showed that the set of metabolites that was up-or downregulated differed for 254 each species. Metabolite changes that were significantly associated with the predictive component for 255 A. thaliana included elevated levels of glucose and suppressed levels of glutamine (gln), arginine (arg), 256 and gamma-aminobutyric acid (GABA) (Fig. 4 and Fig. S2D). Sucrose levels were elevated in D.
257 melanogaster acclimated to thermal fluctuations and alanine (Ala) levels lowered (Fig. 4 and S2C). 258 Lastly, O. cincta that was acclimated to thermal fluctuations had elevated levels of hydroxyphenyl 259 ethanol and 3-hydroxybutyric acid ( Fig. 4 and S2B).  Fig. 3). This suggests a lower ability of these 281 species to induce a thermal plastic response which may relate to the ability of the species' to 282 behaviorally evade stressors in nature by e.g. seeking deeper into the soil column or escaping rapidly by 283 flight to more favorable conditions [33,59,60].

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The magnitude of the acclimation response observed in LT 50 values was manifested in the metabolome

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showing bigger differentiation for A. thaliana exposed to fluctuating and constant temperatures 286 compared to the arthropod species investigated (Fig. 2 and 3 and Table S1). This is well in accordance 287 with the idea that plants, due to their limited mobility, exert a greater metabolite response to 288 environmental fluctuations than invertebrates. Further, we found that the set of metabolites that was 289 elevated or suppressed in response to thermal fluctuations for each species differed, but some of the 290 affected metabolites shared some biochemical properties. For instance, sugar levels were elevated in 291 plants and flies exposed to thermal fluctuations, whereas amino acids were suppressed (Fig. 4). These 292 patterns were not found for collembolans, which showed a markedly different metabolite response than 293 flies and plants.

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Accumulation of soluble sugars, which act as compatible compounds that help stabilizing proteins and 295 membranes and regulating osmotic pressure, is a common low temperature response in invertebrates